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Nutraceuticals capsules, pills, powders, etc. are natural source and plant derived which have proved their benefits to the health. Grape seed extract with resveratrol attributes various properties like anticancer potential, cardio-protective and improving vascular function which has driven the attention of researchers. Grape seed extract was extracted with methanol by reflux extraction this was then screened for antioxidant activity using TLC (Thin layer chromatography). Radical scavenging activity of the sample was assayed quantitatively by spectrophotometric assay. Reverse phase high performance liquid chromatography (RP-HPLC) using isocratic mobile phase system was carried out for measuring quantitatively concentration of resveratrol in sample extract. High radical scavenging activity of sample extract was observed at 0.5 mg/ml concentration and IC50 of the sample extract was found to be 150µg/ml. Evidence of antioxidant activity and effective separation of grape seed extract was observed by TLC. RP-HPLC showed 9.81µg/ml of resveratrol concentration in 50µg/ml concentration of the sample extract using standard resveratrol as a reference. Gas chromatography- Mass spectrometry (GC-MS) did not detect compounds present in sample grape seed extract due to high polarity and low volatility.
Herbal medicines have been used since dawn of humanity. Traditional systems such as Chinese herbal medicine, Indian (Ayurveda) or Japanese (Kampo) medicine have now entered to western industrialized countries where their use is often integrated into conventional medicine (Gurib-Fakim, 2006). Since ancient time's plant source is one of the major sources of drug, it holds many species of plants which holds medicinal value and are yet to be discovered (De Pasquade, 1984). Industrialisation and development of synthetic product from natural plant products which are purified and structurally modified to produce potentially more active and safer drugs are leads and precursors for significant number of synthetic drugs. About 25% of drugs comes from a plant source and 60% of anti-tumour and anti-infectious are already in the market and are of natural origins (Yue-Zhong Shu, 1998). They have shown exponential growth, greatly in the developing countries and are considered safest due to their harmless effect and natural origin .It is observed that herbal supplement have advantageous effects on human body though they have low potency as compared to pharmaceutical drugs but have long term activity when taken on regular basis. (Espin J, et al., 2007).
Grape seed is known for its phenolic compounds like flavonoids (monomeric flavon 3-ols and proanthocyanidins), phenolic acids (gallic acid and ellagic acid) and stibenes (resveratrol & piceid) which are widely used for their antioxidant, radio protective, anti-hyperglycaemic, anti-inflammatory effects( Xu et al, 2009.) and prevention of cataract (Yamakoshi, et al., 2002).It also shows increase in plasma antioxidant capacity and also improves the endothelial function along with blood circulation bringing vasodilation to an individual with high cardiovascular risk and decrease in fluid retention in pre-menopausal women( Espin J, et al.,2007).
Fig 1 Grape seed extract 100mg (http://www.hollandandbarrett.com/pages/categories)
Oligomeric and polymeric flavon-3-ols or condensed tannins also called as proanthocyanidins are one of the most common bioactive constituent in the plant kingdom which finds its place in regular human diet .They provide astringency and flavour to the grape seed extract and are mainly focused due to their effects on vascular system to decrease LDL-cholesterol levels and blood pressure (Williamson and Manach, 2005).Another anticancer potential constituent in grape seed which has attracted the attention of the researchers is resveratrol(3,5,4'-trans-trihydroxystilbene)(Asensi et al., 2002). Found in nearly 72 vegetable source it was a french paradox study in 1992 which proved resveratrol one of the best natural polyphenolic compound (Renaud and De Lorgrril., 1992). It was first isolated in 1940 from Veratrum grandiflorum O. Loes which is usually found with cardioprotective effects. (Espin, et al., 2007). Resveratrol provides chemopreventive effects. In-vivo studies have shown anti-angiogenesis activity of resveratrol inhibiting endothelial cell proliferation. Potential activity of resveratrol was investigated which showed micro vessel formation of rat aorta rings in serum-free fibrin gel cultures in control group and disintegration observed at 40µmolL-1 by resveratrol treatment causing decrease in number of capillary tubes(Cao.Y, et al., 2004).
Fig 2 Effect of resveratrol on serum-free fibrin gel cultures of rat aorta rings; microvessels indicated by arrows. (A) Control (B) 40 µmolL-1 resveratrol (Cao.Y, et al., 2004).
It is also known that resveratrol has got cancer preventive and therapeutic effects. It affects the carcinogenic process by inhibiting the cancer associated gene expression with a little cytotoxic effect and it enters the blood brain barrier which helps in management of malignancies at childhood (Hong shu. X, et al., 2010). They can also sensitize various human breast cancer cell lines like SKBR-3, MCF-7 and many more. It strongly diminishes susceptibility of SKBR-3 cells and may not be suitable for certain types of human cancers (Fukui. M, et al., 2010). In humans it is rapidly absorbed after its oral absorption and is detected in both plasma and urine. It has also been found that resveratrol has a potential use in obesity as well as diabetes (Katarzyna and Tomasz., 2010). It induces apoptic cell death as well as non apoptic cell death in ovarian cancer cell. It is seen that resveratrol reduces the plasma glucose and triglyceride concentration thereby decreases insulinemia. The effect of endocrine function of the pancreas must be well defined to recommend its use in some diabetic complications (Szkudelski. T., 2006). Resveratrol supresses the expression of the pro-inflammatory markers including COX 2 and inducing inducible NO (Nitric oxide) synthase in the both macrophages as well as in cancer lines. Anti-inflammatory effect of the drug has been associated with inhibition of the transcription factor (Kang. O, et al., 2009). Resveratrol is a potent scavenger with strong anti-tumor effects. The radicals and reactive oxygen species in high concentration have harmful effect while lower concentration it can trigger proliferation and activate cytokine signalling by different mechanism (Glafia. P, et al., 2007). The tras-resveratrol is more electron donor and proton in polar media than in gas phase, the planar geometry of 4-o-monoanions are more able to donate proton then the dianions which shows that 4-o-monoanions can be scavenger of free radicals in a basic media. Thus the trans-resveratrol has more contribution towards the free radical studies (Bader, Y. et al., 2007).
Chemistry of Resveratrol
Fig 3 3, 5, 4'-trans-trihydroxystilbene (http://alpinmack.files.wordpress.com/2009/06/pure03resveratrol3d.jpg )
Molecular formula: C14H1203
Molecular mass: 228.24 g mol-1
Resveratrol is a polyphenol phytolexin found in the skin of grapes and seed produced by chemical synthesis from p-coumaroyl CoA and malonyl CoA by an enzyme called stilbene synthase. It is also produced from the seeds of muscadine grapes .Trans-form can undergo isomerisation in presence of UV to give cis-form (LeBlanc and Mark Rene; 2005). Stibene group of compounds possess two aromatic rings which are joined by methylene bridge. Low doses of resveratrol along with other supplements which are present as dietary consitituents produces significant effect. It shows synergize effect with quercetin and ellagic acid found along with natural products for induction of apoptosis in human leukemia cells (Mertens -Talcott and Percival., 2005), with ethanol inhibition of nitric oxide synthase expression (Bradamante, S. et al., 2003), along with vitamin E it prevents lipid peroxidation and also inhibition of HIV-1 replication in cultured T-Lymphocytes (Zhang, SH. et al., 2009). A target enzyme of resveratrol includes various protein enzymes like DNA polymerases, cyclo-oxygenases, and ribonucleotide reductase. In 2001, group of Soleas, Yang and Goldberg were first to investigate the oral administration of resveratrol which achieved high concentration in 30 minutes in plasma blood samples of the subjects (Soleas, et at., 2001). Recent studies have shown 50 % of resveratrol conjugates by sulfation or glucuronide bound to plasma proteins and excreted in urine(53.85%) and fecal (0.3-38%) measured by total radioactivity (Walle, et al., 2004).
1.1. Aim: Investigation of the potential of resveratrol as a standard for the quality assessment of grape seed herbal supplement and assessment of the antioxidant activity of the supplement.
Extraction is the method in which the active constituent is solubilized and removing it with a suitable extracting solvent and it is the most common technique followed in field of analysis. They are of two types: Solid- liquid extraction and liquid- liquid extraction.
In both types there are various methods and devices adopted for the complete separation of one or more constituents from a given mixture. Solid- liquid extraction the active constituent from the solid material is extracted out by using a suitable extracting solvent. A soxhlet apparatus or reflux extraction is used for this method. In liquid- liquid extraction the ratio of activities of a solute species in a pair of two immiscible liquids and equilibrium is a constant. In this method a separating funnel is used where they are separated according to the difference in their partition coefficient. After removing the two phases the solid content in both the phases are determined (Evans, W.C., 2002). Plant materials are extracted using reflux extraction. In this method extraction is performed at a temperature with higher boiling point then solvent system. Components are extracted when dissolved into the solvent, and extraction conditions are set. Method produces a considerable amount of extraction when used for extraction in plants. Extracting compounds are effectively drawn into the solvent from the biomass due to their low initial concentration in the solvent; it is one of the efficient methods for the recovery of big yields of extract (Heinrich et al., 2004).
1.2.2 GC-MS (Gas chromatography-mass spectrometry)
GC-MS is a newer technique which is used for finger printing the different samples by giving detailed and more specific results compared to other methods. A single analysis gives high degree of resolution of any selected biomarkers. The sample is introduced first in the injection port; the temperature should be high enough to vaporize the liquid sample immediately. The carrier gases used in gas chromatography are inert gases like hydrogen and helium. Mass spectroscopy then identifies the spectrum molecules and shifts it to the magnetic field where it breaks into different charges. Ionisation of neutral ions is carried out by electron impact ionisation. GC-MS coupled with mass spectrometry provides extreme powerful analytical results. Capillary gas chromatography along with thousands of theoretical plates can resolve hundreds of molecules into separate peaks and mass spectroscopy provides identification of these samples which makes this method effective combination for chemical analysis (Willard et al 1988). Gas chromatography is common conformational test which ensures proper separation of sample that enters the column, while mass spectroscopy identifies substance by electrically charging the molecule. Development of high-technology fast computers has led to advancement of GC-MS to routine now. This combined method to identify different compounds in the sample was first introduced in 1950 by Roland Gohika and Fred Mclafferty which is now oversimplified and reducing time for analysis (Gohlke, et al., 1993). Mass spectrometer is used as detectors for gas chromatographs, since any compound that can pass through a gas chromatograph are converted into ions in the mass spectrometer.
1.2.3 TLC (Thin layer chromatography)
TLC is used for the separation of different compounds. In this technique small amount of sample or analyte or sometime a mixture of sample are added or spotted on the plate known as TLC plate. The TLC plate consists of stationary phase that is supported by a backing by glass or the plastic sheet. Then the silica gel layer is allowed to dry or activation is done under vacuum chamber by which the water molecules are removed. The sample or the analyte is spotted on the layer and the end-of the plate is kept in the solvent which is mobile phase which moves up and also known as solvent reservoir. The mobile phase moves up the silica gel layer by capillary action against the gravitational force. The analyte or the sample which is spotted gets partitioned or separate themselves between the stationary and mobile phase. Distance that the analyte travels when divided by the distance the mobile phase travel is known as Rf (Retention factor). The visualization methods like UV-lights are used to identify the samples ideally (Ahuja,et al., 2003).
1.2.4 UV Spectroscopy
UV spectroscopy is widely used in dealing with many analytical problems in organic and inorganic chemistry like determination of stable isotopes, ionization phenomena and also in the study of free radicals (Kasture A, et al., 2007). UV absorption is used for a number of stilbenes. Two main bands can usually be distinguished between 308- 336 nm and 281-313 nm, corresponding to the benzene rings. Stilbenes show two distinct bands at 260-280 nm and 300-312 nm in acidic alcoholic solvents. Increasing the PH shifts absorbance of the lower wavelength band of stilbenes and their 2 carboxylic acids to lower wavelengths (John Gorham Motoo tori and Yoshiniori asakawa., 1995)
1.2.5 HPLC (High-performance liquid chromatography)
HPLC is a very important and powerful tool in analysing the complex samples as it helps in the separation from impurities and eliminates the interferences from other complex matrixes. It is also a predominant method which offers a high sensitivity as well as high selectivity (Kasture A, et al., 2007). In this method first a dilute solution of the analyte or the sample is passed through the column which is packed with solid particles. The packing should be small enough to have an efficient separation. The sample preparation time for this method is relatively short and also the qualitative as well as the quantitative data's can be monitored for very high polar samples in a single run. HPLC is also used to assess the quality of the intermediates and also the API (Active pharmaceutical ingredient) being produced. It is an ideal method for the separation of various plants extracts resembling in structure like digitalis, cinchona, liquorice etc. It is also used for ascertaining stability of various pharmaceuticals. It is also used for analysing complex molecules such as antibiotics, and peptide hormones. HPLC is used to detect both immunoreactive and unreactive urinary albumin, used to predict the diabetic neuropathy in patients with diabetes. It is also used in the design of dosage form to give idea about bio-pharmaceutics of the dosage form and the pharmacokinetics of the drugs (Chen Q, et al., 2008).
2. Materials and methods
Grape seed extract capsules were obtained from a Holland & Barrett store in London,UK. Solvent toluene, ethyl acetate, acetic acid, ethanol, dichloromethane and methanol of analytical grade, acetonitrile, acetic acid of HPLC grade were purchased from Fisher Scientific (Loughborough, UK). DPPH powder (2, 2-Diphenyl-1-picrylhydrazyl), standard for quecertin , resveratrol and TLC plate were purchased from the Sigma Aldrich (Dorset, UK)
Powdered extract was separated from capsule shell (gelatine). 2g of powdered supplement was placed into 500ml round bottomed flask and extracted by refluxing twice at 60-700C using heating mantle (Branstead/electrothermal) for 30-35 minutes in 500 ml round bottom flask with methanol (75ml) analytical grade in a fume hood, anti-bumping granules were added to prevent bubbling of the extract. The extract was then allowed to cool for 15 minutes biomass was filtered using Buchner funnel under vacuum by using whatman filter paper (0.2µm pore size, 25mm diameter) and then transferred to 50ml round bottom flask to remove rest of the solvent using Buchi rotary evaporator(R-200) at 400C with coolant as a crushed ice placed in a condenser reservoir, motor was switched on to rotate the flask and vacuum pump was switched on to reduce the solvent to desirable level. Dryness was carried out using N2 gas.
Fig 4 Reflux extraction in fume cupboard and Buchni rotary evaporator(R-200), 8th June 2010, Super lab, Holloway road.
2.2.2 Spectrophotometric assay
The assay is based on the colour change caused by reduction of the DPPH radical which was determined by measuring absorbance at 517 nm. Radical scavenging activity of grape seed extract was assayed by using thermo Electron Corporation (Nicolet evolution 100) spectrophotometer for quantitative analysis. Plant flavonoid quercertin (2mg/ml) in methanol with 10-fold dilution (e.g. 1mg/ml, 0.5mg/ml etc.) of known antioxidant was used as a positive control to compare the results with sample extract. Sample extract (4mg/ml) in methanol with 10-fold dilution (e.g. 2mg/ml, 1mg/ml etc.) was carried out. Cuvettes (1.5ml) were used for the spectrophotometric analysis which contained 10 µL of sample extract and 1ml of 0.004% DPPH solution in methanol, also control was made up by 1ml of DPPH and 10µL methanol in cuvette. All the cuvettes were covered with parafilm and aluminium foil it was then inverted to mix the solution and were left for 30 minutes of reaction time. Spectrophotometer was then set to measure the absorbance at 517 nm. The spectrometer was blanked with methanol. DPPH control followed by sample absorbance reading was determined at 517 nm. Percentage reduction of DPPH activity was calculated by using given below formula. (Braca, A. et al., 2002).
Percentage reduction of DPPH = [Abs of DPPH - Abs of sample/Abs of DPPH] x 100
Determination of antioxidant activity in grape seed extract is based on reaction between chromogenic compound DPPH and antioxidant (AH).DPPH free radical (DPPH.) when reacts with an antioxidant (AH) which donates H atom to reduce DPPH with change in colour from violet to yellow. Reacted radical is absorbed in visible range and intensity of the colour is measured at 517nm. (Molyneux, P., 2003.)
DPPH. + AH â†’ DPPH-H + A.
2.2.3 Thin layer chromatography
Thin layer chromatographic analysis was performed on thin layer silica plate which was cut into (2.5 x 6.5cm) each. Baseline was drawn and extracts was spotted on it by using small glass capillary tubes. Solvent system was allowed to run on TLC plate by using suitable solvent systems. Plate was taken out when mobile phase ran two-third up the plate and was allowed to dry. TLC was observed under UV light. Same procedure was carried out using different solvent system in which the mobile phase of toluene: ethyl acetate: acetic acid (TEA) (80:18:2), ethyl acetate: ethanol: water (8:2:7), hexane: ethyl acetate: ethanol: water (1:8:2:7) (4:5:3:3) and CH2Cl2:CH3OH: Acetic acid was used. The DPPH solution (80µg/ml) was sprayed on the plate using a sprayer and dried with air.
2.2.4 Gas chromatography-Mass spectroscopy
GC-MS analysis was performed on Thermo Electron Corporation coupled with Trace GC Ultra fitted Triplus auto sample. Dried 3mg sample extract was dissolved in 5ml of HPLC graded methanol in centrifuge tubes and tubes were allowed to centrifuge for 30 minutes at 3000 rpm. Supernatant liquid was collected in small vials and gas chromatography was carried out 2Î¼L of each extract sample was injected into the injection port at 250°C using Phenomonex Zebron ZB-5cm fused capillary column (30mÃ-0.25mm, film thickness 0.25 Î¼m). Helium gas was used as carrier gas at flow rate of 1ml/min. Split ratio was 1:10 and temperature of oven was 40°C to 2600C for 5minutes then it was held for 5 minutes at 260°C. Sample concentration for mass was 10mg/ml. Electron impact used was 70eV as ionization mode at 250°C ion source temperature. Samples were scanned from 50-650 mass scan range with scan time of 1.22 second. Thermo Electron Corporation Xcalibur v 1.6 software was used. The graph, molecular formula and molecular structure were searched and compared in NIST Library version 2 search libraries.
2.2.5 RP-HPLC (Reverse phase-high performance liquid chromatography) analysis
RP-HPLC analysis of standard resveratrol and sample methanol extract was performed on the Perkin-Elmer (PE-HPLC system) series 200 auto samplers with Perkin-Elmer series pump. Reverse phase analytical column SN X F14 (Knauer column 4.6 x 110 dimension, eurospher 100-5C18) was used .Standard resveratrol stock solution (100µg/ml) was prepared by diluting 1ml of standard resveratrol 10mg/ml concentration in 100ml volumetric flask with acetonitrile. The stock solution was diluted further with acetonitrile to obtain concentration of 10, 20, 30, 40 and 50µg/ml. Sample methanol extract stock solution was prepared in 100ml volumetric flask by diluting 10mg of sample extract with 100ml acetonitrile. Stock solution was diluted further with acetonitrile to obtain concentration of 10, 20, 30, 40 and 50µg/ml. Column was calibrated with mobile phase with flow rate of 1ml/min, detection wavelength of 282nm and column temperature of 330C was set. Reverse phase HPLC assay was carried out using isocratic system with mobile phase of acetonitrile and 2% acetic acid (40:60 v/v).The injection volume was 20µL was used, solution was filtered through 0.45 µm nylon membrane prior to HPLC injection. Initially total chromatographic analysis time of 30 minutes per sample was set which was reduced to 8 minutes per sample.
3. Results and discussion
The percentage yields of methanol extract which was found to be 2.26g extracted twice with methanol was found to be 53.15%. The methanol extract appeared dark brown in colour in 50 ml round bottom flask which was dried by N2 gas prior to further analysis.
3.1 Antioxidant assay using UV spectrophotometer
*Spectrometric analysis measured at 517nm (NM= Not measured).Values are the mean of triplicate observations.
Serial Dilution No.
Average Standard Quercetin Absorbance
% reduction of DPPH by Sample extract
% reduction of DPPH by standard quercetin.
Average absorbance of DPPH control was observed to be 1.180 for standard and 1.134 for sample extract.
Fig 5 Reduction of DPPH by a standard Quercetin at different concentrations (mg/ml)
Fig 6 Reduction of DPPH by a grape seed extract at different concentrations (mg/ml)
DPPH reduction in the sample was observed at high concentration which gradually decreased with the decrease in concentration which showed increase in radical scavenging activity with increase in concentration of sample extract.IC50 of standard quercetin was found to be 50µg/ml and of sample extract was found to be 150µg/ml. Scavenging activity of DPPH from grape seed extract was drawn in Microsoft Office Excel 2007.
3.2 Qualitative assay using thin layer chromatography
Thin layer chromatography using various solvent systems was carried out spraying reagent DPPH was used to qualitatively analyse the anti-oxidant activity. Brown colour extract changed into yellow spot due to conversion of 2, 2-diphenyl-picrylhydrazyl (purple colour) gets reduced to diphenylpicryl hydrazine (yellow colour) which gives the evidence of antioxidant activity (Braca et al., 2002).
Fig 7 Yellow spot showing antioxidant activity of methanol extract using Toluene: ethyl acetate: acetic acid (80:18:2) as mobile phase.
(Standard Resveratrol) (Sample extract)
Fig 8 TLC plates of standard and sample methanol extract using acetonitrile: methanol: acetic acid (80:15:3) as mobile phase.
Effective separation was observed on sample TLC plate. Retention factor (Rf) of sample extract was calculated to be 0.53 and that of standard resveratrol was found to be 0.21.
3.3 UV detection
The resveratrol spectrum ranged from 260 to 310 nm. The spectrum of resveratrol detection with optimal wavelength (ÊŽmax) of 282nm was used.
Fig 9 UV detection of resveratrol in sample methanol extract
3.4 RP-HPLC (Reverse Phase high performance liquid chromatography)
Fig 10 standard curves, Resveratrol concentration were 20, 30, 40 and 50µg/ml.
Standard curves were constructed by plotting the peak area against concentration of resveratrol. The standard curve for resveratrol showed linear increase over the range 20-50µg/ml. The standard curve was calculated by linear regression, according to the following formula: Y = aX + b, where Y is the peak area ratio of standard drug, a (the slope) and b (the y-intercept) are constants, and X is the resveratrol concentration (µg/ml).Typical values for the regression parameters a, b and r (correlation coefficient) were calculated to be 314597, 3000000 and 0.9939, respectively (n = 5).Concentration of resveratrol in 50µg/ml sample extract was found to be 9.81µg/ml using standard calibration curve.
3.5 Gas chromatography-Mass spectroscopy
Resveratrol present in minute quantities is a polar compound with very low volatility, which makes it difficult to vaporise the extract and to separate on a gas chromatography (GC) column. Derivatization increases volatility and reduce polarity and therefore can improve extraction efficiency, selectivity and detection. There are three different derivatization procedures that are currently used in solid-phase microextraction (SPME) including direct derivatization, derivatization on the SPME fiber and derivatization in the GC injection port. Bis(trimethylsily)trifluoroacetamide (BSTFA) can be employed as derivatizing reagent for resveratrol by using solid-phase micro-extraction (SPME) with on-fiber silylation derivatization. The use of SPME results in a number of advantages like it simplifying sample preparation, increasing reliability, selectivity, sensitivity and reducing the cost and time of analysis.