PROTOCOL FOR
M.PHARM DISSERTATION
SUBMITTED TO THE
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
BANGALORE, KARNATAKA.
BY
NEELUFAR.C.A
M.PHARM PART- I
DEPARTMENT OF PHARMACEUTICAL BIOTECHNOLOGY
2008-2009
UNDER THE GUIDANCE OF
Dr. T. TAMIZH MANI Ph.D.,
PROFESSOR ,
DEPARTMENT OF PHARMACEUTICAL BIOTECHNOLOGY,
BHARATHI COLLEGE OF PHARMACY,
BHARATHI NAGARA.
KARNATAKA-571422.
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
BANGALORE, KARNATAKA.
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1. / Name of the Candidate and Address(In Block Letters) / NEELUFAR.C.A,
H.NO: XI/629
MECKENZIE GARDEN
PATTALAM
COCHIN-1, KERALA
2. / Name of the Institution / BHARATHI COLLEGE OF PHARMACY
BHARATHI NAGARA, MANDYA. 571422
3. / Course of Study and Subject / MASTER OF PHARMACY IN PHARMACEUTICAL BIOTECHNOLOGY.
4. / Date of Admission of Course / 30-06-2008
5. / Title of Topic / BIOCHEMICAL CONSTITUENT VARIATION STUDY AMONG HEALTHY AND DISEASED SOYBEAN SEEDS AND THEIR MANAGEMENT
6. / Brief Resume of the Intended Work
6.1 Need for the study
6.2 Review of the literature
6.3 Objectives of the study /
ENCLOSURE-I
7 /
Materials and Methods
7.1 Source of data
7.2 Method of collection of data
7.3 Does study require any investigations or interventions to conduct on patients or other human or animal? If so, please describe briefly.
7.4 Has ethical clearance been obtained from your institution in case of 7.3
/ENCLOSURE-II
8 / List of References / ENCLOSURE-III
ENCLOSURE-I
6. BRIEF RESUME OF THE INTENDED WORK
6.1 Need For The Study:
Soybean (Glycine max (L)) is commonly called wonder bean since it is an excellent source of nutrients such as proteins, fats, carbohydrates, vitamins and minerals. Soybean is also a major source of phytochemicals such as isoflavones, spingo lipids and phenolic acids and shows anti-oxidant activity. Isoflavones reduce blood cholesterol level, incidence of cancer and osteoporosis. Eating 6.1g of soya protein per day provides 16-20 mg of soya isoflavones. Soybean is used extensively as human food, animal feed and for industrial purposes. All the products of soybean are of dietary importance such as soybean cheese, soybean milk, soybean oil, soybean meal.1
The present research is planned to analyze the biochemical constituents of soybean seeds with different degree of fungal infection over healthy seeds. Fungal diseases cause significant reduction in the protein, phenol, oil content and phytochemicals which has adverse effect for cancer treatment, osteoporosis and many diseases which are associated with blood cholesterol.2
The present research program concentrate on the incidence of seed borne fungus Colletotrichum truncatum, 3 Phomopsis sojae, and Macrophomina phaseolina on soybean seeds. These seed borne fungus affect seed germination, seedling vigour, seed weight and enters into the plant system which intern reduce the phenol, protein, oil content and the phytoconstituents of the seed.4 Hence the main objective of study is to diagnose seed borne diseases and planned to recommend producers, consumers and researchers to analyze nutritional and pharmaceutical value of infected and healthy seeds. The study also planned to recommend the use biopesticide for the management of seed borne fungal diseases
6.2 Review Of Literature:
6
1. Maria John Newton A et al., Reported that soy protein is safe, nutritious and possessing valuable medicinal properties. Soy isoflavones and other essential components present in the soy protein are contributing friendly role on health of the normal individuals and the patient under the risk of Cardio vascular diseases, cancer, osteoporosis and post menopausal problems.2
2. Goulart, Detected the following seed borne fungi on soybean viz. phomopsis spp. C.truncatum, C.Kikuchi, C.Sojina, Rhizostonia solani, S.Sclerotiorum, F.Semitectum and Aspergillus spp. Goulart (2000) during 1995-97 collected 985 seed samples of soybean and analyzed using blotter test.5
3. Lee S.J et al., Comparison of isoflavone concentrations in soybean (Glycine max (L)) sprouts grown under two different light conditions. There were no significant differences in total isoflavone concentrations between the green and yellow sprouts. Significant differences in total isoflavone amounts were observed among the three organs with roots exhibiting the highest total isoflavone concentrations followed by cotyledons and hypocotyls.6
4. Sinclair, Reported that discoloration of soybean seeds caused by various micro organisms and these seeds are source for diagnostic purpose. Discoloration of soybean seeds indicates undesirable physical qualities such as density, shape, size, surface area, volume and also presence of toxic metabolites.7
5. Bhutta et al., Recorded the effect of seed borne fungi on oil content and fatty acid profile in sunflower. It was observed that oil and unsaturated fatty acid content decreased with the presence of seed borne fungi and increased saturated fatty acid concentration.8
6. Hoffman et al., Studied yield and seed quality of soybean cultivars infected with Sclerotia sclerotiorum. Sclerotina stem rot (SSR) most important disease of soybean in USA. The SSR drastically reduces yield, 100 seed weight, seed protein, oil content, visual seed quality and seed germination. In addition, the number of sclerotia in seed samples and seed borne incidence of S.sclerotiorum were determined. Disease incidence was negatively correlated with seed germination, oil, protein content and seed weight.9
7. Hamawaki et al., Reported the seed-borne fungi P.sojae, C.dematium, F.semitectum with the percent incidence of 49.7, 2.1 and 47 in Uberland of Brazil. There was a negative correlation with seed vigour and fungal incidence.10
8. Prabakar, Reported that seed borne pathogens brings about qualitative changes in the physical and chemical properties of seed such as colour, odour, oil content, iodine value, refractive index and protein content of seeds.11
9. Dhingra et al., Soybean seed samples inoculated with spores of Aspergillus rubber were stored for 20-140 days at 250C with moisture content 11.3-17.7%, fungus colonized the seed at all moisture levels. Ergosterol and free fatty acids concentration in seed increased with increase in storage period.12
10. Norskove et al., The disease cycle of soybean anthracnose, caused by Colletotrichum truncatum was investigated. In infected seeds of soybean fungal hyphae generally occur in the seed coat, in case of heavy infection in the embryo as well, but never in the embryo alone. During germination the pathogen is transferred from the seed coat, by colonization of the cotyledons, to the seedling. The pathogen is occasionally present in the superficial stem tissue of the cotyledonary node, but has not been observed growing through the stem.13
3 6.3 Objectives Of The Study:
1. Effect of Colletotrichum truncatum, Macrophomina phaseolina, Phomopsis sojae pathogens on the nutritional qualities of soybean seed.
2. Isolation and characterization of proteins, phenols and oils.
3. Comparative study of proteins, phenols and oils in healthy and fungal infected seeds.
4. Isolation and characterization of phytochemicals.
5. Comparative phytochemical studies of fungal infected and healthy seeds.
6. Management of seed borne disease.
ENCLOSURE-II
7. MATERIALS AND METHODS:
The present work plan to undertake the collection of seed samples from Research Stations and from farmers and then it will be subjected to the following experiments.
Ø Seed germination and seedling vigour done by seed germination test by paper towel method.
Ø Seed health test for major seed borne fungi using standard blotter method.
Ø Determination of 1000 seed weight and seed moisture by using standard procedure of International Standard of Seed Testing14
Ø Isolation of protein, phenol, oil and phytochemicals from healthy and infected seeds.
All the above mentioned tests shall be carried out at department of pharmaceutical biotechnology. The lab is adequately equipped with necessary set up to carry out the works. The following instruments such as Hot air oven, Incubator, Autoclave, Cool centrifuge, HPLC, HPTLC, PCR, Gel Documentation system etc are available in the college lab.
7.1 Source Of Data:
Ø Bharathi College of pharmacy library, Bharathi Nagara.
Ø E-library from Bharathi College of pharmacy library, Bharathi Nagara.
Ø IISC library, Bangalore.
.
7.2 Method Of Collection Of Data:
Data collected from the following journals and related links.
Journals:
Ø Medicinal and Aromatic Plant Abstract.
Ø Journal of Agricultural and Food Chemistry.
Ø Indian Journal of Biotechnology.
Ø Journal of Biosciences, Biotechnology Research Asia.
Ø Journal of Plant Diseases.
Ø Journal of Natural Products.
Ø Indian Journal of Pharmaceutical Education and Research.
Ø Journal of Seed Science and Technology
Related Links:
Ø http://www.google.com
Ø http://www.Rxlist.com
Ø http://www.sciencedirect.com
Ø http://www.pubmed.com
Ø http://www.medline.com
Ø http://www.wekipidia.com
Ø http://www.wileyinterscience.com
Ø http://www.drugbank.com
7.2
.
7.3 Does the study require any investigation or interventions to be conductedon
patients or other humans or animals?
-NO-
7.4 7.4 Has ethical clearance been obtained from your institution in case of 7.3?
-NOT APPLICABLE-
ENCLOSURE-III
8. List Of References:
1. Coplin S. Using soy in every day meals. Journal of Nutraceuticals. Functional and Medical Foods. 2000;2(4):65-70.
2. Maria John Newton A, Karthikeyan M, Ramasamy C. Soy protein-A treatment supportive protein-An overview. Indian Journal of Pharm. Educ. Res. 2008;42(1):12-17.
3. Neergaard E, Tornoc C, Norskov AM. Colletotrichum truncatum in soybean, Studies of seed infection. Seed science and technology 1999;27:911-921.
4. Anwar SA, Abbas SF, Gill MM, Rauff CA, Seed borne fungi of soybean and their effect on seed germination. Pak Jour of Phytopathology. 1995;7(2):184-190.
5. Goulart ACP. Fungi in soybean seed: detection and importance. Documentos EMBRAPA Centro De Pesquisa sgropecaario do oesto 1997;11:58.
6. Lee SJ, Ahn JK, Khanh TD, Chun SC et al. Comparison of isoflavone concentration in soybean sprouts grown under two different light conditions. Journal of Agricultural and Food Chemistry. 2007;55(23):9415-9421.
7. Sinclair JB. Discoloration of soybean seeds an indicator of quality. Plant Dis. 1992;76(11):1087-1091.
8. Bhutta AR, Bhatta MHR, and Ahmad I. Effects of seed-borne fungi on oil content and fatty acid profile in sunflower. Helia. 1997;20(27):67-72.
9. Hoffman DD, Hartman GL, Mueller DS, Leitz RA, Nickell CD, et al. Yield and seed quality of soybean cultivars infected with Sclerotinia Sclerotiorum. Plant Diseases. 1998; 82(7): 826-829.
10. Hamawaki OT, Juliatti FC, Gomes GM, Rodrigues FA, Santos VLM. Evaluation of the physiological and health quality of genotype of soybean genotype seeds in Uberlandia. Minas Gerais. Fitopatologia Brasileira. 2002;27(2):201-205.
11. Prabakar K. Disease management for storage of hardened and pelleted seed. Seed Hardening and Pelleting Technologies for Rainfed/Gardenland Ecosystems. Tamil Nadu Agricultural University, Coimbatore, India. 2003; 70-73
12. Dhingra OD, Jhan G, Napoleao IT. Ergosterol accumulation and oil quality changes in stored soybean invaded by Aspergillus ruber. Mycopathologica. 1998;143(2):85-91.
13. Neergaard E, Tornoc C, and Norskov AM. 1999. Colletotrichum truncatum in soybean. Studies of seed infection. Seed Science and Technology 27:911-921
14. ISTA, International seed testing rules. Seed sciences and technology. ISBN 1999;3:906.
9 / Signature of Candidate
10 / Remarks of Guide / The Present Work Found To Lead New Dimension In The Field Of Pharmacetical Biotechnology.
11 / Name and Designation of
11.1 Guide
11.2 Signature / Dr. T. TAMIZH MANI . Ph.D.,
Professor,
Department of Pharmaceutical Biotechnology,
Bharathi College of Pharmacy,
Bharathi Nagara.
Karnataka-571422
11.3 Co- Guide (if any)
11.4 Signature / Dr. GURUKAR MATHEW.S Ph.D.,
11.5 Head of Department
11.6 Signature / Dr. GURUKAR MATHEW.S Ph.D.,
Professor and Head,
Department of Pharmaceutical Biotechnology,
Bharathi College of Pharmacy,
Bharathi Nagara.
Karnataka-571422.
12 / 12.1 Remarks of the Chairman and Principal
12.2 Signature / Recommended for approval
Prof. Dr. TAMIZH MANI. T
Principal
Bharathi college of pharmacy
Bharathi Nagara
Karnataka-571422