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DETERMINATION OF ANTIOXIDANT FLAVONOIDS AND PHENOLIC COMPOUNDS IN BETEL PLANT BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

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DETERMINATION OF ANTIOXIDANT FLAVONOIDS AND PHENOLIC COMPOUNDS IN BETEL PLANT BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Powered By Docstoc
					                                Mummidivarapu Durgaprasad et al., IJSID 2011, 1 (2), 109-114



                                                                                                 ISSN:2249-5347
                                                                                                           IJSID
                         International Journal of Science Innovations and Discoveries                 An International peer
                                                                                                 Review Journal for Science


Research Article                                                      Available online through www.ijsidonline.info

 DETERMINATION OF ANTIOXIDANT FLAVONOIDS AND PHENOLIC COMPOUNDS IN BETEL
           PLANT BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
Mummidivarapu Durgaprasad, Amarendra. CH, Anusha.G, Balu. Knodeti, A. Durga Prasad, A. Durga
                                Rao, Rambabu. Kuchi*.
                    Dept of P.G Chemistry, D.N.R College, Bhimavaram, Andrapradesh, India.

 Received: 03.08.2011
                                                                          ABSTRACT
 Modified: 19.10.2011
                                            This paper describes the quantitative determination of flavonoids,
 Published: 27.10.2011
                                    Phenolic Antioxidants in Piper betel. The crude extracts of Flavanoid and
 Keywords:
 Betel Plant, Antioxidants,         phenolic compounds were obtained from Betel (Piper betel Linn.)Leaves
 HPLC, 280 nm, Phenolic
 compounds, 354 nm                  and stems. The presence of           Flavanoid and phenolic antioxidants in the
 *Corresponding Author              extracts was determined using the isocratic elution of RP-HPLC with
                                    suitable mobile phases, Zodiac C18 Column . Methanol and HCl extract is
                                    injected for Flavanoids at 354nm and Acetone extract is injected for
                                    Phenolic compounds at 280nm. Phenolic compounds (Fig-2)of (Piper betel
                                    Linn.)was identified. All of the reference compounds, Gallic acid (R.T-
                                    1.67)and Catechin (R.T-3.02), epicatechin (8.10) and querecetin (12.96)
                                    were detected in the extracts . the Flavanoids(Fig-1) of Prunus dulcis was
                                    identified.      All    of     the      reference      compounds,          calycosin-o
                                    glucoside(R.T5.4),Onnin(R.T-8.52),calycosin(R.T-9.47) were detected in the
 Address:

 Name:   Rambabu Kunchi             extracts.

 Place: Bhimavaram, AP, India
 E-mail:
 rambabu.kuchi@gmail.com


                                                    INTRODUCTION


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                            Mummidivarapu Durgaprasad et al., IJSID 2011, 1 (2), 109-114

                                                INTRODUCTION
       Extracts of many plants have been shown to have various degrees of antioxidant activity in
different fats and oils. The antioxidant activity of these plants and their extracts can be attributed to the
presence of flavonoid compounds, phospholipids, tocopherols, and ascorbic acid . The identification of
certain flavanoids and phenolic compounds in almond somehow Provide an alternative method for
medicine and remedies as many studies have proved to alleviate certain ailments in treating diseases. In
these antioxidants mainly Phenolic acids and flavonoids function in plant cells is thought to be defensive,
providing protection against insect, fungal, and viral attacks and consumption by invertebrate and
vertebrate herbivores.
       The Piper betel Linn., which is commonly known as Betel leaf, is consumed for chewing with Betel
nut and lime by some people in Asia. Data on the phenolic compounds of this plant is scarce. Piper betel
Linn. (Piperaceae) is a tropical Asian vine closely related to the common pepper. The leaves are chewed
alone or with other plant materials, including the areca nut (Areca catechu Linn.), and lime. Several
solvent systems have been used to extract antioxidants from Betel leaves. These solvent systems include
ethanol, methanol and chloroform (Amonkar, et al., 1986; Nagabhushan, et al., 1989; Rimando, et al.,
1986). These different solvent extracts of Betel leavespossess antioxidant, antimicrobial and anti-
inflammatory effects. It is noted that a solvent system for extraction is selected according to the purpose
of extraction such aspreparation for analysis. It was evident that Betel leaves contained rich carotenes
(80 IU/g fresh wt.), ascorbic acid (1.94 mg/g fresh wt.) and phenolics. Data on the phenolic compounds of
this plant is related to chavicol (Amonkar, et al., 1986), chavibetol, chavibetol acetate (Rimando, et
al.,1986) and eugenol (Nagabhushan, et al., 1989).Objectives of this study are aimed at determine the
antioxidants of betel plants.
                                                EXPERIMENTAL
Instrumentation
       A Series 200 HPLC system PEAK LC7000 isocratic HPLC with PEAK 7000 delivery system.
Rheodyne manual sample injector with switch (77251),Analytical column Zodiac100-5 C18. 250×4.6mm,
Electronic balance-DENVER (SI234), A manual Rheodyne injector with a 20 μl loop was used for the
injection of sample., PEAK LC software were used.
Collection of plant material
       The stem and leaf Betel were collected from the Forms nearer to DNR College, Bhimavaram, West
Godavari dist, Andhra Pradesh, in the month of the March 2011.This is a most available plant in east
Andhra side.
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Extraction of plant material:
Methanol Extract:- The stem and leaf of Betel (Piper betel Linn.)were shade dried and made to a fine
powder (particle size ~0.25mm). The powder was first defatted with Methanol and then it was further
extracted with methanol. Take another, 1g of powder sample was accurately weighed and taken in to
100ml beaker and added 20ml of Hcl+80ml of methanol to prepare 10mg/ml sample solution
Acetone Extract
       About 1 gram of The powder was dissolved in 95%acetone Solution and stands for overnight.
                                        MATERIALS AND METHODS:
Chemicals:
       All solvents used were of analytical grade. Methanol, Acetone, Hydrochloric acid, The above
chemicals and reagents used were of analytical grade were acquired from the local chemical stores. And
Methanol, water, Acetonitrile, potassium dihydrogen phosphate were of HPLC grade were purchased
from Qualligens
HPLC analysis of the crude extracts.
       For the HPLC finger print analysis of phenolic compounds present in extracts a PEAK LC7000
isocratic HPLC consisting of PEAK 7000 delivery system , LC7000detector, Rheodyne manual sample
injector with switch (77251) and a prepacked Zodiac C18 (4 × 250 mm, 5 μm, Phenomenex) were used.
A flow rate of 1 mL/min, and isocratic elution of methanol- water (50:50, v/v/v) for flavanoids and
Elution of 10mm KH2Po4-methano (70:30, v/v/v) for phenolic acid and injection volume of 20 μL were
applied; whereas the separation of compounds was monitored at 280 and 3540 nm.
       The mobile phase was filtered through 0.22m membrane and degassed using ultrasonicator. The
experiments were carried out at room temperature . Both the standard and sample solutions were
injected and the experiment carried out using the above optimized chromatographic conditions and the
mass spectrum of β- Sitosterol (Figure 1) and the chromatograms of standard and sample solutions
(Figure 2 and 3) were recorded, from which the amount of β-Sitosterol present in the leaf extract was
determined.
                                          RESULT AND DISCUSSION
        Total phenolic and flavonoid contents of different extracts of Betel (Piper betel Linn.) were
examined. High contents of total phenolic compounds and total flavonoids were determined. In this
report, a method based on reversed phase HPLC separation detection was used for Flavanoid and
Phenolic Antioxidants assay in Betel (Piper betel Linn.) . An isocratic system was chosen to minimize the

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                            Mummidivarapu Durgaprasad et al., IJSID 2011, 1 (2), 109-114

variation of the baseline and ghost peaks. The mobile phase, as well as the other chromatographic
conditions, showed high performance in the separation of the flavonoid an Phenolic Antioxidants.
            Method for flavanoids included a isocratic elution of Methanol: Water (50:50 v/v) for 30
min, and a flow rate of 1mL min, at 354nm Detector Wavelength and Zodiac C18 column was used. 20µl
of HCl extracted is injected.With this study the Flavanoids(Fig-1) of Prunus dulcis was identified. All of
the reference compounds, calycosin-o glucoside(R.T5.4),Onnin(R.T-8.52),calycosin(R.T-9.47) were
detected in the extracts.
. In above three compounds eallagic acid was first eluted and it is dominant.Querecetin and calycosin are
in low quantity. Flavonoids are water soluble polyphenolic compounds,which are extremely common and
wide spread in theplant kingdom as their glycosides.
           Method for Phenolic compounds included a isocratic elution of 10MM KH2Po4:
Methanol(70:30 v/v) for 30 min, and a flow rate of 1mL min, at 280nm Detector Wavelength and Zodiac
C18 column was used. 20µl of Acetone extracted is injected.With this study, the Phenolic compounds
(Fig-2)of (Piper betel Linn.)was identified. All of the reference compounds, Gallic acid (R.T-1.67)and
Catechin (R.T-3.02), epicatechin (8.10) and querecetin (12.96) were detected in the extracts( fig-2) .
Phenols comprise the largest group of plants secondary metabolite. Phenolic compounds are commonly
found in both edible and non-edible plants and they have been reported to have multiple biological
effects, including antioxidant property.




                                  Fig: 2. HPLC Chromatogram of Flavanoids
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                                 Fig 2: HPLC Chromatogram of Phenolic compounds
                                                        CONCLUSION
         The crude extracts of phenolic compounds and of Flavanoids obtained from the Betel (Piper betel
Linn) have shown the different Antioxidants in it. Three flavanoid ellagic acid,querecetin,calycosin were
detected in the HCl extracts and Two Phenolic compounds Gallic acid and Catechin were detected in the
Acetone extract. However, the concentrations differ due to the metabolism of the plant in different
conditions. Previous studies found ferulic acid In further studies, the amount of phenolic acids can be
analyzed in different parameters, such as depth, hours of the day, season, and location, in order to
identify the concentration changes of the plant. According to the efficiency of analysis, activity tests of the
plant extracts could be performed for antioxidant activities.
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