EVALUATION OF ROOT EXTRACTS OF “Ximenia americana Linn.” FOR

ANTI ARTHRITIC ACTIVITY

SYNOPSIS FOR REGISTRATIONOf

M.PHARM DISSERTATION

Submitted toRAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

KARNATAKA

In partial fulfillmentof the requirement for the Degree of

Master of pharmacy In Pharmacology

ByMURUGESH.D.M

I.M.PHARM

Under the Guidance ofMr. PAVAN KUMAR.P

Senior LecturerDepartment Of Pharmacology

DAYANANDA SAGAR COLLEGE OF PHARMACY2010-11

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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCESKARNATAKA, BANGALORE.

ANNEXURE - II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. Name of the Candidate and Address

MURUGESH.D.M#1125/1, Maatrukrupa, Opp. Datta clinic, Thyagaraj Nagar, Challakere-577522, Karnataka.

2. Name of the Institution DayanandaSagarCollege of Pharmacy,Kumaraswamy Layout, Bangalore – 560 078,Karnataka, India.

3. Course of Study andSubject

M. Pharmacy-Pharmacology

4. Date of Admission 15th June 2010

5. Title of the Topic:

EVALUATION OF ROOT EXTRACTS OF “XIMENIA AMERICANA LINN.”

FOR ANTI ARTHRITIC ACTIVITY

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6.0 Brief resume of the intended work:

6.1 – Need of the study:

The ancient Greeks explained the swelling of arthritic joints simply. It resulted from the

rheuma of fluid, but the word rheumatism was not introduced into clinical medicine until the

sixteenth century by the Royal French Physician Guillaune de Ballon (Redford, 1980).

Rheumatoid arthritis (RA)is a systemic inflammatory disease of the joints that disables

almosthalf of the affected patients. The etiology of RA is still unknown, but hereditary

factors andpossible infectious agents (bacteria and viruses) are assumed to participate in the

diseaseinitiation. RA is mediated by T cells, predominantly CD4+ T cells, and

proinflammatory cytokines, such as TNF-α and IL-1, are considered responsible for

orchestrating pathogenesis. Using anti-0TNF-α antagonists has resulted in success when

combined with cytostatictherapy. The design of vaccines capable of preventing or reversing

chronic inflammationisof particular interest.Rheumatic diseases are characterized by

inflammation of connective tissue. When the major disturbance is in the joints, the term

arthritis is used: when the primary involvement is in the soft tissues, the term non articular

rheumatism is often employed. The study ofall conditions embraced by theterms arthritis and

rheumatism is called Rheumatology.1

Rheumatoid Arthritis is a common disease (1-2% of adult population) and can be presentat

any age and involve at any joint. It is most common between the ages of 25 – 55 years, and

the most frequent presentation is an insidious, symmetrical polyarthritis. Although systemic

manifestations may be present at the outset, they usually become more as the

diseaseprogresses.

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6.2 –About the plant:

An ethno pharmacological survey of six medicinal plants “Ximeniaamericana” Linn. Family-

Olacaceae is a variable shrubby tree up to 5m high, armed bushy stragglers. Leaves- ovate-

lanceolate, entire, obtuse, Flowers- creamish, interminal and axillary umbellate racemes.

Sepals- 4 or 5 basally connate. Petals- 4 or 5, stamens 8 to 10, ovary 4-locular; ovule 1 per

locule, pendulous, Drupes ellipsoid, 1-seeded. It is found in Senegal to west Cameroon and

is also widely distributed in tropical Africa, America and Asia. In India it is found

inAndhrapradesh and Tamilnadu2,3,4.

In Mali (South Africa)wounds are treated traditionally with Ximenia leaves or bark or by

applying powder. It is used in dysmenorrhoea, ailments, febrifugal schistosomiasis5,6.

With Anonachrysophylla,it is used in the treatment of sleeping sickness and in insect sting7.

It has anti-trypanosomal activity8itself. Widely used in gum trouble9. It shows antiviral10,

anticancer11 and antimicrobial12,13activity. Roots are used in fever, guinea worm infection,

leprosy, jaundice, wound healing, rheumatism and stomatitis14.

Plant contains volatile oils in leaves. The major constituents are benzaldehyde (63.5%),

hydroxybenzyl cyanide (13.00%), and isophorone (3.5%). In plant saponins, cyanogenetic

glycosides, flavanoides and tannins are present. It is an edible plant which is used in skin

care and eye care due to presence of ximenyic acid15.

6.3 – Review of the literature:

1. Irina kochetkova, Theresa Trunkle, Gayle callis and David W. pascual, reported the

Vaccination without Auto-antigen protects against Collagen II –Induced arthritis via

Immune deviation and Regulatory T cells,(2008)1.

2. Dr.K. Madavachetty et.al. reported themorphological features and traditional use of

the roots of this plant is indicated for Rheumatism, jaundice, cathartic, diarrhea,

veneral diseases(2008)2.

3. Dr. K.M. Nadkarni's Indian MateriaMedica., By K. M. Nadkarni, A. K.

NadkarniPopular Prakashan Pvt. Ltd.,. framed the morphological features and

traditional uses ofthis plant3.

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4. Tom Erik Gronhaug, SiljeGlaeserud, Mona Skogsrud, NgoloBallo, Sekau Bah,

DrissaDiallo and BeritSmestad Paulsen (2008) reported Ethnopharmacological

survey of six medicinal plants from Mali, West- Africa. More than sixty medical

indication are reported for the use of these plants in traditional medicine. The most

frequently used plants part is leaves and stem bark4.

5. Diallo D, Sogn C, Samake FB, Paulsen BS, Michaelson TE Keita (2002) reported

wound healing plants in Mali the Bamako region. According to the literature, this

plant is well known for its medicinal properties and wounds are treated with a

decoction of leaves or a combination of a wash with a decoction of bark5.

6. Inngjerdingen K, NergardCecilie S, Diallo D, MounkoroPakuy P, Paulsen Berits

(2004) An Ethnopharmacological survey of plants used for wound healing in

Dogonland, Mali, West-Africa. According to the literature the aim to identify

medicinal plants used in the treatment of wounds. Plants used as first aids, in the

washing of wounds, extraction of pus, as coagulants as well as for infected wounds6.

7. Burkil HM (Ed) (1997) reported the useful plants of west tropical Africa. The root

bark is put into febrifugal medicine and feed in boiled water with crushed bark. The

plant is used in schistosomiasis and with Anona in sleeping sickness7.

8. Maikai V.A., Nok J.A., Adaudi A.O and Klawa (2008) reported an in-vitro

antitrypanosomal activity of aqueous and methanolic crude extracts of stem bark of

Ximeniaamericana on Trypanosoma congolense8.

9. S Ganesan (2008) reported traditional oral care medicinal plants survey of

Tamilnadu. An oral care medicinal plants survey was conducted in different district

of Tamilnadu during the period of 2004-08.Most of plant used to relief tooth ache,

tooth brush and common dental diseases9.

10. Asresk, Bucar F, Kartnig T, Witvrovw M, Pannecouque C, De Clercq E (2001)

reported an antiviral activity against HIV-1 and HIV-2 of ethnobotanically selected

Ethiopian medicinal plant10.

11. Voss C, Eyol E, Frank M, VorderLieth, Berger MR (2006) reported identification

and characterization of riproximin a new type-II ribosome-inactivating protein with

antineoplastic activity from Ximenia americana11.

12. Omer MEFA, Elinima EI (2003) reported an antimicrobial activity of

Ximeniaamericana. According to the literature extract of the bark, leaves, root and

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stem of Ximeniaamericana were tested for their antimicrobial and antifungal

activity12.

13. D.S.Ogunleye and S.F. Ibitoye (2003) reported an antimicrobial activity and

chemical constituent of Ximeniaamericana. The leaves extract was active against the

test organism E. coli. , Pseudomonas aeruginosa and Candida albicans. Tannins,

Flavonoids, Alkaloides, Saponins, Quinones, Starch and Glycoside are present13.

14. Ake and Guinko(1991), reported that the roots are used for treating abdominal pains,

dysentery, inflamed joints and mouth ulcers14.

15. Jean Philippe Mevy, Jean Marie Bessiere, StephaneGreff, Gerard Zombre and

Josetteviano (2006) reported the composition of volatile oil in leaves of

Ximeniaamericana. Volatile oil of leaves was analysed by GC.MS. Major constituent

identified were Benzaldehyde (63.5%), Hydroxybenzylcyanide (13%) and

Isophorone (3.5%).The occurrence of Hydroxybenzylcyanide as well as

Benzaldehyde was discussed in this paper15.

16. Jaljeshpaval et.al. compared the anti-arthritic activities of the plants

JusticiagendarussaBurm. And Withaniasomnifera Linn(2009)16.

17. M. Rasool et.al studied the Antiinflammatory Effect of the IndianAyurvedic Herbal

Formulation Triphala onAdjuvant-induced Arthritis in Mice(2007)17.

18. R. Mythilypriyaet.al., reportedTherapeutic effect of Kalpaamruthaa, a herbal

preparationon adjuvant induced arthritis in wistar rats18.

19. Maikai, V. A.et.al.reported the acute toxicity studies of aqueous stem bark extract

ofXimeniaamericana19.

20. Gerhard H. vogel(Ed) et.al., Drug Discovery and Evaluation of Pharmacological

Assays,concised the methods of various models for arthritis induction20.

21. TiangaTayaSoro, Traore F and Sakande. J, reported the analgesic activity of the

aqueous extract from Ximenia americana21.

6.4 – Objective of the study:

The main objective of the study will beevaluating theanti-arthritic and phytochemical

investigation of root extracts of Ximeniaamericana.

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A. Collection and authentification of roots of Ximeniaamericana Linn.:

The plant will be collected from nearby Tirupathi Forest area2, Andhra Pradesh, India, and

will be authenticated by Dr. K.Madhavachetty, Assistant Professor and botanist from Sri

Venkateshwara University, Tirupathi.

B.Extraction of dried rootswith methanol in a soxhlet apparatus :

The roots will be shade dried and powdered mechanically. Powdered materials were

subjected to successive extraction with petroleum ether, chloroform, methanol and distilled

water in the increasing order of polarity in a soxhlet apparatus8.

C. Preliminary phytochemical screening of crude extracts :

The crude methanol extract will be subjected to preliminary phytochemical screening for

the presence of carbohydrates, flavonoids, tannins and cyanogenetic glycosides etc.

D. Toxicity studies:

Acute toxicity studies are to be carried according to OECD guidelines.

Selection of Dose:The dosage of the extract will be fixed based on the results of acute

toxicity studies.

E. Evaluation of anti arthriticactivity by following methods:

1.Complete Freund’s Adjuvant induced arthritis:

Adjuvant arthritis in rats has been described by Pearsonand Wood (1959) exhibiting many

similarities to humanrheumatoid arthritis. Injections of complete Freund’sadjuvant into the

rat paw induces inflammationas primary lesion with a maximum after 3 to 5 days.Secondary

lesions occur after a delay of approximately11 to 12 days which are characterized by

inflammationof non-injected sites (hindleg, forepaws, ears, noseand tail), a decrease of

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weight and immune responses.

The procedure has been modified by several authorsin order to differentiate between anti-

Inflammatory andimmunosuppressive activity. Anti-inflammatory compounds do not inhibit

secondarylesions, which are prevented or diminished by immunosuppressiveagents. Two

protocols, termed “preventative”(or “prophylactic”) and “therapeutic” (or“established”)

adjuvant arthritis, have gained wide usagefor assessing a drug’s potential anti-arthritic

activity16,18,20.

2.Type II collagen induced arthritis :

As reported by Trentham et al. (1977),intradermal injectionof homologous or heterologous

type II collagenin incomplete Freund’s adjuvant results in an inflammatorypolyarthritis in

rats. The demonstration ofantibodies to collagen in patients with rheumatic

polyarthritissuggests that autoimmunity may contributeto the pathophysiology of synovitis

and joint destruction.Because of the similarities of the symptoms inrats to human disease the

test is considered to be usefulto detect anti-inflammatory and immunosuppressive properties

of test compounds16,18,20.

F.Materials and methods:

1.Complete Freund’s Adjuvant induced arthritis:

The choice of the animal strain has been found to bevery important for the performance of

this test. Wistar-Lewis rats have been proven to be very suitable in contrastto other sub

strains. Male rats with an initial body weight of 130 to 200 g are used. On day 1, they

areinjected into the sub plantar region of the left hind paw with 0.1 ml of complete Freund’s

adjuvant. This consistsof 6 mg mycobacterium butyricum (Difco) beingsuspended in heavy

paraffin oil (Merck) by thoroughlygrinding with mortar and pestle to give a concentrationof 6

mg/ml. Dosing with the test compounds or thestandard is started on the same day and

continued for12 days. Paw volumes of both sides and body weightare recorded on the day of

injection, whereby paw volumeis measured plethysmographicallywith equipmentas described

in the paw oedema tests. On day 5, the volumeof the injected paw is measured again,

indicatingthe primary lesion and the influence of therapeuticagents on this phase. The severity

of the induced adjuvantdisease is followed by measurement of the non-injectedpaw

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(secondary lesions) with a plethysmometer.Purposely, from day 13 to 21, the animals arenot

dosed with the test compound or the standard. Onday 21, the body weight is determined again

and theseverity of the secondary lesions is evaluated visuallyand graded16,18,20.

2.Type II collagen induced arthritis :

Bovine type II collagen is prepared from nasal Septumcartilage, which is cut into small

fragments, frozen inliquid nitrogen, and pulverized in a freezer mill. Proteoglycansare

extracted overnight by stirring 25 g of pulverizedcartilage in 1 liter of 0.2 N NaOH.

Followingcentrifugation at 20 000 g for 30 min, the residue iswashed with 250 ml of

absolute ethanol, the supernatantaspirated, and the residue vacuum dried. Hundred mgpepsin

are added to 150 ml of 0.5 M acetic acid, afterwhich 1.0 g of cartilage is added to reach a

cartilage topepsin ratio of 10 : 1 (w/w). The mixture is stirred 18 hat room temperature and

centrifuged at 20 000 g for 1 h.Acid soluble collagen present in the supernatant is

precipitatedby adding NaCl to reach a final concentrationof 0.9 M, followed by

centrifugation at 20 000 g for 1 h.The precipitate from 1.0 g cartilage is dissolved in100 ml

1.0 N NaCl/0.005 M Tris-HCl, pH 7.5, andstirred for 3 days. Then, the solution is dialyzed

against0.02 M Na2HPO4, pH 9.4, and the precipitate collectedby centrifugation at 30 000 g

for 1 h. The pellet is dissolvedin 0.5 M acetic acid, dialyzed against 6 litres of0.01 M acetic

acid, and lyophilized. All procedures,unless otherwise stated are performed at 4 °C16,18,20.

G. NUMBER OF GROUPS TO BE USED IN THE STUDY

1.Complete Freund’s Adjuvant induced arthritis:

Group 1:Control (saline-0.5 ml/kg)

Group 2: Arthritic group (CFA)

Group 3: Arthritic + herbal extract (First dose)

Group 4: Arthritic + herbal extract (Second dose)

Group 5: Arthritic + Standard allopathic drug (GLUCOSAMINE)

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2.Type II collagen induced arthritis :

Group 1:Control (saline-0.5 ml/kg)

Group 2:Arthritic group (CFA emulsified in Collagen Type II Arthritis(CII)

Group 3: Arthritic + herbal extract (First dose)

Group 4: Arthritic + herbal extract (Second dose)

Group 5: Arthritic + Standard allopathic drug (GLUCOSAMINE)

H. Histopathological studies:Histopathological study carried out to assess the effect of the

extract on Joint of the Hind Paw.

I. Elisa Test:This test is carried for the immunological assay to estimate the level of C-

Reactive Protein16.

J.Statistical analysis:Significance of the difference between mean values were determined

by one-way analysis of variance (ANOVA) followed by the Tukey’s test for multiple

comparison. Significant differences between control and treatment groups were assigned at p

< 0.0517.

Number Of Animals Required:

No of Models : TWO

No. of groups: Five

No. of animal in each group : Six

Total : 2 X 5 X 6 = 60 animals

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7.0 7.1-Source of data :

Journal of Ethnopharmacology, Indian journal of forestry, International journal of

pharmacology, Indian journal of experimental biology, Complementary and alternative

medicine, Phytotherapy research, Fitoterapia, Indian journal of physiology and

pharmacology.

Standard books:

Indian medicinal plants by Kirtikar KR, Basu BD

Indian MateriaMedica

Experimental pharmacology by M.N.Ghosh

PharmacographicaIndica

Medicinal plants research

7.2-Method of collection of the data:

The Experiments will be conducted using different animal models and the data will be

generated from such experimental studies.

7.3:Does the study require any investigations or interventions to be conducted on

Patients or other humans or animals? If so, Please describe briefly.

Yes, the study requires investigation on albino rats.

7.4: Has ethical clearance been obtained from your Institution in case of 7.3?

Yes, the protocol is being submitted to IAEC.

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8.0 REFERENCES:

1. Irina kochetkova, Theresa Trunkle, Gayle callis and David W. pascual, Vaccination

without Auto-antigen protects against Collagen II –Induced arthritis via Immune

deviation and Regulatory T cells,J Immunol. 2008; 181(4): 2741–2752.

2. Dr.K. Madavachetty, K.Sivaji, K.TulsiRao , Flowering Plants of

Chittordistrict,Andhrapradesh, India,Student offset Printers,Ist Edition,65, 2008.

3. Dr. K.M. Nadkarni's Indian MateriaMedica., By K. M. Nadkarni, A. K.

NadkarniPopular Prakashan Pvt. Ltd.,Volume 2,1298.

4. Gronhaug Erik Tom , GlaeserudSilje, Skogsrud Mona, BalloNgolo, Bah Sekau,

DialloDrissa and Paulsen SmestadBerit, Ethno pharmacological survey of six

medicinal plants from Mali, West- Africa. J Ethnobiol and Ethno med 2008; 4:26.

5. D Diallo, C Sogn, FB Samake, BS Paulsen, Keita TE Michaelson, wound healing

plants in Mali the Bamako region. Pharmaceutical Biol2002; 40:117-128.

6. K Inngjerdingen, S CecilieNergard, D Diallo, P PakuyMounkoro, Berits Paulsen., An

Ethnopharmacological survey of plants used for wound healing in Dogonland, Mali,

West-Africa. J Ethno Biol and Etnomed2004; 92:233-234.

7. HM Burkil (Ed), The useful plants of west tropical Africa. Royal Botanic

Gardens1997; 4(2).

8. V.A Maikai., J.A.Nok, A.O Adaudi and Klawa,An in-vitroantitrypanosomal activity

of aqueous and methanolic crude extracts of stem bark of Ximeniaamericana on

Trypanosomacongolense. J Med Plants Res 2008; 2(3):055-058.

9. Ganesan S, Traditional oral care medicinal plants survey of Tamilnadu. Natural

Product Radiance 2008; 7(2):166-172.

10. Asresk, F Bucar, T Kartnig, M Witvrovw, C Pannecouque, E Clercq De,An antiviral

activity against HIV-1 and HIV-2 of ethnobotanically selected Ethiopian medicinal

plant. Phytother Res 2001; 15:62-69.

11. C Voss, E Eyol, M Frank, LiethVorder, MR Berger , Identification and

characterization of riproximin a new type-ц ribosome-inactivating protein with

antineoplastic activity from Ximineaamericana. J Federation of American societies

for ExpBiol2006; 20:1194-1196.

12. MEFA. Omer, EI Elinima, An antimicrobial activity of

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Ximeniaamericana.Fitoterapia2003;74(1-2):122-126.

13. Ogunleye D.S. and Ibitoye S.F. An antimicrobial activity and chemical constituent of

Ximeniaamericana.Trop J Pharm Res 2003; 2(2):239-241.

14. Ake AI, Guinko S (1991). In: Plants used in traditional medicine in West Africa. F.

Hoffman, La Roche Ltd. Basel Switzerland pp.100.

15. Mevy Philippe Jean,Bessiere Marie Jean, GreffStephane,Zombre Gerard

andvianoJosette,The composition of volatile oil in leaves of

Ximeniaamericana.Biochem Systematic and Ecology 2006;34(7):549-553.

16. Jaljeshpaval, SrinivasanKelothKaitheri, Bhagath Kumar Potu, SreejitGovindan, Raju

Suresh Kumar ,SareeshNaduvil Narayanan, SudheerMoorkoth., Comparing the anti-

arthritic activities of the plants JusticiagendarussaBurm. and Withaniasomnifera

Linn, Int J Green Pharm 2009;3:281-4.

17. M. Rasool and E.P.Sabina., Antiinflammatory Effect of the IndianAyurvedic Herbal

Formulation Triphala onAdjuvant-induced Arthritis in Mice, Phytother. Res.

2007;21, 889-894.

18. R. Mythilypriya, P.Shanthi and P.Sachdanandam.,Therapeutic effect of

Kalpaamruthaa, a herbal preparationon adjuvant induced arthritis in wistar rats,

Inflammopharmacology2008;16, 21–35.

19. Maikai, V. A., Kobo, P. I. and Adaudi, A. O.,Acute toxicity studies of aqueous stem

bark extract ofXimeniaamericana,Afr. J. Biotechnol. Vol. 7 (10),1600-1603, 2008.

20. Gerhard H. vogel(Ed), Drug Discovery and Evaluation of Pharmacological Assays,

Springer inc.,IInd Edition,802-806.

21. TiangaTayaSoro, Traore F and Sakande. J, Analgesic activity of the aqueous extract

from Ximeniaamericana.ComptesRendusBiols2009; 332(4):371-337.

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9Signature of the candidate (Murugesh.D.M)

10 Remarks of the Guide: Requested for clearance and approval

11 Name and Designation of:

11. Guide:

11.2 Signature:

Mr.Pavan kumar.PSenior LecturerDept of PharmacologyDayanandaSagarCollege of Pharmacy,Kumaraswamy layout, Bangalore – 560 078.

11.3 Co-Guide:

11.4 Signature

Not applicable

11.5 Head of theDepartment:

11.6 Signature

Dr. Geetha K.M.Associate ProfessorDept of PharmacologyDayanandaSagarCollege of Pharmacy,Kumaraswamy layout, Bangalore – 560 078.

12 12.1 Remarks of the Chairmanand Principal

Recommended for research

12.2 Signature

Dr. V. MuruganProfessor and principalDept of Pharmaceutical ChemistryDayanandaSagarCollege of Pharmacy,Kumaraswamy layout, Bangalore – 560 078.

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