Screening of Anti-Rheumatic, Anti-Inflammatory and Analgesic Activities of Rheumavedic

SCREENING OF ANTI-RHEUMATIC, ANTI-INFLAMMATORY AND ANALGESIC ACTIVITIES OF ‘RHEUMAVEDIC OIL’ POLY HERBAL FORMULATION

SYNOPSIS FOR

M.PHARM DISSERTATION

SUBMITTED TO

RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES

KARNATAKA

SHUBHAPRIYA.K.HEBBAR

I M.PHARM

DEPARTMENT OF PHARMACOLOGY

VISVESWARAPURA INSTITUTE OF

PHARMACEUTICAL SCIENCES

BANGALORE-560070

(2009-2010)

RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES

BANGALORE, KARNATAKA.

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. / Name of the candidate and address(in block letters) /

SHUBHAPRIYA.K.HEBBAR

D/O K. PRAKASH HEBBAR

NANDINI LAYOUT
BANGALORE(DISTRICT)
KARNATAKA

PIN CODE: 560058

2. /

Name of the institution

VISVESWARAPURA INSTITUTE OF PHARMACEUTICAL SCIENCES

3. /

Course of study and subject

MASTER OF PHARMACY IN

PHARMACOLOGY

4. /

Date of the admission

/ 16TH JUNE 2008
5.
6. /

Title of the topic:

“SCREENING OF ANTI-RHEUMATIC, ANTI-INFLAMMATORY AND ANALGESIC ACTIVITIES OF RHEUMAVEDIC OIL - POLY HERBAL FORMULATION”
BRIEF RESUME OF THE INTENDED WORK

6.1 NEED FOR THE STUDY

Arthritis is a joint disorder featuring inflammation. Arthritis literally means inflammation of one or more joints.1 There are over 100 types of arthritis, including osteoarthritis, rheumatoid arthritis, and gout. Therapy for rheumatic pain and other related conditions includes NSAID’s and Disease modifying anti- rheumatic drugs(DMARD’s) like cyclophosphamide, intra muscular gold, sulfasalazine, methotrexate etc. All drugs carry the risk of side effects and arthritic drugs are no exception, the common side effects of anti-arthritic drugs are stomach ulcers, GIT bleeding, kidney damage, liver damage, hypertension etc.
Allopathic medicine even though are specific in their pharmacological and therapeutic actions, cause some severe side effects which are undesirable. So the use of an alternative medicine becomes important here. An alternative medicine like ayurveda, siddha, homeopathy, unani etc, uses ‘herbs’ as medicine. The ancient documents made by CHARAKA, SUSHRUTHA and many other contributors suggests that the ancient science of life provides a safe alternative treatment using herbal drugs with least or no side effects, to treat rheumatic pain and various types of arthritis. Unfortunately, there is “Lack of standardization of these herbal drugs.There is no documentary evidence to support the clinical efficacy of many of these alternative medicines”.
“RHEUMAVEDIC OIL” apoly herbal formulation intended to cure rheumatic pain and other related conditions, contains the extracts of 8 different medicinal plants viz, Gloriosa superba, Commophora mukul, Vitex negundo, Psoralea corylifolia, Sida cordifolia, Asparagus racemosus, Cedrus deodara, Alpinia galangal. Theconstituents of rheumavedic oil are used in folk medicine for the treatment of inflammation and pain associated with arthritis.The present study is taken up to evaluate anti-rheumatic, anti-inflammatory and analgesicactivities of rheumavedic oil in rats and thereby, provide preclinical data to support the clinical efficacy of rheumavedic oil.
7
8. / Sl no / Botanical name / Common name / Uses
1. / Gloriosa superba / Languli / Antirheumatic.Anthelmentic,anti inflammatory.2
2. / Commophora mukul / Guggulu / Anti-inflammatory,antirheumatic, hypolipidemic, and hypo cholestermic.3
3. / Vitex negundo / Nirgundi / Anti-rheumatic, anti- inflammatory.4
4. / Psoralea corylifolia / Bakuchi / Anti-rheumatic and anti-inflammatory.5
5. / Sida cordifolia / Bala / Anti-rheumatic and anti-inflammatory.6
6. / Asparagus racemosus / Shatavari / Anti-rheumatic and immunomodulator.7
7. / Cedrus deodara / Devedaru / Anti inflammatory and anti ulcer.8
8. / Alpinia galangal / Rasna / Immunomodulator, hypoglycemic and cardio protective.8
9. / Rosemarry oil / Base.
6.2REVIEW OF LITERATURE:
The polyherbal formulation “Rheumavedic oil” is developed and provided by Vedic Bio Labs pvt ltd. It contains the extracts of 8 medicinal plants listed below:-
6.3 OBJECTIVE OF THE STUDY
Aim: To evaluate Rheumavedic oil, a poly herbal formulation for anti-arthritic, anti-inflammatory & analgesic activity.
Objectives:

To evaluate the anti-rheumatic, anti-inflammatory and analgesic activity of Rheumavedic oil using Adjuvant induced arthritis model in rats.

2. To study of histopathology of knee joint.
3. To evaluate the anti- inflammatory activity of Rheumavedic oil using Carageenan
Induced hind paw edema in rats.
4. Screening of Central Analgesic activity of Rheumavedic oil using Eddy’s hot plate in
Mice.
5. Screening of Peripheral analgesic activity of Rheumavedic capsules using Acetic acid
Induced writhing in mice.
6. To evaluate the penetration enhancers in rheumavedic oil.
7. To evaluate the synergistic action of rheumavedic oil along with rheumavedic capsules.
MATERIAL AND METHODS
7.1Source of Data :
Experimentalprocedures are collected from various journals and standard textbooks available in the IISc library, RGUHS digital library, library of Visveswarapura Institute of Pharmaceutical Sciences and standard websites.
7.2Method of collection of Data
1)The data will be generated by performing the experiments using animal models like RATS.
2)Standard information is taken from various Journals and standard websites
Web sites:

7.3METHODOLOGY :
A. Screening of Anti arthritic activity 1,9
1)Adjuvant induced arthritis model in rats10.
Female Sprague –Dawley rats weighing 150-200g will be divided into 5 groups of 6 rats each, three animals being housed in labeled cage.
Grouping:
Group I------Control, animals will be administered the vehicle (0.5% W/V SCMC) p.o,
Group II------Arthritic control, animals will be administered the vehicle (0.5% W/V
SCMC) p.o. and arthritis will be induced.
Group III------Animals will be applied rheumavedic oil, and arthritis will be induced.
Group IV------Animals will be applied rheumavedic oil and rheumavedic capsule contents will
be administered orally, and arthritis will be induced.
Group V------Standard group, animals will be applied the reference Standard Indomethacin gel. 11
Induction of arthritis
Arthritis will be induced by a 0.1ml injection of Complete Freund’s Adjuvant emulsion (CFA) into the sub-plantar surface of right hind paw.12
Procedure:
Drugs will be applied topically twice a day, from the day of injection of CFA and continued up to 14th post CFA challenge day. The assessment of following parameters will be done
Paw volume: The change in the inflammatory reaction will be measured using mercury plethysmograph on 0, 4, 7, 14,21day from the day of CFA injection.13
Body Weight: The change in the body weight will be calculated using digital weighing balance on 0, 4, 7, 14,21days from the day of CFA injection.13
2)Anterior cruciate ligament (ACL) transection model in rats.14,15,18
Female Sprague –Dawley rats weighing 130-150g will be divided into 5 groups of 6 rats each, three animals being housed in labeled cage.
Arthritis is induced by surgery
Grouping:
Group I------Control, animals will be administered the vehicle (0.5% W/V SCMC) p.o,
Group II------Arthritic control, animals will be administered the vehicle (0.5% W/V
SCMC) p.o. and arthritis will be induced.
Group III----- Animals will be applied Rheumavedic oil, and arthritis will be induced.
Group IV----- Animals will be applied Rheumavedic oil and Rheumavedic capsule contents will
be administered orally, and arthritis will be induced.
Group V------Standard group, animals will be applied the reference Standard Indomethacin gel.
Procedure:-
a)Healthy mature (tide mark well formed) rats 10 weeks of age will undergo surgical transection of Anterior cruciate ligament (ACL).
b)Animal develops progressive changes on medial side of joints
i) by 2 weeks – proteoglycon loss,
ii) by 4 weeks – areas of fibrillation is seen,
iii) other changes like pannus on anterior tibia, cartilage hypertrophy, large osteophytes
& marginal zone proliferative changes are also seen.
c)After 4 weeks of surgery the screening of drug can be done.
d)29th day onwards the Rheumavedic oil will be applied twice a day for 14 days.
The assessment of following parameters should be done
Paw volume: The change in the inflammatory reaction will be measured using mercury plethysmograph on 29, 33, 36, 43, 50 days from the day of surgery.
Body Weight: The change in the body weight will be calculated using digital weighing balance on 29, 33, 36, 43, 50 day from the days of surgery.
At the end of 50thday rats will be anaesthetized with diethyl ether. Blood will be withdrawn by puncture of retro orbital plexus of eye of all the animals & serum will be separated by centrifugation (Remi).
Estimation of ALT (Reitmen &Frankel method-1957),AST Reitmen &Frankel method-1957)and Total protein (Lowry et, al method -1951) will be done usingstandardRMS kits and semiautoanalyser.
Histopathological assessment:-
After euthanasia on 50th day, the hind paws will be amputated above the knee joint and fixed in 7.4 % formalin solution. The paws will then be decalcified using 10% Nitric acid, embedded in paraffin, and sectioned in a mid-sagittal plane. The sections of articulation of the tarsal joints will be stained with haematoxylin and eosin, and will be examined microscopically. Mononuclear infiltration, pannus formation and bone destruction will be compared between different groups.
B. Screenings of anti-inflammatory activity
Carageenan induced hind paw edema in rats.16,17,18
Albino Wistar rats weighing between 150-200 gms will be divided into 4 groups of 6 rats each; three animals being housed in labeled cage each. Animals will be given a period of time to adjust to the new environment provided with food & water ad libitum.
Grouping:
Group I------Animals will be administered 0.1ml saline p.o.
Group II------Animals will be applied Rheumavedic oil, inflammation due to arthritis will be
induced by Complete Freund’s Adjuvant emulsion.
Group III-----Animals will be applied rheumavedic oil, inflammation will be induced by
Carageenan.
Group IV-----Animals will be applied Rheumavedic oil and Rheumavedic capsule contents will
be administered orally, inflammation will be induced by Carageenan.
Group V-----Animals will be applied standard reference Indomethacin gel.
Procedure:
All rats will be injected with 0.1ml of carageenan (1%) in normal saline into sub planter area of right hind paw. Rheumavedic capsule contents are administered 1hr prior to carageenan injection and Rheumavedic oil is applied.
Paw volume will be measured by mercury plethysmograph at 0, 3, 6, 12, 24 hrs after the carageenan injection.
C. Screening of analgesic activity 12,17,18
a. Eddy’s hot plate method
Swiss – albino mice weighing 20-25g are divided into 4 groups of 6 animals each, three animals being housed in labeled cage each. Animals will be given a period of time to adjust to the new environment provided with food & water ad libitum
Grouping:
Group I------Animals will be administered 0.1ml saline p.o.
Group II------Animals will be applied rheumavedic oil, pain due to arthritis will be induced by
Complete Freund’s Adjuvant emulsion.
Group III-----Animals will be applied rheumavedic oil and rheumavedic capsule contents will
be administered orally, pain due to arthritis will be induced by Complete Freund’s
Adjuvant emulsion.
Group IV-----Animals will be administered standard reference (pethidine sulfate 5 mg / kg).p.o
Procedure:
The time for licking paws or jumping in hot plate will be recorded as response, prior and 30, 60, 90, 120, &150 min after application of rheumavedic oil/ Standard.
b. Acetic acid induced writhing test in mice18
Swiss – albino mice weighing 20-25g are divided into 4 groups of 6 animals each, three animals being housed in labeled cage each. Animals will be given a period of time to adjust to the new environment providing with food & water ad libitum
Grouping:
Group 01: Animals will be administered 0.1ml saline p.o
Group 02: Animals will be applied rheumavedic oil twice a day, pain due to arthritis will be
induced by Complete Freund’s Adjuvant emulsion.
Group 03: Animals will be applied Rheumavedic oil twice a day, pain will be
induced by acetic acid.
Group 04: Animals will be applied Rheumavedic oil twice a day and Rheumavedic capsule
Contents will be administered orally.
Group 05: Animals will be applied standard reference Diclofenac gel.
Procedure:
Writhing will be induced 30 min later by intraperitoneal injection of 1 ml/kg of 0.6% v/v acetic acid in distilled water. The number of writhes will be counted for 30 min immediately after the acetic acid injection.
Following parameter will be evaluated.
% inhibition of abdominal constrictions (Control mean – Treated mean) X 100
between control animals and poly herbal = ------
formulation treated animals using the ratio Control Mean
D. DRUG PENETRATION STUDIES19
Penetration of a drug through skin will be studied using in-vitro model by DIFFUSION CELL.
In-vitro techniques are more commonly used because much of the primary information about the rate and extent of drug penetration, the effects of vehicles and altered environment can be accurately assessed without risk to the animals.
Procedure:-Diffusion Cell19, 20
a)The primary technique of in vitro transdermal drug studies involves skin collected from the target animal species and mounted within a two-chambered diffusion cell.
b)The stratum corneum side of the skin is orientated towards the donor chamber, while the underside is exposed to the receptor chamber.
c)The diffusion cell will be maintained in a water-bath at 32-35o C to keep the skin at a physiological temperature.
d)Radio labeled Rheumavedic oil will be applied and gently rubbed onto samples of skin, which are then placed onto the diffusion apparatus.
e)Drug penetration will be determined by sampling the reservoir over time and calculating cumulative percent recovery.
f)At 1, 8 and 24 hr, 1.0 ml reservoir fluid samples are obtained for scintillation counts, and the sample volume will be replaced in each chamber.
7.4STATISTICAL ANALYSIS
The results will be expressed as mean  SEM for each of the parameters studied.
All the data’s will be analyzed using One-Way ANOVA using Graph pad INSTAT, and Post hock analysis will be done followed by Dunnet’s / Tukey’s test. P≤0.05 will be considered to be statistically significant.
7.4 Does the study require any investigation to be conducted on patients
Or other humans or animals? If so please describe briefly
No, studies do not use anyhuman patients.
Yes, The above study requires investigation on animals. The effect of drug will be studied on
various parameters using rat and mice as animal model.
7.5 Has ethical clearance been obtained from your institution in case of 7.3?
Yes, Ethical clearance certificate is attached.
REFERENCES:

Chamundeeswari D, Vasantha J, Gopalakrishanan S and Sukumar E. Effect of the alcoholic extract of Trewia polycarpa roots on Cathepsin D in arthritic rats. Indian J. Pharm. Educ. 2005; 39(1): 51-3.
Indian Herbal Pharmacopeia Revised edn. 2002 (Mumbai: IndianDrug Manufacturers Association): pp 493–6.
Arora RB, Kapoor V, Gupta SK and Sharma RC. Isolation ofa crystalline steroidal compound from Commiphora mukul andits anti-inflammatory activity; Indian J. Exp. Biol. 1971; 9:403–4.
Svoboda RE. Ayurvedic life health and longevity 1st edition: 1992 ;(New Delhi: Penguin Books); pp 221, 258–25
Singh BB, Mishra LC, Vinjamury SP, Aquilina N, Singh VJand Shepard N. The effectiveness of Sida cordifolia for osteoarthritis of the knee: Altern. Ther.Health Med. 2003;9: 74–79
Jagetia GS,RaoSK, Baliga MS and Babu K. The activity of Psoralea corylifolia; Indian J. Exp. Biol.2004;18 : 561–565
Moharana D. Shatavari: The Ayurvedic Therapy. Orissa review, 2008 ; pp 72-4.
Kokate CK, Purohit AP,Gokhale SB. In: drugs containing glycosides, Pharmacognosy. 39th ed; Nirali Prakashan, Pune, 2007; p 212-20.
Mi-Jung Yeom, et.al., Anti-arrthritic effect of Ephedra sinica stapf herb acupuncture: inhibition of lipopolysaccharide-induced inflammation and adjuvant – induced polyarrthritis. J of Pharmacol Sci.2006; 100: 41-50.
Kawahito Y,et.al., Localizationof quantitative trait loci regulating adjuvant-induced arthritis in rats: evidence for genetic factors common to multiple autoimmune diseases ; J Immunol 1998, 161:4411-9.
Hong-Mei Xu, Wei Wei, Xiao-Yi Jia, Yan Chang and Lei Zhang. Effects and mechanisms of total glucosides of paeony on adjuvant arthritis in rats. J of Ethnopharmacol 2007; 109: 442–8.
Kaneria MS, Naik S R and Kohli R K. Anti-inflammatory, antiarthritic and analgesic activity of an herbal formulation (DRF/AY/4012). Indian J Exp Biol 2007; 45: 278-84.
Jongwon Choi, et.al. Antirheumatoid arthritis effect of Rhus verniciflua and of the active component, Sulfuretin. Planta Med 2003; 69: 899-904.
Hayami T, et al., Characterizationof articular cartilage and subchondral bonechanges in the rat anterior cruciate ligament transactionand meniscectomized models of osteoarthritis.Bone 2006; 38:234–43.
Stoop R, Buma P, van der Kraan PM, et al; Differences in type II collagen degradation betweenperipheral and central cartilage of rat stifle joints aftercranial cruciate ligament transection. Arthritis Rheum. 2000; 43: 2121–31.
Nivasarkar M, et.al. Launaea nudicaulis leaf juice exhibits anti-inflammatory action in acute and chronic inflammation models in rats. Indian Drugs 2002; 39(5): 290-92.
Olumayokun A. Olajide, Modupe Makinde J and Olubusayo Awe. Effects of the aqueous extract of Bridelia ferrugined stem bark on carageenan- induced edema and granuloma tissue formation in rats and mice. J of Ethnopharmacol 1999; 66: 113-7.
Gerhard Vogel H. In: Analgesics & Anti- inflammatory drugs, Drug discovery and evaluation, 2nd ed. Springer- Verlag Publishers Newyork: 2002; pp669-785.

Barry BW. Novel mechanisms and devices to enable successful transdermal drug delivery. Eur J. Pharm. Sci. 2001, 14: 101-14.
Finnin BC. and Morgan TM. Transdermal penetration enhancers: applications, limitations, andpotential. J of Pharm. Sci. 2004; 88: 955-58.