Total Phenolic Content and Antioxidant Activity of Morinda Tinctoria Leaves
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www.ijpsonline.com antinociceptive effect of curcumin. Korean J Pain 2012;25:221-7. 13. Aggarwal BB, Sung B. Pharmacological basis for the role of curcumin in chronic diseases: An age-old spice with modern targets. Trends Accepted 22 March 2015 Pharmacol Sci 2009;30:85-94. Revised 30 October 2014 14. Dewar AM, Clark RA, Singer AJ, Frame MD. Curcumin mediates Received 03 October 2013 both dilation and constriction of peripheral arterioles via adrenergic receptors. J Invest Dermatol 2011;131:1754-60. Indian J Pharm Sci 2015;77(2):222-226 Total Phenolic Content and Antioxidant Activity of Morinda tinctoria Leaves DEEPTI KOLLI, K. R. AMPERAYANI AND UMADEVI PARIMI* Department of Chemistry, GIS, GITAM University, Visakhapatnam‑530 041, India Kolli, et al.: Antioxidant activity of Morinda tinctoria Leaves The antioxidant activity and total phenolic content of Morinda tinctoria leaves was evaluated. The successively extracted leaves of Morinda tinctoria using various solvents was analyzed for their total phenolic content. The extracts were subjected to column chromatography for the isolation of bioactive molecules. In vitro antioxidant activity was evaluated by employing different assays, including 2,2-diphenyl-1-picrylhydrazyl, nitric oxide scavenging assay and phosphomolybdenum reducing power assay. The 2,2-diphenyl-1-picrylhydrazyl radical scavenging efficacy of hexane extract is significant at higher concentration (500 µg/ml- 91.2±0.05%) and the efficacy at lower concentration is more significant for ethyl acetate extract (100 µg/ml ‑ 65.1±0.05%). The total phenolic content was highest in methanol extract (5.30±0.011 µg/mg). Cynarin, a hydroxy cinnamic acid was isolated from chloroform extract; oleuropein, a polyphenolic iridoid was isolated from methanol extract. The results obtained suggeted that Morinda tinctoria leaf extracts possessed antioxidant properties and might offer protection from free radicals. Two compounds, cynarin and oleuropein were reported for the first time fromMorinda tinctoria leaves. Key words: DPPH activity, reducing power, nitric oxide, phenolic content, oleuropein, cynarin Excessive generation of reactive oxygen species (ROS) externally applied to gout to relieve pain. The leaves is involved in structural alterations of cellular are administered internally as a tonic and febrifuge[4]. [1] molecules leading to cytotoxicity and cell death . The n-hexane, dichloromethane and methanol extracts Plant extracts and phytochemical constituents play of the leaves were shown to possess antibacterial and an important role in the prevention of many diseases antifungal activities[5]. The petroleum ether extract and are recognized as natural antioxidants. Phenolic showed anticonvulsant activity[6]. Anticonvulsant, compounds found in vegetables, fruits or medicinal analgesic, antiinflammatory, cytoprotective effect and plants are known for their antioxidant potential and antimicrobial activity of Morinda tinctoria leaves has their role in prevention of human diseases[2]. Morinda been reported[7]. The ashes of Morinda tinctoria leaves tinctoria (Rubiaceae) is distinguished for its traditional are also reported to act as biosorbents in controlling use, phytochemical profiles and therapeutic potential. ammonia pollution in waste water[8]. There are very It is locally known as “Togaru” and commonly few reports on the chemical constituents of the leaves known as Indian mulberry or aal or nunaa in India. of Morinda tinctoria. The objective of the present It is a small tree with immense medicinal properties. work was to investigate the total phenolic content, The plant is well known for its therapeutic use in chemical constituents and antioxidant activity of the traditional systems of medicine like ayurveda and leaf extracts of the plant. The isolated compounds siddha. Literature survey reveals that traditionally were identified as cynarin and oleuropein (fig. 1) their the leaf juice is given orally to children before food structures were determined based on the observed for easy digestion[3]. The expressed juice of leaves is spectroscopic (1H-NMR, 13C-NMR and IR) data and reported data in literature. This is the first report *Address for correspondence for the occurrence of cynarin and oleuropein from E-mail: [email protected] Morinda tinctoria. 226 Indian Journal of Pharmaceutical Sciences March - April 2015 www.ijpsonline.com Fresh leaves of Morinda tinctoria were collected and chloroform:methanol in different ratios. Every from GITAM University campus, Visakhapatnam, 30 ml fraction was collected in different boiling India and a voucher specimen has been deposited tubes and each tube was performed for TLC with at the Botany Department herbarium, Andhra mobile phase hexane:chloroform:methanol (5.5:2:1) University, India (Herbarium no. AU-B.D.H- 21106). so as to analyze the purity of the isolated fractions. All chemicals and solvents used in the present study Fractions with same bands were clubbed together. were of analytical reagent grade and were purchased Six similar fractions were collected. Cynarin was from S. D. Fine Chem, Himedia and Qualigens eluted with chloroform: methanol (6:4) as solvent India. system. The dried fraction was further purified by preparative TLC and characterized by IR, The leaves were washed under tap water and shade NMR (1H and 13C) and LC-MS. dried. The dried leaves were coarsely powdered and stored at 20o until further use. One kilogram powder About 10 g of dried methanol extract was defatted with was extracted with 2000 ml of n-hexane, chloroform, n-hexane for the removal of fatty materials. The extract ethyl acetate and methanol successively using a was then dried adsorbed with silica gel and used for Soxhlet extractor for 48 h each. The extracts were column chromatography. The column was packed with then distilled using a rotary evaporator and evaporated silica gel (60-120 mesh) by wet packing method and to dryness and weighed accurately. The completely then eluted with chloroform:methanol in different ratios dried extracts weighed, 15, 33, 20 and 43.6 g each and the isolated fractions were analyzed on TLC with for hexane, chloroform, ethyl acetate and methanol mobile phase n-hexane:chloroform:methanol (5.5:2:1). extracts, respectively. Similar fractions were combined and dried. Oleuropein was eluted with chloroform:methanol (6:4) solvent About 10 g of the extract was defatted using n-hexane system and further purified by preparative TLC. The for 1 h for the removal of fatty materials. The extract purified compound was characterized by IR, 1H NMR, was then dried and dissolved in chloroform (50 ml) 13C NMR and LC-MS. and mixed with silica gel mesh 60-120 and dried, the dried material was used for column. About 500 g The free radical scavenging activity of the extracts of silica mesh 60-120 (column chromatography was measured in terms of hydrogen donating grade) was kept in oven for 1 h at 105° for or radical scavenging ability using the stable activation. It was cooled in desiccators for 1 h. radical DPPH (2,2-diphenyl-1-picrylhydrazyl). The column was packed up to 2/3 portion with 0.1 mM solution of DPPH in ethanol was activated silica by wet packing procedure. The column prepared and 1.0 ml of this solution was added was then eluted with n-hexane, hexane:chloroform to 3.0 ml of extract solution in water at different concentrations (100-500 µg/ml). The absorbance measured after thirty min is 517 nm. Ascorbic acid was used as a standard. Lower absorbance of the reaction mixture indicates higher free radical scavenging activity. The efficacy to scavenge the DPPH radical was calculated from the formula, DPPH scavenged (%) = (Acontrol-Atest/Acontrol)×100, where Acontrol a is the absorbance of the control, Atest is the absorbance in the presence of the extracts. Nitric oxide radical inhibition is estimated by the use of Griess Illosvoy reaction[9]. Nitric oxide generated from sodium nitroprusside in aqueous solution at physiological pH interacts with oxygen to produce nitrite ions, which were measured by the b method of Garrat. Sodium nitroprusside (5 mM) in Fig. 1: Structures of compounds isolated from Morinda tinctoria leaves phosphate-buffered saline (PBS) was mixed with A. Structure of cynarin and B. structure of oleuropein. 3.0 ml of different concentrations (10-100 µg/ml) of March - April 2015 Indian Journal of Pharmaceutical Sciences 227 www.ijpsonline.com the extracts dissolved in the suitable solvent systems work DPPH scavenging efficacy, nitric oxide radical and incubated at 25° for 150 min. After incubation, scavenging efficacy and reducing power assay were 0.5 ml of the reaction mixture were reacted with successfully used for the evaluation of free radical Griess reagent and allowed to stand for 30 min. scavenging activity (antioxidant activity) of the crude The absorbance of the chromophore formed during extract derived from Morinda tinctoria leaves. Two the diazotization of nitrite with sulphanilamide and compounds cynarin and oleuropein were isolated from subsequent coupling with naphthyl ethylenediamine the chloroform and methanol extracts. was read at 546 nm. The percent scavenging of nitric oxide is calculated using the formula, NO The presence of phenolic compounds like flavonoids scavenged (%) = (Acontrol-Atest/Acontrol)×100. and terpenoids in the leaf extracts of Morinda tinctoria are likely to be responsible for the free The reducing power of leaf extracts was determined radical scavenging activity. Phenolic compounds and according to the method of Oyaizu[10]. Different flavonoids have been reported to be associated with concentrations of plant extracts (25-200 µg/ml) antioxidative action in biological systems, acting as in 1 ml of methanol were mixed with phosphate scavengers of singlet oxygen and free radicals[12,13]. buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferrocyanide (2.5 ml, 1%). The mixture was The structural identification of cynarin and oleuropein incubated at 50° for 20 min. A portion of trichloro were carried out by LC–MS, 1H NMR, 13C NMR acetic acid (10%, 2.5 ml) was added to the mixture and IR.