Rajiv Gandhi University of Health Sciences s44

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE, KARNATAKA.

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. Name of the candidate and Address: MANJULA S

D-26 KPWD QUARTS, JEEVANBHIMANAGAR, BANGALORE-75

2. Name of the institution Government College of Pharmacy, Bangalore-560027.

3. Course of study and subject: Master of Pharmacy in Pharmacognosy.

4. Date of admission to the course 07/07/2010

5. Title of the topic:

“PREPARATION,EVALUATION AND STABILITY STUDIES OF DIFFERENT FORMULATION CONTAINING ANDROGRAPHIS PANICULATA AND PHYLLANTHUS AMARUS ”

1 6. BRIEF RESUME OF THE INTENDED WORK:

6.1: Need for the study:

 In the present 21st century where modern technology and scientific discoveries are ushering remarkable change in over lives, nevertheless, the story of plants as herbal medicines definitely continues to unfold importance, quietly & independently.

 The quality of the drugs from herbal source are dependent on various factors like place of collection, season of collection , time of collection and many more factors can affect to the quality of drugs .

 Quality control is an important wing of pharmaceutical industry. It is a requirement for any drug to be used should meet the standard requirement. It is to be noted that not many details are available regarding the evaluation of herbal drug formulation for most of the formulations.

 It is requirements for all formulation are subjected to stability studies to find out stability of the preparation. All the different formulations available for the synthetic drugs invariably subjected to accelerated stability studies. However it is to be noted that stability studies has not been carried out to ascertain the stability of the preparation it is a requirement for herbal drugs to establish the stability of the standards.

 Establishing the method of preparation, standardisation and stability of the sample is a requirement for formulations containing natural drugs.

2 6.2: REVIEW OF LITERATURE 1. Andrographis paniculata is one the important plant in Ayurvedic system. Different methods such as gravimetry, colorimetric, spectrophotometric and titrametric methods were not useful for quantitative evaluation of andrographolide.HPTLC method of evaluation has been developed. Sample was extracted with alcohol and the ex- tracts were utilized. Separation of compound was established on TLC plates on sili-

ca gel 60F 254 by using ethyl acetate as solvent at 260nm. Calibration was linear in the range of 0.1-1µg and recovery shows reliability. Total andrographolide was found to be 22.90-32.98 in the different samples taken for evaluation1-5. 2. A chemical fingerprint of Andrographis paniculata was developed using HPLC, HPTLC and densitometry. The leaves were extracted with different solvents .The solutions were applied on silica gel 60F 254using different mobile phase for different extract. Scanned at 540 nm. HPLC finger print was developed using different mobile phase extract scanned at 220nm. T he finger print profile for different ex- tracts of Andrographis paniculata was developed 6. 3. A study was carried out to standardize the kalmegh by HPLC determination of andrographolide. The plants were extracted with methanol. The methanolic extract was subjected to HPLC analysis. The andrographolide was estimated using refer- ence standard. The content was calculated by linear regression by employing silica column using chloroform: methanol as mobile phase scanned at 254 nm. The plant shows wide variation in andrographolide content with leaves containing highest 2.39% and seeds the lowest 0.13% 7. 4. A HPLC and HPTLC densitometry study was conducted to determine the andrographolide along with anti oxidant potential for Andrographis paniculata. The methanolic extract of leaves were used to isolate andrographolide and 14-deoxy-11, 12-didedhydro andrographolide. The HPTLC study was done using chloroform: methanol (8:2) as mobile phase at 254nm. The HPLC study was done using methanol: water (6:4) as mobile phase, scanned at 210nm. The recoveries of two components were between 96.5 - 99 % by HPTLC method and 98.1 - 99.2 % by HPLC assay8. 5. A HPTLC method for simultaneous quantification of andrographolide was developed. The methanolic extract was used for isolation of different andrographolide eluted with different mixtures of chloroform: methanol and were characterized by spectral studies. The HPTLC plate was developed using chloroform: toluene:

3 methanol (6:2.5:1.5) at 540nm.The calibration curves were linear in the range 1- 25µg .Methanol was found suitable for the most efficient extraction of andrographolide derivatives10.

6. A study was conducted to standardize Phyllanthus amarus on the basis of its bioac-

tive lignans phyllanthin and hypophyllanthin by using a RP-HPLC and C18 column was employed for isocratic resolution using methanol: water as mobile phase. Esti- mation was done on various parts of the plants like roots, stem, branches, leaves and fruits. The methods has been found to be sensitive, precise and recorded more than 98% recovery amount of lignans. The leaves contain the highest amount of phyllanthin (0.7%w/w) and hypo phyllanthin (0.3%w/w) as compared with other parts of the plant6 .

7. The HPLC method was developed for the estimation of phyllanthin and hypophyllanthin. An isocratic RP HPLC procedure has been adopted using a mixture of PH 2.8 phosphate buffer and acetonitrile as mobile phase. CN column as stationary phase and UV detector. The method developed was relatively better in terms of separation, precise and reproducible for quantifying phyllanthin and hypophyllanthin4 .

8. Two simple and accurate methods to determine Phyllanthin (PTN) and Hypophyl- lanthin (HTN) was developed using spectrophotometry and RPLC. In spectrophotometry, PTN and HTN were quantitated at 259.2 nm and 252.4 nm respectively in methanol. Linearty was seen at 10 to 50 pg/mL for PTN and 4 to 20 pg/mL for HTN. In the LC method, analysis was performed with isocratic elution using a mixture of tetrahydrofuran: water: acetonitrile (10:50:40 ) at 230 nm. Calibration curves were linear in the concentration range 10-100μg/mL for PTN and 5-50μg/mL for HTN. Both methods were validated, and the results were compared statistically12.

9. The methanolic extract of Andrographis paniculata was divided into chloroform, methyl carbonate and methanol soluble fractions. Each fraction was submitted to a series of chromatographic separations individually to yield two new flavonoids, 5,7,20,30-tetramethoxyflavanone and 5-hydroxy-7,20,30-trimethoxyflavone. The structures of these compounds were established with the aid of spectroscopic methods, including analysis by 2D NMR spectroscopy13. 4

6.3: MAIN OBJECTIVES OF THE STUDY:

1. Formulation of individual drug Andrographis paniculata and Phyllanthus amarus. 2. Formulation of drugs containing Andrographis paniculata and Phyllanthus amarus. 3. Evaluation of all formulations for important constituents and other parameters. 4. Stability studies of all formulations for different parameters. 5. Compilation of data.

5 7. MATERIALS AND METHODS:

7.1: Source of the data: The required data will be obtained from:  Electronic data (Internet).  Published research papers.  Review articles from journals.  Library of IISC, Govt. College of pharmacy.  Indian Herbal Pharmacopoeia revised new edition, 2002.  Library of Natural Remedies, Bangalore.

7.2 Methods of collection of the data (including sampling procedure if any) . Collection and authentication of samples from renowned centre in Bangalore. . Extraction of drug will be utilized for preparation of tablet and liquid oral preparation. . Preparation of tablet and liquid oral formulations: Adjuvants are useful in the preparation of formulations is to be short listed. . One of the important factors required for the herbal formulation is the evaluation of different parameters such as uniformity of weight, friability and disintegration test for tablet preparation .Viscosity, leak test for liquids oral preparation has to be established. . Standardization methods: TLC and HPTLC studies will be carried out by using var-

ious mobile phases. The RF value will be calculated. Using HPLC, RT and area un- der the curve were calculated. . . Stability studies for different oral formulation is an important factor. Various parameters such as temperature and pH will be used for stability validation. . Compilation of data collected.

7.3: Does the study require any investigations or intervention to be conducted on Patients other human or animals? If so, please describe briefly: -NO-

7.4: Has ethical clearance been obtained from your institute in case of 7.3 -Not applicable-

6 8. LIST OF REFERENCES: 1. Srivastava SK, Bose PC, Ray GK, Mukherjee B. Ind. J Pharm.1959; 21(8):229 30. 2. Gaind KN, Dar RN, Kaul RN. Ind. J Pharm.1963; 7:225-26. 3. Subba Rao V. Ind. J Pharm.1963; 24:134. 4. Talukdar PB, Banerjee S, Chatterjee PK, Datta AK. Ind. J Chem.1968; 7. 5. Chauhan SK, Singh BP, Kimothi GP. Agarwal S. Determination of andrographolide in Andrographis paniculata by high performance thin layer chromatography. Ind. drugs 1999; 36:130-32. 6. Alpana S, Himanshu M,Ram KV,Madan MG. Chemical fingerprinting of Andrographis paniculata using HPLC ,HPTLC and densitometry. Phytochem. Anal .2004; 15:280-85. 7. Anupam S, Krishan L, Sukhdev SH. Standardization of the Indian crude drug kalmegh by high pressure liquid chromatographic determination of andrographolide. Phytochem. Anal.1992; 3:129-31. 8. Akowuah GA,Zheri I,Norhayati I,Mariam A. HPLC and HPTLC densitometric determination of andrographolide and anti oxidant potential of Andrographis paniculata .J Food comp and Anal.2006;19:118-26. 9. Sudhanshu S,Dharam CJ, Madan MG,Rajendra SB,Hari OM,Ram PS. HPTLC Analysis of hepatoprotective diterpenoids from Andrographis paniculata. Phy- tochem. Anal. 2000; 11:34-36. 10. AnupamS,Ravneet TS, Sukhdev SH.Estimation of phyllanthin & hypophyllanthin by HPLC in Phyllanthus amarus.Pytochem.Anal.1993;4:226-9. 11. Murali B,Amith MS,Dinesh JK,Samiulla DS.J Natural remedies.2001;1:55-9. 12. Pallavi R, Puroshottam P,Rajput SJ.Simultaneous determination of phyllanthin and hypophyllanthin in herbal formulation by derivative spectrometry and liquid chromatography .2009;5(18):151-58 13.. Kotteshwara RV,Vimalamma G,Venkata RC,Yew-Min T.Flavonoids and an- drographolides from Andrographis paniculata.Phytochem.2004;65:2317-21 14. Giddings K.Advances in chromatography .1968; 5:91-6.Indian herbal pharmacopoeia. Revised edition 2002:57-69:281-89 15. Indian pharmacopoeia.2007;3-2044.

9. Signature of the candidate:

7 (MANJULA .S.)

10. Remarks of the guide:

11. Name and designation of

11.1 Guide: Dr. S. SHASHIDHARA HOD.DEPT. OF PHARMACOGNOSY, GOVT. COLLEGE OF PHARMACY, # 2, P. KALINGA RAO ROAD, SUBBAIAH CIRCLE, BANGALORE-560027.

11.2 Signature

11.3 Head of the department Dr. S. SHASHIDHARA, GOVT COLLEGE OF PHARMACY, BANGALORE- 560027.

11.4 Signature

12. 12.1 Remarks of the chairman and principal

12.2 Signature

Dr. S. SHASHIDHARA, PRINCIPAL, GOVT. COLLEGE OF PHARMACY, BANGALORE-560 027