j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 5 3 e6 0

Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/JOPR

Original Article

Validated RP-HPLC method to estimate eugenol

from commercial formulations like Caturjata
Churna, Lavangadi Vati, Jatiphaladi Churna, Sitopaladi
Churna and clove oil
Sagar Saran a,*, Sasikumar Menon a, Sunita Shailajan b, Priyanka Pokharna a
a
b

Institute for Advanced Training and Research in Interdisciplinary Sciences, Sion Koliwada, Sion (E), Mumbai 400022, India
Herbal Research Lab, Ramnarain Ruia College, Matunga (East), Mumbai 400019, India

article info

abstract

Article history:

Eugenol is an important phytochemical bioactive compound of ayurvedic and other mar-

Received 11 August 2012

keted herbal formulations. It shows anti-inflammatory, anti-bacterial and anti-tubercular

Accepted 10 November 2012

activity. Thus, it is a suitable bioactive biomarker to establish the quality of commercial

drug and its formulation. The aim is to develop and validate an efficient and effective RP-

Keywords:

HPLC method for quantification of eugenol from commercial marketed formulations.

Caturjata Churna

Separation was carried out on a cosmosil C18 column, mobile phase composition was

Lavangadi Vati

methanol: distilled water (60:40, v/v) at flow rate of 1 mL/min. Detection was carried out at

Jatiphaladi Churna

215 nm using a photodiode array detector (PDA). Commercial Ayurvedic formulations such

Sitopaladi Churna

as Caturjata Churna, Lavangadi Vati, Jatiphaladi Churna, Sitopaladi Churna and clove oil were

Clove oil

further subjected to RP-HPLC for estimation of eugenol. The RP-HPLC method was validated as per ICH guidelines. The LOD and LOQ level were found to be 25.00 ng/mL and
50.00 ng/mL, respectively. Detector response was found to be linear from 50.00 ng/mL to
50,000.00 ng/mL. The method was found to be simple, accurate, reproducible and rugged.
This method was used to estimate the content of eugenol in commercial formulations and
the method is found suitable for quality assurance and marker based standardization of
ayurvedic formulations containing eugenol.
Copyright 2012, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights
reserved.

1.

Introduction

Medicinal plants have been used since thousands of years

from the beginning of human civilization for its therapeutic
properties, containing inherent active ingredients that has
properties to heal sores, relieve pain, cure diseases1 and
maintenance of overall good health.2 Medicinal properties of

plants provide ample opportunity for development and

obtaining a wide variety of drugs. Therefore should be investigated further to better understand their safety and efficacy
(Figs. 1e4).3
The property of herbal medicine is highly dependent upon
the composition of chemical phytoconstituents in their
extracted final product. There is current need for development

* Corresponding author.
E-mail address: sagar.saran@gmail.com (S. Saran).
0974-6943/$ e see front matter Copyright 2012, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jopr.2012.11.013

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Fig. 1 e Molecular structure of eugenol.

of method for establishing quality control parameters for

ayurvedic formulations owing to variability and complexity of
chemical constituents present in herbal plant based drugs.4
Identification, isolation, purification and characterization of
active ingredients in crude extracts from herbal plants is now
possible relatively easily because of development and implementation of high resolution separating analytical techniques
like RP-HPLC.5,6 Among these bioactive compounds, there has
been current explosion of interest in areas of distilled essential oils from fresh leaves, roots, stems and root sources of
plant parts. These essential oils of plant contain phytochemicals. Among these phytochemicals, the major essential oil
eugenol, a phenolic compound (L-hydroxy-2-methoxy-4allylbenzene) is widely distributed.7 Eugenol can be predominantly extracted from various species and families of
aromatic plants and comprise about 70e85% in many essential oils (Fig. 1).8
Several studies have reported pharmacological mode of
action of eugenol from medicinal plants such as Ocimum
sanctum (leaf), Anethum sowa Roxb (leaf), Pimpinella anisum
Linn. (leaf), Alpinia galanga wild (rhizome), Salvadora persica
Linn. (leaf) and Vetiveria zizanioides (root) in experimental
animal systems9e14 as hepatoprotective agent, vasorelaxing
action,15 as an attractant to fruit fly,16 membrane stabilizing
properties useful in the treatment of neurological, allergic
disorders, anti-tubercular activity,7 and has antinociceptive
potential to be used as dental analgesic.10
Various methods such as HPLC mass spectrophotometry
using offline dansyl chloride derivatization has been carried
for detection of lower limit of eugenol.17,18 Additionally,
HPLCeUV method has been successfully used for determination of eugenol in Syzygium aromaticum Linn (Clove) and

Cinnamomum zeylanicum (cinnamon oils) by using NDBD-F as

a labelling reagent.19 However, these systems are relatively
costly and are increasingly complicated. The use of costly
polymer based columns and absence of organic phase have
contributed to difficulties in developing viable and cheaper
RP-HPLC analysis. Although there are many chromatographic
methods currently utilized for quantification of eugenol from
various fruits, vegetables, leaves etc but virtually not much
work has been validated and used for estimation and quantification of eugenol from commercial formulations. Hence,
an alternative method needs to be developed for determination of such essential oil which is simpler, reliable and offers
results in shorter span of time.
Present study aims in development of reliable, cost effective and validated analytical method for separation and
quantification of eugenol from commercial formulation of
Caturjata Churna, Lavangadi Vati, Jatiphaladi Churna, Sitopaladi
Churna and clove oil like commonly eugenol containing
formulation by HPLC using photodiode array detector. The
estimation of eugenol from ayurvedic formulations can be
useful for quality control analysis and for safety purposes, due
to possible toxic effects.

2.

Materials and methods

2.1.

Chemicals and reagents

Eugenol (99%, C10H12O2) was obtained from Aldrich, USA.

Commercial Ayurvedic formulations (plants) like Caturjata (tvak,
ela, patra, kesera),20 Sitopaladi Churna (Cinnamomum verum zeylanicum-pippali, ela, tvak),20 Lavangadi Vati (lavanga, marica, aksaphala, khadirasara, babbula),20 Jatiphaladi Churna (Cannabis sativajatiphala, lavanga, ela, patra, tvak, nagakesara, candana, tila, tvaksiri, tagara, amla, talisa, pippali, pathya, sthulajiraka),20 and clove
oil (Syzygium aromaticum)20 containing eugenol were purchased
from local markets. HPLC grade methanol was procured from
Merck Specialist Private Limited (Mumbai, India). Distilled water
was prepared in-house using Millipore (Millipore S.A. Molsheim,
France). All other chemicals used were of analytical grade.

Fig. 2 e Representative HPLC chromatogram of (A) standard sample 10 ppm and (B) Caturjata Churna.

j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 5 3 e6 0

2.2.

Preparation of standard solution

A stock solution of 1000 ppm was prepared by accurately

weighing 10 mg of eugenol standard in a 10 mL volumetric
flask and it was further diluted with HPLC grade methanol up
to the mark. The solution was vortexed for 10 s.

2.3.

Preparation of sample solution

1 g of ayurvedic formulations were taken in 10 mL of methnol

and then solvent extraction was performed using a rotary
shaker for 24 h. The tubes were centrifuged at 4000 rpm for
10 min and the solution was filtered with Whatman filter
paper no. 41. The filtrate was collected in polypropylene tubes
and stored at 4
C until further analysis. Furthermore, the
filtrate was given appropriate dilution in mobile phase prior to
injection on to the HPLC system.

2.4.

Chromatographic conditions

The HPLC system used for quantification of eugenol consisted of

a Jasco PU-980 pump, AS-2057 auto sampler and Jasco UV-970
detector. The chromatogram peaks were quantified by means
of PC based Borwin software (Version 1.5). Chromatography
separation for analyte was achieved on cosmosil C18 analytical
column (150 mm  4.6 mm, 5 m) maintained at ambient
temperature. The mobile phase was pumped at a flow rate of
1 mL/min. The mobile phase was filtered through a 0.45 m nylon
membrane filter and degassed in an ultrasonic bath prior to use.
The injection volume was 30 mL, the flow rate was 1.0 mL/min
and a chromatographic peak was detected at 215 nm.

3.

Method validation

The entire experimental analysis was according to the ICH

guidelines and was validated for calibration curve, limit of
detection, limit of quantification, system suitability, precision,
accuracy, solution stability and ruggedness.21 Marketed
commercial formulation samples of Caturjata Churna, Lavangadi Vati, Sitopaladi Churna, Jatiphaladi Churna and clove oil

55

were accurately weighed in weighing balance. Later they were

transferred to Tarson tubes, filled with methanol and kept
overnight on rotator shaker. These tubes were subsequently
centrifuged, filtered and stored in fridge for further HPLC
analysis.

3.1.
(LOQ)

Limit of detection (LOD) and limit of quantification

The limit of detection is defined as the smallest concentration

that can be detected but not necessarily quantified as an exact
value whereas the limit of quantification is termed as the
lowest amount of analyte in the sample that can be quantitatively determined with precision and accuracy that provided
a peak area with signal to noise ratio higher than 10, with
precision (% CV) and accuracy with () 10%.

3.2.

Calibration curve and linearity

Linearity was determined by means of calibration graph. The

graph is further analyzed by using an increasing amount of
each analyte and further evaluated by visual inspection of
a calibration graph. These calibration curves were plotted over
different concentration ranges. The absorbance of the analyte
was determined at 215 nm. Regression equation was calculated by constructing calibration curves by plotting absorbance v/s concentration .The results of linearity ranges, plots
and curves are shown in Fig. 5.

3.3.

System suitability

The system performance parameters of the developed HPLC

method was evaluated by six replicate analysis of the
formulation at a concentration of 10 ppm. The retention time
of their areas were recorded subsequently. Mean area and SD
was calculated to determine relative SD and the criteria is 2%
respectively.

3.4.

Precision and accuracy

Accuracy was determined for the assay method at two levels:

i.e. repeatability and intermediate precision. The repeatability

Fig. 3 e Representative HPLC chromatogram of (C) Lavangadi Vati and (D) Jatiphaladi Churna.

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Fig. 4 e Representative HPLC chromatogram of (E) Sitopaladi Churna and (F) clove oil.

was evaluated by means of intraday variation and intermediate precision was determined by measuring interday variation in the assay method of formulation in six replicate runs.
Accuracy and precision of the method assay was performed
by injecting three samples spiked at 500 ng/mL, 1000 ng/mL
and 5000 ng/mL of drug in the placebo triplicate sets at three
different levels LQC, MQC and HQC respectively for interday
and intraday batch respectively. Mean was determined by,
S.D, CV % and % nominal of three different levels was
calculated.

3.5.

Solution stability

The solution stability of working standard solution of eugenol

was tested at day 0, 24 h and 20 days respectively. The
important criterion for selecting the solution stability is by
comparing per cent area and peak purity of the eugenol from
chromatograms.

3.6.

Ruggedness and robustness

The ruggedness of the method is defined as its capacity to

remain unaffected by minuscule changes in method conditions. The ruggedness was evaluated by deliberate changes in
composition of mobile phase and flow rate.

Analyte Response (cps)

6000000

y = 96149x - 14341
R = 0.996

5000000

4.

Results of RP-HPLC method development

4.1.

Method validation

The principle objective of the proposed research work was to

develop method for analytical quantification of eugenol from
Caturjata Churna, Lavangadi Vati, Jatiphaladi Churna, Sitopaladi
Churna and clove oil and to validate the developed method
according to ICH guidelines for its further estimating pharmaceutical formulation. Based on different validation
parameters used for detection of eugenol from HPLC analysis,
this method offers reliable estimation of eugenol from
commercial formulations.

4.1.1.

4.1.2.

4000000

Method optimization

The project was found to be rapid, simple, accurate and reliable for routine estimation of eugenol in commercial formulations by RP-HPLC. HPLC conditions were optimized to enable
separation of eluted compounds. Methanol: water (60:40, v/v)
was successfully employed as the mobile phase and it gave
symmetry and well resolved peaks for eugenol. The retention
time of eugenol were recorded at 5.91 min and detected at UV
absorbance at 215 nm at flow rate of 1.00 mL/min (Fig. 2A). The
use of PDA detector allows optimum utilization of online UV
spectra to assess peak purity. The peaks recorded with
a retention time in all the chromatograms of eugenol from
ayurvedic formulations resulted to be within the peak purity
limits. These data excludes the presence of significant interference by other plant constituents.

Calibration curve and linearity

A good linearity was successfully achieved in the concentration range of 50.00 ng/mL to 50,000.00 ng/mL. The regression
equation and correlation coefficient was found to
y 96149x  14341 and R2 0.996.

3000000
2000000
1000000

4.1.3.

0
0

10

20
30
40
50
Eugenol concentration (50-5000 ng/mL)

Fig. 5 e Linearity plot of eugenol.

60

Method application

The relative retention time (RRT) and relative peak area (RPA)
of each characteristic from samples related to the reference
peak was calculated for quantifying eugenol from ayurvedic

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formulations: Caturjata Churna, Lavangadi Vati, Jatiphaladi

Churna, Sitopaladi Churna and clove oil. The concentration (mg/
gm) and % CV are shown below in Table 1.

4.1.4. Limit of determination and limit of quantification (LOD

and LOQ)
The LOD and LOQ were determined from both the values of
calibration curve and with signal to noise ratios of 3 and 10
respectively. The LOD and LOQ were found to be 25.00 ng/mL
and 50.00 ng/mL. The acceptance criterion for system suitability is 2% for the per cent coefficient of the variation of the
peak area and retention time of the drug. The values are
depicted in Table 2 which indicated good performance of the
system.

4.1.5.

Precision and accuracy

The precision and accuracy % RSD values for recovery at each

level was not more than 0.2% for accuracy and were within
the acceptable limits to meet the guidelines for analytical
method validation. The accuracy was determined by means of
recovery of the added analytes at three different concentration (low, medium and high level) as well as S.D. of the assays.
The results recorded for accuracy studies mean recovery
values for all ayurvedic formulations were always higher than
85% as indicated in Table 2. The % CV intraday and interday
results were obtained in the values ranging between 0.33e1.21
and 1.08e1.58 individually. The mean assay result for intraday
and interday precision was found to be 103.87% and 104.30%
respectively. Since there was no impurity of peaks in the
chromatograms, the values obtained indicate that solution is
stable for at 24 days at ambient temperature. The accuracy of
both the methods was good with the deviation between the
nominal concentration and calculated concentration well
below the limits of 15%. Thus, intraday and interday precision
and accuracy data indicated that the method is validated,
highly reproducible reliable and satisfactory.

4.1.6.

Stability

Stability of eugenol from Caturjata Churna, Lavangadi Vati,

Jatiphaladi Churna, Sitopaladi Churna and Clove Oil for 12 h and
24 days was evaluated. The experimental conditions were
deliberately altered for determining the robustness of the
assay method and check the reliability of an analysis with
respect to deliberate variations in method parameters. The
method conditions such as flow rate and mobile phase
concentrations were altered and the values are illustrated in
Table 2.

Table 2 e Method validation parameters for

quantification of eugenol from commercial formulations.
Method validation parameters
LOD (ng/mL)
LOQ (ng/mL)
Linear range (ng/mL)
Mean correlation coefficient (r2)
System suitability (% CV, n 6)
Retention time (min)
Accuracy and precision
Intraday
Interday
Stability
Caturjata Churna
Lavangadi Vati
Jatiphaladi Churna
Sitopaladi Churna
Clove oil
Stability
Caturjata Churna
Lavangadi Vati
Jatiphaladi Churna
Sitopaladi Churna
Clove oil
Ruggedness
Mobile phase
(Methanol: d/w, v/v)
59:41 (v/v)
60:40 (v/v)
61:39 (v/v)
Flow rate change (mL/min)
0.9 mL/min
1.0 mL/min
1.1 mL/min

4.1.7.

Sr. no.
1
2
3
4
5

Sample

Concentration (mg/gm)a

% CV







1.5
6.8
6.61
1.86
4.84

Caturjata Churna
Lavangadi Vati
Jatiphaladi Churna
Sitopaladi Churna
Clove Oil

a Mean  S.D., n 3 (batches).

2.30
2.76
0.54
0.33
450.77

0.03
0.17
0.04
0.01
21.84

25
50
50e50000
1
0.88
5.91
(% CV, n [ 3)
0.33e1.21
1.08-1.58
24 h
95.68%
98.27%
92.81%
92.67%
95.26%
20 days
94.95%
97.22%
102.31%
105.75%
100.99%

Retention time (min)

6.3
6.54
5.21
Retention time (min)
6.2
6.33
5

Ruggedness/robustness

Ruggedness was studied by using different composition of

mobile phase and changing flow rate. The retention time
recorded for our parameters was well within the limit 1 min,
which indicated that this method is robust as indicated in
Table 2.

4.1.8.

System suitability

System suitability for six replicate analyses (% CV) was found

to be 0.88 which is completely within the acceptable analytical
range 0.999, which proves the method validated is highly
accurate and sensitive and meets with ICH guidelines.

5.
Table 1 e Application of method to quantification of
eugenol from commercial formulation.

Results

Discussion

Several variations in factors like temperature, storage, packaging, drying, etc affects both the quality of phototherapeutic
agents and their therapeutic value in plant constituents.
Therefore, not only standardization but also method validation is becoming increasing important for routine quality
control analysis of raw materials and for to carry out quality
evaluation of marker substances whose active principle is
unknown.22 Despite the number of studies published on
standardization of in house and marketed herbal medicinal
formulations, our knowledge regarding quantification of

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phytochemicals from commercial ayurvedic formulation to

set quality specification, stability profiles and chemical analysis of analyte of interest is largely unknown mainly due to
lack of simple, reliable and sensitive validated analytical
methods. In this contribution, we developed completely
simple and new experimental chromatographic set up
method for separation and quantification of phytochemical
eugenol from Caturjata Churna, Lavangadi Vati, Sitopaladi
Churna, Jatiphaladi Churna and clove oil based on classical RPHPLC using photodiode array detector (PDA) and methanol:
distilled water (60:40,v/v) as mobile phase.
Lavangadi Vati, an ancient Ayurvedic formulation, has been
known to cure diseases like indigestion, loss of appetite,
cough and acts as a good blood purifier. Owing to its superior
medicinal activity, it is further explored for standardization to
increase the acceptance of this herbal medicine among
patients and physicians.4 Therefore, simultaneous quantification of eugenol along with other phytochemical constituents from marketed Lavangadi Vati technique was carried out
by HPTLC fingerprinting method.4 However, few shortcomings
of the HPTLC method reported include failure to separate and
detect eugenol from other constituents because of interfering
peaks from other plant raw materials and excipients added
during formulation.4 Secondly, this method also needs further
evaluation to ensure batch to batch consistency in quality and
efficacy.4 Moreover, this assay does not claim to be fully
validated for application in standardization of herbs and
herbal formulations. These scientific finding highlights
current urgent need of reliable, sensitive analytical technique
method validation for meeting current demands of pharmaceutical Industries, as per ICH guidelines. In this view, RPHPLC method described in this research article provides
fully validated methodology for quantification of eugenol
from Lavangadi Vati which can be safely extended for further
detecting concentration of eugenol present in plant herbal
formulations within shorter run time (Rt 5.91 min) and easy
separation of other plant constituents present in formulation.
Therefore, this method provides ample opportunities, which
can be extended into quantification of plant phytochemicals,
checking authenticity of other herbal formulations and facilitating routine quality control analysis of commercial ayurvedic formulations, containing Lavangadi Vati (Fig. 3C).
Caturjata Churna is polyherbal ayurvedic formulation used
for treatment of cold and cough.23 Several studies such as thin
layer chromatography and HPTLC fingerprinting after post
column derivatization with vanillin-sulphuric acid have been
carried out for standardization, quantification and quality
control analysis of in house and marketed formulations of
Caturjata Churna to determine its potent therapeutic efficacy in
herbal medicines.23 However, this technique offers several
shortcomings like it involves relatively high reagent
consumption and are difficult for high sensitivity analysis.
Another method has been shown to be validated in separating
and quantifying eugenol from clove and cinnamon oils by
HPLCeUV analysis after pre-column derivatization and use of
fluorescent labelling reagents.20 However, this method
involves use of NBD-F labelling fluorescent reagents which is
highly toxic and expensive. Secondly, retention time recorded
was 12.1 min for eugenol which is more time consuming
process. Third major disadvantage of this methodology

include possibility of derivatizing reagents mixing directly

with samples (analyte) of interest and the reaction efficacy
easily influenced by coexisting components present in
formulations during analysis. In conclusion, such reagents
require cumbersome reactions that may also require heating
protocols or methods along with post reaction clean up. On
the other hand, this paper successfully reports quantification
and separation of eugenol from Caturjata Churna without the
use of derivatizing reagents, albeit expensive fluorescent
reagents and produces very accurate and highly sensitive
results. Hence, further research was needed to validate and
produce reliable results which can be stretched to set quality
specifications for composition and concentration of phytoconstituents needed for herbal medicines. Thus, we have fully
validated RP-HPLC method, which can be used reliably for
estimation of eugenol and other phytochemicals, with high
reproducible results and be easily employed for detecting the
difference in quality control parameters and set specifications
for plant phytoconstituents (Fig. 2B).
With exponential increase and demand of Ayurvedic remedies for treatment of cough, bronchitis, asthma, cold, coryza and
other respiratory disorders,24 Sitopaladi Churna has become very
popular herbal medicine because they exhibit no side effects
when consumed in prescribed dose. Its principle formulation
ingredients is also explicitly very well described in Ayurvedic
formulatory of India.20 In addition, comparative organoleptic
analysis ash value, macroscopic properties,25Ayurvedic inhouse formulation standardization and pharmacognostic
characters of Sitopaladi Churna26 was also established. However,
to meet pharmaceutical demands, validation is needed for
identification, purity, stability data and scientific based
evidence about efficacy of Sitopaladi Churna formulations to
produce results which are reliable, accurate and reproducible.
UV-spectrophotometric fingerprint method developed has been
developed for simultaneous estimation of phytochemical
piperine from Sitopaladi Churna.24 The method described in this
paper was completely validated for estimation of eugenol from
commercial ayurvedic formulation like Sitopaladi Churna and
thus can be extended in routine quality control analysis to check
batch to batch variation for drug approval. However, UV method
reported, poses various issues like inadequate sensitivity and
narrow dynamic linear range. Thus, this method proves very
challenging to be reliable for quantitative and semi quantitative
analysis for separation of main active ingredients (markers
compounds) present in Ayurvedic formulations. Due to strong
UV absorbance involved in UV spectrophotometers, the
preservatives, polymetric matrices, cross interference with
other active components, detector saturation with polysorbate
solutions are potential sources of interference in producing
reliable data for direct spectrophotometric analysis. Hence,
further research was needed to validate and produce reliable
results which can be stretched to set quality specifications for
composition and concentration of phytoconstituents in herbal
medicines. We present completely new experimental analytical
validated RP-HPLC method with properly selected Column like
C18, mobile phase concentration (60:40) methanol: distilled
water and detector set at 215 nm for quantification of eugenol
from Sitopaladi Churna with reliable and reproducible results.
Therefore, with increasing emphasis on reliable product quality
control requirements for drug formulation and standardization,

j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 5 3 e6 0

proposed RP-HPLC method developed and validated in this

paper can be successfully exploited for successfully quantifying
even low sample concentrations of eugenol from Sitopaladi
Churna. This method also confirms reliable separation and
quantification of analytes of interest without interference from
excipients, preservatives and dissolution media respectively
(Fig. 4E).
Enteric bacterial species like Salmonella sp and S. aureus are
a major infectious agents contributing to health problems like
diarrhoeal infections in developing countries and are primarily
responsible for mortality around 6 million children annually.27 It
has also been reported that many bacterial pathogens have
acquired immunity against synthetic drugs. Additionally, alternative synthetic drugs produced are very expensive, produce
adverse side effects and therefore, an alternative approach is
needed for formulating ayurvedic drugs having potent antibacterial properties. Recent finding confirms Jatiphaladi Churna
as having strong anti-bacterial activity in inhibiting and preventing chronic enteric bacterial infections using disc diffusion
method.27 Currently no reported analytical validation data is
available which can be further carried for routine quality control
analysis in formulation for this Churna. The analytical separation
technique validated in this paper demonstrates for the very first
time quantification and separation of eugenol (anti-bacterial
and antioxidant) phytochemical from Jatiphaladi Churna with
very short retention time (Fig. 3D). This finding can be further
used for critical simultaneous quantification of other marker
compounds such as active markers (possess therapeutic
activity) from Jatiphaladi Churna and other marketed herbal
medicines, thus facilitating easy separation and detection of
phytochemicals for development of herbal medicines against
multidrug resistant microbial pathogens.
Eugenol present in clove oil has been proved to possess
anti-microbial activity against bacteria species such as S.
aureus ATCC25923, K. pneumoniae species, etc.28 Gas chromatography mass spectrophotometer (GCeMS) technique has
been used for detection of eugenol. In principle, the main
shortcomings of this technique for quantification of phytochemical are that the results are not of very high resolution,
difficult to record and not automated. GCeMS operates at high
temperature and this may affect the stability of thermally
labile phytochemical constituents in herbal formulations. On
the other hand, validated RP-HPLC method developed in this
paper for detection of detection of eugenol was found to be
highly sensitive and flexible technique. This was evident from
Ruggedness validation parameter data, in which chromatographic conditions such as Mobile phase concentration and
Flow rate change were deliberately changed without use of
any heating protocols and need of high temperature. The
retention time recorded completely satisfy the acceptance
criteria 1% (Table 2). Thus, the validated analytical chromatographic method reported is highly rugged, sensitive,
requires less retention time and is not affected by minuscule
changes in the chromatographic conditions (Fig. 4F).
Fishes get easily get spoilt at room temperature and
therefore, increasing the lifespan of fishes is now big issue in
food technology industry.29 Eugenol has scientifically been
proven to have anti-microbial activity because of it significant
anti-oxidant capacity and has currently acquired large
interest among food scientists to incorporate this

59

phytochemical as natural anti-microbial agent in the form of

natural preservative in extending shelf life of fishes. This
report is further supported by research carried out for
studying the effect of 0.5% of eugenol oil on fresh carp, Cyprinus carpio L. fillets during storage in fish industry.29 This
breakthrough research suggests very high demand for isolation and quantification of eugenol from herbal formulations.
With increasing human population food requirements and
growing interest in need of animal protein sources from
fishes, there is high demand for development of analytical
method which can easily separate and quantify eugenol from
other plant interfering constituents, to be safely used in food
preservation industry worldwide. Thus, validated RP-HPLC
method demonstrated in this paper quantifies micrograms of
eugenol in short span of time and is thus highly sensitive. This
method will definitely aid in quantifying, separating potential
anti-microbial commercial phytochemical like eugenol and
provide highly reproducible data for quality control analysis in
food technology related industries.
In conclusion, solvent extraction methods by RP-HPLC PDA
detection method was developed and validated for quantitative
estimation of eugenol from Ayurvedic formulations of Caturjata
Churna, Lavangadi Vati, Sitopaladi Churna, Jatiphaladi Churna and
Clove oil successfully. The developed analytical chromatographic method offered adequate calibration curve/linearity,
LOD, LOQ, system suitability, precision, accuracy, solution
stability, robustness method application and has been fully
validated as per ICH guidelines. This method can be successfully applied for quality control of herbal medicines containing
eugenol to screen toxic botanicals, microbial toxins, pesticides,
fumigation, foreign organic matter, fingerprinting/marker
compound for identification and standardization of botanical
drugs containing eugenol. This will aid in identification of
chemical constituents marker compounds such as chemical
and active marker compounds that possess therapeutic activity
of the herbal drug which are major constituents of plant
materials, identifying herbal materials and standardize botanic
preparations during all aspects of manufacturing process.

Conflicts of interest
All authors have none to declare.

Acknowledgements
The authors gratefully acknowledge the technical assistance
rendered by Mandar Mhatre, Sreenath Nandakumar Nair,
Varun Satose, Ashish Singh, Naresh Shejawal,Kavita Mhatre,
Dipti Singh, Santosh Daval and Karan Sarvaiya in completing
this project.

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