PROPHYLACTIC AND CURATIVE PROPERTY OF ANTIUROLITHIATIC ACTIVITY OF LEAVES OF MORINGA OLEIFERA LAM

ON EXPERIMENTALLY INDUCED UROLITHIATIC RATS

M-PHARM DISSERTATION PROTOCOL SUBMITTED TO THE

RAJIV GANDHI UNIVERSITY OF HEALTHSCIENCES, KARNATAKA, BENGALURU

BY

Mr. MOHAMMED SAMEER B.Pharm

UNDER THE GUIDANCE OF

PROF. ITTAGI SHANMUKHA M.Pharm.,(Ph.D).

P. G. DEPARTMENT OF PHARMACOLOGYS. C. S. COLLEGE OF PHARMACY

HARAPANAHALLI-5831312012-13

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’sS. C. S. College of Pharmacy,Harapanahalli – 583131 (Davangere dist.) Karnataka

03 Course of the StudyBranch

M. Pharm., Pharmacology

04 Date of Admission to course 16.08.2012

05 Title of the Topic

Prophylactic and curative property of antiurolithiatic activity of leaves of Moringa oleifera lam on experimentally induced urolithiatic rats

06

Brief resume of the intended work6.1. Need for the Study Enclosure – I

6.2. Review of the Literature Enclosure – II

6.3. Objective of the Study Enclosure – III

07

Materials and Methods7.1. 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/1999 CPCSEA.(Copies enclosed)

08 List of References (About 4 – 6) Enclosure – VII

09 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 oleifera Lam.

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 pharmacyHarapanahalli

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-I06. Brief resume of Intended Work

6.1 Need for the study.

Urolithiasis (from Greek oûron, "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-II6.2 Review of Literature:

Moringa oleifera Lam. (Family: Moringaceae) is a small or middle sized tree, about

10 m in height, cultivated throughout India.

Commonly known as:  

1) Drumstick in English, 

2) Saragvo in Gujrati, 

3) Soanjna in Hindi, 

4) Sajna in Bengali, 

5) Nugge in Kannada, 

6) Sigru in malyalam, 

7) Shevga in Marathi,

8)  Shobhanjana in Sanskrit, 

9) Munaga in Telgu and 

10) Murungai in 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. oleifera Lam “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 minerals including 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

Subkingdom Tracheobionta

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

glycosides 4-[(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 contains d-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, wax Flavonoids-quercetin, kaempferol, isoquercitrin, rhamnetin, kaempferitrin

Minerals- 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 flavonoids Presence 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 al15 have 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 al16 explained 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 al18 proved that the leaf juice of Sesbania grandiflora

showed significant antiurolithiatic activity against calcium oxalate-type stones and also

exhibited antioxidant properties.

ENCLOSURE –III6.3 Objectives of the study:

Since there is incomplete phytochemical and pharmacological profile, so it is planned

to undertake 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 phytoconstituents 19, 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 content 21:-

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 content 21:-

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 tannins 22:-

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

a. Male Wistar albino rats weighing between 150-/200 g will be used in this study.

b. 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.

c. Ethylene glycol and ammonium chloride will be used to induce hyperoxaluria to

assess the antiurolithiatic activity in albino rats.

d. Fifty four animals will be randomly divided into seven groups as

Group1,2,3,4,5,6,and 7containing 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

a. Male Wistar albino rats weighing between 150-/200 g will be used in this study.

b. 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.

c. Sodium oxalate and calculi producing diet will be used to induce hyperoxaluria to

assess the antiurolithiatic activity in albino rats.

d. 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 Anti urolithiatic activity requires 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.

7.4 Has 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:

01.Pearle MS, Calhoun EA and Curhan GC (2007). "Chapter 8: Urolithiasis". In Litwin, MS;

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.http://kidney.niddk.nih.gov/statistics/uda/Urolithiasis-Chapter08.pdf.

Retrieved 2011-06-04

02. IIodigwe EE,Akahpa2* ,Nwuru,Cs.Evaluation of the acute and sub chronic toxicities of

eathanol leaf extract of Spathodea campanulata P Beauv.2010,3,17-21.

http://www.healthy-synergics.com.

03.Hossein Hosseinzodeh, Ali-Roza khooi, Zahra khashayavmanesh, Vahideh motamed- 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 UrolithiasisAlternative or complementary? Plant Med. 2009;75:1095-1103

06. Kaur T, Bijarnia RK, Singla SK, Tandon C. In vivo efficacy of Trachyspermum ammianticalcifying 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". http://www.ars- grin.gov/cgi-bin/npgs/html/taxon.pl?24597

11.Pandey et al. Medicinal Aromatic Plants 2012, 1:1http://dx.doi.org/10.4172/map.1000101

12.Bose B (1980) Enhancement of nodulation of Vigna mungo by ethanolic extract

of Moringa leaves – a new report. Nat Acad Sci Lett 3: 103–104

13.Punjani BL (2010) Herbal folk medicines used for urinary complaints in tribal pockets of

Northeast Gujarat. Indian Journal of Traditional Knowledge 9: 126-130.

14. Cáceres A, Saravia A, Rizzo S, Zabala L, De Leon E, et al. (1992)

Pharmacologic properties of Moringa oleifera ,Diuretic activity. J Ethnopharmacol 36: 233-237

15. Karadi RV, Gadge NV, Alagawadi KR, Savadi RV. Effect of Moringa oleifera Lam. root-wood on ethylene glycol induced urolithiasis in rats. J Ethnopharmacol

2006;105:306–11.

16. Bahuguna Y, Rawat MSM, Juyal V, Gupta V. Antilithiatic effect of flowers of Jasminum auriculatum vahl. Int J Green Pharmacy 2009;07:155-8.

17. Betanabhatla KS, Christina AJM, Sundar BS, Selvakumar S, Saravanan KS.Antilithiatic activityof Hibiscus sabdariffa linn. On ethylene glycol-induced

lithiasis in rats. Natural product radiance 2009;8:43-7.

18. Doddola S, Pasupulathi H, Koganti B, Prasad KVSRG. Evaluation of Sesbania grandiflora for antiurolithiatic and antioxidant properties. J Nat Med 2008;62:300-7. 20.

Khandelwal KR. Practical Pharmacognosy techniques and experiments, 2nd ed. Pune:

Nirali Prakashan 2000: 149-156.

19. . Kokate CK. Practical Pharmacognosy 4th ed. New Delhi: Vallabha prakashan, 1999:

149- 156.

20. Khandelwal KR. Practical Pharmacognosy techniques and experiments, 2nd ed. Pune: Nirali Prakashan 2000: 149-156.

21. Shanmukha I, Harshil Patel, Jignesh Patel, Riyazunnisa. Quantification of Total phenol and Flavonoids of Delonix regia Flowers. International Journal of ChemTech Research. 2011;1(3):280-3

22. Urve Paaver, Vallo Matto, Ani Raal. Total tannin content in distinct Quercus robur L. galls. Journal of Medicinal Plants Research. 2010;4(8):702-5

23. Karadi RV, Gadge NB, Alagawadi KR, Savadi RV. Effect of Moringa oleifera Lam. root-wood on ethylene glycol induced urolithiasis in rats. J Ethnopharmacol 2006; 105: 306-311.

24. Shukkur M F, Devarajan A, Periandavan K, Ramasamy S, Palaninathan V. Prophylactic role of phycocyanion: a study of oxalate mediated renal cell injury. Chemico-Biological Interactions 2004;149:1-7.