Evaluation of Psychopharmacological and Biochemical Activities on Photo-Detoxified Venom

“Evaluation of psychopharmacological and biochemical activities on photo- detoxified venom sac extract of Vespa orientalis venom”

SYNOPSIS FOR M.PHARM DISSERTATION

SUBMITTED TO

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES BENGALORE, KARNATAKA

SUBMITTED BY

MAHESH BABU TUDICHERLA 1st YEAR M.PHARM DEPARTMENT OF PHARMACOLOGY P.E.S COLLEGE OF PHARMACY BANGALORE – 560050 (2009-2011)

1 RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES BANGALORE, KARNATAKA. ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION

1.0 NAME AND ADDRESS OF THE CANDIDATE. MAHESH BABU TUDICHERLA LOCAL ADDRESS #808, NEW BLOCK, PESIT BOYS HOSTEL, BSK III Stage, BANGALORE 85

PERMANENT ADDRESS #87-394-2, KAMALA NAGAR, B CAMP, KURNOOL-2, A.P

2.0 NAME OF THE INSTITUTION. P.E.S. COLLEGE OF PHARMACY Hanumanthnagar, 50 feet road, B.S.K 1st stage, Bangalore – 50.

3.0 COURSE OF STUDY AND MASTER OF PHARMACY SUBJECT. PHARMACOLOGY.

4.0 DATE OF ADMISSION TO 28th May 2010 COURSE.

5.0 TITLE OF THE TOPIC:

“ Evaluation of psychopharmacological and biochemical activities on photo-detoxified venom sac extract of Vespa orientalis venom”

2 6.0 6.1 NEED FOR THE STUDY: The venom of social wasp Vespa orientalis is known to contain several active constituents, used by wasp to paralyse the prey and a defensive armamentarium. Venom of solitary wasp Vespa troplea have been shown (to attack Drosophila larval neuromuscular junction presynaptically and post synaptically23. The chemical constituents present in these venom are acetyl choline, serotonin, adrenaline, nor-adrenaline, dopamine, kinins, and high molecular weight

compounds like phospholopase-A2, hyaluronidase, histidine decarboxylase, acid alkaline, and neutral DNAase, poly and disaccharides, and several polycationic peptide and protein acting together to produce bilolgical effects29. The social wasps are found in abundance at bee hives. The venom of Vespa orientalis present in the southern Indian subcontinents is not extensively studied for their pharmacological properties. In our efforts to explore the therapeutics of naturally occurring active constituents, systematic investigation of venom of Vespa orientalis to know the significance of several active constituents producing constellation of effects, will contribute. Evaluation of photooxidized species from Venom sac extract (VSE) will be helpful to understand implication of specific alterations using various psychopharmacological models in rats having treatment protocol. Previous studies on hornet envenomation suggested that anti cholinesterase like activity present in basic venom fractions caused specific CNS effect in dog, cat, and mice11. Photooxidation of snake venoms in UV-light at deferent time extents in presence of sensitizer methylene blue resulted in detoxification of venom, and lead to significant changes with no loss of antigencity30. Further Photooxidation of echis carninatus venom had shown to resulted antidepressant and CNS stimulant activity following intracerebral injection31. The aim of the present work is to investigate the possible psychopharmacological effect of photooxidized Vespa orientalis venom protein (PVOVP) using various animal models

6.2REVIEW OF THE LITERATURE:

The Vespa orientalis belongs to the genera vespidae, it is a social type of wasp. The venom secreted by these wasp was used to paralyze the prey. The venom of Vespa orientalis consists of active constituents like Acetylcholine. Histamine, 5-HT, Adrenaline and Nor-adrenaline and kinins4. It was reported that amines secreted from the venoms of Vespa orientalis was found to potentiate the pain producing action of Bradykinin24. The venom also possess various enzyme such as hyaluronidase, protease4 and deoxyribonuclease (acid neutral and alkaline). A small peptide Orientotoxin obtained from these giant hornet Vepsa orientalis found to have potent hemolutic Phospholipase-A activity21. The venom also have been shown to possess anticoagulant13, hyperglycemic11, hypotensive and bronchoconstriction (Kaplinsky et al. 1974), cause increase in the permeability of microcircular vessels4, and disruption of the brain electrical activity in cats (Ishay et al . 1974b). Venom of Vespa orientalis has been shown to cause paralysis of the striated muscle in cat, rat and mouse4. Contracts guinea pig ileum9 and that of frog rectus muscle12. A peptide obtained from the venom called Mastoparan found to possess mast cell degranualation releasing histamine21. The venom of Vespa orientalis possess a low molecular weight peptide HR1 and HR2 which degranulate rat mast cells similar to MCD peptide of honey bee venom21. The active principle isolated from the Vespa orientalis “Orientotoxin” have been shown to possess potent hemolytic activity21. An amphipathic peptide “Mastoparan which i8s a mast cell degranulating peptide with 13 amino acid residue isolated from giant hornet Vespa orientalis. This peptide has the capability of causing degranulation of the rat mast cells there by initiating histamine release1 The venom has various pharmacological properties like it decreases the mean blood pressure after intravenous administration in dogs. This hypotensive effect elicited by Vespa orientalis coule be due to presence of histamine, Acetylcholine, and serotonin and was not abolished in the presence of antagonists of acetylcholine, histamine, catecholamines, 5-HT indicating that non receptor mediated fall in Blood pressure16 Joshua et al (1971) and Joshua and Ishay (1973) reported that Vespa orientalis venom produced

4 lyses at concentration from 0.75-1.5 ug/ml. of human, guinea pig, cat, rabbit, rat, and mouse erythrocytes. The study of anticoagulant activity of venom sac extract (VSE) showed that the incubation of human plasma or fibrinogen with VSE and prolonged recalcification showed the presence of clear-cut antithromboplastic activity15. Vespa orientalis could induce hyperglycemia in cats, the factor responsible for this action could be due a protein or a protein bound substance10. Intradermal injection of Vespa orientalis venom increases the permeability of microcirculatory vessels after injection of 5-10 ug of venom into rats and rabbits 4. This venom elicited an immediate bronchoconstriction in guinea pig at a dose of 5-25 ug/kg, iv, which may be due to histamine and acetylcholine release4 and in dog, tachyapnea after i.v injection. Injection of Vespa orientalis venom at a dose of 200-500 µg/kg. Into ispilateral femoral artery induced twitches of cat log muscle gradually dimished and disappear and similar action of paralysis in cat muscle gradually dimished and disappear and similar action of paralysis in cat muscle gradually dimished and disappear and similar action of paralysis in cat muscle seems be to derived from the central and peripheral effects of venom. The central effect could be effect on spinal motoneurons and peripheral effect could be a manifested by neuromuscular blockade4 The lethal factor of molecular weight 43 k Da was extracted from the venom of Vespa orientalis19. The toxic protein isolated from wasp Vespa orientalis with typical antiacetylcholinestrase activity which induced CNS manifestations such as salivation, vomiting, convulsions, pinpoint pupil, hind limb paralysis and death due to respiratory paralysis after 4 days11. Hornetin (32 k Da, LD50 0.92 ug/g/mouse) a neurotoxin isolated from wasp, Vespa flavitarsus, which showed presynaptic and musculotropic effects on mammalian and avian neuromuscular junction8.

RESEARCH QUESTION  Comparative evaluation of activity profile of Vespa orientalis venom, photooxidized species with reference to various hydrolytic enzymes namely, Phospholipase-A2 , Proteases, hyalurodinase, 5’ – Ribonucleotidase, Phosphodiesterase, and activity profile namely, hemolytic activity, anti-cholinesterase activity, smooth muscle contractility using

5 guinea pig ileum for histamine and rat fundus strip for 5-HT.

 Determination of minimum lethal dose.

 Psychopharmacological studies of venom and its photooxidized venom products for its effects in rats on memory and learning in rats.

6.3 OBJECTIVE OF THE STUDY:

1. It is aimed to evaluate comparatively enzyme and activity profile of the venom of Vespa orientalis, and its photo-oxidized species.

2. To investigate the psychopharmacological effects of venom and its photooxidized species on memory and learning in rats by using acetylcholine, dopamine, dopamine, 5-HT antagonist

7.0 MATERIALS AND METHODS :

7.1 SOURCE OF DATA :

Whole experiment is planned to generate data from the laboratories studies. Experiment will be performed as described in the standard procedures, may be obtained from standard journals and text books available within the college or from other institutes.

IISc library, Bangalore. PESCP library, Bangalore. RGUHS digital library (Helinet), Bangalore. Websites: www.ncbi.nlm.nih.gov/pubmed/ www.google.com www.ijp-online.com www.sciencedirect.com Will be used to obtain related information regarding this research protocol.

6 7.2 METHODS OF COLLECTION OF THE DATA (including sampling procedure if any):

METHOD OF COLLECTION OF DATA: Laboratory based studies includes:

1. METHOD OF VENOM COLLECTION:

Venom from Vespa orientalis will be collected by squeezing the venom sac. The pooled venom will be homogenized, centrifuged and supernatant is stored at 15 °C-20 °C according to the method with modification4. 2. METHODS FOR DETERMINATION OF ENZYMATIC ACTIVITIES:

A: Assay of proteolytic activity: The proteolytic activity was assayed by using casein and denatured hemoglobin as a substrate. Casein digestion: A casein suspension was incubated with 0.5 ml of varying concentration of venom at 37 C for 15 minutes. The reaction mixture was stopped by adding 0.1 ml of 0.44M Trichloroacetic acid. After standing for 30 minutes, it was filtered and to the filtrate added 2.5ml of 0.4M sodium carbonate and 0.5ml of Folin-ciocateu reagent are added. The optical density was measured at 660 nm. Denatured hemoplobin: 1gm of hemoglobin in 40 ml water was denatured byheating for 30 minutes at 800C and the pH of the solution was adjusted to 8.5 with sodium hydroxide and the same assay procedure was carried out as described above by replacing casein with hemoglobin. B. Assay of Phospholipase-A activity It is assayed by the trubidimetic methos using egg yolk as a substracte (Marrinetti. 1965). A fresh egg yolk suspension was prepared in 0.9% sodium chloride. This suspension was diluted with 0.9% sodium chloride in such a way that the absorbance at 925 nm was 0.9% sodium chloride in such a way that the absorbance at 925 nm was around 1.2.varying concentration Of venom (0.1 ml) was incubated with 2 ml of this egg yolk suspension at 37 c for 20 minutes .The reaction was stopped by adding 3 ml of ice-cold 0.9%sodium chloride and optical density

7 was measured at 925 nm against 0.9% sodium chloride C.Assay of 5` nucleotdiase : The 5`nucleotidase activity was determined by20 with modification25 using adenosine Monophosphate (AMP) as a substrate .the reaction mixture containing0.1 ml of 1M glycine buffer at pH9,0.1ml of 0.1 magnesium chloride ,0.3ml of 0.01M AMP and 0.1kl of venom solution of varying concentration the reaction mixture was incubated at 37 c for 15 minutes, and liberated phosphate was measured by colorimetric method5 D. Assay of phosphodiestrate activity25 It was determined by using calcium-di-nitrophenylphosphate as Substrate .the reaction mixture contained 0.11 of 0.1M tris-hcl buffer at pH 8.9,1.2ml of 0.001M calcium-dinitrophenylphosphate ,0.2ml of venom solution of varying concentration and distilled water to make 3ml .the reaction mixture was incubated at 37 c for 30 mintues and reaction was stopped by adding 3ml of 0.05N

Sodium hydroxide .the absorbance was measured at 400nm against blank. E.Assay of Hyaluronidase: 1% sheep red blood cells were incubated with venom in tris-buffer (0.01M pH 7.4) saline at 37 c for 60 minutes22. The hemolytic action was stopped by adding cooled tris-buffer saline (4 c).the degree of hemolysis was determined by measurement of release of hemoglobin at wavelength of 540 nm. F.Assay of Hyaluronidase: Hyaluronidase activity will be performed using turbidimetric27.

II. PHARMACOLOGICAL EXPERIMENTS: 8.0 A. Toxicity studies: Determination of MLD by various route of administration to mice (oral, iv or im)

B. The dose of the venom requirement 1/10th for treatment protocol of venom for psychopharmacological studies in rats as follows:

Psychopharmacological models:

8 1.Open field Test : An open field test apparatus consists of circular arena (wall height 27:diameter 84cm ) with 25 houses equipped with light and sound (78 dB)sources .The rats will be placed individually in the circular open arena of apparatus .When rats move from one segment to another ,one ambulation (simple stereotype ) score will re recorded .similarly when rat stand on its hind limbs with or without support of wall, one rearing (complex stereotypy)score will be recorded. Urination and defecation are observed.

2.elevate plus maze: This apparatus consists of two open (47X14cm) and two closed arms (4X14X37 cm) facing each other with open roof. the entire maze is elevated at height of 47 cm. the rats will be placed individually at end of the open arm and time taken to move from open to closed arm will be recorded (transfer latency).For the study effect of on learning (acquisition), the sample will be administered 15-30 minutes prior to trial, and the effect on retention (memory) will be studied (by administering 30 minutes after first day trial).

3.Passive avoidance test :- This passive avoidance task is a one trial fear-motivated avoidance task in which the mouse learns to refrain from stepping through a door to an apparently safer but previously punished dark compartment. Training chamber consist of a rectangular chamber divided into 2 compartments. One compartment is lighted by an overhead stimulus light and the other is black so as to remain dark. The two compartments are separated by an automatic guillotine door and each has a grid floor placed through which a foot shock can be delivered.

Procedure Habituation On habituation day mouse is placed in the lighted compartment, facing away from the dark compartment and allowed to explore for 30 seconds. After 30 seconds the door is raised and the mouse is allowed to explore freely. When the mouse enters the dark compartment with all four paws, the guillotine door is closed, and the latency to enter is recorded (from the time the door is lifted). Once the mouse crosses to the darkened chamber the door closed and the mouse is 9 10