Rajiv Gandhi University of Health Sciences s37

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

BANGALORE, KARNATAKA

ANNEXURE –ІІ

REGISTRATION OF SUBJECT FOR DISSERTATION

1.0 NAME OF THE CANDIDATE D.T.VISHRUTA ADDRESS D/O D.C.THYAGARAJ # 156, NEAR ANJENEYA TEMPLE, DOMLUR, BANGALORE-570061.

2.0 NAME OF THE INSTITUTION Al-AMEEN COLLEGE OF PHARMACY BANGALORE.

3.0 COURSE OF STUDY & M.PHARMA (Part-I) SUBJECT PHARMACEUTICAL CHEMISTRY

4.0 DATE OF ADMISSION JUNE 2011

5.0 TITLE OF THE TOPIC:

METHOD DEVELOPMENT AND VALIDATION OF ESTIMATION OF ROSAMARIC ACID AND EUGENOL FROM AERIAL PARTS OF OCIMUM SANCTUM AND IT’S EXTRACTS BY HPLC.

6.0 BRIEF RESUME OF THE INTENDED WORK:

6.1: NEED OF STUDY:

METHOD DEVELOPMENT:

Method development involves three main criteria.

 Sample and standard preparation  Setting the chromatographic conditions  Standardization of the analytical method

A time frame should be allotted to each of these steps to ensure that all three steps are given adequate importance as generally analysts tend to focus too much on the Chromatographic conditions and tend to neglect the other two steps which are equally vital for the development of an accurate and robust method.

In order to develop an accurate and robust method one must

 Method objectives must be defined and the chemistry of the analytes and the drug product must be understood  Preliminary HPLC conditions should be developed to achieve minimally acceptable separations. These HPLC conditions will be used for all subsequent method development experiments.  A suitable sample preparation scheme for the drug product must be developed.  The appropriate standardization method must be determined and relative response factors should be used in calculations.  The method performance with different conditions, different instrument setups and different samples should be understood. The weaknesses of the method should be identified and the method has to be optimized through experimental design. METHOD VALIDATION: Method validation is the process which is used to confirm that the analytical procedure employed for a specific test is suitable for its intended use. Results from method validation can be used to judge the quality, reliability and consistency of analytical results; it is an integral part of any good analytical practice.

Analytical methods need to be validated or revalidated:

 before their introduction into routine use;  whenever the conditions change for which the method has been validated (e.g., an instrument with different characteristics or samples with a different matrix); and  Whenever the method is changed and the change is outside the original scope of the method.

The USP has published specific guidelines for method validation for compound evaluation. USP defines eight steps for validation:  Accuracy  Precision  Specificity  Limit of detection  Limit of quantitation  Linearity and range  Ruggedness  Robustness

The method validation can be carried out for methods using High Performance Liquid Chromatographic instrument, which is a form of column chromatography used frequently in biochemistry and analytical to separate, identify, and quantify compounds. HPLC utilizes a pump that moves the mobile phase through the column, a column that holds chromatographic packing material (stationary phase) and a detector that shows the retention times of the molecules. Retention time varies depending on the interactions between the stationary phase, the molecules being analyzed, and the solvent used.

Traditional medicine has many natural herbal preparations with versatile medicinal properties, which deserve detailed investigation for effective drug development. Chemical constituents of these traditional medicines are safe, have less side effects and so their isolation and characterization is of much importance in the modern era. In the last few decades, herbal medicine has been found to have some impressive credentials. To bring these herbal preparations to the market we need to have standardized raw materials, extracts and drug preparations. Hence there is need for the exploration of the potentially rich traditional medicines.

Ocimum sanctum(tenuiflorum) is an erect, herbaceous, multibranched, softhaired annual with purple or crimson flowers. It is commonly called “Tulsi” or “Holy Basil” Ocimum sanctum shows the presence of active constituents in its crude extract, which are responsible for blood sugar lowering activity, antispasmodic, antioxidant and analgesic activities. Aqueous decoction of tulsi leaves is also used to treat gastric and hepatic disorders. Rosamarinic acid and eugenol were obtained in the extract of the plant.

Rosamarinic acid, isolated from tulsi shows antiviral, antibacterial, antiinflammatory and antioxidant activity. It is also a potential anxiolytic as it acts as a GABA transaminase inhibitor.It is currently being studied for it’s use in Alzheimer’s disease. It is also being used as an effective intervention in case of allergic asthma.

OH O COOH

HO O OH

HO STRUCTURE OF ROSAMARINIC ACID Eugenol, a volatile oil obtained from tulsi shows a local antiseptic and analgesic (in dentistry) and anesthetic activity. Eugenol can be combined with zinc oxide to form a material known as zinc oxide eugenol, which has restorative and prosthodontic applications in dentistry. Eugenol kills certain human colon cancer cell lines in vitro.

O STRUCTURE OF EUGENOL

Since rosamarinic acid and eugenol have the above said activities we are aiming at developing a method for the simultaneous quantification of rosamarinic acid and eugenol. Hence need of the study is to carry out the method development and validation of estimation of rosamarinic acid and eugenol from the aerial parts and extracts of Ocimum sanctum by HPLC.

7.1):LITERATURE SURVEY: 1) Prakash.P, Neelu Gupta have reviewed the therapeutic uses of Ocimum sanctum with 7.0 a note on Eugenol and it’s pharmacological actions. 2) Shanmuga Sundaram R. and co-workers have developed a HPLC method for the determination of Rosamarinic acid and ursolic acid in the leaves of the ethanol extract of Ocimum sanctum and validated it. 3) Lukmanul Hakkim.F and co-workers have developed a method for the production of rosamarinic acid in Ocimum sanctum cell suspension cultures by the influence of growth regulators. 4) Lukmanul Hakkim.F and co-workers have investigated the antioxidant property of eight selected Ocimum species and their secondary metabolites. The total phenolic content was estimated and the quantification of individual phenolic compounds was done using HPLC. 5) Sook Young Lee and co-workers have carried out the production of rosamarinic acid from transformed root cultures of Nepeta cataria L and the identification of rosamarinic acid in the extracts was done using HPLC by comparing the retention times and spectral characteristics to those from a single HPLC run of a known rosamarinic acid standard. 6) Bruno.. Zelic and co-workers have recovered and purified rosamarinic acid from Rosemary using Electrodialysis. The quantification of rosamarinic acid was done using HPLC. 7) Vera Canelas and Cristina Teixeira da Costa have carried out the quantitative HPLC analysis of Rosamarinic acid in extracts of Melissa officinalis and spectrophotometric measurement of their antioxidant activities. 8) Robert A.Miller and co-workers have carried out the quantitative analysis of eugenol by HPLC and purified the samples using preparative HPLC. 9) Wei-Chun Chang carried out the analysis of eugenol from the essential oil of Eugenia caryophyllata by HPLC and against the proliferation of cervical cancer cells.

10) San Myint and co-workers carried out the separation and identification of Eugenol in ethanol Extract of Cloves by Reversed-Phase HPLC. 11) Pattanayak.P and co-workers have reviewed the therapeutic applications of Ocimum sanctum and it’s pharmacological activity. 12) Toth, J.Mrlianova and co established the importance of Rosamarinic acid which is an important phenolic active compound of Lemon Balm (Melissa officinalis) 13) Arun Kumar HS and co-workers have evaluated the efficiency of Zeetress in preventing the salicinomycin toxicity in broiler chicks. 14) Anju has tested the ethanolic extract of the leaves of Ocimum sanctum for anti-stress, adaptogenic activity in mice using swim endurance test and cold restraint stress. 15) Shankar mondal and co-workers have reviewed the pharmacological activities and therapeutic uses of Ocimum sanctum 16) Lalit mohan and co-workers have reviewed the pharmacological activities and therapeutic uses of Ocimum sanctum

17) Timomi Shiga and co-workers have evaluated the effect of light quality on rosmarinic acid content and antioxidant activity of sweet basil, Ocimum basilicum L. 18) Kirti Thakur and K.S. Pitre have studied the anti-inflammatory activity of Eugenol that was extracted from Ocimum sanctum leaves.

7.2):OBJECTIVES OF THE STUDY:

 Improved method development of estimation of rosamarinic acid and eugenol from Ocimum sanctum leaves and its extracts.  Method validation of the developed method of the estimation of rosamarinic acid and eugenol from Ocimum sanctum extracts by HPLC.

MATERIALS AND METHODS:

8.1: SOURCE OF DATA:

 References from library - Al-Ameen College of Pharmacy, Bangalore. 8.0  References from Internet Browsing.  www.sciencedirect.com  www.google.com  Instrument: HPLC.  Phytochemistry journals.  Journal of agriculture and food chemistry.

8.2)METHOD DEVELOPMENT:

Analytical experiments will be planned and executed at Natural remedy Bangalore. The following instrument is proposed to be used for collection of data.

 High performance liquid chromatograph.

8.3)METHODS AND INSTRUMENTATION:

 The HPLC instrument for validation of estimation of Rosamarinic acid and Eugenol content in Tulsi.

8.4)METHODOLOGY:

 All experiments will be carried out in the Natural remedies Bangalore.

8.5) DOES THE STUDY REQUIRE ANY INVESTIGATION TO BE CONDUCTED ON PATIENTS OR ANIMALS?

8.6) HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION IN CASE OF 7.3?

-NOT APPLICABLE

REFERENCES:

1) Prakash P, Neelu Gupta. Therapeutic uses of Ocimum sanctum Linn (Tulsi) with a note on Eugenol and it’s pharmacological actions: A short review.Indian J Physiol 9.0 Pharmacol. 2005; 49(2): 125-131 2) Shanmuga Sundaram R, Gowtham L, Ramanathan M, Manikandan P, Venugopal, Kamalakannan D, Nayak BS. Quantification of Bioactive Principles in Indian Traditional Herb Ocimum sanctum Linn. (Holy Basil) Leaves by High Performance Liquid Chromatography. Asian Jour. of Biomedical and Pharmaceutical Sciences. 2011; 1(3): 22-28 3) Lukmanul Hakkim F, Sivan kalyani, Mohammed Essa, Girija S, Hyuk Song. Production of rosmarinic acid in Ocimum sanctum (L.) cell suspension cultures by the influence of growth regulators. Int J Biol Med Res. 2011; 2(4): 1158 -1161 4) Lukmanul Hakkim F, Girija Arivazhagan, Boopathy R. Antioxidant property of selected Ocimum species and their secondary metabolite content. Journal of Medicinal Plants Research. 2008 September; Vol. 2(9): 250-257 5) Sook Young Lee, Chung Yeol Lee, Seok Hyun Eom, Yong Kyoung Kim, Nam I Park, Sang Un Park. Rosmarinic acid production from transformed root cultures of Nepeta cataria L. Scientific Research and Essays. 2010 May; Vol. 5(10): 1122-1126 6) Bruno Zelić, Majda Hadolin, Davorin Bauman, Ðurđa Vasić-Rački. Recovery and Purification of Rosmarinic Acid from Rosemary Using Electrodialysis. Acta Chim. Slov. 2005; 52: 126–130 7) Vera Canelas, Cristina Teixeira da Costa. Quantitative HPLC analysis of rosmarinic acid in extracts of Melissa officinalis and spectrophotometric measurement of their antioxidant activities. J.Chem. Educ. 2007; 84(9): 1502-1520 8) Robert A Miller, Norman E Bussell, Charles K Ricketts, Howard Jordi. Analysis and Purification of Eugenol. J Dent Res. 1979 April; 58(4): 1394-1400 9) Wei-Chun Chang, Meen-Woon Hsiao, Hsi-Chin Wu, Yin-Yi Chang, Yao-Ching Hung, Je-Chiuan Ye. The analysis of eugenol from the essential oil of Eugenia caryophyllata by HPLC and against the proliferation of cervical cancer cells. Journal of Medicinal Plants Research. 2011 April; Vol. 5(7): 1121-1127 10) San Myint, Wan Ramli Wan Daud, Abu Bakar Mohamad, Abdul Amir H Kadhum. Separation and Identification of Eugenol in Ethanol Extract of Cloves by Reversed- Phase High-Performance Liquid Chromatography. JAOCS. 1995; 72: 1231-1233 11) Pattanayak P, Behera P, Panda SK. Ocimum sanctum Linn.- A reservoir plant for therapeutic applications: An overview. Pharmacognosy Reviews.2008 Jan- June;4(10): 95 -105

12) Tóth J, Mrlianová M, Tekel’ová D, Korenová M. Rosamarinic Acid – An Important Phenolic active compound of Lemon balm (Melissa Officinalis L.). . Acta Fac. Pharm. Univ. Comenianae. 2003; 50: 139-146 13) Arun Kumar HS, Manjunath HS, Reddy KS, Reddy R. Zeetress(a polyherbal formulation of Withania somnifera, O.sanctum and E. officinalis) prevents salinomycin toxicity in broiler chicks. Phcog Mag. 2005 Jul-Oct; 1(3): 109-113 14) Anju. Adaptogenic and anti-stress activity of Ocimum sanctum in mice. RJPBCS. 2011 Jul-Sept; 2(3): 670-678 15) Shankar Mondal, Bijay R. Mirdha, Sushil C. Mahapatra. The Science behind sacredness of Tulsi(Ocimum sanctum Linn). Indian J Physiol Pharmacol. 2009; 53 (4): 291–306 16) Lalit Mohan, Amberkar MV, Meena Kumari. Ocimum Sanctum Linn (Tulsi) - An Overview. International Journal of Pharmaceutical Sciences Review and Research. 2011 March – April; 7(1): 51-53 17) Tomomi Shiga, Kazuhiro Shoji, Hiroaki Shimada, Shin-nosuke Hashida, Fumiyuki Goto, Toshihiro Yoshihara. Effect of light quality on rosmarinic acid content and antioxidant activity of sweet basil, Ocimum basilicum L. Plant Biotechnology. 2009; 26, 255–259 18) Kirti Thakur, Pitre KS. Anti-inflammatory Activity of extracted Eugenol from Ocimum sanctum L. leaves. Rasayan J. Chem. 2009; 2(2): 472-474

Signature of the candidate: 10. Remarks of the Guide: Recommended for Research 11. Name and Designation of: 12. 12.1. Institutional Guide: Dr.MONICA KACHROO Asst.professor Department of Pharmaceutical Chemistry, Al-Ameen College of Pharmacy, Hosur Road, Bangalore-560 027. 12.2. Signature:

12.3. Co-Guide Mr.B.Murali. 12.4. Signature:

12.5. Head of the Department: Dr. Gopal Krishna Rao Professor and Head Department of Pharmaceutical Chemistry, Al-Ameen College of Pharmacy, Hosur Road, Bangalore -560 027. 12.6. Signature

13. 13.1. Remarks of the Principal Forwarded to the University for approval.

13.2. Signature

Prof. B. G. Shivananda Principal Al-Ameen college of pharmacy Bangalore