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Journal of Research in Biology ISSN No: Print: 2231 6280; Online: 2231- 6299

An International Scientific Research Journal

Original Research

Estimates of sensitivity, specificity, false rates and expected proportion of

population testing positive in screening tests
Authors: ABSTRACT:
Journal of Research in Biology

Oyeka Ikewelugo Cyprian

Anaene 1, Okeh Uchechukwu
Marius 2, Igwebuike Victor
Onyiaorah3, Adaora Amaoge
Onyiaorah4 and Chilota
Chibuife Efobi 5 This paper proposes and presents indices used as measures to evaluate or
Institution: assess results obtained from diagnostic screening tests. These indices include
1. Department of Applied sensitivity, specificity, prevalence rates and false rates. We here present statistical
Statistics, Nnamdi Azikiwe methods for estimating these rates and for testing hypotheses concerning them. An
University, Awka Nigeria. estimate of the proportion of a population expected to test positive in a diagnostic
2. Department of Industrial screening test is also provided. Further interest is also to estimate the sensitivity and
Mathematics and Applied specificity of the test and then the false rates as functions of sensitivity and specificity
Statistics, Ebonyi State given knowledge or availability of an estimate of the prevalence rate of a condition in
University Abakaliki, Nigeria.
a population. The indices proposed ranges from -1 to 1 inclusively and therefore
3. Department of enables the researcher to determine if an association exists and if it exists between
Histopathology, Nnamdi test results and condition as well as whether it is positive and direct or negative and
Azikiwe University Teaching
indirect which will serve as an advantage over the traditional methods. The proposed
Hospital Nnewi Anambra State,
Nigeria. indices provide estimates of the test statistic. When the proposed measures are
applied, results indicate that it is easier to interpret and understand more than those
4. Department of obtained using the traditional approaches. In addition, the proposed measure is
Opthalmology, Enugu State
University Teaching Hospital shown to be at least as efficient and hence as powerful as the traditional methods
Park lane Enugu State, Nigeria. when applied to sample data.
5. Department of Haematology,
University of Port Harcourt
Teaching Hospital, Port
Harcourt, Rivers State Nigeria.

Corresponding author: Keywords:

Okeh Uchechukwu Marius Traditional odds ratio, prevalence, sensitivity, specificity, false rates.

Article Citation:
Email Id:
Oyeka Ikewelugo Cyprian Anaene, Okeh Uchechukwu Marius, Igwebuike Victor
Onyiaorah, Adaora Amaoge Onyiaorah and Chilota Chibuife Efobi
Estimates of Sensitivity, Specificity, False Rates and Expected Proportion of Population
Testing Positive in Screening Tests
Web Address: Journal of Research in Biology (2014) 4(8): 1498-1504
Received: 06 Nov 2013 Accepted: 15 Jan 2014 Published: 15 Nov 2014

This article is governed by the Creative Commons Attribution License (

licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

1498-1504 | JRB | 2014 | Vol 4 | No 8

Journal of Research in Biology
An International
Scientific Research Journal
Anaene et al., 2014

INTRODUCTION previous results from a gold standard test to actually

In diagnostic screening tests indices used as have a certain condition in nature from a population and
measures to evaluate or assess results obtained include, also takes a second random sample of n.2 subjects from
sensitivity and specificity of the test and if the prevalence the same population Keeping in mind that known or
rate of a condition of interest in a population is known or believed not to actually have the same condition in
can be estimated from a previous study, also the false nature, thus giving a total random sample of size
positive and false negative rates of the test as well as the n=n..=n.1+n.2 subjects to be studied. It is always treated
proportion of the population expected to test positive to to confirm through a diagnostic screening test for
the condition (Fleiss, 1973; Pepe, 2003). Hence research whether or not each sampled subjects have or does not
interest is often in statistical methods for estimating have the condition of interest. Further interest is also to
sensitivity, specificity, false rates and the proportion of a estimate the sensitivity and specificity of the test and
population expected to test positive to a condition in then the false rates as functions of sensitivity and
these screening tests. The sensitivity of a test is the specificity given knowledge or availability of an estimate
proportion of subjects testing positive among the subjects of the prevalence rate of a condition in a population.
known or believed to actually have a condition in nature, Now suppose B and B are respectively the
while the specificity of a test is the proportion of subjects events that a randomly selected subject from a
who actually test negative to a condition among the population has and does not have a condition in nature.
subjects known or believed not to actually have the Also let A and A be respectively the events that the
condition in nature. False positive rate of a test is the randomly selected subject tests positive, and negative to
proportion of subjects who are known or believed not to the condition in the test. We here assume that the
actually have a condition in nature among the subjects prevalence rate P(B) of the condition in the population is
testing positive, while false negative rate is the either known or can be reliably estimated from previous
proportion of subjects who are known or believed to studies. The results of such a screening test may be
actually have a condition in nature among the subjects presented in the form of a four fold Table (Table 1).
who never-the-less test negative (Fleiss,1973;Greenberg In Table 1 above, of the n=n.. sample subjects
et al., 2001;Linn, 2004).Sensitivity and Specificity of a studied, n.1 subjects are known or believed to have the
test are independent of the population being studied and condition in nature, that is in B and n.2 are known or
hence independent of the prevalence rate of a condition B and
believed not to have the condition in nature, that is B .
in the population. False rates of a test on the other hand Also n1. subjects respond positive that is in A and n2.
are functions of the prevalence rate of a condition in a subjects respond negative, thatAisand
in A . Of the n.1
population and hence are dependent on the population of subjects in B, n11 subjects actually have the condition
interest (Fleiss, 1973;Linn, 2004). and test positive that is in AB and n21 subjects actually
We here present statistical methods for have the condition but test negative, that is in AB .
estimating these rates and for testing hypotheses Of the n.2 subjects who are known or believed not to
concerning them. An estimate of the proportion of a have the condition in nature,n12 subjects who do not have
population expected to test positive in a diagnostic the condition test positive, that is in A B w h i l e
screening test is also provided. n22 subjects who do not have the condition in nature also
Given that a researcher collects a random sample test negative that is in A B. In an actual screening
of n.1 subjects known or believed, perhaps on the basis of test usually only the total sample size n=n..,n.1 subjects
1499 Journal of Research in Biology (2014) 4(8): 1498-1504
Anaene et al., 2014

Table 1.Format for Presentation of Results of a Diagnostic Screening Test

Screening Test Results Condition Present Condition Absent Total
(B) B (ni.)
Positive (A) n11 n12 n1.
Negative () n21 n22 n2.
Total (n.j) n.1 n.2 n..(=n)

in B, n11 subjects in AB,n.2 subjects in B

a n d n 2 2 known or believed not to have the condition in nature.
subjects in A B are observed and actually known. Notationally, we have that
The values n12 in AB and n21 in AB are not P(A) = P (AB) + P (AB ) 3
known and hence also are n1. and n2., the overall number Now to develop sample estimates of these indices,
of subjects who would test positive and negative sensitivity for instance, we may let,
respectively in the screening test. Hence only the known 1, if the ith randomly selected and screened
values namely total sample size n, the number of subject known or believed to actually
have a condition in nature tests positive
subjects, n.1 known to have the condition in nature, n11,
0, otherwise 4
the number of subjects who test positive among these for i 1, 2,...n.1 subjects
known to have the condition in nature, the number of Let
subjects n.2 known not to have the condition in nature 1=1 P (ui1 =P1) ui1 1 5
and n22 subjects who test negative among the subjects and
known not to have the condition in nature are used here n1.
Wi =
W ui1
to estimate the required indices and test statistics. Now 1
i 1 6
the sensitivity (Se) and specificity (Sp) of a screening
test expressed in terms of conditional probabilities or Now the expected value and variances of ui1 are
specific rates of events A and B are respectively E ui1 : Var ui1 1
1 1 1 7
Se P ( A / B ); Sp P( A / B ) 1 1
The higher Se and Sp are more sensitive and Similarly the expected value and variance of are
specific is the screening test, the lower these rates, the respectively
weaker are the sensitivity and specificity of the test. The E W1
E (ui1 ) n.1 ;Var W1
Var (ui1 ) n.1 1 8
1 1 1
i 1 i 1 8
false positive rate and the false negative rate of a
screening test also expressed in terms of conditional Now 1 is the probability that a randomly
probabilities or specific rates of events A and B are selected and screened subject known or believed to have
respectively a condition in nature in a population tests positive; that is
1 P( A / B ) P( B ) 1 P( A / B ) P ( B ) the proportion of subjects testing positive among the
F ve P( B / A) ; F ve P( B / A) 2 2
P( A) P( A) subjects in the population known or believed to actually
Where P(A) consists of the probability of have a condition in nature. This is in fact a measure of
composition of the events AB and which is the the sensitivity Se of the screening test. The sample
probability of the union of events that a randomly estimate of 1 is
selected subject tests positive and is known or believed W1 f
1 Se 9
n.1 n.1
to have a condition in nature or tests positive and is

Journal of Research in Biology (2014) 4(8): 1498-1504 1500

Anaene et al., 2014

Where f+ is the number of subjects who test condition given that the subject is known or believed not
positive among subjects in the population known or to actually have the condition in nature. In other words,
believed to have the condition of interest in nature. In 2 is the proportion of subjects testing negative among
other words, f is the number of 1s in the frequency the population of subject known or believed not to have a
distribution of the n.1values of 1s and 0s in ui1,for i=1,2, condition in nature. Thus 2 is actually a measure of the
,n.1.Hence f =n11 of Table 1. specificity Sp of the screening test. Its sample estimate is
The corresponding variance of is from equation (8) from equation (18)

Var W1 1 (1 1 ) (1 Se
Se ) W2 f
Var 1
Var Se 2 10 Se 19
n.11 n.1 n.1 10 n.2 n.2
A researcher may sometimes wish to test a null Where f is the number of subjects whose test
hypothesis that sensitivity of a screening test is at most negative among the n.2 subjects in the sampled
some value 10 = Se0 . That is the null hypothesis, population known or believed not to have a condition in
H 0 : Se Seo versus H1 : Se Seo(0 Seo 1) 11other words f - is the total number of 1s in the
nature. In
10 11
This null hypothesis may be tested using the test statistic frequency distribution of the n.2 values of 0s and 1s in
ui2,for i=1,2,n.2.Thus f - = n22 in Table 1. The variance
n.1 Se Seo 1 10
W1 n.1 Seo n.1
Var W1 (1
Se )
Se 1 (1 1 ) 12
of equation 2 Sp (18)
Which under Ho has approximately the chi-square Var (W2 ) 2 (1 2 ) (1 Sp
Sp )

Var 2 Var Sp
n.22 n.2 n.2 20 20
distribution with one (1) degree of freedom for
sufficiently large n.1.the null hypothesis Ho is rejected at A researcher may also wish to test a null hypothesis that
the level of significance if specificity Sp of a diagnostic screening test is at least
2 2 some value That is the null hypothesis
1 ;1 13 13
Ho : Sp Spo versus H1 : Sp Spo, (0 Sp 1)
Similarly to develop a sample estimate of the specificity This null hypothesis is tested using test statistic
Sp of a screening test, we may let W2 n.2 Spo
n.2 Sp Spo
n.2 2 20
22 22
1, if the ith randomly selected and screened Var W2 (1
Sp )
Sp 2 (1 2 )
subject in the population is known or believed not to actually
ui 2
have a condition in nature tests negative
Which under Ho has approximately the chi-
0, otherwise 14 square distribution with one (1) degree of freedom for
for i 1, 2,...n.2 subjects
sufficiently large n.2. The null hypothesis Ho is rejected
Define at the level of significance if equation (13) is satisfied,
2 P ui 2 1 otherwise Ho is accepted.15
And To develop sample estimate of the proportion of
W2 ui 2 16 a population expected to
16test positive to a condition in a
i 1
diagnostic screening test, we note that when expressed in
terms of conditional probability using Bayes rule
E ui 2 2 ;Var ui 2 2 1 2 17 17
equation (3) becomes
P( A) P( A / B).P( B) P( A / B ).P( B ) P( A / B).P( B) 1 P( A / B ) 1 P( B) 23
E W2 n.2 2 ;Var W2 n.2 2 (1 2 ) 18 18
Or when expressed in terms of sensitivity Se and
Note that 2 is the probability that a randomly specificity Sp of the screening test and prevalence rate
selected and screened subject tests negative to the P(B) of a condition in a population becomes
1501 Journal of Research in Biology (2014) 4(8): 1498-1504
Anaene et al., 2014
P( A) SeP( B) (1 Sp).P( B) 1 P( A / B) 1 P( A / B) P( A / B) .P( B) 1 Sp 1 Se Sp .P( B) 24 The null hypothesis Ho is expected at the level of
The sampled estimate of P(A) is using equation ( 9) and significance if equation (13) is satisfied; otherwise Ho is
equation (19) in equation (24) accepted.
P ( A) P( A) ( B)
SeP ).P( B )
(1 Sp
1 Sp
1 Se .P( B)
Sp 25 The researcher may also wish to obtain sample estimates
The corresponding sample variance is of false rates in a diagnostic screening test if the
)( P( B))
Var ( P( A)) Var (Se 2 ) P(( B))
Var (Sp 2 )
2P( B).P( B).Cov SeSp 26 prevalence rate P(B) of a condition in a population is
It is easily shown that known or can be determined.
W1 W2 Now from equations (2) and (25), the sample estimate of
; Sp
Cov ( Se ) Cov ; 0
n.1 n.2 false positive rate in terms of sample estimates of
To prove this it is sufficient to show that sensitivity and specificity and the known or estimated
Cov ui1 ; ui 2 0 prevalence rate is
Now ).P( B ) 1
Sp 1 P( B)
(1 Sp
F ve 31
Cov ui1 ; ui 2 E ui1 .ui 2 E (ui1 ).E (ui 2 ) E ui1 .ui1 .P( B) 31
1. 2 .
P( A) 1 Sp 1 Se Sp

Now ui1 .ui1can assume only the values 1 and 0 . Similarly the sample estimate of false negative rate is
It assumes the value 1 if ui1 and ui2both assume the value from equations (2) and (25)
1 with probability it assumes the value 0 if assumes (1 ).P( B)
Se 1 P( B)

the values 1 and ui2 assumes the value 0 or ui1 assumes
P ( A)
Sp 1
Se .P( B)
Sp 32
the value 0 and ui2 assumes the value 1 with probability Where e and p are given in equation (30).
1(1-2) - 2(1-1) Hence Finally with further interest the researcher may use some
Cov ui1 ; ui 2 1 2 1 2 0 so that elementary calculus or apply Fiellers convenience
(1 Se
Se ) Sp
(1 Sp ) 1 (1 1 ) 2 (1 2 ) Theory to obtain approximate estimates of the variances
) Var ( Sp
Var ( P( A)) Var ( Se ) 27
n.1 n.2 n.1 n.2
of F ve and F ve and also test any desired hypotheses.
The researcher may also wish to test the null
hypothesis that the proportion P(A) of subjects in a ILLUSTRATIVE EXAMPLE
population expected to test positive to a condition in a It a clinician is collecting a random sample of 98
diagnostic screening test is at most some value Po(A). subjects from a certain population; twelve of whom are
That is the null hypothesis doubted for having prostrate cancer and 86 of whom are
Ho : P( A) Po( A) versus H1 : P( A) Po( A), (0 Po( A) 1) 28 assumed not to28have the disease. The clinicians interest
This null hypothesis is tested using the test statistic is to confirm through a diagnosis screening test whether
2 or not each of the sampled subjects are actually prostrate
P ( A) Po( A)
Var P ( A) 29 cancer positive or negative. The results of the screening
test are presented in Table 2.
Which under Ho has approximately the chi-square Now from Table 2 we have that the sample
distribution with one (1) degree of freedom where P(A) estimate of the sensitivity and specificity of the test are
and Var (P(A)) are given by equations (25) and (26) respectively
respectively and from Table 1 f
n f
n 98 4
Se 8.167 0.041 0.335
n.1 n.1 12
f1 n11 f2 n22 and
Se 1 ; Sp 2 30 30
n.1 n.1 n.2 n.2 n f
98 84
f n 98

Sp 1.140 0.857 0.977.
n.2 n.2 86

Journal of Research in Biology (2014) 4(8): 1498-1504 1502

Anaene et al., 2014

These results show that the screening test is low Table 2: Result of Prostrate Cancer Screening Test
in sensitivity but has high specificity. Clinical diagnosis Present (B) Absent B
Now from equations (13) and (14) the sample estimates Prostrate Cancer n11=f + +=4 n12=f+ - =4 n1.= 6
of are respectively Positive (A) n21=f - +=4 n22=f - - =4 n2.=92
12 0.335 86 0.977 Negative () n.1=12 n.2=86 n..=n=98
12 1 0.335 86 1 0.977 expected to be correctly informed. This probably makes
98 more difficulty in understanding than the simple
Hence from equations (15) and (17) we have that information conveyed by the simple difference in rates,
0.898 0.102 0.796 0.796, namely, the proportion of subjects
With estimated variance obtained from equations (16) testing positive among subjects who have prostrate
and (18) as cancer or testing negative among subjects who do not
1 (0.796) 2 have prostrate cancer is 79.6 percent higher than the
Var ( ) Var ( ) 0.004
98 proportion of subjects testing positive among subjects
Hence the test statistic of no association between who do not have prostrate cancer or testing negative
screening test results and state of nature or condition among subjects who have the disease.
(Prostrate Cancer) of equation (19) are obtained from 1 1 1 1 1 1 1 1
Se(O) O 21.00
equation (20) as n11 n12 n21 n22 4 2 8 84
2 (21.00)(0.942) 19.782
158.500( P value 0.0000)
0.004 0.004 This measure of the error of O namely 19.782
Which with one (1) degree of freedom is highly is clearly much larger than the error of only
statistically significant indicating a strong degree of Se 0.004 0.063 of the estimated value of for our
association between screening test results and state of sample data. The chi-square test statistic for the
nature or condition (presence of Prostrate cancer in significance of O is
the population). Also since 0.796 n(n11n22n12 n21)2 98(94)(84)-(2)(8)2
X2= = =17.616 (p-value=0.0000)
is positive, the association is positive and direct. n1.n2.n1.n2 (6)(92)(12)(86)
It is commendable to compare the present results with Which is also statistically significant again leading to a
what would have been obtained if we have used the rejection of the null hypothesis of no association.
traditional odds ratio to analyze the data of Table 2. In However, the proposed method and the traditional odds
spite of odds ratios short comings as already pointed out ratio approach explained here (both) lead to a rejection of
above, when used in the analysis of screening test results. the null hypothesis, the relative sizes of the calculated
The sample estimate of the traditional odds ratio for the chi-square values suggest that the traditional odds ratio
data of Table 2 is method is less efficient and likely to lead to an
n11 n22 (4)(84) acceptance of a false null hypothesis (Type II Error)
O 21.00
n12 n21 (2)(8) more frequently and hence is likely to be less powerful
This means, for every subject who has prostrate cancer than the proposed method.
among tested subjects and erroneously informed that
they are free of the disease (21 subjects) among those
tested and found to have prostrate cancer would be
1503 Journal of Research in Biology (2014) 4(8): 1498-1504
Anaene et al., 2014

In this paper, we proposed, developed and
presented a statistical method for measuring the strength
of association between test results and state of nature or
condition in a population expressed to a diagnostic
screening test. The proposed measure is based on only
the sensitivity and specificity of the screening test which
are independent of the population of interest and
estimated using only observed sample values.
The proposed measure which ranges from -1 to 1
can be used to establish whether an association is strong
and direct, strong and indirect or zero estimates of the
standard error. Test statistics for the significance of the
proposed measure are provided. The proposed measure
of association is shown to be easier to interpret and
explain than the traditional odds ratio, and the sample
data used suggest that the measure is at least as efficient
and powerful as the traditional odds ratio.

Baron JA and Sorensen HT. 2010. Clinical
epidemiology, in Teaching Epidemiology: A guide for
teachers in epidemiology, public health and clinical
medicine. eds. Olsen J; Saracci R and Trichopoulos D;
Oxford: Oxford University Press, 237-249.

Fleiss JL. 1973. Statistical Method for Rates and

Proportions. John Wiley, New York.

Greenberg RS, Daniels SR, Flanders WD, Eley JW

and Boring JR. 2001. Medical Epidemiology, London:
Submit your articles online at
Lange-McGraw- Hill.
Pepe MS. 2003. The Statistical Evaluation of Medical Easy online submission
Complete Peer review
Tests for Classification and Prediction. Oxford statistical Affordable Charges
series 28, Oxford: University Press, U.K. Quick processing
Extensive indexing
Shai Linn. 2004. A new Conceptual Approach to You retain your copyright
teaching the interpretation of clinical tests. Journal of
statistics education 12(3).

Journal of Research in Biology (2014) 4(8): 1498-1504 1504

Journal of Research in Biology ISSN No: Print: 2231 6280; Online: 2231- 6299

An International Scientific Research Journal

Original Research

Ferulic acid modulates ultraviolet-B radiation mediated inflammatory

signaling in human dermal fibroblasts
Journal of Research in Biology

Authors: ABSTRACT:
Kanagalakshmi A1, Ultraviolet B (UVB 290-320 nm) participate in the development of the
Agilan B1, Mohana S1, cutaneous inflammatory response which includes a cascade of events that involves
Ananthakrishnan D2, increased expression of cyclooxygenase-2 (COX-2), release of tumor necrosis factor-
Velmurugan D2, alpha (TNF-) and other inflammatory cytokines. Peroxisome proliferator-activated
Karthikeyan R1, receptors (PPAR/) are considered to be potential targets for photo protection
Ganesan M1, Srithar G1 because they inhibit UVB mediated inflammatory responses. In this study, we
and Rajendra Prasad N1* investigated the effect of ferulic acid on UVB-radiation induced expression of TNF-
and COX-2 in human dermal fibroblasts (HDFa). Further, the action of ferulic acid on
PPAR/ activation and its binding interaction with these proteins were analyzed by
1. Department of
Biochemistry and induced fit docking. We found that onetime UVB exposure (19.8 mJ/cm 2) showed
Biotechnology, Annamalai significantly increased the expressions of COX-2 and TNF- in HDFa after 4 h post-
University, Annamalainagar irradiation when compared to the control cells. Ferulic acid pretreatment for 30 min
608 002, India. before UVB exposure prevented UVB-induced overexpression of these inflammatory
markers. It has also been found that ferulic acid activates PPAR/ expressions in
2. Bioinformatics HDFa. Further, induced fit docking analysis showed that there was a greater binding
Infrastructure Facility interaction of ferulic acid with PPAR than PPAR. Thus, ferulic acid exhibits
(BIF),University of Madras, beneficial effects against UVB-induced inflammatory responses probably through
Chennai-25 down-regulating COX-2 and TNF- expressions and activating PPAR/ agonists.

Corresponding author: Keywords:

Rajendra Prasad N Ultraviolet B radiation, Ferulic acid, Human dermal fibroblasts, Inflammatory
markers, Photoprotection

Article Citation:
Email Id:
Kanagalakshmi A, Agilan B, Mohana S, Ananthakrishnan D, Velmurugan D,
Karthikeyan R, Ganesan M, Srithar G and Rajendra Prasad N.
Ferulic acid modulates ultraviolet-B radiation mediated inflammatory signaling in
human dermal fibroblasts
Journal of Research in Biology (2014) 4(8):1505-1515
Web Address: Dates:
documents/RA0488.pdf Received: 06 Oct 2014 Accepted: 25 Oct 2014 Published: 17 Nov 2014

This article is governed by the Creative Commons Attribution License (

licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

Journal of Research in Biology 1505-1515 | JRB | 2014 | Vol 4 | No 8

An International
Scientific Research Journal
Kanagalakshmi et al., 2014

INTRODUCTION damages by their antioxidant, anti-inflammatory and

Epidemiological studies have showed that immunomodulatory actions (Ramachandran and Prasad
ultraviolet (UV) radiation exposure mediates several 2008). Ferulic acid (3-methoxy-4-hydroxycinnamic acid)
damaging effects that include melanoma and non- is a naturally occuring phenolic compound derived from
melanoma skin cancers (Afaq and Santosh, 2012). the phenylpropanoid pathway. It is commonly abundant
Although UVB radiation (280320 nm) be a small in fruits, vegetables and Cereals. (Prasad et al., 2011).
portion of sun light that reaches the earth, it is considered Ferulic acid recovers the antioxidant cell defense
to be a most deleterious agent because it can penetrate system and stimulates cytoprotective enzymes due to its
the skin to a depth of 160180 m and alters the skin resonance-stabilized phenoxy radical structure such as
architecture (Chilampalli et al., 2011; Gregoris et al., phenolic nucleus and unsaturated side chain (Picone
2011). UVB is a strong pro-inflammatory agent with et al., 2009). Ferulic acid can block the penetration of
profound effects on skin in part through its ability to UV radiation into the epidermis. This sunscreen ability
stimulate cytokine production. UVB exposure leads to of ferulic acid can reduce UV-induced erythema (Saija
activation of many cytokines such as cyclooxygenase-2 et al., 2000; Oresajo et al., 2008). Moreover, ferulic acid
(COX-2), tumor necrosis factor-a (TNF-) and could exert beneficial therapeutic effects of free radical-
interleukin-6 (IL-6), (Alexia et al., 2003; Kondo et al., related syndromes such as neurodegenerative disorders,
1993). These cytokines support the development of the cancer, cardiovascular diseases and diabetes (Barone
UVB-induced cutaneous inflammatory responses that is et al., 2009). Recently, we found that ferulic acid inhibits
observed in the skin as sunburn and erythema (Kondo UVB mediated ROS generation, TBARS levels and
1999). apoptosis in human dermal fibroblasts (Kanagalakshmi
Peroxisome proliferator-activated receptor and Prasad, 2014). In the present study, we examined the
(PPARa/) regulates inflammatory signaling and beneficial effect of ferulic acid on the UVB mediated
cytokine activation in different experimental systems inflammatory responses by targeting PPAR/ agonists
(Zhang et al., 2004). PPARs belong to the nuclear in human dermal fibroblasts.
receptor super family, a family of ligand activated
transcriptional factors and it consists of three isotypes MATERIALS AND METHDOS
(PPAR, and ). PPARs function as ligand dependent Chemicals
transcription factors and can heterodimerize with retinoid HDFa cells were procured from Invitrogen
X receptors and then bind to PPAR-responsive elements Bioservices, India. Low Serum Growth Supplement, fetal
(PPRE) in target gene promoters, which usually leads to bovine serum (FBS), human epidermal growth factor,
transcriptional activation. Moreover, PPARs inhibits fibroblast growth factor, heparin, trypsin-EDTA and were
inflammatory gene expression in experimental models obtained from Invitrogen Bioservices, India. Ferulic acid,
(Ricote et al., 1998). Previous studies provide strong monoclonal antibodies anti-TNF, anti-COX-2, -actin
evidence for the role of PPAR in controlling anti-mouse and goat anti-mouse IgG-HRP polyclonal
inflammation and suggest their potential as therapeutic antibody were purchased from Sigma chemical Co., St.
targets for inflammatory diseases (Kim et al., 2012). Louis, MO, USA. Bovine serum albumins (BSA), radio
Dietary phytochemicals offer exciting platforms immune precipitation assay (RIPA) buffer were
for the management of UV related disorders. Dietary purchased from Himedia, Mumbai. All other analytical
phytochemicals modulate UVB radiation-mediated
1506 Journal of Research in Biology (2014) 4(8): 1505-1515
Kanagalakshmi et al., 2014

grade chemicals, solvents and reagents were purchased used for photoprotection experiments (Ramachandran
from SD Fine Chemical, Mumbai. et al., 2010).
Culturing human skin fibroblasts Irradiation procedure
HDFa cells were maintained at 37C under 5% HDFa cells were washed twice with PBS and
CO2 condition in medium-106 supplemented with UVB irradiated in a thin layer of medium without FBS. A
2% v/v fetal bovine serum, 1 g/ml hydrocortisone, battery of TL 20 W/20 fluorescent tubes (Heber
10 ng/ml human epidermal growth factor, 3 ng/ml basic Scientific, Chennai, India) was used as UVB source,
fibroblast growth factor, 10 g/ml heparin and which possess a wavelength range of 290320 nm,
antibiotics. The HDFa cells were cultured to grow for peaked at 312 nm, and with an intensity of 2.2 mW/ cm2
7 days to obtain the maximum confluence for for 9 min. The total UVB radiation exposure was
experiments. Then, HDFa cells were harvested using 19.8 mJ/ cm2, with an average value of 1.52 103 mJ/
trypsin-EDTA, subcultured and the remaining cells were cell. Immediately after UVB exposure, the HDFa cells
used for photo protection experiments (Ramachandran were kept at 37C for 4 h at in 5% CO2 environment.
et al., 2010). Irradiated HDFa cells were then washed with PBS, and
Study design transferred to sterile centrifuge tubes for biochemical
Cultured fibroblasts were divided into four analysis (Kanagalakshmi and Prasad 2014).
groups as follows: Western blot analysis for pro-inflammatory markers
Group 1: Normal fibroblasts without any treatment; expression
Group 2: Normal fibroblasts with 40 g/mL of FA; Western blot analysis was carried out for TNF
Group 3: UVB-irradiated fibroblasts; and COX-2 expressions in ferulic acid plus UVB-
Group 4: UVB-irradiated fibroblasts pretreated with 40 irradiated HDFa. The results were normalized to -actin
g/mL of FA. gene expression. Treated HDFa cells were washed with
Treatment of the HDFa cells PBS and detached using 0.25% trypsin/EDTA solution.
Thirty minutes before UVB exposure, 40 g/mL Cell suspensions were centrifuged and the pellets were
of ferulic acid was added to the HDFa cells. Trypan blue lysed with an ice-cold lysis RIPA buffer containing a
dye exclusion test was carried out to find out the toxicity protease inhibitor cocktail (SigmaAldrich, St. Louis,
and suitability of 40 g/mL of ferulic acid for MO, USA) for 30 min. The lysate was centrifuged at
photoprotection studies. Before UVB exposure, the 4C at 13,000 rpm for 10 min and the supernatant was
HDFa cells were washed once with PBS solution. Mock- used to determine protein concentration using Nanodrop
irradiated HDFa showed no viability changes over the 2000 (Thermo Scientific, USA). Cell extracts containing
30 min period of incubation (HDFa cells were 50 g of proteins were subjected to electrophoresis on
maintained at 37C under 5% CO2 condition in medium- 12% SDS-PAGE gel and transferred to a PVDF
106 supplemented with 2% v/v fetal bovine serum, 1 g/ membrane using transblot semi-dry apparatus (Biorad,
ml hydrocortisone, 10 ng/ml human epidermal growth USA). PVDF membranes were blocked with non-fat
factor, 3 ng/ml basic fibroblast growth factor, 10 g/ml milk (5% (w/v) for 6 h and then incubated overnight with
heparin and antibiotics. The HDFa cells were cultured to TNF and COX-2 antibodies (Sigma-Aldrich, USA), in
grow for 7 days to obtain the maximum confluence for blocking solution at 37C. Then the membranes were
experiments. Then, HDFa cells were harvested using washed with TBST thrice with 10 min interval and
trypsin-EDTA, subcultured and the remaining cells were incubated with secondary antibody (diluted 1:2000) in

Journal of Research in Biology (2014) 4(8): 1505-1515 1507

Kanagalakshmi et al., 2014

blocking solution for 2 h at 37C. Then, the PVDF Ligprep 2.3 module (Schrdinger, USA) was
membranes were washed with TBST thrice with 10 min employed for ferulic acid preparation. The
interval and the developed bands were detected using a three dimensional crystal structure of PPAR / (PDB Id:
DAB solution. The images were acquired by Image 1K7L/ PDB Id: 3DZY) and Cox-2 (PDB Id: 6COX) were
Studio software (LI-COR, USA) (Ramachandran et al., downloaded from the Protein Data Bank (PDB)
2012). ( Protein preparation wizard of
RNA isolation and real-time quantitative PCR. Schrodingers was used for PPAR/ and COX-2
The total RNA was extracted from the HDFa preparation. Non-hydrogen atoms were minimized until
cells using RNeasy Mini kit (Qiagen, USA) as per the the average root mean square deviation reached default
protocol recommended by the manufacturer. The mRNA value of 0.3. Sitemap 2.3 was used to understand
expression of PPAR/ in HDFa cells was determined binding site in the ligand binding domain (LBD) of the
using real-time PCR, as described previously (Sharma PPAR/ and COX-2 (Schrodinger Suite 2009).
and Katiyar, 2010). RNA purification and quantity was Induced fit docking (IFD) was performed to
analyzed by nanodrop 2000 (Thermo Scientific, USA). predict ferulic acid binding modes and structural
Experiments were run in triplicate to confirm movements in the LBD region of PPAR/ and COX-2
amplification integrity. Manufacturer-synthesized primer using Glide and Prime modules. The prepared proteins
pairs were used to measure the mRNA expression level were loaded in the workstation and the Grid values were
of PPARs. PCR cyclic condition 25C for 10 min; 42C calculated about 20 in order to cover all the active site
for 50 min; 75C for 15 min were used for cDNA amino acids. The Vander Waals radii of nonpolar amino
synthesis. The cyclic condition used for amplification acids and ligand atoms were scaled by a default value of
was 95C for 2 sec; 55C for 15 sec; and 68C for 20 sec 0.50. About 20 conformational images were created and
as prescribed by the primers manufacturer. The analyzed for the best conformational pose based on the
expression levels of genes were normalized to docking score and glide energy.
18S mRNA expression level. The cyclic threshold (Ct)
for positivity of real-time PCR was determined based on RESULTS
negative controls. Ferulic acid inhibits UVB-induced TNF- and COX-2
Molecular docking expressions in HDFa
Molecular docking was performed on Red Hat Western blot analysis shows that there was an
Enterprise Linux EL5 workstation using Maestro overexpression of TNF- and COX-2 in the UVB
(Schrodinger LLC 2009, USA). GLIDE5.5 searches exposed HDFa (Figure 1). It indicates inflammatory
were performed for understanding docking interactions responses in HDFa cells as compared with control HDFa
between ferulic acid and PPAR/. All molecular cells. TNF- and COX-2 expression levels were
modeling was carried out using OPLSAA (Optimized significantly down-regulated in ferulic acid pretreated
Potential Liquid Simulation for All Atom) force field plus UVB irradiated HDFa (Figure 1).
(Glide, 2009). PyMOL (DeLano WL, 2002) software Ferulic acid activates PPAR/ mRNA expression in
employed for the analysis of hydrogen bond interactions. HDFa
Hydrophobic interactions were analyzed between protein Quantitative Real Time-PCR analyses were
and ligand using Ligplot software (Wallace AC, 1995). adopted to analyze the activation of PPAR/ mRNA
expression in ferulic acid and/or UVB-irradiated HDFa
1508 Journal of Research in Biology (2014) 4(8): 1505-1515
Kanagalakshmi et al., 2014

cells (Figure 2A,B). In this study mRNA levels of The amino acid residues such as Phe 273, Cys 276 and
PPAR/ were down-regulated in UVB-exposed HDFa. Ile 354 showed hydrophobic bond interactions with
Whereas, FA treatment prevented the UVB induced loss ferulic acid and the co-crystallized ligand (Figure 3).
of PPAR/ and up-regulated these mRNA expression in The energy score of PPAR with ligand retinoic
HDFa cells. acid was -49.18 (kcal/mol) and with the ferulic acid it
Molecular docking with PPAR/ was -40.44 kcal/mol. PPAR possess a common
Ferulic acid was docked against PPAR (PDB hydrogen bond interaction (Arg 316) with retinoic acid
code: 1K7L). The glide energy score, docking score and and ferulic acid. The amino acid residues such as Ala
hydrogen bond interactions of PPAR with its 272, Ile 268, Leu 326, Leu 309, Phe 313 and Ile 310
cocrystallized ligand 2-(1-methyl-3-oxo-3-phenyl- showed hydrophobic interactions with ferulic acid and
propylamino)-3-{4-[2-(5-methyl- retinoic acid. The aminoacid Ala 271 was interacting
2-phenyl-oxazol -4-yl)-ethoxy]- phenyl}-propionic acid with ferulic acid through hydrogen bonding and
was shown in the table. 1. The energy score of PPAR interacting with retinoic acid through hydrophobic
with cocrystallized ligand was -86.02 (kcal/mol) and interaction (Figure 4).
with ferulic acid was -39.31 (kcal/mol). PPAR has a Molecular docking with COX-2
common hydrogen bond interaction (Tyr 464 and The energy score of COX-2 with the
Ser 280) with co-crystallized ligand and ferulic acid. co-crystallized ligand 1-Phenylsulfonamide-3-

Table. 1. Induced fit docking results of PPAR, PPAR and Cox-2 with their cocrystallized ligands and ferulic acid.
Induced-fit docking was carried out using Schrodinger software. Ferulic acid interacts with PPAR, PPAR and COX-2
through hydrogen bonding and hydrophobic interactions. Ferulic acid possess greater binding interaction with PPAR
(glide energy -40.44) than PPAR (glide energy 39.31) when compared with their corresponding cocrystallized ligands.

Docking Score Glide Energy Hydrophobic Bond Hydrogen-Bond
Protein Compound/Ligand between donor
(Kcal/mol) (Kcal/mol) Interactions Interactions
and acceptor (A)

Gln 277, Ile 272,

2-(1-methyl-3-oxo-3- Ile 339, Phe 273,
phenyl-propylamino)-3 Leu 254, Cys 275, Leu Tyr 464 (O-H-O) 3.02
- {4-[2-(5-methyl-2- 347, Met 355, Leu 347,
-16.22 -86.02 His 440 (N-H-O) 2.98
phenyl-oxazol-4-yl)- Met 330, Val 332, Phe Ser 280 (O-H-O) 2.72
PPAR ethoxy]- phenyl}- 351, Leu 321, Ile 354,
propionic acid Cys 276
Phe 273, Cys 276, Leu Tyr 464 (O-H-O) 2.88
Ferulic acid -8.15 -39.31 460 and Ile 354 Ser 280 (O-H-O) 2.74
Tyr 314 (O-H-O) 2.79
Phe 346, Cys 432,
Ile 324, Ile 310, Ala 327 (N-H-O) 3.15
Phe 313, Leu 326, Arg 316 (N-H-O) 2.88
Retinoic Acid -13.93 -49.18
leu 309, ile 268, Arg 316 (N-H-O) 2.86
PPAR ala 272, Ala 271. Gln 275 (N-H-O) 2.62
Ala 272, Ile 310, Phe Asn 306 (O-H-O) 2.74
Ferulic acid -7.75 -40.44 313, Leu 309, Cys 269, Ala 271 (O-H-O) 2.69
Ile 268, Leu 326 Arg 316 (N-H-O) 3.04
Val 349, Ser 530, Ala
1-Phenylsulfonamide- Tyr 355 (O-H-N) 3.08
527, Gly 526, Val 523,
3-Trifluoromethyl-5- His 90 (N-H-O) 3.31
-11.68 -62.09 Ala 516, Leu 352, Ser
Parabromophenylpyra- Gln 192 (O-H-O) 3.25
Cox-2 zole Phe 518 (N-H-O) 3.32
Trp 387, Phe 518,Leu Tyr 385 (O-H-O) 3.22
Ferulic acid -7.95 -37.82 384, Ser353 and Val His 90 (N-H-O) 2.70
523 Leu 352 (O-H-O) 2.86

Journal of Research in Biology (2014) 4(8): 1505-1515 1509

Kanagalakshmi et al., 2014

Figure 1. Effect of ferulic acid on UVB-induced activation of TNF and COX-2 in HDFa
cells. HDFa cells were exposed to UVB (19.8 mJ/cm2) with or without ferulic acid for 30
min. Cells were harvested at 4 h after UVB exposure, and the expression of TNF and
COX-2 were analyzed by Western blot. ferulic acid treatment down-regulated these
protein expressions when compared with UVB control group. The graph represents the
quantification results normalized to -actin levels.

Trifluoromethyl-5-Parabromophenylpyrazole was pro-inflammatory mediators and infiltration of

-62.09 kcal/mol and with ferulic acid the energy score inflammatory cells (Lee et al., 2013). Previous studies
was found to be -37.82 kcal/mol. COX-2 posses a have demonstrated that UVB-induced oxidative stress
common hydrogen bond interaction (His 90) with plays a critical role in the induction of proinflammatroy
co-crystallized ligand and Ferulic acid. The amino acid cytokines including TNF- expression (Ramachandran
Leu 352 was interacting with ferulic acid through et al., 2012). TNF- can promote both DNA damage and
hydrogen bonding and interacting with co-crystallized activation of nuclear factor kB (NF- kB) inducing the
ligand through hydrophobic interaction. The amino acid formation of sunburned cells and leading to
such as Ser 353 and Val 523 shows hydrophobic bond photodamage in the skin (Muthusamy and Piva, 2010).
interactions with ferulic acid and the co-crystallized In the present study, TNF- was over expresed in the
ligand (Figure 5). UVB irradiated HDFa cells when compared to control
cells. The pre-administration of anti-inflammatory agents
DISCUSSION was found to be an effective strategy for preventing
UVB (280320 nm) causes acute inflammatory UVB-irradiation induced skin inflammation. In this
skin damages including erythema, production of
1510 Journal of Research in Biology (2014) 4(8): 1505-1515
Kanagalakshmi et al., 2014

Figure 2. Ferulic acid activated UVB-induced PPAR/ mRNA expression in cultured human dermal
fibroblasts. Total RNAs were prepared after UV irradiation and subjected to real-time PCR. A), mRNA
expression of PPAR. B), mRNA expression of PPAR.

study, ferulic acid treatment diminished the UVB- acid can be converted to eicosonoids, forming multiple
induced overexpression of TNF- in HDFa. prostaglandins through the COX-2 pathway and
Previous studies have indicated that UVB ultimately leading to skin cell death. The expression of
mediated ROS over production and TNF activation COX-2 has been used as inflammatory marker for
resulted in cPLA2 synthesis and increasing the formation evaluating UVB irradiation induced skin inflammation.
of arachidonic acid. This overproduction of arachidonic In this report, we observed that UVB exposure increased

Figure 3. Binding interaction of ferulic acid and cocrystallized ligand with

PPAR. Ligplot image showing hydrogen bonding and hydrophobic
interactions of PPAR with its cocrystallized ligand (A) and ferulic acid (C).
Pymol view of LBD region of PPAR with cocrystallized ligand (B) and
ferulic acid (D). Ferulic acid has similar binding sites as compared with the
cocrystallized ligand on the LBD region of PPAR.

Journal of Research in Biology (2014) 4(8): 1505-1515 1511

Kanagalakshmi et al., 2014

Figure 4. Binding interaction of ferulic acid and retinoic acid with PPAR.
Ligplot image showing hydrogen bonding and hydrophobic interactions of
PPAR with its cocrystallized ligand retinoic acid (A) and ferulic acid (C). Pymol
view of LBD region of PPAR with cocrystallized ligand (B) and ferulic acid (D
Ferulic acid has similar binding sites as compared with the cocrystallized ligand
on the LBD region of PPAR.

COX-2 protein expression in HDFa and ferulic acid inflammation. In this study, ferulic acid was found to be
pretreatment restored the UVB induced expression of an activator of PPAR and subsequently alleviates UVB
COX-2 in HDFa. The inhibition of COX-2 expression by induced inflammatory markers expression. PPAR
ferulic acid may be caused by its phenol function, which possess a small polar and a hydrophobic residue in the
is associated with decreased anti-radical activity (Lee LBD that form hydrogen bonds and hydrophobic binding
et al., 2013). The hydrogen-donating ability of ferulic interactions with ferulic acid and the cocrystallized
acid was previously reported to correlate with COX-2 ligand. Arg 316 present in the LBD region of PPAR
inhibitory activity (Hirata et al., 2005). Previously, we was a common amino acid that interacts with both ferulic
showed ferulic acid scavenges free radicals and restored acid and the cocrystallized ligand. Ferulic acid also
UVB induced oxidative events (Prasad et al., 2007). activates PPAR and the amino acid Tyr 464 and
Molecular docking results show that ferulic acid directly Ser 280 were the common amino acids that are interact
interacts with COX-2 protein. with ferulic acid and the cocrystallized ligand. Further,
It was reported that expression and activation of induced fit docking analysis showed that there was a
PPAR blocks inflammation were induced by cytokine greater binding interaction of ferulic acid with PPAR
production (Hirsch 2003, Blanquart 2003, Grimble than PPAR. The binding interaction of these proteins
2002). PPARs regulate important cellular functions, are due to increase in polarity, number of OH groups
including cell differentiation, proliferation, and present in the LBD, position of the OH group in the

1512 Journal of Research in Biology (2014) 4(8): 1505-1515

Kanagalakshmi et al., 2014

Figure 5. Binding interactions of ferulic acid and the cocrystallized ligand

(1-Phenylsulfonamide-3-Trifluoromethyl-5-Parabromophenylpyrazole) with COX-2. Ligplot
image showing hydrogen bonding and hydrophobic interactions of COX-2 with its
cocrystallized ligand retinoic acid (A) and ferulic acid (C). Pymol view of interaction of LBD
region of COX-2 with cocrystallized ligand (B) and ferulic acid (D). Ferulic acid has similar
binding sites as compared with the cocrystallized ligand on the LBD region of COX-2.

ferulic acid, based on the steric effect/inductive effect of skin cancer. In: Sarkar, F.H. (Ed.) Nutraceuticals and
the ferulic acid. Ligand exposure is also considered to be Cancer. Springer Publishers . chapter 14 295321.
one of the reasons for strong binding interaction. A
Alexia GL, Richard LN, Sophie Gangloff C, Moncef
recent study by Waku et al. (2009) shows that ligand
G. 2003. Differential regulation of TNF- alpha, IL-6 and
interact with LBD region and thereby activating PPAR
IL-10 in UVB-irradiated human keratinocytes via cyclic
conformations. Covalent interactions stimulate alteration
AMP/protein kinase A pathway. Cytokine, 23 (4-5) : 138
of the side-chain network nearby created covalent bond
to create diverse transcriptional strengths. Thus, ferulic
acid exhibits beneficial effects against UVB-induced Barone E, Calabrese V, Mancuso C. 2009. Ferulic acid
inflammatory responses probably through down- and its therapeutic potential as a hormetin for age-related
regulating COX-2 and TNF- expressions and activating diseases. Biogerontology 10 (2): 97108
PPAR / agonists.
Blanquart C, Barbier O, Fruchart JC, Staels B,
Glineur C. 2003. Peroxisome proliferator activated
receptors: regulation of transcriptional activities and
The work was partly supported by University
rules in inflammation. The journal of steroid
Grants Commission, India (File No. 42-641/2013)
biochemistry and molecular biology. 85 (2-5): 267273.

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Uccella A, Barbuzzi T, Paolino D, Bonina F. 2000. In Easy online submission
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Journal of Research in Biology (2014) 4(8): 1505-1515 1515

Journal of Research in Biology ISSN No: Print: 2231 6280; Online: 2231- 6299

An International Scientific Research Journal

Original Research

Determining an effective way to control weeds in the Olam Palm, Kango

(Gabon) using herbicide treatment
Authors: ABSTRACT:
Journal of Research in Biology

Nguema Ndoutoumou P*,

Mbeang Beyeme AM, The fight against weeds in forest areas is a major constraint to
Mouele Balimbi J and agricultural production; indeed, the loss of production there are around 25%. The
Leboussy Ayenengoye SU use of herbicides for control remains common. This study aims to propose an
appropriate herbicide against weeds in the young palms of central Gabon region.
The protocol is based on the observation and analysis of the effects of
three herbicide formulations (T1 = 30 ml Finish + 2g Ally; T2 = 50 ml Roundup +
2g Ally and T3 =100 ml of "mixing 150 ml of Finish + 150 ml water 2g + Ally") on
the dominant weeds and their impact on the growth of palm trees. The data are
Institution: collected on the time taken for drying of weeds, time of recurrence of weeds, the
University of Science and growth of leaves and changing the size of the arrows.
Technology of Masuku,
National Higher Institute of
The treatment T2 is the most effective way to induce speed drying of
Agronomy and weeds, their period of resurgence and growth of oil palm seedling. However, the
Biotechnology (INSAB). species Marantochloa purpurea Ridl., perennial broadleaf weed is the most common
Poto-Poto BP 99 Franceville, resistant. This characteristic is related to the length of the leaves protecting the base
Gabon. of the screw-to-weed herbicide and screw to advanced growth stage. In addition, this
species has a higher resistance compared to grasses (Centotheca lappacea L. and
Paspalum conjugatum Berg).
In conclusion, treatment with the molecules of glyphosate and
metsulfuron methyl are most suitable for weed control in the palm of OLAM Palm,

Corresponding author: Keywords:

Nguema Ndoutoumou P Palm oil, efficiency, weeds, herbicides, growth, dryness, growth

Email Id: Article Citation:

Nguema Ndoutoumou P, Mbeang Beyeme AM, Mouele Balimbi J and Leboussy
Ayenengoye SU
Determining an effective way to control weeds in the palm of Olam Palm Kango
(Gabon) herbicide treatment
Journal of Research in Biology (2014) 4(8): 1516-1525

Received: 21 Oct 2014 Accepted: 04 Nov 2014 Published: 29 Nov 2014
Web Address: This article is governed by the Creative Commons Attribution License (
documents/RA0487.pdf licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

1516-1525| JRB | 2014 | Vol 4 | No 8

Journal of Research in Biology
An International
Scientific Research Journal
Ndoutoumou et al., 2014

INTRODUCTION these products, the glyphosate concentration is between

The oil palm (Elaeis guineensis Jacq.) is a 120 g/l and 480 g/l, but the most common is 360 g/l
perennial plant belonging to the monocot family- (Lemercier, 2009). These herbicides are foliar,
Arecaceae from Africa (Coulibaly, 1999). systemic, non- selective and effective in controlling
According to Hekimian et al. (2002), it is grasses.
mainly grown for its fruit which has fats that are Finally, herbicides having metsulfuron methyl
extracted for multipurpose with a higher yield compared allow the inhibition of cell division in the shoots and
to peanut and soybean. Fruit pulp provides edible oil, roots. They disrupt the physiology of the plant. The
fats and soaps. Almond oil palm gives exploitable ensuing block, prevents the growth of the plant
amount of yield in nature. The cake obtained after the resulting in early senescence. It is rapidly absorbed by
extraction of these oils is used for animal feed. Excess plants at the roots and foliage level (Tissut et al., 2006;
sap of palm oil are collected in jars, ferment quickly Pipon, 2013).
and give the palm wine. Its leaves are used for many In Gabon, the company Olam plans for the
purposes: roofs, fences, straw etc., (Pantzaris, 1988; production of 25,000 tons of oil for the country to
Jacquemard, 1995; Ofosu-Budu and Sarpong, 2013 ). become the leading palm oil producer in Africa by 2020.
This tropical plant with continued growth The plantations are still young. Several parameters,
requires a constant annual climate condition; any including weed competition can undermine these
deviation from the optimum leads to a decrease in the forecasts.
production (Hekimian et al., 2002). Indeed, production losses due to weeds affect
According to Adje and Affoyon (1994) and global agricultural production; the developing countries
Traore et al. (2010), in tropics, weeds are a major brake are the most affected targets as they cant invest in high
on development in crop productivity through the volumes. Loss rates are higher (25% of production) when
phenomena of direct competition for the various inputs. compared to developed countries (5%), (Adou and Ipou
The man had to fight as soon as he began to cultivate Ipou, 2007).
plants for their needs. For the effectiveness of this The application of herbicides has become a
struggle, it is important to have a good knowledge of common way to fight against weeds in the modern
weeds and herbicides (Montegut and Merlier, 1982; agricultural practice (Mischler et al., 2007; Bertonnier
Hornus et al. 1990; Dore, 2008). According to these et al., 2012). The significance of this study lies precisely
previously tested authors, weeds dissociate into three based on the fact that the competition weeds with the
groups, including grasses, sedges and broadleaf crop may limit its growth. It is the question of
weeds. This classification joins those by Barnes (1990), determining impact of three formulations of herbicides
Johnson (1997) and Traore et al. (2005 and 2010). on weeds in the dominant Olam Palm Kango and then to
Weeding in a palm grove is basically to measure their impact on the growth of young oil palms.
eliminate weeds around the palm to reduce or limit It is therefore proposed to check the Olam Palm Kango
weed competition on the one hand, and to prevent the effective herbicide treatment for getting best suited to
cover crop cluttering the palm on the other hand. The combat the expectations for the conduct of the palm.
weeding may be chemical or manual. In general,
chemical weed control is achieved with Roundup or
Finish, where the active ingredient is glyphosate base. In
1517 Journal of Research in Biology (2014) 4(8): 1516-1525
Ndoutoumou et al., 2014

MATERIALS AND METHODS Finish. It is a selective herbicide for pre and post-
Site Characterization emergence that control broadleaf weeds, annual weeds
The site Olam Palm Kango is located on the and some grasses.
002' latitude and 1012' longitude. The experimental 100 ml beaker was used to contain the amounts
plot is located at 003' South latitude and 011' east of herbicides removed from their original packaging,
longitude. using a 20 ml syringe. A backpack sprayer with a
It is an area of rainforest where a succession of capacity of 15 liters was used for processing around the
hills and plateaus are seen. The climate is equatorial palm while a 200 liters drum was used as a reserve for
marked by two dry seasons and two rainy seasons in the the preparation of boiled water.
year. The months of March, April and December are
endowed with heavy rainfall above 300 mm average. METHODS
These are also the months which record the highest Selection and identification of weeds
temperatures (average above 27C). The driest months Site selection was taken into account for
are June, July and August. determining the diversity of weed flora in the oil palm
Samples analyzed using a 52 cm soil pit shows plantation region. Weeds were selected following the
that the soil is acidic and ferralitic kind. The texture of enumeration of different species in the plot. The
the soil is sandy loam. following species have been identified with their
Plant and technical equipment attendance: Centotheca lappacea (MH1, 72.2%),
Five weeds were identified in the experimental Marantochloa purpurea (MH2, 55.5%), Paspalum
plot. For the purposes of collecting and analyzing data, conjugatum (MH3, 83.3%), Dissotis rotundifolia (MH4,
they are coded as MH (weed) and assigned a number 75%) and Mikania micrantha (MH5, 25%). These rates
from one to five. The following species are the dominant correspond to the number of times, the weeds were found
weed in the palm of Olam Palm Kango: around 36 palms of the experimental site.
Centotheca lappacea L. (Grass) = MH1 Species identification was done using a
Marantochloa purpurea (Ridl.) Milne-Redh. magnifying glass and utilized the work of Johnson
(Marantaceae) = MH2 (1997) for characters comparison. A final notice was
Paspalum conjugatum Berg (Grass) = MH3 given by the National Herbarium of Gabon.
Dissotis rotundifolia (Sm.) Triana (Mlastomatace) = MH4 Treatments and experimental design
Mikania micrantha Kunth (Asteraceae) = MH5 The test was conducted according to an
The study covers a total of 36 oil palm experimental device, completely randomized in blocks at
(Elaeis guineensis Jacq.), aged two years. These palms once. It consists of three blocks (B1, B2 and B3) and
are derived from crosses of improved varieties. three experimental units per block. Each unit has four
Preliminary work on the plantation took place a year in palm trees. Treatments are randomly assigned within the
advance. blocks.
Herbicides used in this study are: Roundup Weeds identified were subjected to the following
(glyphosate 360 g/l), the Finish (glyphosate 360 g/l) and experimental treatments:
Ally (metsulfuronmthyle). Ally is usually used in T1 : 30 ml Finish + 2g Ally
combination with other herbicides. In the experiment, it T2 : 50 ml Roundup + 2g Ally
is sometimes associated with Roundup but now with

Journal of Research in Biology (2014) 4(8): 1516-1525 1518

Ndoutoumou et al., 2014

T3 : 100ml mixture "150ml Finish + 150ml Data collection is weekly for the different
water + 2g Ally." parameters studied. Analyses of the size of the boom, the
The mixtures were made in a water container number of leaves and respawning of weeds were made
of 15 liter. These treatments are common in the Olam using the XLSTAT software. Duncan test was used for
Palm Kango. making comparison between means.
Observed parameters and analysis For the number of leaves and the size of the
Drying weed arrow, an analysis of variance on repeated measurements
Drying weed corresponds to the period of first was made, thereby enabling monitoring of the evolution
yellowing weed in the perimeter of the spray. of these parameters until the end of the experiment. For
Observations are made to the total drying of various the respawn of weeds, analysis of variance (ANOVA)
weeds. Five stages of dryness were selected: was performed.
The Resistance (R): No treatment effect on weeds; For the statistical analysis of time taken for
Yellowing (Y): The beginning of desiccation; drying weed, the five levels of dryness were scored from
The Significant Yellowing (YS): Transition between 0 to 4, respectively for levels R, J, JS, DS and D; then the
yellowing and drying of weeds; observations were transformed into log values (X + 1)
The Desiccation (D): Onset of senescence of weeds. and compared using analysis of variance (ANOVA) and
The Significant Dryness (DS): Total desiccation of weeds. on repeated measurements separated by the LSD at 5%
Recurrence weed
when the difference was significant. This analysis was
Recurrence weed is the observation period for
performed using SAS software.
recurrence of any weed on the treated plot. Shades are
made according to the identified flora.
The growth parameters of the palm on one hand
Drying time of weed
are related to the number of sheets issued by the palm
Figures 1 to 4 present the effects of treatments
plant, and on the other hand, the size of the arrow until
T1, T2 and T3 on weeds MH1, MH2, MH4 and MH5, over
maturity. Finally, the time required for the arrows to
becoming true leaves was also determined.

1.8 1.4
1.6 1.2
Level of desiccation

Level of desiccation


1 0.8
MH2 T1 MH2 T1
0.8 0.6
0.6 MH2 T2 MH2 T2
0.4 MH2 T3 MH2 T3

0 0

Time (weeks) Time (weeks)

Figure 1 Evolution of drying MH1 Figure 2 Evolution of drying MH2

(Centotheca lappacea ) among treatments. (Marantochloa purpurea ) among treatments.

1519 Journal of Research in Biology (2014) 4(8): 1516-1525

Ndoutoumou et al., 2014

The effect of the three treatments (T 1, T2, T3) are The overall results showed that the most resistant
identical in MH1 (Centotheca lappacea), where the weed is MH2 (Marantochloa purpurea). In addition, the
superposition of the curves representing that effect over treatment T2 seems to be more effective in the fight
time (Figure 1). against these weeds.
The effect of treatments on Centotheca lappacea From a statistical point of view, if we take into
(MH1), Paspalum conjugatum Berg (MH3) and Mikania account the probability effect of different sources of
micrantha (MH5) was achieved after three weeks to the variation, it appears at first as a highly significant
last level of dryness (DS). As these are short-cycle difference in response between the three treatments given
species mainly following vegetative propagation, they to five weed species. This result seems to be that the life
undergo rapid drying out three weeks after the different cycle of annual grasses are thus relatively short. More
treatments. recent to this, they reproduce only sexually, unlike
Dissotis rotundifolia (MH4) reacts differently to broadleaf weeds (Marantochloa purpurea) and have
other weeds (MH1, MH3 and MH5) with respect to the longer life cycles.
three treatments. Indeed, in the sixth and seventh weeks, In the second step, there is a significant
all the three treatments have the same behavior. These difference between three formulations applied to the
results are explained by the fact that Dissotis rotundifolia herbicides. The treatments do not work with the same
is a perennial broadleaf and therefore more resistant to effectiveness on weeds. This difference is explained by
herbicides compared to grasses. the fact that Roundup does not contain metsulfuron
Formulations T1 and T3 in the same way act on methyl while the Finish contains enough active form of
Marantochloa purpurea (MH2). Their effectiveness on this adjuvant.
this weed is less compared to that of T 2 with which they Finally, the probability of interaction (treatment
will reach a higher level of drying (JS) after three weeks. x species) confirms the effect of differences between the
In general, Marantochloa purpurea is resistant to these data for three treatments or behavior differences in weed
three treatments. It is a plant with large leaves and species which meet these three species treatments.
rhizomes, where foliage limit absorption of the herbicide The speed of drying out is higher in grasses than
and rhizomes promote regeneration. broadleaf weeds. Similarly, in general, the time and

Level desiccation

Level desiccation

1 MH2 T1
MH2 T1 MH2 T2
0.4 MH2 T2 MH2 T3
0.2 MH2 T3

Time (weeks) Time (weeks)

Figure 3 Evolution of drying MH4 Figure 4 Evolution of drying MH5

(Dissotis rotundifolia) among treatments. (Mikania micrantha) among treatments.

Journal of Research in Biology (2014) 4(8): 1516-1525 1520

Ndoutoumou et al., 2014

Size (m)

Time (enjours)

Figure 5 Evolution of the size of the arrow.

dryness is greater when the treatment is accelerated with Number of sheets

T2. Analysis of the differences between treatments Growth in the number of leaves is not significant
revealed that for the "drying time of weed" parameter, during the period of observation. Indeed, this parameter
treatment T2 is much better than T3. does not vary between treatments received by palm
Treatment T1 occupies an intermediate position plants. However, experimental units having received the
between T2 and T3 in the analysis of interactions (species treatment T2 exhibit better growth compared with the
x treatment). Its effectiveness on Marantochloa purpurea other units which received other two treatments. Indeed,
remains lower compared to the effect of treatment T 2 of the growth of the arrows cause an increase in the number
the same species. The treatment T2 has a better efficacy of leaves; ultimately, the application of different
on weed control treatments than T 1 and T3. herbicides on weed formulations allow the increase of
Size of the arrow palm leaves.
The curves of the three treatments have a Respawn weed
constant gradual pace. However, regarding the Treatments followed by T1, T2 and T3 are
processing curve of T2 (Roundup + Ally) it is above the significantly different at the 5% level.
curves representing experimental units having received There are significant differences between
treatment with T1and T3. treatments T1 and T2, on one hand, and between
Indeed, the reduction of competition enable treatments T1 and T3, on the other hand, while the effects
palms to increase their export of mineral, water and soil of treatments on the respawn of weeds are similar to
as well as their ability to capture light for photosynthesis.
Table 1 Analysis of the differences between
This promotes the growth and development of the palm treatments for the respawn of weeds.
of the arrows. All treatments allow a better development
Treatments Means and standard
of the size of the arrows. In addition, weeds can be a deviations
habitat for some palm pests. Removing them allows T3 94.08a 23.12
expelling the pests of cultivated areas. Although various
T2 90.16a 10.69
treatments have a positive impact on the growth of the
size of the arrows of palms; treatment T 2 proves to be the T1 73.16b 09.99

1521 Journal of Research in Biology (2014) 4(8): 1516-1525

Ndoutoumou et al., 2014

treatments T3 and T2. DISCUSSION

The mean time for recurrence of palms receiving Resistance to treatment
treatment T2 and T3 treatments are very close. The effect In terms of drug resistance, it would be wise to
of treatment T2 and T3 on weeds can greatly delay the treat weeds in stage two or three leaves, so the treatment
time to recurrence weeds. Treatments T 2 and T3 are frequency is quarterly. The different treatments were
effective in delaying the onset of weeds and promote the applied while weeds had overtaken the juvenile stage
harmonious growth of the oil palm. Nevertheless, one development. In fact, the observed resistance of Dissotis
can notice that the standard deviation of the respawn rotundifolia could be explained. Indeed, early weed
units receiving treatment T 3 is very high (23.12). control is important for effective control and yields. This
Application of the treatment T 3 is obtained promptly to is consistent with the observations of Johnson (1997).
recur weeds. The treatment T 3 does not allow time to The conduct of palm monoculture promotes
obtain a balanced and stable recurrence. Against by weed resistance vis--vis herbicides (Mangara et al.,
standard deviation units, receiving treatment T 2 is low 2010). Marantochloa purpurea is the only weed that
and therefore stable (10,69), this treatment allows for truly has broad leaves and rhizomes. Indeed, perennials
respawn close to the average. Indeed, the Finish contains are much more resistant to herbicides than grasses
not only glyphosate as the active ingredient but also as (Johnson, 1997).
metsulfuron methyl builder. It is therefore possible that Finish, Round up and Ally are the main herbicide
these two substances in certain concentrations reduce the used, they contain glyphosate (active ingredient) at a
effectiveness of the fight against weeds. While in the concentration of 360 g/l. Some weeds such
treatment T2, the only source of the product is Marantochloa purpurea may acquire resistance to
Metsulfuron methyl Ally as Roundup has only glyphosate due to the repeated use of the same herbicide.
glyphosate as active ingredient. The treatment T 2 seems Indeed, the work of Baylis (2000) showed that this fact
more stable and allows for respawn which is almost promotes weed resistance.
uniform. Determining the type of weeds is essential to
Respawned weed and palms which received the effectively fight against their impact on culture.
treatment T1 is 73 days on average. This duration is less Perennial weeds have a high resistance compared to the
than three months. It is significantly lower than the other. Resistance of Marantochloa purpurea is also
respawn of weeds in units that have received treatments explained by the fact that it has a long life cycle.
T2 and T3. The respawn in units that received the Marantochloa purpurea can be propagated vegetatively,
herbicide formulations T2 and T3 are 90 days and unlike species Paspalum conjugatum, Centotheca
94 days respectively and correspond to three months on lappacea and Mikania micrantha which can reproduce
average. that sexually. This faculty of reproduction gives
All treatments can slow the recurrence of weeds, Marantochloa purpurea a better resilience compared to
although the treatment T3 and T2 are used to obtain other weeds, such as stated by Aubert and Glachant
respawn of longer weeds. Indeed, the stage of weed (2009).
growth is a key factor for weed control in an effective Effectiveness of treatment T2 compared to T1 and T3
way. The treatment T2 (Roundup + Ally) is better
compared to the other two treatments. Indeed, it only
contains glyphosate Roundup outside its adjuvants. In

Journal of Research in Biology (2014) 4(8): 1516-1525 1522

Ndoutoumou et al., 2014

Finish, it contains not only glyphosate but also three treatments. The treatment T 2 is significantly
metsulfuron-methyl which is a sulfonylurea substance. different from the other two treatments. It has a long
The combination of the Ally and Finish increased respawn time of weeds, a different curve drying weeds
the content of metsulfuron-methyl mixture. It thus seems over other treatments and a greater influence on the
clear that the presence of metsulfuron-methyl in the growth parameters of oil palm. This treatment combines
solution is a factor limiting the effectiveness of the the molecules of glyphosate (Roundup) and metsulfuron
herbicide treatment. These results confirm the resistance methyl (Ally) which is most suitable for weed control in
shown by weeds that deal with sulfonylurea herbicides the palm of Olam Palm Kango.
substances, up to 60-100%, according to Rodriguez These results would be improved by taking into
(2005) and Chauvel and Guillemain (2013). account the identification of other weeds present in the
Foliar and root herbicides should be applied field, the combination of herbicide treatment in round
earlier. Indeed, three weeks after herbicide treatment, weeding around the palm trees, the quarterly frequency
different weeds have very different spectra of activities of herbicide treatment and the use of cover crop
in accordance with the work of Tissut et al. (2006). (Pueraria javanica) with its various beneficial effects on
The Roundup association and Ally (T 2) is a the culture.
combination of herbicides that can fight against a wide
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Journal of Research in Biology ISSN No: Print: 2231 6280; Online: 2231- 6299

An International Scientific Research Journal

Original Research

Application of multivariate principal component analysis on dimensional

reduction of milk composition variables
Authors: ABSTRACT:
Journal of Research in Biology

Alphonsus C1, Akpa GN1,

Nwagu BI2, Abdullahi I2,
Zanna M3, Ayigun AE3,
Opoola E3, Anos KU3, Variable selection and dimension reduction are major prerequisites for
Olaiya O3 and Olayinka- reliable multivariate regression analysis. Most a times, many variables used as
Babawale OI3
independent variables in a multiple regression display high degree of correlations. This
problem is known as multicollinearity. Absence of multicollinearity is essential for
1. Animal Science multiple regression models, because parameters estimated using multi-collinear data
Department, Ahmadu Bello are unstable and can change with slight change in data, hence are unreliable for
University, Zaria, Nigeria. predicting the future. This paper presents the application of Principal Component
Analysis (PCA) on the dimension reduction of milk composition variables. The
2. National Animal application of PCA successfully reduced the dimension of the milk composition
Production Research variables, by grouping the 17 milk composition variables into five principal
Institute, Shika-Zaria
components (PCs) that were uncorrelated and independent of each other, and
3. Kabba College of explained about 92.38% of the total variation in the milk composition variables.
Agriculture, Ahmadu Bello
University, Kabba, Nigeria

Corresponding author: Keywords:

Alphonsus C Principal component analysis, eigenvalues, communality

Email Id: Article Citation:

Alphonsus C, Akpa GN, Nwagu BI, Abdullahi I, Zanna M, Ayigun AE, Opoola E,
Anos KU, Olaiya O and Olayinka-Babawale OI
Application of multivariate principal component analysis on dimensional reduction of
milk composition variables
Web Address: Journal of Research in Biology (2014) 4(8): 1526-1533
documents/RA0489.pdf Dates:
Received: 27 Oct 2014 Accepted: 15 Nov 2014 Published: 03 Dec 2014

This article is governed by the Creative Commons Attribution License (

licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

1526-1533| JRB | 2014 | Vol 4 | No 8

Journal of Research in Biology
An International
Scientific Research Journal
Alphonsus et al., 2014

INTRODUCTION used to reduce the number of predictive variables as well

In recent times, many scientist, especially in the as solving the problem of multicollinearity (Bair et al.,
field of dairy science have postulated the use of milk 2006). It transforms the original independent variables
composition variables as a tool for monitoring and into newly uncorrelated variables called Principal
evaluation of energy balance (Friggens et al., 2007; Components (PCs) (Lafi and Kaneene, 1992), so that
Lovendahl et al., 2010; Alphonsus, 2014), health each PC is a linear combination of all the original
(Hansen et al., 2000; Pryce et al., 2001; Invartsen et al., independent variables. It looks for a few linear
2003; Cejna and Chiladek, 2005), fertility (Harris and combinations of variables that can best be used to
Pryce, 2004; Fahey, 2008) and nutritional status summarize the data without loosing information of the
(Kuterovac et al., 2005; Alphonsus et al., 2013) of dairy original variables (Lafi and Kaneen, 1992; Bair et al.,
cows. One way of validating this hypothesis is to assess 2006)
the relationship between the milk composition variables This study therefore attempted to apply the
and the parameters in question through multiple principle of Principal Component Analysis (PCA) on
regression analysis. However, the drawback in applying variable selection and dimension reduction of milk
multiple regression analysis to the milk composition composition variables
variables is that most of the milk composition variables
are highly correlated (Lovendahl et al., 2010; Alphonsus MATERIALS AND METHODS
and Essien, 2012). Experimental site
A high degree of correlation among the Data for this study were collected from 13
predictive variables increases the variance in estimates of primiparous and 47 multiparous Friesian x Bunaji dairy
the regression parameters (Yu, 2010). This problem is cows, at the dairy herd of National Animal Production
known as multicollinearity (Kleinbaum et al., 1998; Research Institute (NAPRI) Shika-Zaria, located
Fekedulegn et al., 2002; Leahy, 2001; Yu, 2008). between latitude 11 and 12N at an altitude of 640m
The problem with multicollinearity is that it above sea level, and lies within the Northern Guinea
compromises the basic assumption of multiple regression Savannah Zone (Oni et al., 2001). The cows were
that state that the predictive variables are uncorrelated managed during the rainy season on both natural and
and independent of each other and parameters estimated paddocksown pasture, while during the dry season they
using multi-collinear data are unstable and can change were fed hay and /or silage supplemented with
with slight change in data, hence are unreliable for concentrate mixture of undelinted cotton seed cake and
predicting the future. When predictors suffer from grinded maize. They had access to water and salt lick ad-
multicollinearity, using multiple regressions may lead to libitum. Unrestricted grazing was allowed under the
inflation of regression coefficients. These coefficients supervision of herdsmen for 7 9 hours per day
could fluctuate in signs and magnitude as a result of a (Alphonsus et al., 2013)
slight change in the dependent variables (Fekedulegn Milk composition measures
et al., 2002). Cows were milked twice daily (morning and
Therefore, the first step to counteract this evening) and milk yield was recorded on daily basis. The
problem of multicollinearity is the use of Principal milk sampled for the determination of fat, protein and
Component Analysis (PCA). Principal component lactose percentages were taken once per week starting
analysis is a multivariate statistical tool that is commonly from 4 days postpartum to the end of each lactation.
1527 Journal of Research in Biology (2014) 4(8):1526-1533
Alphonsus et al., 2014

The milk samples were frozen immediately after The principal component analysis was run using
collection and stored at -20 C until analysed (Alphonsus PROC Factor SAS software (SAS, 2002).
et al., 2013). The milk composition analysis was carried RESULTS AND DISCUSSION
out at the Food Science and Technology Laboratory of Correlation matrix of the milk composition variables
Institute of Agricultural Research (IAR) in Ahmadu The correlation matrix shows high degree of
Bello University, Zaria-Nigeria. The yield values and the correlation among the milk composition variables (Table
ratios were derived from the percentage values of fat, 1). This strong correlation among the measured variables
protein and lactose (Friggens et al., 2007 Lvendahl et is called multicollinearity (Kleinbaum et al., 1998;
al., 2010). The following milk composition measures Vaughan and Berry, 2005). Multicollinearity is a serious
were calculated: Milk Fat Content (MFC), Milk Protein problem in multiple regression analysis because it
Content (MPC), Milk Lactose Content (MLC), Milk Fat violates the basic assumption of regression that requires
Yield (MFY), Milk Protein Yield (MPY), Milk Lactose the predictors to be independent and uncorrelated with
Yield (MLY), Fat-Protein Ratio (FPR), Fat-Lactose each others. It also compromise the integrity and
Ratio (FLR), Protein - Lactose Ratio (PLR), change in reliability of the regression models (Kleinbaum et al.,
Milk Yield (dMY), change in Milk Protein Content 1998; Maitra and Yan, 2008).
(dMPC), change in Milk Fat Content (dMFC), change in The problem of multicollinearity is as a result of
Milk Lactose Content (dMLC), change in Fat Protein redundancy of some variables. Redundancy in this case
Ratio (dFPR), change in Fat Lactose Ratio (dFLR) and means that some of the variables are strongly correlated
change in Protein-Lactose Ratio (dPLR). with one another, possibly because they are measuring
Statistical Analysis the same characteristic (
The correlation matrix of all the milk publishing/publicat/chaps/55). For example, the
composition variables was first run using PROC CORR correlations between the milk composition yield
procedure of SAS (2000) to determine the level of the variables (MFY, MPY, MLY) were very strong (r =
collinearity among milk composition variables. 0.943 to 0.989). Likewise, the correlations between the
Principal component analysis rate of change d in milk composition variables (dMY,
Principal component analysis is a method for dMFC, dMPC, dMLC) were very strong ranging from
transforming the variables in a multivariate data set 0.980 to 0.992, and a lot of others. Therefore, given this
X2, X2,.Xn, into new variables, Y1, Y2,..Yn, apparent redundancy, it is likely that these correlated
which are uncorrelated with each other and account for variables are measuring the same construct or have the
decreasing proportions of the total variance of the same characteristics. Therefore, it could be possible to
original variables, defined as reduce these collinear variables into smaller number of
Y1 = P11X1 + P12X2 +. +P1nXn composite variable (artificial variables) called Principal
Y2 = P21 X1 + P22X2 + + P2nXn Components (PCs) that are independent and account for
Y3 = Pn1X1 + Pn2X2 + . + PnnXn most of the variation in the milk composition variables.
With the coefficient being chosen so that The PCs can then be used for subsequent multiple
Y1, Y2, .. Yn account for decreasing proportion of regression analysis. One way of achieving this is the use
the total variance of the original variables X1, X2 ..Xn of Principal Component Analysis (PCA).
(Lafi and Kaneene, 1992). Principal Component Analysis
The measured milk composition variables were

Journal of Research in Biology (2014) 4(8): 1526-1533 1528

Table 1: Correlation co-efficients among milk yield and milk composition variables used for prediction of Energy Balance (EB)

variables MY
MFC -0.264 -
MPC -0.195 0.352 -
MLC -0.321 0.853 0.305 -
MFY 0.956 0.025 -0.054 -0.078 -
MPY 0.986 -0.189 -0.029 -0.275 0.966 -
MLY 0.939 0.019 -0.089 0.014 0.988 0.943 -
FPR -0.177 0.853 -0.079 0.773 0.063 0.191 0.078 -
FLR 0.183 0.044 0.169 -0.484 0.203 0.218 0.016 -0.048 -
PLR 0.240 -0.669 0.162 -0.889 0.056 0.272 -0.057 -0.841 0.579 -
dMY -0.669 0.037 0.232 -0.058 -0.681 -0.645 -0.728 -0.095 0.161 0.171 -
dMFC -0.674 -0.056 0.120 -0.085 -0.714 -0.668 -0.742 -0.133 0.056 0.144 0.980 -
dMPC -0.671 0.000 0.117 -0.068 -0.695 -0.666 -0.734 -0.118 0.118 0.123 0.985 0.989 -
dMLC -0.653 0.021 0.187 -0.084 -0.671 -0.634 -0.723 0.183 0.183 0.176 0.992 0.983 0.985 -
dFPR -0.182 -0.388 -0.002 -0.154 -0.279 -0.183 -0.220 -0.352 -0.352 -0.152 0.240 0.345 0.212 0.246 -
dFLR -0.129 -0.433 -0.336 0.017 -0.254 -0.196 -0.126 -0.284 -0.691 0.171 -0.021 0.142 0.063 -0.036 0.605 -
dPLR 0.070 -0.061 -0.391 0.147 0.038 -0.005 0.115 0.165 -0.385 -0.363 -0.297 -0.23 0.459 -0.321 -0.427 0.459
milk composition variables indicated by the following: Average Daily Milk Yield (ADMY), Milk Fat Content (MFC), Milk Protein Content (MPC), Milk
Lactose Content (MLC), Milk Fat Yield (MFY), Milk Protein Yield (MPY), Milk Lactose Yield (MLY), Fat Protein Ratio (FPR), Fat Lactose Ratio (FLR),
Protein Lactose Ratio (PLR). Variable abbreviations starting with d are the current minus the previous values of milk measures in question. Yield values are
in kilogram per day (kg/day), content values are in percentages (%) and ratios are unitless. The measures used were group mean averages.
cummulative percentages of variation explained with increasing number of PC indicated

Journal of Research in Biology (2014) 4(8): 1526-1533

Alphonsus et al., 2014
Alphonsus et al., 2014
Table 2: Relationships among milk composition measures1 expressed as loadings in a
principal component analysis.
Items a Principal components (PCs) h
Variable explained2 38.88 60.01 75.00 85.30 92.38 -
Average Daily Milk Yield -0.34 0.93 -0.02 0.00 -0.00 99.81
Milk Fat Content (MFC) 0.02 0.13 0.85 -0.23 0.42 99.96
Milk Protein Content (MPC) -0.04 0.15 0.05 0.07 0.98 99.96
Milk Lactose Content (MLC) 0.05 0.13 0.82 0.08 0.37 99.89
Milk Fat Yield (MFY) -0.33 0.92 0.17 -0.05 0.08 99.88
Milk Protein Yield (MPY) -0.34 0.93 -0.01 -0.01 0.14 99.90
Milk Lactose Yield (MLY) -0.34 0.91 0.19 0.01 0.07 99.83
Fat-Protein Ratio (FPR) 0.05 0.04 0.90 -0.27 -0.25 99.97
Fat-Lactose Ratio (FLR) 0.10 -0.04 -0.15 -0.48 -0.05 99.97
Protein-Lactose Ratio (PLR) 0.03 -0.06 -0.86 -0.09 0.17 99.86
dMY 0.94 -0.33 0.01 -0.06 0.01 99.40
dMFC 0.94 -0.32 -0.02 0.06 -0.03 99.77
dMPC 0.94 -0.32 0.02 -0.06 -0.04 88.81
dMLC 0.95 -0.31 -0.01 -0.07 -0.02 99.77
dFPR -0.04 0.03 -0.25 0.81 0.04 99.94
dFLR -0.04 -0.05 -0.08 0.92 -0.03 99.95
dPLR -0.01 -0.09 0.22 0.02 -0.09 99.96
% variance 38.88 21.20 14.92 10.30 07.08 -
Eigen values 6.610 3.604 2.536 1.751 1.204 -
Variable abbreviations starting with d are the change variables signifying current minus
the previous values of milk measures in question. Yield values are in kilogram per day (kg/
day), content values are in percentages (%) and ratios are unitless.
cummulative percentages of variation explained with increasing number of PC indicated
percentage variance explained by each principal components
h= communality estimates is a variance in observed variables acounted for by a common

subjected to Principal Component Analysis (PCA) using component if the factor loading was 0.50 or greater.
one as a prior communality estimate. The principal axis Using these criteria, it was obvious that the change d
method was used to extract the components, and this was in milk composition variables (dMY, dMFC, dMPC,
followed by varimax (orthogonal) rotation. Only the first dMLC) loaded heavily on the first Principal Component
five components accounted for a meaningful amount of (PC) which were subsequently labeled change
the total variance (92.38%) in the milk composition component. Also, the four milk composition yield
variables. Also using eigenvalue criteria of one, variables (ADMY, MFY, MPY, MLY) loaded heavily on
it was obvious that the first five components displayed the second PC and were labeled yield component.
eigenvalues equal to or greater than one. Therefore, the Other variables like MFC, MLC, FPR and FLR loaded
first five principal components were retained and used heavily on the third PC and were labeled mixed
for rotation and interpretation. The milk composition component. Change in Fat-Protein Ratio (dFPR) and
variables and the corresponding factor loadings are Fat-Lactose Ratio (dFLR) loaded heavily on the fourth
presented in Table 2. In interpreting the rotated factor PC and were labeled change in fat ratio component.
pattern, an item was said to load heavily on a given The last PC had only one variable (MPC) heavily loaded

Journal of Research in Biology (2014) 4(8): 1526-1533 1530

Alphonsus et al., 2014

Table 3: Pearson correlation between the Principal components and milk composition variables

Variables i Principal Components (PCs)

Average daily milk yield -0.340 0.938** -0.016 0.000 0.004
Milk fat content (mFc) 0.018 0.135 0.853** -0.233 0.317
Milk protein content (mPc) -0.043 0.146 0.059 0.015 0.981**

Milk lactose content(mLc) -0.052 0.128 0.825 0.085 0.373

Milk fat yield (mFy) -0.327 0.923** 0.168 -0.053 0.079

Milk protein yield (mPy) -0.341 0.928** 0.012 -0.005 0.137

Milk lactose yield (mLy) 10.342 0.909** 0.193 0.014 0.078

Fat-protein ratio (FPR) 0.046 0.039 0.900** -0.267 -0.246

Fat-lactose ratio (FLR) 0.098 -0.036 -0.153 -0.476 -0.046
Protein-lactose ratio (PLR) 0.029 -0.061 -0.859** -0.085 0.172
dmy 0.939** -0.328 0.008 -0.057 0.009
dmFc 0.942** -0.322 -0.021 0.069 -0.030
dmPc 0.941** -0.316 0.021 -0.062 -0.039
dmLc 0.944** -0.307 -0.008 -0.069 -0.022
dFPR -0.041 0.027 -0.254 0.811** 0.043
dFLR -0.044 -0.055 -0.082 0.921** -0.028
dPLR -0.005 -0.091 0.221 0.049 -0.087
PC1 1.000 0.000 0.000 0.000 0.000
PC2 0.000 1.000 0.000 0.000 0.000
PC3 0.000 0.000 1.000 0.000 0.000
PC4 0.000 0.000 0.000 1.000 0.000
PC5 0.000 0.000 0.000 0.000 1.000
Variable abbreviations starting with d are the current minus the previous values of milk measures in
question. Yield values are in kilogram per day (kg/day), content values are in percentages (%) and rati-
os are unitless. The measures used were group mean averages. ** = P < 0.001

on it, suggesting that MPC is not strongly correlated with on the PCs (the best loading of each variable is indicated
any of the measured milk composition variables (as can by the bolded values). Each variable loaded only on one
be verified in Table 1) and could therefore be treated as component. No variable loaded heavily on more than one
independent variable in subsequent multivariate analysis. PC. This suggested that the milk composition variables
Since PCs are labeled according to the size of can be reduced into smaller composite variable without
their variances, the first Principal Component (PC) losing much of the information.
explained larger amount of variation (38.88%) among The PCs displayed varying degrees of
the variables, while the last PC explained the least correlations with the milk composition variables
(07.08%). Also, the eigenvalues followed the same trend (Table 3) and the correlation structure was similar to the
as the percentage variance explained by each of the PCs. loading pattern of the milk composition variables on the
The communality estimates, which tells us how much of PCs. Thus, confirming the loading pattern of the
the variance in each of the original variables is explained principal component analysis (Table 2). However, the
by the extracted PC was very high ranging from 83.30 to correlation among the PCs was zero. This shows that the
99.71%. There was a clear grouping of the measured Principal component analysis resulted in orthogonal
variables evident by the loading pattern of the variables solution whereby the PCs extracted were completely

1531 Journal of Research in Biology (2014) 4(8): 1526-1533

Alphonsus et al., 2014

Table 4: Descriptive statistics of the principal components

Principal components N Means S.D Min Max
PC1 60 0.00 1.000 -2.544 3.102
PC2 60 0.00 1.000 -3.001 2.573
PC3 60 0.00 1.000 -2.626 2.036
PC4 60 0.00 1.000 -5.045 2.563
PC5 60 0.00 1.000 -4.104 2.085
N= animals, S.D = standard deviation, Min =minimum, Max = maximum

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Journal of Research in Biology ISSN No: Print: 2231 6280; Online: 2231- 6299

An International Scientific Research Journal

Original Research

Diversity and conservation status of water birds at Upper lake,

Bhopal A Ramsar site in central India
Authors: ABSTRACT:
Journal of Research in Biology

Hina Veerwal,
Satish Balapure and Wetlands support considerable biodiversity including water birds. In central
Vipin Vyas India, the Upper lake of Bhopal is designated as a Ramsar site and IBA status
concerning to its diverse avifauna. At present, a detailed study regarding avian
diversity of entire Upper lake is lacking. Considering the increasing population
Institution: pressure leading to land use changes in immediate catchment of Upper lake and
Department of affecting the lake ecology, such monitoring is essential. The present work has been
Environmental Sciences carried out from June 2010 to June 2012. Monthly observations of birds were made
and Limnology, Barkatullah with the aim to identify and enlist various species of water birds of Upper lake. Total
University, Bhopal. 68 species of water birds belonging to 14 families are reported with Anatidae as the
most dominating family with 16 species. Of these, 43 species were migratory and 25
species were residents. Also, 11 important species in terms of their conservation
Corresponding author: status are reported. Of these, eight species of Near Threatened status, two species of
Vipin Vyas Vulnerable status and one species of Endangered status are reported. The presence of
internationally important birds, migratory, local migratory and resident species of
birds in this area indicates the importance of Upper Lake as a year round habitat for
water birds. The increasing human population in the area is placing strain on this
valuable inland freshwater resource and the related avifauna and thus more
conservation efforts are desperately needed.

Upper Lake, wetland, water birds, diversity, conservation status

Email Id:

Web Address: Article Citation:
documents/RA0485.pdf Hina Veerwal, Satish Balapure and Vipin Vyas
Diversity and conservation status of water birds at Upper lake, Bhopal A Ramsar site
in central India
Journal of Research in Biology (2014) 4(8): 1534-1542

Received: 15 Oct 2014 Accepted:04 Nov 2014 Published: 03 Dec 2014

This article is governed by the Creative Commons Attribution License (

licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

Journal of Research in Biology 1534-1542| JRB | 2014 | Vol 4 | No 8

An International
Scientific Research Journal
Veerwal et al., 2014

INTRODUCTION grounds in the northern regions. In central India, the

Wetlands are shallow water areas which act as Upper lake of Bhopal is an important water bird abode
transitional zones between terrestrial and aquatic systems for resident species as well as an equally prominent
(Cowardin et al., 1979; Mitsch and Gosselink, 1986) and staging and wintering site for numerous migratory
support considerable biodiversity of organisms (Dudgeon species. It is designated as a Ramsar site and IBA status
et al., 2006). Wetlands hold immense ecological in concern to the fact that it supports a distinct and ample
significance by providing suitable habitat to a vast population of water birds around the year. A variety of
variety of faunal diversity. Water birds generally occupy water birds like cormorants, egrets and herons, storks
the position of top level consumers in aquatic food chain and ibises, crane, ducks, jacanas, lapwings, stilts,
and thus any changes in the habitat and food availability sandpipers, gulls and terns, kingfishers etc. find refuge in
directly affects them. Water birds are thus considered as this lake (Vyas, 1992) emphasizing the overall
indicators of wetland habitat conditions (Kushlan, 1978). importance of this water body. The avian species
Among the several kinds of wetlands such as marshes, richness supported by Upper lake is largely due to the
lagoons, bogs, fens, mangroves and other open water presence of high food availability which attracts avifauna
bodies etc., urban lakes are at a large risk of habitat to settle here (Vyas et al., 2010). At present, a detailed
degradation due to the prevailing anthropogenic pressure study regarding avian diversity of entire area of Upper
in the surroundings which in turn affects the avian lake is lacking which is essential considering the
diversity supported by them. increasing population pressure leading to changes in the
India possesses wide-ranging wetland habitats landuse of immediate catchment of Upper lake and
that support numerous water birds, many of which are affecting the lake ecology. Thus, the present work has
migratory visiting the subcontinent from their breeding been carried out with the aim to identify and enlist

Map Showing Sampling Stations of Upper Lake

S8 S6 S5



S1- Prempura Ghat S2- VanVihar
S3- Takia Island S4- Kamla Park
S5- Inlet A S6- Inlet B
S7- KhanuGaon S8- BehtaGaon
S9- Deep Zone Urban S10-Deep Zone Rural
S11- BishenKhedi S12- GohraGaon

Upper Lake

Figure 1 Map of the study area showing all the sampling stations

1535 Journal of Research in Biology (2014) 4(8): 1534-1542

Veerwal et al., 2014

Figure 2 Upper Lake of Bhopal

various species of water birds visiting and residing in the sampling points, total 12 in number, were identified
Upper lake which may provide a baseline for the future carefully keeping in mind the subject of habitat features
management of avian fauna in the area. and avifaunal occurrence and also such limitations as
approachability (Figure 1). Study was conducted with
MATERIALS AND METHODS special reference to diversity of birds. Monthly
The Upper lake is an east-westerly elongated observations of birds were made during the study where
urban lake which receives water from the river Kolans birds were observed within 300 m transect using Nikon
and from the direct rains, both during rainfall months Binoculars of 1050 magnification. Identification of the
which is the main source of drinking water for the water birds was done using standard taxonomic keys (Ali
residents of Bhopal. This lake was formed by and Ripley, 1988; Ali, 2002) and the checklist was
constructing an earthen dam across the Kolans river in prepared as per Manakadan and Pittie (2001). On foot
the 11th century by Raja Bhoj of Dhar. The catchment surveys were done by walking across selected tracks
area of the lake is 361 sq. km and the water spread area along the lake margins and the lake area was covered
is 30.72 km at FTL, its mean depth is 3.16 m while using boats. Information from the local community and
maximum depth is 11.64 m. The excess water from on site observations were recorded on every visit and
Upper lake flows into Kaliasot River which further meets accumulated at the end of the study. The status of the
Betwa River and finally gets drained into the Yamuna birds is categorized as Resident (R), Migratory (M) and
river. The altitude of Upper lake is about 503 m above Resident Migratory (RM) after Ali (2002) and the
mean sea level and it is situated at 23 16 N latitude and threatened status is taken according to the Bird Life
77 25 E longitude. It is an east westerly elongated International, (2014).
shallow lake bordered by human settlements on the
eastern and northern boundaries while its western RESULTS AND DISCUSSION
margins are intensively used for agriculture, on the The lake ecology of Upper Lake is under
southern margin lies Van Vihar National Park. It has constant threat due to various on going developmental
irregular margins that support dense growth of activities in the immediate catchment. In the urban
macrophytes and sustains well diversified aquatic flora surroundings, a vast increase in developmental activities
and fauna. and related waste dumping is observed whereas in the
The study was conducted from June 2010 to June rural areas, major changes in cropping patterns, including
2012 at the Upper Lake of Bhopal (Figure 1 and 2). The extensive use of chemical fertilizers and pesticides has

Journal of Research in Biology (2014) 4(8): 1534-1542 1536

Veerwal et al., 2014

Table 1 The residence and conservation status of bird diversity of Upper lake

S.No Family Scientific Name Common Name Status

Residence Conservation
1 Tachybaptus ruficollis Little Grebe R LC
2 Phalacrocorax carbo Great Cormorant RM LC
3 Phalacrocorax fuscicollis Indian Shag RM LC
4 Phalacrocorax niger Little Cormorant RM LC
5 Anhinga melanogaster Darter RM NT
6 Casmerodius albus Large Egret RM LC
7 Egretta garzetta Little Egret R LC
8 Mesophoyx intermedia Median Egret RM LC
9 Bubulcus ibis Cattle Egret RM LC
10 Ardea cinerea Grey Heron RM LC
11 Ardea purpurea Purple Heron RM LC
12 Butorides striatus Little Green Heron R LC
13 Nycticorax nycticorax Black-crowned Night-Heron R LC
14 Ardeola grayii Indian Pond-Heron R LC
15 Ixobrychus cinnamomeus Chestnut Bittern RM LC
16 Mycteria leucocephala Painted Stork RM NT
17 Anastomus oscitans Asian Openbill-Stork R LC
18 Ciconia episcopus White-Necked Stork R V
19 Ciconia ciconia European White Stork M LC
20 Ciconia nigra Black Stork M LC
21 Ephippiorhynchus asiaticus Black-Necked Stork R NT
22 Threskiornis melanocephalus Oriental White Ibis R NT
23 Pseudibis papillosa Black Ibis R LC
24 Plegadis falcinellus Glossy Ibis RM LC
25 Platalea leucorodia Eurasian Spoonbill RM LC
26 Anser indicus Bar-headed Goose RM LC
27 Tadorna ferruginea Brahminy Shelduck RM LC

28 Sarkidiornis melanotos Comb Duck R LC

29 Dendrocygna javanica Lesser Whistling-Duck R LC

30 Anas acuta Northern Pintail M LC
31 Anas crecca Common Teal M LC
32 Anas poecilorhyncha Spot-billed Duck RM LC
33 Anas platyrhynchos Mallard RM LC
34 Anas strepera Gadwall M LC

become a common practice. The entry of pesticides from the rural margins and untreated sewage from both urban

1537 Journal of Research in Biology (2014) 4(8): 1534-1542

Veerwal et al., 2014

36 Anas clypeata Northern Shoveller M LC

37 Anas querquedula Garganey M LC
38 Rhodonessa rufina Red-crested Pochard M LC
39 Aythya ferina Common Pochard M LC
40 Aythya nyroca Ferruginous Pochard RM NT
41 Nettapus coromandelianus Cotton Teal R LC
42 Grus antigone Sarus Crane R V
43 Amaurornis phoenicurus White-breasted Waterhen R LC
44 Gallinula chloropus Common Moorhen RM LC
45 Porphyrio porphyrio Purple Moorhen R LC
46 Fulica atra Common Coot RM LC
47 Metopidius indicus Bronze-winged Jacana R LC
48 Hydrophasianus chirurgus Pheasant-tailed Jacana R LC
49 Vanellus indicus Red-wattled Lapwing R LC
50 Charadrius dubius Little Ringed Plover RM LC
51 Charadrius alexandrinus Kentish Plover RM LC
52 Tringa totanus Common Redshank RM LC
53 Tringa nebularia Common Greenshank M LC
54 Actitis hypoleucos Common Sandpiper RM LC
55 Tringa stagnatilis Marsh Sandpiper M LC
56 Tringa glareola Wood Sandpiper M LC
57 Calidris temminckii Temmincks Stint M LC
58 Numenius arquata Eurasian Curlew M NT
59 Limosa limosa Black-tailed Godwit M NT
60 Rostratula benghalensis Greater Painted-Snipe R LC
61 Gallinago gallinago Common Snipe RM LC
62 Himantopus himantopus Black-winged Stilt R LC
63 Sterna aurantia River Tern R NT
64 Sterna acuticauda Black-bellied Tern R E
65 Larus brunnicephalus Brown-headed Gull RM LC
66 Ceryle rudis Lesser Pied Kingfisher R LC
67 Alcedo atthis Small Blue Kingfisher RM LC
68 Halcyon smyrnensis White breasted Kingfisher R LC
Residenoncern; NT Near Threatened; V Vulnerable; E Endangeredce Status :R Resident; RM Resident
Migratory; M MigratoryConservation Status : LC Least

and rural surroundings is severely affecting water quality surrounding villages which is seriously degrading the
of the lake (Nandi, 2003). As a result, this water bird natural water quality and thus habitat of water birds (ii)
habitat is under immense pressure, which if not managed uninterrupted effluent discharge leading to high nutrient
and conserved properly, may deteriorate to a level where levels which may lead to increased eutrophication (iii)
its suitability to water birds may decline irreversibly. The sedimentation leading to reduced water storage that may
Upper lake supports many ecosystem services on which influence the open water area required by waterfowl (iv)
many taxa depend. Thus changes in its wetland structure livestock grazing that disturbs the water birds (v)
will undoubtedly also affect the related biodiversity unmanaged tourism and religious activities also
especially the associated avifauna. The major threats to adversely affect bird population.
avifauna in the area are (i) intense agriculture in the A list of birds recorded from Upper lake along

Journal of Research in Biology (2014) 4(8): 1534-1542 1538

Veerwal et al., 2014

with their common names, residence and conservation higher during post monsoon and winter months. Deshkar
status is reported in Table 1. In the present study, 68 et al. (2010) have reported similar observations on
species of water birds belonging to 7 orders and 14 seasonal variations in species richness of birds. The high
families are reported at Upper lake. Among these, the diversity during winter is due to arrival of migratory
most dominating family with 16 species is Anatidae as birds during this season and minimum during summer as
also observed by Vyas (1992; Vyas et al., 2010) in during this season all the migratory birds leave the area
Upper lake and Balapure et al. (2012) in Barna reservoir and return to their home ground. Least number of avian
of M.P. The second dominant family in the present study species was recorded in summer and monsoon which
was Charadriidae with eleven species, Ardeidae with ten may be due to departure of winter visitors, local
species, Ciconiidae with six species, Phalacrocoracidae, migration of resident birds, drying of sites making
Threskiornithidae and Rallidae each with four species, habitats unsuitable for birds in summer season and the
Laridae and Alcedinidae each with three species, commencement of heavy rains in monsoon season. Such
Jacanidae and Rostratulidae each with two species while behaviour is also reported by Surana et al. (2007) in
Podicipitidae, Gruidae and Recurvirostridae were all Chimdilake of Nepal. Out of the total 68 species of
represented by a single species each. Kumar and Gupta waterbirds, 43 species (63.24 %) were migratory birds
(2009; 2013) have also noted that family Anatidae while 25 species (36.76 %) were resident birds. Of all the
dominated the wetland bird community at Kurukshetra 43 migratory birds, 27 species (62.79 %) were local
and Chhilchhila Wildlife Sanctuary, Haryana migrants and 16 species (37.21 %) were long distance
respectively. Verma (2009) reported 68 waterbird species migrants. Chinchkhede and Kedar (2012) reported 54
Bharatpur, Bundh Baretha Reservoir where Anatidae, residents, 9 local migrants and 13 winter migrants among
Scolopacidae, and Ardeidae were recorded as the total observed 76 water bird species in and around the
dominant families. Total 39 water bird species belonging Koradi lake of Nagpur. Among all the 68 water bird
to 16 families were reported by Das and Saikia (2011) species using Upper Lake, 11 important species in terms
from Deeporbeel of Assam. Twenty eight species of of their conservation status are reported (IUCN, 2014).
water birds were reported in three lakes viz. Rajura, Of these 11 species, 8 species of Near Threatened status,
Godada and Dhanora lakes of Buldhana district in 2 species of Vulnerable status and 1 species of
Maharashtra, India by Joshi (2012). Endangered status are reported in this study. These
The waterbird diversity was observed to be Near-threatened species are: Black-Necked Stork
(Ephippiorhynchus asiaticus), Black-headed Ibis
(Threskiornis melanocephalus) and River Tern (Sterna
aurantia) which are all resident water birds; Darter
(Anhinga melanogaster) (Figure 4), Painted Stork
(Mycteria leucocephala) and Ferruginous Pochard
(Aythya nyroca) which are resident migratory water
birds; Black tailed Godwit (Limosa limosa) and Eurasian
Curlew (Numenius arquata) which are migrant species of
water birds. Among the vulnerable species were 2 water
birds - White necked Stork (Ciconia episcopus) and
Figure 3 SARUS CRANE at Upper lake of Bhopal Sarus Crane (Grus antigone) (Figure 3) which are both

1539 Journal of Research in Biology (2014) 4(8): 1534-1542

Veerwal et al., 2014

resident birds. One water bird species reported in the lake with proper perching sites where the birds could
endangered category is the Black-bellied Tern (Sterna assemble to consume their prey which were for the most
acuticauda) which is a resident bird. Asian Openbill part small fishes.
Stork (Anastomus oscitans) (Figure 5) is a resident water
bird of least concern. CONCLUSION
Presence of such important birds as regular Upper lake is an ideal residence for innumerable
visitors as well as some residents in this lake highlights water birds with different needs. The presence of
the importance of this area as a bird haven. Water birds migratory birds, local migratory and resident species of
like Black-necked Stork and White necked Stork were birds equally utilizing this area as their abode for
found along the shoreline areas of the lake with moderate continuing various lifecycle activities indicates the
tree canopy which allowed these birds proper roosting importance of Upper Lake as a year round habitat for
sites as well as protection from direct human disturbance. water birds.
Maheswaran and Rahmani (2001) have reported that However, identification of this lake as a Ramsar
higher water level (> 60 cm) is not suitable for wading site and IBA is alone not sufficient to conserve the entire
birds, including the Black-necked Stork even though the biodiversity. In the present state of ecologically
patch has more prey species. Sarus Crane and Black- unmanaged development, full protection to all the
headed Ibis preferred marshy borders of the wetland with existing habitats should be given with special attention
more affinity towards surrounding agricultural fields. during the migratory period. Since the lake is a shared
Thus, these species demonstrated a tendency to endure resource, a common property to all the occupants
human presence up to some level. Darter and Painted including animals and human being alike, it is imperative
storks were found preferring areas with dense to protect or conserve the entire ecosystem. For this, the
surrounding vegetation but with least disturbance and most notorious component of this arrangement and the
moderate water depth. The conversion of wetlands into principal stakeholder involved in the deterioration
agricultural fields is altering the preferred habitats of process i.e. the human community must be educated
birds and thus negatively impacting their distribution about the importance of conserving the lake. With an
(Del Hoyo et al., 1996; Bird Life International, 2014). increasing human population placing strain on the
The terns were mostly observed at deep water zones of valuable inland freshwater resources and the related

Figure 4 DARTER at Upper lake of Bhopal Figure 5 OPENBILL-STORK at Upper lake, Bhopal

Journal of Research in Biology (2014) 4(8): 1534-1542 1540

Veerwal et al., 2014
avifauna due to rapid conversion of rural areas to urban Del Hoyo J, Elliott A and Sargatal J. 1996. Handbook
lands, by creating awareness in local people through of the birds of the world, vol. 3: Hoatzin to Auks. Lynx
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Deshkar S, Rathod J and Padate G. 2010. Avifaunal
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Commission (UGC), Government of India, New Delhi, Z, Knowler DJ, Leveque C, Naiman RJ, Prieur-
for providing financial assistance in the form of award of Richard AH, Soto D, Stiassny ML and Sullivan CA.
Junior/ Senior Research Fellowship to Hina Veerwal 2006. Freshwater biodiversity: importance, threats, status
which made completion of this work possible. and conservation challenges. Biological Reviews. 81(2):
Joshi PS. 2012. An annotated checklist of aquatic
Ali S. 2002. The Book of Indian Birds. Bombay Natural
avifauna of Rajura, Godada and Dhanora lakes of
History Society. Oxford University Press, 326.
Buldhana district of (M. S.) India. Science Research
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birds of India and Pakistan together with those of
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Bangladesh, Nepal, Bhutan and Sri Lanka. Oxford
abundance of wetland birds around Kurukshetra, India.
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diversity in Barna \wetland of Narmada basin in central
of Chhilchhila Wildlife Sanctuary, Haryana, India.
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Journal of Research in Biology ISSN No: Print: 2231 6280; Online: 2231- 6299

An International Scientific Research Journal

Original Research

A review on ethnomedicinal, phytochemical and pharmacological studies

of Geodorum densiflorum (Lam.) Schltr. - an endangered orchid
Journal of Research in Biology

Authors: ABSTRACT:
Keerthiga M* and
Anand SP.

From the primitive period, medicinal plants have been used in treating
Institution: human diseases and the traditional herbal medicines have owned its distinct place in
1. Department of
medication because of their pharmacological importance. Geodorum densiflorum is a
Biotechnology, National
medicinally important endangered orchid which is widely used in the orient.
College (Autonomous),
Tiruchirappalli 01, Flavonoids, alkaloids and terpenoids, the primary constituents of G. densiflorum are
Tamil Nadu. mainly believed to be responsible for its wide therapeutic actions. The orchid was
traditionally recommended for various ailments such as wound healing, skin diseases,
2. PG and Research dysentery, diabetes, improving fertility in men, to cure carbuncles and to regularize
Department of Botany, menstrual cycle in women. The present review provides comprehensive information
National College on plant, ethnomedicinal, phytochemical and pharmacology profile and current
(Autonomous), research prospects of the orchid.
Tiruchirappalli 01, Tamil

Corresponding author: Keywords:

Keerthiga M. Orchidaceae, Ethnomedicinal uses, Geodorum densiflorum

Article Citation:
Email Id:
Keerthiga M and Anand SP.
A review on ethnomedicinal, phytochemical and pharmacological studies of
Geodorum densiflorum (Lam.) Schltr. - an endangered orchid
Journal of Research in Biology (2014) 4(8):1543-1548
Web Address: Received:22 Aug 2014 Accepted: 12 Sep 2014 Published: 03 Dec 2014
This article is governed by the Creative Commons Attribution License (
licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

1543-1548| JRB | 2014 | Vol 4 | No 8

Journal of Research in Biology
An International
Scientific Research Journal
Keerthiga and Anand, 2014

Orchids belonging to the family Orchidaceae Intensive phytochemical investigations of the
display diversified in their floral morphology compared leaves and pseudobulb of orchid have resulted in the
to other angiospermic plants. Eventhough the family is presence of flavonoids, terpenoids, alkaloids and
characterised as highly advanced group, they are at risk steroids. The isolated compounds display some of the
for slight changes in environmental conditions will cause interesting pharmacological activities, such as
the orchids as endangered one. Approximately 25,000 to antibacterial, cytotoxicity, antioxidant, thrombolytic,
35,000 species within 600 800 genera of Orchidaceae analgesic, sedative and anxiolytic properties. Despite
were distributed throughout the world (Chugh et al., number of studies were reported on the evaluation of
2009). In India, Orchidaceae form 9% of our flora and biologically active components and their
about 1,300 species with 140 genera was found in pharmacological action, yet these studies were still
Himalayas as their native (Yonzone et al., 2008). inadequate. Although there have been several claims
Orchidaceae plants will grow in variety of habitats and regarding the bioactive compounds which involved in the
are mostly annual and perennial herbs and live either as biological actions of the plant, more scientific research
epiphytes, lithophytes and terrestrial. The people are are needed to justify the therapeutic value of this orchid.
attracted by orchids because of their high ornamental The present review gives a detailed account of the plant,
value but nowadays they are known for its medicinal ethnomedicinal uses, phytochemical and pharmacology
importance. In Chinese Materia Medica, a total of 365 profile along with the current research potential of the
plants including several orchids are grown primarily as orchid.
ornamentals, many of which are now used as herbal Systematic Description
medicines, food and other cultural components by tribes Kingdom - Plantae
in different parts of the world. Division - Tracheophyta
Geodorum densiflorum (Lam.) Schltr. belongs to Class - Magnoliopsida
the family Orchidaceae, an endangered terrestrial orchid Order - Asparagales
(Datta et al., 1999) commonly known as Nodding Family - Orchidaceae
Swamp orchid. It is a glabrous and deciduous plant Genus - Geodorum
which grows during spring and summer, becoming Species -densiflorum (Lam.) Schltr. (ITIS Report,
deciduous in winter (Ramirez, 2007) and is widely
distributed in the tropical forests of south India Plant Description
(Sudhakor et al., 2005) and in Bangladesh. Terrestrial herb forming small clumps, 33-50 cm
G.densiflorum is a medicinal orchid plant that has been tall. Pseudobulbs subglobose, crowded, subterranean to
traditionally used for the treatment of various diseases. partially emergent, erect, ovoid, 2-4 x 3-3.6 cm, fleshy,
The root paste is used as an insecticide and wound often yellowish, enclosed by scarious sheaths. Stem
healing medicine. The pseudobulb is used to regularize erect, 8 cm long, slender. Leaves
menstrual cycle (Dash et al., 2008), for the treatment of 3-5, erect to prostrate, apical; enclosed in sheathing
diabetes (Patil et al., 2005, Roy et al., 2002), and it is bracts forming a pseudostem; 10-32 x 4-9 cm, suberect,
applied externally to cure carbuncles (Nath et al., 2011), lanceolate, lanceolate-elliptic, or oblong-elliptic, acute to
etc. It is also possessing antimicrobial and antidiabetic acuminate, petiolate, blade; petiole 5-22 cm long, dark
property (Saleha et al., 2010). green to yellowish, pleated, margins entire to undulate
1544 Journal of Research in Biology (2014) 4(8): 1543-1548
Keerthiga and Anand, 2014

margins entire to undulate, apex acute to acuminate. are made into powder and 5 gm of the powder mixed
Inflorescence: shorter than the leaves, compact, with 200 ml cow milk is given orally twice a day for 15
subglobose axillary raceme, 200-300 mm tall, nodding in days or till it is cured (Tiwari et al., 2012).
flower, erect in bud and at seed dispersal; pendent, 8-10 Tubers are cleaned, powdered and taken with honey to
flowered; pedicels 5-10mm long. Flowers: 0.9-1.2 cm increase sperm density (Rahmatullah et al., 2010).
long, white to pale green with yellow and purple Rhizomes are consumed along with honey for two to
markings. Sepals and petals similar, gaping, linear- three months as a treatment for impotency (Patil et al.,
oblong, ovate-elliptic, acute, 5 to 7 veined. Dorsal sepal 2004).
lanceolate, lateral sepals free, lanceolate. Petals similar Phytochemical profile
to lateral sepals. Lip 1-1.3 x 0.8-1.1 cm, pink with dark G. densiflorum was investigated for the
red veins boat shaped; apex obscurely 2 rounded lobes, phytochemical chemical constituents of methanol, ethyl
margins weakly undulate; Column 3-4 mm long, weakly acetate and petroleum ether extracts of the roots which
curved, with a foot. Capsules dehiscent, obovoid, revealed the presence of carbohydrate, alkaloid,
pendant. Fruit elongated. (Figure 1, 2 and 3). glycoside and steroid (Habib et al., 2011).
Ethnobotanical Uses Phytochemical screening of G. densiflorum shows
To regularize menstrual cycle in women, the root paste presence of alkaloids, steroids, carbohydrates,
of G. densiflorum was taken orally by mixing along with flavonoids, tannin and saponins in different extract such
two drops of ghee and 5 ml of honey (Dash et al., as water, ethanol and chloroform and were reported
2008). having various pharmacological activities (Keerthiga et
Australians use the gum obtained from root stock for al., 2014).
joining parts of musical instruments. Fresh root stocks Various major, minor and trace elements
were crushed and rubbed on cattle to kill flies (Yonzone concentration of G. densiflorum were studied for
et al., 2011). determining pharmacological action against various
Tuber powder mixed with cow ghee is employed to diseases. Among the twenty six vital elements, the plant
treat dysentery (Mohammad, 2011). had maximum concentration of phosphorus. As stated by
Tuber is used to treat impotency in men. Dried tubers Hossen et al. (2014), Phosphorus participates in DNA

Figure 1. Habit of Geodorum densiflorum Figure 2. Vegetative stage of Geodorum densiflorum

Journal of Research in Biology (2014) 4(8): 1543-1548 1545

Keerthiga and Anand, 2014

Oxide (NO) scavenging activity showed that the

petroleum ether extract had high antioxidant capacity
than other extract. Reducing power and CUPRAC assay
revealed that ethyl acetate extract was having high
reducing activity (Habib et al., 2011).
Cytotoxic Activity
In vivo cytotoxicity studies were subjected and a
concentration dependent increase in percent mortality
was produced by the extracts. The study revealed the
presence of cytotoxic principles in these extractives
(Habib et al., 2011).
Figure 3. Flowers of Geodorum densiflorum (Lam.) The cytotoxic activities of crude extract were
determined using brine shrimp lethality bioassay and on
synthesis, energy metabolism and calcium absorption comparison to vincristine sulfate as standard, ethyl
and utilization. acetate extract showed a significant cytotoxic activity
Pharmacological profile (Hossain et al., 2012).
Antibacterial activity Thrombolytic activity
In vitro antibacterial study of three different Thrombolytic activity of five traditional
extracts using water, ethanol and chloroform solvents medicinal plants was evaluated from which
with various concentrations was tested for their G. densiflorum whole plant with different solvents viz:
bioactivity. Maximum antibacterial activities were ethanol, petroleum ether and ethyl acetate shows
recorded for ethanolic extract against Staphylococcus moderate to negligible amount of clot lysis, i.e. 25.31%,
aureus followed by Klebsiella pneumoniae but water 29.82%, and 19.06% (Hossen et al., 2014).
extracts didnt show any significant antibacterial activity
(Keerthiga et al., 2014) Analgesic activity
The antibacterial screening of extracts revealed Khatun et al. (2013) evaluated peripheral
that methanolic extract showed significant activity analgesic activity by conducting acetic acid induced
against both Gram positive and Gram negative bacterium writhing test. The n-hexane and dichloromethane soluble
than other extracts (Akter et al., 2010). Previous extract of the root of G. densiflorum was used for
investigation on the antibacterial activity of G. analgesic activity in mice. At higher dose (400mg/
densiflorum against thirteen pathogenic bacteria was kg, p.o.), both extracts exhibited significantly (p<0.001)
reported that ethyl acetate extract displayed the highest moderate analgesic property compared to that of
inhibitory actions (Habib et al., 2011). diclofenac sodium which might be due to interaction
Antioxidant activity with the central and/or peripheral opiate system.
G. densiflorum (Lam) Schltr. was investigated Sedative activity
for the antioxidant property of methanol, ethyl acetate Extracts from G. densiflorum using n-hexane and
and petroleum ether extracts of the roots. In vitro dichloromethane was assessed by hole cross and open
antioxidant activity of the extracts was performed using field tests. Both extracts at doses of 200mg/kg and
various methods. DPPH radical scavenging and Nitric 400mg/kg, p.o. showed statistically significant (p<0.001

1546 Journal of Research in Biology (2014) 4(8): 1543-1548

Keerthiga and Anand, 2014

and p<0.01) suppression of locomotor and exploratory

Datta KB, Kanjilal B and Desarker D. 1999. Artificial
behaviours of mice compared to that of diazepam in the seed technology: Development of a protocol
hole cross test. In the open field test, all test samples in Geodorum densiflorum (Lam.) Schltr. An endangered
except 400mg/kg dose of dichloromethane soluble root orchid. Current Science. 76: 1142-1145.
extract showed same results that were observed in the Farjana Khatun, Nishat Nasrin, Shammee Monira,
hole cross test. The study done by Khatun et al. (2013) Muhammad Asaduzzaman and Apurba Sarker Apu.
revealed the sedative activity of the medicinal orchid. 2013. Assessment of neuropharmacological and
analgesic potentials of Geodorum densiflorum (Lam.)
Anxiolytic property
Schltr. root extracts in experimental animals.
The root of G. densiflorum was reported to
Pharmacologyonline. 3: 16-22
possess anxiolytic activity in both n-hexane and
Habib M R, Rana M S, Hasan M R, Imam M Z,
dichloromethane extracts as confirmed by the hole board
Hasan S M R and Saha A. 2011. Antioxidant,
test (Khatun et al., 2013). cytotoxicity and antibacterial potential of different
extract of Geodorum densiflorum (Lam.) Schltr. root.
CONCLUSION Journal of Herbal Medicine and Toxicology. 5 (1): 63-
It is concluded that the plant extract possesses 70 .

antibacterial and antioxidant property and it can be used
effectively as a natural tool for treating different SingleRptsearch_topic=all&search_value=Geodorum+
infectious diseases. The orchid is also reported to possess densiflorum&search_kingdom=every&search_span=exac
cytotoxic, thrombolytic, sedative, analgesic and tly_for&categories=All&source=html&search_credRatin
anxiolytic activities. The present review provided g=All (Accessed on July 10, 2012).

combined information about ethnomedicinal, Keerthiga M and Anand SP. 2014. Physicochemical,
phytochemical and pharmacological studies on G. preliminary phytochemical analysis and antibacterial
activity against clinical pathogens of medicinally
densiflorum. It is necessary to carry out more clinical and
important orchid Geodorum densiflorum (Lam) Schltr.
pharmacological studies by the researchers to exploit the
International Journal of Pharmacy and Pharmaceutical
potential of this plant. Further investigations to find out Sciences. 6(8): 558-561
novel drug lead compounds of industrial importance
Lokman Hossen Md, Azharul Islam SM, Md. Joynal
could be a new platform for future researchers. Abedin, Shirin Akter, Rasel OF, M. Monjur Ahasan,
Rajada Khatun and Ashrafun Nahar Monika 2014.
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Dilruba Nasrin, Rasheda Ahmed, Md Mahbubur
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plants used by the Kavirajes of Daulatdia Ghat, Kushtia
district, Bangladesh. American-Eurasian Journal of
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Akter. 2010. Antimicrobial activity of different extracts
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Yonzone R., Lama D, Bhujel RB and Rai S. 2011.
Intraspecific colour variation in population of
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1548 Journal of Research in Biology (2014) 4(8): 1543-1548

Journal of Research in Biology ISSN No: Print: 2231 6280; Online: 2231- 6299

An International Scientific Research Journal

Original Research

Phytochemical and antibacterial studies on the aqueous extract

of Eucalyptus gomphocephala DC
Authors: ABSTRACT:
Journal of Research in Biology

Hayate Bouharb1 ,
Khalid El Badaoui1,
Ali Amechrouq2 and This work aims to find out the molecule responsible for the good activity
Jalila El Amri1. against the strains of Pseudomonas aeruginosa reflected in a pre work done on the
phytochemical study of aqueous extract of Eucalyptus gomphocephala. We have
Institution: initially processed the quantitative colorimetric determination by a UV-Vis
1. Laboratory spectrophotometer for total polyphenols and flavonoids and a qualitative analysis by
of Environment and Health, high performance liquid chromatography (HPLC) coupled with mass spectroscopy.
Faculty of Science, Quantitative determinations of total polyphenols by the Folin-Ciocalteu reagent and
University Moulay Ismail, flavonoids by AlCl3 method revealed the richness of this extract in total polyphenols.
BP 11201 Zitoune Meknes, Qualitative analysis by HPLC / ESI-MS revealed the presence of gallic acid. This
Morocco. molecule was tested by the agar diffusion method and the macrodilution method in
2. Laboratory of liquid medium, which showed greater activity than the aqueous extract. The results
Chemistry and Molecular obtained in this study suggest that the gallic acid may be used in the treatment of
Substances , Faculty of infections caused by Pseudomonas aeruginosa .
Science, University Moulay .
Ismail, BP 11201 Zitoune,
Meknes, Morocco
Corresponding author: Eucalyptus gomphocephala, quantitative analysis, qualitative analysis,
Hayate Bouharb aqueous extract, gallic acid, Pseudomonas aeruginosa

Article Citation:
Hayate Bouharb, Khalid El Badaoui, Ali Amechrouq and Jalila El Amri.
Email Id: Phytochemical and antibacterial studies on the aqueous extract of Eucalyptus
gomphocephala DC
Journal of Research in Biology (2014) 4(8): 1549-1556

Received: 22 Nov 2014 Accepted: 29 Nov 2014 Published: 20 Dec 2014

Web Address: This article is governed by the Creative Commons Attribution License ( licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and
documents/RA0494.pdf reproduction in all medium, provided the original work is properly cited.

1549-1556| JRB | 2014 | Vol 4 | No 8

Journal of Research in Biology
An International
Scientific Research Journal
Bouharb et al., 2014

INTRODUCTION variety of bioactive molecules (Ferrari, 2002).

The genus Eucalyptus is native to Australia The purpose of this work is the phytochemical
where there are over 750 species (Elliot and Jones, study of aqueous extract of Eucalyptus gomphocephala
1983; Singh et al., 1999). These kinds of species which showed good activity against strains of
have the particularity to grow rapidly and are used to Pseudomonas aeruginosa (Bouharb et al., 2014) and
drain the swampy soil. They are also used for timber to identify some compounds for highlighting the
and firewood. Their essential oils are used in molecule responsible for the antibacterial activity.
pharmaceutical and cosmetic industries for the
manufacture of various products (Hyodo et al., 1992) MATERIALS AND METHODS
The genus Eucalyptus is known to be a rich Plant material
source of bioactive natural products, including terpenes, The plant was harvested in summer during the
tannins, flavonoids and derivatives of phloroglucinol month of July. The harvest was carried out at mid-day,
(Sing et al., 1999).The eucalyptol (1.8 cineole) is the the collection was casually done by selecting adult tree
principle active essential oil of the genus Eucalyptus that leaves on a same tree. These are dried in shade and
posses different pharmacological actions (IPD, 1996). ground finely to make the plant material suitable for
The introduction of Eucalyptus in Morocco elucidating the bioactive compound. The identification
dates back to the early 19 century. They are spread of the plant has been made to the National Forestry
over more than 200,000 ha, or 41% of the total area of School of Engineering (Sale).
artificial plantations, most of which is occupied by the Preparation of the aqueous extract
E. gomphocephala and E. camaldulensis (80% of the 100g of powder was extracted by heat reflux for
planted area) (Marien, 1993). two hours in water and then filtration and evaporation
The Eucalyptus gomphocephala is a plant very was carried out in a rotary evaporator at 60C; the
interesting because it grows well on limestone, upto residue obtained is kept still until its use.
the level of 45 meters (Boudy, 1952). The plant division Phytochemical study
has many beneficial qualities and economic uses. It is Quantitative analysis
used in the national industrial wood production division Content of total polyphenols
and also as a honey plant. It was widely used in Polyphenols are estimated by the Folin-Ciocalteu
traditional medicine for its healing properties. In (Wong et al., 2006). 1 ml aqueous extract dissolved in
Morocco, this plant is used in the treatment of cold, distilled water was added to 1 ml of Folin-Ciocalteu
bronchitis and as a fumigant against microbes. reagent and diluted 10 times. After 4 min, 800 l of
The leaves of E. gomphocephala were reported sodium carbonate solution (75g / l) was added in a
to have biological activities including antioxidant and volumetric flask of 25ml. The absorbance was measured
cytotoxic activities (Alsayed et al., 2010), antitumor at 765 nm after 2h of incubation. The concentration of
activity (Alsayed et al, 2012), insecticidal activity polyphenols were detected from the calibration ranges
(Barbouche et al., 2007; Guendouz et al., 2006), established with gallic acid (0-200g / ml) and are
antibacterial (Bouharb et al., 2014) and so on. expressed in milligrams of gallic acid equivalent per
The study of the chemistry of plants is still a gram of dry matter MS (EAG mg / g) .
burning issue despite its age. This is mainly due to the Determination of Flavonoids
fact that the plant kingdom is a major source of immense The method of aluminum trichloride (Bahorun
1550 Journal of Research in Biology (2014) 4(8): 1549-1556
Bouharb et al., 2014

et al., 1996) was used to quantify the flavonoids of Table 1: Identification of chemical profile of
the aqueous extract
various extracts. Quercetin was used as a standard (0.1
g / l prepared in methanol). 1 ml of extract was added to N Temps de rtention Aire (%)
0.1 ml of the solution of AlCl3 (10% in methanol). 20 ml 1 2.44 18.79
2 3.36 81.21
distilled water was added to it and mixed with methanol
% Total 100
in a volumetric flask of 50ml. After 10 min, the
absorbance was read at 430 nm. The concentration of phase flow rate was 0.5 ml / min. The temperature was
flavonoids were detected from the calibration range adjusted to 40 C (Kuntie et al., 2007).
established with quercetin (0-35 g / ml) and are Antibacterial tests
expressed in milligrams of quercetin equivalents per In vitro inhibition of bacterial growth of
gram of dry matter (EQ mg / g). Pseudomonas aeruginosa by the aqueous extract of E.
Qualitative Analysis gomphocephala was investigated by solid medium
Work on this section were performed on a diffusion method and macrodilution method was done in
HPLC chromatographic system coupled to mass liquid medium (Bouharb et al., 2014). In this study, we
spectrum in the laboratory of UATRS (CNRST, Rabat). have tested the activity of pure molecule of gallic acid
The HPLC used was LC Surveyor thermo-electron (10mg / ml) against the bacterial strain.
brand, equipped with low-pressure quaternary pump with
integrated degasser in isocratic mode. The separated RESULTS AND DISCUSSION
molecules were then detected by two systems: a Photo Quantitative study
Diode Array spectrophotometer (PDA) and Surveyor For there are many important biological
(spectral range from 190 to 800 nm) followed by a mass activities in polyphenols and flavonoids, we have chosen
spectrometer (LCQ Advantage MAX) comprising an ion them among different phytochemicals for quantification.
trap with ESI ionization. The ionization of samples was Two straight calibration (Fig. 1 and 2) were
made using negative polarity. The mass range was set plotted for this purpose which are made with the
at: 50-2000. To analyze the aqueous extract of standard solutions at different concentrations. The
Eucalyptus, HPLC-RP-C18 was used. The stationary amounts of polyphenols and related flavonoids have
phase was a column (125 x 4.6mm) whereas, the mobile been reported in milligrams equivalent of the standard
phase was methanol / water (60/40), and the mobile used per gram of dry matter (ms) (mg EE / ms g) and

Figure 1 : Calibration curve of gallic acid for the determination of total polyphenols

Journal of Research in Biology (2014) 4(8): 1549-1556 1551

Bouharb et al., 2014

Figure 2: Calibration curve of quercetin for determination of flavonoids

determined by the type of equation: y = ax + b. The coefficient of determination (R2 = 0.995) (Figure 2).
assay of total polyphenols was made according to the The contents of total polyphenols and flavonoids in
method of Folin-Ciocalteu reagent. Gallic acid was used the aqueous extract of leaves of Eucalyptus
as a standard. The flavonoids assay was performed gomphocephala (mg / g of DM) are:
according to the method of aluminum trichloride and Polyphenol content: 10.64 1.94 mg / g DM
quercetin was used as a standard. Flavonoid content: 0.49 0.004 mg / g DM
The only work done on the levels of phenolic
Calibration curve for the determination of total
compounds in the species studied is that of Alsayed et al.
(2012). A comparison between the two studies is
This curve is established using gallic acid as
difficult because it is important to emphasize that the
reference. The formula for the linear regression of the
use of original plant is distinctly different from
curve y = 0.0048 x + 0.0198 is used with a correlation
geographical and climate zone as well as different
coefficient R2 = 0.991 (Figure 1).
extraction and assay methods which reduce the
Calibration curve for the determination of flavonoids
reliability of a comparison between the two studies.
The reference compound used in the preparation
Recent studies have shown that the level of phenolic
of this curve is quercetin. The formula for the linear
compounds change in a considerable way from one
regression of the curve is y = 0.0732*-0.0811 with a

Figure 3: Chromatogram of aqueous extract of Eucalyptus gomphocephala

1552 Journal of Research in Biology (2014) 4(8): 1549-1556

Bouharb et al., 2014

Figure 4: Mass spectrum of gallic acid in negative mode

species to another and within the same species (Ksouri et nature of organs) (Maisuthisakul et al., 2007) and the
al., 2012), because of external factors (temperature, period of storage (Aganga and mosase, 2001).
climate, etc.,) (Ksouri et al., 2008), genetic (variety and Qualitative Study
the origin of species) (Ebrahimzadeh et al., 2008), The aqueous extract of E. gomphocephala was
physiological (the degree of maturation of the plant, analyzed by HPLC / ESI-MS to obtain information

Figure 5: Mass Spectrum of gallic acid in the aqueous extract in negative mode

Journal of Research in Biology (2014) 4(8): 1549-1556 1553

Bouharb et al., 2014

on the chemical nature of its constituents. Both standards seeds. Animal Feed Science and Technology. 91(1-2):
were used: gallic acid and quercetin. The results of this 107-113
study showed the absence of quercetin and the presence
Al-Sayed E, Martiskainen O, Bobrowska-
of large quantities of gallic acid (81.21%) (Table 1).
Hgerstrand M, Sinkkonen J, Trnquist K, Pihlaja
Antibacterial tests
K, Ayoub N and Singab AN. 2010. Phenolic
The results of antibacterial tests (Bouharb et
compounds from Eucalyptus gomphocephala with
al., 2014), from the aqueous extract of E.
potential cytotoxic and antioxidant activities. Natural
gomphocephala showed good activity against all
Product Communications. 5(10): 1639-1642
strains of P. aeruginosa with inhibition zones of 12 to 18
mm and the minimum inhibitory concentration (MIC) Al-Sayed E, Singab A, Ayoub N, Martiskainen O,
between 6.25 and 12.5 mg / ml. Sinkkonen J and Pihlaja K. 2012. HPLCPDAESI
The remarkable presence of gallic acid in the MS/MS profiling and chemopreventive potential of
aqueous extract prompted us to test this molecule Eucalyptus gomphocephala DC. Food Chemistry. 133
against P. aeruginosa. The results showed higher (3): 10171024
activity than the aqueous extract with MICs of 2.5 mg /
Bahorun T, Gressier B, Trotin F, Brunet C, Dine T,
ml. Gallic acid was demonstrated effectively by several
Vasseur J, Cazin JC, Pinkas, M, Luyckx M and
studies as antibacterial and especially against P.
Cazin M. 1996. Oxygen species scavenging activity of
aeruginosa. Ikuro et al. (2000) showed that the gallic
phenolic extracts from hawthron fresh plant organs and
acid and its esters were evaluated as inhibitors of the
pharmaceutical preparations. Arzneimittel-Forschung. 46
enzyme p-Hydroxybenzoate recombinant Hydroxylase
(11): 1086-1089.
(PHBH) a flavin-dependent monooxygenase
Nicotinamide Adenine Dinucloeotide Phosphate Barbouche N, Jeridi S and Ben Dhaou S. 2007.
(NADPH) from P. aeruginosa . Premkumar et al. (2010, Alternative de lutte biologique contre Sitophilus
2011) also has demonstrated antibacterial effect of the oryzae (Coleoptera : curculionidae) par lutilisation des
combination of polyphenolic compounds and gallic acid huiles essentielles et des poudres de plantes. Rev. INAT.
with antibiotics. Revue de l'Institut National Agronomique de Tunisie
(TN) 22(1): 85-96.
Boudy P. 1952. Guide du forestier en Afrique du nord.
In this study, gallic acid was shown to be a
Ed. La Maison Rustique. Paris. 505
promising molecule against P. aeruginosa. It is
recommended to do further research with different Bouharb H, K EL Badaoui, T Zair, H Shisseh, S
solvents and extraction methods on the Eucalyptus Chakir and Alaoui T. 2014. Antibacterial Evaluation
gomphocephala for identifying more active molecules. and Phytochemical Screening of Eucalyptus
gomphocephala DC against Pseudomonas aeruginosa.
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1556 Journal of Research in Biology (2014) 4(8): 1549-1556

Journal of Research in Biology ISSN No: Print: 2231 6280; Online: 2231- 6299

An International Scientific Research Journal

Original Research

A comparative hydrobiological study on Authoorangal channel and

Srivaikuntam channel and their role as sustainable water resources
Journal of Research in Biology

A hydrobiological study conducted in two different river channels of two

Authors: different taluks of south Tamil Nadu showed that the concentrations of Physico-
Esther Isabella Eucharista F
chemical parameters like turbidity, temperature, pH, dissolved oxygen, electrical
and Mohanraj Ebenezer
conductivity, total dissolved solids, total hardness, total alkalinity, calcium,
magnesium, sodium, potassium, chloride, sulphate, carbonate, bicarbonate, total
coliform and faecal coliform were deteriorating water quality rapidly due to intense
and excessive amounts of fertilizers, manures, pesticides and insecticides. In the
present investigation among the two channels, maximum values of the physical
parameters with a mean of turbidity 6.50.070 NTU, temperature 33.90.707 0C, pH
Institution: 8.70.707, dissolved oxygen 7.90.707 mg/l, electrical conductivity 3.080.632 ds/m,
PG and Research
total dissolved solids 15.7 3.162mg/l, total hardness 25.66.324 mg/l, and total
Department of Zoology,
alkalinity 50.11.140 mg/l was recorded. The maximum values of the chemical
St. Johns College,
Palayamkottai - 627002, parameters with a mean of calcium 10.01.702 mg/l, magnesium 11.60.378 mg/l,
Tamil Nadu. sodium 8.482.828 mg/l, potassium 0.230.012 mg/l, chloride 16.12.607 mg/l,
sulphate 11.93.224 mg/l, carbonate 0.30.070 mg/l and bicarbonate 2.80.050 mg/l
was noticed. The maximum values of the coliform parameters with a mean of total
coliform bacteria 3900.707 (MPN/100 ml) and faecal coliform 370.707 (MPN/100
ml) was also recorded. The results of this study point out the fact that the water
quality of both the channel waters are slowly deteriorating.

Corresponding author: Physico-chemical and coliform parameters, Authoorangal and Srivaikuntam
Esther Isabella Eucharista F.
river channels

Email Id:
Article Citation:
Esther Isabella Eucharista F. and Mohanraj Ebenezer
A comparative hydrobiological study on Authoorangal channel and Srivaikuntam
channel and their role as sustainable water resources
Journal of Research in Biology (2014) 4(8): 1557-1567

Web Address: Received: 09 Aug 2014 Accepted: 4 Dec 2014 Published: 26 Dec 2014
This article is governed by the Creative Commons Attribution License (
licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

1557-1567 | JRB | 2014 | Vol 4 | No 8

Journal of Research in Biology
An International
Scientific Research Journal
Eucharista and Ebenezer, 2014

INTRODUCTION essential in monitoring the physico-chemical parameters

Fresh water is an important natural resource of Authoorangal channel and Srivaikuntam channel for
necessary for the survival of all ecosystems. In India, the welfare of surrounding human settlements. The
ponds, lakes, rivers and ground water are used for present study is an attempt to assess the water quality
domestic and agricultural purposes (Hemant Pathak et with the following objectives:
al., 2011). Agriculture is the chief source of livelihood Collection of baseline data on the structure and
for the use of agriculture a great effect on the flow of function of the Authoorangal channel and Srivaikuntam
fresh water is necessary. The potable water should be channel.
free from infectious agents and chemical constituents, Assessment of seasonal changes in physico-chemical
pleasant to taste, odour and usable for domestic purposes parameters.
(Saini et al., 2010). Physico-chemical parameters play a Analysis of seasonal fluctuations in the microbes.
significant role in determining the distributional pattern
and quantitative abundance of aquatic ecosystem MATERIALS AND METHODS
(Sayeswara et al., 2011). The physical and chemical Study area of Authoorangal Channel
properties of fresh water bodies are based upon the The Authoorangal channel has thirteen channel
geomorphological and weathering processes (Sahni and inlets. It has the width of about 6m broad and 1.5m depth
Yadav, 2012). (Figure 1). The channel is getting water from
The presence of safe and reliable drinking water Thamirabarani river. Further, it is passing through
is a significant prerequisite for a stable community (Sen Authoor and Sethukavaithan villages. In about 460
et al., 2011). Due to the growth of population and man- acres, the water is utilized for agricultural purposes. It
made activities, the quality of water is deteriorating belongs to Tiruchendur taluk.
everywhere. For this purpose, it is important to know Study area of North main channel (Srivaikuntam)
about different physico-chemical characteristics of water. The North main channel (Srivaikuntam) is spread
There has been no report on physico-chemical over a distance of 87 and 82 acres (Figure 2). The
parameters assessment in the Authoorangal channel and channel has the width of 30 feet and 6 feet depth. It
Srivaikuntam channel. Hence, a periodical assessment is consists of two channel inlets namely Mukoot and

Figure 1: Sampling Site Authoorangal Figure 2: Sampling Site Srivaikuntam

1558 Journal of Research in Biology (2014) 4(8): 1557-1567

Eucharista and Ebenezer, 2014
Table 1: Monthly variations of physical parameters of Authoorangal channel in the year of 2011
Turbidity Tempera-
Month pH DO (mg/l) EC (ds/m) TDS (mg/l) TH (mg/l) TA (mg/l)
(NTU) ture 0C
January 4.00.252 21.10.935 7.30.196 7.90. 326 1.20. 078 11.00. 465 10.10.345 22.20. 677
February 4.40. 307 22.40. 196 7.90. 183 7.10. 111 3.00.058 11.10. 160 15.50.316 25.70. 080
March 4.10.040 23.50.223 7.70.066 7.30.094 0.490.029 11.50.353 4.30.050 28.00.948
April 4.00.707 23.70.241 7.70.230 6.60.215 0.490.010 11.30.330 3.30.134 26.20.322
May 3.90.037 25.90.583 8.50.612 5.60.168 0.310.005 12.00.400 6.60.276 33.30.583
June 4.00.316 25.70.241 7.80.193 5.50.067 1.270.013 12.30.083 4.10.031 29.80.083
July 4.30.221 24.50.223 7.40.126 5.80.126 0.270.023 13.10.333 3.00.707 30.30.377
August 3.40.141 24.81.414 7.40.126 6.30.134 0.300.044 13.23.049 16.23.391 33.21.140
September 3.40.178 24.90.707 7.32.302 5.72.024 0.210.032 12.31.898 13.01.140 40.26.324
October 4.51.414 23.76.324 7.90.707 7.60.707 3.080.632 14.90.707 25.66.324 45.512.747
November 5.91.303 22.23.224 7.71.140 7.91.140 2.020.707 15.73.162 23.93.178 50.11.140
December 6.20.317 21.11.000 8.00.707 7.20.707 0.210.010 14.31.303 3.11.000 30.40.100

Thamiraparani river. Further, the water is passing cleaned polythene two liter bottles without any air bubbles
through Varadharajapuram, Appankoil and Kaspa and after rinsing it with the sample waer in morning hours
villages. It belongs to Srivaikuntam taluk. between 8 am to 10 am. The temperature was recorded on
Water analysis the sot by mercury thermometer. The turbidity was
The two channels have been selected for
measured on spot by Nephelometer. The other parameters
investigation on the basis of geographical locations.
such as pH, Dissolved Oxygen (DO), Electrical
Samplings were made at monthly intervals from January
Conductivity (EC), Total Dissolved Solids (TDS), Total
2011 to December 2011. Samples were collected in
Hardness (TH), Total Alkalinity (TA), calcium,

Table 2: Seasonal variations of chemical and coliform parameters of Authoorangal channel in the year
Total Faecal
Ca Mg Na K Cl SO4 (mg/ CO3 HCO3 Col Col
(mg/l) (mg/l) (mg/l) (mg/l) (mg/l) l) (mg/l) (mg/l) (MPN/ (MPN/
100ml) 100ml)
January 8.20.31 5.50.37 6.45000.14 0.2100.01 13.50.50 4.20.09 0.000.00 2.50.29 2408.94 210.58

February 9.90.36 11.60.18 8.10000.20 0.2300.01 16.00.15 0.00.00 0.240.05 2.70.13 2203.53 200.67

March 2.90.04 1.30.08 0.69560.00 0.0250.00 2.30.13 0.00.00 0.000.00 2.60.16 2302.02 221.14

April 2.60.15 1.20.18 0.59520.00 0.2200.03 2.20.32 0.00.00 0.000.00 2.50.15 2406.51 250.53

May 1.80.37 0.60.07 0.65210.13 0.0510.00 1.40.18 0.00.00 0.200.03 1.50.20 2507.07 230.66

June 2.90.07 451.58 0.66230.00 0.0520.00 2.30.03 3.00.31 0.000.00 2.80.05 2707.07 252.23

July 0.90.08 1.30.09 0.52170.00 0.0250.00 1.00.07 0.00.00 0.000.00 1.70.17 2643.40 242.28

August 2.80.70 1.50.35 0.68560.00 0.0270.00 2.20.31 2.50.70 0.200.07 2.60.32 2771.14 293.11

2.80.70 1.30.32 0.56210.03 0.2300.03 2.30.32 2.60.33 0.000.00 2.60.33 27631.63 263.30

October 10.01.7 11.63.16 8.48002.82 0.2100.03 16.12.60 11.93.22 0.200.07 2.70.70 27031.62 273.22

9.80. 1.0 10.20.7 7.31000.70 0.2000.07 14.20.70 5.50.63 0.000.00 2.60.33 2610.70 213.17
2.00.70 3.00.70 0.56500.00 0.0280.00 2.50.07 0.00.00 0.200.07 2.80.31 2560.70 2601.00

1559 Journal of Research in Biology (2014) 4(8): 1557-1567

Eucharista and Ebenezer, 2014

magnesium, sodium, potassium and chloride, were month of December (Table 3). In this investigation, the
analyzed in the laboratory as per the standard methods temperature values were maximum during summer and
described by APHA (2005). Microbial studies were minimum during winter. Sonawane (2011) reported that
analyzed by MPN method described by Papen and Van the maximum value was recorded as 21.07 1.49 oC
Berg (1998). during summer; the minimum value was recorded as
Statistical analysis 12.152.95oC during winter. In the present study, the
Results obtained were subjected to statistical maximum value recorded during summer may be due to
analysis using SPSS (11.5). low water level, more solar radiation and more
evaporation. The minimum value recorded during winter
RESULTS AND DISCUSSION may be due to high water level, less solar radiation and
Turbidity less evaporation.
The present study indicated that the highest value Hydrogen Ion Concentration (pH)
of turbidity was 6.2 0.317 NTU in the month of The acidity of water is gauged by its pH, which
December 2011 in the Authoorangal channel; Similarly is a measure of the concentration of the hydrogen ion
the lowest value of turbidity was reported as 3.4 0.141 (H+). During the study period January December 2011,
NTU in the months of both August and September 2011. the maximum value of pH was recorded as 8.50.612
In the Srivaikuntam channel, the highest value of during summer in the month of May 2011 in the
turbidity was reported as 6.5 0.070 NTU in the month Authoorangal channel whereas the minimum value was
of August 2011. On the other hand, the lowest value of recorded as 7.30.196 in the months of January and
turbidity was showed as 2.9 0.316 NTU in the month of September 2011 (Table 1). The maximum pH value of
September 2011 (Table 3). The present investigations Srivaikuntam channel was recorded as 8.7 0.707 in the
reported the highest concentration of turbidity was due to month of February 2011. The minimum value was
the interaction of agricultural fertilizers, manures, recorded as 7.30.707 in the months of January and
insecticides and pesticides from the nearby banana field. September 2011 (Table 3). The present study revealed
Water Temperature that the pH was alkaline during summer season in
The sun is the source of heat that warms most Authoorangal channel, on the other hand pH was alkaline
water and its effect depends on the angle at which it during winter season in Srivaikuntam channel. In the
strikes the surface. There may also be a direct exchange present investigation, higher values may be due to
of heat between air and water and between substratum accumulation of ions owing to evaporation and
and water (Macan, 1974). In the present investigation, pesticides, insecticides from the nearby banana field.
the temperature values in the station Authoorangal Pathak et al. (2012) reported that the higher values may
channel from January to December 2011 were recorded. be due to accumulation of ions owing to evaporation,
The maximum value of water temperature was recorded biological turnover and interaction with sediments.
as 25.9 0.583oC in the month of May 2011. The Dissolved Oxygen (DO)
minimum value was recorded as 21.11.00 C in the Dissolved oxygen is essential to the respiratory
months of December and January (Table 1). In the metabolism of most aquatic organisms. Natural sources
station Srivaikuntam channel, the maximum value was of dissolved oxygen are derived from the atmosphere or
recorded as 33.90.707 C in the month of May. The through photosynthetic production by aquatic plants. In
minimum value was recorded as 10.10.707 C in the the analysis of dissolved oxygen during the study period,

1560 Journal of Research in Biology (2014) 4(8): 1557-1567

Eucharista and Ebenezer, 2014

the maximum value was recorded as 7.90.326 mg/l in continuous use will result in salinity hazard, with
the Authoorangal channel during the months of January ultimate effect on plant growth.
and November (Table 1). The minimum value was Total Dissolved Solids (TDS)
recorded as 5.50.067 mg/l in the month of June. In the In this investigation, the maximum value of TDS
Srivaikuntam channel, the maximum value was recorded was noticed as 15.73.162 mg/l in the Authoorangal
as 7.90.707 mg/l during the months of August and channel in the month of November, whereas the
November. The minimum value was recorded as minimum value was noticed as 11.00.465 mg/l in the
5.90.707 mg/l in the month of May (Table 3). The month of January. The analysis of Sirvaikuntam channel
present investigation indicated the dissolved oxygen revealed the maximum value of TDS as 14.01.000 mg/l
level concentration was high during rainy season. High in the month of May. The minimum value of TDS was
concentration of DO was due to the photosynthetic 9.1 0.707mg/l in the month of February (Table 3). The
activity of algal flora. Bhuiyan and Gupta (2007) present study revealed the highest concentration of total
reported that the dissolved oxygen is mainly regulated by dissolved solids which may be due to the surface run off
photosynthetic activity of algal flora. The minimum and agricultural run off from the nearby banana field.
concentration level of DO was due to the depletion of Sonawane (2011) reported that the higher concentration
water and abundance of nutrients in the Authoorangal of total dissolved solids ranged between 502 mg/l and
channel and Srivaikuntam channel. Sonawane (2011) 8626.25 mg/l. The ground water chemistry changes when
reported that minimum concentration is probably due to the water flows through the subsurface geological
the decomposition of organic matter. environment having overall change in the major ions and
Electrical Conductivity (EC) dissolved solids.
In the analysis of electrical conductivity during Total Hardness (TH)
the period of January to December 2011, the maximum The hardness of water is generally due to the
value was 3.080.632 ds/m in the Authoorangal channel presence of calcium and Magnesium in the water. The
during the month of October. The minimum value was study recorded the maximum value of total hardness in
recorded as 0.20.032 ds/m in the months of September the Authoorangal channel as 25.66.324 mg/l in the
and December. In the Srivaikuntam channel, the month of October 2011. Similarly, the minimum value of
maximum value was recorded as 0.410.007 ds/m in the hardness was 3.00.707 mg/l in the month of July 2011
months of February and May. The minimum value was (Table 1). But in the Srivaikuntam channel, the
recorded as 0.20.031 ds/m in the month of November maximum value of hardness was revealed as 13.90.707
(Table 3). This study reported that the electrical mg/l in the month of November 2011. The minimum
conductivity was high due to the surface run off during value of total hardness was noticed as 3.00.707 mg/l in
the rainy season in the Authoorangal channel. The study the month of July 2011 (Table 3). The present
indicated that the EC was high during the months of May investigation noticed the highest concentration of total
and February in the Srivaikuntam channel which may be hardness reflected by the nature of the agricultural
due to the greater content of ions in the water and fertilizers, manures, insecticides and pesticides. Saini et
dissolved solids. Dutta and Chowhan (2009) reported al. (2010) reported that the hardness of water reflects the
that water of higher conductivity may be used with nature of the geological formation with which it has been
suitable amendments and precautions, but under normal contacted.
conditions, they are harmful to the soil structure and their

1561 Journal of Research in Biology (2014) 4(8): 1557-1567

Eucharista and Ebenezer, 2014

Total Alkalinity (TA) 1974). The values of calcium ions were given in Table 2
The present study indicated the highest for Authoorangal channel which represented the highest
concentration of TA in the Authoorangal channel as value of 10.01.702 mg/l in the month of October 2011.
50.11.140 mg/l in the month of November 2011, Similarly, the lowest value was 0.90.086 mg/l in the
whereas the lowest concentration of TA was 22.20.677 month of July 2011. Considering the Srivaikuntam
mg/l in the month of January 2011. The analysis of channel, the analysis of calcium ions was recorded as
Srivaikuntam channel showed the highest concentration 3.60.633 mg/l in the month of March 2011 as the
of TA as 45.10.317 mg/l in the month of November highest value. The lowest value was recorded as
2011 and the lowest concentration of TA as 20.2 0.80.031mg/l in the month of May 2011. The
0.707mg/l in the month of January (Table 3). The present precipitation of agricultural pesticides, fertilizers and
study showed the presence of the highest concentration manures from the banana field represented the highest
of total alkalinity which was due to the surface run off calcium concentration. Samson and Elangovan (2011)
from banana field which contains the highest reported that water more or less in equilibrium with the
concentration of calcium, carbonates and bicarbonates materials in the drainage basin is characterized by higher
ions during rainy season. Sahni and Silotia (2011) concentration of calcium.
reported that the alkalinity values correlate positively Magnesium
with the pattern of rainfall and this implies surface run The magnesium ion in the Authoorangal channel
off from the Mansagar lake. was recorded as 11.60.378mg/l in the months of
Calcium February and October 2011 is the highest value.
Waters that emerge from the earth are highly Similarly, the lowest value was 0.60.070mg/l in the
charged with calcium bicarbonate and flow away as month of May 2011 (Table 2). In the Srivaikuntam
streams which may deposit calcium carbonate (Macan, channel, the highest value of magnesium ion was

Table 3: Monthly variations of physical parameters of Srivaikuntam channel in the year of 2011

Turbidity Tempera- DO TDS
Month pH EC (ds/m) TH (mg/l) TA (mg/l)
(NTU) ture 0C (mg/l) (mg/l)
January 3.00.707 13.11.000 7.30.707 7.10.707 0.260.007 9.20.707 6.10.317 20.20.707

February 3.00.707 12.40.707 8.70.707 7.30.633 0.410.007 9.10.707 5.51.000 22.70.707

March 4.10.707 15.01.000 7.80.707 7.60.707 0.350.007 10.50.707 4.30.707 23.00.707

April 4.00.707 23.70.070 7.70.070 6.60.317 0.310.007 10.30.635 3.30.707 21.20.707

May 3.60.317 33.90.707 8.40.707 5.90.707 0.410.007 14.01.000 6.60.317 23.30.317

June 3.10.707 33.70.070 7.70.633 6.50.707 0.270.007 12.30.322 4.10.707 22.80.317

July 3.50.707 29.50.633 7.60.707 6.80.707 0.270.007 13.10.317 3.00.707 31.30.317

August 6.50.070 27.80.633 7.40.707 7.90.707 0.390.007 13.20.317 4.20.707 30.20.707

September 2.90.316 26.80.707 7.30.707 7.70.317 0.210.007 12.70.317 6.00.707 39.20.317

October 3.30.317 17.00.707 7.90.707 7.60.317 0.280.010 13.90.707 8.60.317 37.50.317

November 3.20.317 15.20.317 7.70.317 7.90.707 0.20.031 10.70.633 13.90.707 45.10.317

December 3.60.317 10.10.707 7.60.317 7.20.317 0.290.007 12.30.317 13.10.707 23.40.317

1562 Journal of Research in Biology (2014) 4(8): 1557-1567

Eucharista and Ebenezer, 2014
1.60.317 mg/l in the month of October 2011. Similarly, to the disposal of waste water.
the lowest value of magnesium ion was 0.050.007 mg/l Chloride
in the month of December 2011. The present study Chloride is one of the most widely used
revealed the highest magnesium concentration due to disinfectants. The highest concentration of chloride in
leachates from the banana field during rainy season. the Authoorangal channel was 16.12.607 mg/l during
Sonawane (2011) reported that the main source of October 2011. Likewise, the lowest value was recorded
magnesium concentration is obtained from the leachates as 100.070 mg/l in the month of July 2011 (Table 2).
of rocks in the catchments. With reference to the Srivaikuntam channel, the highest
Sodium concentration of chloride analyzed was 1.90.318 mg/l
The present analysis of sodium ion revealed the in the month of March 2011. Similarly, the lowest
highest value as 8.482.828mg/l in the month of October concentration of chloride was analyzed as 1.00.00 mg/l
2011. The lowest value of sodium ion 0.520.007mg/l in in the months of July and December 2011. The present
the month of July 2011 was recorded in the study depicted that the highest concentration of chloride
Authoorangal channel (Table 2). Similar results were (16.12.607 mg/l) may be due to the water sources that
reported in the Srivaikutam channel (Table 4). The come from organic wastes and refuse of leachates.
highest concentration of sodium ion in both channels Prabakaran et al. (2013) reported that the high
represented the agricultural fertilizers, manures and the concentration of chloride may be due to anthropogenic
detergents used by the humans for bathing and washing activities, sewage contamination and decomposition of
clothes. Mary Kensa (2011) reported that the main organic wastes.
source of sodium is obtained from soaps and detergents, Sulphate
used by the local residents for bathing and washing Sulphate occurs in certain igneous rock minerals
purposes. of the field spathoid group, but the most extensive and
Potassium important occurrences are in the evaporate sediments.
The present study recorded the highest value of The highest concentration of sulphate in the
potassium ion in the Authoorangal channel as Authoorangal channel was found as 11.93.224 mg/l in
(0.230.012 mg/l) in the months of February and the month of October 2011 (Table 2). While comparing
September 2011 (Table 2). Likewise, the lowest value of with the Srivaikuntam channel, the highest concentration
potassium ion was (0.0250.0009 mg/l) in the months of of sulphate was reported as 11.90.707 mg/l during
March and July 2011 (Table 1). In proportion to October 2011 which is similar to the Authoorangal
Srivaikuntam channel, the highest value of potassium ion channel. The highest concentration of sulphate in the
(0.23 0.007 mg/l) was recorded in the month of present investigation revealed the deposition of
September 2011. The lowest value of potassium ion was agricultural fertilizers which are drained into the water
recorded as 0.022 0.007 mg/l in the month of April body from the catchment area. Suresh et al. (2013)
2011. The present investigations reported that the highest reported that sulphate enter into the water body from the
concentration of potassium may be due to wash out of catchment area through surface run off. Since the study
slurry from the agriculture fields and sewage run off. area is bordered by agricultural lands where sulphate
Gopalkrushna (2011) reported that the major source of fertilizers are used in plenty, relatively higher
potassium in natural fresh water is due to weathering of concentrations of sulphate observed could be attributed
rocks but the quantities increase in the polluted water due to the run off from these agricultural lands.

1563 Journal of Research in Biology (2014) 4(8): 1557-1567

Eucharista and Ebenezer, 2014

Carbonate Authoorangal channel was found as 2.80.050 mg/l in

The present study investigated the highest the months of June and December 2011(Table 2) but the
concentration of carbonate in the Authoorangal channel lowest concentration was 1.50.203 mg/l in the month of
as 0.20.050 mg/l (Table 2) and was uniformly May 2011. With reference to the Srivaikuntam channel,
distributed over the month of February, May, August, the highest concentration of bicarbonate was recorded as
October and December 2011. The Srivaikuntam channel 2.20.633 mg/l in the months of January, April, August,
revealed the highest concentration of carbonate as September, November and December 2011 whereas the
0.30.070 mg/l in the months of February and April lowest concentration of bicarbonate recorded was
2011. The present investigation manifested the highest 1.50.317 mg/l in the month of May 2011. The present
concentration of carbonate which was due to the surface findings revealed the highest concentration of
run off from the agricultural fertilizers, manures and bicarbonate which may be due to the leachates from the
pesticides as precipitation to where it exits the watershed. agricultural run off and human activities. Sonawane
Vasanthy and Velmurugan (2009) reported the presence (2011) reported that the highest concentration of
of temporary (carbonates and bicarbonates of Ca and bicarbonates may be attributed to the rate of organic
Mg) and also permanent hardness (sulphates, phosphates, decomposition during which Co2 is liberated, which
nitrates, chlorides of Ca and Mg). This may be attributed reacts with water to form HCo 3.
to the geological reasons and surface run-off. Total Coliform Bacteria
Bicarbonate The analysis of total coliform bacteria revealed
The highest concentration of bicarbonate in the the highest count as 2771.140 (MPN / 100ml) in the

Table 4: Seasonal variations of chemical and coliform parameters of Srivaikuntam channel in the year of
Total Faecal
Ca Mg Na Cl SO4 Co3 Hco3 Col Col
Month K (mg/l)
(mg/l) (mg/l) (mg/l) (mg/l) (mg/l) (mg/l) (mg/l) (MPN (MPN/
/100ml) 100ml)
January 2.20.31 1.50.31 6.450.31 0.10.00 1.50.31 4.20.63 0.0000.00 2.20.63 2590.70 210.70

February 2.90.70 0.700.07 8.100.70 0.100.00 1.60.31 0.0000.00 0.30.37 2.10.70 2551.00 201.00

March 3.60.63 0.000.00 0.690.00 0.020.00 1.90.31 0.0000.00 0.0000.00 2.10.70 2570.70 270.70

April 2.60.31 1.20.31 0.590.00 0.020.00 1.20.31 0.0000.00 0.30.07 2.20.07 2570.70 250.70

May 0.80.03 0.60.07 0.650.00 0.050.00 1.70.32 0.0000.00 0.0000.00 1.50.31 3000.70 230.70

June 2.90.70 1.50.31 0.660.00 0.050.00 1.30.31 3.00.70 0.0000.00 2.10.70 2591.14 291.14

July 1.30.31 1.30.07 0.520.00 0.070.00 1.00.00 0.0000.00 0.0000.00 1.70.07 2550.70 230.70

August 2.80.31 1.50.31 0.680.00 0.020.00 1.20.31 2.50.31 0.20.03 2.20.31 2530.70 270.70

September 2.80.31 1.30.31 0.560.00 0.230.00 1.30.31 2.60.31 0.0000.00 2.20.31 2551.00 251.00

October 3.00.70 1.60.31 8.480.31 0.210.00 1.10.31 11.90.70 0.20.03 2.10.31 3500.70 300.70

November 2.80.31 1.20.31 7.310.31 0.200.03 1.20.31 5.50.31 0.0000.00 2.20.31 3900.70 370.70

December 2.00.70 0.050.00 0.560.00 0.050.00 1.00.00 0.0000.00 0.0000.00 2.20.31 3000.70 291.14

1564 Journal of Research in Biology (2014) 4(8): 1557-1567

Eucharista and Ebenezer, 2014

month of August 2011 in the Authoorangal channel. The showed the highest counts (500 to 700 MPN 100 mL-1)
lowest counts of total coliform bacteria was recorded as of fecal coliforms during the rainy season in 2000. These
2203.535 (MPN/ 100ml) in the month of February 2011 sites receive large amounts of organic matter from non-
(Table 2). In the Srivaikuntam channel, the highest treated effluents from the farms and small towns
counts of total coliform bacteria was recorded as surrounding the region.
3900.707 (MPN/ 100ml) in the month of November
2011 (Table 4). Similarly, the lowest counts revealed as CONCLUSION
2530.707 (MPN/100ml) in the month of August 2011. From the above investigations, it may be
The present study revealed the highest counts of total concluded that most of the physico-chemical parameters
coliform which may be due to physico-chemical viz. turbidity, temperature, pH, dissolved oxygen,
parameters along with the agricultural decomposition of electrical conductivity, total dissolved solids, total
organic matter from the catchment area. Sharma et al. hardness, total alkalinity, calcium, magnesium, sodium,
(2013) reported that all physico-chemical parameters of potassium, chloride, sulphate, carbonate, bicarbonate,
sediments showed significant relationship with bacterial total coliform and faecal coliform were found within the
count. Dark brown color of sediments, organic carbon World Health Organization limits in the Authoorangal
(1.62%) and high organic matter (2.79%) showed high channel and Srivaikuntam channel. The findings clearly
content of organic waste in the bottom sediments. pH of indicated that both the channel water bodies are slowly
the sediment sample showed alkaline nature (8.1) and deteriorating due to discharge of agricultural fertilizers,
most of the isolated bacteria grow at slight pollution and manures, pesticides and insecticides from the nearby
helped in the growth of pollution indicator bacteria such banana field. The manifested results calls for the need to
as Escherichia coli, Pseudomonas sp., Enterobacter sp. conserve manage and restore the water bodies. Intensive
etc. efforts such as regular monitoring, systematic assessment
Faecal Coliform Bacteria can save the human health and provide sustainable
During the study, the highest counts of fecal environment. There is a need for awareness among the
coliform bacteria was shown to be 293.114 (MPN/ people to maintain the significance of highest quality and
100ml) in the month of August 2011, in the station purity levels of both Authoorangal and Srivaikuntam
namely Authoorangal channel (Table 2). The lowest channel water bodies.
count of faecal coliform bacteria was found to be
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Journal of Research in Biology ISSN No: Print: 2231 6280; Online: 2231- 6299

An International Scientific Research Journal

Original research

Ethnobotanical study of medicinal plants in the region El Hajeb

(central Morocco)
Authors: ABSTRACT:
Journal of Research in Biology

Jalila El Amri1, Khalid El

Badaoui1, Touria Zair2, As a part of enhancement of the natural heritage of the region Meknes-
Hayate Bouharb1, Said Tafilalet, an ethnobotanical study was conducted in the El Hajeb region. This study
Chakir1and Taj El Molk describes the different uses of medicinal plants by the local population and their
Alaoui1 impact on the degradation of plant biodiversity. And it also helped to create a
catalogue of different species in the region. On the basis of questionnaire prepared, a
survey was conducted among the local population and herbalists operating in the field
1. Laboratory of
Environment and Health, of medicinal and aromatic plant intermediaries. The analysis of results from 220
Faculty of Sciences, question cards, using data processing technique has enabled us:-
University Moulay Ismail , To identify 80 species distributed in 40 families with a significant representation
BP 11201 Zitoune, Meknes, of the Lamiaceae family
Morocco. To represent the frequency of use of cash by the local population surveyed at the
study area
2. Laboratory of Chemistry To represent the frequency of use by age, gender, and according to the school.
of Bioactive Molecules and Despite the richness of the studied medicinal and aromatic plants in this area, the
Environment , Faculty of local population does not benefit from its natural resources.
Sciences , University
Moulay Ismail, Zitoune BP
11201, Meknes, Morocco.
Corresponding author: Morocco, El Hajeb, medicinal plants, ethnobotany, Lamiaceae
Jalila El Amri
AC: Circulatory apparatus; AD: Digestive System; AR : Respiratory; AU: Urinary; AG: Genital Apparatus;
AA: Hearing Aids; AV: Device Image; SN: Nervous System; SQ: Skeleton; P: Skin
Part used
PS: Underground Party; T: stem; F: Leaf; TF: Stem + Leaf; FL: Flower; FR: Fruit; PE: Whole plant
Method of Preparation
Inf: Infusion; D: Decoction; C: poultice; M: maceration; Inh: Inhalation; F: Friction; Inj: Injection; P: Powder;
N: Nature; D: Miscellaneous
(***): Abundant; (**): Means; (*): Rare ; (0): none

Email Id: Article Citation:

Jalila El Amri, Khalid El Badaoui, Touria Zair, Hayate Bouharb, Said Chakir and Taj El
Molk Alaoui
Ethnobotanical study of medicinal plants in the region El Hajeb (central Morocco)
Journal of Research in Biology (2014) 4(8): 1568 1580
Web Address: Dates: Received: 20 Nov 2014 Accepted: 05 Dec 2014 Published: 27 Dec 2014
This article is governed by the Creative Commons Attribution License (
licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

1568-1580 | JRB | 2014 | Vol 4 | No 8

Journal of Research in Biology
An International
Scientific Research Journal
El Amri et al., 2014

INTRODUCTION the said region and the types of diseases that affect
The ethnobotanical and ethnopharmacological human beings.
study of Moroccan medicinal plants show the relative
importance of such plants in the health system in MATERIALS AND METHODS
Morocco. The first serious study of the Moroccan Description of the study area
medicinal plants dated back in 1978 when book was The province of El Hajeb, relatively new, spreads
published entitled Traditional Medicine and Toxicology over an area of 2,22,000 ha. It is limited:
west-Saharan Africa by Jamal Bellakhdar. This book To the north is the province of Meknes
was an undeniable contribution of Moroccan To the south is the province of Ifrane
pharmacopoeia. Other studies have focused on regional To the east is the province of Fez and Sefrou province
medicinal flora, the most important of which were To the west is the province of Khmisset
conducted by Bellakhdar et al. (1987) in the region of The administrative division consists of three
Tata (southern Morocco), Benabid and Bellakhdar circles, five cadats, twelve rural communes and four
(1987) in the region of Rif and Kahouadji (1995) in urban municipalities.
Morocco oriental and Hmamouchi and Agoumi The total population was estimated in 2004 as
(1993) have reported studies from 2,40,436 with 64% rural and 36% urban inhabitants. The
Morocco (Central plateau). Other studies have focused number of households was estimated as 30,326 and the
on the study of biological activity of some Moroccan number of farmers has been estimated as 18,800.
medicinal plants (Abdelrhafour et al., 1993 a and b; The area is characterized by semi-arid to sub-
Bammi et al., 2000; Remmal, 1994). humid climate with a average annual rainfall of 520 mm.
In 1997, Bellakhdar published a document of The minimum temperature reached was 2.8 C, while the
crucial importance entitled, Traditional Moroccan maximum temperature reached was 38.2 C.
Pharmacopoeia, a bibliographic research on ancient Farming is the main source of population in as given
arabic texts of medical material, developed by North below
African. Andalusian authors have demonstrated that the Total area: 2,22,000 ha
Moroccan pharmacopoeia showed a remarkable SAU: 1,46,000 ha
continuity with the knowledge of the ancients, at least as
Bour: 1,25,500 ha
regards the nature of the remedies, since 77.7% of what
Irrigated: 44,700 ha
it employs are already mentioned in the texts taken as a
Forests: 31,300 ha
reference (Bammi et al., 2000) .
UAA consists mainly of cereal crops (78,000 ha),
Our laboratory has been trying for
market gardening and arboriculture 44.00 ha (22,000 ha)
years conducting floristic and ethnobotanical research of
medicinal plants in the region of Mekns-Tafilalet, to
The choice of the region
enhance these natural resources. In this sense, an
The choice of the study has been focused on the
ethnobotanical study was conducted at the area of El
region El Hajeb for the following reasons:
Hajeb, a province of the Meknes-Tafilalet region.
The geographical position of the mass if that forms
This study is to invent medicinal plants, for
the transition between the Rif Mountains and the
identifying the different ways of use and to understand
Atlas chain means.
the close relationship between plant species described in
1569 Journal of Research in Biology (2014) 4(8): 1568 1580
El Amri et al., 2014

The abundance and diversity of plants in the of El Hajeb and describe their methods of valuation by
spontaneous state in this region. the local population.
The existence of traditional know-how on aromatic and
medicinal plants. RESULTS AND DISCUSSION
Knowledge of the area as we live in El Hajeb since The ethnobotanical information was included on
childhood. raw data sheets and transferred to a database after
Collection of plants collection, processed and analysed to obtain standardized
Collection of medicinal species was made on data for the following areas:
land by a random sampling method. Taxonomic Frequency of use of medicinal and aromatic plants by
identification was performed on comparison with the the local population surveyed at the study area: El
herbarium specimens of the vegetation in the area and Hajeb
verified by Mohamed, El Hajeb scientific institute in a) Frequency of use of PAM by gender:
Rabat Agdal. The WFP operation in this region affects both
The choice of investigators men and women, although women have more knowledge
For the choice of the survey population, we about the species in relation to men (56% against 44%).
considered the following criteria: The data processing and the graph were shown on Figure
Age: Age between 19 and 70 years 1. The results presented in the graph confirms the results
Gender: Heterogeneity of respondents (women / men) of other ethnobotanical work at the national level, which
Level of activity performed: Local population, farmers, says that more women are holders of traditional herbal
herbalists, intermediate and healers. knowledge (Bellakhdar, 1997).
Ethnobotanical surveys
To meet the goals of the ethnobotanical study,
surveys were conducted and exploratory outings were
done to familiarize themselves with the terrain, the local
vocabulary and recognition of species of WFP in the
region, their spatial distribution, and abundance. This
work lasted eight months.
On the basis of a questionnaire, a series of
surveys was carried out with the local population,
herbalists and intermediaries; 220 records on
questionnaires were collected, of which 110 listings were
b) Frequency of use by age:
from the local population, 80 from the souks: City El
The ethnobotanical study revealed differences in
Hajeb, Ait Boubidmane (14 km), Agourai (30km), Ain
the age of the study population regarding the use of PAM
Lhnach (20 km) and 30 from intermediaries, herbalists,
as shown in the graph in Figure 2.
farmers and traders.
A wide range of El Hajeb people over 50 years
Statistical analysis
of age, have a greater frequency of use of medicinal
Data collected on raw data sheets were processed
plants (50%) compared to other age groups: [40-50], [ 30
by MS excel; this helped to establish a herbal monograph
-40], [20-30], [<20], with frequencies of use 20%, 16%,

Journal of Research in Biology (2014) 4(8): 1568 1580 1570

El Amri et al., 2014

10%, 4%, respectively; This shows that knowledge of people with primary level schooling - a percentage of use
users of WFP is usually acquired following a long of significant WFP is 20%, while those with the level of
experience and passed from one generation to another, secondary and university studies are found to be very
but now the transmission is in danger, because it is not little users of PAM (Figure 3).
always assured. 3. Frequency of medicinal plants according to their
There is also some loss of information on the origin
WFP because of the reduction of plant resources of the Most species of WFP in El Hajeb are
region and the distrust of some people, particularly characteristics of the region and are spontaneous from
young people who tend to know little about the virtues of the forests and Douars with a frequency of 83.75%. Also
plants and be reluctant to their use in traditional crops such as caraway, anise, spearmint, fenugreek,
lemongrass and garlic, do not have a low frequency of

medicine. availability - 16.25% (Figure 4).

2. Frequency of use of PAM by level of schooling 4. Distribution of different plant parts used
El Hajeb is a region which has a lower level of From the survey, the local population uses
schooling. Illiteracy reached a high level, especially in different parts of the plant in the treatment of diseases; it
women with a higher percentage compared to men (HCP, can be the leaves, stems, flowers, fruit, bark of roots,
2014). tubers and rhizomes.
It should be noted that at this region most users The graph given in Figure 5 shows the most
of PAM are illiterate (68% of the population). However, commonly used plant parts in the area of El Hajeb. Stems

1571 Journal of Research in Biology (2014) 4(8): 1568 1580

El Amri et al., 2014

were used by 33% of people followed by the stems with mint (Mentha suaveolens), oregano (Origanum
leaves (22%), fruit (20%) and underground parts (15%). compactum), pennyroyal (Mentha pulegium L), white
5. WFP and their methods of preparation horehound (Marrubium vulgare L.) with a frequency of
The method of preparation of the crude drug operation between 70% and 90%. Other species such
from a designated body of the plant is very diverse as German chamomile (Camomilla recutita L.),
(infusion, maceration, decoction, bouillon powder etc). Paronychia argenta, oleander (Nerium oleander),
The results shown in graph in Figure 6 are as follows: Corrigiola telephifolia, Euphorbia helioscopia,
Decoction is the most common method of Mercurialis annua, Malva sylvestris and Calendula
preparation with a percentage of 45%, followed by the officinalis represent frequencies between 40% and 60%

infusion preparation (19%), and other modes are less (Figure 7).
than 9%. 7. Botanical families represented in the study area
6. PAM very common in the region According to the survey and graph in Figure
Computer analysis revealed the most common 8, we find that families Lamiaceae and Asteraceae are
PAM in the study area tel que thyme the most represented with 11 and 10 species,
(Thymus ciliatus), round-leaved respectively, followed by the Apiaceae family (7),

Journal of Research in Biology (2014) 4(8): 1568 1580 1572

El Amri et al., 2014

liliaceae (6), Fabaceae, and Solanaceae grasses argenta, Camomilla recutilla L., Marrubium vulgare.L
respectively (3), other families are less than two. The and Mentha suaveolens.
lamiaceae are flavouring plants. WFP belonging to this 9. Distribution of WFP as pathologies
family are most commonly used for self-medication in According to the statistical analysis, we are
the region. These results confirm those of other successful in revealing various pathologies most
ethnobotanical studies throughout the Moroccan territory frequently encountered in the El Hajeb region (Figure
(Bellakhdar , 1997). 10) .
8. The frequency of medicinal plants mostly used by Dermatoses occur by a percentage of 27%
the local population followed by the digestive tract (22%), nervous system
From the ethnobotanical study conducted it is (14%), urogenital tract (14%), hormonal and circulatory
possible to highlight 13 most used medicinal plants by system (9%), and other disorders less than 5%.
the local population (Figure 9). 10. The number of species listed according to the
Plantago psyllium plant is most commonly used; most common diseases
when it is not among the most common plants in the The number of species found in the study area
region, it is due to its effectiveness in treating wounds, that treats a given disease were given in Figure 11. The
followed by plants that are common as Thymus ciliatus, most common diseases in the study area are skin
Origanum compactum, Mentha pulegium, Paronychia diseases. 18 species treat these diseases, most of them are

1573 Journal of Research in Biology (2014) 4(8): 1568 1580

El Amri et al., 2014

healing, the most represented are Plantago psyllium with antiseptic, stomachic and antitussive. Over-harvesting
a percentage of 25.17%, followed by Corrigiola led to their extinction which leads us to adopt a
telephifolia (14.42%), Euphorbia helioscopia (13.13%) , management approach to the protection and preservation
Rosmarinus officinalis (13.10%), Nerium oleander of natural resources.
(12.15%), Solanum sodomaeum L. (8%), Teucrium The ethnobotanical study revealed that the most
capitatum .L (7.33%) and Silene vulgaris (6.7% ). common disease prevalent in this area is dermatitis.
For diseases of the digestive system, 14 species Foliage is the most used plant organ and decoction is the
are used, followed by 15 species for the nervous system, most dominant mode. Also skin diseases are treated
5 species for respiratory and 4 species for the urogenital locally which allowed us to describe the different uses of
tract problem (Figure 12). medicinal plants by the local population. This study
It is found that 15 species treat diseases of the helped us to understand the close relationship between
gastrointestinal tract, the most represented are plant species and described the different types of
primarily Thymus ciliatus, Euphorbia helioscopia, pathologies affecting population. Thus two plants used
Origanum compactum, Solanum sodomeum L., Silefia both for skin diseases and diseases of the intestine were
vulgaris, Teucrium capitalum. L, Ceratonia siliqua, chosen for future studies.
Arbutus unedo, Ajuga iva, Helosciadium nodiflorum , It is noted, that the exploitation of wild plants is
Carum carvi L and Pimpinella anisum L not developed neither technically nor economically.
and last Ziziphus lotus. Therefore, local people do not benefit from the
According to Figures 12 and 13, it was found development of natural resources and are not said to be
that two plants were used for curing both dermatoses and as top managers.
digestive tract diseases. Both plants were chosen to study For sustainable management of aromatic and
their antimicrobial effect. medicinal resources and better utilization of medicinal
and aromatic plants at the local level, Provincial
CONCLUSION Agriculture Officer (PAO) with others proposed the
This study identified the aromatic and medicinal following steps:
plants that exist in the region of El Hajeb and their An organization of local people together
various uses by the local population. The integration of rural women in the exploitation of
The results of this study showed that the area medicinal and aromatic plants
holds significant potential in spontaneous WFP crop and Improved incomes of men and women
toxic. El Hajeb region has a low level of schooling. It Creating self-employment
should be noted that at this region most of the users of Improved quality of products of medicinal and
PAM are illiterate. aromatic plants
The information acquired from questionnaires Promotion markets for WFP nationally and
sheets and floristic surveys conducted in the field, helped internationally
us to catalog 80 plant species belonging to 40 families.
An organization from the sale of products derived
The most common medicinal and aromatic plants
from PAM (dried plants, floral waters and essential
widely used in the study area are thyme, mint round
leaves, oregano and white horehound. These species
contain essential oils used mainly as a carminative,

Journal of Research in Biology (2014) 4(8): 1568-1580 1574

El Amri et al., 2014

Appendix 1: Photo of some of the most used plants in the region El Hajeb

Thymus ciliatus (Le thym) Nerium oleander (laurier rose) Mentha pulegium (La menthe pouliot)

Inula viscosa (Linule visqueuse) Marrubium vulgare L. (Le marrube Laurus nobilis L. (Le laurier noble)

Origanum compactum (Lorigan) Papaver rhoeas (Le coquelicot) Salvia officinalis L. (La sauge)

Calendula officinalis (Le souci offici- Camomilla recutita ( L.) (Camomille Lippia Triphylla (La verveine)

1575 Journal of Research in Biology (2014) 4(8): 1568 1580

El Amri et al., 2014

Appendix 1: Photo of some of the most used plants in the region El Hajeb (Contd)

Mentha longifolia L. (Menthe Rumex acetosa L. (Loseille)

Urtica dioica L. (Lortie)

Lavandula dentata (Lavande sauvage) Carum carvi L. (Le carvi)

Appendix 2 : Lot picture ( investigators and users )

Journal of Research in Biology (2014) 4(8): 1568-1580 1576

El Amri et al., 2014

Appendix 3: Landscape picture of the region of El Hajeb

Appendix 4 : Field pictures (users, herbalists and healers )

User 1 User 2 Herbalist 1

Herbalist 2 Herbalist 3 Healer

1577 Journal of Research in Biology (2014) 4(8): 1568-1580
El Amri et al., 2014

Table I : Catalog of aromatic and medicinal plants of the region of El Hajeb

Family Botanical Name Use No species Toxic Plants Abundant Spontaneous

Name vernacular cited by Yes / no or
Bellakh- cultivated
dar plant
- To treat
Thymus zitra abdominal Yes Spontaneous
ciliatus pain plant
- As a flavor in
-To treat fever,
Mentha mersita headache, Yes Spontaneous
lamiaces suaveolens typhoid plant
- As a flavor in
Flavor milk,
tea and
Mentha manta coffee Enough Spontaneous
longifolia - Treating plant
zaatar - Gastralgia
Origanum - Flavor bread Enough Spontaneous
compactum plant
Mentha L. Ber Fliyou cough,
pulegium headache and Yes Spontaneous
chills plant
- Flavor meals,
lamiaces burns
Salvia salmia - Flavor tea Spontaneous
officinalis L. - Antidiabetic Not plant
Mentha viridis Nanaa - Flavor tea
L. -Headache. + Yes Cultivated
Teucrium L. Lkhiyata Furuncle,
capitatium healing, + Yes Spontaneous
gastroenteritis plant
Rosma- Azir dysmenorrhea,
rinusofficinali skin care, coo- not Spontaneous
s L. ling, headache plant
Calendula jemmra - Freezing
officinalis L. them, acne Yes Spontaneous
Anthemis babnouj Belly aches,
Aste- nobilis hair care and Enough Spontaneous
raceae facial plant
Inula mgrmn Tuberculosis, Yes Spontaneous
viscosa pneumonia, plant
Atractylis Ddd Fumigation Deadly com- not Spontaneous
gummifera L. against mon in Mo- plant
microbes and rocco: Root
insects, (Bellakhdar,
abscess 1997)
Scolymus l-gernina -Consumption Yes Spontaneous
hispanicus l. plant
Echinops Taskra or -Consumption Yes Spontaneous
Aste- Chawkate plant
spinosus L.
raceae elhmar

Journal of Research in Biology (2014) 4(8): 1568-1580 1578

El Amri et al., 2014
Paronychia Harras el Kidney - Yes Spontaneous
Argenta Hjar Lithiases plant
Anacyclus Krae Djaja -gastralgie not Spontaneous
clavatus plant

Rutaceae Ruta FIJL Fever, Yes Spontaneous

angustifo- typhoid, plant
lia headaches
Apiaceae Carum karwiy difficiles- Yes Cultivated
carvi L. antispasmodic
Apiaceae Eryngium
ilicifolium boumghiz Gastroenteri- not Spontaneous
al tis plant
Apiaceae Daucus bouzfour headache not Spontaneous
crinitus plant

- Digestive
Lily Pimpinella Habat problems + Yes Cultivated
anisum L. Hlawa - Flavor bread

Corrigiola Serghina -Eczma, + not Spontaneous

telephiifo- Hemorrhoids plant
Apocyna- Nerium Dafla cooling, Very toxic enough Spontaneous
ces oleander headache ac- (Kingsbury, plant
ne, burns 1964)
Lauraceae nobilis L. Asa Musa - Flavor Not Spontaneous
sauces plant
-Cooling Toxic to
Papave- Papaver bel- -Toux + animals Yes Spontaneous
raceae rhoeas naaman -laryngite (Cooper plant
Lippia Lwiza - Flavor tea,
Ver- triphylla fever, + Not Cultivated
benaceae headache

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An International Scientific Research Journal


Puntius nigronotus, a new fish species (cypriniformes; cyprinidae) from

Kerala, India
Journal of Research in Biology

Mathews Plamoottil
Puntius nigronotus, a new fish species of the family Cyprinidae, is described
from Mananthavady River of Kerala, India. It is distinguished from its congeners by the
following combination of characters: body deeper, maxillary barbels shorter and never
Institution: reach nostrils, dorsal fin shorter, 9 branched rays in dorsal fin and 6 branched rays in
Assistant Professor in anal fin, last unbranched dorsal fin ray feebly ossified and flexible, 27 lateral line
Zoology, Govt. College, scales, pre pelvic distance greater, caudal peduncle wide and lateral line straight.
Chavara, Kollam, Kerala,

Western Ghats, Mananthavady River, New description, Puntius viridis

Corresponding author:
Mathews Plamoottil

Email Id:

Article Citation:
Mathews Plamoottil
Puntius nigronotus, a new fish species (cypriniformes; cyprinidae) from Kerala, India
Journal of Research in Biology (2014) 4(8): 1581-1588

Web Address:
Received: 06 July 2014 Accepted: 29 Nov 2014 Published: 31 Dec 2014
This article is governed by the Creative Commons Attribution License (
licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

1581-1588| JRB | 2014 | Vol 4 | No 8

Journal of Research in Biology
An International
Scientific Research Journal
Plamoottil, 2014

INTRODUCTION rakers and presence of a post epiphyseal fontanelle. The

Species of genus Puntius are the most common present fish described from the Mananthavady River of
cyprinid fishes of India. Jayaram (1991) revised the Wayanad, Kerala, India, carries characters of the genus
Puntius species of India and classified the genus into ten Puntius but bears many features to separate it from its
groups and fourteen complexes. Pethiyagoda et al. relative species. So it is described here as a new species
(2012) created five genera namely Puntius, Dawkinsia, Puntius nigronotus.
Systomus, Haludaria and Pethia based on taxonomic
analysis of 30 putative species of Indian and Sri Lankan MATERIALS AND METHODS
Puntius species. Methods used are those of Jayaram (2002);
Puntius species are characterized by the absence subunits of the head are presented as percentage of Head
of rostral barbels, smooth last unbranched dorsal fin ray, Length (HL); head length and measurements of body
presence of free uroneural, simple and accumulate gill parts are given as percentage of Standard Length (SL).

Figure 1. A fresh specimen of Puntius nigronotus, Holotype, ZSI FF 5285, 82.3 mm SL

Figure 2. Head region of Puntius nigronotus Figure 3. Dorsal fin of P. nigronotus

1582 Journal of Research in Biology (2014) 4(8): 1581-1588
Plamoottil, 2014

Table 1. Meristic Counts of Puntius nigronotus sp. nov. and its relative species
SL. Counts P. nigronotus P. viridis P. parrah P. madhusoodani P. chola P. dorsalis P. sophore
No. ZSI FF 4934 CRG/SAC 456- ZSI/ ZSI/F2730, ZSI FF
ZSI/ WGRC/ (n=4) 459 F2203, ZSI/ 4938
Holotype, IR/2382, 2383, (n=4) 4009 SRC4954 (n= 2)
ZSI FF 5285 ZSI FF 4932 (n=2) (n=3)
(n= 8)
Scale Counts
1 LLS 27 25- 26 25 25- 26 26 25-26 25

2 PDS 11 9 8 9 9 9 9

3 PRPLS 10 5 6 6 5-6 5-6 5

4 PRAS 18 10- 12 14 14 12-13 11-13 13

5 CPS 10 9-10 10 10 11-12 9-10 10

6 LL/D 5 5- 5 5 4 4-5 4-5 5

7 LL/V 3 3 3 3 3-3 2 3

8 LL/A 4 3 3 3 3 3 4

9 L/TR 5/3 5-5/3 5/4 5/3 5/4 5/2 5/4

Fin Ray Counts

10 D ii, 9 iii, 8 iii, 8 iii,7 iii,8 iii, 8 iii, 8

11 P i, 15 i, 14 i, 14 i, 14 i, 13-16 i, 14-15 i, 13- 14

12 V i, 8 i, 8 i, 8 ii, 8 i, 8 i, 7 i, 8

13 A ii, 6 iii, 5 ii, 5 ii, 6 iii, 5 iii, 5 iii, 5

14 C ii, 17 18- 19 19 19 19 17 18

Abbreviations used: Caudal Peduncle; LL/D- scales between lateral

line and dorsal fin; LL/V- scales between lateral
BDD Body depth at dorsal fin origin; IOW
line and ventral fin; LL/A- scales between
Inter Orbital Width; HD Head Depth; LLS
lateral line and anal fin; L/TR Lateral
Lateral Line Scales; PDS Pre Dorsal Scales;
Transverse Scales; D Dorsal fin rays; P
PRPLS Pre Pelvic Scales; PRAS Pre Anal
Pectoral fin rays; V Ventral fin rays; A Anal
Scales; CPS Circum Peduncular Scales; LCP
fin rays; C Caudal fin rays. ZSI/ WGRC-
Length of Caudal Peduncle; DCP Depth of
Zoological Survey of India, Western Ghats
Regional Centre, Kozhikode; ZSI- Zoological
Survey of India, Kolkata; CRG/SAC-
Conservation Research Group, St. Alberts
college, Cochin; ZSI/SRC- Zoological Survey
of India, Southern Regional Centre, Chennai;
UOK/AQB- department of aquatic biology and
fisheries, University of Kerala.
Figure 4. Puntius viridis, Paratype, ZSI FF 4932

Journal of Research in Biology (2014) 4(8): 1581-1588 1583

Plamoottil, 2014

Table 2. Comparison of morphometric characters of P. nigronotus and its congeners

SL. P. viridis ZSI/ P. parrah P. madhusoodani
N0. Characters P. nigronotus WGRC/IR/2382, ZSI FF CRG/SAC 456- 459,
2383, ZSI FF 4934 (n= 4)
ZSI FF 5285 4932 (n= 8) (n=4)
1 Total length (mm) 105.1 91.2-103 86.5- 102.0 90.5- 118.3
2 Standard Length (mm) 82.3 72-81 65.5- 78.0 67.6- -91.4
% SL
3 Head length 29.5 26.4- 31.1 25.6-26.0 27.5-29.5
4 Head depth 22.1 19.7- 22.9 21.6-24.0 20.7-23.1
5 Head width 17.0 15.8- 17.8 15.4-17.6 15.0-16.7
6 Body depth at dorsal fin 35.8 31.5- 33.8 32.1- 33.1 34.5-36.2
7 Body depth at anal fin 23.7 21.1- 23.9 23.7-24.4 22.1-23.7
8 Body width at dorsal fin 20.0 16.2- 19.1 17.3-19.7 17.6-19.1
9 Body width at anal fin 12.2 10.8- 13.2 13.4- 15.2 11.7-14.5
10 Pre occipital distance 18.5 18.9- 23.0 20.5- 24.3 18.9-22.9
11 Distance from occiput to dorsal front 33.8 30.4- 31.7 24.3- 29.8 29.0-32.9
12 Pre-dorsal length 54.7 48.2- 54.8 50.0-52.1 49.3-50.6
13 Post-dorsal length 51.0 48.2- 54.8 48.7- 53.5 50.2-58.6
14 Pre-pectoral length 27.0 25.8- 29.7 27.0-28.2 26.2- 28.9
15 Pre-pelvic length 52.6 47.9- 50.0 47.2- 51.3 46.5-50.3
16 Pre-anal length 75.1 72.2- 76.6 70.3- 74.4 67.6-74.3
17 Length of dorsal fin 21.1 22.4- 26.5 22.1-24.4 25.2-28.7
18 Length of pectoral fin 19.4 16.7- 19.7 17.6-19.8 17.7- 19.1
19 Length of pelvic fin 17.9 17.3-20.3 20.3-21.4 20.7- 21.1
20 Length of anal fin 16.2 14.8- 18.9 13.3-16.8 19.2-21.5
21 Length of caudal fin 27.7 29.3- 30.0 28.4- 32.1 24.8- 27.0
22 Length of base of dorsal fin 18.2 17.6- 19.2 18.0-21.0 19.0-20.0
23 Length of base of anal fin 8.7 9.8- 11.1 12.0-15.4 9.0-12.0
24 Length of base of pectoral fin 5.1 4.1- 5.3 3.3-4.2 3.7- 4.1
25 Length of base of pelvic fin 4.9 5.0- 6.9 4.2-5.4 6.0-7.1
26 Length of base of caudal 14.6 13.5- 14.2 12.2-14.1 12.4- 13.8
27 Length of caudal peduncle 17.6 16.3- 17.8 19.1-21.2 12.6-17.5
28 Depth of caudal peduncle 14.0 13.5-14.5 12.9-13.5 12.8-14.6
29 LCP/DCP 79.3 77.0- 88.0 63.6-74.3 73.1- 84.6
30 Width of caudal peduncle 9.1 5.5- 7.4 4.1- 5.4 6.2-.6.6
31 Distance from pectoral to pelvic fin 25.7 21.0-21.6 20.4-20.9 22.8-25.0
32 Distance from pelvic to anal fin 24.3 23.8- 25.0 24.3- 26.8 25.0-28.9
33 Distance from anal to caudal fin 26.4 25.9- 27.5 27.7-29.6 25.5-27.0
34 Distance from anal to vent 0 2.6- 4.1 0 4.8-6.6
35 Distance from ventral to vent 22.5 19.1- 22.8 23.0-25.6 22.4-23.4
% HL
36 Head depth 74.9 68.2- 80.0 84.2-89.5 95.0-100.0
37 Head width 57.6 56.5- 63.2 60.0- 68.4 55.0-61.9
38 Eye diameter 32.1 26.1- 31.6 32.5-36.8 27.5-33.3
39 Inter orbital width 32.1 31.6- 40.9 42.1- 42.5 37.5-41.9
40 Inter narial width 23.0 23.9- 28.9 23.5-30.0 25.0-28.6
41 Snout length 24.7 22.7- 31.8 26.3-30.0 28.6-30.0
42 Width of gape of mouth 22.6 23.0- 27.3 28.9-30.0 25.0-27.6
43 Length of maxillary barbels 12.3 13.0- 21.1 15.0-17.6 14.3-15.0

RESULTS AND DISCUSSION Plamoottil, 1 January 2012.

Puntius nigronotus sp. nov. Diagnosis
(Figures 1- 3; Table 1 and 2) Puntius nigronotus can be differentiated from all
Holotype: ZSI FF 5285, 82.3 mm SL, India: Kerala, its congeners in having blackish dorsal side, shorter
Mananthavady River, Wayanad, coll. Mathews maxillary barbels (12.3 % HL), shorter width of mouth

1584 Journal of Research in Biology (2014) 4(8): 1581-1588

Plamoottil, 2014

Figure 5. Puntius parrah, ZSI FF 4934, coll. Plamoottil Figure 6. Puntius chola, ZSI FF 2203, coll. Hora

gape (22.6 5 % HL), a higher body (BDD 35.8 % SL), a eyes than to snout tip; jaws equal; barbels one pair
row of elongated tiny black dots present on dorsal fin, maxillaries only, shorter than orbit, feeble and roughly
the latter is shorter (21.1 % SL) and with 9 branched reach the lower margin of orbit but never reach nostrils;
rays, last unbranched dorsal fin ray feebly ossified and mouth terminal, straight and protruding; width of gape of
flexible, 6 branched rays in anal fin, 27 lateral line mouth equal to inter narial distance; operculum soft and
scales, greater pre pelvic distance (52.6 % SL), wide flexible.
caudal peduncle (9.1 % SL) and straight lateral line. Dorsal fin originates considerably behind the
Description pectoral tip, a little in front of ventral origin and nearer to
General body shape and appearance is shown in caudal fin base than snout tip; its upper margin fairly
Figure 1- 3. Meristic counts are shown in Table 1 and concave; dorsal fin with two undivided and nine
morphometric data in Table 2. Body laterally branched rays; first ray short and hard; second ray feebly
compressed; dorsal profile convex; region from dorsal ossified and flexible, tip a little filamentous; its inner
front to occiput a little bent, after sinking down very margin slightly roughened but not serrated; last branched
slightly goes straight to snout tip; post dorsal region ray divided to root; pectoral tip never reach ventral
slightly concave. Ventral profile from the base of origin; ventral fin originates just behind dorsal fin origin
pectoral fin to tip of pelvic fin straight, then abruptly and considerably behind pectoral tip; its tip never reach
concave and then goes straight to caudal base. Eyes anal fin origin and vent; two axillary scales present on
situated considerably behind and above the angle of either side of base of ventral, one above the other, of this
jaws, protruding above the surface of head and distinctly the upper one soft and delicate and form 2 in length of
visible from below ventral side; inter orbital region ventral fin; lower one short but more fleshy; anal fin
slightly convex; nostrils, in short tube, situated nearer to roughly rectangular, outer margin fairly concave, its tip

Figure 7. Puntius dorsalis, ZSI FF 2730, coll. Day Figure 8. Puntius madhusoodani, Paratype, CRG- SAC 459

Journal of Research in Biology (2014) 4(8):1581-1588 1585

Plamoottil, 2014

(2013) in having 27 lateral line scales (vs. 25/26 in P.

viridis), 11 pre dorsal scales (vs. 9), 9 branched rays in
dorsal fin (vs. 8), deeper body (35.8 % SL vs. 31.5-
33.8), greater distance (25.7 % SL vs. 21.0- 21.6) from
pectoral fin to pelvic fin, no distance from anal fin origin
to vent (vs. 2.6- 4.1 % SL) and wider caudal peduncle
(9.1 % SL vs. 5.5- 7.4). P. nigronotus differs from P.
parrah Day (1865, 1878, 1889) in having closely located
Figure 9. Puntius sophore, ZSI FF 4938, coll. Plamoottil orbits (IOW 32.1 vs. 42.1- 42.5 % HL in P. parrah),
never reach caudal base; no distance between anal fin shorter maxillary barbels (12.3 % HL vs. 15.0- 17.6),
origin and vent; no prominent ridge on the base of anal lower (HD 74.9 % HL vs.84.2- 89.5) and longer head
fin and dorsal fin; unbranched and branched rays of anal (29.5 % SL vs. 25.6- 26.0) and greater number of
fin soft; caudal lobes equal. Scales thin and soft; lateral branched dorsal fin rays (9 vs. 8) and lateral line scales
line straight and passes through the middle of body. (28 vs. 25).
Lateral line scales 27. The new fish differs from Puntius chola
Colouration (Hamilton, 1822) in having 11 pre dorsal scales (vs. 9 in
In live condition, back side deep black; upper P. chola), 10 pre pelvic scales (vs. 5/6), 27 lateral line
lateral side blackish brown; lower lateral side silvery scales (vs. 26), 9 branched rays in dorsal fin (vs. 8), 6
white; dorsal fin pale orange red; pectoral, pelvic and branched rays in anal fin (vs. 5) and 4 scales between
anal fins whitish yellow; caudal fin dirty yellow; minute lateral line and anal fin (vs. 3). Puntius nigronotus
bluish black dots present on operculum; a few tiny differs from P. dorsalis Jerdon (1849) in many meristic
elongated black spots present on dorsal fin rays; caudal and morphometric characters. In P. dorsalis, a black
blotch small, diffuse and concentrated on one scale as an spot present at the posterior portion of the base of dorsal
aggregation of bluish black dots. After preservation in fin (vs. absent in new species), 2 scales present
formalin back and upper lateral side turned to reddish between lateral line and ventral fin (vs. 3), snout longer
brown and lower lateral side yellowish white and fins (31.8-37.1 % HL vs. 24.7), dorsal fin with 8 branched
became hyaline. rays (vs. 9) and lateral line with 25/26 scales (vs. 27).
Etymology: The specific name nigronotus denotes the The new species can also be easily distinguished
color of the new fish. In Greek nigra means black and from Puntius madhusoodani Kumar et al. (2011) in
notus means back; refers to the blackish dorsal side many taxonomic characters. In P. madhusoodani 4
of the fish. scales present between lateral line and dorsal fin (vs.
Distribution: 5), pre dorsal scales 9 (vs.11), dorsal fin longer (25.2-
Currently known only from the type locality in Kerala. 28.7 % SL vs. 21.1), dorsal fin with 7 branched rays (vs.
Comparisons 9), lateral line scales 27 (vs. 25/26), snout longer (28.6-
Puntius nigronotus shows similarity to Puntius 30 % HL vs. 24.7), head deeper (head depth 95.0- 100.0
viridis, P. parrah and P. madhusoodani of Kerala and % HL vs. 74.9) and anal fin longer (19.2- 21.5 % SL vs.
Puntius dorsalis of Chennai and Puntius chola and 16.2). The new species can be distinguished from
P. sophore of River Ganges (Figure 4- 9). The new fish Puntius sophore (Hamilton, 1822) in having one pair of
differs from Puntius viridis Plamoottil and Abraham maxillary barbels (vs. absent), smaller eyes (32.1 % HL

1586 Journal of Research in Biology (2014) 4(8): 1581-1588

Plamoottil, 2014

vs. 34.7- 36.0), lower head (head depth at occiput 74.9 % SRC/F 4954, coll. M.B. Reghunathan; undated, 1
HL vs. 80.3- 86.7), lateral line scales 27 (vs. 25), 11 pre example, Madras, ZSI/F 2730, coll. Francis Day;
dorsal scales (vs. 9) and 9 branched rays in dorsal fin (vs. undated, 1 example, 53 mm SL, Tunga River at
8). Shimoga, ZSI/F 12320/1, coll. H.S. Rao; undated, 5
examples, 55- 62 mm SL, Cauvery River, Coorg,
CONCLUSION Karnataka, ZSI/F 12319/1, coll. C.R. Narayan Rao;
Puntius nigronotus is a unique fish having Puntius parrah: 10.01. 2012, 4 examples, 65.5-
greater number of branched dorsal fin rays than all other 78.0 mm SL, Arattupuzha, Karuvannoor River,
relative species; its lateral line scales and pre dorsal Irinjalakuda, Kerala, ZSI FF 4934, coll. Mathews
scales are also greater in number than its congeners; their Plamoottil; 15.12.1994; 1 example, 60 mm SL, Kuruva
last unbranched dorsal fin ray feebly ossified and Island, Wayanad, ZSI/WGRC/IR/742, coll. C.
flexible; it was believed that branched rays in dorsal fin Radhakrishnan; 24.03.1997, 1 example, 44 mm SL,
of Puntius species are only eight (Pethiyagoda et al., Parambikulam WLS, ZSI/WGRC/IR/10696, coll. K. C.
2012); the present fish is an exception to this; Puntius Gopi; 10.8.2001, 2 examples, 100.0- 103.0 mm SL,
nigronotus have nine branched rays in dorsal fin and Achankoil River, UOK/AQB/F/ 102, coll. Bijukumar;
additionally its last branched ray is divided to root; it is undated, 1 example, Karuvannoor River, Kerala, ZSI/F
expected that more aspects of its biology will be revealed 2718 Syntype, coll. Francis Day; 08.05. 1977, 6
in near future based on studies on more number of examples, 71 mm- 94 mm SL, Cauvery River at
specimens. Chunchanakatte, ZSI/SRC Uncat, coll. K. C. Jayaram.
Comparative material examined Puntius chola: 08.11.1939, 1 example, 41.5 mm
Puntius viridis: Holotype, 21.08.2011, 81 mm SL, Soni Gaon Bheel, Lokpa, Batipara, Assam, ZSI/F
SL, Kallumkal, Manimala River, Kerala, India, 2203, coll. S.L. Hora; 1963, 1 example, 54 mm SL,
9200N, 76300E, coll. Mathews Plamoottil, ZSI/ Sukla Talai, Jhalwar, Rajasthan, ZSI/F 4009/2, coll. N.
WGRC/IR/2382. Paratypes, 21.08.2011, 5 examples, 72- Majumdar & R.N. Bhargava; 18.03.1958, 2 examples,
76 mm SL, Kallumkal, Manimala River, Kerala, India, 32.5- 55 mm SL, Raxanal, Bihar, ZSI/F/2804/2, coll.
9200N, 76300E, coll. Mathews Plamoottil, ZSI/ Keval Singh; 3 examples, 50- 62 mm SL, Rajastan,
WGRC/ IR/2383; 10. 10. 2012, 2 examples, 63- 74 mm ZSI/F/4379/2, coll. Birla college, Pilani; 1 example, 71
SL, Manimala River at Kallumkal, Kerala, coll. Mathews mm SL, Mahanadi Irrigation Canal, Rudri, Orissa, ZSI/F
Plamoottil, ZSI FF 4932. 13082/1, coll. H.S. Rao.
Puntius dorsalis: 27.10.95, 1 example, 62 mm Puntius madhusoodani: 17.11.2010, Holotype,
SL, Thunakadavu dam, Parambikulam wild life 91.43mm SL, Manimala River, near Thirumoolapuram,
sanctuary, Kerala, ZSI/WGRC/IR 8466, coll. P.M. Thiruvalla, Kerala, CRG-SAC 456, coll. K.
Sureshan, identified by K.C. Gopi; 23.2.2000, 2 Krishnakumar; 17. 11. 2010, 3 examples, 67.6 -
examples, 56- 63 mm SL, Pampa River at Parumala, 80.91mm SL, Manimala River, near
Kerala, ZSI/WGRC/IR/10379, coll. K. C. Gopi; 11.02. Thirumoolapuram,Thiruvalla, Pathanamthitta District,
58; 1 example, 53 mm SL, Isteri tank, 7 miles north west CRG-SAC 457 459 paratypes, coll. K. Krishnakumar
of Pondicherry, ZSI/F 2801, coll. A.G.K. Menon; 16.02. and Benno Pereira.
1996, 2 examples, 52- 53 mm SL, Sethumadai canal, Puntius sophore: 10.05.2012, 2 examples, 58-
Indira Gandhi Wild Life sanctuary, Tamil nadu, ZSI/ 59 mm SL, Serrampore, River Ganges, Kolkata, ZSI FF

Journal of Research in Biology (2014) 4(8): 1581-1588 1587

Plamoottil, 2014

4938, Coll. Mathews Plamoottil; 20.06. 1963, 4

Jayaram KC. 2002. Fundamentals of Fish Taxonomy.
examples, 62.5- 70.0 mm SL, Sukla Talai, Jhalawar,
Narendra Publishing House, Delhi. 174 p.
Rajasthan, ZSI/F 4008/2, coll. N. Majumdar & R. N.
Bhargava; 24.10.1939, 1 example, 40 mm SL, Jerdon TC. 1849. On the freshwater fishes of southern
Suwannee River, east of Hazaribagh Barhi Road, ZSI/F India, Madras Journal of Literature and Science.15 (2):
13827, H.S. Rao; 22.06.1963, 4 examples, 66- 102 mm 302- 346.
SL, Gadhuli Talai, Shergarh, Rajasthan, ZSI/F 4023, SE
Kumar KK, Pereira FGB and Radhakrishnan KV.
Rajastan Survey of ZSI; 30.06.1983, 4, examples, 58.0-
2011. Puntius madhusoodani (Teleostei: Cyprinidae), a
67.5 mm SL, Talbi, N. of Bimmal Railway station, ZSI/F
new species of barb from Manimala River, Kerala, South
4029/2, S. E. Rajasthan Survey of ZSI.
India, Biosystematica. 5 (2): 31- 37.

ACKNOWLEDGMENTS Pethiyagoda R, Meegaskumbura M and Maduwage

The author acknowledges University Grants K. 2012. A synopsis of the South Asian fishes referred
Commission of India for sanctioning Faculty to Puntius (Pisces: Cyprinidae). Ichthyological
Development Programme to undergo this research. I am Exploration of Freshwaters. 23 (1): 69-95.
grateful to anonymous reviewers for their comments that
Plamoottil M and Abraham NP. 2013. Puntius viridis
helped to improve the manuscript.
(Cypriniformes, Cyprinidae), a new fish species from
Kerala, India, Journal of Research in Biology. 3 (7):
Day F. 1865. The Fishes of Malabar. Bernard Quaritch,
London. 293.

Day F. 1878. The fishes of India: being a natural history

of the fishes known to inhabit the seas and fresh waters
of India, Burma, and Ceylon. William and Norgate,
London. (suppl.) 556-574.

Day F. 1889. Fauna of British India including Ceylon

and Burma. Edited by W. T. Blanford. Fishes, by Francis
Day. 2, 8: London: Taylor and Francis, 1889. Fishes. I,
Taylor and Francis, London. 209-334.
Submit your articles online at
Hamilton F. 1822. An Account of Fishes found in the Advantages
River Ganges and its branches. Edinburgh: Printed for Easy online submission
Complete Peer review
Archibald Constable and Company and Hurst, Robinson,
Affordable Charges
and Co. 90, Cheapside, London. 312-389. Quick processing
Extensive indexing
Jayaram KC. 1991. Revision of the genus Puntius You retain your copyright
Hamilton (Pisces: Cypriniformes, Cyprinidae,
Cyprininae). from Indian region. Records of Zoological

Survey of India, Occasional Paper No. 135: 178.

1588 Journal of Research in Biology (2014) 4(8): 1581-1588

Journal of Research in Biology ISSN No: Print: 2231 6280; Online: 2231- 6299

An International Scientific Research Journal


Ethnobotanical plants used as curatives for skin diseases in a Cauvery river

stretch, Namakkal district, Tamil Nadu, India
Authors: ABSTRACT:
Journal of Research in Biology

Thirumaran G1,
Ganesan CM2,
Nandakumar K1
and Paulsamy S2 The study area covers about 15 km stretch in the perennial river bed, Cauvery
which supports semi evergreen to dry deciduous type of forests. Ethnobotanical
information on 24 plant species was recorded during the extensive field survey carried
1. PG and Research out in this stretch during 2012-2013. The information covers botanical names,
Department of Botany, vernacular names, family, plant parts used and the mode of usage.
Kandaswami Kandars
College, Velur, Namakkal,
Tamil Nadu, India.

2. Department of Botany,
Kongunadu Arts and Science
College, Coimbatore, Tamil
Nadu, India Keywords:
Cauvery river basin, Medicinal plants, Traditional uses

Corresponding author:
Thirumaran. G

Email Id:

Article Citation:
Thirumaran G, Ganesan CM, Nandakumar K and Paulsamy S.
Ethnobotanical plants used as curatives for skin diseases in a Cauvery river stretch,
Namakkal district, Tamil Nadu, India
Web Address: Journal of Research in Biology (2014) 4(8): 1589-1594
documents/RA0493.pdf Dates:
Received: 21 Nov 2014 Accepted: 29 Nov 2014 Published: 31 Dec 2014

This article is governed by the Creative Commons Attribution License (

licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

1589-1594| JRB | 2014 | Vol 4 | No 8

Journal of Research in Biology
An International
Scientific Research Journal
Thirumaran et al., 2014


Plants are proved to be more reliable for Survey and collection of ethnomedicinal plants
therapeutic uses. Still 70-80% of the people all over the used by the local people of Namakkal district
world rely on plants for their health care (Shanley and (Pandamangalam, Nanjai Edayar and Mohanur) were
Luz, 2003). India, the mega diversity nation, harbours made over a period of 12 months (2012-2013). Frequent
about 3000-3500 (15%) medicinal plants, out of 20000 field trips were undertaken to the study areas for
available in the world. At global level, 90% of these collecting information regarding the medicinal plants
species are growing in various climatic areas (Farombi, used by them from the elderly people. Experienced
2003). Studies on medicinal plants availability, healers are interrogated intensively to bring out the
biochemical compounds, mechanism of their action etc., information about the medicinal plants in various aspects
are being made in India since few decades intensively. In viz., plant parts used, medicinal uses, mode of
Tamil Nadu, many vegetation are being attempted for administration and the doses to be prescribed. The plant
this study by many workers (Banu et al., 2007; specimens were collected for taxonomic identification
Murugesan et al., 2007; Ignacimuthu et al., 2008; from different parts of the study area. Identification was
Arunachalam et al., 2009; Balakrishnan et al., 2009; made with the help of The Flora of Presidency of
Ayyanar et al., 2010; Maruthapandian and Mohan, 2010; Madras (Gamble, 1915- 1935) and The Flora of Tamil
Shanmugam et al., 2011 and 2012). Nadu Carnatic (Mathew, 1983) and finally confirmed by
The riparian ecosystem at Cauvery stretch in comparing with the authenticated specimens in the
Tamil Nadu is known to harbor many medicinal plants of Herbarium of Botanical Survey of India (Southern
local uses. However, no documentation has been made in Circle) Coimbatore district, Tamil Nadu. Voucher
this line. Therefore, the present study was aimed to specimens were deposited in the Herbarium of the
collect information on traditional uses of medicinal Research Department of Botany, Kandaswami Kandars
plants used in preparation of herbal drugs for the College, Velur, Namakkal, Tamil Nadu. Indian
treatment of skin diseases by local people living in the Medicinal Plants (Kirtikar and Basu, 2012) and The
nearby villages of Cauvery river, Namakkal District, Wealth of India, (2006) were referred for further
Tamil Nadu. information on medicinal uses.
STUDY AREA Enumeration
The Cauvery river stretching between Nanjai In the following enumeration, the plants are
Edayar and Mohanur area of Namakkal district Tamil arranged alphabetically as per botanical name, local
Nadu, is situated at 1104 L and 7803 E. The altitude name in parenthesis family name and a brief note on
is 130.45 above MSL. The soil is mostly sandy with plant parts, mode of utilization, dosage etc.
slightly acidic pH. The local people of nearby villages, in Aegle marmelos (L.) Corr. Serr. (Vilvam) Family:
addition to western medicine also use herbal plants for Rutaceae.
their day to day life, as per the prescription made by One fresh fruit is crushed with seeds of
healers. The present study was undertaken to document Strychnos nux vomica and Pongamia pinnata and boiled
the traditional uses of medicinal plants available in this with coconut oil for fifteen minutes. This extract is
stretch which comprises grassy patches, man-made applied on the affected parts twice a day for three days to
plantations and semi evergreen and dry deciduous treat scabies.
1590 Journal of Research in Biology (2014) 4(8): 1589-1594
Thirumaran et al., 2014

Calotropis gigantea (L.) R.Br. (Erukku) Family: wounds.

Asclepiadaceae. Cynodon dactylon L. Pers. (Arugam pull) Family:
Paste prepared from leaf powder of this species Poaceae.
is applied externally once a day for three days to cure the Five grams of fresh leaves is made into paste
lesions of eczema. with little amount of turmeric (Curcuma longa) and few
Cassia alata L. (Seemai agathi) Family: drops of water. This paste is applied externally twice a
Caesalpiniaceae. day for ten days to treat dermatophytosis.
Ten grams of fresh leaf is applied over infected Datura innoxia Mill. (Vellai umathai) Family:
skin twice a day for fifteen days to treat ringworm Solanaceae.
infection. Paste prepared from dried leaf powder with
Cassia fistula L. (Sarakonrai) Fa mily: coconut oil is applied externally once a day, continuously
Caesalpiniaceae. for eight to ten days to get relief from wounds.
About five grams of the powder of flower is Euphorbia tirucalli L. (Elaikalli) Family:
made into paste with lemon juice and applied externally Euphorbiaceae.
on the skin to treat eczema and freckles. Five milliliters of milky latex is externally
Celosia argentea L. (Kolikondai) Family: applied once a day for a period of two days to treat
Amaranthaceae. arthritis and cracked foot.
A paste is prepared from five grams root with Ficus religiosa L. (Arasu) Family: Moraceae.
water. This paste is applied externally once a day for ten About three grams of shade dried powder of bark
days to treat wounds. is mixed with water and taken orally once a day for ten
Clerodendron inerme (L.) Gaertn. Fruct. days to treat psoriasis.
(Naaraseengi) Family: Verbenaceae. Ficus racemosa L. (Athi) Family: Moraceae.
About two grams of fresh leaves is made into A decoction is prepared from ten grams of bark
paste with water and applied externally on the skin once powder mixed with water. This decoction is applied
a day, for one week to treat psoriasis. externally once a day for six days on the wounds.
Cocos nucifera L. (Thennai maram) Family: Lawsonia inermis L. (Maruthaani) Family:
Areaceae. Lythraceae.
A thick paste prepared from five milliliters of oil About fifty grams of bark powder is made into a
and two grams of turmeric (Curcuma longa) powder is paste with coconut oil. This paste is applied externally
applied externally twice a day for a period of three days once a day for seven days to treat skin diseases.
to treat cuts and wounds. Madhuca longifolia (Koen) Macbr. (Illuppai) Family:
Commelina benghalensis L. (Neerchedi) Family: Sapotaceae.
Commelinaceae. A decoction is prepared by boiling a hundred
Leaf paste prepared is applied once a day on the grams of stem bark and five hundred milliliters of water.
wounds for healing and to remove the poisonous spines This decoction is taken orally once a day, for one week
that had struck accidently on the body parts. to get relief from wounds and skin diseases.
Curcuma longa L. (Manjal) Family: Zingiberaceae. Melia dubia Cav. (Malai vembu) Family: Meliaceae.
A sufficient amount of dried powder of rhizome The fruits are made into paste and applied
is applied externally twice a day for three days to treat externally once a day, for a period of one week as

Journal of Research in Biology (2014) 4(8):1589-1594 1591

Thirumaran et al., 2014

Senna alata Melia dubia Calotropis gigantea

Euphorbia tirucalli Pedalium murex Ficus religiosa

Curcuma longa Datura inoxia Mirabilis jalapa

Figure 1. Some common medicinal plant species of Cauvery riparian ecosystem

ointment to get cure from skin diseases. Pedalium murex L. (Aanai nerunjil) Family:
Mirabilis jalapa L. (Anthimalli) Family: Pedaliaceae.
Nyctaginaceae. Two milliliters of juice is prepared and mixed
About six grams of fresh leaves are made into with five milliliters of water. This juice is taken orally
paste with water. This paste is applied externally twice a once a day for fifteen days to treat psoriasis.
day, for three days to treat swellings in the skin. Pongamia pinnata (L.) Pierre. (Punga maram)
Euphorbia hirta L. (Arisi poondu) Family: Family: Fabaceae.
Euphorbiaceae. Ten grams of seed powder are boiled with
The latex obtained from the leaves is applied coconut oil and the extract is applied over the skin to
externally once a day for three days to treat pimples in treat skin diseases.
the face (Acne vulgaris). Hundred grams of bark powder are boiled with

1592 Journal of Research in Biology (2014) 4(8): 1589-1594

Thirumaran et al., 2014

Caesalpiniaceae (2 species). They were using these

plants to treat various types of skin diseases, scabies,
eczema, wounds, acne vulgaris, ringworm infection,
freckles, psoriasis, cracked foot, swellings, rashes, etc.
Among different plant parts used, the leaves are
most commonly prescribed. They use the plant parts in
various forms, either raw or paste, powder, decoction and
juice for curing various skin disorders. In the above
mentioned data, 14 plants are used in the form of paste,
8 plants are used in the form of powder, decoction and
juice. The latex is used as such in 2 plants. External
application is also prescribed for the treatment of certain
Figure 2. Pie chart describing the percent plant species
used for various ailments skin diseases. The investigated 24 plant species can treat/
cure as many as 7 different skin diseases. About 9 plants
two hundred milliliters of coconut oil and the oil extract are used for wounds healing followed by 4 plants for
is applied externally once a day to treat ringworm psoriasis, 3 plants for eczema, 2 plants for scabies, 2
infection, rashes, scabies, eczema and psoriasis. plants for acne vulgaris, ringworm and healing the
Solanum torvum Sw. (Sundai) Family: Solanaceae. cracked foot.
The paste prepared from five grams of fresh root The healers have preferred to prepare
with water is applied externally to get cure from chronic the formulations by combining several plants for their
wounds. synergetic effect to heal the ailments. The
Thespesia populnea (L.) Sol. Ex Correa (Poovarasu) medicinal plants prescribed by the healers need to be
Family: Malvaceae. systematically screened for their phytochemicals and
Two grams of senescent leaves are made into thus the scientific validation may help in the discovery of
paste and applied externally twice a day for one week to new drugs from these medicinal plants.
get cure from chronic wounds.
Tridax procumbens L. (Thaneerpundu) Family: CONCLUSION
Asteraceae. From the study it is suggested that Cauvery river
Ten grams of whole plant are made into paste. stretch in Namakkal district, Tamil Nadu is a potential
This paste is applied externally twice a day for one week ecosystem of medicinal plants as it is endowed with
to treat cuts and wounds. many medicinal plants which are prescribed most
commonly by the local healers for various ailments. In-
RESULTS AND DISCUSSION depth studies are suggested all over the Cauvery river
The present study revealed that local people of banks in Tamil Nadu to explore the medicinal plants.
Namakkal district in the nearby villages of Cauvery Phytochemical and pharmacological investigation should
stretch were using 24 species of medicinally important also be made to have the scientific validation of such
plants belonging to 20 families. The most commonly valuable bioresources and confirm our traditional
used families were Solanaceae (2 species), knowledge on medicinal plants.
Euphorbiaceae (2 species), Moraceae (2 species) and

Journal of Research in Biology (2014) 4(8): 1589-1594 1593

Thirumaran et al., 2014

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1594 Journal of Research in Biology (2014) 4(8): 1589-1594

Journal of Research in Biology ISSN No: Print: 2231 6280; Online: 2231- 6299

An International Scientific Research Journal


Anesthetic efficacy of clove oil and its impact on hematological and

biochemical changes in Channa striatus (Bloch, 1793)
Authors: ABSTRACT:
Journal of Research in Biology

Jeyasheela P, Haniffa MA Channa striatus is one among the dominant group of air breathing
and Kavitha K freshwater fishes in Southeast Asian countries. In the present study, fish anesthetic
clove oil was used to study the hematological and biochemical changes at different
concentration (400 ppm, 450 ppm and 500 ppm) and time interval (0 h, 1h and 24 h)
in C. striatus. The induction and recovery time was noted for each treatment groups.
Institution: Erythrocyte count (T/L), Hemoglobin and Hematocrit values showed elevated levels
Centre for Aquaculture when compared to control. RBC, Hb and Ht values significantly increased 1 h after
Research and Extension anesthesia and returned to normal after 24 h. Anesthetic treated fishes exhibited
(CARE), St.Xaviers College marked decrease in WBCs when compared to control group. The rest of the indices
(Autonomous), (MCV, MCH, MCHC, Lymphocytes, Monocytes, Neutrophils) were at comparable levels
Palayamkottai, in all groups. The anesthetic treated fishes were found to show a significant increase
Tamil Nadu-627005, India in the concentration of glucose. The rest of the indices (TP, ALB, GLOB, ALT, AST) were
at comparable levels in all groups. Results of the study suggested that the use of clove
oil at the concentrations of 400, 450 and 500 ppm does not cause irreversible damage
on the blood parameters as well as biochemical profile in C. striatus.
Corresponding author:
Haniffa MA
C. striatus, anesthesia, induction and recovery time

Email Id:
Article Citation:
Jeyasheela P, Haniffa MA and Kavitha K
Anesthetic efficacy of clove oil and its impact on hematological and biochemical
changes in Channa striatus (Bloch, 1793)
Web Address: Journal of Research in Biology (2014) 4(8): 1595-1603
documents/RA0497.pdf Dates:
Received: 09 Oct 2014 Accepted: 11 Nov 2014 Published: 31 Dec 2014

This article is governed by the Creative Commons Attribution License (

licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

1595-1603 | JRB | 2014 | Vol 4 | No 8

Journal of Research in Biology
An International
Scientific Research Journal
Jeyasheela, 2014

INTRODUCTION: induction and recovery periods and mortality are also

Murrels, commonly called snakeheads are studied.
important air breathing freshwater fishes and are highly
regarded as food fish in the South and Southeast Asian MATERIALS AND METHODS:
countries. They belong to the family Channidae The striped murrel, C. striatus acclimatized in
(Ophiocephalidae) (Wee et al., 1982). Murrels can be cement tanks (15mX3mX2m) for a period of two weeks
kept alive for several hours outside water under slight at CARE Aquafarm were used for this study. During this
moist condition which facilitates transportation to distant period, the fishes were fed twice a day with chicken
markets in good condition. Even then often fishermen intestine. Forty fishes (29.7 1.69 cm and 232 14 g)
meet heavy loss due to death of murrels during transport. were selected and were divided into four groups each
Hence, sedation or use of anesthetics can be beneficial with 10 fishes based on the concentration of clove oil
to calm excitable fish during bulk transportation of fish (group I: control [0ppm], group II: 400ppm, group III:
stocks, especially over long distances and high density. 450 ppm and group IV: 500 ppm). Test fishes were
In aquaculture practices and research activities, starved for 24 hours prior to the experiment and
fish handling are a common source of stress. Hence, a mortality rate (if any) was recorded regularly throughout
variety of anesthetics are used mainly in order to reduce the course of the study. Stock solution of clove oil was
stress level and to prevent fish injury/ death during their prepared by dissolving 1 ml of clove oil in 9 ml of tap
handling. The most commonly used fish anesthetics are water. The test fishes were exposed to the respective
tricaine methanesulfonate (MS-222), benzocaine (ethyl concentrations and they were anesthetized.
paraaminobenzoate) (Kiessling et al., 2009), The induction and recovery period of fishes as a
2-phenoxyethanol, metomidate (Weber, 2009), and function of concentration were recorded. Blood was
carbon dioxide (Pirhonen and Schreck, 2003). sampled from caudal vein of the anesthetized C. striatus
Clove oil is a dark - brown liquid, a distillate of with heparin coated syringe. To stabilize the blood
flowers, stalks and leaves of the clove tree Eugenia samples, aqueous solution of heparin sodium salt (5000
aromatica (Soto and Burhanuddin, 1995) having a U/ml) was added to blood (Svobodova et al., 1991). The
mild anesthetic effect on human (Nagababu and blood sample was divided into two aliquots; one part was
Lakshmaiah, 1992; Taylor and Roberts, 1999) and transferred to a 2 ml heparinized tube and stored in
fish (Ross and Ross, 2008). Keene et al. (1998) refrigerator prior to hematological analyses. The other
showed that the clove oil i s much less e x p e n s i v e part of aliquots was transferred to 1.5 ml microcentrifuge
than other chemicals including MS222 and tubes and centrifuged for 15 min. at 4C. The plasma
recommended the same for fish transport. was removed and transferred to another 1.5 ml microtube
Anesthesia may affect blood parameters and and stored frozen at 70C for biochemical analyses
hemolyse tissues (McKnight, 1966). Since, (Fast et al., 2008).
hemodynamic is closely related to response of animal to Hemogram was established by estimation of total
external environment (Fernandes and Mason, 2003). The erythrocyte count (RBC), total white blood cell count
goal of this study was to assess efficacy of clove oil as an (WBC), hematocrit (Ht), hemoglobin concentration (Hb),
anesthetic through the measurement of multiple blood erythrocyte indices (MCV, MCH, MCHC) and white
parameters of striped murrel Channa striatus. The blood cell differential count (Campbell, 2004).
effect of different concentrations of clove oil on Biochemical indices of blood plasma included glucose
1596 Journal of Research in Biology (2014) 4(8): 1595-1603
Jeyasheela, 2014

Time (min)

Concentration (ppm)

Figure 1: Effect of Clove oil on Induction and Recovery time of C. striatus

(GLU), Total Protein (TP), Albumin (ALB), Globulin complete when the fish lost its response to external
(GLOB), Aspartate amino Transferase (AST), and stimuli. As per the results obtained, the optimum
Alanine amino Transferase (ALT). Statistical differences concentration to anesthetize C. striatus was found to be
between groups if any at each time point (0 h, 1 h 450 ppm. The fishes underwent anesthesia through six
and 24 h) were tested using SPSS software and data stages as described by Bowser (2001). The first stage of
were presented a s m e a n SD. anesthesia was light sedation which involved: Slight loss
of reactivity to external stimuli; opercular rate slightly
RESULT AND DISCUSSION: decreased with normal equilibrium. This was followed
Water quality parameters were measured and by the next stage i.e., deep sedation involving total loss
recorded as pH 7.0, chloride 200 ppm, total hardness of reactivity to all but strong external stimuli; slight
525 ppm, fluoride 0.5 mg/l, iron 0.5 mg/l, residual decrease in opercular rate with normal equilibrium. The
chlorine 0 mg/l and nitrate 0.45 mg/l. signs of next stage included: Partial loss of muscle tone
All the fish exposed to different concentrations and swimming was erratic; increased opercular rate;
of clove oil recovered well and returned to normal reactivity only to strong tactile and vibration stimuli.
behaviour with respect to feeding, surfacing activity, Total loss of muscle tone and equilibrium; slow but
swimming and respond to external stimuli after the regular opercular rate; loss of spinal reflexes especially
anesthetic treatment. Furthermore, no mortality was loss of equilibrium observed in the fourth stage. The
noticed within 48 h following recovery from anesthesia. changes that were noticed in the fifth stage were total
The technique anesthesia by immersion wa s loss of reactivity; opercular movements were slow and
ap p li ed to provide the active ingredient into the fish irregular, heart rate was very slow and loss of all reflexes
gills through water flow to travel through bloodstream was noticed. The final stage during anesthesia was
to the central nervous system. Thus, the fish goes medullary collapse (stage of asphyxia) and the
through several stages of anesthesia viz ; light sedation, anesthetized fishes showed opercular movements to
deep sedation, partial loss of equilibrium, total loss of cease; cardiac arrest followed quickly. The time duration
equilibrium, loss of reflex reactivity and ultimately for each stage was shown in the figure 1.
medullary collapse as described by Bowser (2001). Increase in clove oil concentration resulted in
Induction of anesthesia was assumed to be decrease in induction time whereas the recovery time

Journal of Research in Biology (2014) 4(8): 1595-1603 1597

Jeyasheela, 2014

increased significantly. However, the recovery and were put into induction tray containing clove oil, they
induction time was also concentration dependent. The became excited and hypermotile followed by bubbling;
mean induction time for C. striatus 400, 450 and 500 the gill and fin movements progressively decreased, the
ppm clove oil was found to be 7.10 1.10 min., 2.36 fish lost equilibrium and started swimming laterally.
0.42 min. and 2.020.52 min. respectively. The mean Finally, the fish became immobile with full loss of
recovery time for each concentration was 5.211.1 min., equilibrium and consciousness. After transfer to recovery
3.200.40 min. and 6.381.15 min. respectively. The tray, reappearance of gill movement was noticed first.
induction time was longer at the lower dose whereas it is This was followed by fin and then tail movement. The
quick and short at higher dose. In the same manner, the fish started moving laterally. Gradually full equilibrium
recovery time was quick in lower dose whereas it elapsed was regained and normal behaviour was restored at
for a longer duration at higher dose. For morphological 5.211.1 min., 3.200.40 min. and 6.381.15 min. as a
evaluations, biopsy and stripping, long handling periods function of 400 ppm, 450 ppm and 500 ppm clove oil
are required and hence, anesthetizing with clove oil respectively. Similar, behavioural changes during
would be an added advantage which produces longer induction and recovery as a function of anesthesia have
recovery time (Seol et al., 2007; Park et al., 2009). been reported by McFarland (1960).
It was observed that if the exposure was Hematological and biochemical profiles of blood
prolonged until the fish become anesthetized; the are necessary to provide vital information about internal
recovery was concentration-independent and lasted for environment of organism. Erythrocyte count (T/L)
about four minutes. In the present study, the tested showed increased values in anesthetic treated groups
concentrations met the efficacy criteria specified by (1.18-1.21) when compared to control (1.14) (Table 1).
Marking and Meyer (1985) and hence, dose of 450 ppm Similarly, Hemoglobin and Hematocrit also showed
of clove oil can be suggested for transport or grading of elevated levels in anesthetic induced fishes (Hb: 59.93
C. striatus. This finding is also in accordance with the 64.826 g/dl and Ht: 0.179 0.194 l/l) as compared to
results reported by Woody et al. (2002) where higher control (Hb: 59.58 63.24 g/dl and Ht: 0.178 0.189 l/l
dose produced a rapid and uniform response in all size respectively). RBC, Hb and Ht values significantly
classes, suggesting that a dose of 400 ppm might be well increased 1 h after anesthesia and returned to normal 24
over the effective concentration for sockeye salmon h post anesthesia.
(Oncorhynchus nerka). Anesthetic treated fishes exhibited marked
Similar observations were made by Inoue et al. decrease in WBCs (0.876 1.109 g/L) when compared
(2003) on juveniles of matrinxa, Brycon cephalus, to control group (0.981 1.138 g/L) (Table 1). The rest
where, the recovery in the experiment with a prolonged of the indices (MCV, MCH, MCHC, Lymphocytes,
exposure was longer at all concentrations, but with 30 Monocytes, Neutrophils) were at comparable levels in all
and 40 mg /L, it was still below 10 min. Our results groups. Results of the study suggested that the use of
showed that increasing the anesthetic dose significantly clove oil at the concentrations of 400, 450 and 500 ppm
decreased induction time but prolonged recovery time. does not cause irreversible damage on the blood
This was in agreement with reports of Salmo salar smolts parameters in C. striatus.
(Iversen et al., 2003), Cyprinus carpio (Velisek et al., Tort et al. (2002) reported clove oil altering
2005) and Silurus glanis (Velisek et al., 2006). hematocrit concentrations in rainbow trout. Velisek et al.
As reported by Matin et al. (2009), when the fish (2005) observed the same in rainbow trout and carp, both

1598 Journal of Research in Biology (2014) 4(8): 1595-1603

Table 1: Effect of Clove Oil on Hematological Indices of C. striatus
S. Hematological Control 400 ppm 450 ppm 500 ppm
No. Indices
0h 1h 24 h 0h 1h 24 h 0h 1h 24 h 0h 1h 24 h
1 Erythrocyte 1.180.07 1.140.08 1.140.05 1.180.08 1.200.09 1.180.06 1.190.09 1.200.12 1.180.05 1.190.06 1.210.07 1.180.08
Jeyasheela, 2014

(RBC) (T/L)
2 Hb (g/dl) 59.331.2 59.351.72 59.501.28 59.601.32 63.240.84 60.230.30 59.930.89 64.280.81 58.391.21 60.220.53 64.830.60 60.370.42

3 Ht (l/l) 0.170.003 0.140.005 0.160.004 0.180.004 0.190.003 0.180.30 0.180.003 0.190.002 0.180.004 0.180.002 0.190.002 0.180.001

4 MCV (fl) 151.918.98 156.2012.36 156.538.90 150.5910.23 157.7621.01 152.676.98 150.8017.26 160.4321.57 148.316.37 151.858.68 159.9337.22 153.2612.58

5 MCH (pg) 50.582.98 52.084.09 52.212.99 50.423.47 52.797.08 51.092.34 50.495.72 53.437.20 49.482.10 50.522.92 53.4412.36 51.124.12

6 MCHC (g/L) 332.960.53 333.440.52 333.550.69 334.820.71 334.600.63 334.630.52 334.800.34 333.050.44 333.630.3 332.270.45 334.150.57 333.550.53

7 Leukocytes 1.070.01 1.140.02 0.980.01 0.990.02 0.920.02 0.930.02 0.960.01 0.900.02 1.110.02 0.910.17 0.880.26 0.980.02
(WBC) (g/L)
8 Lymphocytes 83.43.05 83.62.41 84.12.71 81.61.14 81.21.14 83.11.58 821.58 80.41.14 81.80.84 811.58 80.61.14 82.41.14
9 Neutrophils 14.41.58 14.02.08 13.51.14 16.60.71 16.01.48 14.91.52 13.41.14 15.60.89 131.58 13.81.30 14.80.84 11.81.10

Journal of Research in Biology (2014) 4(8): 1595-1603

10 Monocytes % 2.20.84 2.41.14 2.41.52 1.80.84 2.80.84 2.01.0 1.60.89 3.01.22 2.20.84 1.20.45 2.60.89 2.00.71

Table 2: Effect of Clove Oil on Blood Plasma Indices of C. striatus

S.No Biochemical Control 400 ppm 450 ppm 500 ppm

0h 1h 24 h 0h 1h 24 h 0h 1h 24 h 0h 1h 24 h

1 Glucose 6.130.22 6.490.18 6.410.14 7.280.10 8.420.21 6.410.07 7.200.11 7.510.25 6.250.18 7.320.10 7.620.07 6.310.21

2 Total Protein 69.080.18 68.290.42 69.310.49 70.380.71 71.300.42 69.00.54 70.250.51 71.220.31 69.150.58 69.40.53 70.220.57 69.800.99
3 Albumin 4.090.19 4.510.28 4.240.40 6.260.32 5.320.50 5.110.25 7.960.30 7.080.42 5.380.23 13.110.61 15.370.24 11.330.37

4 Globulin 64.990.27 63.780.49 65.070.86 64.120.72 65.850.36 63.890.35 62.290.36 64.540.93 63.770.67 56.291.09 54.840.72 58.460.87
5 AST (IU/L) 8.330.28 6.60.45 7.340.44 7.600.35 5.040.20 8.310.33 6.270.27 8.180.30 14.140.32 12.330.33 16.260.45 18.180.26

6 ALP (IU/L) 12.440.38 14.520.36 13.420.39 8.590.36 12.630.29 19.850.34 13.790.33 10.490.30 13.390.31 16.520.33 21.540.15 23.070.26

Jeyasheela, 2014
of them also exhibited significantly increased plasma system was evaluated by the changes in White Blood
glucose concentrations after longer exposure periods (10 Cell (WBC). It showed a decline trend associated with
min.). arresting in anesthetic. Increase in plasma cortisol
The increase in RBC level after anesthetic concentration which is a glucocorticoid hormone, is also
treatment showed that fish's body is kept in touch with acting as an immunosuppressive (Fast et al., 2008), so it
insufficient gas when anesthetized. When oxygen could suppress humoral factors and lead in declining
becomes a limiting factor, RBCs increased for carrying circulating WBC along with elevating cortisol.
more oxygen to the cells. Hemoglobin (Hb) is an The anesthetic treated fishes were found to show
effective index of taking more oxygen in the blood and a significant increase in the concentration of glucose i.e.,
hence Hb increased for obtaining more oxygen; the 7.196 7.318 mmol/l at 0 h and 7.514 8.422 mmol/l at
average volume value (MCV) with a red blood cell 1 h following anesthetic treatment. The glucose values of
increases after anesthetization and showed that dissolved anesthetic treated fishes returned back to control values
oxygen amount decrease in fish's blood. So MCV is after 24 h post treatment (6.134 6.486 mmol/l). The
strengthened by holding more oxygen, and keeping the rest of the indices (TP, ALB, GLOB, ALT, AST) were at
basic physiological function to fish's body; the average comparable levels in all groups (Table 2).
concentration of hemoglobin of red blood cells (MCHC) These results revealed the significance of
increase after anesthetization and show that dissolved exposure time and dosage on some physiological
oxygen amount is decreasing in fish's blood, so MCHC indicators of anaesthetized fish. Post-exposure mortality
increase for combining more oxygen (Wu et al., 2000). and lack of biochemical alteration in the present study
The respiratory actions lower resulting in could be due to the short induction time after which
reduced O2 for circulation for breathing and survival blood was collected. These results indicated the need to
creating a hypoxic environment which results study possible physiological changes occurring in
physiological changes altering the blood factors like different fish species exposed to different doses of clove
glucose and Hematocrit (Ht). Hyperglycemia (increased essence.
blood glucose), elevated HB and Ht were similar to other Plasma cortisol as well as glucose is a
fishes anesthetized by Pirhonen and Schreck (2003); physiological indicator of stress in fishes and their
Park (2009); Gomes et al. (2001). Reports by Velisek et interactive effects on metabolism during recovery from
al. (2005) on Cyprinus carpio and Oncorhynchus mykiss stress have recently become a subject of more intense
suggests that clove oil anesthesia at 30 mg/l study (Tytler and Hawkins, 1981; Woody, 2002; Weber
concentration and 10 min exposure did not produce any et al., 2009). Clove oil was found to block the activity of
marked changes in the blood parameters after 24 h. cortisol, although not completely, in Brycon cephalus
However Sudagar et al. (2009), reported that a 7-min (Inoue et al., 2005). Although the mechanism is not well
exposure to clove powder resulted in significant known, Iverson et al. (2003) suggested that it blocks
reversible increase in Ht, Hb and RBC immediately after transmission of impulses to the Hypothalamus-Pituitary
anesthesia in Roach Rutilus rutilus. Interregnal axis (HPI).
Increase in the numbers of leukocyte (WBC) In our experiments with murrel, an increase in
increase after anesthetization showed the unexpected blood plasma glucose immediately after clove oil
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1600 Journal of Research in Biology (2014) 4(8): 1595-1603

Jeyasheela, 2014

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Journal of Research in Biology ISSN No: Print: 2231 6280; Online: 2231- 6299

An International Scientific Research Journal

Original Research

Genetic variations of Adiantum incisum Forssk. revealed by ISSR markers

in the Western Ghats of Tamil Nadu, India
Authors: ABSTRACT:
Journal of Research in Biology

Abiya Chelliah D1,

John De Britto A2 and Inter Simple Sequence Repeats (ISSR) markers were used to measure the
Selvin Samuel A1
levels of genetic variation and patterns of the population structure within and among
the five populations of Adiantum incisum, a terrestrial fern in India. For this purpose, a
detailed study was conducted in three replicates at 2011-14 season in the collection
Institution: points of Western Ghats, South India. Five wild A. incisum accessions (maiden hair)
1. Department of Botany, were evaluated for genotyping experiment. Results showed a significant variation
St. Johns College,
Palayamkottai - 627 002, among genotypes and were classified based on this variation in four groups by genetic
Tamil Nadu. cluster analysis. In the experiment, five ISSR primers amplified 63 polymorphic bands.
The genetic identity data among genotypes were calculated and varied from 0.4603 to
2. Department of Botany,
St. Xaviers College, 0.7460. The percentage of polymorphism showed superior genotype that could be
Palayamkottai - 627 002, used for the conservation of species. ISSR proved to be a helpful marker for genotype
Tamil Nadu. identification prediction within a closed group of inter specific population in the study
Corresponding author:
Abiya Chelliah D Keywords:
ISSR analysis, Adiantum incisum Forssk, genetic variation, southern Western Ghats

Email Id:
Article Citation:
Abiya Chelliah D, John De Britto A and Selvin Samuel A.
Web Address: Genetic variations of Adiantum incisum Forssk. revealed by ISSR markers in the Western
Ghats of Tamil Nadu, India
Journal of Research in Biology (2014) 4(8): 1604 1610
Received: 10 Oct 2014 Accepted: 15 Nov 2014 Published: 31 Dec 2014

This article is governed by the Creative Commons Attribution License (

licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

1604 1610 | JRB | 2014 | Vol 4 | No 8

Journal of Research in Biology
An International
Scientific Research Journal
Abiya et al., 2014

INTRODUCTION possessing these capabilities will have a selective

The Indian subcontinent is endowed with an
advantage over other genotypes when colonizing new
amazing array of herbal plants which constitute the main
and distant habitats. This advantage is becoming more
resource base of the health care system in the country.
important in a world increasingly under the pressure of
Its rich vegetation wealth and diversity is undoubtedly
climate change and fragmentation of natural habitats.
due to the immense variety of the climate and altitudinal
Various studies on plants and animals have
variations coupled with the various ecological habitats.
shown that individuals with higher dispersal capacities
The Western Ghats of peninsular India is of great
tend to be found with greater frequency towards species
phyto-geographical importance which constitutes one of
range limits and that these enhanced capacities tend to
the 34 global biodiversity hotspots along with Sri Lanka,
have a genetic basis. Likewise, inbreeding rates often
on account of exceptional levels of plant endemism and
increase towards range margins.
higher levels of habitat loss.
Genetic diversity measurements are important for
The flora of Western Ghats comprises about
considering conservation of particular species. A decline
12,000 species ranging from unicellular cyanobacteria to
in genetic variation can undermine the ability of an
angiosperms. In this spectrum, the flowering plants
organism to respond to natural selection and
constitute about 27% of Indian flora with 4000 species of
consequently limits its evolutionary potential. Small
which, about 1500 species are endemic. Apart from
populations are often subjected to the loss of alleles
harbouring a rich diversity of the angiosperm flora, the
through genetic drift, or random fluctuations in allele
Western Ghats are also a rich repository of cryptogams
frequency. Thus, any study in genetic diversity of
such as pteridophytes, bryophytes, lichens, fungi and
Adiantum incisum has to address the above issues.
algae. The Western Ghats along with the Himalayas,
DNA markers have proved valuable in crop
Eastern Ghats and parts of Central India forms a major
breeding, especially in studies on genetic diversity and
centre for the distribution of the ferns and fern-allies
gene mapping. The commonly used Polymerase Chain
(MoEF, 2014).
Reaction (PCR) based DNA marker system are Random
Ferns in Western Ghats of South India, south of
Amplified Polymorphic DNA (RAPD), Amplified
Palghat gap constitute about one third of the fern flora of
Fragment Length Polymorphic (AFLP) and more
India. Most of them occur on streams and stream banks
recently Simple Sequences Repeats (SSRs) or
in evergreen forests and shoals above 800m while some
microsatellite (Gupta and Varshney, 2000). The major
occur on exposed roadsides and clearings. (Manickam,
limitations of these methods are low reproducibility of
RAPD, high cost of AFLP and the need to know the
In spite of its relatively infrequent occurrence,
flanking sequences to develop species specific primers
long distance colonization is of disproportionate
for SSR polymorphism.
importance to species range expansions. Long-distance
ISSR is such a DNA based marker system which
colonization requires plant species to possess a distinct
could be used for screening genetic variability. Changes
set of capabilities, not only related to the dispersal of
in DNA sequences and single base substitutions
propagules, but also to plant and population
including DNA conformation changes can be detected as
establishment upon arrival. This involves di-spore
shifts in electrophoretic mobility using these techniques.
characteristics, plant ontogenetic and morphological
In this present study, A. incisum Forssk, were
traits, as well as reproductive strategies. Genotypes
collected from Western Ghats of Tamil Nadu. A. incisum
1605 Journal of Research in Biology (2014) 4(8): 1604 1610
Abiya et al., 2014

Forssk, is found in the plains and lower slopes of the Table 1. Place of collection of the plants and their accession ID
hills of Punjab, Rajasthan, West Bengal, Tamil Nadu and S.No Species Accession ID Location

Maharashtra. It is used to cure hemicrania, cough and 1. POP 1 Kothayar

fever; it is applied externally for skin diseases and used 2. POP 2 Gundar
Adiantum inci-
as a substitute for A. capillus-veneris. This fern yields 3. sum POP 3 Thirugurankudi
4. POP 4 Kodaikanal
adiantone, isoadiantone, fernene, hentriacontane,
5. POP 5 Kadana dam
hentriacontanone-16 and beta-sitosterol on extraction
using different solvents. The plant extract of Adiantum ISSR amplification
incisum Forssk. (Adiantaceae) is also used in the ISSR amplification reactions were carried out in
treatment of cough, diabetes, and skin diseases 25-l volume containing 50 ng template DNA, 0.5 U Taq
(Manickam, 1995). DNA polymerase, 10 mM dNTP, 10 M primer in 1
The confusion prevailing at its species level is a reaction buffer that contained 10 mM Tris-HCl (pH 8.3),
great menace to the Pteridologists who try to identify it 50 mM KCl, 2.5 mM MgCl2, and 0.01% gelatine
for its valuable usages. The morphology remains similar
Table 2. ISSR primers for study species and their sequences
with that of the closely related species except minute
S. No Name of the Primer Sequences
characteristics with its chromosomal nature and primer
chemistry stands unique. Thus, designing a specific 1 B07 (CG)5AG
strategy for its species identification in spite its variation 2 B09 (ATG)3CA
3 G06 (GC)5CA
stands as a credential task irrefutably. Also, the superior
4 G04 (AT)6GC
genotype of the species was identified so that the 5 L03 (GC)4AT
conservation of the species made easy with special
initiative. (Williams et al., 1990). Amplifications were performed
in an Eppendorf Master Cycler gradient. Amplification
conditions were one cycle at 94C for 4 min, and 94C
Study area
for 30 s, 55C for 45 s, followed by stepwise reduction of
Five Western Ghats regions viz., Kothayar,
1C for the first five cycles, and 72C for 2 min. In
Gundar, Thirugarankudi, Kodaikanal and Kadana Dam
subsequent 35 cycles, annealing temperature was
are selected for the study due to the availability of these
maintained at 50C, followed by one cycle of 7 min at
three species uniformly with no particular order.
72C. Amplified products were loaded on 2% agarose
DNA isolation
Table 3. Overall genetic variation statistics for all loci in
A. incisum samples were collected from the
Adiantum incisum
Western Ghats of Tamil Nadu, India. DNA was extracted
S. No. Parameters Values
from young leaves using the method described by
Dellaporta et al. (1983). The DNA isolated was purified 1. Observed numbers of 1.7778
using Phenol- Chloroform method and the concentration
2. Effective numbers of alleles 1.5887
of the DNA samples were determined using UV-
3. Neis (1973) gene diversity 0.3276
Spectrophotometer at the optical density of 260 nm and
4. Shannons Information Index 0.4741
280 nm; the DNA samples were diluted to 25 ng l1 for (Lewontin, 1972)
PCR amplification. 5. Overall percentage of polymorphism 77.78

Journal of Research in Biology (2014) 4(8): 1604 1610 1606

Abiya et al,, 2014

gel and separated in 1 TBE buffer at 75 V. The gels reproducible, informative and easily scorable ISSR
were visualized under UV after staining with ethidium profiles. A total of 315 bands were scored, out of which
bromide and documented using a gel documentation and 180 were polymorphic bands and the number of bands
Table 4. ISSR Profile of A. incisum using selected primers ranged from 09 to 20 per primer (Table 4). The genetic
distance between the population ranged from 0.2930 to
S. ISSR Total Total Percentage
No. Primers Number Number of of 0.7758 and the genetic identity ranged from 0.4603 to
of Bands Polymorphic Polymor-
Bands phism (%) Table 6. Nei's original measures of genetic identity and
1 B07 63 20 31.74 genetic distance in Adiantum incisum
2 B09 63 09 14.28 POP 1 2 3 4 5
3 G04 63 12 19.04 ID
4 G06 63 10 15.87 1 ***** 0.5873 0.6190 0.4921 0.6190
5 L03 63 12 19.04
2 0.5322 ***** 0.4603 0.6190 0.4921
Total 99.97
3 0.4796 0.7758 ***** 0.6190 0.7460
4 0.7091 0.4796 0.4796 ***** 0.6508
image analysis system. The primers used for the ISSR
5 0.4796 0.7091 0.2930 0.4296 *****
analysis are listed out in Table 2.
Data Analysis 0.7460 (Table 06). The overall observed and effective
The gels from ISSR analysis were visualized at
numbers of alleles were 1.7778 and 1.5887 respectively
Table 5. Distance between and population length in and overall genetic diversity was 0.3276 (Table 3; Fig 2).
Adiantum incisum Forssk. The Shannans information index was found to be
Between And Length 0.4741. The overall percentage of polymorphism was
4 3 3.66294 77.78 (Table 3; Table 5).
3 pop1 26.61084 The number of polymorphic loci and percentage
3 pop2 26.61084 of polymorphism was calculated by using the software
4 2 7.54538 POPGENE package version 1.3.2. Among these five

2 1 8.07904 populations, populations 1, 4, and 5 (Kothayar,

Kodaikanal and Kadana Dam) showed high
1 pop3 14.64936
polymorphism. Considering these three populations,
1 pop5 14.64936
population 1 (Kothayar) showed highest polymorphism
2 pop4 22.72839
(Fig 1).
Hence, among the five accessions of A. incisum
gel documentation system (Alpha Imager 1200). Based
in the Kothayar accession (Pop 1) is considered as
on the primary data (presence or absence of bands) and
superior genotype, due to the high percentage of
pair wise genetic distance between samples was
polymorphism in ISSR analysis.
calculated using NTSYS and POPGENE packages Figure 1. UPGMA dendrogram of A. incisum based on Neis
Genetic distance derived using ISSR markers


Analysis of five accessions of A. incisum Forssk.
revealed 63 polymorphic loci. Ten primers were
analyzed of which five primers (Table 2). generated

1607 Journal of Research in Biology (2014) 4(8): 1604 1610

Abiya et al., 2014

Dendrogram was drawn based on Nei's (1972) the predominant source of genetic variation than asexual
with the consideration of genetic distance. The reproduction (Mes, 1998; Van Der Hulst, 2000; Kjolner
et al., 2006). This finding is in good agreement with
Fig 2. Summary of genetic variation statistics for all loci in
Adiantum incisum other previous studies, which have nearly all used
Summary of genic variation statistics for all loci allozyme markers to infer the mating systems operating
L03-11 B07-2
in natural populations of ferns.
L03-10 B07-4
L03-9 B07-5
L03-8 B07-6
L03-7 B07-7
L03-6 B07-8

Allozyme studies showed that populations of

L03-5 B07-9
L03-4 B07-10
L03-3 B07-11
L03-2 B07-12
diploid homosporous ferns are usually dominated by
G06-9 B07-15
G06-8 B07-16 Gene Diversity sexual random mating or outcrossing (Soltis and Soltis,
G06-7 B07-17
Mean Natural Selection Coefficient
1987; Ranker and Geiger, 2008). Secondly, gene flow
G06-4 B07-20
from the neighboring populations would slow the
G06-1 B09-3
B09-5 diversification but only via sexual reproduction. Similar
G04-10 B09-6
G04-9 B09-7

patterns have been identified in a number of other fern

G04-8 B09-8
G04-7 B09-9
G04-5 G04-1
G04-4 G04-3

methodology followed is UPGMA based on neighbour species (Ranker, 1992; Ranker and Geiger, 2008)
joining method of PHYLIP Version 3.5. Rumsey, 1999; Chen et al., 2010).
The dendrogram of A. incisum Forssk. (Fig 1) The reason for this genetic variation is most
produced 2 clusters. Cluster 2 was larger, containing likely arisen from differences in the DNA contents of the
population 3, 5 and 4. Here, population 3 and 5 were progenitor species. This suggestion is consistent with the
closely related together than population 4. In the first occurrence of diploid apomicts in other ferns, including
cluster, population 1 and 2 formed a separate clade. It taxa of the Adiantum incisum (Pravin, 2005). The clades
was understood that there was considerable amount of are constructed through Popgene 2.1 and related lineages
genetic variability between the populations 1, 2 and 3, 4, show strong patterns of reticulate evolution and higher
5 of A. incisum Forssk. the genetic diversity of these species, greater is their
The morphological variations were counter viability in the environment.
confirmed by the genetic variations present in the plants However, when evolutionary relationships were
through ISSR markers. Specific primers that mediate considered using Phylogenetically Independent Contrasts
differentiation between the species were identified (PICs), no significant correlation was found. The
successfully. The genetic relationship exemplified by the discrepancy between analyses is interesting, and
molecular markers via DNA fingerprinting shows their although the significance of the raw data should not be
closeness and relativity. Out of ten primers, five revealed discounted, it does highlight the importance of using
consistent banding pattern and thus revealed variability PICs to determine the evolutionary association of
within the species. statistically non-independent traits (Garland, 1992).
The identification of a clear systematic Previous authors have pointed out that this
relationship thus paves a way for better understanding of inference should be restricted to very close relatives, and
the species in their position. the distinction between diploids and their autoploid
ISSR markers were used to compare genetic offspring (Moran, 1982; Barrington, 1986).
differentiation within and between the selected species. Our findings are consistent with suggestions that
Analysis of genetic variability indicated all the five the high chromosome numbers and conserved
populations examined, sexual recombination had been chromosome sizes reported for many homosporous ferns

Journal of Research in Biology (2014) 4(8): 1604 1610 1608

Abiya et al., 2014

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