Download Annexure – II

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 PHARMACY
IN
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: 571448
KARNATAKA, INDIA.
NATIONAL COLLEGE OF
PHARMACY,
BALRAJ-URS ROAD,
SHIMOGA-577 201
KARNATAKA-INDIA
02
Name of the Institution
03
Course of the Study
Branch
M.PHARM. (PHARMACOLOGY)
04
Date of Admission to course
O1/08/2013
05
06
07
08
Title of the Topic
PHARMACOLOGY AND
TOXICOLOGICAL STUDIES OF THE
PLANT BARK PTEROCARPUS
MARSUPIUM.
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
Materials and Methods
7.1. Source of data
Enclosure – IV
7.2. Methods of collection of data
7.3. Does the study require any
Investigations on animals?
If yes give details
7.4. Has ethical clearance been
obtained form your institution
in case of 7.3.
Enclosure – V
List of References (About 4 – 6)
Enclosure – VII
Enclosure – VI
Enclosure – VI – A
09
10
Signature of the Candidate
Remarks of the Guide
Name and Designation of
(in Block Letters)
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.
Mr. VEERASHEKAR.T.
M.Pharm
11.1. Guide
Lecturer
National College of Pharmacy,
Balraj-Urs Road,
Shimoga-577 201
Karnataka-INDIA
11.2. Signature
11
11.3. Head of the Department
Dr. I. J. KUPPAST
M.Pharm , Ph.D , F.I.C.
HEAD, DEPARTMENT OF PHARMACOLGY
National College of Pharmacy,
Balraj-Urs Road,
Shimoga-577 201
Karnataka-INDIA
11.4. Signature
Remarks of the Principal
12
The present study is permitted to perform in the
Pharmacology Department laboratory of our
Institution and the study protocol has been
approved by IAEC.
12.1. Signature
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, antianaphylactic 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+, clexcreted 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
List of references:
1. http://evolvingwellness.com/posts/984/the-value-and-importance-of-plants-inmedicine/ (accessed on 28-04-2012).
2. http://www.botanical-online.com/theimportanceofplants.html (accessed on 28-042012).
3. http://en.wikipedia.org/wiki/herbalism (accessed on 28-04-2012).
4. Kokate C K. Text book of Pharmacognosy, Nirali Prakashan, New Delhi. Ed.4. p.
3-4.
5. Chopra, RN; Nayar, RL and Chopra, IC (1956), “Glossary of Indian Medicinal
Plants”, Council of scientific and Industrial research, New Delhi, 78.
6. Manish devgun, Arun nandha, Ansari, Pterocarpus marsupium-A comprehensive
review phytochemistry 2009; 3(6):359-363.
7. Anonymous (1969), “The Wealth of India: A Dictionary of Raw Material and
Industrial Products”, Vol. VII, Council of scientific and Industrial Research, New
Delhi, 303-305.
8. Varghese, E (1996), “A Case Study among the Kharias of Central India”, Deep
publication, New Delhi, 164.
9. Pullaiah, T (1999), “Medicinal Plants of Andhra Pradesh (India)”, Regency
Publication, New Delhi, 165.
10. Mankani, KL; Krishna, V; Manjunatha, BK et al. (2005), “Evaluation of
hepatoprotective activity of stem bark of Pterocarpus marsupium”, Indian J of
Pharmacol., 37(3), 165-168.
11. Mohanta, RK; Raout, SD and Sahu, HK (2006), “Ethnomedicinal plant resources of
simplipal biosphere reserve of Orrisa, India”, Zoos Print Journal, 21(8), 2372-2374.
12. Prusti, AB and Behera, KK (2007), “Ethnobotanical Exploration of Malkangiri
District of Orissa, India”, Ethnobotanical Leaflets”, 1, 12-15.
13. K.L. Mankani, V. Krishna, B.K. Manjunatha, S.M. Vidya, S.D. Jagadeesh singh,
Y.N. Manohara, Anees-Ur Raheman, K.R. Avinash Studies on hepatoproptective
activity of stem bark of Pterocarpus marsupium Roxb. International journal of
pharmacology 2005; 37(3): 165-168.
14. M. Manickam, M. Ramanathan, M.A. Farboodniay Jahromi, JPN. Chansouria and
A.B. Ray studies on Antihyperglycemic activity of phenolics from Pterocarpus
marsupium. Journal of natural products 1997; 60(6):609-610.
15. Arpita sikdar, Anirban Biswas, Sanjib Bhattacharya, Moulisha Biswas studies on
analgesic activity of Pterocarpus marsupium. Journal of Advance pharmacological
education and research 2013; 3(1):42-45.
16. Anupama.A. Suralkar, Gayatri.S. Vidya, Abhijeet.R.Borate, Asha.S.Jadhav and
Kuldeep.K.Gaikwad studies on Anti-allergic, Antianaphylactic and mast cell
stabilization activity of Pterocarpus marsupium. International journal of research in
pharmaceutical and biomedical sciences 2012; 3(4): 1691-1697.
17. Akansha Mishra, Rohit Shrivastava, Swayam prakash Srivastava, Sudeep Gautam,
Akhilesh Kumar Tamrakar, Rakesh Maurya, Arvind K Srivastava studied the Antidiabetic activity of heart wood of Pterocarpus marsupium. Indian journal of
Experiment biology 2013;51:363-374.
18. Mohire N.C., Salunkhe V.R., Bhise. S.B., Yadav.A.V., studies on cardiotonic
activity of aqueous extract of heart wood of Pterocarpus marsupium. Indian journal
of experimental biololgy 2013;45(6):532-537.
19. Bhupendra chauhan, A.Mrendra kumar chaudary studies on memory enhancing
activity of methanolic extract of Pterocarpus marsupium. Phytopharmacology
2011;2(1):72-80.
20. Udaysing hari patil & Dattatraya.k. Gaikwada studies on phytochemical screening
and microbial activity of stem bark of Pterocarpus marsupium. International
journal of pharm.science and research 2011;2(1):36-40.
21. Kachhawa. JBS. Sharma.N., Tyagi.S. Gupta.R.S. Sharma.K.K. studies the
evaluation of antibacterial activity of Pterocarpus marsupium. International journal
of pharmacy and pharmaceutical sciences 2012;1(1):67-68.
22. Mohammed Rageed, Md. Usman, Pathan Ekbal Khan, Jain Bharath.V., Pawar
sandeep.R., studies the antiinflammatory activity of Pterocarpus marsupium stem
bark.Journal of pharmaceutical and scientific innovation 2012;1(2):21-25.
23. Edwin.S., Joshi.S.B., Jain.D.C. the leaves of plumbago zeylanica linn, acetone and
ethanolic extracts of leaves of plumbago zeylanica have an anti-fertility
activity.2009;14(3):233-239.
24. Kakjing Radul Falang, Mary ogonnaya uguru, noel nenman wannang, Iliya hosea
azi, Nwoye chamaka carried out studies on antiulcer activity of the aqueous extract
of the roots of Plumbago zeylanica linn. Scholars research library, 2012;2(5):563567.
25. Agbaje.E.D. and Adeniran.J.O carried out the studies on some gastrointestinal
effect of the aqueous Root extract of plumbago zeylanica(lead Wort). African
journal of Bio medical research , 2009;12(1):63-68.
26. Vishnukanta, A.C. Rana evaluation of anti convulsant of plumbago zeylanica linn
2010;3(1):76-78.
27. S.K. Gupta Drug Screening Methods, vol 1. New Delhi: Jaypee Brothers Medical
Publishers, 2004. 92 and 95.
28. Tripathi.K.D. Essential of Medicinal Pharmacology. 6th ed. New Delhi: Jaypee
Brothers Medical Publisher; 2008. 184.
29. Drug discovery Evaluation by H. Gerhaed Vogel, 2nd ed. Newyork.