Hindawi Publishing Corporation ISRN Biotechnology Volume 2013, Article ID 450948, 10 pages http://dx.doi.org/10.5402/2013/450948 Research Article Simultaneous Extraction Optimization and Analysis of Flavonoids from the Flowers of Tabernaemontana heyneana by High Performance Liquid Chromatography Coupled to Diode Array Detector and Electron Spray Ionization/Mass Spectrometry Thiyagarajan Sathishkumar,1 Ramakrishnan Baskar,1 Mohan Aravind,1 Suryanarayanan Tilak,1 Sri Deepthi,1 and Vellalore Maruthachalam Bharathikumar2 1 Department of Biotechnology, Kumaraguru College of Technology, Coimbatore 641049, India 2 Department of Biochemistry, University of Saskatchewan, Saskatoon, SK, Canada S7N 5E5 Correspondence should be addressed to iyagarajan Sathishkumar; [email protected] Received 24 June 2012; Accepted 9 August 2012 Academic Editors: Y. H. Cheong, H. Kakeshita, W. A. Kues, and D. Pant Copyright © 2013 iyagarajan Sathishkumar et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Flavonoids are exploited as antioxidants, antimicrobial, antithrombogenic, antiviral, and antihypercholesterolemic agents. Normally, conventional extraction techniques like soxhlet or shake �ask methods provide low yield of �avonoids with structural loss, and thereby, these techniques may be considered as inefficient. In this regard, an attempt was made to optimize the �avonoid extraction using orthogonal design of experiment and subsequent structural elucidation by high-performance liquid chromatography-diode array detector-electron spray ionization/mass spectrometry (HPLC-DAD-ESI/MS) techniques. e shake �ask method of �avonoid extraction was observed to provide a yield of (mg/g tissue). With the two different solvents, namely, ethanol and ethyl acetate, tried for the extraction optimization of �avonoid, ethanol (80.1 mg/g tissue) has been proved better than ethyl acetate (20.5 mg/g tissue). e optimal conditions of the1.2 extraction ± 0.13 of �avonoid were found to be 85 C, 3 hours with a material ratio of 1 : 20, 75 ethanol, and 1 cycle of extraction. About seven different phenolics like robinin, quercetin,∘ rutin, sinapoyl-hexoside, dicaffeic acid, and two unknown compounds were identi�ed for the �rst time in the �owers of T. heyneana. e study has also concluded that L%orthogonal design of experiment is an effective method for the extraction of �avonoid than the shake �ask method. 16 1. Introduction survival and proper functioning of plants. ey provide protection against herbivores, microorganisms, and com- Herbal or medicinal plant products, in various forms, have petitors, regulate growth (e.g., delaying seed germination been used to treat different illness for many hundreds of years until an appropriate time), and control pollination, fertiliza- across the world. About 70–80 of the world population, tion, and rhizosphere environment [3]. e main secondary particularly in the developing countries, rely on noncon- metabolite present in plants includes lignins, �avonoid, ventional medicine in their primary% healthcare [1]. India phenols, alkaloids, amino acid derivatives, organic acids, has a rich �ora that is widely distributed throughout the terpenoids, steroids, and sugar derivatives. Among different country, and a large number of Indian medicinal plants are phytochemicals, �avonoid exerts a wide range of biochem- attributed with various pharmacological activities, because ical and pharmacological properties, including free radical of diversi�ed class of phytochemicals, but still, the efficacy scavenging, inhibitors of lipid peroxidation, antimicrobial, of these plants are yet to be scienti�cally documented [2]. antiviral, antioxidant, antithrombogenic, hepatoprotectivity, In general, phytochemical constituents are essential for the and nephroprotectivity [4]. 2 ISRN Biotechnology T 1: Different variables for optimal extraction of �avonoids from the �owers of T. heyneana (aqueous ethanol as extraction solvent, 45-L design). 16 A B C D E Levels Temp. ( C) Ext. tim. (hrs) Ethanol ( ) Material ratio (g : mL) No. of ext. cycles 1 55 ∘ 1 65 1 : 05 1 2 65 2 75 % 1 : 10 2 3 75 3 85 1 : 15 3 4 85 4 95 1 : 20 4 Plants of Apocynaceae family (Dogbane) are rich in heyneana. is paper reports about the development of alkaloids or glycosides, especially in seeds and latex. Some extraction optimal process and high-performance liquid species are valuable sources of medicine, insecticides, �bers, chromatography-diode array detector coupled with electron and rubber [5]. is botanical family includes 4555 species, spray ionization/mass spectrometry (HPLC/DAD-ESI/MS) distributed in 415 genera [6], and the genus Tabernaemonana method for characterizing the chromatographic �ngerprint- is included under this family that consists of shrubs or small ing of �avonoid and other possible polyphenolic compounds. trees. Tabernaemontana heyneana Wall. (syn. Ervatamia heyneana) is included in the oldest script Amarakosam or 2. Materials and Methods Namalingkanusasanum written by Amarasshimhan some- where in between the �rst and sixth century AD [7]. It is 2.1. Chemicals. Ethanol, methanol, ethyl acetate, chloroform, known as kundalam paalai in Tamil, possesses curative pro- heptane, acetone, AlCl , ammonium hydroxide, rutin, and perties against venereal diseases, gonorrhoea, respiratory quercetin were obtained from SD Fine-Chem. Ltd., India. problems, nervous disorders, diabetes, chronic bronchitis, For HPLC analysis, HPLC3 grade acetonitrile from SD Fine- rheumatism, cardiotonic ailments, and snake bite [8, 9]. Chem. Ltd., India and formic acid from Merck, Darmstadt, Preliminary phytochemical screening of the ethanolic extract Germany were obtained. For TLC analysis, silica gel G was of the roots of T. heyneana Wall. revealed the presence obtained from Merck, Darmstadt, Germany. of alkaloids, sterols, triterpenoids, resins, and �avonoids 60 [10]. Sathishkumar et al. [11] have proved the presence of quercetin and rutin related �avonoids in the leaves of T. 2.2. Plant Material. e �owers were collected from the heyneana. Reports are available for the therapeutic effect of medicinal garden of Kumaraguru College of Technology, �ower �uice (mixed along with coconut oil) against burning Coimbatore, India. e species was identi�ed and con�rmed sensation of eyes and improved vision [12]. at Botanical Survey of India (BSI), Southern Circle, Coim- batore, India (BSI/SC/5/23/06-07/Tech. 478). About 5 g of Extraction is a very important process for production air-dried fresh �owers was dissolved in 50 mL of the solvent of �avonoid concentrate from rich sources. e traditional (methanol, ethanol, distilled water, chloroform, heptane, extraction methods possess several limitations such as time and acetone) and kept in an orbital shaker for overnight consuming, laborious, low selectivity, and yield as well as uti- (shake �ask method). e residue was reextracted under the lization of large amount of organic solvents [13]. At present, same conditions. e obtained extracts were �ltered with there is a renewed interest in developing new processes based Whatman number 1 �lter paper, and the �ltrate was used for on the use of different variables like temperature, solvent total �avonoid estimation. modi�ers, and material ratio for the extraction of low molecular weight components that may be environmen- tally friendly and benign. Previous research documentation 2.3. Experimental Design of Extraction Process. e main authenticates that temperature-assisted, enzyme-assisted, factors that affect the extraction of �avonoid like temperature, supercritical-�uid-based, and semibionic-based extractions extraction time, material ratio (weight of the �owers (g): are superior over conventional soxhlet-mediated extraction volume of the solvent (mL)), solvent modi�er ( ), and the [14]. It is quite complex to predict the suitable experimental number of extraction cycles were investigated. e factors conditions for a given separation task, and therefore good and the experimental design were slightly modi�ed% according experimental design becomes signi�cantly important. Such to the type of the solvent used. e optimum extraction experiments are oen executed in the form of orthogonal conditions were determined by L (4 ) orthogonal design arrays. Because of the cost efficiency, experimenters always of experiment using ethanol as extracting5 solvent (Table 1). consider as many factors as possible in a design with mini- For ethyl acetate-mediated optimization,16 L (3 ) orthogonal mum number of runs in order to make the design saturated design of experiment was adopted (Table 2).4 A single- [15]. factor analysis of variance (one-way ANOVA)9 was adopted Although the roots and leaves have been reported to investigate the effect of each factor in the extraction of to have �avonoids, no scienti�c information is available �avonoid. GraphPad Prism 5 trial version soware was used about the �avonoid content in �owers of Tabernaemontana to carry out the statistical analysis. ISRN Biotechnology 3 T 2: Di�erent variables for optimal extraction of �avonoids from the �owers of T. heyneana (ethyl acetate as extraction solvent, 34-L design). 9 A B C D Levels Temp. ( C) Ext. tim. (hrs) Material ratio (g : mL) No. of ext. cycles 1 60 ∘ 1 1 : 10 1 2 70 2 1 : 15 2 3 80 3 1 : 20 3 2.4. Estimation of Total Flavonoid Content (TFC) by Alu- of mobile phase A and 88 of mobile phase B for 5 min.