Pharmacognostical, Phytochemical and Pharmacological activity studies (Diuretic and Antiurolithiatic) on the root of

Mitragyna parvifolia (Roxb.)Korth

SYNOPSIS FOR

M. PHARM. DISSERTATION

SUBMITTED TO

RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES,

BANGALORE, KARNATAKA.

SUBMITTED BY

R. LAKSHMI RAMYA

I- M. PHARM (2012-2013) PHARMACOGNOSY

DEPARTMENT OF PHARMACOGNOSY

M.S.RAMAIAHCOLLEGE OF PHARMACY

BANGALORE- 560054

1. / NAME OF THE CANDIDATE AND ADDRESS / R. LAKSHMI RAMYA
D/O R. BHASKARA RAO
No#448C 7/1, 8th CROSS HMT LAYOUT
MATHIKERE, BANGALORE – 560054.
2. / NAME OF THE INSTITUTION / M.S.RAMAIAHCOLLEGE OF PHARMACY, BANGALORE-560054.
3. / COURSE OF STUDY AND SUBJECT / M.PHARMACY
PHARMACOGNOSY
4. / DATE OF ADMISSION / 28-04-2012
5. / TITLE OF THE TOPIC / PHARMACOGNOSTICAL, PHYTOCHEMICAL AND PHARMACOLOGICAL (DIURETIC & ANTI UROLITHIATIC) ACTIVITY STUDIES ON ROOT OF Mitragyna parvifolia (Roxb.) Korth.
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12 / BRIEF RESUME OF THE INTENDED WORK
NEED FOR STUDY
Mankind all over the world mainly depends upon plant kingdom to meet all the needs of medicine for alleviating ailments since ancient times. WHO estimated that 80% of population depends on traditional medicine, mostly derived from plant drugs for their primary health care needs1.
The role of medicinal plants as primary tools in the preservation of health as well as prevention and management of diseases is realized with alarming concern in recent days. India is well known as the “Emporium of medicinal plants”; the use of plants to treat various diseases in India dates back to the times of Rigveda (3500 to 1800Bc)2.
Ayurveda is a traditional healing system of India with its origin firmly rooted in culture of Indian subcontinent3. According to Ayurveda there is no plant which is non medicinal 4.
Kadamba is an important Ayurvedic drug; it possesses bitter, astringent, febrifuge, expectorant properties. Stem bark is used in inflammations, haemoptysis, fever, strangury, cough, eyediseases, wounds, ulcers and debility. Root is used as diuretic and in gravels, and in urinary calculi5.
The accepted botanical source of Kadamba is Neolamarckia cadamba (Roxb.) Boiss. Its synonyms are Anthocephalus cadamba Miq and Anthocephalus chinensis (Lam.) A. Rich.Mitragyna parvifolia (Roxb.) Korth is used as the substitute for kadamba6,7.
Mitragyna parvifolia (Roxb.) Korth belongs to family Rubiaceae. It is commonly called as Leichhardt tree, cheesewood. It is distributed in all districts of India and in Srilanka, Myanmar(Burma)8. It is found in all dry deciduous forests and in evergreen forests 9.
Root bark of Mitragyna parvifolia (Roxb.)Korth. contains isorhynchophylline and rhynchophylline. Leaves contain alkaloids angustine tetrahydroalstonine, akuammigine, pteropodine, isopteropodine, speciophylline, uncarine F, rotundifoline, isorotundifoline, khirsultine, dihydrocornyantheine and demethoxy isohortiamine 10.
Diuretics are the drugs which cause a net loss of sodium ions and water from the body resulting in an increase of secretion of urine from kidney11. Several plants are reported to show significant diuretic activity such as Tribulus terrestris, Boerhaavia diffusa, Trichosanthus lobata, Tinospora cardifolia.
The present investigation is aimed to carry out Pharmacognostical, Phytochemical and Pharmacological activity (Diuretic and Urolithiatic) of the root of Mitragyna parvifolia (Roxb.)Korth. It is found that no similar work has been carried out so far on the root of species especially with regard to Diuretic and Urolithiatic activity. Pharmacognostical studies include exomorphology of plant, macro & microscopical studies of the root including macerate studies, UV analysis, Histochemical studies, Flourescence analysis and chromatography (HPTLC) studies.
REVIEW OF LITERATURE
Mitragyna parvifolia (Roxb.) Korth belongs to family Rubiaceae13. Mitragyna parvifolia (Roxb.) Korth is a large deciduous tree14.The vernacular names of Mitragyna parvifolia are kadavala (Kannada), kaddam (Hindi), Bhoomi kadamba(Sanskrit), Battagenupu (Telugu)15.
The phytoconstituents reported in Mitragyna parvifolia root bark are isorhynchophylline and rhynchophylline16. Leaves constituteTetra hydroalstonine, akuammigine, pterodine, isoteropodine, speciophylline and uncarine F, Corynantheidol(allo) and dihydro corynantheol17,18. Other constituents isolated are 9-methoxyrhynchophylline N-oxide19. Antioxidant and Anti inflammatory activities ofMitragyna parvifolia leaves extract was reported20.
Anti convulsant activity of Mitragyna parvifolia leaves extract was reported21.
Invitro investigation of Anthelmintic activity of Mitragyna parvifolia(Rubiaceae) was reported22.
Anti inflammatory and anti nociceptive activity of Mitragyna parvifolia was reported23.
Anti microbial activity of Mitragyna parvifolia barks and Butea monosperma leaves extract against human pathogenic microbial strains was reported24.
MAIN OBJECTIVE OF STUDY:
The main objective of the proposed work is to evaluate the Pharmacognostical, Phytochemical and Diuretic and anti urolithiatic activity studies on the root of Mitragyna parvifolia.
The scheme of proposed work is as follows:
a)To study Taxonomical characters of plant which helps in the Identification of plant.
b)To understand the macro and microscopical characters of drug besides carrying out Phytochemical studies including HPTLC analysis. This helps in evolving diagnostic characters for the identification of drugs and also contribute towards Pharmacopoeial standards.
c)To carry out acute toxicity studies on alcohol and aqueous root extracts of Mitragynaparvifolia & to evaluate the diuretic and anti urolithiatic activity of the drug.
SOURCES OF DATA:
Data will be acquired from Books, both the National and International journals and also from other reputed libraries.
The day to day development in the area will be updated by conducting literature survey through e – publishing and Helinet provided by RGUHS.
METHOD OF COLLECTION OF DATA:
The whole study is divided into different phases.
Phase I
Field and laboratory based studies:
Field work:
The selected plant and the root part will be collected from the forests of South India. The voucher specimen will be collected and deposited at the herbarium/ museum of the department.
Phase II
Laboratory work:
  1. Taxonomical Studies:
The plant material will be identified by using various floras25,26.
  1. Pharmacognostical studies:
Free hand sections will be taken following Wallis.27
Microscopical studies including macerate studies and Histochemical tests will be carried out following Evans.28
Quantitative microscopy for measurements of tissues will be done by using Micro Image Lite analysis software. Using Photomicrography images will be captured on computer.
Physical constants of drug will be determined as per Indian Pharmacopeia.The chromatographic and HPTLC studies will be carried out following Krebs et al29 and flouresence studies will be carried out following Kokoski et al30.
  1. Preliminary phytochemical investigation:
The preliminary phytochemical investigation of the root extracts will be carried out as per Harborne31, after successive solvent extraction with petroleum ether, benzene, chloroform, 95% v/v ethanol, water using soxhlet apparatus. All the chemicals and reagents proposed to be used will be of analytical grade.
Phase III
Pharmacological studies:
Animals: Albino Wistar rats of either sex will be used in the study.
Inclusion criteria:
Albino Wistar rats of either sex
Age 1½ -2 months
Weight range 170-200g.
Exclusion criteria:
Diseased animals
Other strains of albino rats
Wistar albino rats outside the weight range and age mentioned above.
Pharmacological studies will be carried out using aqueous and alcohol extracts of M.parvifolia roots.
Preparation of the extracts
  1. Aqueous extract
Powdered shade dried roots of M.parvifoliawill be extracted by maceration with chloroform water (0.25% v/v of chloroform in distilled water) followed by filtration and concentration of the extract to dryness under reduced pressure. The aqueous extract for pharmacological studies will be prepared by dissolving the extract in distilled water.
2. Alcohol extract
Powdered shade dried roots of M.parvifolia will be extracted with 95% v/v ethanol in soxhlet apparatus by continuous hot extraction. The alcohol extract will be filtered and concentrated to dryness under reduced pressure. The alcoholic extract for pharmacological studies will be suspended in a suitable suspending agent.
The total alcohol and aqueous extracts of the root of M.parvifolia will be subjected to the following pharmacological studies
a)Acute toxicity of the aqueous and alcohol extracts of roots of M. parvifolia
b)Diuretic activity ofthe aqueous and alcohol extracts of roots of M.parvifolia
c)Anti urolithiatic activity of the aqueous and alcohol extracts of roots of M.parvifolia
a)Acute toxicity studies:
OECD guidelines32 will be followed for the determination of minimum lethal dose of alcohol and aqueous extracts of the roots of M.parvifolia. Wistar albino rats weighing 170-200g will beadministered with the extracts p.o. up to the dose of 2000 mg/kg and will be observed for any symptoms of toxicity and/or mortality for 48h. Further the animals will be under regular observation for a week.
b)Diuretic activity:
Diuretic activityof the alcohol and aqueous extracts of roots of M. parvifolia will be evaluated bythe method described by
Lipschitz et al33 .The alcohol and aqueous extracts will be suspended in a suitable vehicle for oral administration. Furosemide (25mg/kgb.w) will be used as the standard diuretic agent.
Animals will be fasted and deprived of food and water for 18h prior to and during the experiment. Thirty six healthy male rats will be selected and are divided in to six groups consisting of six rats each. The treatment pattern will be as follows.
Group 1 (control): Vehicle control
Group 2 (standard): Furosemide (25mg/kg b.w) p.o.
Group 3,4: Test groups with alcohol extract p.o.
Group 5,6: Test groups with aqueous extract p.o.
All the animals will be hydrated with 25ml/kg of distilled water prior to the drug administration. Immediately after dosing, animals will be placed in metabolic cages specially designed to separate urine and faeces. Urine will be collected in a measuring cylinder up to 5h. During this period animals will be deprived of food and water.
The diuretic activity will be evaluated by measuring various parameters34 such as
  • Total urine volume
  • Urine concentration of Na+, K+,Cl- ions
  • Serum urea
  • Serum creatinine
  • Urinary creatinine
  • GFR
  • Urine PH
The results will be subjected to statistical analysis by one way ANOVA followed by Tukey Krammer multiple comparision test.
c) Anti Urolithiatic activity35,36,37:
Urolithiasis will be induced by the addition of ammonium chloride (AC) (2%w/v) & ethylene glycol (EG) (0.75%v/v) into drinking water and fed to animals for 10 days.
42 healthy male adult albino Wistarstrain rats weighing 140-200g will be divided in to seven groups. Treatment period is 10 days.
Group 1: Normal rats willbe fed with rat chow and water for 10 days.
Group 2: Positive control(normal diet + water containing EG 0.75% v/v and AC 2% w/vfor 10 days).
Group 3: (Standard): Normal diet + water containing EG 0.75% v/v & AC 2% w/v + Cystone (750mg/kg) for 10 days.
Group 4, 5: Test groups with 2 doses of aqueous extract will be fed with normal diet + water containing EG 0.75% v/v & AC 2% w/v for 10 days.
Group 6,7: Test groups with 2 doses of alcohol extract + normal diet + water containing EG 0.75% v/v AC 2% w/v for 10 days.
Anti urolithiatic activity is evaluated by
a)Urine and Blood sampling:
Urine samples collected over 24 hwill be centrifuged and examined under light microscope for the presence of oxalate micro crystals, followed by the estimation of
  • Calcium
  • Uric acid
  • Oxalate
  • Glycolate
Blood samples will be collected by retro orbital puncturing under light ether anaesthesia before they are sacrificed. Serum will be separated from the blood collected and analyzed for
  • Creatinine
  • Calcium
Histopathology studies will be performed for 1 animal from each group.
The results will be subjected to statistical analysis by one way ANOVA followed by Tukey Krammer multiple comparison test.
Does the study require any investigation to be conducted on patients or other humans or animals? If so, please describe briefly.
Yes, Animal experiments on rats will be required for studying acute toxicity, diuretic and urolithiatic activity.
Has ethical clearance been obtained from your institution in case of 7.3?
Our Institution’s Animal Ethics Committee meeting has been convened. Ethics Committee Clearance will be obtained and the same will be submitted in due course.
LIST OF REFERENCES:
  1. Kalia A.N Industrial Pharmacognosy, CBS publishers and distributors 2003;2.
  2. Medicinal Plants of India vol-1 Karnataka, S.N.Yoganarasimhan, Interline publishing limited, Bangalore. Pgno:314.
  3. Elizabeth M.Williamson, major herbs of Ayurveda, Dabur research foundation and Dabur Ayurvet limited.
  4. Sharma PC, Yelne MB and Dennis TJ. Database on medicinal plants used in ayurveda, 2nd ed. Delhi, Central council for research in Ayurveda and Siddha 2000;1(9);4.
  5. Sharma PC, Yelne MB and Dennis TJ. Database on medicinal plants used in ayurveda, 4th ed. Delhi, Central council for research in Ayurveda and Siddha .pg no: 242.
  6. The Ayurvedic formulary of India vol.2, Government of India. Ministry of health and family planning, Department of health.
  7. Medicinal plants of Tamilnadu - vol.2, S.N. Yoganarasimhan, Interline publishers pgno: 357.
  8. Medicinal plants of Tamilnadu – vol.2, S.N.Yoganarasimhan, Interline publishers pgno: 357.
  9. Flora of Presidency of Madras, J S Gamble vol.2;pgno:585.
  10. Medicinal plants of Tamilnadu – vol.2, S.N. Yoganarasimhan, Interline publishers pgno: 357.
  11. Agarwal SL, Agarwal S, Pharmacology for undergraduate students (2nd edn). New delhi: CBS Publishers and distributor;2000;130
  1. Sharma PV Dravyaguna Vijnana.vol.2, Varanasi; oriental publishers a distributors; 2005; 630,651,658,734,761.
  2. Flora of presidency of Bombay ;Theodore, Cooke vol-1 pgno: 581-582.
  3. Flora of presidency of madras ; J.S.Gamble vol-2 pgno:585.
  4. Botanical and Vernacular names – Magadi R.Gurudeva. pgno: 279.
  5. Journal tropical of medicinal plants vol-12 no.2 Dec 2011.
  6. Compendium of Indian Medicinal plants vol-2 (1970-1979),Ram P.Rastogi, B.N.Mehrotra, Central Drug Research Institute Lucknow and National Institute of Science Communication New delhi 1999.
  7. Planta medica 1973,24,13.
  8. Phytochemistry 1973,12,2043.
  9. D.Kaushik etal. Der pharmacia letter: 2009 1(1): 75-82.
  10. A Badgujar vishal.B and Surana sanjay - Veterinary world vol-3(7); 326-328.
  11. Kaushik etal. Pharmacology online 3; 101-106(2009).
  12. Asian journal of medical sciences 1(3); 97-99, 2009. ISSN: 2040-8773- Maxwell scientific org. 2009.
  13. Antimicrobial activity of Mitragyna parvifolia barks and Butea monosperma leaves International journal Drug development and research, Oct – Dec 2011.5(4); 141-147.
  14. Flora of presidency of madras ; J.S.Gamble vol-2 pgno:585.
  15. Flora of presidency of Bombay ;Theodore, Cooke vol-1 pgno: 581-582.
  16. WallisTE. Text book of Pharmacognosy,New Delhi CBS Publishers and Distributors;1985; 355-358.
  17. Evans WC. Trease and Evans Pharmacognosy, 15th ed.,London Saunders; 2002;137.
  18. Krebs KG, Heunsen D,Wimmer H.Thin layer chromatography, A laboratory hand book, 2nd ed., London ELBS; 1969; 204-255,855-909.
  19. Kokoski CJ, Kokoski RJ, Slama FJ. Flourescence of powdered vegetable drugs under ultraviolet radiation. J.Amer. pharm1958 Oct; XLVII (10): 715-717.
  20. Harborne JB. Phytochemical methods, A Guide to modern techniques of plant analysis, 3rd., London Chapman and Hall; 1998:60-66,74-87.
  21. iccvam.niehs.nih.gov/Supp Docs/Fed Docs/OECD/OECD_GL423.
  22. . Lipschitz WL, Hadidan Z, Kerpscar A(1943), Bioassay of diuretics, J. pharm Exp Ther.79: 97-110.
  23. Der. pharmacia Lettre, 2011:3(4) 207-214. (http;/scholars research library.com/archieve.html).
  24. Chow FHC, Hamar DW, Udall RH (1974). Prevention of oxalate and phosphate lithiasis by alanine. Invest Urol. 12:50-54.
  25. Metzer RP, Sauerheber RD, lyous SA, Westall JR (1975) The effect of streptozotocin diabetes on the levels of glycolate and lactate excreted in rat urine. Arch Biochem Biophys. 169:555-558.
  26. Colkins VP (1943) Microdetermination of glycolic and oxalic acids. Analyt Chem. 15:762-766.
Signature of the Candidate:
Remarks of the Guide:
Name and Designation:
11.1) Guide Dr. V.Madhavan. M. Pharm. Ph.D.
Principal & HOD
Department of Pharmacognosy
M. S. Ramaiah College of Pharmacy
Bangalore.
11.2) Signature:
11.3) Co-Guide: Mrs. Anita Murali, M pharm
Professor (Department of Pharmacology),
M.S. Ramaiah College of Pharmacy,
Bangalore-54..
11.4) Signature:
11.5) Head of the Department: Dr. V.Madhavan. M. Pharm. Ph.D.
Principal & HOD
Department of Pharmacognosy
M. S. Ramaiah College of Pharmacy
Bangalore.
11.6) Signature:
12.1) Remarks of the Principal:
12.2) Signature:

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