DEVELOPMENT OF AN ANALYTICAL METHOD TO DETERMINE EUGENOL CONCENTRATIONS IN ALCOHOLIC EXTRACTS OF DIFFERENT SPECIES OF OCIMUM’S

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, a sensitive and reliable quantitative Ultra-violet spectroscopy and high-performance liquid chromatographic method has been established for quantification of the compound in the leaves of the plant. A methanol extract of the powder of dried leaves of Ocimum , was spotted on the Merck aluminium plate precoated silica gel F254 with 0.2 mm thickness. Meoh:Chloroform ( 95:5) and MeOH: H2O: ACN (50:25:75) 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 sanctum linn contains higher amount of Eugenol.


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. 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.

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 (238-39-40/BOT/SAFIA/11) was deposited in Department of Pharmacognosy TIT college of Pharmacy, Bhopal, Madhya Pradesh. [5][6][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.

Estimation of Eugenol contents 2.2.1 Thin layer chromatography profile of eugenol and sample preparation for Thin layer chromatography
Standard preparation [6][7][8][14][15][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. 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.

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.

UV analysis of eugenol contained in:
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 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. Hence % of Eugenol contains in Ocimum canum l = 11.57 /5000 × 100 = 0.23%

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 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% Bhopal for providing unceasing encouragement, precious and erudite suggestions and directions, constant and untiring guidance alongwith the freedom of work that he gave me. To work under the guidance of such a persons has been a great and inexplicablie experience.