EVALUATION OF FRUIT EXTRACT OF “Morinda citrifolia.” FOR

ANTI ARTHRITIC AND ANTI DIABETIC ACTIVITY

SYNOPSIS FOR REGISTRATION

Of

M.PHARM DISSERTATION

Submitted to
RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES,
KARNATAKA

In partial fulfillment
of the requirement for the Degree of
Master of pharmacy inPharmacology
By
RASHMI HUGGISHETTAR
I.M.PHARM
Under the Guidance of
Mr. PAVAN KUMAR.P
Senior Lecturer
Department Of Pharmacology

DAYANANDA SAGAR COLLEGE OF PHARMACY
2011-12

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA, 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 / DayanandaSagarCollege of Pharmacy,
Kumaraswamy Layout,
Bangalore – 560 078,
Karnataka, India.
3. / Course of Study and
Subject / M. Pharmacy-Pharmacology
4. / Date of Admission / 28 July 2010
  1. 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 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 disease initiation. RA is mediated by T cells, predominantly CD4+ T cells, and proinflammatorycytokines, 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 inflammationis 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 componentshave been identified in theNoni plant such asscopoletin, octoanoic acid, potassium, vitamin C,terpenoids, alkaloids, anthraquinones (such asnordamnacanthal, morindone, rubiadin, and rubiadin-1-methyl ether, anthraquinone glycoside), b-sitosterol,carotene, vitamin A, flavone glycosides, linoleic acid,Alizarin, amino acids, acubin, L-asperuloside, caproicacid, 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 Polynesiansutilized the whole Noni plant in various combinationsfor herbal remedies. The fruit juice is in high demandin alternative medicine for different kinds of illnessessuch as, arthritis, diabetes, high blood pressure, muscleaches and pains, menstrual difficulties, headaches, gastric ulcers5, heartdisease6, AIDS, cancers, sprains, mentaldepression,antioxidant7, senility, poor digestion, atherosclerosis,blood vessel problems, and drug addiction,. Scientificevidence of the benefits of the Noni fruit Juice is limitedbut there is some anecdotal evidence for successfultreatment 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 MateriaMedica., By K. M. Nadkarni, A. K. Nadkarni Popular Prakashan Pvt. Ltd., framed the morphological features and traditional uses ofthis plant.
  4. Wang Mian-Ying et .al.,Morinda citrifolia (Noni): A literature review andrecent 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 LinnFruit Extract having antiulcer activity.(2009)
  6. Anwarul Hassan Gilani et. al,reported Antispasmodic and vasodilator activities of Morinda citrifoliaroot 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 morindacitrifoliaLeaf 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., reportedTherapeutic effect ofKalpaamruthaa, 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 IndianAyurvedic Herbal Formulation Triphala onAdjuvant-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) .
  1. Dinesh Kumar et.al., Antidiabetic activity of methanolic bark extract of Albizia odoratissimaBenth. in alloxan induced diabetic albino mice.(2001)
  2. In intranational journal of endocrinology, Mohammed Fazil Ahmed, reported Antidiabetic activity of Vinca rosea Extracts in Alloxan-Induced Diabetic Rats.(2010).
  3. A.Conforti,P, et al., has reported anti-inflammatory activity of monomethoxypolyethylene glycol superoxide dismutase on adjuant arthritis in rats(1991)15.
  4. 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.
  5. 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 Pearsonand Wood (1959) exhibiting many similarities to human rheumatoid arthritis. Injections of complete Freund’s adjuvant 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 inflammation of non-injected sites (hindleg, forepaws, ears, noseand 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 immunosuppressiveagents. Two protocols, termed “preventative”(or “prophylactic”) and “therapeutic” (or“established”) adjuvant arthritis, have gained wide usagefor 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 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 weight are recorded on the day of injection, whereby paw volumeis measured plethysmographically with equipmentas described in the paw oedema tests. On day 5, the volume of the injected paw is measured again, indicatingthe primary lesion and the influence of therapeuticagents on this phase. The severity of the induced adjuvant disease is followed by measurement of the non-injectedpaw (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 graded8,9,10.
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 : one
No. 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 albinomice (6–7 weeks old) were used. Animalswere caged in uniform hygienic conditions and fed with standard pellet diet and water adlibitum 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 :



Standard books:
Indian MateriaMedica
Experimental pharmacology by M.N.Ghosh
Medicinal plants research
7.1Method 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 onPatients 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 arthritis via Immune deviation and Regulatory T cells.J Immunol 2008; 181(4): 2741–52.
  2. Wikipedia free encyclopedia. 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, SUChen, Afak Palu,Gary Anderson,Morinda citrifolia (Noni): A literature review and recentadvances in Noni research. Acta Pharmacol Sin 2002; 23(12):1127 -41.
  5. MuralidharanP, SrikanthJ. 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 ofMorinda citrifoliaroot extract are mediated through blockade of voltage dependent caliumchannels.BMC Complementary and Alternative Medicine2010; (10):1186/1472-6882-10-2.
  7. Vijaykumar Pandurang Rasal, Arulmozhi Sinnathambi, Purnima Ashok,Sridhar Yeshmaina.Wound Healing and Antioxidant Activities of MorindacitrifoliaLeaf Extract in Rats.IJPT2008;7:49-52.
  8. 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;3:281-84.
  9. Mythilypriya R, Shanthi P and Sachdanandam P. Therapeutic effect of Kalpaamruthaa, a herbal preparationon adjuvant induced arthritis in wistar rats.Inflammopharmacology2008;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 IndianAyurvedic Herbal Formulation Triphala onAdjuvant-induced Arthritis in Mice.Phytother Res2007;21:889-94.
  12. Ajikumaran S Nair, Shylesh BS, Gopakumar B, Subramoniam A.Anti-diabetes and hypoglycaemic properties ofHemionitis arifolia (Burm.) Moore in rats.Jethnopharmcol2006;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 roseaextracts in alloxan induced diabetic rats.J Endocrinol2010;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 res1991; 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 ethnopharmacol2005; 97:219-25.