IDENTIFICATION AND QUANTIFICATION OF PHENOLIC COMPOUNDS FROM OPERCULINA TURPETHUM (L.) SILVA MANSO LEAF BY HPLC METHODHTML Full Text
IDENTIFICATION AND QUANTIFICATION OF PHENOLIC COMPOUNDS FROM OPERCULINA TURPETHUM (L.) SILVA MANSO LEAF BY HPLC METHOD
Anju Vijayakumari Jalaj* and Puthuparampil Madhavan Radhamany
Department of Botany, University of Kerala, Thiruvananthapuram- 695581, Kerala, India.
ABSTRACT: Plants served as potent sources of bioactive compounds since ages. The understanding of phytochemical constitution of each and every plant on earth is the preliminary and the most important step in drug discovery. As Operculina turpethum(L.) Silva Manso is traditionally used for various ailments, the understanding of phytochemical composition of the plant is very essential. The present study was an attempt to identify the phenolic compounds present in the leaf of the plant O. turpethum. Total phenol and flavonoid content was determined from the methanol extract of the leaf and the extract was subjected to HPLC analysis with seven standard compounds. A gradient mobile phase comprising acetonitrile and 0.1% phosphoric acid was used to get the phenolic profile. The quantity of each compound present in the extract was calculated from the peak area and expressed in mg/ ml of sample injected. The first mobile phase revealed the presence of compounds viz., coumarin, ferulic acid, catechin, salicylic acid, rutin, vanillin and gallic acid. Among the seven identified phenolics, coumarin was present in highest concentration (0.34mg/ml of sample injected) and vaniilin was present in least amount (0.002mg/ml of sample injected). All the compounds identified are reported to have pharmacological activities. This report is the first report on the identification of phytochemicals from the O. turpethum leaf
HPLC, salicylic acid, coumarin
INTRODUCTION: Plants produce amazing diversity of low molecular weight compounds through various secondary biochemical pathways, often in response to specific environmental stimuli such as herbivore-induced damage, pathogen Attacks or nutrient depravation 1, 2. These secondary metabolites can be unique to specific species or genera and do not play any role in the plant’s primary metabolic requirements 3. They are structurally diverse and show differential occurrence among plant species. So they are used as tools in chemotaxonomic studies.
Based on their biosynthetic origin, plant secondary metabolites are classified into three major groups- phenolics, terpenoids and alkaloids 4. Phenolic compounds are widely distributed groups of natural products in the plant kingdom. They range from simple, low molecular weight, single aromatic- ringed compounds to large and complex tannins and polyphenols. Based on the number of carbon atoms in the basic skeleton, they are classified as benzoquinones, naphthoquinones, stilbenes, flavonoids, norlignans, lignans and condensed tannins.
The role of phenolic compounds in plants include provide defence against a range of attacks from browsing animals, bacteria, fungi and insects, inhibit growth of competing plants, provide protection against the damaging effects of UV radiation, promote the growth of pollen tubes in the style of flowers and impart colour to the stem, leaves, flowers and fruits. The plant phenolics are viewed as potential sources of new natural drugs, antibiotics, insecticides and herbicides 5. Phenolic compounds act as antiviral, antitumor, biocidal and bioactive agents 6. Qualitative and quantitative analysis of phenolic compounds found in any part of the plant is an essential prerequisite for testing efficacy of the particular plant part in treating various ailments.
Operculina turpethum (L.) is a large perennial twiner of the family Convolvulaceae. It is a rare and endangered medicinal plant of peninsular India, threatened by overexploitation 7. It is also known as Indian Jalap and occasionally cultivated in botanical gardens as an ornamental plant. The Sanskrit name Trivrit and the Malayalam name Triputa is assigned to the plant due to the presence of three layered bark 8. The leaf of the plant possesses antimicrobial properties 9 and fresh juice of the leaf is used for treating corneal opacity and conjunctivitis 10. The ancient literature provides many clinical properties of the plant but they were not yet fully scientifically evaluated. As the phytochemical composition of a plant reflects biological use of the plant, understanding of the chemical profile of the plant is the very first step in any drug discovery process.
As per early reports, the leaf of the plant is rich in secondary metabolites such as flavonoids, cardiac glycosides and terpenoids 11. But there is no report regarding the specific phenolic, terpenoid or glycosidic compounds present in the leaf. So the present work is designed to identify and quantify phenolic compounds present in leaf methanolic extract of O. turpethum by High Performance Liquid Chromatography (HPLC).
MATERIALS AND METHODS:
Procurement of plant material:
Operculina turpethum was collected from Pangappara, a place in Thiruvananthapuram district of Kerala, India. The material was verified and authenticated by Dr. G. Valsala Devi, Curator, Department of Botany, University of Kerala, Kariavattom and a Voucher specimen was deposited in the Department herbarium (KUBH 6987).
Preparation of leaf extract and standard solutions:
For extract preparation, leaves of the plant were collected, cleaned and shade dried for one month. The dried leaves were powdered using a blender and fine powder was used for extraction. Accurately weighed (10g) powder was continuously extracted with methanol in a soxhlet extractor for 48 hours. After extraction, the extract was concentrated on a rotary evaporator and dried to a constant weight. The weight of extract was measured and kept at 50C until use.
The seven standards used for the study include catechin, gallic acid, rutin, coumarin, ferulic acid, salicylic acid and vanillin. All the standard compounds were dissolved in one millilitre of HPLC grade methanol and this was passed through Whatman Nylon Membrane Filter before injecting it in the column.
Spectroscopic quantification of phenols and flavonoids:
Total phenol and flavonoid contents were estimated from the methanol extract according standard procedures 12, 13.
The methanolic Extract and seven phenolic standards were subjected to High Performance Liquid Chromatography using 600 series HPLC pump and 2487 dual wavelength UV detector-254 and 360 nm of Waters company, USA, having Reprobond C18 column-4.6x250mm and 7725 Rheodyne injector. Sample volume of 20 microlitre each was injected in all cases and flow rate of 1.0 ml/min was maintained. The data analysis was done using Empower 2 software. The compounds were eluted by employing the following method.
Gradient elution of two solvents such as acetonitrile (solvent A) and 0.1% phosphoric acid in water (solvent B) were used for the detection of catechin, gallic acid, rutin, coumarin, Ferulic acid, salicylic acid and vanillin 14. The total run time of the program was 30 minutes. The gradient program was begun with 85% B and was held at this concentration for the first 12 minutes. This was followed by 75% eluent B for the next 10 minutes with a linear gradient and after which its concentration was again increased to 85% B for the next 8 minutes.
RESULTS AND DISCUSSION: The total phenol and flavonoid content in the methanol extract was determined and result is presented in Table1. Chromatograms of phenolic standards catechin, gallic acid, rutin, coumarin, Ferulic acid, salicylic acid and vanillin having retention times 6.873, 3.571, 20.498, 23.497, 15.787, 25.080 and 11.161 respectively were obtained using mobile gradient phase of acetonitrile and 0.1% phosphoric acid in water for 30 minutes run time when each chemical was individually analyzed. The result obtained is shown in Fig. (1 – 7). The chromatogram of methanol extract (Fig.8) showed the presence of tested standards. The quantity of each identified compound in the extract and other characters of them are presented in Table 2. The predominant unidentified peaks in the chromatogram of the extract need to be identified by the use of other advanced methods.
Natural phenolic compounds play an important role in cancer prevention and treatment. They are reported to possess antioxidant, anticarcinogenic, or antimutagenic and anti-inflammatory effects15. Flavonoids are known to inhibit production of heat shock proteins in several malignant cell lines, including breast cancer, leukemia and colon cancer16.
The identified phenolic compounds were reported to possess several biological activities. Salicylic acid and its derivatives showed analgesic, antipyretic, cytotoxic and anti-inflammatory activities 17. They are used extensively for the relief of headache, inflammation, arthritis pain, and some are employed in the treatment of heart attacks and strokes in the elderly 18. Gallic acid and its derivatives also showed numerous pharmacological activities such as antioxidant, apoptotic and antimicrobial effects 19. Coumarins, commonly known as benzopyrones act as antimicrobial, anti-tumoural, anti-HIV agents, CNS activators and enzyme inhibitors 20. Ferulic acids are proved as platelet aggregation (PA)-inhibitory, tyrosinase-inhibitory, angiotensin converting enzyme (ACE)-inhibitory compounds and they exhibited superoxide dismutase (SOD)-like activities 21.
The flavonoid compound rutin is reported to possess hepatoprotective activities 22. In addition, these compounds possess antimicrobial and antioxidant properties 23. Catechin possess antiviral properties 24, 25. So the present study revealed the presence of potent medicinal principles in O. turpethum and adds a data source for utilizing real healing potentials of the plant.
The identified phenolic compounds were reported to possess several biological activities. There are more phenolic compounds to be identified in the plant. The wide range of bioactivities reported for the identified compounds make the plant a potent source of antioxidant, antimicrobial, anticancer and hepatoprotective drugs.
Value (mg/ml equivalents of standards)
Total phenol content
1.89 ± 0.01
Total flavonoid content
1.0 ± 0.00
TABLE 2: COMPOUNDS IDENTIFIED IN HPLC
|Molecular formula||Molecular weight
|Gallic Acid||Trihydroxy benzoic acid||3.554||0.15||C7H6O5||170.12|
|Ferulic Acid||Hydroxy cinnamic acid||15.724||0.01||C10H10O4||194.18|
|Salicylic acid||Monohydroxy benzoic acid||24.911||0.28||C7H6O3||138.121|
ACKNOWLEDGEMENTS: We are thankful to Kerala State Council for Science, Technology and Environment for providing fund and Bio-Medical Technology wing of Sree Chithra Thirunal Institute for Medical Science and Technology for providing lab facilities.
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How to cite this article:
Jalaj AV and Radhamany PM: Identification and Quantification of Phenolic Compounds from Operculina Turpethum (L.) Silva Manso Leaf by HPLC Method. Int J Pharm Sci Res 2016; 7(4): 1656-61.doi: 10.13040/IJPSR.0975-8232.7(4).1656-61.
All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
Anju Vijayakumari Jalaj* and Puthuparampil Madhavan Radhamany
Department of Botany, University of Kerala, Thiruvananthapuram, Kerala, India.
06 November, 2015
29 December, 2015
26 January, 2016
01 April, 2016