Oxidation Communications 37, No 2, 533542 (2014)

Antioxidants in biological systems

ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF Leontice

eversmannii ROOTS
Q. U. REHMANa, I. H. BUKHARIa*, N. RASOOLa, T. H. BOKHARIa,
N. ASLAMa, M. RIAZb, M. KHURRAM SHAHZAD QURESHIa,
R. B. TAREENc
a

Department of Chemistry, Government College University, 38 000 Faisalabad,

Pakistan
E-mail: pdiftikhar@yahoo.com
b
Department of Chemistry, University of Sargodha, Women Campus,
38 000 Faisalabad, Pakistan
c
Department of Botany University of Baluchistan, Quetta, Baluchistan, Pakistan
ABSTRACT
Nature is a very important source of medicinal agents from the early times of human
races. Plants do not only show medicinal properties but also contain secondary metabolites which are responsible for the medicinal characteristics of plants. Extracts of
plants are used to cure infectious diseases from the ancient times. Extracts of plants
do not show adverse effects as compared to the synthetic drugs. Keeping in view the
medicinal importance of plants the present research project was designed to investigate
biological activities of Leontice eversmannii roots by using different techniques. As a
therapeutic agent there is limited information published on the biological activities of
this plant. Antifungal and antibacterial activities were also studied and these studies
revealed that Leontice eversmannii roots exhibited excellent antimicrobial potential.
Antioxidant activities were also examined by using different latest reported method
in literature. Total avonoid contents, total phenolic contents and DPPH free radical
scavenging were also determined. Overall research revealed that Leontice eversmannii
can be used for medicinal purposes.
Keywords: Leontice eversmannii, antioxidant and antimicrobial activities.
AIMS AND BACKGROUND
The use of plants as medications belonged to early man. Ancient Chinese, Indians,
and North Africans civilisations provided written evidence of man knowledge in
consuming plants for the treatment of various infectious and dangerous diseases. In
*

For correspondence.

533

ancient times of Greece, for example, some scholars classied plants in a denite
order and gave detailed explanations of medicinal plants, thus made the identication
process of plants effective. Theophrastus has been considered as the father of botany
by some scholars and botanists. He explained the characteristics of medicinal plants
and forecasted the probability of discovering avonoids in plants1. In some countries
local herbs and shrubs are used in herbal medicines to get rid of diseases and also for
the prevention from diseases24.
Leontice eversmannii belongs to the Berberidaceae family. This family comprises
of about 14 genera which contains herbs and shrubs. Majority of members of Berberidaceae family are of medicinal values having therapeutic applications and are used
for the cure of infectious diseases. Plants of this family are used for the treatment of
rheumatism, dropsy, colic, sore throat, hepatitis, cramps, epilepsy, hysterics and for the
inammation of uterus. Some plants of this family also have antispasmodic, diuretic
and diaphoretic, parturifacient and expectorant effects5,6. It has been reported that genus
Leontice has numerous important medicinal values. In the herbal or folk medicines
of Turkey, tubers of these plants are used effectively for the treatment of epilepsy
and brain diseases, rheumatism and joint pain710. Anti-inammatory, analgesic and
sedative effects were manifested when these extracts were applied to the biological
systems. A number of compounds of medicinal importance along with the alkaloid
and avones have been isolated from these plants11,12. Tubers of genus Leontice are
used for the treatment of tuberculosis, eczema and diseases of cardiovascular system
effectively13,14. As per literature survey no reports have been available regarding
antioxidant, antimicrobial activity of Leontice eversmannii extract and fractions. So
antioxidant and antimicrobial activities of Leontice eversmannii roots were determined
by using different parameters and techniques.
EXPERIMENTAL
The plant for the research project was selected on the foundations of intensive review
and ethnopharmacological survey. The Leontice eversmannii roots were selected for
research project. The plant was collected from the Quetta region and recognised by
Dr. Rasool Bakhsh Tareen, from the Department of Botany, University of Balochistan,
Quetta, Balochistan, Pakistan.
Sample preparation. To remove dust specks and other superuous matter the roots of
Leontice eversmannii were washed with cold water. The roots were dried under the
shade and ground into a ne powder form. This powdered form sample was soaked
in 100% methanol for 7 days at room temperature. The plant material was extracted
3 times. The extract obtained as a result of this procedure was concentrated with the
help of rotary evaporator and was regarded as methanol extract. By using solvent
extraction method, different fractions were made from methanol extract with the help
of separating funnel with different solvents like n-hexane, chloroform, ethyl acetate
and n-butanol on the polarity bases. Extracts were ltered, evaporated, concentrated
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and freed of solvent under reduced pressure using a vacuum rotary evaporator at 50C
approximately. The whole process was repeated 3 times to obtain ample amounts of
extracts. The semi-dried, crude, concentrated extracts were weighed to evaluate the
yield and stored at 4C till analysis14.
Phytochemical screening. In order to determine phytochemical constituents of Leontice
eversmannii roots different chemical tests were performed on powdered plant material
and dried extracts by following already reported procedures15.
Evaluation of antioxidant activity. The evaluation of antioxidant activity of Leontice
eversmannii roots was done by using the following antioxidant assays:
determination of total phenolic contents (TPC). The total phenolic contents of
different fractions of Leontice eversmannii roots extract were determined by applying
reported method16;
determination of total avonoid content (TFC). The total phenolic contents of
different fractions of Leontice eversmannii roots extract were determined by applying
reported method in Ref. 16;
determination of free DPPH radical scavenging assay. The free 1,1-diphenyl2-picrylhydrazyl radical (DPPH) scavenging of different fractions of Leontice eversmannii roots extract was determined by applying reported method17.
Antimicrobial assay by zone of inhibition and minimum inhibitory concentration
(MIC). The antimicrobial activities of the extract and all factions of Leontice eversmannii roots were tested against some microorganisms, comprising 4 strains of
bacteria and three strains of fungi. Streptococcus, Bacillus subtilis, Escherichia coli
and Staphylococcus were bacterial strains while strains of fungus were E. candida,
Aspergillus avus and Aspergillus niger. Determination of the antimicrobial activity
with reference to the minimum inhibitory concentration (MIC) of the extract and
fractions was also made by using the above-mentioned infective strains according to
the method described in Ref. 18.
RESULTS AND DISCUSSION
Phytochemical analysis. Phytochemical analysis of the Leontice eversmannii roots
indicated the presence of alkaloids, avonoids, glycosides, tannins, whereas phlobatannins and steroids were absent. Previous investigations of Leontice eversmannii showed
the presence of alkaloids and saponins in it and they were isolated and characterised
by using column chromatography and different spectroscopic techniques12. Lupanin
and taspine along with many types of saponins have been isolated from the plants of
the family Berberidaceae. In recent years different types of triterpene saponins isolated
from the tubers of Leontice smirnowii a member of Berberidaceae family19. After
performing different types of qualitative tests for the determination of phytochemicals,
it was revealed that alkaloids were almost present in fractions/extracts of Leontice
eversmannii roots, but saponins were present only in the methanol, aqueous and ethyl
535

acetate extracts. Tannins were found in the extracts of methanol and residue only. It was
observed that falvonoids, tannins, and saponins were present in highly polar solvents
as compared to the other solvents20. Flavonoids were present in all extracts except in
n-hexane. Results of different experiments revealed the presence of phytochemical
constituents in the roots of Leontice eversmannii and results are shown in Table 1.
Table 1. Phytochemical analysis of various extracts of Leontice eversmannii roots

Sample name
Methanol
n-Hexane
Chloroform
Ethyl acetate
n-Butanol
Aqueous

Phloba- Terpe- Saponins Glyco- Steroids Fla- Tannins

tannins noids
sides
vonoids

+
+
+

+
+

+
+

+
+
+

+
+

Alkaloids
+

+
+
+
+

Note: absence () and presence (+).

Percentage yield of extracts. The percentage yield of different extracts and fractions
of Leontice eversmannii roots was found in the range of 1.1221.45 g/100 g of ne
dry powder. The maximum yield was found by absolute methanol extract which is
21.45 g; it was a good yield but minimum yield was given by chloroform extract
1.12 g. The percentage yield of different extract of fractions of Leontice eversmannii
roots are shown in the decreasing order as follows: methanol > aqueous > n-hexane >
n-butanol > ethyl acetate > chloroform. The results of average percentage of triplicate
are shown in Table 2.
Table 2. Percentage yield, total phenolic, avonoid contents, free radical scavenging activity and %
inhibition in linoleic acid system of Leontice eversmannii roots

Sample
Methanol
n-Butanol
Chloroform
Ethyl acetate
n-Hexane
Aqueous
BHT

% age Yield
(g/100 g)
21.470.65
3.010.08
2.220.07
1.520.04
1.000.01
12.000.08

TFC (mg/100 g) TPC (mg/100 g)

107.201.01
33.210.06
31.530.02
8.260.09
2.640.01
43.670.65

115.930.45
16.700.09
13.720.26
32.030.04
11.350.01
45.820.08

IC50 (g/ml)
9.840.02
18.250.03
10.930.01
13.980.01
14.430.03
12.100.02
8.910.01

Evaluation of antioxidant activity. It was reported earlier that extracts of genus Leontice has antioxidant potential along with important phenolic contents21,22. To evaluate
antioxidant potential of Leontice eversmannii roots different kinds of experiments
were performed and were elaborated as follows:

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 total phenolic contents (TPC). Total phenolic contents shown by the extracts and
fractions of Leontice eversmannii roots were in the range of 11.35115.93 mg/100 g
of all extracts. The TPC values shown by the plant for different extracts of fractions
were as follows: methanol (115.93), n-hexane (11.35), chloroform (13.72), ethyl
acetate (32.03), n-butanol (16.70) and aqueous (45.82). The order of TPC values of
different fractions can be shown as given below:
methanol > aqueous > ethyl acetate > n-butanol > chloroform > n-hexane

The maximum value of total phenolics contents was shown by methanol extract
as it has maximum values out of all extracts of fractions whereas aqueous, n-butanol
and ethyl acetate showed signicant phenolic contents as compared to the methanol
extracts. The chloroform and n-hexane extracts showed minimum phenolic contents
whereas n-hexane fraction was found in the lower limit. The average results of TPC
values as triplicate are shown in Table 2;
total avonoid contents (TFC). The different extracts and fractions of Leontice eversmannii roots showed total avonoid contents values were in the range of
2.64107.2 mg/100 g of all extract. The TPC values shown by the plant for different
extracts of fractions were as follows: methanol (107.2), n-hexane (2.64), chloroform
(31.53), ethyl acetate (8.26), n-butanol (33.21) and aqueous (43.67). The order of
TFC values of different fractions: methanol > aqueous > n-butanol > chloroform >
ethyl acetate > n-hexane.
The maximum value of total avonoid contents was shown by methanol extract
as it has maximum values out of all extracts of fractions whereas aqueous, n-butanol
and chloroform showed signicant avonoid contents as compared to the methanolic
extracts. The ethyl acetate and n-hexane extracts showed minimum avonoid contents
whereas n-hexane fraction was found in the lower limit. The average results of TFC
values as triplicate are shown in Table 2.
DPPH free radical scavenging. The IC50 assay was applied on the extracts and fractions of Leontice eversmannii roots, to check the DPPH free radical scavenging and
its range was from 9.84 to 18.25%. A methanol extract and aqueous extract of root
samples exhibited maximum free radical scavenging activity and so their values of IC50
were lower when compared to all other extracts of fractions of Leontice eversmannii
roots. It was assessed that the range of IC50 value for roots were for n-hexane fraction showed the highest value (18.25), followed by chloroform (10.93%), aqueous
(12.10%), ethyl acetate (13.98%), n-butanol (14.43%), and absolute methanol (9.84%)
fractions. The order of DPPH free radical scavenging, according to the activities of
extracts, was arranged as follows:
n-hexane > n-butanol > ethyl acetate > aqueous > chloroform > methanol.

The IC50 values of extracts and fractions of Leontice eversmannii roots results
are shown in Table 2.

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Antimicrobial activity of Leontice eversmannii roots. Some other studies have also
shown the use of natural sources for antimicrobial studies2325. Hence the extracts of
fractions of Leontice eversmannii roots were used to determine the antibacterial and
antifungal activities by using different strains of bacteria and fungi. The results, thus
obtained were discussed below.
Antibacterial activity of Leontice eversmannii roots. Antibacterial activity of different
extracts of Leontice eversmannii roots was performed against 4 different bacterial
strains with the help of samples of different extracts of fraction by using antibacterial
assays. Hence the results obtained are shown in tabular form against each bacterial
strain. The inhibition zone was noted in the millimetre scale. In all the extracts of
fractions of Leontice eversmannii roots, excellent antibacterial activity against E. coli,
B. cerus, Streptococcus and Staphylococcus was shown by the sample containing
methanol extract. All the other remaining fractions also show better antibacterial activities but after methanol; aqueous extract of fraction shows good antibacterial activities against E. coli, B. cerus, Streptococcus and Staphylococcus. Samples comprising
extracts/fraction of chloroform and ethyl acetate show overall minimum antibacterial
activities against the above-mentioned 4 bacterial strains. Sample of n-hexane fraction
also showed signicant antibacterial activities against E. coli, B. cerus, Streptococcus
and Staphylococcus. The order of antibacterial potential of extracts of different fractions against pathogenic bacterial strains can be arranged as follows:
methanol > aqueous > n-butanol > n-hexane > ethyl acetate > chloroform.

The overall excellent antibacterial activities were shown against B. cerus by all the
extracts/fractions of Leontice eversmannii roots and were for methanol (20.670.58),
n-butanol (20.330.57), ethyl acetate (13.170.29), chloroform (12.330.29), n-hexane
(15.170.76) and aqueous (17.330.57). The results as the mean of triplicate experiments are shown in Table 3.
Table 3. Antibacterial activity of Leontice eversmannii roots

Samples of
extracts
Methanol
n-Butanol
Ethyl acetate
Chloroform
n-Hexane
Aqueous
Rifampicin

E. coli
15.170.29
10.670.57
11.100.17
11.170.29
13.330.57
14.500.50
25.670.57

Zones of inhibition of bacterial strains (mm)

B. cerus
Streptococcus
Staphylococcus
20.670.58
18.170.28
20.500.50
20.330.57
17.330.57
11.830.76
13.170.29
15.330.57
11.330.57
12.330.29
12.170.28
12.500.50
15.170.76
11.500.50
10.330.57
17.330.57
12.160.76
15.330.57
25.670.57
25.670.57
25.670.57

Antifungal activity of Leontice eversmannii roots. Antifungal activity of different extracts of Leontice eversmannii roots was performed against 3 different fungal strains
with the help of samples of different extracts of fraction by using antifungal assays.
538

Hence the results obtained are shown in tabular form against each bacterial strain.
The inhibition zone was noted in the millimetre scale. In all the extracts of fractions
of Leontice eversmannii roots, excellent antifungal activity against A. niger, A. avus
and E. candida was shown by the sample containing extract of n-butanol fractions.
After the n-butanol, extracts of methanol and ethyl acetate showed good antifungal
activities against the above-mentioned strains of pathogenic funguses. However, the
sample comprising extracts of the n-hexane and aqueous fractions could not show
any inhibition of the growth of E. candida fungus. Samples of extracts/fractions of
chloroform also show signicant inhibition of fungal growth in the culture media
against the all 3 strains of fungi. The order of antibacterial potential of extracts of
different fractions against pathogenic bacterial strains can be arranged as:
n-butanol > methanol > ethyl acetate > n-hexane > aqueous > chloroform.

The overall excellent antifungal activities were shown against A. avus by all
the extracts/fractions of Leontice eversmannii roots for methanol (18.50.50), nbutanol (20.830.76), ethyl acetate (17.50.50), chloroform (13.670.58), n-hexane
(16.330.58) and aqueous (15.330.58). The results as the mean of triplicate experiments are shown in Table 4.
Table 4. Antifungal activity of Leontice eversmannii roots

Samples of extracts
Methanol
n-Butanol
Ethyl acetate
Chloroform
n-Hexane
Aqueous
Fungone

Zones of inhibition of fungal strains (mm)

A. niger
A. avus
E. candida
15.170.58
18.500.50
16.700.57
21.160.29
20.830.76
15.170.73
11.670.57
17.500.50
13.830.29
16.170.76
13.670.58
10.330.57
13.330.29
16.330.58

15.330.57
15.330.58

26.040.58
26.040.58
26.040.58

Minimum inhibitory concentration (MIC) against fungi. MIC values were opposite
to the antimicrobial values. During the antifungal assay n-butanol extract showed
the maximum antifungal activity against A. niger as 21.160.29 and its minimum
inhibitory concentration was observed as 1.730.01 which described that the assay
of n-butanol extract of Leontice eversmannii roots could only inhibit the growth of
fungus at this concentration. Same pattern was observed in methanol as it showed
maximum inhibition of A. avus and its inhibition zone against this strain was at
18.50.50 and its observed MIC value was 2.100.01 which was its minimum value
of inhibition. In case of chloroform fraction, maximum antifungal activity was shown
at 16.170.76 against A. niger but its MIC value was 1.800.02 which showed that
it inhibited the growth of fungus at this concentration only. It was observed that in
ethyl acetate fraction maximum antifungal activity was observed at 17.50.50 against
A. avus, its MIC was 1.450.01 which exhibited that it could inhibit the growth of
539

fungus at this concentration. The results as the mean of triplicate experiments are
shown in Table 5.
Table 5. MIC of antifungal activity of Leontice eversmannii roots

Samples of extracts
A. niger
1.500.01
1.730.01
1.760.01
1.800.02
1.900.01
1.750.02
0.810.01

Methanol
n-Butanol
Ethyl acetate
Chloroform
n-Hexane
Aqueous
Fungone

Fungal strains
A. avus
2.100.01
1.360.01
1.45 0.01
1.600.02
1.920.01
1.610.01
0.780.01

E. candida
1.900.01
1.670.01
1.980.01
1.740.01

0.890.01

Minimum inhibitory concentration (MIC) against bacteria. Bacterial strains were

also subjected for the determination of minimum inhibitory concentration. MIC was
determined against 4 pathogenic bacterial strains E. coli, B. cerus, Streptococcus and
Staphylococcus. During the antibacterial assay extract of methanol showed the maximum antibacterial activity against B. cerus and was observed as 20.670.58 and its
minimum inhibitory concentration was observed as 1.900.01 which described that
the methanol extract of Leontice eversmannii roots could only inhibit the growth of
bacteria at this concentration. Same pattern was observed in n-butanol as it showed
maximum inhibition against B. cerus and its inhibition zone against this strain was
at 20.330.57 and its observed MIC value was 1.760.01 which was its minimum
value for inhibition. In case of aqueous extract/fraction maximum antibacterial activity was exhibited at 17.330.57 against B. cerus but its MIC value was 1.950.01
which showed that it inhibited the growth of bacteria at this concentration only. It
was observed that in n-hexane fraction maximum antibacterial activity was observed
at 15.170.76 against B. cerus and its MIC was 1.720.01 which exhibited that it
could inhibit the growth of bacteria at this concentration. The results as the mean of
triplicate experiments were shown in Table 6.
Table 6. MIC of antibacterial activity of Leontice eversmannii roots

Samples of
extracts
Methanol
n-Butanol
Ethyl acetate
Chloroform
n-Hexane
Aqueous
Ciprooxacin

540

E. coli
1.720.01
1.980.01
1.870.01
1.890.01
2.000.02
1.780.01
1.020.01

Bacterial strains
B. cerus
Streptococcus
1.900.01
1.610.01
1.760.01
1.680.01
1.960.01
1.550.01
1.740.02
1.650.01
1.720.01
2.010.01
1.950.01
1.880.01
0.760.01
0.860.10

Staphylococcus
1.730.01
1.910.01
1.900.01
1.710..02
1.680.02
2.050.57
0.900.01

CONCLUSIONS
The research was focused on study of the antioxidant and antimicrobial activities
of the Leontice eversmannii roots. The results of the total phenolic contents, total
avonoid contents, DPPH free radical scavenging assay and percentage inhibition in
linoleic acid system showed that the plant has active antioxidant components in its
chemical composition. Phytochemical analysis revealed that alkaloids, tannins, avonoids, terpenoids and saponins are also present in it. It is concluded that Leontice
eversmannii roots have very active component and showed an excellent antioxidant,
antibacterial, antifungal activities. In short, the plant has many bioactive and secondary
metabolites which can be used for the synthesis of drugs against the diseases provoked
by pathogens and related to some oxidants.
ACKNOWLEDGEMENTS
We are highly thankful to Dr. Rasool Bakhsh Tareen, Department of Botany, University of Balochistan, Quetta, Balochistan, Pakistan, for suggesting and recognising
the plant.
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Received 7 November 2013
Revised 29 December 2013

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