EVALUATI

ON OF FRUIT EXTRACT OF “Morinda citrifolia.” FOR

ANTI ARTHRITIC AND ANTI DIABETIC 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

ByRASHMI HUGGISHETTAR

I.M.PHARM

Under the Guidance ofMr. PAVAN KUMAR.P

Senior LecturerDepartment Of Pharmacology

DAYANANDA SAGAR COLLEGE OF PHARMACY2011-12

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCESKARNATAKA, BANGALORE.

ANNEXURE - II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. Name of the Candidate And Address

Rashmi Huggishettar# 34, silver town gokul road, Hubli : 580030

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

3. Course of Study andSubject

M. Pharmacy-Pharmacology

4. Date of Admission 28 July 2010

5. Title of the Topic:

EVALUATION OF FRUIT EXTRACT OF “Morinda citrifolia.” FOR

ANTI ARTHRITIC AND ANTI DIABETIC ACTIVITY

6.0 Brief resume of the intended work:

6.1 – Need of the study:

A . RHEUMATOID ARTHRITIS

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

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

factors and possible infectious agents (bacteria and viruses) are assumed to participate

in the disease initiation. 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-TNF-α antagonists has resulted in success when

combined with cytostatic therapy. The design of vaccines capable of preventing or

reversing chronic inflammation is of 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 of all conditions

embraced by the terms arthritis and rheumatism is called Rheumatology.1

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

present at 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 disease progresses.

B . DIABETES MELLITUS

Diabetes mellitus, often simply referred to as diabetes, is a group of metabolic diseases

in which a person has high blood sugar, either the body does not produce enough

insulin, or because cells do not respond to the insulin that is produced. This high blood

sugar produces the classical symptoms of polyurea (frequent urination), polydipsia

(increased thirst) and polgia (increased hunger).

There are main types of diabetes:

Type 1 diabetes: result from the body failure to produce insulin, and

presently requires the person to inject insulin. (Also referred to as insulin

dependent diabetes mellitus, IDDM for short, and juvenile diabetes.)

Type 2 diabetes: result from insulin resistance, a condition in which cells

fail to use insulin properly, sometimes combined with an absolute insulin

deficiency.(Formerly referred to as non-insulin-dependent diabetes

mellitus, NIDDM for short, and adult-onset diabetes.)

Gestational diabetes: is when pregnant women, who have never had

diabetes before, have a high blood glucose level during pregnancy. It may

precede development of type 2 diabetes mellitus.

Other forms of diabetes mellitus include congenital diabetes, which is due to genetic

defects of insulin secretion, cystic fibrosis-related diabetes include by high doses of

glucocorticoids, and several forms of monogenic diabetes.

Diabetes without proper treatments can cause many complications. Acute

complications include cardiovascular diabetes chronic renal failure, rectal damage.

Adequate treatment of diabetes is thus important, as well as blood pressure control and

lifestyle such as smoking cessation and maintaining a healthy body weight.

As of 2000 at least 171 million people worldwide have diabetes, or 2.8% of the

population. Type 2 diabetes is by far the most common, affecting 90 to 95% of the U.S.

diabetes population2.

6.2 –Plant Profile :

An ethno pharmacological survey of medicinal plants “Morinda cetrifolia” Linn.

Family-Rubiaceae3 .common name great morinda, indian mulberry, beach mulberry

and noni. Morinda citrifolia is a small evergreen tree that grows to a height of 10-12

feet. The fruits are used in the manufacture of fruit drinks, medicines and dyes.

In India the tree is predominantly grown in coastal Kerala, Karnataka, Tamil Nadu,

Orissa, Andaman and Nicobar Islands. It can grow up to 9 m tall, and has large, simple,

dark green, shiny and deeply veined leaves. The plant flowers and fruits all year round

and produces a small white flower. The fruit is a multiple fruit that has a pungent odor

when ripening, and is hence also known as cheese fruit or even vomit fruit. It is oval

and reaches 4-7 cm in size. At first green, the fruit turns yellow then almost white as it

ripens. It contains many seeds4.

Major components A number of major components have been identified in the Noni

plant such as scopoletin, octoanoic acid, potassium, vitamin C, terpenoids, alkaloids,

anthraquinones (such as nordamnacanthal, morindone, rubiadin, and rubiadin-1-methyl

ether, anthraquinone glycoside), b-sitosterol, carotene, vitamin A, flavone glycosides,

linoleic acid, Alizarin, amino acids, acubin, L-asperuloside, caproic acid, caprylic acid,

ursolic acid, rutin, and a putative proxeronine.

Retired biochemist, Ralph Heinicke, states that the Noni fruit contains a natural

precursor for Xeronine that he named Proxeronine. Proxeronine is converted to the

alkaloid, Xeronine, in the body by an enzyme he calls Proxeroninase4.

Medicinal use of Noni plant The Polynesians utilized the whole Noni plant in various

combinations for herbal remedies. The fruit juice is in high demand in alternative

medicine for different kinds of illnesses such as, arthritis, diabetes, high blood

pressure, muscle aches and pains, menstrual difficulties, headaches, gastric ulcers5,

heart disease6, AIDS, cancers, sprains, mental depression, antioxidant7, senility, poor

digestion, atherosclerosis, blood vessel problems, and drug addiction,. Scientific

evidence of the benefits of the Noni fruit Juice is limited but there is some anecdotal

evidence for successful treatment of colds and influenza.

6.3 – Review of the literature:

1. Irina kochetkova, et.al., reported the Vaccination without Auto-antigen protects

against Collagen II –Induced arthritis via Immune deviation and Regulatory T

cells,(2008).

2. From Wikipedia, the free encyclopedia , the introduction and pathophysiology

of diabetes mellitus.

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

Nadkarni Popular Prakashan Pvt. Ltd., framed the morphological features and

traditional uses of this plant.

4. Wang Mian-Ying et .al., Morinda citrifolia (Noni): A literature review and

recent advances in Noni research, has reported the chemical constituents and

therapeutic uses of noni.(2002)

5. In the journal of scientific research, P. Muralidharan1 and J. Srikanth , reported

that Morinda Citrifolia Linn Fruit Extract having antiulcer activity.(2009)

6. Anwarul Hassan Gilani et. al,reported Antispasmodic and vasodilator activities

of Morinda citrifolia root extract are mediated through blockade of voltage

dependent calcium channel (2010).

7. In iranian journal of pharmacology and therapeutics ,Vijaykumar pandurang

rasal et.al , reported wound healing and antioxidant activities of morinda

citrifolia Leaf Extract in Rats.(2008).

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

JusticiagendarussaBurm. And Withaniasomnifera Linn (2009).

9. R. Mythilypriya, et.al., reported Therapeutic effect of Kalpaamruthaa, a herbal

preparationon adjuvant induced arthritis in wistar rats (2008).

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

Pharmacological Assays,concised the methods of various models for arthritis

induction.

11. M. Rasool et.al., reported the Antiinflammatory effect of the Indian Ayurvedic

Herbal Formulation Triphala on Adjuvant-induced Arthritis in Mice (2007).

12. S. Ajikumaran Nair et.al.,reported the Anti-diabetes, anthypoglycemic

properties of Hemionitis arifolia (Burm) Moore in rats (2006) .

13. Dinesh Kumar et.al., Antidiabetic activity of methanolic bark extract of Albizia

odoratissima Benth. in alloxan induced diabetic albino mice.(2001)

14. In intranational journal of endocrinology, Mohammed Fazil Ahmed, reported

Antidiabetic activity of Vinca rosea Extracts in Alloxan-Induced Diabetic Rats.

(2010).

15. A.Conforti,P, et al., has reported anti-inflammatory activity of

monomethoxypolyethylene glycol superoxide dismutase on adjuant arthritis in

rats(1991)15.

16. Mingxing Liu, Jing Dong et.al, reported anti-inflammatory effect of triptolide

loaded poly(D,L- lactic acid) nanoparticles on adjuant-induced arthritis in rats.

(2005)16.

17. In the journal of ethnopharmacology, Jung Bong Ju et.al, reported the ethanolic

aqueous extracts from Chinese juniper berrier for hypoglycaemic and

hypolipidemic effects in alloxan-induced diabetic in rats.(2008)17.

6.4 – Objective of the study:

The main objective of the study is to evaluate the anti-arthritic and anti-diabetic

activity of fruit extracts of Morinda cetrifolia.

Collection and authentification of fruits of Morinda cetrifolia Linn.:

The fruits will be collected and authenticated by botanist.

Extraction :

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

subjected to successive extraction with petroleum ether, chloroform, aqueous alcoholic

and methanol by increasing order of polarity using soxhlet apparatus.

Preliminary phytochemical screening of crude extracts :

The crude extracts will be subjected to preliminary phytochemical analysis for the

presence of phytoconstituents.

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.

6.5 – Evaluation of anti arthritic activity by fallowing method:

Complete Freund’s Adjuvant induced arthritis:

Adjuvant arthritis in rats has been described by Pearson and Wood (1959) exhibiting

many similarities to human rheumatoid arthritis. Injections of complete Freund’s

adjuvant into the rat paw induces inflammation as 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 inflammation of non-injected sites (hind leg, forepaws,

ears, nose and tail), a decrease of weight and immune responses.

The procedure has been modified by several authors in order to differentiate between

anti-Inflammatory and immunosuppressive activity. Anti-inflammatory compounds do

not inhibit secondary lesions, which are prevented or diminished by

immunosuppressive agents. Two protocols, termed “preventative” (or “prophylactic”)

and “therapeutic” (or “established”) adjuvant arthritis, have gained wide usage for

assessing a drug’s potential anti-arthritic activity8,9,10.

Materials and methods:

Complete Freund’s Adjuvant induced arthritis:

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

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

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

they are injected into the sub plantar region of the left hind paw with 0.1 ml of complete

Freund’s adjuvant. This consists of 6 mg mycobacterium butyricum (Difco) being

suspended in heavy paraffin oil (Merck) by thoroughly grinding with mortar and pestle

to give a concentration of 6 mg/ml. Dosing with the test compounds or the standard is

started on the same day and continued for12 days. Paw volumes of both sides and body

weight are recorded on the day of injection, whereby paw volume is measured

plethysmographically with equipment as described in the paw oedema tests. On day 5,

the volume of the injected paw is measured again, indicating the primary lesion and the

influence of therapeutic agents on this phase. The severity of the induced adjuvant

disease is followed by measurement of the non-injected paw (secondary lesions) with a

plethysmometer. Purposely, from day 13 to 21, the animals are not dosed with the test

compound or the standard. On day 21, the body weight is determined

again and these verity of the secondary lesions is evaluated visually and graded 8,9,10 .

Number of groups to be used in the study :

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

Group 2: Arthritic group (CFA)

Group 3: Arthritic + herbal extract (Low dose)

Group 4: Arthritic + herbal extract (High dose)

Group 5: Arthritic + Standard allopathic drug (GLUCOSAMINE SULPHATE)

Parameters for anti-arthritic activity:

1. paw volume

2. body weight

3. copper level estimation

4. hematological estimation

Histopathological studies : Histopathological study carried out to assess the effect

of the extract on Joint of the Hind Paw.

Elisa Test: This test is carried for the immunological assay to estimate the level of

C-Reactive Protein8.

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 <

0.0511.

Number Of Animals Required :

No of Models : oneNo. of groups : Five

No. of animal in each group : Six

Total : 5 X 6 = 30 animals

6.6–Evaluation of anti-diabetes activity by alloxone induced method:

Experimental animals: Inbred Wistar rats (150–200 g weight) and Swiss albino mice (6–7 weeks old) were

used. Animals were caged in uniform hygienic conditions and fed with standard pellet

diet and water ad libitum as per the guide lines of Institute Animal Ethics Committee.

Oral glucose tolerance test :

The effect of extract was evaluated on the glucose (2g/kg) loaded normal mice. Blood

sample was collected from the tail vein at the time intervals of 0, 15, 30, 45, 60, 75, 90,

105, 120 min. the percentage change in the blood glucose level were monitored at

various time intervals after single administration of the extract12,13,14.

Preparation of glucose solution :

The 20% w/v glucose solution was prepared by dissolving 20 g glucose in 100 mL of

distilled water.

Induction of experimental diabetes :

Hyperglycemia was induced by injecting alloxan hydrate at a dose of 150 mg/kg

intraperitonally. The animals are kept under observation. After 48 h, the blood glucose

level was checked before and 72 h after alloxan injection to confirm the development

of diabetes. The diabetes animals were stabilized for 5 days and experiment was started

on the next day. only the animal which showed blood glucose level >250 mg/dl were

separated and used for study12,13,14 .

Parameters estimated: The serum separated from the blood will be used to estimate biochemical parameters

such as blood glucose, cholesterol, triglycerides, urea, creatinine, serum glutamic

oxaloacetic transaminase(SGOT), serum glutamic pyruvic transaminase (SGPT) and

alkaline phosphatase13.

Method of collecting blood samples:Blood samples were collected on 28th day and centrifuged.

Number of groups to be used in the study :Group 1: normal healthy control given by vehicle (Tween 80, 1%w/v)

Group 2: serves as diabetes control receiving only vehicle

Group 3: diabetic rats receiving extract (Low dose)

Group 4: diabetic rats receiving extract (High dose)

Group 5: diabetes rats receiving standard drug.

Number of animals required:No. of groups 05

No. of animals in each group 06

Total 5 X 6 = 30

Statistical analysis :The Dennett’s test was employed for statistical comparison. P<0.05 were considered

significant in relation to control and standard. All values are presented as mean SEM13.

Source of data : Journal of Ethnopharmacology, Indian journal of forestry, International journal of

pharmacology, Indian journal of experimental biology, Indian journal of physiology

and pharmacology.

Web site : www.traditional.tree.org

http://www.chinaPhar.com

www.pubmed.com

www.sciencedirect.com

Standard books:Indian Materia Medica

Experimental pharmacology by M.N.Ghosh

Medicinal plants research

7.1 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.

7.2: 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.

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

of 7.3? Yes, the protocol is being submitted to IAEC.

REFERENCES:1. Irina kochetkova, Theresa Trunkle, Gayle callis and David Pascual.

Vaccination without Auto-antigen protects against Collagen II –Induced

8.0

7.0

arthritis via Immune deviation and Regulatory T cells. J Immunol 2008; 181(4):

2741–52.

2. Wikipedia free encyclopedia. http://en.wikipedia.org/wiki/Diabetes-mellitus.

(Visited on 18 october 2011).

3. Nadkarni KM, Nadkarni AK, Chopra RN . Indian MateriaMedica, 1st vol,

Mumbai: Popular Prakashan Pvt. Ltd; 2002.

4. Wang Mian Ying, Brett J West, Jarakae C Jensen, Diane Nowicki, SU Chen ,

Afak Palu, Gary Anderson, Morinda citrifolia (Noni): A literature review and

recent advances in Noni research. Acta Pharmacol Sin 2002; 23(12):1127 -41.

5. Muralidharan P, Srikanth J. Antiulcer Activity of Morinda Citrifolia Linn Fruit

Extract . J Sci Res 2009; 1(2):345-352.

6. Anwarul Hassan Gilani , Saf-ur Rehman Mandukhali, Javeid Iqbal, Masoom

and Najeeb. Antispasmodic and vasodilator activities of Morinda citrifolia root

extract are mediated through blockade of voltage dependent calium channels.

BMC Complementary and Alternative Medicine 2010; (10):1186/1472-6882-

10-2.

7. Vijaykumar Pandurang Rasal, Arulmozhi Sinnathambi, Purnima Ashok,Sridhar

Yeshmaina. Wound Healing and Antioxidant Activities of Morindacitrifolia

Leaf Extract in Rats. IJPT 2008; 7:49-52.

8. Jaljeshpaval, Srinivasan Keloth Kaitheri, 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; 3:281-84.

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

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

Inflammopharmacology 2008; 16:21–35.

10. Gerhard H. Vogel(Ed), Drug Discovery and Evaluation of Pharmacological

Assays, Springer inc., IInd Edition;2002.

11. Rasool M, Sabina EP. Antiinflammatory Effect of the Indian Ayurvedic Herbal

Formulation Triphala on Adjuvant-induced Arthritis in Mice. Phytother Res

2007; 21: 889-94.

12. Ajikumaran S Nair, Shylesh BS, Gopakumar B, Subramoniam A. Anti-diabetes and hypoglycaemic properties of

Hemionitis arifolia (Burm.) Moore in rats. J ethnopharmcol 2006; 106: 192–

197.

13. Dinesh Kumar, Sunil Kumar, Sonia Kohli, Renu Arya, Jyoti Gupta.

Antidiabetic activity of methanolic bark extract of Albizia odoratissima Benth

in alloxan induced diabetic albino mice. Asi pac j trop med 2011; 900-03.

14. Mohammed Fazil Ahmed, Syed Mohammed Kazim, Syed Safiullah Ghori,

Syeda Sughra Mehjabeen, Shaik Rasheed Ahmed, Shaik Mehboob Ali,

Mohammed Ibrahim. Antidiabetic activity of Vinka rosea extracts in alloxan

induced diabetic rats. J Endocrinol 2010; 2010:6 .

15. Conforti A, Caliceti P, Sartore L, Schiavon O, Veronese F, Velo G P. Anti

inflammatory activity of Monomethoxypolyethelene glycol superoxide

dismutase on adjuant arthritis in rats. pharmaco res 1991; 23:51-56.

16. Mingxing Liu, Jing Dong, Yajiang Yang, Xingliang Yang, Huibi Xu. Anti-

inflammatory effects of triploide loaded poly (D,L-lactic acid) nanoparticles on

adjuent induced arthritis in rats. J ethnopharmacol 2005; 97:219-25.

17. Jung Bong Ju, Ji Su Kim, Chang Won Choi, Hae Kyung Lee, Tae-Kyun Oh,

Sei Chang Kim. Comparison between ethanolic and aqueous extracts from

Chinese juniper berries for hypoglycaemic and hypolipidemiceffects in alloxan-

induced diabetic rats. J ethnopharmacol 2008; 115:110-15.

9 Signature of the candidate (Rashmi Huggishettar)

10 Remarks of the Guide: Requested for clearance and approval

11 Name and Designation of

11.1 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

Mrs. 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.