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Enclosure – I 6 Brief resume of the intended work: 1 Need for the study


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ENCLOSURE – I
6 Brief resume of the intended work:

6.1 Need for the study:
The kidneys are organs that filter wastes such as urea from the blood and excrete them along with water as urine and nephrology, the medical field that studies the kidneys and diseases of the kidney1. The kidneys excrete a variety of waste products produced by metabolism including the nitrogenous wastes such as urea from protein catabolism, uric acid from nucleic acid metabolism and water. The kidneys are one of the major organs involved in acid-base balance, regulation of electrolyte concentrations, control blood volume, and ultimately regulates blood pressure2.

Stone formation in the kidney is one of the oldest and most widely spread disease known to mankind. Urinary calculi have been found in the tombs of Egyptian mommies dating back to 4000BC. Reference to stone formation in kidneys is made in the early Sanskrit document in India between 3000 to 2000BC. Kidney stones, also called as renal calculi, are solid concretions i.e crystal aggregations of dissolved minerals in urine and the calculi typically form inside the kidneys or ureters3.

The solutes involved are oxlates, phosphates, urates and uric acid further stones usually consist of more than one substance deposted in layers. After 30 years of age these are more common in males and most originate in collecting tubules or in renal papillae and then pass into the renal pelvis where they may increase in size. Some become too large to pass through the urethra and may abstruct the outflow of urine causing renal failure. Others pass to the bladder and are either excreted are increased in size and obstruct the urethral opening. Predisposing factors for stone formation includes dehydration, pH of urine, infection and metabolic conditions4.
ENCLOSURE – II

6.2 Review of Literature:

Melia azedarach Linn is a tree found common in western Himalayas and cultivated in some parts of India.
Plant Description5,6:

Synonyms: Kondavepa, Turkavepa, Mahanimba, bakayam

Family: Meliaceae

Parts used: Leaves, Root-bark, Fruit or Berry, Seeds, Flowers, Oil and Gum.

Chemical Constituents: It contains resinous substance without alkaloidal properties, sugars and tannins.

Medicinal Uses:

Melia azedarach Linn is used as antilithic, cathartic, emetic, emmenagogue, antihelminthitic, diuretic, astringent, stomachic and for nervous headache. Leaves and bark are used in leprosy, as insecticides, for scrofula and other skin diseases. Seeds are used in rheumatism, typhoid fever and gum is used for spleenic enlargement. Decoction of bark is used in malaria. Since ancient times so many plants and their specified parts are used for kidney stones. Melia azedarach Linn is one such plant mentioned in ayurvedic texts for the treatment in above conditions.

Earlier the plant has been studied for its antimicrobial7, antifungal8, antiviral9, antifeedant10, pregnancy interceptive11, ovicidal and larvicidal activity12.

In the recent past herbal extracts of Tribulus terrestris and Bergenia ligulata13 and root-wood extracts of Moringa oleifera14 are proved to have anti-lithic activity.

ENCLOSURE – III


6.3 Main Objective of the Study:

The main objective of the proposed work is to evaluate anti-lithic activity of leaf extracts of Melia azedarach Linn in different experimental models in rats.




In Phase-I:


  • Preparation of different extracts of Melia azedarach Linn using leaves.

  • To investigate preliminary phytochemical constituents present in those extracts.

  • Determination of LD50 and dose selection as low, medium, high doses for the intended study.

In Phase-II: To evaluate anti-lithic activity of the leaf extracts in various experimental animal models like

  • Ethylene glycol induced kidney stone in rats.

  • Sodium glycolate induced kidney stones in rats.

  • Hydroxyproline induced kidney stones in rats

  • Vitamin-C induced kidney stones in rats.

It is also planned to evaluate the following parameters in the above models.


  • Quantitative estimation of calcium and oxalate in urine.

  • Urinary pH.

  • Volume of the Urinary Output.

  • Estimation of Total Urinary protein.

  • Estimation of renal SOD, CAT, GSH15 levels.

  • Histopathological study of kidneys.

ENCLOSURE-IV



7. Materials and Methods:

7.1 Source of the data:

Whole work is planned to generate data from laboratory based experimental animal models as described in various National/International journals and books available with our college and other reputed institutions of India and through e-publishing and Helinet of RGUHS, Bangalore.



ENCLOSURE-V



7.2 Methods of collection of the data (including sampling procedure if any):

The whole data is divided into the following three phases:



Phase-I:

  1. Preparation of leaf extracts of Melia azedarach using different solvents:

Leaf powder of Melia azedarach Linn will be successively extracted with petroleum ether, ethanol and methanol. Each time before extracting with the next solvent, the marc will be dried in air-oven below 500c. Finally the marc will be macerated with chloroform water (i.e.; chloroform acts as a preservative) for 24 hrs to obtain the aqueous extract. Each extract will be concentrated by distilling off the solvent and then evaporating to dryness on a water bath.


  1. Preliminary Phytochemical Screening:

The preliminary phytochemical investigations will be carried out with the leaf extracts of Melia azedarach Linn for qualitative identification of various phytoconstituents.


  1. Determination of LD50 of leaf extracts of Melia azedarach Linn:

The acute toxicity of Melia azedarach Linn will be determined by using albino mice of either sex (20-25 g), maintained under standard conditions. The animals will be fasted for 3 hrs prior to the experiment. Animals will be administered with single dose of leaf extracts of Melia azedarach Linn and observed for its mortality upto 48 hrs study period (Short term toxicity). Based on the short-term toxicity profile, the next dose will be determined as per OECD guidelines No: 425. From the LD50 dose 1/20,1/10 and 1/5 doses are to be selected and considered as low, medium and high dose respectively16 for carrying out the proposed study.
Phase-II:

7.2:1 Determination of Anti-lithic activity:

  1. Ethylene glycol induced kidney stones17,18:

Albino rats weighing between 150-200g each group containing six animals will be divided into five groups.


Group A: Normal animals treated with vehicle only.

Group B: Toxicant Control treated with 0.75% ethylene glycol in place of drinking water.

Group C: Standard treated with Potassium magnesium citrate.

Group D: Low dose of Melia azedarach Linn

Group E: Medium dose of Melia azedarach Linn

Group F: High dose of Melia azedarach Linn



Experimental Procedure:

Albino rats weighing between (150-200g) will be divided into six groups of six rats in each. Group A served as normal control, which will be given with vehicle only. Group B served as toxicant control, given with 0.75% ethylene glycol in drinking water and Group C served as standard, given with potassium magnesium citrate.

Animals in Groups D, E, and F will be treated with three different doses (low, medium and high) of leaf extracts of Melia azedarach Linn. To Groups B, C, D, E and F will be intoxicated with 0.75% ethylene glycol in drinking water for 8 weeks. On the 56th day, urine will be collected and observed for the changes in physical parameters of urine and the urinary clearance of calcium and oxalate.

  1. Sodium glycolate induced kidney stones19:


Albino rats weighing between 150-200g each group containing six animals will be divided into five groups.
Group A: Normal animals treated with vehicle only.

Group B: Toxicant control treated with 3% Sodium glycolate in saline.

Group C: Standard treated with Potassium magnesium citrate.

Group D: Low dose of Melia azedarach Linn

Group E: Medium dose of Melia azedarach Linn

Group F: High dose of Melia azedarach Linn



Experimental Procedure:

Adult male Albino rats weighing between (100-120g) will be divided into six groups of six rats in each. Group A served as normal control, which will be given with vehicle only. Group B served as toxicant control, given with 3% sodium glycolate in saline (0.9 % NaCl), Group C with potassium magnesium citrate, which served as standard. Animals in Groups D, E, and F will be treated with three different doses (low, medium and high) of leaf extract of Melia azedarach Linn. Groups B, C, D, E and F will be intoxicated with 3% sodium glycolate in saline (0.9 % NaCl) at a dose of 3 ml daily for 4 weeks. After 4 weeks calcium oxalates crystals formed will be detected by physical examination in the bladder or kidney in the rats and quantitative estimation of calcium and oxalates will be carried out.



  1. Hydroxyproline induced kidney stones20:

Albino rats weighing between 150-200g each group containing six animals will be divided into five groups.


Group A: Normal animals treated with vehicle only.

Group B: Toxicant control treated with Hydroxyproline 2.5 g/kg.

Group C: Standard treated with Potassium magnesium citrate.

Group D: Low dose of Melia azedarach Linn

Group E: Medium dose of Melia azedarach Linn

Group F: High dose of Melia azedarach Linn



Experimental Procedure:

Adult male Albino rats weighing between (150-200g) will be divided into six groups of six rats in each. Group A will be served as normal control, which will be given with vehicle only. Group B served as toxicant control given with Hydroxyproline at a dose of 2.5 g/kg, Group C served as standard treated with Potassium magnesium citrate. Animals in Groups D, E, and F will be treated with three different doses (low, medium and high) of leaf extract of Melia azedarach Linn. Groups B, C, D, E and F will be intoxicated with single dose 2.5 g/kg of Hydroxyproline. After 24 hrs urine samples will be collected and subjected for estimation of calcium and oxalate crystals


  1. Vitamin-C induced kidney stones21:

Albino rats weighing between 150-200g each group containing six animals will be divided into five groups.


Group A: Normal animals treated with vehicle only.

Group B: Toxicant control treated with vitamin-c 0.4g/kg.

Group C: Standard treated with Potassium magnesium citrate.

Group D: Low dose of Melia azedarach Linn

Group E: Medium dose of Melia azedarach Linn

Group F: High dose of Melia azedarach Linn



Experimental Procedure:

Adult male Albino rats weighing between (150-200g) will be divided into six groups of six rats in each. Group A will be served as normal control, which will be given with vehicle only. Group B served as toxicant control given with vitamin-c at a dose of 0.4g/kg for 15 days. Group C served as standard treated with Potassium magnesium citrate. Animals in Groups D, E, and F will be treated with three different doses (low, medium and high) of leaf extract of Melia azedarach Linn. Groups B, C, D, E and F will be administered with 0.4g/kg of vitamin-c for 15 days. After 15 days urine samples will be collected and subjected for estimation of calcium and oxalate crystals.



STATISTICAL ANALYSIS

All values will be expressed as mean ± SEM from 6 animals. Results are to be subjected for statistical analysis using one-way ANOVA (analysis of variance) followed by Post hoc test (Dunnett’s ‘t’ test). P<0.05*, 0.01**, 0.001*** will be considered as statistically significant.



ENCLOSURE-VI

REFERENCES:





  1. http://en.wikipedia.org/wiki/Kidney

  2. http://en.wikipedia.org/wiki/Kidney_stones

  3. Prasad K.V.S.R.G, Sujatha D, Bharathi K. Herbal drugs in urolithiasis. Pharmacognosy Review 2007; 1(1): 175-179.

  4. Ross and Wilson. Anatomy and physiology in health and illness. 9th ed. Churchill Livington Publishers; 1998: 354.

  5. Nadkarni A K. Indian materia medica. Popular Prakashan Bombay 2000; Vol 1: 784-785.

  6. Kertikar and Basu. Indian medicinal plants.2nd ed. Periodical Experts Book Agency; 1991; Vol 1: 542-545.

  7. Khan M R, Kihara M and Omoloso A D. Antimicrobial activity of Horsfieldia helwigii and Melia azedarach. Fitoterapia 2001; 72 (4): Pages 423-427.

  8. María C. Carpinella, Gabriela G. Herrero, Ruben A. Alonso, Sara M. Palacios. Antifungal activity of Melia azedarach fruit extract. Fitoterapia 1999; 70(3): 296-298.

  9. Monica B. Wachsman, Elsa B. Damonte, Celia E. Coto, Ramon A. de Torres. Antiviral effects of Melia azedarach L. leaves extracts on Sindbis virus-infected cells. Antiviral Research 1987;  8(1): 1-12.

  10. Defago M, Valladares G, Banchio E, Carpinella C, Palacios S. Insecticide and antifeedant activity of different plant parts of Melia azedarach on Xanthogaleruca luteola. Fitoterapia 2006; 77(7-8): 500-505.

  11. Govind Keshri, Malini Bajpai, Vijai Lakshmi, Bachu Sreenivasulu Setty, Gopal Gupta. Role of energy metabolism in the pregnancy interceptive action of Ferula asafoetida and Melia azedarach extracts in rat. Contraception 2004; 70 (5): 429-432.

  12. Maciel M V, Morais S M, Bevilaqua C M L, Camurça-Vasconcelos A L F, Costa C T C, Castro C M S. Ovicidal and larvicidal activity of Melia azedarach extracts on Haemonchus contortus. Veterin Parasitol  2006; 140 (1-2): 98-104.

  13. Joshi V S, Parekh B B, Joshi M J, Vaidya A B. Herbal extracts of Tribulus terrestris and Bergenia ligulata inhibit growth of calcium oxalate monohydrate crystals in vitro. J Crystal Growth 2005; 2759(1-2): 1403-1408.

  14. Karadi R V, Gadge N B, Alagawadi K R, Savadi R V.Effect of Moringa oleifera Lam. Root-wood on ethylene glycol induced urolithiasis in rats. J Ethnopharmacol 2006;105(1-2):306-311.

  15. Htroshi F, Rizzo A, Sankari S, Biese I, Westermarcj T, Veijalainan P. Liver enzymes activities of rats exposed to ochratoxin A and T-2 toxin with antioxidants.Bull.Environ.Contam. Toxicol 2000; 64; 586-592.

  16. OECD 2001guidelines acute oral toxicity. “Environmental health and safety monograph series on testing and adjustment No” 425.

  17. Saeed R. Khan, Joanne M, Johnson, Ammon B. Peck, and Janet G. Cornelius. Expression of Ostepontin in rat kidneys: induction during ethylene glycol induced calcium oxalate nephrolithiasis. J Urol 2002; 168(9): 1173-1181.

  18. Ho-Shiang Huang, Ming-Chieh M A, Jun Chen, Chau-Fong Chen. Changes in the oxidant-anti oxidant balance in the kidney of rats with nephrolithiasis induced by ethylene glycol. J Urol 2002; 167(6): 2584-2593.

  19. Prasanna Shama K, Satyanarayan D, Subramanyam EVS, Vijayanarayana K, Jennifer Fernandes. Anti-lithic activity of ethanolic extract of Hibiscus rosa sinensis Linn in albino rats. J Pharm and Chem 2007; 1(1).

  20. Anand R, Patnaik G K, Kamal Roy. Bhaduri A P.Anti-oxaluric and anti-calciuric activity of lupeol derivatives. Ind J Pharmacol 1995; 27: 265-268.

  21. Foods that harm and foods that heal. A-Z guide to safe and healthy eating. The Readers Digest Association Limited, London, 1st ed. 1996; 107.


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