International Journal of Research in Pharmacology & Pharmacotherapeutics

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1 International Journal of Research in Pharmacology & Pharmacotherapeutics Available online at Print ISSN: Online ISSN: IJRPP Volume 2 Issue Research article Phytochemical screening and Polyphenol estimation by HPLC of Terminalia arjuna Ramakrishna U.V 1, *, Dinesh kumar B 2, Sukesh Narayan Sinha 2, Ratna Priya 2 *, 1 Department of Pharmaceutical Analysis & Quality Assurance, Bapatla College of Pharmacy, Bapatla, A.P. India. 2 Food and Drug Toxicology Research Centre, National institute of nutrition, Tarnaka, Hyderabad, A.P., India. ABSTRACT Medicinal Plants, as a source of remedies, are widely used as alternative therapeutic tool for the prevention or treatment of many diseases. Our main objective was to evaluate the components in Terminalia arjun samples and to evaluate the amount of polyphenols in the Terminalia arjuna dosage form using High Performance Liquid Chromatography. KEYWORDS: Terminalia arjuna, Proximate Analysis, HPLC. INTRODUCTION [1,2,3] Arjuna consists of dried stem bark of Terminalia arjuna, found as naturally growing plant in dense forests. The thick, white-to-pinkish-grey bark has been used in India s native Ayurveda for over three centuries, primarily as a cardiac tonic. Clinical evaluation of this botanical medicine indicates it can be of benefit in the treatment of coronary artery diseases, heart failure and possibly hypercholesterolemia. It has also been found to be antiviral and anti-mutagenic. Terminalia s active constituents include tannins, cardenolide, triterpenoidsaponins (arjunic acid, arjunolic acid, arjungenin, arjunglycosides), flavonoids (arjunone, arjunolone, luteolin), gallic acid, ellagic acid, oligomericproanthocyanidins (OPCs), phytosterols, calcium, magnesium, zinc and copper.[4] MATERIALS AND METHODS Proximate Analysis of the Test Samples of [7, 8, 9, 10, 18] Terminalia arjuna SCIENTIFIC CLASSIFICATION Kingdom Division Class Order Family Genus Species Plantae Magnoliophyta Magnoliopsida Myrtales Combretaceae Terminalia T.arjuna The analysis of samples in 50% methanol and water and moisture content were determined by standard procedures: * Corresponding author: Ramakrishna U.V address: uv.ramakrishna@gmail.com ~ 471 ~

2 Ramakrishna U.V, et al / Int. J. of Res. in Pharmacology and Pharmacotherapeutics Vol-2(3) 2013 [ ] Determination of Foreign matter Foreign matter was visualized by unaided eye and then separated, weighed and expressed as DRUG TABLET 0.0 CAPSULE 0.0 POWDER POWDER CHOORNAM 0.0 BARK 0.1 percentage of total weight as per standard procedure mentioned in WHO Library. FOREIGN MATTER (%w/w) Determination of Moisture Content 1gm of powdered drug was weighed into a weighed flat and thin porcelain dish and dried in oven at 100ºC. Porcelain dishes are cooled PRODUCT MOISTURE CONTENT (%w/w) TABLET 0.01 CAPSULE 0.05 POWDER POWDER CHOORNAM 0.01 BARK 0.01 in desiccators. The loss in weight is recorded as moisture. Determination of ash Different ash values like total ash, water soluble ash and acid insoluble ash were determined as per standard procedure mentioned in WHO library. Determination of extractable matter Different extractive values like water soluble extractive and alcohol soluble extractive value were Drug PRODUCT WEIGHT OF ASH (%) TABLET 6% CAPSULE 4.3% POWDER-1 4.6% POWDER-2 8.7% CHOORNAM 8.3% BARK 8% Water soluble extractives (%w/w) TABLET CAPSULE POWDER POWDER CHOORNAM BARK determined as per standard procedure mentioned in WHO library. Alcohol soluble extractives (%w/w) ~ 472 ~

3 Ramakrishna U.V, et al / Int. J. of Res. in Pharmacology and Pharmacotherapeutics Vol-2(3) 2013 [ ] Determination of tannins 0.5 ml of extract solution, 1-2 drops of ferric chloride solution were added. Depending on the colour obtained the presence or absence of tannins (gallic/catecholic) was determined (Iyengar, 1995). DRUG TABLET 1.3 CAPSULE 1.8 POWDER POWDER CHOORNAM 0.6 BARK 0.6 Determination of Swelling Index The swelling index was the volume in ml taken up by the swelling of 1g of plant material under specific conditions. The index was determined with method described by WHO. SWELLING INDEX Determination of foaming index About 1gm of the plant material was reduced to a coarse powder, weighed accurately and transferred to 500ml conical flask containing 100ml of boiling water. It is maintained at moderate boiling for 30min, cooled and filtered into 100ml volumetric flask. Sufficient water was added through filter paper to dilute to volume. The decoction was DRUG TABLET CAPSULE POWDER-1 POWDER-2 CHOORNAM BARK Determination of microorganism The presence or absence of pathogenic bacteria was assessed using selective media such as MacConkey Agar and CLED agar. No pathogenic bacteria were detected in test substances using the above media. Test for Phenols 1gm of drug is taken and 10 ml of water is added and shaken vigoursly. 1ml of solution is taken in a test tube and few drops of dilute nitric acid solution is added. Colour changes from reddish to yellow colour showing presence of phenols. Test for Alkaloids 0.5gm of sample was wormed with 10ml of 2% sulphuric acid for 2minutes and filtered 1ml portion was treated with few drops of Dragendroff s reagent, orange brown precipitate was observedshowing presence of alkaloids. Test for Saponins poured into 10 stoppered test tubes in successive portions of 1ml, 2ml, 3ml, etc. up to 10ml. the test tubes are stoppered and shaken in length wise motion for 15sec, 2 shakes per second. It was allowed to stand for 15 min, and height of the foam measured and foam index calculated. The index was determined with the method which described by WHO. HEIGHT OF FOAM 4.0 cm 4.0 cm 3.5 cm 2.5 cm 2.0 cm 3.0 cm Ethanolic and water extracts are diluted to 1ml separately with distilled water to 20ml and shaken in a graduated cylinder for 15min and subjected to foam test. Thin-layer chromatography Sample Preparation To 200mg of test sample, 10ml of water was added and the solution was stirred using a stir bar for 2min. Centrifugation was done at 1500 rpm for 15 minutes. The top layer collected was used as sample for TLC. TLC Analysis Silica Gel-G was used for TLC analysis. Solvent system employed consisted of Chloroform : Ethyl Acetate : Formic acid (5:4:1). System suitability factors such as reproducibility were performed. The mobile phase was optimised using silica gel G and then on precoated plates. After complete run, R f values of different pigments were determined. ~ 473 ~

4 Ramakrishna U.V, et al / Int. J. of Res. in Pharmacology and Pharmacotherapeutics Vol-2(3) 2013 [ ] PRODUCT TABLET CAPSULE CHOORNAM R f VALUE PRODUCT POEDER-1 POWDER-2 BARK R f VALUE ~ 474 ~

5 Ramakrishna U.V, et al / Int. J. of Res. in Pharmacology and Pharmacotherapeutics Vol-2(3) 2013 [ ] HPLC Chromatograms of Polyphenols in various Dosage Forms of Terminalia arjuna INSTRUMENT Dionex (Rapid separation liquid chromatography) COLUMN Waters spherisorb, C18150mm x 4.6µm ODS2 SOFTWARE Chromeleon DETECTORS DAD (Diode Array Detector) MOBILE PHASE Acetonitrile: Methanol: Dichloromethane (700:100:200) DILUENTS: Chloroform FLOW RATE: 0.5ml/min [6, 21, 22] PROCEDURE Standard solutions were injected into the system under the standard conditions mentioned above and the chromatograms were recorded. Different sample solutions were prepared and injected into the system under the same chromatographic conditions. The R.T of the peaks in the sample chromatograms is compared with that of standard chromatogram to identify the compounds. After identifying the components, the amount of the components as calculated. Amount of the components present in the sample are calculated. POLYPHENOLS CONTENT (mg/100gm) SAMPLE NAME GALLIC ACID 3,4, DI HYDROXY BENZOIC ACID CAAFFEIC ACID COUMARIC ACID ELLAGIC ACID CHLOROGENIC ACID TABLET CAPSULE POWDER POWDER CHOORNAM BARK The amount of Gallic acid was found to me higher in Choornam and Bark followed by Powder-1.The amount of 3, 4, DIHYDROXY BENZOIC ACIDwas found to me higher in Bark and Powder- 2 followed by Capsule. The amount of CAAFFEIC ACID was found to me higher in Bark followed by Powder-2. The amount of COUMARIC ACID was found to me higher in Bark followed by Powder-1. The amount of COUMARIC ACID was found to me higher in Bark followed by Powder-1. The amount of ELLAGIC ACIDwas found to mehigher in Choornam followed by Bark. The amount of CHLOROGENIC ACID was found to me higher in Powder-2 followed by Bark. ~ 475 ~

6 TABLET Ramakrishna U.V, et al / Int. J. of Res. in Pharmacology and Pharmacotherapeutics Vol-2(3) 2013 [ ] POLYPHENOLS TABLET HPLC COMPONENT STANDARD SAMPLE CONTENT mg/100rm Gallic Acid ,4 Dihydroxy Benzoic Acid Caffeic Acid Coumaeic Acid Ellagic Acid Chlorogenic Acid Tablet was found to have a decent amount of Ellagic Acid and all the other components were found to be low. ~ 476 ~

7 CAPSULE Ramakrishna U.V, et al / Int. J. of Res. in Pharmacology and Pharmacotherapeutics Vol-2(3) 2013 [ ].POLYPHENOLS CAPSULE HPLC COMPONENT STANDARD SAMPLE CONTENT mg/100rm Gallic Acid ,4 Dihydroxy Benzoic Acid Caffeic Acid Coumaeic Acid Ellagic Acid Chlorogenic Acid Capsule was found to have a good amount of 3,4, DIHYDROXY BENZOIC ACID and ELLAGIC ACID, a decent amont of GALLIC ACID and CHLOROGENIC ACID and the other components were found to be low. POLYPHENOLS POWDER-1 HPLC ~ 477 ~

8 POWDER-2 POWDER-1 Ramakrishna U.V, et al / Int. J. of Res. in Pharmacology and Pharmacotherapeutics Vol-2(3) 2013 [ ] COMPONENT STANDARD SAMPLE CONTENT mg/100rm Gallic Acid 3,4 Dihydroxy Benzoic Acid Caffeic Acid Coumaeic Acid Ellagic Acid Chlorogenic Acid Powder-1 was found to have a good amount of GALLIC ACID and the other components were found to be low. POLYPHENOLS POWDER-2 HPLC COMPONENT STANDARD SAMPLE CONTENT mg/100rm Gallic Acid ,4 Dihydroxy Benzoic Acid Caffeic Acid Coumaeic Acid Ellagic Acid Chlorogenic Acid ~ 478 ~

9 CHOORNAM Ramakrishna U.V, et al / Int. J. of Res. in Pharmacology and Pharmacotherapeutics Vol-2(3) 2013 [ ] Powder-2 was found to have a good amount of 3,4, Dihydroxy Benzoic Acid and Chlorogenic Acid, a decent amount of Gallic Acid and the other components were found to be low. POLYPHENOLS CHOORNAM HPLC COMPONENT STANDARD SAMPLE CONTENT mg/100rm Gallic Acid 3,4 Dihydroxy Benzoic Acid Caffeic Acid Coumaeic Acid Ellagic Acid Chlorogenic Acid Choornam was found to have a higher amount of Gallic Acid, good amount of 3,4, Dihydroxy Benzoic Acid, Ellagic Acid And Chlorogenic Acid, and the other components were found to be low. ~ 479 ~

10 BARK Ramakrishna U.V, et al / Int. J. of Res. in Pharmacology and Pharmacotherapeutics Vol-2(3) 2013 [ ] POLYPHENOLS BARK HPLC COMPONENT STANDARD SAMPLE CONTENT mg/100rm Gallic Acid ,4 Dihydroxy Benzoic Acid Caffeic Acid Coumaeic Acid Ellagic Acid Chlorogenic Acid Bark was found to have a higher amount of Gallic Acid, good amount of 3,4, Dihydroxy Benzoic Acid, Ellagic Acid and Chlorogenic Acid, and the other components were found to be medially low. RESULTS AND DISCUSSION The Various Phytochemical Screening parameters has been performed, Such as Test for Alkaloids, phenols, Saponins, Tannins, Determination of Foreign matter, Swelling Index, Ash values, Extractive values, Foaming index, Moisture content, Microorganisms and TLC has been performed HPLC of the polyphenols in various brands of Terminalia arjuna were performed on Dionex RPLC (Rapid Separation Liquid Chromatography) instrument, which has chromelon software installed. The column used for the analysis was Waters SpheriSorb C18 150mm 4.6 5µm ODS2 Column with a flow rate of 0.5ml/min. The detector used to detect the samples was DAD (Diode Array Detector) 450nm. Chloroform was used as the diluents and the mobile phase used was ~ 480 ~

11 Ramakrishna U.V, et al / Int. J. of Res. in Pharmacology and Pharmacotherapeutics Vol-2(3) 2013 [ ] Acetonitrile:Methanol: Dichloromethane in the ratio 700:100: 200. Standard solutions were injected into the system under the standard conditions mentioned above and the chromatograms were recorded. Different sample solutions were prepared and injected into the system under the same chromatographic conditions. The R.T of the peaks in the sample chromatograms is compared with that of standard chromatogram to identify the compounds. After identifying the components, the amount of the components as calculated. Amount of the components present in the sample are calculated. The amount of Gallic acid was found to me higher in Choornam and Bark followed by Tablet. The amount of 3,4, Dihydroxy Benzoic Acid was found to me higher in Bark and Powder-2 followed by Capsule. The amount of Caaffeic Acid was found to me higher in Powder-2 followed by Bark. The amount of Coumaric Acid was found to me higher in Bark followed by Powder-1. The amount of Coumaric Acid was found to me higher in Bark followed by Powder-1. The amount of Ellagic Acid was found to me higher in Choornam followed by Bark. The amount of Chlorogenic Acid was found to me higher in Powder-2 followed by Bark. After all the analysis and calculations it was found that BARK was found to be higher in most polyphenols followed by Choornam. SUMMARY AND CONCLUSION The phytochemical screening of the samples has been done and Rf values of the samples using TLC (Thin Layer Chromatography) were determined. HPLC of the polyphenols in various brands of Terminalia arjuna were performed on Dionex RPLC (Rapid Separation Liquid Chromatography) instrument, which has chromelon software installed. The column used for the analysis was Waters SpheriSorb C18 150mm 4.6 5µm ODS2 Column with a flow rate of 0.5 ml/min. The detector used to detect the samples was DAD (Diode Array Detector) 450 nm. Chloroform was used as the diluents and the mobile phase used was Acetonitrile: Methanol: Dichloromethane in the ratio 700: 100: 200. After all the analysis and calculations it was found that BARK was found to be higher in most polyphenols followed by Choornam. REFERENCES [1] Bone K. Clinical Applications of Ayurvedic and Chinese Herbs. Warwick, Queensland, Australia. Phytotherapy Press; 1996: [2] Kapoor LD. Handbook of Ayurvedic Medicinal Plants. Boca Raton, FL. CRC Press; 1990: [3] Munasinghe TC, Seneviratne CK, Thabrew MI, Abeysekera AM. Antiradical and anilipoperoxidative effects of some plant extracts used by Sri Lanken traditional medical practitioners for cardioprotection. Phytother Res 2001;15: [4] Zheng, W., and Wang, S.Y. (2001). Antioxidant activity and phenol compounds in selected herbs. J. Agricul. Food Chem., 49(11): [5] Ashok D.B. Vaidya and Thomas P.A. Devasagayam. Current Status of Herbal Drugs in India: An Overview. J ClinBiochemNutr July; 41(1): [6] Uma R. LAL, Shailendra M. TRIPATHI, Sanjay M. JACHAK, Kamlesh K. BHUTANI, Inder P. SINGH *, HPLC Analysis and Standardization of Arjunarishta An AyurvedicCardioprotective Formulation Sci Pharm. 2009; 77: [7] Yun-Fang Chen1,2, Hsiao-Yuh Roan1,2, Chong-Kuei Lii3, Yuan-Ching Huang1,2, and Tsu-Shing Wang1,2* Relationship between antioxidant and antiglycation ability of saponins, polyphenols, and polysaccharides in Chinese herbal medicines used to treat diabetes,, Journal of Medicinal Plants Research Vol. 5(11), pp , 4 June, 2011, ISSN [8] Alam MS, Kaur G, Ali A, Hamid H, Ali M, Athar M. Two new bioactive oleananetriterpene glycosides from Terminalia arjuna. Nat Prod Res. 2008;22(14): [9] Sharma PC, Yelne MB, Dennis TJ. Database on Medicinal Plants in Ayurveda,Vol.3,Central Council for Research in Ayurveda & Siddha, New Delhi; 2 nd ed; [10] Chaudhari M, Mengi S. Evaluation of Phytoconstituents of Terminalia arjuna for wound healing activity in rats. Phytother Res. 2006;20(9): ~ 481 ~

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