SIMULTANEOUS DETERMINATION OF PHENOLIC COMPOUNDS IN CATHARANTHUS ROSEUS LEAVES BY HPLC METHOD

Received on 24 October, 2013; received in revised form, 04 December, 2013; accepted, 16 February, 2014; published 01 March, 2014

SIMULTANEOUS DETERMINATION OF PHENOLIC COMPOUNDS IN CATHARANTHUS ROSEUS LEAVES BY HPLC METHOD

A. Srinivasa Rao*¹ and Mohammed Fazil Ahmed ²

Bhaskar Pharmacy College, Yeknapally, Moinabad (Mandal), R.R (Dist), Hyderabad 500075, Andhra Pradesh, India

Nizam Institute of Pharmacy & Research Center, Deshmukhi, Pochampally (M), NearRamoji Film City, Nalgonda -508284, Andhra Pradesh, India

ABSTRACT: In Catharanthus roseus, Rutin, Quercetin and Kaempferol are the most important phenolic compounds with antioxidant activity. For simultaneous determination of Rutin, Quercetin and Kaempferol in Catharanthus roseus a simple and rapid HPLC method was developed. Phosphate buffer (pH=5.8) and solvent Acetonitrile are used in ratio of 55: 45 as mobile phase. Rutin, Quercetin and Kaempferol were determined by HPLC by using Athena C18 column and UV absorbance were measured at 254 nm. Retention time (Rt) of standards, Rutin, Quercetin and Kaempferol was found to be 2.357, 6.093 and 9.373 respectively, while in Catharanthus roseus the Retention times of Rutin, Quercetin and Kaempferol are 2.403, 6.143 and 8.903 are found to be matching with standards Retention time values. Thus for quantitative estimation of phenolic compounds, this HPLC method was found to be simple and convenient.

Catharanthus roseus, HPLC, Phenolic compounds, Quercetin, Rutin and Kaempferol

INTRODUCTION:Antioxidants always play important roles in delaying the development of chronic diseases such as cardiovascular diseases (CVD), cancer, atherosclerosis, inflammatory bowel syndrome and Alzheimer's diseases ¹.

Oxidative stress caused by an unbalance between pro-oxidants and antioxidants. Reactive oxygen species (ROS) are divided in two main classes consisted of non-radical species (H₂O₂) or free radical forms (O₂^-, OH^-, and OH₂.).

Accumulation of high concentrations of ROS is potentially detrimental to plants cells causing damage to valuable biomolecules like DNA, proteins, lipids, chlorophyll, membrane etc. ².

Antioxidants are radical scavengers, which protect the human body against free radicals that may cause pathological conditions such as anaemia, asthma, arthritis, inflammation, neuro-degeneration, Parkinson’s diseases, mongolism, ageing process and perhaps dementias ^{3, 4}.

Antioxidants are believed to  play a very important  role  in  the  body defense system against reactive  oxygen species  (ROS),  which are  the  harmful by products  generated  during  normal  cell  aerobic  respiration ⁵. The importance of antioxidants in health and disease is now recognized by every branch of medicine and biological science ⁶.

Catharanthus roseus or Vinca rosea belongs to the family Apocynaceae; Catharanthus roseus has a variety of medicinal properties, such as antibacterial ⁷, antifungal ⁸ and antiviral ⁹. A variety of different alkaloids is present in C. roseus: more than 130 different compounds have been reported including about 100 monoterpenoid indole Alkaloids ¹⁰.The flower petals, seeds and other parts of the C. roseus exhibit antioxidant properties ^{11, 12}.

Phenolic compounds play important role in plant driven antioxidant activity which is due to their redox properties, permit them to act as reducing agents, hydrogen donors, singlet oxygen quenchers or metal chelators. Phenolics belong to  a  large heterogeneous group of secondary  plant metabolites  that  are  widespread  in  the  plant  kingdom  and  have  multiple  applications  in  food,  cosmetic  and pharmaceutical  industries ^{13, 14}.

The present study was designed to for simultaneous determination of Rutin, Quercetin and Kaempferol in ethanolic leaves extract of Catharanthus roseus by high-performance 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 Catharanthus roseus leaves was obtained from Mount Opera Garden, near Ramoji Film City, Hyderabad. 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 Catharanthus roseus were dried under shade and powdered in a mechanical grinder.

The leaves powders of Catharanthus roseus, 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 desiccator and it was used for subsequent experiments. Preliminary photochemical screening revealed the presence of Polyphenols, flavonoids 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 of Catharanthus roseus were dissolved in 25ml of mobile phase as above as standard preparation.

Chromatographic conditions: 

Flow rate                     : 1 ml/min

Detection                    : 254 nm

Injection quantity        : 0.02ml

Column used               : Athena C18 250X 4.6

Column temperature   : 35°C

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 Kaempferolin Catharanthus roseus (C1): 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. 1-3 and Table 1). While the retention time (Rt) of Rutin, Quercetin and Kaempferol in Catharanthus roseus (C1)extract, was found to be 2.420, 6.160 and 9.463 respectively (Fig. 4 and Table 2), which are matching with standards Rt values respectively. The amount of rutin, quercetin and kaempferol in Catharanthus roseus (C1) was found to be 21.59%, 0.12 % and 0.028 % 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.

FIG: 1 HPLC CHROMATOGRAM OF STANDARD RUTIN

FIG. 2: HPLC CHROMATOGRAM OF STANDARD QUERCETIN

FIG. 3: HPLC CHROMATOGRAM OF STANDARD KAEMPFEROL

FIG. 4: HPLC CHROMATOGRAM OF EXTRACT CATHARANTHUS ROSEUS (C1)

TABLE 1: RETENTION TIME, HEIGHT AND % AREA OF STANDARDS RUTIN, QUERCETIN AND KAEMPFEROL

TABLE: 2 RETENTION TIME, HEIGHT AND % AREA OF RUTIN, QUERCETIN AND KAEMPFEROL IN EXTRACT OF CATHARANTHUS ROSEUS (C1):

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 Catharanthus roseus. The cost and Running time per analysis are found to be low relatively in comparison with other methods. Hence this method can be applied for the Quantitative analysis of rutin, Quercetin and Kaempferol. Furthermore, the method can be used as quality control for phenolic compounds and was found to be simple, rapid and efficient.

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