Physico-Chemical and Phytochemical Evaluation of Dendrobium ...

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701_________________________________________Research ArticlePhysico-Chemical and Phytochemical Evaluation of Dendrobiummacraei RootsChetankumar N. Prajapati 1 , Natvarlal M. Patel 21B. K. Mody Government Pharmacy College, Polytechnic Campus, Near Aji Dam,Rajkot-360 003, Gujarat, India.2Shri B. M. Shah College of Pharmaceutical Education and Research, Modasa-383315 Gujarat, India____________________________________________________________________________________________________ABSTRACTPresent study was undertaken for development of physico-chemical parameters of Dendrobium macraei Lindl.,traditionally known as Jivanti a jeevana tonic that boost energy level of the body according to Ayurveda,belonging to family Orchidaceae. Study comprises physico-chemical, phytochemical evaluation to confirmpurity and authenticity of Dendrobium macraei roots using standard methods. Results revealed the presenceof carbohydrates, coumarins, alkaloids, phytosterols, flavonoids and phenolic compounds in the methanolextracts of Dendrobium macraei roots. This study will help for setting down pharmacopoeial standards indetermining the quality and purity of Dendrobium macraei roots.Key Words: Jivanti, D. macraei, physico-chemical, phytochemical, extractive values, ash values.Various formulations with Jivanti as chiefINTRODUCTIONDendrobium macraei Lindl. (Synonyms:majority of authors as the genuine Jivanti. 1,18,19,20Dendrobium fimbriatum Bl. 1 , 2 , Desmotrichum ingredient are Jivantyadya ghrita, Rativallabhafimbriatum, 3 , 4 , 5 Ephemerantha macraei Lindl. 6 , modaka, Godhumadya ghrita, Amritprasha ghrita,Dendrobium nodosum Dalz. 7 , 8 , Flickingeria Jivantyadi Leha, Brihat aswagandha ghrita,nodosa (Dalz.) Seiden f.) 4 belonging to family Brihachchhagaladya ghrita and ShatavaryadiOrchidaceae is commonly known as Swarna ghrita 21 .Jivanti, Radarudi (Guj.), Jivanti (Mar.) Jivanti(Sansk.), Jibai (Bengali) and Jivanti (Hindi) 9 , 10 . Itis an epiphyte with creepy rhizome and pendulousstem. Branches are golden yellow and end with a 5– 6 cm long pseudobulb. The roots are 0.2- 0.4 cmin diameter with soft velamen coating. 11 The plantis an epiphyte found in Sikkim Himalaya at analtitude of 7000-8000 ft., Khasia mountains ataltitude of 4000 ft., Konkan, Ram Ghat, Dalzelli,MATERIALS AND METHODSPlant collectionDried plants of Swarna Jivanti (D. macraei) wereprocured from market (LV Gandhi Store) inAmdavad in the month of January 2009.After taxonomic verification and authentication bythe taxonomist at Botanical Survey of India, Pune(voucher specimen number 154704), a voucherNilgiri Hills, Srilanka and Java. 12 The plant is specimen (BMCPER/VS/0903) was deposited incooling, astringent to the bowels, tonic, the Department of Pharmacognosy, Shri B. M.aphrodisiac, expectorant; useful in asthma, Shah College of Pharmaceutical Education andbronchitis, ‘tridosha’, throat troubles, fevers,burning sensations, biliousness, diseases of the eyeand the blood. 13 , 14Jibantine, resinous principles- alpha and betajibantic acid and diosgenin derivatives likedenfigenin and defuscin as steroids are reported aschief constituents in D. macraei 15 .research, Modasa.The roots, after removal of stems, leaves,pseudobulbs and other adhering material, weredried at room temperature for 15-20 days undershade, powdered with grinder to 40# and kept inairtight container at room temperature for furtherstudy.Different plants used under one common name‘Jivanti’ are Sarcostemma brevistigma W& A,Desmotrichum fimbriatum Auct, Cimicifuga foetidaLinn, Trema orientalis (Linn) Blume, Wattakakavolubilis Linn., Holostemma annulare K. Schum &Physicochemical evaluationsMoisture content 22An accurately weighed (3 g) shade-dried rootpowder of D. macraei was and taken in a tarredLeptadenia reticulata (Retz.) Wt & Arn.1,16,17glass bottle. The crude drug was heated at 105ºC inAmongst these plants L. reticulata is considered by an oven till a constant weight. Percentage moistureVol. 4 (1) Jan– Mar 2013 www.ijrpbsonline.com 75

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701junction of two liquids indicate presence ofcarbohydrates.(ii) Fehling testTo the mixture of 1 ml Fehling A and 1 ml FehlingB solutions, (boiled for 1 min) add equal volume oftest solution. After heating it on boiling water-bathfor 5-10 minutes formation of red precipitatesindicate presence of carbohydrates.(iii) Keller Killiani testTo 2 ml extract add glacial acetic acid, one drop5% FeCl 3 and conc. H 2 SO 4 . Reddish brown colourappearing at junction of the two liquid layers andupper layer if appear bluish green, indicatespresence of 2-deoxy sugars.Tests for proteins and amino acids 24100 mg of methanol extract of D. macraei root wasdissolved in 10 mL of water and filtered. Filtratewas used to test the presence of proteins and aminoacids.(i) Millon’s testTo 2 ml of filtrate add 2 ml of Millon’s reagent in atest tube and heat in a water bath for 5 minutes.After cooling add few drops of NaNO 2 solution.Formation of white precipitates turning to red uponheating indicates presence of proteins and aminoacids.(ii) Ninhydrin testTo 2 ml of filtrate add 2-3 drops of Ninhydrinreagent in a test tube and boil for 2 minutes.Formation of distinct blue colour indicatespresence of amino acids.(iii) Biuret testTreat 2 ml of filtrate with 2 ml of 10% sodiumhydroxide solution in a test tube and heat for 10minutes. Then add a drop of 7% of copper sulphatein the above solution. Distinct violet colourationindicates presence of proteins.Tests for glycosides 25The aqueous extract of D. macraei was prepared bycold maceration with 3% methanol-water for 7days with occasional shaking.(i) Legal testTake 1 mL filtrate in a test tube. To it add 3 mLsodium nitroprusside in pyridine and KOH inmethanol. If the alkaline layer turns to blue itindicates presence of cardiac glycosides.(ii) Keller-killiani testTake 1 mL filtrate; shake with 1mL of glacialacetic acid containing traces of ferric chloride.Carefully add 1 mL of concentrated sulphuric acidby the side of test tubes. Blue colouration in aceticacid layer or red color at the junction of the twoliquids indicates presence of glycosides.Tests for anthraquinone glycoside 26(i) Borntrager’s testPrepare ether extract of root powder of the drugs.Add ammonia to the filtered ethereal extract. If theaqueous layer shows pink red or violet color aftershaking then anthraquinone glycosides are present.(ii) Modified Borntrager’s testAdd ferric chloride and dilute HCl to the aqueoussolution of drug and heat, cool and filter. Filtrate isshaken with ether or any other organic solvent. Theethereal extract is then shaken with diluteammonia. If the aqueous layer shows rose-pink tocherry red color then anthraquinone glycosides areconfirmed.Tests for sterols and triterpenoids 17(i) Liberman Buchardt testMoisten 1 g powdered drug with 1.0 mL of aceticanhydride and 2 drops of sulphuric acid on a cleantile. The powder after mixing well and the colorgained by the root powder is to be observed.Purple to violet colouration will indicate presenceof sterols and triterpenoids.(ii) Salkowski reactionTo 2 ml of methanolic extract, 2 mL chloroformand 2 mL concentrated H 2 SO 4 are to be added andshaken well. Chloroform layer appearing red andacid layer showing greenish yellow fluorescenceconfirms presence of sterols and triterpenoids.Tests for saponins 27,28(i) Froth testShake vigorously 0.1 g of powder of drug with5 mL of distilled water in a test tube for 30second and keep aside for 20 min. Persistentfrothing indicates presence of saponins.(ii) Haemolytic ZoneMix 0.5 mL blood with gelatin solution (3 ggelatin powder dissolved in 100 mL of 0.85%NaCl solution) at 60˚C and taken on a glassslide. Place a thick section of plant on it.Observation of haemolytic zone confirms thepresence of saponins.29, 30Tests for Coumarins(i) Test with ammoniaTo a drop of ammonia on a filter paper add a dropof aqueous extract of the plants. Greenfluorescence indicates presence of coumarins.(ii) Test with hydroxylamine hydrochlorideTreat the ethereal extract of the drugs with onedrop of saturated alcoholic hydroxylaminehydrochloride and a drop of alcoholic KOH. Heat,cool and acidify with 0.5 N hydrochloric acid andadd a drop of 1 %w/v FeCl 3 . Distinct violetcoloration indicates presence of coumarins.Tests for flavonoids 31(i) Shinoda testOne g of powdered drug was extracted with 10mlof ethanol (95 %v/v) for 15 min on a boiling waterbath and filtered. To the filtrate was added a smallpiece of magnesium ribbon and 3 to 4 drops ofVol. 4 (1) Jan– Mar 2013 www.ijrpbsonline.com 77

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701concentrated sulphuric acid. Formation of red colorindicates presence of flavonoids.(ii) Fluroscence testExtract 1 g powder of D. macraei roots with 15 mLmethanol for 2 min. on a boiling water bath. Filterwhile hot and evaporate to dryness. To the residueadd 0.3 ml boric acid solution (3 %w/v) and 1 mloxalic acid solution (10 %w/v). Evaporate themixture to dryness and dissolve the residue in 10mL ether. The ethereal layer showing greenishfluorescence under UV light indicates presence offlavonoids.Tests for tannins 32,33,34For following tests, aqueous extract of D. macraeiroot powder (10 g) was prepared by refluxing with50 mL water for about 1h on water bath.(i) Test with gelatinAdd 2-3 mL of aqueous extract to 1 %w/wgelatin solution containing NaCl. Formation ofheavy white precipitates indicates presence oftannins.(ii) Reaction with lead acetateTo the aqueous extract of drug add 2 ml of 10%w/w solution of lead acetate. Precipitation ifobtained indicates presence of tannins.Tests for phenolic compounds 35,36(i) Test with FeCl 3To methanolic extracts of powdered drug add adrop of freshly prepared FeCl 3 solution. Brownishgreen color indicates presence of phenolics.(ii) Test with Folin ciocalteu reagentTo a drop of methanolic extract of roots add a dropof Folin ciocalteu reagent. Bluish green colorindicates presence of phenolics.22, 37Test for alkaloids(i) Dragendroff’s testExtract 1 g powdered drug with 20mL alcoholby refluxing for 15 min and filter; evaporatethe filtrate to dryness. Dissolve the residue in15 mL 2N H 2 SO 4 and filter. After makingalkaline, extract the filtrate with chloroform.Treat the residue with Dragendroff’s reagent.Development of orange precipitates indicatespresence of alkaloids.(ii) Hager’s testDissolve 100 mg of methanol extract in 10 ml ofdilute hydrochloric acid (0.1 N) and filter. Treattwo ml of the filtrate with Hager’s reagent;formation of yellow coloured precipitates indicatespresence of alkaloids.STATISTICAL ANALYSISThe values are represented as mean ± S.D. (n=3)and results were analyzed using ANOVA, followedby Dunnett’s test where P

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701Table 1: Physico-chemical parameters for D.macraei rootsSr. No Quality Parameters Results (% w/w)1. Moisture content 5.54±0.5382. Ash valuea. Total ash value 4.84±0.273b. Acid insoluble ash 2.05±0.875c. Water soluble ash 0.70±0.8423. Extractive valuea. Water soluble extractive 4.7±0.378b. Alcohol soluble extractive 8.2±0.287c. Acetone soluble extractive 7.5±0.364d. Chloroform extractive 4.7±0.072e. Petroleum ether extractive 0.3±0.679Mean± SD, Standard deviation (SD); Numberof readings (n)=3Table 2: Phytochemcial Analysis of D. macraeirootsSr. No. Tests Results1 Carbohydrates +2 Proteins and amino acids -3 Glycosides -4 Anthraquinone Glycosides -5 Saponin Glycosides -6 Steroids +7 Coumarins +8 Flavonoids +9 Tannins -10 Phenolics +11 Alkaloids ++ = Present, - = Absent.REFERENCES1. Shivrajan VV, Balchandran I, ayurvedic Drugsand their plant sources, IBH publishing co.pvt.ltd, new delhi.pp.1994;195-197.2. Bailey LH, The Standard Cyclopedia ofHorticulture, Vol. I, Mcmillan, New York. pp.1950;983.3. Watt G, A Dictionary of The Economic Productsof India, Vol. V, 2nd reprint, Periodical experts,Delhi. pp.1972; 30-31.4. Mehrotra B N, Ashwal B S, Bisht B S,Companion to Chopra’s Glossary of IndianMedicinal Plants, Bishen Singh Mahendra PalSingh, Dehradun. pp. 1987;91.5. The Wealth of India, Raw Materials, Vol. III, D-E, CSIR, New Delhi. pp.1952; 43-44.6. Mehrotra B N, Ashwal B S, Bisht B S,Companion to Chopra’s Glossary of IndianMedicinal Plants, Bishen Singh Mahendra PalSingh, Dehradun. pp. 1987;51, 60.7. Hooker J D and C. B. – K. C. S. I. (1985): TheFlora of British India, Vol. IV, Authority of Sec.Of State for India in Council, L. Reeve and Co.,5 Henrietta Street, Covent Garden. The MustonCompany, London. Pp1985; 417.8. Gupta A K, Quality Standards of IndianMedicinal Plants, Vol-3, ICMR, New Delhi2003; 236-245.9. Chopra R N, Indigenous Drugs of India, 2nd Ed,U. N. Dhar & Sons Pvt. Ltd., Calcutta. Pp1958;512.10. Kirtikar K R, Basu L M, Indian MedicinalPlants, Vol. II, 2nd Ed, Lalit Mohan Basu,Allahabad. pp. 1973; 2401-2402.11. Santapau H, Kapadia Z, The Orchids ofBombay, Govt. of India, The Manager ofPublication, Civil Lines, New Delhi. pp. 1966;680-682.12. Gamble J S, Flora of the Presidency ofMadras, Vol. III, CEC Fischer, Botanical Surveyof India, Calcutta. pp. 1957; 597-598.13. Sharma P C, Yelne M B, Dennis T J, Databaseon Medicinal Plants used in Ayurveda, Vol II,CCRAS, Dept of ISHM& H. Ministry of H&FW.Govt of India, New Delhi. pp.1999; 270-276.14. Sharma R K, Dhyani S K, Shankar V SomeUseful and Medicinal Plants of the District ofDehradun and Siwalik, J Sci Res Pl Med1979;1(1): 87.15. The Wealth of India, Raw Materials, Vol. III,D-E, CSIR, New Delhi. pp. 1952;43-44.16. Mehrotra S, Rawat A K S, Khatoon S,Puchangadam P, Adulteration and substitution inHerbal Drugs- A Review, Recent Progress inMedicinal Plants, Vol. 7. Ethnomedicine andPharmacognosy II, Texas , U.S.A. 2003; 18017. Dhar B, Billore K V, Gupta O P, AnEvaluation of Some Controversial AyurvedicDrugs of Indian Pharmaceutical Industry, RecentProgress in Medicinal Plants, Vol I, Texas,U.S.A. 2002; 297-31418. Vaidya B G, Some controversial Drugs ofIndian Medicine VIII, J. Res. Ind. Med, 1975;10:319. Gupta R C, Kapoor L D, PharmacognosticalStudies on Jeevanti; Bull of Bot Survy 1971; 13:5320. Uniyal M R, Tiwary J, Market Study ofControversial Drugs, J. Res. Indian Med. Yoga.Homeo.,1979; 14(1): 5521. Sharma P C, Yelne M B, Dennis T J,Database on Medicinal Plants used in AyurvedaVol II, CCRAS, Dept of ISHM& H. Ministry ofH&FW. Govt .of India, New Delhi. 1999; 270-276.Vol. 4 (1) Jan– Mar 2013 www.ijrpbsonline.com 79

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