WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Amit et al. World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 2.786

Volume 3, Issue 10, 1358-1368. Research Article ISSN 2278 – 4357

ANALYTICAL METHOD DEVELOPMENT FOR DETERMINATION

OF EUGENOL CONCENTRATIONS IN SELECTED SPECIES OF
OCIMUM

*Amit Joshi 1, A.K. Pathak1, Mukul Tailang2

1
Department of Pharmacy, Barkatullah University, Bhopal (M.P.) India
2
Department of Pharmacy, Jiwaji University, Gwalior (M.P.) India

ABSTRACT
Article Received on
20 August 2014, Eugenol is used as a flavor in the food industry, has a variety of
Revised on 09 September
2014, biological activity, and can serve as a biomarker. Because eugenol is
Accepted on 29 September
2014 present in the leaves of Ocimum, which are used as a herbal medicines,
a sensitive and reliable quantitative Ultra-violet spectroscopy and high-
performance liquid chromatographic method has been established for
*Correspondence for
Author quantification of the compound in the leaves of the plant. A methanol
Amit Joshi extract of the powder of dried leaves of Ocimum, was spotted on the
Department of Pharmacy, Merck aluminium plate precoated silica gel F254 with 0.2 mm
Barkatullah University,
thickness. Meoh:Chloroform (95:5) and MeOH: H 2O: ACN (50:25:75)
Bhopal (M.P.) India
used as mobile phase to isolate the eugenol and to prepare the sample
for UV and HPLC analysis respectively. The UV and HPLC method
proposed for the quantitative monitoring of eugenol in Ocimum leaf powder is rapid, simple,
and precise. Hence from that UV and HPLC analysis it was conclude that the Ocimum
sanctum linn contains higher amount of Eugenol.

Keywords: Ocimum; Ultra-violet spectroscopy; high-performance liquid chromatography;

Merck aluminium plate precoated silica gel.

1. INTRODUCTION
Medicinal properties of Ocimum are known for thousand years to various civilizations of the
world. This medicinal herb is considered as a sacred plant by the Hindus in the Indian
subcontinent. Scientific explorations of traditional belief of medicinal properties of ocimum
have got momentum mostly after the middle of the 20th century.

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There are three types of tulsi mentioned in ayurvedic texts – Rama or green leaf tulsi (O.
gratissimum), Shyama or krishna or purple leaf tulsi (O. sanctum) and Vana or wild leaf tulsi
(O.canum). Although, all three types of Tulsi have their uses in ayurveda, the Rama and
Krishna are the most widely used.

Eugenol is used as a flavor in the food industry, has a variety of biological activity, and can
serve as a biomarker. Because eugenol is present in the leaves of Ocimum, which are used as
a herbal medicines. Sophisticated chromatographic and spectrophotometric techniques are
used for isolation, qualitative and quantitative estimation of eugenol from extracts of different
species of Tulsi.

2. MATERIALS AND METHODS

There are three varieties of Tulsi namely, Rama Tulsi (Ocimum gratissimum), Krishna Tulsi
(Ocimum sanctum) and Vana Tulsi (Ocimum canum).leaves were collected from local area of
Bhopal Madhya Pradesh India and authenticated at Safia college of Science Bhopal, Madhya
Pradesh The voucher specimen was deposited in Department of Pharmacy Barkatullah
University, Bhopal, Madhya Pradesh.

2.1 Extraction of plant material [5-7, 10]: The collected, cleaned leaves of ocimum were used
for the extraction process. 200g of powder of Leaves were macerated with Methanol, shaking
frequently during first 6 hours and allowing stand for 18 hours. The extracts were filtered
through what Mann filter paper to remove any impurities if present.

The extracts were concentrated by vacuum distillation to reduce the volume 1/10. The
concentrated extracts were transferred to 100 ml beaker and to removing solvent were
evaporated on the water bath. The dried extracts were packed and labeled in air tight
container for the further studies.

2.2 Estimation of Eugenol contents

2.2.1 Thin layer chromatography profile of eugenol and sample preparation for Thin
layer Chromatography
Standard preparation[6-8,14-16]: Around 2 ml of 99% eugenol was taken in a clean and dry test
tube. The standard eugenol was spotted on the Merck aluminium plate precoated silica gel
F254 with 0.2 mm thickness.

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Preparation of mobile phase: 95% of methanol was mixed with 5% of chloroform by ultra
sonication and taken around 100 ml in a chromatographic chamber. The chamber was
saturated for 30 mins to avoid edge effect.

Method: After chamber saturation the spotted plate was placed in the chamber. Then the
mobile phase was allow running, the present eugenol in the spot according to its affinity
towards mobile phase moved and its retardation factor (Rf) was calculated.

Preparation of sample for Thin layer chromatography: 175mg of each extract was drawn
and dissolved in 10 ml of solvent (Meoh:Chloroform 95:5) So 1 ml of solution Contains 17.5
mg of extracts. This 1ml from the above solution was applied to the silica gel percolated
plate(For Thin layer Chromatography) in the form of band and all the procedure was
maintained as like as standard.

2.3 UV analysis
2.3.1 Determination of λ max: Solvent was prepared by mixing 95 parts of methanol with 5
parts of chloroform with proper sonication. Since standard eugenol contains 99%, hence 99
gm in 100ml and 1000 µg contained in 1.01 ml.

1.01ml-----dissolved up to 10 ml of solvent to give--100 µg / ml of eugenol.

From the above 10ml contains 100 µg of eugenol 1ml was drawn and again dissolved up to
10 ml of solvent to give 10 µg / ml of eugenol. Again From the above 10ml contains 10 µg of
eugenol 1ml was drawn and dissolved up to 10 ml of solvent to give 1 µg / ml of eugenol.The
prepared three concentrations (1 µg / ml, 10 µg / ml, 100 µg / ml) of eugenol was then carried
out for determination of λ max.

2.3.2 Preparation of standard curve: From 99% eugenol stock solution was prepared. that is
10.1 ml of solution contains 99% was dissolved in 10 ml of solvent to give 1000µg. from that
stock solution different Eli quotes of 1,2,3,4,8,10,20 & 25µg/ml was prepared and scanned in
UV. The corresponding absorbances were noted. Then a calibration curve was plotted by
taking Absorbance vs. Concentration.

2.3.3 UV analysis of eugenol

Ocimum gratissimum linn: By comparing the Rf value of standard eugenol from Thin layer
chromatography, the band of eugenol obtained from extract of Ocimum gratissimum was
identified and scrub out. The scrub silica gel containing eugenol was thoroughly mixed with

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5 ml of solvent and filtered. Then the solution was taken for UV Analysis.

Ocimum Sanctum linn: By comparing the Rf value of standard eugenol from Thin layer
chromatography, the band of eugenol obtained from extract of Ocimum Sanctum was
identified and scrub out. The scrub silica gel containing eugenol was thoroughly mixed with
5 ml of solvent and filtered. Then the solution was taken for UV Analysis.

Ocimum canum linn: By comparing the Rf value of standard eugenol from Thin layer
chromatography, the band of eugenol obtained from extract of Ocimum canum was identified
and scrub out. The scrub silica gel containing eugenol was thoroughly mixed with 5 ml of
solvent and filtered. Then the solution was taken for UV Analysis.

2.4 HPLC analysis

2.4.1 Development of method: After all trial and error, the following developed method
having 50 parts of methanol, 25 parts of water and 75 parts of acetonitrile was fit strong for
eugenol in a Isocratic system and this method was developed for both standard eugenol and
eugenol present in alcoholic extracts of different species of ocimum.

2.4.2 Calibration of eugenol by HPLC: 1.01ml of 99% v/v eugenol when diluted up to 10 ml
of solvent (MeOH: H2O: ACN, 50:25:75) to give100 µg / ml of eugenol. From this stock
solution aliquots of 25, 50, 75, 100, 125nano gram per ml of eugenol were prepared. After
sufficient 30 min sonication and filtration through 0.45 micron filter paper those solutions
were injected to HPLC for Calibration.

3. RESULT
3.1 quantitative estimation of eugenol by uv :The concentrations of eugenol can be
determined by UV- VIS spectrophotometer by putting the absorbances of eugenol obtained
from various extracts of ocimum in the standard calibration curve.

From the spectrum of fig.2 it was found that the λ max of eugenol (Peak-2) is 281 nm.

Ocimum gratissimum linn: In fig.3 peak of eugenol of Ocimum gratissimum l. having

absorbance 0.456 A at near about the λ max of standard eugenol. It was found from
calibration curve 7.26 µg of Eugenol present in 1ml of Solvent. Hence 5ml of solution
contains = 7.26×5=36.3 µg =0.036 mg So 1ml of Solvent which Contains 17.5 mg of extract
contains 0.036 mg of Eugenol. Hence 5000mg (5gm) of Extract contains = 0.036/17.5×5000

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mg of Eugenol=10.38 mg of Eugenol. Hence % of Eugenol contains in Ocimum gratissimum

= 10.38 /5000 × 100 = 0.20%.

Table 1. Calibration Table of Eugenol Table 2. Calibration Table of Eugenol (HPLC)

(UV)

Concentration Concentration
Absorbance Peak area
µg/ml ng/ml

0 0
25 1290

5 0.322
50 2680
10 0.623 75 3976
100 5356
15 0.922
125 6678
20 1.272

25 1.552

Fig . 1: Standard Curve of Eugenol Fig. 2: UV Spectrum of Standard Eugenol

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Fig. 3: UV Analysis of Ocimum Gratissimum Fig. 4: UV Spectrum of Ocimum

sanctum

Fig. 5: UV Spectrum of Ocimum Canum Linn Fig. 6: HPLC Calibration Curve of

Eugenol

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Fig. 7: HPLC Chromatogram of Eugenol

Fig. 8: HPLC Chromatogram of Ocimum Gratissimum Linn

Fig. 9: HPLC Chromatogram of Ocimum Sanctum Linn

Fig. 10: HPLC Chromatogram of Canum Linn

Ocimum sanctum linn: In fig.4 peak of eugenol of Ocimum sanctum l. having absorbance
0.678 A at near about the λ max of standard eugenol. It was found from calibration curve
9.74 µg of Eugenol present in 1ml of Solvent. Hence 5ml of solution contains = 9.74×5= 48.7

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µg = 0.0487 mg So 1ml of Solvent which Contains 17.5 mg of extract contains 0.0487 mg of

Eugenol. Hence 5000mg (5gm) of Extract contains = 0.0487 /17.5×5000 mg of
Eugenol=13.91 mg of Eugenol. Hence % of Eugenol contains in Ocimum sanctum l.= 13.91
/5000 × 100 = 0.27%.

Ocimum canum linn: In fig.5 peak of eugenol of Ocimum canum l having absorbance 0.540
A at near about the λ max of standard eugenol. It was found from calibration curve 8.10 µg of
Eugenol present in 1ml of Solvent. Hence 5ml of solution contains = 8.10×5= 40.5 µg =
0.0405 mg so 1ml of Solvent which Contains 17.5 mg of extract contains 0.0405 mg of
Eugenol. Hence 5000mg (5gm) of Extract contains = 0.0405 /17.5×5000 mg of Eugenol=
11.57 mg of Eugenol. Hence % of Eugenol contains in Ocimum canum l = 11.57 /5000 × 100
= 0.23%

3.2 Quantitative estimation of eugenol by HPLC method

The concentrations of eugenol isolated by thin layer chromatography from alcoholic extracts
of different species of ocimum can be determined by putting the peak areas of eugenol in the
standard calibration curve of HPLC.

The fig.7 shows the spectrum of standard eugenol (Peak-1) at Rt 12.79.

Ocimum gratissimum lin: In Fig.8 spectrum peak no 9 is the peak of eugenol of Ocimum
gratissimum l at near about the Rt of standard eugenol and having peak area 1535.96.
175 mg of Extract was dissolved in 10ml of solvent. So 1 ml of solution Contains 17.5 mg of
extracts. This 1ml was taken as a band in silica gel precoated plate (For thin layer
chromatography). The developed Eugenol band was scrub out & dissolved in again 2ml of
Solvent and taken for sonication and filtration then from this 2 µl was injected to HPLC. It
was found from calibration curve, 29.5 ng of Eugenol present in 2 µl of Solution. Hence 2ml
of solution contains = 0.029 mg. So 1ml of Solvent which Contains 17.5 mg of extract
contains 0.029 mg of Eugenol. Hence 5000mg (5gm) of Extract contains = 0.029/17.5 ×5000
= 8.28 mg of Eugenol Hence % of Eugenol contains in Ocimum gratissimum =
8.28/5000×100 = 0.16%

Ocimum sanctum linn: In Fig.9 spectrum peak no 8 is the peak of eugenol of Ocimum
sanctum l. at near about the Rt of standard eugenol and having peak area 1849.

175 mg of Extract was dissolved in 10ml of solvent. So 1 ml of solution Contains 17.5 mg of

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extracts. This 1ml was taken as a band in silica gel precoated plate (For thin layer
chromatography). The developed Eugenol band was scrub out & dissolved in again 2ml of
Solvent and taken for sonication and filtration then from this 2 µl was injected to HPLC. It
was found from calibration curve, 35.3 ng of Eugenol present in 2 µl of Solution. Hence 2ml
of solution contains = 0.035mg. So 1ml of Solvent which Contains 17.5 mg of extract
contains 0.035mg of Eugenol. Hence 5000mg (5gm) of Extract contains =
0.035mg/17.5×5000 = 10.08 mg of Eugenol Hence % of Eugenol contains in Ocimum
sanctum l. = 10.08 /5000×100 = 0.20%.

Ocimum canum lin: In Fig.10 spectrum peak no 9 is the peak of eugenol of Ocimum canum
l. at near about the Rt of standard eugenol and having peak area 1598.79.

175 mg of Extract was dissolved in 10ml of solvent. So 1 ml of solution Contains 17.5 mg of

extracts. This 1ml was taken as a band in silica gel precoated plate (For thin layer
chromatography). The developed Eugenol band was scrub out & dissolved in again 2ml of
Solvent and taken for sonication and filtration then from this 2 µl was injected to HPLC. It
was found from calibration curve, 30.6 ng of Eugenol present in 2 µl of Solution. Hence 2ml
of solution contains = 0.030mg. So 1ml of Solvent which Contains 17.5 mg of extract
contains 0.030mg of Eugenol. Hence 5000mg (5gm) of Extract contains = 0.030/17.5×5000=
8.5 mg of Eugenol Hence % of Eugenol contains in Ocimum canum l = 8.5/5000 ×100 =
0.17%.

DISCUSSION
The result from the UV analysis it was conclude that the Ocimum gratissimum linn contains
10.38 mg (0.20%) of Eugenol. Ocimum sanctum linn contains 13.91 mg (0.27%) of Eugenol.
Ocimum americanum linn contains 11.57 mg (0.23%) of Eugenol From 5gm of Extracts.
And from the HPLC analysis it was conclude that the.Ocimum gratissimum linn contains 8.28
mg (0.16%) of Eugenol.Ocimum sanctum linn contains 10.08 mg (0.20%) of Eugenol.
Ocimum canum linn contains 8.5 mg (0.17%) of Eugenol From 5gm of Extract.
The result obtain from UV and HPLC analysis shows that the, Ocimum sanctum linn contains
higher amount of eugenol and established Ultra-violet spectroscopy and high-performance
liquid chromatographic method for quatification of phenolic compound or eugenol in leaves
of the plants has been sensitive and reliable.

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ACKNOWLEDGEMENTS
I wish to extend my sincere thanks to my Respected Guide Dr. Mukul Tailang professor
Jiwaji University, Gwalior and my Co-Guide Dr.A K Pathak Professor & Head, Department
of Pharmacy Barkatullah University , Bhopal for providing unceasing encouragement,
precious and erudite suggestions and directions, constant and untiring guidance along with
the freedom of work that he gave me.

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