Simultaneous Determination of Phenolic Compounds in Melia Azedarach . Linn Leaves by High-Performance Liquid Chromatography

Rutin, Quercetin and Kaempferol are the most important phenolic compounds of Melia azedarach. A simple and rapid HPLC method was developed for simultaneous determination of Rutin, Quercetin and Kaempferol in ethanolic leaves extract of Melia azedarach. Acetonitrile and Phosphate buffer (pH=5.8) in ratio of 55: 45 used as mobile phase. The phenolic compounds were determined by Athena C18 column type at 254 nm with flow rate of 1 ml/min. Retention time of standards, Rutin, Quercetin and Kaempferol were found to be 2.357, 6.093 and 9.373 respectively. While the Retention times of Rutin, Quercetin and Kaempferol in Melia azedarach are 2.403, 6.143 and 8.903 which are found to be matching with standards retention time values respectively. Thus this HPLC method was found to be convenient and simple for quantitative analysis of phenolic compounds in Melia azedarach. designed to for simultaneous determination of Rutin, Quercetin and Kaemp- ferol in ethanolic leaves extract of Melia azedarach by high-performance liquid chromatography.


INTRODUCTION
Plants are potential sources of natural antioxidants. It produces various antioxidative compounds to counteract reactive oxygen species (ROS) in order to survive. 1 ROS, which include free radicals such as superoxide anion radicals (O 2 -), hydroxyl radicals (OH -) and non free-radical species such as H 2 O 2 and singled oxygen ( 1 O 2 ), are various forms of activated oxygen. These free radicals responsible for cellular injury and aging process. 2 Plant phenolics are commonly found in both edible and non-edible plants, and have been reported to have multiple biological effects, including antioxidant activity. The antioxidant activity of phenolics is mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donators, and singlet oxygen quenchers. In addition, they have a metal chelation potential. 3 It is an established fact that polyphenolic compounds possess remarkable antioxidant activities which are present quite commonly in the plant family Meliaceae. Recently, interest has increased considerably in finding naturally occurring antioxidants for use in foods or medicinal materials to replace synthetic antioxidants. 4 Therefore; the need exists for safe, economic, powerful and natural antioxidants to replace these synthetic ones.
Rutin is the rhamnoglucoside of the flavonoid quercetin, and found in many plants 5 and used for treatment of various diseases related to the vascular. 6 Quercetin is a flavonol, it is plant derived flavonoid used as a nutritional supplement found in fruits and vegetables. Quercetin is thought to have potent antioxidant, Antidiabetic and anti tumour, and antiviral, anti inflammatory benefits. 7 Quercetin is mainly found in many often consumed foods include green apple, onion, green tea, lemon as well as many seeds, flowers, barks, and leaves. 8 Kaempferol occurs naturally in a variety of fruits, vegetables, wine and tea. Kaempferol can be isolated from tea, broccoli, witch-hazel, propslis, grapefruit, and other plant source. 9 kaempferol is one of the most important flavonoids that inhibit heart, spinal cord, and brain disease. Itinhibits both oxidative susceptibility of low density lipoprotein (LDL) in vitro,and platelet aggregation. 10 Melia azedarach Linn. ( Family: Meliaceae) is a shrub or small evergreen and medium-sized deciduous tree.It has been useful in fever, thirst, nausea, vomiting, and skin diseases. 11,12 Leaves and fruits showed antifeedant activity. 13,14 The stem extracts showed larval mortality 15 and insecticidal activity. The plant has also showed antifungal 16 , antibacterial 17 ,cytotoxic 18 , antimalarial 19 , anthelmintic 20 , antilithic 21 and antifertility activity. 22 The present study was designed to for simultaneous determination of Rutin, Quercetin and Kaempferol in ethanolic leaves extract of Melia azedarach by highperformance liquid chromatography.

MATERIALS AND METHOD Reagents and Materials:
All chemicals and solvents used were of analytical grade. The standard Rutin and Quercetin were purchased from Yucca Enterprises,MumbI (purity >97%).The standard Kaempferol MP Biomedicals, Mumb(purity >97%). Phosphate buffer (pH=5.8) and solvent Acetonitrile used as mobile phase were obtained from S.D.Fine Chem Limited, Mumbai. The column type was, Athena C18 250X 4.6 (CNW Technology).

Plant material:
The basic plant material of Melia azedarach leaves was obtain from Zaheerabab,Medak Dist. The plant was identified and authenticated by Department of Botany and Research office (Botanist) Anwar-ul-loom college of Pharmacy, Hyderabad.

Extraction of plant material for HPTLC analysis:
The leaves of Melia azedarach were dried under shade and powdered in a mechanical grinder. The leaves powders of Melia azedarach, weight about (250 g) were individually packed in the thimble of soxhlet apparatus and extracted with ethanol at 55°C for 18 h. The extract was concentrated to get dry residue and stored in the dessicator and it was used for subsequent experiments. Preliminary photochemical screening revealed the presence of Polyphenols, flavanoids and glycosides.
Preparation of standard and sample solutions Phosphate buffer (pH=5.8) and solvent Acetonitrile used as mobile phase.10 mg of Standard Rutin and Quercetin were dissolved in 25ml of mobile phase, while 15mg of kaempferol were dissolved in 25ml of mobile phase and 10 mg of Sample solution of extract Melia azedarach were dissolved in 25ml of mobile phase as above as standard preparation.

ReseaRch PaPeR
Mobile phase ration : 55: 45 % v/v Mobile phase : Phosphate buffer (pH=5.8) and Acetonitrile The operating temperature was maintained at room temperature. Identification of the compounds was achieved by comparison with retention times of standards with the samples.

Assay formula
RESULTS AND DISCUSSION Quantification of Rutin, Quercetin and Kaempferol in Melia azedarach: The retention time (Rt) of standards Rutin, Quercetin and Kaempferol were found to be 2.357, 6.093 and 9.373 with 100% area (Fig:). While the retention time (Rt) of Rutin, Quercetin and Kaempferol in Melia azedarach extract, was found to be 2.403, 6.143 and 8.903 respectively (Fig: 5.10) which are matching with standards Rt values respectively. The amount of rutin, quercetin and kaempferol in ethanolic leaves extract of Melia azedarach was found to be 10.4%, 0.07% and 0.11 % w/v respectively.The mobile phase include acetonitrile and Phosphate buffer (pH=5.8) were tested and the results showed the good resolution and good peaks shape.

CONCLUSION
The HPLC in-house analytical methods were developed, which was found to be excellent technique for simultaneous determination of rutin,Quercetin and Kaempferol in ethanolic leaves extract of Melia azedarach . The cost and Running time per analysis are found to be relatively low in comparison with other methods. Hence this method can be apply for the Quantitative analysis of rutin, Quercetin and Kaempferol. Furthermore, the method can be used as quality control for phenoloic compounds and was found to be efficient, simple and rapid.