Download “Evaluation of antioxidant and hepatoprotective effect of Herbal

“Evaluation of and hepatoprotective and antibacterial activity of
aqueous alcoholic (70%) extract of Rhodomyrtus tomentosa (Aiton)
Hassk.”
Synopsis for registration of M.Pharma dissertation.
Submitted to
Rajiv Gandhi University of Health Sciences, Bangalore– 560 041
KARNATAKA
In partial fulfillment
of the requirement for the Degree of
MASTER OF PHARMACY
IN
PHARMACOLOGY
Submitted by
Vinaykumar Patil
I. M.Pharm
Under the Guidance of
Geetha.K.M
Associate professor & Head of the Department
Department of Pharmacology
DAYANANDA SAGAR COLLEGE OF PHARMACY BANGALORE-560078
2009
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
BANGALORE - 560 041, KARNATAKA
ANNEXURE - II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1.
Name of the Candidate
and Address
Vinaykumar Patil
S/o G S Patil
At: Hossalli
Post: Aladakatti
Tal/Dist: Haveri.
2.
Name of the Institution
Dayananda Sagar College Of Pharmacy
Shavige Malleshwara Hills
Kumaraswamy layout,
Bangalore – 560 078
3.
Course of Study and
Subject
Master of Pharmacy in Pharmacology
4.
Date of Admission
19th June 2009
5.
Title of the Topic:“Evaluation of hepatoprotective and antibacterial activity of aqueous
alcoholic (70%) extract of Rhodomyrtus tomentosa (Aiton) Hassk.”
6.0
Brief resume of the intended work:
6.1 – Need of the study:
Medicinal plants are interesting as a source of pharmacologically active compounds.
Many plants have been used by the world population for their basic health care needs
including the treatment of infections. It has been recognized by WHO that they are the
main medicinal source used to treat infectious diseases in most developing countries and
also become more important in a number of developed countries. It is of interest that
scientific and clinical studies for alternative treatment of infections with medicinal plants
as a source of antibacterial compounds have increased worldwide. There is an increasing
concern that bacteria are becoming resistant to clinically used drugs and there is a high
demand to discover new antibiotics to fight against resistant bacterial species. Many
studies try to find alternative ways to reduce and prevent the problem of antibiotic
resistance in bacteria. A number of plants may have a strong activity and valuable
medical potential to be developed into an effective drug. Such plant may substitute
antibiotic consumption or decrease antibiotic resistant bacteria. Rhodomyrtus tomentosa
(Aiton) Hassk. Is an evergreen shrub native to Southeast Asia. This plant is one of the
components in traditional medicines used to treat urinary tract infections for example
rhodomyrtone1 with an amount of 0.3% (dry weight). Previous studies demonstrated that
the leaf extract from R.tomentosa possessed significant antibacterial activity against
Gram-positive bacteria. Therefore, the aim of this study was to further investigate the
antibacterial activities of leaf extract from R.tomentosa against other different strains of
bacteria.
The liver is an important organ concerned with the biochemical functions in the human
body. It detoxifies and synthesizes substances; therefore damage to the liver caused by
hepatotoxic agents is of serious matter. There is an ever-increasing need of an agent,
which could protect it from such damage. In view of severe undesirable side effects of
synthetic agents, there is growing focus to follow systemic research methodology and to
scientifically evaluate the basis for traditional herbal medicines, which are claimed to
possess hepatoprotective activity. Hepatotoxicity is a potentially serious adverse effect of
currently used anti-tubercular chemotherapeutic regimens containing isoniazid,
rifampicin, and pyrazinamide. Adverse effects of anti-tubercular therapy are sometimes
potentiated by multiple drug regimens. Isoniazid, rifampicin and pyrazinamide are each
potentially hepatotoxic, thus when administered in combination their toxic effect is at
least additional. Isoniazid causes liver damage due to its reactive metabolites generated
from acetylhydrazine. Rifampicin is an enzyme inducer and enhances formation of
reactive metabolites which impair the uptake of bilirubin and cause acute cellular
necrosis. Pyrazinamide also has the potential to produce hepatocellular damage. An
increased level
of
aspartate transaminase,
alkaline phosphatase
and alanine
aminotransferase in serum of antitubecular drug treated rats is due to leakage of these
enzymes from liver as a result of tissue damage.
About the plant:
Rhodomyrtus tomentosa is a flowering plant in the family of myrtaceae, native to
southern and souteastern asia, from India, east to southern China, Taiwan and the
Philippines, and south to Malaysia and Sulawesi. It grows in coasts, natural forest,
riparian zones, wetlands, moist and wet forests, bog margins, from sea level up to 2400 m
elevation.
It is often used in traditional Thai medicine. Extracts and pure compounds
(rhodomyrtone)1 from this plant have been proved to possess antibacterial activity. There
have also been reports that it has anti-hepatitis2 properties. Extracts have also acted as a
blood platelet aggregation inhibitor and calcium antagonist. In addition, it is used in
formulations
of
skin-whitening,
anti-aging
and
skin-beautifying
agents.
Acylphloroglucinol2, anthracene glycosides3, flavonoids, tannins and triterpenes have
been identified from this plant.
Based on the above said ethno medical uses and chemical constituents present in the
plant, the present work would be done to evaluate the antibacterial activity against and
the hepatoprotective activity of 70% aqueous alcoholic extract of Rhodomyrtus
tomentosa
against antitubercular drugs
induced hepatotoxicity in rats.
(Isoniazid, Rifampicin and Pyrazinamide)
6.2 – Review of the literature:
Herbal drugs play a major role in the treatment of bacterial infections and hepatic
disorders. A number of
medicinal plants and their formulations are used to cure
bacterial infections hepatic disorders in the traditional medicine.
1. Surasak Limsuwana et. al (2009) reported Rhodomyrtone: A new candidate as
natural antibacterial drug from Rhodomyrtus tomentosa.
2. Asadhawut
Hiranrat,
Wilawan
Mahabusarakam
(2008)
identified
New
acylphloroglucinols from the leaves of Rhodomyrtus tomentosa.
3. Nguyen Huu Tung et. al (2009) reported New Anthracene Glycosides from
Rhodomyrtus tomentosa Stimulate Osteoblastic Differentiation of MC3T3-E1
Cells.
4. V. Kuetea et. al (2008)4 reported Antimicrobial activity of the crude extracts and
compounds from Ficus chlamydocarpa and Ficus cordata (Moraceae):
5. Veronique Seidel1, Peter W. Taylor (2004) 5 reported In vitro activity of extracts
and constituents of Pelagonium against rapidly growing mycobacteria.
6
6. J.I. Murilloa et. al (2003)
reported Antimycobacterial flavones from
Haplopappus sonorensis.
7.
Tulin Askun et. al (2009)
7
reported In vitro activity of methanol extracts of
plants used as spices against Mycobacterium tuberculosis and other bacteria.
8. Shahani.S (1999)8 reported hepatoprotective efficacy of APCL-a polyherbal
formulation in vivo in rats.
9. Berkowitz.FE et. al (1998)9 showed acute liver failure caused by isoniazid in
a child receiving carbamazepine.
10. Ramachandran G and P. Gurumurthy P (2002)10 reported
toxic
effect
of
rifampicin and isoniazid on cytochrome P-450 in mycobacteria.
11. Saraswathy SD et. al (1998)11 reported hepatoprotective effect of of Liv 100
against anti-tubercular drugs induced hepatotoxicity in rats.
12. Vijaya padma V et. al (1998)12 reported hepatoprotective effect of Liv 52 on
anti-tubercular drugs induced hepatotoxicity in rats.
13. Tahaoglu K et. al (2001)13 reported that isoniazid, rifampicin and pyrazinamide
are each potentially hepatotoxic, thus when administered in combination.
14. Achliya GS et. al (2004)14 reported hepatoprotective effect of Amalkadi ghrita
against carbon tetrachloride-induced hepatic damage in rats
15. Rao GM et. al (2006)15 reported hepatoprotective effect of rubiadin, a major
constituent of Rubia cardifolia Linn.
16. Askgaard D S et. al (1995)16 reported hepatotoxicity caused by the combined
action of isoniazid and rifampicin.
17. Khanna BK (1983)17 reported hepatitis during isoniazid, pyrazinamide and
rifampicin therapy.
18. Yanpallewar S (2003)18 reported the effect of Azadirachta indica on paracetamolinduced hepatic damage in albino rats.
19. Agrawal S(2004)19 showed hepatoprotective activity of Eclipta alba
against
paracetamol induced hepatocellular damage in mice.
6.3 – Objective of the Study:
Extracts and pure compounds from this plant have been proved to possess antibacterial
activity. There have also been reports that it has anti-hepatitis properties. Extracts have
also acted as a blood platelet aggregation inhibitor and calcium antagonist. In addition, it
is used in formulations of skin-whitening, anti-aging and skin-beautifying agents.
Acylphloroglucinol, anthracene glycosides, flavonoids, tannins and triterpenes have been
identified from this plant. Recent studies have been reported for their antioxidant
activity also. It has been reported that hepatotoxicity is associated with the generation of
free radicals or reactive oxygen species (ROS).
The present investigation is undertaken to study the antibacterial activity of aqueous
alcoholic (70%) extract of Rhodomyrtus tomentosa (Aiton) Hassk against different strains
of various bacteria’s and hepatoprotective effect against antitubercular drug induced
hepatotoxicity in rats.
Thus, the aim and objective of the present investigation are

Collection and authentication of leaves of Rhodomyrtus tomentosa (Aiton) Hassk

Extraction of leaves with aqueous alcohol ( 70%) in a soxhlet apparatus

Carrying out preliminary Phyotochemical screening to identify phytochemical
constituents.

Evaluation of Hepatoprotective activity against antitubercular drugs induced
hepatotoxicity using various parameters such as
(A) Biochemical Parameters.
1) Aspartate amino transferase (AST)
2) Alanine amino transferase (ALT)
3) Alkaline Phosphatase (ALP)
4) Gamma Glutamate transpeptidase (GGT)
5) Total bilirubin.
6) Total protein.
(B) Antioxidant studies.
1) Lipid peroxidation(LPO)
2) Superoxide dismutase(SOD)
3) Catalase.
4) Glutathione peroxidase(GPx)

Evaluate the antibacterial activity against gram positive microorganisms.
7.0
Materials and Methods:
7.1 Source of Data:
Whole work is planned to generate data from laboratory studies i.e. experiments are
performed as described in references. Journal of pharmacy and pharmacology,
International Journal of Pharmacology, Indian Journal of Pharmacology, Indian Journal
of Experimental Biology, Complementary and Alternative medicine, Phytotherapy
Research, Fitoterapia, Indian Journal Physiology and Pharmacology, Journal of Ethno
pharmacology, International Journal of Antimicrobial Agents, Food Chemistry, Indian
Journal of
Tuberculosis, International Journal of Tuberculosis, Tetrahedron,
Phytomedicine, Pharmacognosy Magazine, Chemical abstract, Internet browsing and
Standard books like Wealth of India, Agro’s dictionary of Medicinal Plants etc.
7.2 Method of collection of data:
The plant will be
collected from Nilgiris, Ottakkalamand, Tamil Nadu, India.
Authenticated by Dr. Rajan, botanist from Tamilnadu agricultural university. All drugs,
chemicals and solvents used in the study would be procured from reputed manufacturers
like Merck,BDK etc. Estimation of serum parameters will be carried out by using standard
kits or biological method.
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, Approved by CPSCEA.
7.5-Method:
Plant extraction:
Leaves are air dried at room temperature. Extracts of leaves of Rhodomyrtus tomentosa
(Aiton) Hassk will be extracted with 70% aqueous alcohol by soxhlet extraction. The
extract will be dried in vaccum at 40 oc. All stocks will be stored at 20 oc.
Preliminary phytochemical screening:
The extract will be screened for the presence of various secondary metabolites such as
acylphloroglucinol, anthracene glycosides, flavonoids, tannins and triterpenes.
Animals:
Adult male albino rats of Wistar strain weighing 120-l50g were used for the experiment.
They were maintained in clean, sterile, polypropylene cages and maintained under
standard conditions i.e. 26 ± 2 °C and relative humidity 44 - 56% and 10 h light: 14 h
dark cycles each day for one week before and during the experiments. All animals were
fed standard rodent pellet diet and drinking water. All studies were performed in
accordance with the guide for the care and use of laboratory animals, as adopted and
promulgated by the Institutional Animal Care Committee, CPCSEA, India.
Experimental design:
Hepatoprotective activity: The rats were divided into 4 groups of 6 animals each.
Group I served as normal control. Group II rats were administered a combination of three
antitubercular drugs (INH-7.5 mg/kg body wt., RMP-10 mg/kg body wt. and PZA-35
mg/kg body wt. for 45 days by intragastric administration)11 in sterile saline. Group III
rats will be given 70% aqueous alcoholic extract of Rhodomyrtus tomentosa (Dose will
be decided on acute toxic studies). Group IV rats will be administered both 70% aqueous
alcoholic extract of rhodomyrtus tomentosa and the combination of three antitubercular
drugs (INH+RMP+PZA) at the above mentioned dosage for 45 days. After the
experimental period, the rats will be sacrificed by cervical decapitation. Blood will be
collected and the serum will be used for the assay of marker enzymes (lactate
dehydrogenase (LDH), aspartate amino transferase (AST), alanine amino transferase
(ALT) and alkaline phosphatase (ALP)). Immediately after the sacrifice, the liver will be
dissected out, washed in ice-cold saline, weighed and a homogenate will be prepared in
0.1 M Tris- HCl buffer (pH7.4). The homogenate will be centrifuged and the supernatant
used for the assay of marker enzymes superoxide dismutase (SOD), catalase (CAT),
glutathione peroxidase (GPX) and
glutathione-S-transferase (GST). Lipid peroxide
levels will be estimated in serum and liver in terms of TBA reactants using 1, 1, 3,3’
tetramethoxy propane as the standard. Protein will be determined with Folin –Phenol
reagent. A portion of liver was fixed in 10% formalin for histopathological studies.
Histopathological studies:
Liver slices fixed for 12 hrs in Bouin’s solution were processed for paraffin embedding
following standard micro techniques20. 5μm sections of liver grained with alum
haematoxylin and eosin were observed microscopically for histopathological changes.
Antibacterial activity:
A broth microdilution method1 will be carried out according to Clinical and Laboratory
Standards Institute Guidelines(CLSI 2006). The minimum inhibitory concentration
(MIC) will be
recorded as the lowest concentration that produced a complete
suppression of visible growth. Aliquots from the broth with no growth were plated on to
blood agar (BA) plates by using a sterile loop and incubated at 37oC for 24h. The
minimum bactericidal concentration (MBC) is defined as the lowest concentration of the
extracts completely preventing bacterial growth.
8.0
List of References:
1. Surasak Limsuwana, Erik N.Trip, Thijs R.H.M.Kouwen, Sjouke Piersma,
Asadhawut Hiranrat, Wilawan Mahabusarakam et al; Rhodomyrtone: A new
candidate
as
natural
antibacterial
drug
from
Rhodomyrtus
tomentosa;
Phytomedicine,16(2009)645–651.
2. Asadhawut Hiranrat, Wilawan Mahabusarakam; New acylphloroglucinols from
the leaves of Rhodomyrtus tomentosa; Tetrahedron, 64 (2008) 11193–11197.
3. Nguyen Huu Tung, Yan Ding, Eun Mi Choi, Phan Van Kiem, Chau Van Minh,
and Young Ho Kim; New Anthracene Glycosides from Rhodomyrtus tomentosa
Stimulate Osteoblastic Differentiation of MC3T3-E1 Cells; Arch Pharm Res, Vol
32, No 4, 515-520, 2009.
4. V. Kuetea, B. Ngamenib, C.C. Fotso Simoc, R. Kengap Tankeuc, B. Tchaleu
Ngadjuib, J.J.M. Meyer et al; Antimicrobial activity of the crude extracts and
compounds from Ficus chlamydocarpa and Ficus cordata (Moraceae); J.
Ethnopharmaco, 120 (2008) 17–24.
5. Veronique Seidel, Peter W. Taylor: In vitro activity of extracts and constituents of
Pelagonium against rapidly growing mycobacteria; Int J.Antimicrobial Agents 23
(2004) 613–619.
6. J.I. Murilloa, R. Encarnacio´n-Dimayugaa, J. Malmstrømb, C. Christophersenb,
S.G. Franzblauc: Antimycobacterial flavones from Haplopappus sonorensis;
Fitoterapia, 74 (2003) 226–230.
7. Tulin Askun a, Gulendam Tumen , Fatih Satil , Mustafa Ates; In vitro activity of
methanol extracts of plants used as spices against Mycobacterium tuberculosis
and other bacteria; Food Chem, 116 (2009) 289–294.re
8. Shahani S.; Evaluation of hepatoprotective efficacy of APCL-a polyherbal
formulation in vivo in rats; Indian Drugs, 1999; 36: 628-31.
9. Berkowitz FE, Henderson SL, Fajman N, Schoen B and
M.Naughton; Acute
liver failure caused by isoniazid in a child receiving carbamazepine; Int J Tuberc,
Lung Dis.1998; 2(7): 603-06.
10. Ramachandran G and Gurumurthy P; Effect of rifampicin and isoniazid on
cytochrome P-450 in mycobacteria; Ind J. Med, Res.2002; 116:140-44.
11. Saraswathy SD, Suja V and Shyamaladevi CS; Effect of Liv 100 against
anti-tubercular drugs induced hepatotoxicity in rats; Ind J. Pharmacol, 1998;
30:233-38.
12. Vijaya padma V, Suja V and Shyamaladevi CS; Hepatoprotective effect of
Liv52 on anti-tubercular drugs induced hepatotoxicity in rats; Fitoterpia,
1998;6:519-20.
13. Tahaoglu K, Atac G, Sevim T, Torum T, Yozicioglu O and Horzum G. The
management of antituberculosis drug induced hepatotoxicity. Int J Tuberc, Lung
Dis 2001;5(1):65-69.
14. Achliya GS, Wadodkar SG and Dorle AK.; Evaluation of hepatoprotective
effect of
Amalkadi Ghrita
against
carbon tetrachloride-induced
hepatic
damage in rats; J. Ethnopharmacol, 2004 90: 229-32.
15. Asha VV, Akhila S, Wills PJ and Subramonium A; Further studies on the
antihepatotoxic
activity
of
Phyllanthus
maderaspatensis
Linn.;
J.
Ethnopharmaco, 2004; 92: 67-70.
16. G. M. Rao GM, Rao CV, Pushpangadan P and Shirwaikar A; Hepatoprotective
effect of rubiadin, a major constituent of Rubia cardifolia Linn; J.
Ethnopharmaco,2006; 103:484-90.
17. Askgaard SD, Wilcke T and Dossing M; Hepatotoxicity caused by the
combined action of isoniazid and rifampicin; Thorax,1995;50(2): 213-214.
18. Khanna B K; Hepatitis during isoniazid, pyrazinamide and rifampicin therapy; Int
J Tuberc, 1983; 30(3):104-6.
19. S . Yanpallewar S; Effect of Azadirachta indica on paracetamol-induced hepatic
damage in albino rats; Phytomedicine, 2003 ;10(5), 391 - 96 .
20. Galigher AE and Kozloff EN; Essential Practical Micro technique, 2nd edn, 1971,
Lea and Febiger, Philadelphia, p.77.
21. Narayana Das Prajapati, Dr. V Kumar; Agro’s dictionary of Medicinal
Plants.2003; Page No. 289.
9
Signature of the candidate
(Vinaykumar Patil)
10
Remarks of the Guide:
Recommended for research
11
Name and Designation of:
11.1 Guide:
Geetha.K.M
Associate Professor & Head of the Dept.
Dept of Pharmacology
Dayananda Sagar College of Pharmacy,
Kumaraswamy layout, Bangalore – 78.
11.2 Signature:
11.3 Co-Guide:
Not applicable
11.4 Signature
11.5 Head of the Department:
Geetha.K.M
Associate Professor & Head of the Dept.
Dept of Pharmacology
Dayananda Sagar College of Pharmacy,
Kumaraswamy layout, Bangalore –78.
11.6 Signature
12
12.1 Remarks of the Chairman and Recommended for research
Principal
Dr. V. Murugan
Professor and principal
Dept of Pharmaceutical Chemistry
Dayananda Sagar College of Pharmacy,
Kumaraswamy layout, Bangalore –78.
12.2 Signature