Rajiv Gandhi University of Health Sciences Karnataka, Bengaluru

“PHARMACOLOGICAL AND TOXICOLOGICAL STUDIES OF THE PLANT BARK PTEROCARPUS MARSUPIUM ”

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

In partial fulfillment of the requirement for the award of

MASTER OF PHARMACYIN

PHARMACOLOGY

Mr. DHARSHAN.S.

Department of Pharmacology,National College of Pharmacy,

Balraj-Urs Road,Shimoga-577 201

Karnataka-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

Mr. DHARSHAN.S#12 ‘E’ BLOCK, B.G. NAGAR,NAGAMANGALA(TQ),MANDYA(DIST).PIN CODE: 571448KARNATAKA, INDIA.

02 Name of the Institution

NATIONAL COLLEGE OF PHARMACY,BALRAJ-URS ROAD,SHIMOGA-577 201KARNATAKA-INDIA

03 Course of the StudyBranch M.PHARM. (PHARMACOLOGY)

04 Date of Admission to course O1/08/2013

05 Title of the Topic

PHARMACOLOGY AND TOXICOLOGICAL STUDIES OF THE PLANT BARK PTEROCARPUS MARSUPIUM.

06

Brief resume of the intended work6.1. 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 Methods7.1. Source of data Enclosure – IV

7.2. Methods of collection of data Enclosure – V7.3. 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

Mr. VEERASHEKAR.T. M.Pharm

LecturerNational College of Pharmacy,Balraj-Urs Road,Shimoga-577 201Karnataka-INDIA

11.2. Signature

11.3. Head of the Department Dr. I. J. KUPPAST M.Pharm , Ph.D , F.I.C.

HEAD, DEPARTMENT OF PHARMACOLGYNational College of Pharmacy,Balraj-Urs Road,Shimoga-577 201Karnataka-INDIA

11.4. Signature

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 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 while being toxic to plant predators turn out 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.

Plants from the genus Pterocarpus Marsupium have been used in traditional

medicine by many cultures. Different stem bark samples(Apical bark, Middle bark, &

Mature inner bark) were analyzed with respect to phytoconstituents total reducing

sugar, amylose, amylopectin, starch, crude fibers, crude protein, total polyphenols,

water soluble tannins, total flavonoids, total alkaloids, nitrate, total ash value

constituents have been reported as the major phyto-constituents of the Pterocarpus

Marsupium species5.

Scientific classification:

Synonym: IndianKino, Bijasal, Vijayasagar, Bibla.

Family: Fabaceae

Domain: Eukaryota

Kingdom: Plantae

Subkingdom: Viridaeplantae

Phylum: Magnoliophyta

Subphylum: Euphyllophytina

Class: Magnoliopsida

Subclass: Rosidae

Super order: Fabanae

Order: Fabales

Genus: Pterocarpus

Speies: Marsupium6

Pterocarous marsupium belongs to Fabaceae family is a deciduous tree,

commonly called as Indian Kino Tree or Malabar Kino. The bark exudes a red gummy

substances called ‘Gum Kino’. When injured, Gum Kino is used in the treatment of

polyurea & inordinate night sweat and phthisis pulmonalis. The gum is used in the

toothache5. Bark is useful in vitiated condition of kapha and pitta, elephantiasis,

erysipelas,urethrorrhea,rectalgia,opthalmopathy,hemorrhages,dysentry,cough,grayness

of hair. Aqueous infusions of the bark posses antidiabetic potential7. Pterocarpus

Marsupium is distributed in deciduous forest throughout the India8.

Bark is useful in urinary discharge and piles. The Gum Kino is externally

applied to Leucorrhoea9. Tribal people residing in the Jodhalal forest of Karnataka use

stem bark to treat the wounds, fever, stomach ache, diabetes and elephantiasis10.

The powdered bark is mixed with schleichera aleosa and taken with cold water

to treat dysentery11. The juice of the bark is applied in the mouth12.

The claim of antiulcer, anticonvulsant and diuretic activity of the bark of the

plant Pterocarpus marsupium has not been studied so far scientifically. Hence the

present study is aimed to investigate antiulcer, anticonvulsant, diuretic activity and

toxicological studies of barks of the plant Pterocarpus marsupium.

Enclosure – II

6.2. Review of literature:

1) K.L. Mankani, et.al (2005) carried out the studies on evaluation of hepatoprotective

activity of stem bark of Pterocarpus marsupium Roxb. and the result has been

concluded that the methanol extract of the stem bark of Pterocarpus marupium

possess significant hepatoprotective activity13.

2) M.Manickam, et.al (1997) carried out studies on antihyperglycemic activity of

phenolics from Pterocarpus marsupium and the result has been conclude that the

Pterocarpus marsupium significantly lowered the blood glucouse level of

hyperglycemic rats14.

3) Arpita Sikdar, Anirban Biswas, Sanjib Bhattacharya, Moulisha Biswas (2013)

carried out studies on assement of analgesic activity of Pterocarpus marsupium leaf

extracts in swiss albino mice and the preliminary study has been shown marked

analgesic activity of Pterocarpus marsupium leaf in swiss albino mice15.

4) Anupama.A. Suralkar*, et.al (2012) carried out studies on anti-allergic, anti-

anaphylactic and mast cell stabilization activity of Pterocorpus marsupium and the

result suggest us that Pterocarpus marsupium may prove to be potential therapeutic

drug for treating allergic diseases such as allergic asthama16.

5) Akansha Mishra, et.al (2013) studied the anti-diabetic activity of heart wood of

Pterocarpus marsupium and the result has been shown that significant

improvement on oral glucose tolerance port sucrose lood in normal rats17.

6) Mohire N.C., Salunkhe V.R., Bhise S.B., Yadav A.V.,(2013) carried out studies on

cardiotonic activity of aqueous extract of heartwood of Pterocarpus marsupium and

the present results indicated that a significant increases in height of force of

contraction with decreases in heart rate has been reported18.

7) Bhupendra Chauhan, A. Mrendra Kumar Chaudary (2011) carried out studies on

memory enhancing activity of methanolic extract of Pterocarpus marsupium, and it

has been shown promise as a memory enhancing agent in all the lab models19.

8) Udaysing hari patil and Dattatraya.K. Gaikwad (2011) carried out studies on

phytochemical screening and microbial activity of stem bark of Pterocarpus

marsupium and the result has been shown that stem bark was effecient in inhibiting

growth of bacteria20.

9) Kachhawa.JBS, Sharma.N., Tyagi.S., Gupta.R.S., Sharma.K.K. (2012) studies the

evaluation of antibacterial activity of Pterocarpus marsupium and the results has

been shown that highly significant activity against the bacteria21.

10) Mohammed Rageed, et.al. (2012) studied the anti-inflammatory activity of

Pterocarpus marsupium stem bark on albino rats and the study has been concluded

that the flavonoids present in the stem bark is responsible for anti-inflammatory

activity22.

Enclosure – III

6.3. Objectives of study:

1. The bark of the plant Pterocarpus marsupium will be collected from the local

area of Shimoga district, Karnataka and authenticated by the botanist.

2. Bark of Pterocarpus marsupium will be dried and powdered

3. Extraction and fractionation of the bark using different solvents.

4. Phytochemical investigations of extracts.

5. Screening of antiulcer, anticonvulsant and diuretic activity.

6. Study of LD50 of various extracts of plant bark of pterocarpus marsupium.

7. Result will be analyzed by ANOVA test.

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 Pterocarpus marsupium will be collected from the local areas of

Shimoga district, Karnataka. The bark has to be cut into pieces and dried. Then

the dried bark material will be pulverized separately into coarse powder by a

mechanical grinder. The resulting powder will be used for extraction by

soxhalation.

2. The obtained extract is subjected to phytochemical analysis.

3. Screening of Pharmacological activities i.e.- antiulcer, anticonvulsant, diuretic

activity.

4. Evaluation of Toxicological studies.

Antiulcer activity will be carried out with Wistar albino rats of both

sexes (150-200 gm.). Rats will be fed standard rats pallet diet.

I. Pyloric Ligation method

Rats will be divided into five groups of six animals.

Group I- control – will receive vehicle.

Group II- III, IV- will receive plant aqueous, ethanolic and chloroform extract

respectively.

Group V - standard drug (Omeprazole 20mg/kg b.w.).

In this method Albino rats will be fasted in individual cages for 24 hours, and

then standard drug, plant extracts and control vehicle will be administered 30 min.

prior to pylorus ligation under light ether anaesthesia and abdomen will be opened

and pylorus will be ligated. The abdomen will then be sutured, after 4 hours the

animals will be sacrificed, the abdomen will be opened and oesophageal end of the

stomach will be tied and separated from the body and gastric juice will be collected

in to graduated centrifugation tube and centrifuge at 1000 rpm for 10 min. and

gastric volume will be noted and pH will be recorded by pH meter. The supernatant

contents will be subjected to analysis for total and free acidity. The stomach will be

opened to observe ulcer and severity will be graded.

II. Indomethacine Induced Ulcer Method:-

Wister Albino Rats weighing 150-200 gm. will be used and randomly allotted in

to five groups of six animals.

Group I – (control). Indomethacine 20mg/kg body weight will be given orally,

after 24 hours of fasting, and then after 4 hours animal will be killed and

histological changes will be observed.

Group II, III & IV – (Treated). aqueous, ethanolic & chloroform extracts of bark

will also be given respectively orally for 5 days once daily. After completion of 5

days, rats will be kept for 24 hours fasting and then Indomethacine (20 mg/kg) will

be given orally and after 4 fours animals will be sacrificed and study will be done.

Group V – (standard) Omeprazole 20mg/kg will be administered intra peritoneally

as a standard for 6 days. On 6th day, animals will be fasted and Indomethacine will

be administered 4 hours after Omeprazole administration. Rats will be killed after 6

hours, stomach dissected out then ulcer index and histological changes will be

observed.

Anticonvulsant activity will be carried out with healthy adult albino mice of

either sex of wister strain. The test is based on inducing epilepsy by electric shock

and chemical induced seizure.

Electroconvulsions: Mice are used for the experiment. For each stimulus

intensity, male mice (18-30)gm, in groups of 8-10 are used. Corneal or ear

electrodes are used to provide electrical stimulation from a stimulator that either

delivers constant current or constant voltage at a frequency of 50-60/sec for 0.2 sec

duration. Rectangular pulses are better than sinusoidal pulses in inducing seizures,

though either of the two can be used for stimulation. Threshold is usually

determined as the current or voltage inducing hind limb extension in 50% of the

animals, i.e. CC50, and CV50 or EV50, respectively. Control thersholds in mice are

about 6-9 mA(CC50) or 90-140V (CV50 or EV50) depending on strain, age and

method of stimulation (thresholds determined via ear electrodes are lower than via

corneal electrodes).

Pentylenetetrazol (PTZ) induced Convulsion: 8-10 mice are used per

threshold determination. One percent solution of PTZ is administered by continuous

i.v. infusion at the rate of 0.3ml/min. The animal develops seizures in the following

order: one or more isolated jerks (first twiich) followed by maximal tonic clonic

seizures after a certain time lag. Dose for the production of generalized clonic

seizures with loss of righting reflex is preferably taken as an endpoint. Threshold is

calculated as the mean dose of PTZ that induces seizures in the group tested and is

about 50 mg/kg for clonic seizures and 90 mg/kg for maximal tonic-clonic seizures

in mice.27

Screening Of Diuretic Activity:

The activity was carried out by Lipschitz et.al. method. Albino rats

of Wistar strain weighing about 150-200gm will be categorized into groups as

control, standard and test containing 6 animals per each group. The test is based on

water and sodium excretion in test animals and compared to rats treated with high

dose of urea. In this method 0.9% sodium chloride solution per 100kg body weight

was given by gavage. Furosemide in the dose of 100mg/kg will be given

interperitoneally for the standard group of animals. The urine excretion is recorded

after 5hrs. The observed parameters were total urine volume for 5hrs, Na+, k+, cl-

excreted in urine. The concentration of electrolytes in urine volume is expressed in

mml/100gm/5hrs. Na+, K+, Cl- conc. were measured by using flame photometry.28&29

The diuretic activity is concluded by comparing the values of test, standard and

control groups. The results obtained will be expressed using one way ANOVA.

Toxicological studies will be carried out as per the standard procedure (as per

OECD guidelines-425) and the results are compared with treated and control group.

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, the study of the pharmacological and

toxicological effects of areal parts of Pterocarpus marsupium will be carried out on the

Wistar albino mice and rats.

Enclosure-VI A

7.4. Has ethical clearance been obtained from your institution?

Ethical clearance is provided by the Institution.

Clearance number: NCP/IAEC/CL/234/2013-14.

Enclosure –VII

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