Proforma for registration of subjects for dissertation




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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA BANGALORE
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION



Name of the Candidate and Address (In block letters)

RAJNISH KUMAR SINGH

S/O SRI RAVINDRA NATH SINGH MADHUBAN (DIST- MAU) (U. P.) (221603)





Name of the Institution

Luqman College of Pharmacy, gULBARGA – 585 102.




Course of Study and Subject

M.PharmA. (PharmacOLOGY)



Date of Admission to Course

22/05/2009



Title of the Topic

ANTIHYPERGLYCEMIC EFFECT OF IPOMOEA CARNEA LEAVES IN STREPTOZOTOCIN INDUCED DIABETIC ANIMALS.


6. BRIEF RESUME OF THE INTENDED WORK



6.1 Need for the study:

Diabetes is a serious illness with multiple complications and premature mortality, accounting at least 10% of total health expenditure in many countries1. Diabetes prevalence among all age groups is projected to rise from 171 million in 2000 to 366 million in 20302. Diabetes mellitus (DM) is one such type which is a major health problem around the globe in recent time, Asia and Africa are the most viable areas where the disease is feared to raise 2-3 folds. DM is a metabolic disorder characterized by hyperglycemia with impairment of carbohydrate, fat and protein metabolism3.

Hyperglycemic condition causes increase glycosylation leading to biochemical and morphological abnormalities due to altered protein structure which over a period of time develops diabetic complications such as nephropathy, retinopathy, neuropathy and cardiomyopathy4. Traditional medicine derived mainly from plants play major role in the management of diabetes mellitus5.

World health organization (WHO) has recommended the evaluation of traditional plant treatments for diabetes as they are effective, non-toxic, with less or no side effect and are considered to be excellent candidates for oral therapy6. Recently there are many medicinal plants possessing experimental and clinical antidiabetic activity that have been used in traditional systems of medicine7.

The present work was under taken to explore the antidiabetic potential of the plant Ipomoea carnea (convolvulaceae). Ipomoea carnea was introduced to Egypt for ornamental purpose8. It was recorded as a neutralized species along canals and drains, road sides, waste lands and field edges in the Nile Delta9, 10. It has been reported that the pharmacological significance was noted due to the presence of various bioactive compounds in the Ipomoea carnea such as glycosides11, alkaloids11, reducing sugars11, flavones glycoside11, flavonoids12 and tannins 12.

Recent studies on diabetes claims that the flavonoids possess antidiabetic activity13, however the literature reveals no scientific data on antihyperglycemic effect of Ipomoea carnea leaves. In view of this, the present study is taken up to investigate the possible antidiabetic activity of Ipomoea carnea leaves in diabetic animals.






6.2 Review of the literature:

Ipomoea carnea (convolvulaceae) is widely distributed throughout the American tropics, Argentina, Brazil and Bolivia14, 15. This plant species has also been reported from India, West-Pakistan and Srilanka16, 17,18,19,20.

Ipomoea carnea which is glory species grows to a height of 6m on terrestrial land, but shorter in the aquatic habitats. The stem is thick and develops into a solid trunk over several years with many branches from base. The stem is erect, woody, hairy, more or less cylindrical in shape and greenish in color, and bearing alternate leaves. It attains 1.25-2.75m long and 0.5-0.8cm diameter. The fresh stem is somewhat flexible, but the dry one breaks with a fibrous fracture exposing a whitish green interior with hollow internodes and solid nodes. The internodes measure 3.5-6.0cm in length21, 22.

The leaf is simple, alternate, exstipulate and petiolate. Petiole is cylindrical, attains 4.0-7.5cm length and 2.5-3.0mm diameter. The leaf blade is cordate with symmetric base, measures 13-23cm in length and 5.5-9.5cm in width, with entire margin and reticulate pinnate venation, slightly hairy on both surfaces, the upper surface is dull green and the lower one is paler. Leaf plasticity related to light and moisture conditions. Shaded leaves may grow larger than leaves fully exposed to sunlight. In aquatic conditions differences between sun and shade leaves appeared to be higher than in dry conditions23, 24, 15.

Flowers of this plant are axial, solitary or arranged in monochasium scropioid cymose inflorescence. The pedicel is green in colour, erect, cylindrical, solitary slightly pubescent, measures 1.5-2.2cm long and 0.15-0.20cm diameter. The calyx is persistent, consisting of 5 free quincunial sepals, ovate in shape, with entire margin, symmetric base and acute apex, green in colour, nearly glabrous, measure 0.5-0.7cm long and 0.6-0.7cm width. The corolla is formed of 5 united petals (sympetalous), delicate, pinkish white in colour, with 5 pink to violet coloured strands in the regions of cohesion with each other. The mouth of the corolla has an entire margin, with slight conspicuous depressions at the points of the cohesion of the petals, measure 5.2-6.0cm long and 1.6-1.8cm width at its mouth8.



The literature survey reveals that the Ipomoea carnea leaves possess various bioactive compounds such as glycosides11, alkaloids11, reducing sugars11, flavonoids12, tannins12, hydrocarbons25, fatty acid25, esters25, and alcohol25. The leaves of this plant showed the presence of thirteen compounds which include hexadecanoic acid, steric acid, 1,2 diethyl phthalate, n-octadecanol, octacosane, hexatriacontane, tetraacontane, 3-diethylamino-1 propanol etc25.
Medicinal uses:

  1. Its ash is used for the treatment of skin disease26.

  2. The milky juice of this plant is used for the treatment of leucoderma26.

  3. It is used for the treatment of polluted tanks26.






LIST OF LITERATURE REVIEW OF IPOMOEA CARNEA:

Reports from modern literature:

  1. Ipomoea carnea leafy latex is exhibited to have wound healing activity12.

  2. Aqueous extract of Ipomoea carnea reported to have immunomodulatory activity27.

  3. The leaves of Ipomoea carnea said to have central nervous system depressant activity28.

  4. Ipomoea carnea leaves have said to possess sedative-hypnotic and muscle relaxant properties28.

  5. Ipomoea carnea makes guinea pigs a valuable animal model for the reproduction of induced alpha-mannosidosis29.

  6. Ipomoea fistulosa Linn (sub-species of Ipomoea carnea) reported to have in vitro antioxidant activity30.

  7. Synergistic effect of insecticides with plant extracts of Ipomoea carnea is reported against malerial vector, Anopheles stephens31.

  8. Antimicrobial activity of metal complexes prepared from the leaf proteins of Ipomoea carnea were reported32.

Review of literature, till date, regarding Ipomoea carnea was carried out by chemical abstract, biological abstract, medicinal abstract and other national and international scientific journals. The different parts of Ipomoea carnea have been screened for various pharmacological activities but there is no report regarding the anti-hyperglycemic activity of Ipomoea carnea leaves so far. Earlier studies reveals that flavonoids are responsible for antidiabetic activity13 and upon literature survey leafy latex of Ipomoea carnea is said possess flavonoids12 and tannins12. So present study is planned to investigate the possible antihyperglycemic activity of Ipomoea carnea leaves in diabetic animal model. Hence this study is essential and justifiable.



6.3 OBJECTIVES OF THE STUDY:

The literature review reveals that antihyperglycemic activities of Ipomoea carnea leaves have not been reported. In view of this, the present study is aimed to investigate the antihyperglycemic activity of the Ipomoea carnea leaves with the following objectives:



  1. Identification and authentication of the plant material.

  2. Extraction of Ipomoea carnea leaves with suitable solvents, such as petroleum ether, chloroform, ethanol and water.

  3. To carryout preliminary phytochemical analysis of crude extracts for the detection of the type of phytoconstituents present.

  4. To study the acute toxicity for determination of LD50 of the extract in mice.

  5. To evaluate the effect of Ipomoea carnea leaf extract for the hypoglycemic and antidiabetic activity in healthy and streptozotocin induced diabetes in albino rats and further for its hypoglycemic activity in healthy rabbits.



7. Materials & methods:




7.1 Source of data:

Whole work is aimed to generate data from the laboratory that is experiments on animals. Albino rats, mice and rabbit will be used for this purpose.

The scheme of proposed work is as follows:


  1. Collection of leaves and preservation.

  2. Shade drying, coarse powdering of leaves.

  3. To prepare extracts from suitable solvents.

  4. Preliminary phytochemical investigation of various extracts.

  5. To establish the pharmacological activities of various extracts.




7.2 Methods of collection of Data:

Plant Material : The Ipomoea carnea leaves are found throughout India.

The whole study is divided in 4 phases.



Phase I:

  1. Preparation of various solvent extracts:

It is planned to dry the leaves under shade at room temperature and pulverized. Than the powder obtained is subject to successive soxhlet extraction with the solvents with increasing order of polarity i.e. petroleum ether (60-80oc), chloroform (59.5-61.5oc), ethanol (64.5-65.5oc) and water. If further required the shade- dried powder is extracted directly with ethanol (hydro-alcoholic extract). The extract is allowed to concentrate under reduced pressure (bath temperature 5oc) and store in air tight container in refrigerator below 10oc . All these extracts are used for biological investigations and in vivo studies, after subjecting it to preliminary qualitative phytochemical analysis.

The solution of various solvent extracts of Ipomoea carnea leaves is planned for the following investigation.

2. Preliminary phytochemical screening 33, 34.

It is planned to carry out the preliminary phytochemical investigation of different extracts of Ipomoea carnea leaves for detection of various phytochemical by following standard method described in practical pharmacognosy by C.K. Kokate and R.K. Khandelwal.


Phase Ii:

Determination of Acute toxicity studies (LD50)35.

It is further planned to study the acute toxicity of solvent extract of Ipomoea carnea leaves in albino mice of either sex (20-30gm). Fixed dose method (OECD guideline number 420) of CPCSEA will be adopted for toxicity studies.


Phase IiI:

Anti-diabetic activity in rats 36, 37, 38.

Fasting blood glucose is planned to determine in rats (weighing 180-250gm) after depriving food for 16 hrs with free access of drinking water. Hyperglycemia is induced by a single intraperitonial injection of streptozotocin (50mg/kg) in buffer (pH 4.5). After 5 days of streptozotocin injection, the hyperglycemic rats (glucose level > 250 mg/dl) are separated and divided into different groups comprising of 6 rats each for the hyperglycemic study. Blood samples are collected from tail vein at different intervals from healthy and diabetic rats.



In Healthy Rats (Hypoglycemic activity):

Group I - Served as normal control and treated with vehicle.

Group II - Healthy rats + standard drug (glibenclamide; 10 mg/kg p.o.)

Group III - Healthy rats+ extracts (Selective dose of extract. p.o.)



In Diabetic Rats:

Group I - Served as diabetic control + streptozotocin and vehicle (aqueous gum acacia)

Group II - Diabetic Rats + standard drug (glibenclamide; 10 mg/kg p.o.)

Group III - Diabetic Rats + extracts (Selective dose of extract. p.o.)

Similarly the hypoglycemic and antihyperglycemic activity of other extracts is planned to carry in healthy and diabetic rats in different groups respectively.

Fasting blood glucose level is estimated by GOD/POD method by using semi auto analyser (BCA 201)39, 40.



Phase IV:

Hypoglycemic activity in rabbits 41.

Healthy rabbits (albino) of either sex weighing 2-3kg are fed in a standard pellet diet and water ad libitum. The rabbits are divided into different groups comprising of 6 each and fasted for 12 hrs for the hypoglycemic study. Blood samples are collected from ear marginal vein at different intervals from healthy rabbits.



In Healthy Rabbits (Hypoglycemic activity) :

Group I - Served as normal control and treat with vehicle.

Group II - Healthy rabbits + standard drug (glibenclamide; 5 mg/kg p.o.)

Group III - Healthy rabbits+ extracts (Selective dose of extract. p.o.)

Similarly the hypoglycemic activity of other extracts is planned to carry in healthy rabbits in different groups of animals.

The standard drug glibenclamide (5 mg/kg) is administered orally.

Fasting blood glucose level is estimated by GOD/POD method by using semi auto analyser (BCA 201)39, 40.
INCLUSION CRITERIA:

Normal and healthy animals weighing between 180-250gm for rats, 20-30gm mice and 2-3kg rabbits will be included in the study.





EXCLUSION CRITERIA:

The albino rats, mice and rabbit which do not fall the above mentioned weights are excluded from study.


STATISTICAL ANALYSIS:

The data obtained from the above findings for antihyperglycemic activity will be subjected to statistical analysis using one way ANOVA followed by Dunnett’s ‘t’ test.




7.3 Does the study require any investigation or intervention to be conducted on patients or other humans or animals? If so, please describe briefly.

Yes, the above study requires investigation on animals like albino mice, rats and rabbits.





7.4 Has ethical clearance been obtained from your institution in Case of 7.3?
Yes, the present study is approved from Institutional Animal Ethics Committee (registration number 346/CPCSEA; and IAEC copy enclosed).

08. List of References:


  1. King H, Aubert RE, Herman WH. Global burden of diabetes 1995-2025: prevalence, neumerical stimates, and projections. Diabetes care 1998; 21: 1414-1431.

  2. Amos AF, McCarty DJ, Zimmet P. The rising global burden of diabetes and its complications: estimates and projection to the year 2010. Diabetic Medicine 1997; 14: 1-85.

  3. Alberti KG, Zimmet PZ. New diagnostic criteria and classification of diabetes-again? Diabetic medicine 1998; 15: 535-536.

  4. Arky R A. Clinical correlates of metabolic derangements of Diabetes Mellitus. In: kozak, G.P. (Ed.), Complications of diabetes mellitus. Philadelphia; W.B. Saunders 1982: 16-20p.

  5. Ahmed I, Adeghate E, Cummings E, Sharma AK, Singh J. Beneficial effects and mechanism of action of Momordica charantia juice in the treatment of streptozotocin- induced diabetes mellitus in rat. Mol. Cell. Biochem 2004; 261: 63-70.

  6. Day C. Traditional plant treatments for diabetes mellitus: pharmaceutical foods. Br. J. Nutr 1998; 80: 203-208.

  7. Mankil J, Moonsoo P, Hyun CL, Yoon-Ho K, Eun SK, Sang KK. Antidiabetic agents from medicinal plants. Curr. Med. Chem 2006; 13: 1203-1218.

  8. Afifi MS, Amer MMA and El-Khayat SA. Macro-and micro morphology of Ipomoea carnea Jacq. Growing in Egypt. Part I. Leaf and flower. Mansoura J. of Pharmaceutical Science 1988a 3: 41-57.

  9. Boulos L. Flora of Egypt, Checklist. Cairo; Al- Hadara Publishing 1995: 283p.

  10. El-Sheikh M.A. Ruderal Plant Communities of the Nile Delta Region. Ph.D. Thesis, Tanta University, Tanta 1996: 189p.

  11. Tirkey K, Yadava RP, Mandal TK and Banerjee NL. The pharmacology of Ipomoea carnea. Indian Veterinarian Journal 1988; 65: 206-210.

  12. Ambiga S, Narayanan R, Gowri D, Sukumar d and Madhavan S. Evaluation of wound healing activity of Ipomoea carnea Jacq. Ancient science of Life 2007; 26 (3&4): 45 – 51.

  13. Fenglin Li, Qingwang Li, Dawei Gao and Yong Peng. The optimal extraction parameters and anti-diabetic activity of flavonoids from ipomoea batatas leaf. Afr.J.Trad.CAM 2009; 6(2): 195-202.

  14. Shinners LH. Convolvulaceae. In: Correll, D.S. and Johnston, M.C. (eds.). Manual of the Vascular Plants of Texas, Texas; Renner 1970: 1241-1261p.

  15. Frey R. Ipomoea carnea ssp. fistulosa (Martius ex Choisy) Austin: taxonomy, biology and ecology reviewed and inquired. Tropical Ecology 1995; 36(1): 21-48.

  16. Bhattacharyya PK. A note on two species of Ipomoea, namely I. carnea Jacq. and I. fistulosa Mart. ex Choisy in Eastern Asia. Journal of the Bombay Natural History Society 1976; 73: 317-320.

  17. Mahapata AK. A brief survey of some unrecorded, less known and threatened plant species of Sundarban of West Bengal. Bulletin of the Botanical Society of Bengal 1978; 32: 54-58.

  18. Sharma M. Aquatic and marshy angiosperms of Punjab. Bulletin of the Botanical Society of Bengal 1978; 31: 52-60.

  19. Austin DF and Ghazanfar S. Convolvulaceae. In: Nasir, E. and Ali, S.I. (eds.) Flora of West Pakistan. Agricultural Research Council, Islamabad 1979: 1-64p.

  20. Dassanayake MD and Fosberg FR. A Revised Handbook to The Flora of Ceylon, Vol 1. Amerind Publishing Co. Pvt. Ltd., New Delhi 1980: 508p.

  21. Afifi MS, Amer MMA and El-Khayat SA. Macro-and micro morphology of Ipomoea carnea Jacq. Growing in Egypt. Part II. Stem and root. Mansoura Journal of Pharmaceutical Science 1988b; 4: 88-97.

  22. Chaudhuri H, Ramaprabhu T and Ramachandran V. Ipomoea carnea Jacq. A new aquatic weed problem in India. Journal of Aquatic Plant Management 1994; 32: 37-38.

  23. Keeler KH. Ipomoea carnea Jacq. (Convolvulaceae) in Costa Rica. Brenesia 1975; 5: 1-5.

  24. Cook CDK. Ipomoea fistulosa: A new problem for India. Aquaphyte 1987; 7(1): 12.

  25. Vaishali Adsul, Eliza Khativora, Manik Kulkarni, Amuruta Tambe, Pushpa Pawar, Nirmala Deshpande. GC-MS Study of fatty acid, esters, alcohol from the leaves of Ipomoea carnea. Int. J. pharmatech Res 2009; 1(4): 1224-1226.

  26. Oudhia P. Botanical.com/site/column-poudhia/83-mites,html.

  27. Hueza IM, Fonseca ESM, Paulino CA, Haraguchi M and Górniak SL. Evaluation of immunomodulatory activity of Ipomoea carnea on peritoneal cells of rats. Journal of Ethnopharmacology 2003; 87( 2-3): 181-186.

  28. Ehattacharya SK, Ray A8, Dasgupta B. Central nervous system depressant activity of Ipomea carnea Jacq. Ind. J. pharmac 1975; 7 (4): 31-34.

  29. Cholich LA, Gimeno EJ, Teibler PG, Jorge NL, Acosta de Pérez OC. The guinea pig as an animal model for Ipomoea carnea induced alpha-mannosidosis. Taxacon international society on toxinology 2009; 54: 3.

  30. Kalpesh Gaur, Kori ML, Tyagi LK, Nema RK, Sharma CS and Priyanka Tripathi In-Vitro Antioxidant Activity of Leaves of Ipomoea fistulosa Linn. Academic Journal of Plant Sciences 2009; 2 (2): 60-64.

  31. Kuppusamy AT. Synergistic effect of insecticides with plant extracts against malarial vector; Anopheles stephensi. Trends Life Sci 1992; 7(1): 39-42.

  32. Agarwal R K, Uppadhay RK. Antimicrobial activity of metal complexes prepared from the leaf proteins of I. carnea Jacq. Indian Drugs Phar.Ind 1979; 14(2): 23-25.

  33. Kokate CK. Practical pharmacognosy 4th ed. New Delhi; Vallabha Prakashan 1999: 169p.

  34. Khandelwal KR. Practical pharmacognosy. 11th ed. Pune; Nirali Prakashan 2004: 149p.

  35. Mrs. Prema Veeraraghavan. Expert consultant, CPCSEA, OECD guide line No. 420, 2000.

  36. Mukhtar HM, Ansari SH, Ali M, Naved T, Bhat ZA. Anti-hyperglycemic activity of Psidium guajava barks extract. J Nat Rem 2004; 4(2): 150-54.

  37. Joy KL, Kuttan R. Anti-diabetic activity of Picrorrhiza kurroa extract. J Ethnopharmcol 1999; 167: 143-148.

  38. Sridevi S, Chary MG, Krishna DR, Prakash Diwan V. Pharmacodynamic evaluation of transdermal drug delivery System of glibenclamide in rats. Indian Journal of Pharmacology 2000; 32: 309-312.

  39. Tietz NW. Clinical guide to laboratory tests, Philadelphia, W.B. Saunders Co. 1976.

  40. Trinder P. Ann. Clin. Biochem 1969; 6: 24p.

  41. Mohammed Habibuddin, Hassan Ali Daghiriri, Touseef Humaira, Mohammed Saeed Al Qahtani, Ali Al Hasan Hefzi. Antidiabetic effect of alcoholic extract of Caralluma sinaica L. on streptozotocin – induced diabetic rabbits. J. of Enthopharmacology 2008; 117: 215- 220.





9.


Signature of Candidate


RAJNISH KUMAR SINGH


10.


Remarks of the Guide

The study is highly justifiable and is feasible to work in the institution. This work may throw light on the leaves of avenue plant i.e. Ipomoea carnea.


11.


Name & Designation of (in block letters)








11.1 Guide

Md. SAIFUDDIN KHALID

M.PHARM

ASST. PROFESSOR

DEPT. OF PHARMACOLOGY

LUQMAN COLLEGE OF PHARMACY,

P & T COLONY,OLD JEWARGI ROAD,

GULBARGA-585102 ( KARNATAKA).





11.2 Signature








11.3 Co-Guide


----





11.4 Signature


----

12.


12.1 Remarks of the Chairman & Principal

We will provide all the necessary facilities required for the proposed research work. So recommended for registration.








12.2 Signature





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