PROPHYLACTIC AND CURATIVE PROPERTY OF ANTIUROLITHIATIC ACTIVITYOF LEAVES OFMORINGA OLEIFERALAMON EXPERIMENTALLY INDUCED UROLITHIATIC RATS
M-PHARM DISSERTATION PROTOCOL
SUBMITTED TO THE
RAJIVGANDHIUNIVERSITY OF HEALTH
SCIENCES, KARNATAKA, BENGALURU
BY
Mr. MOHAMMED SAMEER
B.Pharm
UNDER THE GUIDANCE OF
PROF. ITTAGI SHANMUKHA
M.Pharm.,(Ph.D).
P. G. DEPARTMENT OF PHARMACOLOGY
S. C. S. COLLEGE OF PHARMACY
HARAPANAHALLI-583131
2012-13
Rajiv Gandhi University of Health Sciences, KARNATAKA, Bengaluru.
Annexure – II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
01 / Name and Address of the Candidate / Mohammed Sameer s/o Md moosa, # 125/1 Joshi keri near National school Harapanahalli-583131KARNATAKA (STATE)
02 / Name of the Institution / T. M. A. E. Society’s
S. C. S. College of Pharmacy,
Harapanahalli – 583131
(Davangere dist.) Karnataka
03 / Course of the Study
Branch / M. Pharm.,
Pharmacology
04 / Date of Admission to course / 16.08.2012
05 / Title of the Topic / Prophylactic and curative property of antiurolithiatic activityof leaves of Moringa oleifera lam on experimentally induced urolithiatic rats
06 /
Brief resume of the intended work
6.1. Need for the Study /Enclosure – I
6.2. Review of the Literature / Enclosure – II6.3. Objective of the Study
/ Enclosure – III07 /
Materials and Methods
7.1. Source of data /Enclosure – IV
7.2. Methods of collection of data
/ Enclosure – V7.3. Does the study require any
Investigations on animals?
If yes give details / Enclosure – VI
7.4. Has ethical clearance been
Obtained form your institution
In case of 7.3. / Yes, Registration No: 157/PO/C/1999cpCsea.
(Copies enclosed)
08 /
List of References (About 4 – 6)
/ Enclosure – VII09 /
Signature of the Candidate
/ (Mr.MOHAMMED SAMEER)10 /
Remarks of the Guide
/ The study is highly justifiable and is feasible to work in the institution. This work may through light on the therapeutic utility on the leaves of Moringa oleiferaLam.11 /
Name and Designation of
(In Block Letters)11.1. Guide
11.2.Signature
11.3.Co-Guide (if any)
11.4.Signature
11.5. Head of the Department
11.6. Signature / PROF. ITTAGI SHANMUKHA
M. Pharm., (Ph.D)
Professor
___
___
Prof. A. Veerana Gouda, M.Pharm.
Head. P.G.Dept of pharmacology
S.C.S College of pharmacy
Harapanahalli
12 /
Remarks of the Principal
12.1. Signature / The present study was permitted to work in the institution and animal ethical committee permission is granted to it.(Dr. R. Nagendra Rao)
ENCLOSURE-I
06. Brief resume of Intended Work
6.1 Need for the study.
Urolithiasis (from Greekoûron, "urine" and lithos, "stone") is the condition where urinary calculi are formed or located anywhere in the urinary system, or the process of forming stones in the kidney, bladder, and/or ureters (urinary tract)1. Urolithiasis is one of the most common urological disorders and has afflicted humans since time immemorial. It is an intricate clinical problem and is a major cause of illness in humans2. It is defined as the presence of one or more calculi in any location within the urinary Tract3.The disease affects 5% to 10% of the population in developed countries with a peak incidence between 40 and 50 years of age4. Men are three times more likely to be affected than women and the lifetime risk of developing a calculus in a Caucasian man is nearly 20%. It has been reported that 91% of the urinary calculi contain calcium in some form, while 8% and 1% are composed of uric acid and cystine, respectively. The calcium-containing calculi consist of pure or various amount of calcium components such as calcium oxalate monohydrate, apatite, calcium hydrogen phosphate and calcium carbonate. In men, 70% to 80% of the calculi contain either calcium oxalate alone or in combination with apatite4. The etiology of this disorder is multifactorial and is strongly related to dietary lifestyle habits or practices. Increased rates of hypertension and obesity, which are linked to nephrolithiasis, also contribute to an increase in stone formation5..The medical management of urolithiasis mainly involves techniques like extracorporeal shock wave lithotripsy and percutaneous nephrolithotomy, however, the prevention of recurrence of stone formation is not assured. Besides, these treatments cause undesirable side effects such as hemorrhage, hypertension, tubular necrosis and subsequent fibrosis of the kidney leading to cell injury and recurrence of renal stone formation. So, It is worthwhile to look for an alternative for the management of urolithiasis, therefore phytotherapy is being sought6.Hence the present study is taken for prophylactic and curative property of antiurolithiatic activity of leaves of moringa oleifera lam (M. oleifera)on experimentally induced urolithiatic rats
ENCLOSURE-II
6.2 Review of Literature:
Moringa oleiferaLam.(Family: Moringaceae) is a small or middle sized tree, about 10 m in height, cultivated throughout India.
Commonly known as:
1)Drumstickin English,
2)Saragvoin Gujrati,
3)Soanjnain Hindi,
4)Sajnain Bengali,
5)Nuggein Kannada,
6)Sigruinmalyalam,
7)ShevgainMarathi,
8)Shobhanjanain Sanskrit,
9)Munagain Telguand
10)Murungaiin Tamil7.
Description:
Moringa oleifera Lam. (M. oleifera) is a highly valued plant, distributed in many countries of the tropics and subtropics. Short, slender, deciduous, perennial tree, to about 10 m tall; rather slender with drooping branches; branches and stems brittle, with corky bark; leaves feathery, pale green, compound, tripinnate, 30–60 cm long, with many small leaflets, 1.3–2 cm long, 0.6–0.3 cm wide, lateral ones somewhat elliptic, terminal one obovate and slightly larger than the lateral ones; flowers fragrant, white or creamy-white, 2.5 cm in diameter, borne in sprays, with 5 at the top of the flower; stamens yellow; pods pendulous, brown, triangular, splitting lengthwise into 3 parts when dry, 30–120 cm long, 1.8 cm wide, containing about 20 seeds embedded in the pith, pod tapering at both ends, 9-ribbed; seeds dark brown, with 3 papery wings. Main root thick. Fruit production in March and April in Sri Lanka8.
M. oleiferaLam “one of the most amazing trees God has created”,almost every part
of drumstick viz. bark,root, fruit, flowers, leaves,seed and gum is a rich repository of proteins, vitamins and mineralsincluding potassium, calcium, phosphorus, iron, folic acid as well as βcarotene. Leaves can be eaten fresh, cooked or stored as dry powder formany months without refrigeration, without loss of nutritional value.Almost all the parts of this plant have been used for various ailments in the indigenous medicine of South Asia9 .
Scientific Classification10:
Kingdom Plantae
SubkingdomTracheobionta
Super division Spermatophyta
Division Magnoliophyta Class Eudicots Subclass Rosidsae Order Brassicales Family Moringaceae Genus Moringa Species oleifera
Chemical constituents11:
The plant contains phytoconstituents such as Aurantiamide acetate (a rare dipeptide), 1,3 dibenzyl urea, Vanillin,β-sitosterol,β-sitostenone, 4-hydroxymellein,octacosanoic acid,Alkaloids- moringine, moringinine,Nitrile glycosides- niazirin, niazirinin, three mustard oil glycosides4-[(4'-O-acetyl-α-L-rhamnosyloxy)benzyl]isothiocyanate, niaziminin A, B.Growth promoters, Phenolic acids-gallic,chlorogenic,ellagic, ferulic acid,Flavonoids- kaempferol, quercetin, rutin;Ascorbic acGlycosides- thiocarbamate, isothiocyanate12,Glycosides- thiocarbamate, isothiocyanate two new compounds, O-[2'-hydroxy-3'-(2"-heptenyloxy)]-propyl undecanoate,O-ethyl-4-[(α-L-rhamnosyloxy)-benzyl] carbamate,Methyl p-hydroxybenzoate, β-sitosterol have also been isolated, The polysaccharide containsd-galactose, 6-O-Me-d-galactose, d-galacturonic acid, l-arabinose, l-rhamnose.Plant hormones- auxins, cytokinin, Amino acids,sucrose,d-glucose, traces of alkaloids, waxFlavonoids-quercetin, kaempferol, isoquercitrin,rhamnetin, kaempferitrinMinerals- potassium, calcium,O-ethyl-4-(α-L-rhamnosyloxy)benzyl carbamate, 4(α-L-rhamnosyloxy)benzyl isothiocyanate, 4(α-L-rhamnosyloxy)benzylglucosinolate, niazimicin, 3–O-(6’-O-oleoyl-beta-D-glucopyranosyl)-β-sitosterol,β-sitosterol-3-O-β-D-glucopyranoside, niazirin, β-sitosterol, glycerol-1-(9-octadecanoate), isothiocyanates,thiocarbamates and flavonoidsPresence of a hemagglutin is also reported.
The literature survey reviewed that various parts of this plant such as leaves, roots, seed, bark, fruit, flowers and immature pods act as cardiac and circulatory stimulants, possess antitumor, antipyretic, anti-inflammatory, antiulcer, antispasmodic, diuretic, antihypertensive, cholesterol lowering, antioxidant, antidiabetic, hepatoprotective, antibacterial and antifungal activities, and are being employed for the treatment of different ailments in the indigenous system of medicine13,14.
Karadi et al15have explained that, the ethylene glycol feeding resulted in hyperoxaluria as well as increased renal excretion of calcium and phosphate. Supplementation with aqueous and alcoholic extract of Moringa oleifera root-wood significantly reduced the elevated urinary oxalate, showing a regulatory action on endogenous oxalate synthesis. The increased deposition of stone forming constituents in the kidneys of calculogenic rats was also significantly lowered by curative and preventive treatment using aqueous and alcoholic extracts15.Recently, Bahuguna et al16explained the same activity on Jasminum auriculatum flowers.Bashir et al17 have reported about the underlying mechanism of antiurolithic effect in Bergenia ligulata rhizome against calcium oxalate stones; mediated possibly through a combination of calcium oxalate crystal inhibitory, diuretic, antioxidant and hypermagneseuric effects, rationalize its medicinal use for urinary stone disease. Doddola et al18proved that the leaf juice of Sesbania grandiflora showed significant antiurolithiatic activity against calcium oxalate-type stones and also exhibited antioxidant properties.
ENCLOSURE –III
6.3 Objectives of the study:
Sincethere is incomplete phytochemical and pharmacological profile, so it is planned toundertake a study on the leaves of this plant with the following objectives.
01.To prepare various extracts (petroleum ether, chloroform, hydro Alcoholic, and aqueous extract) by successive extraction technique.
02.To identify the type of phytoconstituents present in the leaves extract.
03.Quantitative determination of total phenol, flavonoids and tannin content present in the leaves extract by spectrophometry method.
04.To screen the leaves extract for the antiurolithiatic activity on various
(Ethylene glycol, Sodium oxalate induced) experimental models in albino rats.
ENCLOSURE – IV
7. Material & methods:
7.1 Source of data:
Whole work is planned to generate data from laboratory i.e., experiments on animals. The rat will be used for this purpose. Standard analytical procedures will be adopted for estimation of biochemical parameters like Urine analysis, sodium, potassium, calcium,magnesium, oxalate and uric acid will be determined using biochemical kits in a semiautoanalyser. The pH of the urine is determined by pH meter.
Histopathology studies will be performed after sacrificing the animal.It is also planned to use the available literature for interpreting the data.
ENCLOSURE – V
7.2 Method of collection of data:
The whole study is divided into four phases to generate data. Following are four phases.
Phase I:Preparation of extract and Identification of phytoconstituents19, 20:-
The leaves extract will be prepared by successive soxhlation i.e. extracting dried powder with the solvents of increasing order of polarity i.e. Pet. ether (60-80), chloroform (59.5-61.5), 70% ethanol (64.5-65.5) and water. Extracts will be concentrated under reduced pressure.
Phase II:Experimental design
Quantitative determination of total phenol, flavonoid and tannin content by Spectrophotometry:
Quantification of total phenolic content21:-
The total phenolic content of the leaf extract of Moringa oleifera lam.will be determined by taking aliquots of the extracts into 10ml glass tube and the volume will be made up to 3ml with distilled water. Then 0.5ml of Folin ciocalteau reagent (1:1 with distilled water) and 2 ml sodium carbonate (20%) will be added subsequently in each test tube. A blue color will be developed in each test tube because the phenols will undergo complex redox reaction with phosphomolibdic acid in Folin ciocalteau reagent in alkaline medium. This results in a blue colored complex, molybdenum blue. The test solutions will be warmed for 1min, cooled and the absorbance will be measured at 650nm using known concentration of catechol. The concentrations of phenols in the test samples will be calculated from the calibration plot and expressed as mg catechol equivalent of phenol per gram of sample.
Quantification of total flavonoid content21:-
To determine the total flavonoidal content, the stock solutions of extract will be prepared with ethanol to a suitable concentration for analysis. For determination of total flavonoidal content, aliquots of each extract will be pipetted out in series of test tubes and the volume will be made up to 1ml with distilled water. Sodium nitrite (5%; 0.3ml) will be added to each test tube and incubated for 5minutes at room temperature. Aluminium chloride solution (10%; 0.06ml) will be added and incubated for 5minutes at room temperature. Sodium hydroxide (1M; 0.25ml) will be added and total volume will be made up to 3ml with distilled water. Absorbance will be measured at 510nm against a reagent blank using U.V. spectrometer and concentration of flavonoids in the test sample will be determined and expressed as mg equivalent per gram of sample.
Quantification of tannins22:-
The tannins will be identified using FeCl3 and gelatin tests. For this purpose, 0.1g of flowers extract will be transferred to a 100ml flask. 50ml of water will be added and boiled for 30min. After filtration with cotton filter, the filtrate will be transferred to a 500ml volumetric flask and the volume will be made up to the mark with distilled water. 0.5 ml aliquots will be transferred to the vials, 1ml 1% K3Fe(CN)6 and 1 ml of 1% FeCl3 will be added and the volume will be made up to 10ml with distilled water. After 5 min the solution will be measured calorimetrically at 720nm. The total content of tannins present in the plant extract will be obtained from standard calibration curve which will be made by taking the tannic acid as standard.
Antiurolithiatic activity:
1. Ethylene glycol induced urolithiasis: 23
- Male Wistar albino rats weighing between 150-/200 g will be used in this study.
- The rats will be housed in an environmentally controlled room with a 12 h light-dark cycle, and free access to normal rat chow and tap water.
- Ethylene glycol and ammonium chloride will be used to induce hyperoxaluria to assess the antiurolithiatic activity in albino rats.
- Fifty four animals will be randomly divided into seven groups as Group1,2,3,4,5,6,and7containing six animals in each as follows:
Group1: Normal control. Group2: Urolithiatic control.
Group 3: Cystone (750 mg/kg).
Group 4: Curative group (Low dose of extract)
Group 5: Curative group (Higher dose of extract)
Group 6: Preventive group (Low dose of extract)
Group 7: preventive group (Higher dose of extract)
Except Group I all rats will be given ethylene glycol in the drinking water to a final concentration of 0.75%, with 2% ammonium chloride for 15 days, Curative group will receive hydro alcoholic extract of Moringa Oleifera Lam from 15th day till 28th day and preventive group will receive extract from 1st day till 28th day. All extracts will be given once daily by oral route. Urine samples (24 h) will be collected at 0, 7, 14, 21 and 28 days.
2. Sodium oxalate induced urolithiasis:24
- Male Wistar albino rats weighing between 150-/200 g will be used in this study.
- The rats will be housed in an environmentally controlled room with a 12 h light dark cycle, and free access to normal rat chow and tap water.
- Sodium oxalate and calculi producing diet will be used to induce hyperoxaluria to assess the antiurolithiatic activity in albino rats.
- Fifty four animals will be randomly divided into nine groups as Group 1, 2, 3, 4, 5, 6, and 7 containing six animals in each as follows:
Group 1: Normal control. Group2: Urolithiatic control.
Group3: Cystone (750 mg/kg).
Group 4: Curative group (Low dose of extract)
Group 5: Curative group (Higher dose of extract)
Group 6: Preventive group (Low dose of extract)
Group 7: preventive group (Higher dose of extract)
Sodium oxalate induced urolithatic model in rat will be used to assess the effect of hydro alcoholic extract of Moringa Oleifera Lam. The study is designed to find out the effect of hydro alcoholic extract of Moringa Oleifera Lam on therapeutic usage against sodium oxalate induced urolithiasis. All rats will be housed in metabolic cages individually for entire duration of the experiment.
Phase IV:
Histopathological studies of kidney.
Statistical analysis.
The study design, criteria and plan of work are outlined as below: --
Inclusion criteria for the selection of animals:--
Sex: - male Age- Adult animals.
Weight: -150-200 grams Health condition: - Healthy
Exclusion criteria:- Any animal not conforming with above criteria are not selected for the experiment.
Study sampling:- Each model of Antiurolithiatic activityrequires nine groups of six animals each.
Duration of the study: Ten months
Total number of animals required:
a) Wistar rats = 66
A) To carry out antiurolithiatic activity:
a)Adult male Wistar albino rats =66
Parameters to be evaluated:
Urine analysis (pH, calcium, phosphate, magnesium, oxalate)
Serum analysis (Calcium, phosphorus, creatinine and urea)
Assay of tissue enzyme: All the animals will be sacrificed at the end of the treatment period. Kidney homogenates will be prepared and the following enzyme levels will be analyzed using suitable methods(Glycolate oxidase, Lactate dehydrogenase, Inorganic pyrophosphatase, Acid phosphatase, Alkaline phosphatase.)
Statistical analysis: The results obtained from the above investigation will be
Subjected to statistical analysis using one way ANOVA followed by Tukey-
Kramer Multiple Comparisons test.
ENCLOSURE – VI
7.3 Does the study require any investigation or interventions to be conducted on patients or other humans or animals? If so, please describe briefly.
Yes, Albino rats will be used for the Prophylactic and curative property of antiurolithiatic activity.
7.4Has ethical clearance been obtained from your institution in case of 7.3?
Yes, the present study is approved from Institutional Animal Ethics Committee
(IAEC Certificate enclosed. Ref. No-SCSCOP/626/8/2012-13 dated 07.01.2013)
ENCLOSURE – VII
8.0List of references:
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Saigal, CS. Urologic Diseases in America (NIH Publication No. 07–5512). Bethesda,
Maryland: US Department of Health and Human Services, Public Health Service, National
Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases.
pp.283–319.
Retrieved 2011-06-04
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eathanol leaf extract ofSpathodea campanulata P Beauv.2010,3,17-21.
03.Hossein Hosseinzodeh, Ali-Roza khooi, Zahra khashayavmanesh,
Vahidehmotamed- shariaty.
Antiurolithiatic activity of PinusEldarica Medw. Fruit aqueous extract in
Rats. Urology Journal. 2010; 7(4):232-37.
04.Yadav et al., IJPSR, 2011; 2(6): 1412-1420 ISSN: 0975-8232
05. Butterweck V. Khan SR.Herbal medicines in the management of Urolithiasis
Alternative or complementary? Plant Med. 2009;75:1095-1103
06. Kaur T, Bijarnia RK, Singla SK, Tandon C. In vivo efficacy of Trachyspermum ammi
anticalcifying protein in urolithiatic rat model. J Ethnopharmacol 2009;126:459–62
07.Tarafder CR, Ethno-gynecology in relation to plants, 2. Plants used for abortion, J Econ
Taxon Bot, 1983, 4(2), 507-516.
08.James A.Duke 1983 Hand book of Energy crop
09.Burkill, J.H. 1966. A dictionary of economic products of the Malay peninsula. Art
Printing Works, Kuala Lumpur. 2 vols.
10.From Wikipedia, the free encyclopedia"USDA GRIN Taxonomy".