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  • 1. 1 A research proposal on Phytochemical Screening and Evaluation of In-Vitro Antioxidant and In-Vivo Hepatoprotective Activity of leaves of Urtica dioica Plant Extract Submitted to Central Institute Of Science and Technology Sangumchowk-34, New Baneshwor Kathmandu, Nepal Submitte by Ranjit Pandey Chhabi Lal Chaudhary May, 2013 ranjitpandey17@gmail.com +977-9849464685
  • 2. 2 Contents 1 PROJECT SUMMARY..............................................................................................................................4 1.1 Research Design (Qualitative) ......................................................................................................4 1.2 Investigators..................................................................................................................................5 1.3 Institution Address........................................................................................................................5 1.4 Reseaech Title ...............................................................................................................................5 1.5 Estimated Budged to complete the Project..................................................................................5 1.6 Duration ........................................................................................................................................5 2 RESEARCH PLAN ...................................................................................................................................6 2.1 Introduction ..................................................................................................................................6 2.2 Statement of the Problem.............................................................................................................7 2.3 Study Site and its Justification.......................................................................................................7 3 Objectives..............................................................................................................................................7 3.1 General..........................................................................................................................................7 3.2 Specific ..........................................................................................................................................7 3.2.1 Collect and identify the proposed plant ...............................................................................8 3.2.2 Prepare and preserve herbaria and the authentic sample of the collected plants . ............8 3.2.3 Perform the 50% ethanol (soxhlet) extraction of the collected samples..............................8 3.2.4 Perform the phytochemical screening of sample .................................................................8 3.2.5 Evaluate antioxidant and hepatoprotactive activities of the extract of collected samples.8 4 Rationale of the study...........................................................................................................................8 5 Materials and Methods.........................................................................................................................8 5.1 Apparatus......................................................................................................................................8 5.2 Chemicals and Reagents ...............................................................................................................9 5.3 Plant Materials..............................................................................................................................9 5.3.1 Extraction..............................................................................................................................9 5.4 Animals..........................................................................................................................................9 5.5 Phytochemical screening ..............................................................................................................9 5.5.1 Detection of alkaloids: ........................................................................................................10 5.5.2 Detection of carbohydrates: ...............................................................................................10 5.5.3 Detection of glycosides:......................................................................................................11 5.5.4 Detection of saponins .........................................................................................................11 5.5.5 Detection of phytosterols....................................................................................................12
  • 3. 3 5.5.6 Detection of fixed oils & fats...............................................................................................12 5.5.7 Detection of resins..............................................................................................................13 5.5.8 Detection of phenols...........................................................................................................13 5.5.9 Detection of tannins............................................................................................................13 5.5.10 Detection of flavonoids.......................................................................................................13 5.5.11 Detection of proteins and aminoacids................................................................................14 5.5.12 Detection of diterpenes ......................................................................................................14 6 Anti-oxidant activity............................................................................................................................15 7 Acute toxicity Study ............................................................................................................................15 8 Hepatoprotective activity....................................................................................................................15 9 Statistical analysis ...............................................................................................................................16 10 Limitations of the study ..................................................................................................................16 11 Programe schedule .........................................................................................................................17 12 Expected outcome ..........................................................................................................................18 12.1 Scientific outcomes:....................................................................................................................18 12.2 Social outcome............................................................................................................................18 12.3 Economic outcomes....................................................................................................................18 13 Financial Plan ..................................................................................................................................19
  • 4. 4 1 PROJECT SUMMARY 1.1 Research Design (Qualitative) Plan collection and Drying Processing and Extraction (50% ethanol), prreservation Phytochemical Screening Alkaloid carbohydrates Glycosides Tannin Flavonoid Terpenoid In-vitro antioxidant DPPH radical scavenging activity In-vivo Hepatoprotective (50 rats) G-1 Control Test G-2 Standard (+ve Control) Silymarin G-3 Standard (-ve Control) Only solvent G-4 ( x mg/ kg/p.o.) G-5 ( 2x mg/ kg/p.o.) G-6 ( 4x mg/ kg/p.o.) G-7 ( 8x mg/ kg/p.o.) Inducing Liver damage by using CCL4 for 7 days Only solvent for 7 days ( Each group contain 5 Albino Rats) Blood sample will be drawn and Laboratory test ( LFT )
  • 5. 5 1.2 Type ( Experimental test ) 1.3 Investigators Ranjit Pandey Chhabi Lal Chaudhary 1.4 Institution Address Central Institute Of Science and Technology Sangumchowk-34, New Baneshwor Kathmandu, Nepal 1.5 Reseaech Title Phytochemical Screening and Evaluation of In-Vitro Antioxidant Ant and In-Vivo Hepatoprotective Activity of leaves of Urtica dioica Plant Extract 1.6 Estimated Budged to complete the Project Rs: 38,500 1.7 Duration Two and half months.
  • 6. 6 2 RESEARCH PLAN 2.1 Introduction Himalayan range of Nepal has vast plant diversity from south 59m to north 8848m which contain wide important to supplement modern allopathic medicine as well as traditional believe of ethno medicinal therapy. Majority of Nepalese population still depends upon indigenous use of medicinal plants and play vital role in health and economy[1]. Plants have been used to treat or prevent illness since before recorded history of sacred Vedas dating back between 3500 B.C and 800 B.C give many references of medicinal plants. According to world health organization (WHO), more than 80% of the world’s population relies on traditional medicines for their primary health care needs[2]. Urtica dioica L. (Urticaceae) is a traditional Ayurvedic herb and also known as Vrishchhiyaa-shaaka. It is also known as Bichu Butti in Hindi and Punjabi, Anjuraa in Unani, Shisuun in folk language (Kumaon) and Stinging Nettle in English.. The plant is an annual and perennial herb available in many South Asian Countries and in the Indian subcontinent and has been known in the world as a medicinal herb for a long time. It is also found in Assam, Arunachal Pradesh and Himachal Pradesh. The plant is used traditionally as diuretic, emmenagogue, blood purifier and as liver tonic[3] . Phytochemical screening is the qualitative analysis of plant chemical constituents like alkaloids, carbohydrates, phenols, flavonoid, tannins etc[4]. The phytochemical research based on ethno- pharmacological information is generally considered an effective approach in the discovery of new drug. Knowledge of the chemical constituents of plants is desirable, not only for the discovery of therapeutic agents, but also because such information may be of value in disclosing new sources of such economic materials as tannins, oils, gums, precursors for the synthesis of complex chemical substances[2]. Anti-oxidant activity of the plant is analyzed chemically in the laboratory by using different chemical reagents like Diphenyl picryl Hydrazyl (DPPH) while hepatoprotective activity is evaluated by using different animal models like rats with CCl4 induced liver injury[5]. Hepatic system is one of the largest organs in the human body and major site for metabolism detoxification and excretion of various endogenous and exogenously administered substances like xenobiotic and pollutants etc. Liver diseases are one of the major health problems in the world caused by toxic chemicals, autoimmune disorders, infections and excess consumption of alcohol. The hepatotoxic chemicals can induce lipid peroxidation and oxidative damages. It is prone to be attacked by the free radicals and cell necrosis results hepatic damage[6, 7]. Hepato toxins inhibits anti-oxidation system like superoxide dismutase (SOD), tissue glutathione (GSH) etc. and increases the various liver enzymes (SGPT, SGOT, ALP, serum bilirubin, cholesterol etc.) indicate liver injury[7]. Antioxidants may offer resistance against oxidative stress by scavenging free radicals, inhibiting lipid peroxidation and by other mechanisms and thereby help
  • 7. 7 in preventing the free radical induced liver diseases[5]. Modern medicine have little to offer alleviation of hepatic diseases and it is chiefly plant based preparation which are employed for their treatment of liver disorder. Therefore, many ethno medicinal plants are tested for its potential antioxidant and hepatoprotactive liver damage in experimental animal model. Where CCl4 is widely used chemical for liver damage in hepatoprotactive study[6]. 2.2 Statement of the Problem • Hepatic disorders are common now days due to different xenobiotic and chemicals. • No more allopathic medicine is available for liver disorder. • Treatment of infection with liver disorder (e.g. jaundice) is main concern. 2.3 Study Site and its Justification The study will be conducted within the premise of Central Institute Of Science Technology Sangumchowk-34, New Baneshwor ,Kathmandu as we have full access to the laboratory and expert in Natural product chemistry and Pharmacology. 3 Objectives 3.1 General To perform Phytochemical Screening and Evaluation of In-Vitro Antioxidant and In-Vivo Hepatoprotective activity. 3.2 Specific The specific objectives of the present research project are to :
  • 8. 8 3.2.1 Collect and identify the proposed plant 3.2.2 Prepare and preserve herbaria and the authentic sample of the collected plants . 3.2.3 Perform the 50% ethanol (soxhlet) extraction of the collected samples 3.2.4 Perform the phytochemical screening of sample 3.2.5 Evaluate antioxidant and hepatoprotactive activities of the extract of collected samples. 4 Rationale of the study U. dioica has some etnnomedicinal uses including liver tonic. Literature review suggest very few researches have been conducted on this plant. More than that research on its clinical use has not been found till date. That encourages us to conduct the project on this plant. All of these reasons inspire us to conduct the research on this plant. 5 Materials and Methods 5.1 Apparatus Knife, Grinder, Refrigerator, Centrifuge, Soxhlet, Water bath, Petridis, Whatman Filter Paper, Beakers (50 ml, 100 ml, 250 ml, 500 ml, 1000 ml), Measuring cylinder (10 ml, 50 ml, 100 ml), pipette ( 0.5 ml,1 ml, 2 ml, 5 ml, 10 ml), Micropipette, Syringe, scissor, Test tubes.
  • 9. 9 5.2 Chemicals and Reagents Ethanol, Carbon Tetrachloride, Silymarine, Ascorbic Acid, Chloroform, Acetone, NaCl, NaOH, copper acetate solution, Hydrochloric acid, Mayer’s reagent (Potassium mercuric iodide), Wagner’s reagent (iodine, potassium iodide), Hager’s reagent (picric acid solution), ) Molisch’s reagent (alcoholic α-naphthol Solution, Conc. Sulphuric acid), Benedict’s reagent, Fehling’s reagent, Modified Borntrager’s reagent (ferric chloride solution ,Benzene), Legal’s reagent( Nitroprusside in pyredine, methanolic alkali), 5.3 Plant Materials Fresh leaves of Urtica dioica will be collected in the month of May from Kathmandu district of Sundarijal and will authenticated by National Herberium Godawary, Lalitpur, Nepal. Herbarium of plant specimen will be deposite at Central Institute Of Science and Technology ( pharmacognosy Lab.), Sangumchowk-34, New BaneshworKathmandu, Nepal. The leaves will be shade dried and crushed to corse powder. 5.3.1 Extraction Powdered crude drug will be extracted by using soxhlet apparatus with the help of 50% Ethanol for 16 hours. Extractive will make concentrated by evaporating the solvent on water bath and store in refrigerator. 5.4 Animals Healthy albino rats (120-150g) of either sex will procure from Department of Plant Resources, Thapathali, Kathmandu, Nepal and will keep in standard plastic animal case in groups of five animals with 12 hours light and dark cycle. The rats will feed n standard rat chow and will provide water ad libitum. Prior to inisialization of experimentation the animals will acclimatize to Laboratori conditions for a week. The experiments will carry out according to guidelines of ‘Nepal Health and research councils’ (NHRC) Kathmandu, and procedures will approve by…… 5.5 Phytochemical screening Phytochemical examinations will be carried out for 50% ethanolic extracts as per the standard methods. (Roopashree et al., 2008)
  • 10. 10 5.5.1 Detection of alkaloids: Extracts will be dissolved individually in dilute Hydrochloric acid and filtered. The filtrates will be used to test for the presence of alkaloids. 5.5.1.1 Mayer’s Test: Filtrates will be treated with Mayer’s reagent (Potassium Mercuric iodide). Formation of a yellow cream precipitate indicates the presence of Alkaloids. 5.5.1.2 Wagner’s test: Filtrates will be treated with Wagner’s reagent (Iodine in Potassium iodide). Formation of brown/reddish brown precipitate indicates the Presence of alkaloids. 5.5.1.3 Hager’s test: Filtrates will be treated with Hager’s reagent (saturated picric acid solution). Formation of yellow colored precipitate indicates the presence of alkaloids. 5.5.2 Detection of carbohydrates: Extracts will be dissolved individually in 5 ml distilled water and filtered. The filtrates will be used to test for the presence of carbohydrates. 5.5.2.1 Molisch’s Test: Filtrates will be treated with 2 drops of alcoholic α-naphthol solution in a test tube and 2 ml of Conc. Sulphuric acid will be added carefully along the sides of the test tube. Formation of violet ring at the junction indicates the presence of Carbohydrates.
  • 11. 11 5.5.2.2 Benedict’s test: Filtrates will be treated with Benedict’s reagent and heated on water bath. Formation of orange red precipitate indicates the presence of reducing sugars. 5.5.2.3 Fehling’s test: Filtrates will be hydrolyzed with dil. HCl, neutralized with alkali and heated with Fehlings A & B solutions. Formation of red precipitate indicates the presence of reducing sugars. 5.5.3 Detection of glycosides: Extracts will be hydrolyzed with dil. HCl, and then subjected to test for glycosides. 5.5.3.1 Modified Borntrager’s Test: Extracts will be treated with Ferric Chloride solution and immersed in boiling water for about 5 minutes. The mixture will be cooled and shaken with an equal volume of benzene. The benzene layer will be separated and treated with ammonia solution. Formation of rose-pink colour in the ammonical layer indicates the presence of anthranol glycosides. 5.5.3.2 Legal’s test: Extracts will be treated with sodium nitroprusside in pyridine and methanolic alkali. Formation of pink to blood red colour indicates the presence of cardiac glycosides. 5.5.4 Detection of saponins 5.5.4.1 Froth Test: Extracts will be diluted with distilled water to 20ml and this will be shaken in a graduated cylinder for 15 minutes. Formation of 1 cm layer of foam indicates the presence of saponins.
  • 12. 12 5.5.4.2 Foam test: Small amount of extract will be shaken with little quantity of water. If foam produced persists for ten minutes it indicates the presence of saponins. 5.5.5 Detection of phytosterols 5.5.5.1 Salkowski’s Test: Extracts will be treated with chloroform and filtered. The filtrates will be treated with few drops of conc. Sulphuric acid, shaken and will be allowed to stand. Appearance of golden yellow colour indicates the presence of triterpenes. 5.5.5.2 Libermann Burchard’s test: Extracts will be treated with chloroform and filtered. The filtrates will be treated with few drops of acetic anhydride, boiled and cooled. Conc. Sulphuric acid will be added carefully along the sides of the test tube. Formation of brown ring at the junction indicates the presence of phytosterols. 5.5.5.3 Tshugajeu test : Extracts will be treated with chloroform and filtered. Excess of acetyl chloride and a pinch of Zinc Chloride will be added, kept aside for some time till the reaction will be complete and then warmed on water bath. Appearance of eosin red colour indicates the presence of triterpenes. 5.5.6 Detection of fixed oils & fats Stain Test: Small quantities of extracts will be pressed between two filter papers. An oily stain on filter paper indicates the presence of fixed oil.
  • 13. 13 5.5.7 Detection of resins Acetone-water Test: Extracts will be treated with acetone. Small amount of water will be added and shaken. Appearance of turbidity indicates the presence of resins. 5.5.8 Detection of phenols. Ferric Chloride Test: Extracts will be treated with few drops of ferric chloride solution. Formation of bluish black colour indicates the presence of phenols. 5.5.9 Detection of tannins Gelatin Test: To the extract, 1% gelatin solution containing sodium chloride will be added. Formation of white precipitate indicates the presence of tannins. 5.5.10 Detection of flavonoids 5.5.10.1 Alkaline Reagent Test: Extracts will be treated with few drops of sodium hydroxide solution. Formation of intense yellow color, which becomes colorless on addition of dilute acid, indicates the presence of flavonoids. 5.5.10.2 Lead acetate Test: Extracts will be treated with few drops of lead acetate solution. Formation of yellow color precipitate indicates the presence of flavonoids.
  • 14. 14 5.5.10.3 Shinoda Test: To the alcoholic solution of extracts, a few fragments of magnesium ribbon and Conc.HCl will be added. Appearance of magenta colour after few minutes indicates presence of flavonoids. 5.5.10.4 Zinc hydrochloric acid reduction Test: To the alcoholic solution of extracts, a pinch of Zinc dust and Conc.HCl will be added. Appearance of magenta colour (purplish-pink) after few minutes indicates presence of flavonoids. 5.5.11 Detection of proteins and aminoacids 5.5.11.1 Xanthoproteic Test: The extracts will be treated with few drops of concentrated Nitric acid solution. Formation of yellow colour indicates the presence of proteins. 5.5.11.2 Biuret Test: The extracts will be treated with 1 ml of 10% sodium hydroxide solution and heated. To this a drop of 0.7% copper sulphate solution will be added. Formation of purplish violet colour indicates the presence of proteins. 5.5.12 Detection of diterpenes Copper acetate Test: Extracts will be dissolved in water and treated with few drops of copper acetate solution. Formation of emerald green colour indicates diterpenes.
  • 15. 15 6 Anti-oxidant activity DPPH radical scavenging activity The DPPH radical scavenging activity will be measured by spectrophotometric method. 1mL of ethanolic solution of extract of various concentrations (25–800 mg/mL) were mixed with 1mL of ethanolic solution of DPPH (200 mM). Similarly 1mL ethanolic solutions of ascorbic acid (200 mg/mL) was mixed with 1mL of DPPH solution. A mixture of 1mL of ethanol and 1mL of ethanolic solution of DPPH (200 mM) served as control. After mixing, all the solutions were incubated in dark for 20 minutes and absorbance was measured at 517 nm [9]. The experiments were performed in triplicate and percent scavenging activity was calculated as follows; Scavenging % =Absorbance of control – absorbance of test x 100 Absorbance of control 7 Acute toxicity Study Acute toxicity studies of all the extracts will be performed in rats (Wister strain) as per NHRC guidelines by slight modification of the method (Wakte,P.S et.al. 2012,(3)). The extracts will be administered in rats orally at the single dose of xmg/kg. After treatment, the animals will be observed for behavior changes and their mortalityfor 48 hours. In case of no mortality, the procedure will be repeated for a higher dose of extracts. Hepatoprotective activity will be carried out at 1/10th of maximum dose of extracts used in acute toxicity. 8 Hepatoprotective activity Hepatoprotective activity of U.diocia leaves extract will be determined by using slight modification of method (Qureshi, M.N. et al, 2010(7); Ellandal, R.2011(1)) by carbon tetrachloride induce hepatoprotective rat model. Rats will be divided into seven groups (G1-G7) each comprising five rats. Before treatment, the rats will be fasted overnight with free access to water. Group1will be served as vehicle control and will be received starch 1%v/v in distilled water (5 ml/kg p.o.) for 14 days. Group2 will be served as toxic control and will be administered vehicle daily and carbon tetrachloride in liquid paraffin 1:1v/v (0.7 ml/kg i.p.) one alternative day for 14 days. Group 3 will be served as positive control and will be administered silymarin (100mg/kg, po, daily) and groups 4, 5, 6 and 7 will be administered ethanolic extract of U.dioica (x, 2x,4x and 8x mg/kg, po, daily) along with carbon tetrachloride in liquid paraffin 1:1 v/v
  • 16. 16 (0.7mL/kg, ip) on alternate days for 14 days. At the end of treatment, Rats will be decapitated under light ether anesthesia and The blood samples will be collected and separately into sterilized dry centrifuge tubes will be allowed to coagulate for 30 min at 37°C. The clear serum will be separated at 2500 rpm for 10 min and biochemical investigations will be carried out to assess liver function viz., total bilirubin, SGOT,SGPT,ALP in reference laboratory, New Baneshwor, KTM. 9 Statistical analysis Data will be reported as mean ± SEM. Statistical analysis of data will be carried out by one way ANOVA comparing all test groups versus control using graph-pad software V5.0. 10 Limitations of the study While phytochemical screening some compound present in very trace amount cannot be detected by simple laboratory reaction and advanced equipmet necessary which cannot be performed in our laboratory.
  • 17. 17 11 Programe schedule This project work is estimated to be completed within 7 weeks (around 2 months) and the time period is scheduled as follows Program Schedule 1-10 days 11-20 days 21-30 days 31-40 days 41-50 days Literature review proposal writing Collection and drying of plant material Extraction and processing of extractive and performing the laboratory analysis Result evaluation, validation and interpretation Thesis preparation and defense
  • 18. 18 12 Expected outcome 12.1 Scientific outcomes: This will provide a great contribution to the scientific field and provides great help in the further study of these plants. 12.2 Social outcome In Nepalese society, there is less awareness about these plant species among the people. Many of them do not know about the medicinal uses of these plants. After the completion of the research project, people will be interested in the cultivation, collection and conversion of these species. 12.3 Economic outcomes In developing countries like Nepal, such studies will help in the development of economic status and such unexplored medicines will be utilized for the improvement of health status of people.
  • 19. 19 13 Financial Plan Total expenditure of the project work is estimated as follows: S. No. Particulars Cost (NRs.) 1. Chemicals and reagents 15000.00 2 Solvents 3000.00 3 Albino rats 4000.00 4 Rat diet 3000.00 5 Crude drugs 1500.00 6 Correspondences 2000.00 7 Books and journals 1000.00 8 Training (mouse handling) 5000.00 9 Printing and publishing 2000.00 10 Miscellaneous 2000.00 Total 38500.00
  • 20. 20 1. Gaire B. P. and S. L.;, Medicinal Plant Diversity and their Pharmacological Aspects of Nepal Himalayas. Pharmacognosy Journal, 2011. 3(25). 2. Chhetri H. P., et al., PHYTOCHEMICAL AND ANTIMICROBIAL EVALUATIONS OF SOME MEDICINAL PLANTS OF NEPAL. KATHMANDU UNIVERSITY JOURNAL OF SCIENCE, ENGINEERING AND TECHNOLOGY, 2008. 1(5): p. 49-54. 3. Kataki, M.S., et al., Antioxidant, Hepatoprotective, and Anthelmintic Activities of Methanol Extract of Urtica urensis L. Leaves. Pharmaceutical Crops, 2012. 3: p. 38-49. 4. Tiwari P., et al., Phytochemical screening and Extraction: A Review. INTERNATIONALE PHARMACEUTICASCIENCIA, 2011. 1(1). 5. Qureshi M. N., et al., In-vitro Antioxidant and In-vivo Hepatoprotective activity of Leucas ciliata leaves. Records of Natural Products 2010. 4(2): p. 124-130. 6. Shashank T., et al., Evaluation of Hepatoprotective activity of Stem Bark of Homalium zeylanicum in Rats. International Journal of PharmTech Research, 2011. 3(3): p. 1630-1634. 7. Kumar P., et al., anti-oxident and hepatoprotective activity of tubers of Momordica tuberosa Cogn. against CCl4 induced liver injury in rats. indian journal of experimental biology, 2008. 46: p. 510-513.