To conclude, this study suggests that Capsicum annum species, contains potential antioxidant and antiinflammatory compounds which could be tested as drug candidates against oxidative and inflammation-related pathological processes. However, further studies should becontinued to obtain appropriate information about the role ofgreen capsicum to isolate the active principles, elucidate their structures and determine theirpharmacological activities.
We are extremely grateful to the Executive Director of The Oxford Group of Institutions and Department of Biochemistry, Department of Statistics and Vision Group on Science and Technology (VGST) Govt. of Karnataka for funding.
Phytochemicals are secondary metabolites of plants which are non-nutritive chemicals that have protective or disease preventive properties. These metabolites are said to exhibit structural similarities to that of the intermediary molecules of animal metabolism. Hence these molecules can interact in a similar mode and may be responsible for the desirable pharmacological properties such as antioxidant and anti-inflammatory effects1 . Phytochemicals have been considered to be of crucial nutritional importance in the prevention of chronic diseases2 .The major phytochemicals from fruits, vegetables and medicinal plants which are in extensive study for pharmacological actions include polyphenols (derived from carbohydrates), terpenes (a group of lipids), flavonoidsand alkaloids (derived from amino acids)3 . Antioxidants exert their therapeutic role by scavenging free radicals4, 5 and thereby could also inhibit the process of lipid peroxidation, thus inhibiting the LOX pathway. Hence, finding a single molecule having both antioxidant and anti-inflammatory activities would be of great therapeutic importance. These phytonutrients that are good for health also produce bright colours in vegetables and fruits and act as pigments also. Thus colours of fruits and vegetables can help guide the dietary levels of antioxidants as well as anti-inflammatory agents, which is extremely important for good health balance6 .Red fruits and vegetablespredominantly contain phytonutrients such as lycopene, anthocyanins which arealso responsible for their colour. Orange and Yellow fruits and vegetables contain antioxidants like beta-carotene, zeaxanthin, vitamin C etc. Green vegetables contain the pigment chlorophyll and other phytonutrients like lutein, zeaxanthin, calcium, folate, calcium, which are potent antioxidants as well as anti-inflammatory compounds. Capsicum annuum (Family Solanaceae) is a native to Americas but now cultivated worldwide. As in other chili varieties, capsicums too have several cultivar types. These bell peppers range from green, red, yellow and even purple. Green peppers ripen to yellow and red if left on the vine. Most of the differences in colour stem from time of harvest and degree of ripening. In addition to use as spices and food vegetables, capsicum has also found use in medicines. Studies have been reported on the capsicum sps related to antioxidant properties (phenolic compounds), 7, 8,and 9 but not clear with respect to different coloured fresh fruits of Capsicum. As Capsicum is easily cultivated, economically feasible in the Indian market and a prominent ingredient of the Indian cuisine, particularly in the growing fast-food industries, the fresh fruit in different colors have been chosen for this study. Green, yellow and red varieties of capsicum are taken on dry weight basis in the present study to compare their antioxidant as well as anti inflammatory activity in vitro. Our results suggest that green capsicum has the most significant antioxidant and anti- inflammatory activity when compared to yellow and red varieties.
2.1 Extraction of vegetables The three different coloured fresh Capsicum annum species (red, green and yellow) were purchased from a local market. 20 gm of each variety were weighed and aqueous extraction was carried out by homogenization, centrifugation followed by lyophilization. The yield was calculated and expressed as % of w/w. All the experiments were performed in triplicates.Total phenolics, reducing power, DPPH assay and LOX enzyme inhibition were estimated fromaqueous extracts of red, yellow and green varieties of Capsicum at 100?g concentration. 2.2Invitroantioxidant activity of the extracts 2.2.1 Total phenolic estimation The concentrations of total phenolics in the aqueous extracts of all the varieties were determined spectrophotometrically at 725 nm by the FolinCiocalteauassay as per the method of Harbertson and Spayd10 . 2.2.2 Reducing power assay The reducing powers of all the extracts were determined according to the method of Oyaizu11 . 2.2.3 DPPH scavenging activity Determination of antioxidant activity by the DPPH method12 was done for all the aqueous extracts at 100 ?g concentrations. The absorbance or radical scavenging activity was expressed using formula: % of radical scavenging activity = [(Control OD-sample OD)/ (Control OD)] X 100 2.3 Anti-inflammatory effect 2.3.1 Extraction and purification of LOX enzyme from soya bean Soy LOX-1 was purified from defatted soya flour according to the method of Axelrod13 . 2.3.2 Precipitation To the above clarified solution, ammonium sulfate precipitation and dialyzed. 2.3.3 Protein estimation The concentration of the protein in the dialyzed sample was calculated using the formula as per the method of Layne, E14and UV spectrophotometrically. Protein concentration (mg/ml) = (1.55 x A280 - 0.76 x A260) 2.3.4 Lipoxygenase assay The bioassay was performed according to the procedure of Axelrod13 . 2.3.5 Inhibition of lipoxygenase activity The inhibitory assay contains 1mg of the sample extract along with other ingredients as mentioned in the procedure according to Axelrod13. The % inhibitions for different samples were determined15using the formula, % inhibition = Control O.D-Test O.D/Control O.D x 100. 2.4 Statistical Analysis Results were calculated as the mean ? SD (standarddeviation) for each sample. Statistical analysis was done with oneway analysis of variance using SPSS 17. Asignificant difference was judged to exist at a level of p< 0.01
3.1 Percentage yield The percentage yield (w/w) of aqueous extract of the three Capsicum annuum species, red, yellow and green calculated as per the method of Rivillas16were 2.55,4.2 and 1.8 respectively. 3.2 Antioxidant activity 3.2.1 Determination of total phenolics by FolinCiocalteau assay The total phenolic content in the aqueous extracts of red,yellow and green capsicum were found to be 1.7913 ?gGAEm/?l? 0.0000, 2.5159?g GAEm/?l ? 0.0502 and 3.2985?g GAEm/?l? 0.1004 (Gallic Acid Equivalent mean) respectively. Higher content of phenolics in green Capsicum indicates higher antioxidant property17.This finding also supports Deepa.N.et.al7 report, which suggests that all the antioxidant constituents like phenolics, ascorbic acid and carotenoids and AOX, when expressed on the dry weight basis, declined in majority of the genotypes during maturity to red stage. 3.2.2 Determination of reducing power In the reducing power assay, the presence of antioxidants in the sample would result in the reduction of Fe3+ to Fe2+ by donating an electron. The amount of Fe2+ complex formed can then be monitored by measuring the intensity of Perl?s blue developed at 700 nm. Increase in absorbance of the reaction mixture would reflect a greater reducing power of the sample. Antioxidant effect often correlates with reductive activity18 .Figure-I shows significant reducing power of the greencapsicum compared to the other varieties which could probably be attributed to the high concentration of antioxidant compounds in the green extract. 3.2.3 Determination of antioxidant activity by DPPH method DPPH, a free radical was used for assessing antioxidant activity and the reduction of thisradical by an antioxidant results in a loss of absorption at 517 nm. Thus the degree of discoloration of the solution containing the aqueous extract indicates the scavenging efficiency of the added extracts. The radical scavenging activities of the samples were expressed in terms of % inhibition of DPPH. Correlation between free radical scavenging activity and the phenolic content have also been reported for various fruits and vegetables19, 20.Highest free radical scavenging activity was again observed (Fig-II) in the aqueous extract of green capsicum (92 %) followed by yellow capsicum (76 %) and red capsicum (67%). 3.3 Inhibition of lipoxygenase Lipoxygenases could be inhibited by antioxidants via chelation of its non-heme bound iron21 or by reduction of its ferric form22. Antioxidants are beneficial for chronic inflammatory conditions, antihyperglycemia23 and thus can act as anti-inflammatory molecules by scavenging the free radicals24.Our results showed higher % of LOX inhibition bygreen capsicum (46.12 %) followed by yellow (44.09 %) and red (32.18 %),featuring anti-inflammatory property in them.
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