Rajiv Gandhi University of Health Sciences

Rajiv Gandhi University of Health Sciences

Karnataka, Bengaluru

“PHARMACOLOGICAL AND TOXICOLOGICAL STUDIES OF FRUITS OF LUFFA ACUTANGULA.ROXB ON RATS”

A Protocol submitted to Rajiv Gandhi University of Health Sciences Karnataka, Bengaluru

In partial fulfillment of the requirement for the award of

MASTER OF PHARMACY

IN

PHARMACOLOGY

KANAGARLA. N. P. GOWTHAM

Department of Pharmacology,

National College of Pharmacy,

Balraj-Urs Road,

Shimoga-577 201Karnataka-INDIA

Rajiv Gandhi University of Health Sciences, Karnataka, Bengaluru

Annexure – II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

01

Name and Address of the Candidate

K. N. P. GOWTHAM

S/O M. V. B. P. KUTUMBA RAO

DOOR NO: 8-149,

MALLAYA PETA

RAJAHMUNDRY-533105

02

Name of the Institution

NATIONAL COLLEGE OF PHARMACY,

BALRAJ-URS ROAD,

SHIMOGA-577 201KARNATAKA-INDIA

03

Course of the Study

Branch

M. Pharmacy, (Pharmacology)

04

Date of Admission to course

04-10-2010

05

Title of the Topic

“PHARMACOLOGICAL AND TOXICOLOGICAL STUDIES OF FRUITS OF LUFFA ACUTANGULA.ROXB ON RATS.”

06

Brief resume of the intended work

6.1. Need for the Study

Enclosure – I

6.2. Review of the Literature

Enclosure – II

6.3. Objective of the Study

Enclosure – III

07

Materials and Methods

7.1. Source of data

Enclosure – IV

7.2. Methods of collection of data

Enclosure – V

7.3. Does the study require any

Investigations on animals?

If yes give details

Enclosure – VI

7.4. Has ethical clearance been

obtained form your institution

in case of 7.3.

Enclosure – VI – A

08

List of References (About 4 – 6)

Enclosure – VII

09

Signature of the Candidate

10

Remarks of the Guide

The present research work is original and not published in any of the journals. This work can be carried out in our Pharmacology Department laboratory.

11

Name and Designation of

(in Block Letters)

11.1. Guide

11.2.Signature

11.3.Co-Guide (if any)

11.4.Signature

11.5. Head of the Department

11.6. Signature

Dr. I. J. KUPPAST

M.Pharm ,Ph.D , F.I.C.

PROFESSOR

National College of Pharmacy,

Balraj-Urs Road,

Shimoga-577 201

Karnataka-INDIA

N. A.

N. A.

Dr. K. L. MANKANI

M.Sc , Ph.D

Head of the Department

National College of Pharmacy,

Balraj-Urs Road,

Shimoga-577 201

Karnataka-INDIA

12

Remarks of the Principal

12.1. Signature

The present study is permitted to perform in the Pharmacology Department laboratory of our Institution and the study protocol has been approved by IAEC.

Principal

Enclosure – I

Brief resume of intended work:

6.1. Need for study:

Plants have had and still have a key role in history of life on earth. Plants have been part of our lives since the beginning of time; we get numerous products from plants, most of them not only beneficial but also crucial to our existence1. Before the onset of synthetic era, man was completely dependent on medicinal herbs for prevention and treatment of diseases2. Plants have evolved the ability to synthesize chemical compounds that help them, defend against attack from a wide variety of predators such as insects, fungi, herbivorous mammals. By chance, some of these compounds whilst being toxic to plant predators turnout to have beneficial effects when used to treat human diseases3. The use of plants to heal or combat illness is as old as humankind. In the present scenario, the demand for herbal products is growing throughout the world and major pharmaceutical companies are currently conducting extensive research on plant materials for their potential medicinal value4.

Luffa acutangula.ROXB is believed to have originated in India, where wild types still occur, but has now spread pantropically to all areas with a high rainfall. It is cultivated and locally naturalized in West Africa, from Sierra Leone to Nigeria. It is cultivated from the coastal areas to the semi-dry savanna, e.g. in Sierra Leone, Ghana, Benin and Nigeria.

Luffa acutangula.ROXB belongs to family Cucurbitaceae. Luffa acutangula is a large monoious climber. It is one of the vegetable plants with medicinal property. The stem of Luffa acutangula is 5 angled glabrous stem and trifid tendrils. Leaves are orbicular-cordate, palmately 5-7 lobed, scabrous on both sides with prominent veins and vein lets. The leaf juice is applied to skin infections such as eczema. Leaf sap is used as an eye wash to cure conjunctivitis, where as in some parts of West Africa leaf extract is applied on sores caused by guinea worms to kill the parasite. Flowers are yellow, males arranged in 12-20 flowered axilary racemes. Female flowers are solitary, arranged in axils of males. Ovary is strongly ribbed. Fruits are obovate, pale yellow brown in color, 4-10 cm long and 2-4 cm broad and outer surface being covered with 8-10 prominent longitudinal ribs. Fruit is divided into 3 chambers; inner part is fibrous and easily detachable as a whole from the outer part. Taste is bitter and transverse section shows papillose epidermis with striated thick cuticle. Immature fruits are used in cooking and also in soups and sauces. Young fruits are eaten raw by cultivators and small fruits are sometimes pickled. Seeds are black, ovoid-oblong, much compressed and winged. Seeds yield an edible oil that is, however, sometimes bitter and toxic. The fruits and seeds are sometimes used in herbal preparation for the treatment of venereal diseases, particularly gonorrhea. From the literature survey it is said to possess ribosome inactivating protein [Luffin A and B] and terpenes with immunomodulatory, anti-HIV, other pharmacological actions viz. Antidiabetic, antioxidant, anticancer. Other uses like as an ornamental climber for enclosure, mature dried fruits processed into sponges and used for scrubbing the body or for domestic purposes like washing utensils and as filters for local drinks such as palm wine. Industrial use is made of these fibers for making hats5-6.

The claim of anti-ulcer and hypolipidaemic activity and toxicological studies of fruits has not been studied so far scientifically. Hence the present study is aimed to investigate anti-ulcer and hypolipidaemic activity and toxicological studies of fruits of Luffa acutangula.ROXB.

Enclosure – II

6.2. Review of literature:

1. The head space volatile compounds of flowers of ridge gourd and bitter gourds were analyzed by solid phase micro extraction [SPME] coupled with Capillary Gas Chromatography/Mass Spectrometry [GC-MS]. In ridge gourd 16 volatiles were identified and 9 were tentatively identified which are of trans β-ocimene7.

2. Hepatoprotective activity of Luffa acutangula against CCl4 and Rifampicin induced liver toxicity in rats: a biochemical and histopathological evaluation8.

3. Studies on the constituents of Luffa acutangula.ROXB. Structures of acutosides A—G oleanane type triterpene saponins isolated from herb9.

4. X-ray sequence and crystal structure of luffaculin-1, a novel type 1 ribosome inactivating protein10.

5. Two proteins with ribosome inactivating, cytotoxic and abortifacient activities from seeds of Luffa acutangula. ROEM [Cucurbitaceae] 11.

6. Protein with abortifacient, ribosome inactivating, immuno- modulatory, anti-tumor and anti-AIDS activities from Cucurbitaceae plants12.

7. Apoptosis inducing activity of Luffa acutangula fruit in leukemia cells13.

8. A Chitotetrose specific lectin from Luffa acutangula Physico-chemical properties and the assignment of sugars in lectin binding site14.

9. Isolation, macromolecular properties and combining site of a chito-oligosaccharide specific lectin from exudates of ridge gourd15.

10. Relationships of Aulacophora beetles feeding behavior with Cucurbitaceae type in host crop16.

11. Luffangulin, a novel ribosome inactivating peptide from ridge gourd seeds17.

12. Crystallization and preliminary X-ray analysis of luffaculin, a ribosome inactivating protein from sponge gourd seeds18.

13. Trypsin inhibitors from ridged gourd seeds: purification, properties and amino acid sequence19.

14. Antimicrobial activity of whole plant of Luffa cylindrica against some common pathogenic micro-organisms20.

15. Combining ability studies in ridge gourd21.

16. Comparative anti-diabetic activity of some herbal plant extracts22.

17. Studies on the anti-microbial and anti-fungal activities of the ethanolic extract of Luffa cylindrica fruit23.

18. Luffa acutangula.ROXB. Tea promotes developmental toxicity to rats24.

19. PAF receptor binding antagonist activity of Malaysian medicinal plant25.

20. Ethno medicines for jaundice used in tribal area of North Maharashtra26.

21. Pharmacological potential of plants used in treatment of piles27.

22. Antioxidant activity of 5 vegetables- traditionally consumed by south Asian migrants in Bradford, Yorkshire, UK28.

23. Bilateral thalamic necrosis following ingestion of ridge gourd infested with coelomycete fungi [diploid] 29.

Enclosure – III

6.3. Objectives of study:

1. Collection and authentication of Luffa acutangula.ROXB.

2. Drying of fruits of Luffa acutangula.ROXB.

3. Extraction of whole plant using different solvents.

4. Phytochemical investigations of extracts.

5. Screening of anti-ulcer and hypolipidaemic activity.

6. Evaluation of Toxicological studies.

Enclosure – IV

MATERIALS AND METHODS:

7.1. Source of data:

The required data will be obtained from:

1. Electronic data [internet].

2. Published Research Papers.

3. Review and Research Articles from Journal.

4. Library, National College of Pharmacy, Shimoga, Karnataka, India.

Enclosure – V

7.2. Method of collection of data:

1. The plant of Luffa acutangula will be collected from local areas of Shimoga district, Karnataka.

2. Screening of Pharmacological activities – i.e., anti-ulcer and hypolipidaemic activity.

3. Evaluation of Toxicological studies.

4. Pharmacological studies include– hypolipidaemic activity and anti-ulcer activity.

Hypolipidaemic activity will be carried out with obese rats; the serum cholesterol level is examined after a period of time. Activity will be concluded by comparing the results of control, standard and test samples.

Anti-ulcer activity will be carried out either by pyloric ligation or by drug induction method. Ulcer Index will be calculated in the control and treated group.

Toxicological studies will be carried out as per the standard procedure as per OECD and finally the results are compared with treated and control group.

The results will be analyzed by ANOVA test.

Enclosure – VI

7.3. Does the study require any investigation or intervention to be conducted on patients or other humans or animals?

As per the standard procedure, to study the pharmacological and toxicological effects of fruits of Luffa acutangula will be carried out on the Wistar albino rats.

7.4. Has ethical clearance been obtained from your institution?

Ethical clearance is provided by the Institution.

Clearance number: NCP/IAEC/CL/22/05/2011-12.

Enclosure –VII

List of references:

1. http://evolvingwellness.com/posts/984/the-value-and-importance-of-plants-in-medicine/

2. http://www.botanical-online.com/theimportanceofplants.htm

3. http://en.wikipedia.org/wiki/herbalism

4. Kokate C K. Text book of Pharmacognosy, Nirali Prakashan, New Delhi. Ed.4. p. 3-4.

5. http://database.prota.org/dbtw-wpd/exec/dbtwpub.dll?AC=QBE_QUERY&BU=http://database.prota.org/search.htm&TN=PROTAB~1&QB0=AND&QF0=Species+Code&QI0=Luffa+acutangula&RF=Webdisplay

6. Jyothi V, Srinath Ambati and Asha Jyothi V, “The Pharmacognostic, Phytochem-ical and pharmacological profile of Luffa acutangula”, International Journal of Pharmacy and Technology; 2010, 2(4): 512-524.

7. Fernando L N and Grun I U, “Head space-SPME analysis of volatiles of the ridge guard [Luffa acutangula] and bitter guard flowers”, Flavour and Fragnance Journal; 2001, 16(4): 289-293.

8. Vishal B Jadhar, Vishnu N Thakare, Apuman A Suralkar, Avinash D Deshpandae and Suresh R Naik, “Hepatoprotective activity against CCl4 and Rifampicin induced liver toxicity in rats”, Indian Journal of Experimental Biology; 2010, 48(8): 822-829.

9. Naga O T, Tanaka R, Iwase Y, Hanazono H and Okabe H, “Studies on the constituents of Luffa acutangula.ROXB structures of acutosides A—G oleanane type triterpene, saponins isolated from herb”, Chemical and Pharmaceutical Bulletin(Tokyo); 1991, 39(3): 599-606.

10. Hou X, Chen M, Chen L, Meehan E J, Xie J and Haung M, “X-ray sequence and crystal structure of luffaculin-1, a novel type 1 ribosome inactivating protein”, BMC structural biology; 2007, 7: 29.

11. Ng T B, Wong R N and Yeung H W, “Two proteins with ribosome inactivating, cytotoxic and abortifacient activities from seeds of Luffa acutangula.ROEM [Cucurbitaceae]”, Biochemistry International; 1992, 27(2): 197-207.

12. Ng T B, Chan W Y and Yeung H W, “Protein with abortifacient, ribosome inactivating, immuno-modulatory, anti-tumor and anti-AIDS activities from Cucurbitaceae plants”, General Pharmacology; 1992, 23(4): 597.

13. Purushotham Reddy B, Raghuram Reddy A, Srinivas Reddy B, Venkata Mohan S and Sarma P N, “Apoptosis inducing activity of Luffa acutangula fruit in leukemia cells”, International Journal of Pharmaceutical Research and Development; 2010, 2(10): 109-122.

14. Vella Reddy Anatharam, Sankhavaram R Patanjali and Avadesha Surolia, “A Chitotetrose specific lectin from Luffa acutangula physico-chemical properties and the assignment of sugars in lectin binding site”, Proc. Int. Symp. Biomol. Struct. Interactions, Supplement Journal of Bioscience; 1985, 8(1,2): 403-411.

15. Vellareddy Anantharam, Sankhavaram R Patanjali, M Joginadha Swamy, Ashik R Sanadi, Irwin J Goldstein and Avadesh Surolia, “Isolation, macromolecular properties and combining site of a chito-oligosaccharide specific lectin from exudates of ridge gourd”, The Journal of Biological Chemistry; 1986, 261(31): 14621-14627.

16. Yang X, Kong C, Liang W, Zhang M and Hu F, “Relationships of Aulacophora beetles feeding behavior with Cucurbitaceae type in host crop”, Yengyongsheng Taixue Bao; 2005, 16(7): 1326-1329.

17. Wang H and Ng T B, “Luffangulin, a novel ribosome inactivating peptide from ridge gourd seeds”, Life Science; 2002, 70(8): 899-906.

18. Ma Q, Yao G, Wus, LiH, LiJ and Dong Y, “Crystallization and preliminary X-ray analysis of luffaculin, a ribosome inactivating protein from sponge gourd seeds”, Acta Crystallogr D Biol Crystallogr; 2000, 56(2): 185-186.

19. Haidar U C, Saha S K, Beavis R C and Sinha N K, “Trypsin inhibitors from ridged gourd seeds: purification, properties and amino acid sequence”, Journal of Protein Chemistry; 1996, 15(2): 177-184.

20. Indumathy R, Satheesh Kumar D, Kolagani Pallavi and Sashikala Devi G, “Antimicrobial activity of whole plant of Luffa cylindrica against some common pathogenic micro-organisms”, International Journal of Pharmaceutical Sciences and Drug Research; 2011, 3(1): 29-31.

21. Rao Y N and Reddy P V, “Combining ability studies in ridge gourd”, International Journal of Tropical Agriculture; 2000, 18(2): 141-146.

22. Patil Priyanka S, Patel M M and Bhavasar C J, “Comparative anti-diabetic activity of some herbal plant extracts”, An International Journal of Pharmaceutical Sciences; 2010, 1(1): 12-19.

23. Sashikala Devi G, Kottai Muthu A, Satheesh Kumar D, Rekha S, Indumathy and Nandhini R, “Studies on the anti-microbial and anti-fungal activities of the ethanolic extract of Luffa cylindrica fruit”, International Journal of Drug Development and Research; 2009, 1(1):105-109.

24. Luciana Cristina Borges Fernandes, Luiz Augusto Vieira Cordeiro and Benito Soto-Blanco, “Luffa acutangula.ROXB. Tea promotes developmental toxicity to rats”, Journal of Animal and Veterinary Advances; 2010, 9(8): 1255-1258.

25. Jantan I, Rafi I A and Jalil J, “PAF receptor binding antagonist activity of Malaysian medicinal plant”, Phytomedicine: International Journal of Phytotherapy and Phytopharmacology; 2005, 12(1, 2): 88-92.

26. Badgujarr S B and Patil M B, “Ethno medicines for jaundice used in tribal area of North Maharashtra”, Natural Product Radiance; 2008, 7(1): 79-81.

27. Lather A, Gupta V, Garg S, Singh A and Sachdeva K, “Pharmacological potential of plants used in treatment of piles”, Journal of Natura Conscientia; 2011, 2(1): 255-265.

28. Ansari N M, Houlihan L, Hussain B and Pieroni A, “Antioxidant activity of 5 vegetables- traditionally consumed by South Asian migrants in Bradford, Yorkshire, UK”, Phytotherapy Research; 2005, 19: 907-911.

29. De Souza A, Naruencar K P, Fernandes Y and Arun G, “Bilateral thalamic necrosis following ingestion of ridge gourd infested with coelomycete fungi [diploid]”, Journal of Neurological Science; 2010, 295(1,2): 104-106.