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EVALUATION OF FRUIT EXTRACT OF “Morinda citrifolia.” FOR
ANTI ARTHRITIC AND ANTI DIABETIC ACTIVITY
SYNOPSIS FOR REGISTRATION
Of
M.PHARM DISSERTATION
Submitted to
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
KARNATAKA
In partial fulfillment
of the requirement for the Degree of
Master of pharmacy in Pharmacology
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
Rashmi Huggishettar
# 34, silver town gokul road,
Hubli : 580030
1.
Name of the Candidate
And Address
2.
Name of the Institution
Dayananda Sagar College 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
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
: 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 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.
7.0 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.
8.0
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. 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
SreejitGovindan,
Raju
Keloth
Suresh
Kaitheri,
Kumar,
Bhagath
Kumar
SareeshNaduvil
Potu,
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. Antidiabetes 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. Antiinflammatory 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 alloxaninduced 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:
Mr.Pavan kumar.P
Senior Lecturer
Dept of Pharmacology
DayanandaSagarCollege of Pharmacy,
Kumaraswamy layout, Bangalore – 560 078.
11.2 Signature:
11.3 Co-Guide:
Not applicable
11.4 Signature
11.5 Head of theDepartment:
Mrs. Geetha K.M.
Associate Professor
Dept of Pharmacology
DayanandaSagarCollege of Pharmacy,
Kumaraswamy layout, Bangalore – 560 078.
11.6 Signature
12
12.1 Remarks of the Chairmanand
Principal
Recommended for research
Dr. V. Murugan
Professor and principal
Dept of Pharmaceutical Chemistry
DayanandaSagarCollege of Pharmacy,
Kumaraswamy layout, Bangalore – 560 078.
12.2 Signature