STUDY OF PHYTOCHEMICAL ANALYSIS AND ANTIOXIDANT ACTIVITY OF SPINACH OLERACEA L PLANT LEAVESHTML Full Text
STUDY OF PHYTOCHEMICAL ANALYSIS AND ANTIOXIDANT ACTIVITY OF SPINACH OLERACEA L PLANT LEAVES
Sowparthani Kaliyaperumal * and M. Radhika
Department of Biotechnology, Valliammal College for Women, Chennai - 600102, Tamil Nadu, India.
ABSTRACT: Spinach is a leafy green flowering plant. The leaves are a common edible vegetable consumed by fresh leaves. The bioactive components present in the Spinach oleracea plant leaves are known to be responsible for its medicinal properties. The present study was undertaken to compare the effect of different extraction solvents to extract the active components like carbohydrates, tannin, flavonoid, phenol, carbohydrate from the dried leaves of Spinach oleracea plant. The extraction solvents used were Ethyl acetate, Petroleum ether and Methanol. Phytochemical analysis, total flavonoid concentration, total phenol concentration and antioxidant activity have been identified to compare the efficiency of different extraction solvents. The results shows that using methanol as an extraction solvent works best for the extraction of various active phytochemicals, the total flavonoid and phenols concentration and the antoxidant activity showed the effective scavenging activity. This is the report that directly compares the three extraction solvents for the extraction of active components from the Spinach oleracea leaves and shows that method should be the solvent of choice.
Spinach oleracea, Methanol, Ethyl acetate, Petroleum ether, Flavonoid
INTRODUCTION: Spinach is a green leaves vegetable and is grown in most parts of the world. Spinach leaves are best source of dietary magnesium. They help in energy metabolism, maintaining muscle, nerve function, maintaining heart rhythm, a healthy immune system and blood pressure 1, 2. Several studies on the chemical composition of leafy vegetables have shown that, they contain enormous amount of micronutrients, several agronomic advantages and economic value. They also contain some chemical compounds that are having important medicinal uses, human well-being and healthy lifestyle. Some of the vegetables are also reported to cure more than one health problem.
The medicinal values of vegetables and fruits are believed to be dictated by their phytochemical and other chemical constituents 3. The nutritional value of Spinach indicates it to be a very nutrient dense food. It is low in calories yet very high in vitamins, minerals and other phytonutrients. Spinach is also packed with a number of antioxidants like poly-phenols, flavonoids and carotenoids, which are shown to possess anti inflammatory effects, anti mutagenic potential, anti neoplastic effects as well as chemo preventive activities 4, 5.
Antioxidant compounds in food play an important role as a health protecting factor. Plant sourced food antioxidants like vitamin C, vitamin E, carotenes, phenolic acids, phytate and phyto-estrogens have been recognized as having the potential to reduce disease risk. Most of the anti-oxidant compounds in a typical diet are derived from plant sources and belong to various classes of compounds with a wide variety of physical and chemical properties. Some compounds such as gallates which have strong antioxidant activity, while others such as the monophenols are weak antioxidants. The main characterstic of an antioxidant is its ability to trap free radicals 6.
MATERIALS AND METHODS:
Collection of Plant Sample: The Spinach oleracea leaves were collected from Tamil Nadu. The plant was identified and authenticated. Fresh plant material was washed with water, air dried and then blended fine powder Fig. 1 and 2.
FIG. 1: SPINACH OLERACEA
FIG. 2: SPINACH OLERACEA (LEAVES POWDER)
Extraction Preparation: The leaves Spinach oleracea was dried in shade for 12days. The dried samples were grind into a coarse powder using a motor and pestle. The extraction with different solvents like Ethyl acetate, Petrolium ether and Methanol were done using Soxhlet apparatus. For every 100 ml for each solvent, 25 g of crushed leaves powder was used for soxhlet extraction. After extraction for 3 consecutive days the crude liquids were placed in water bath at 55 ºC for excess solvent evaporation
Phytochemical Analysis: Phytochemical screening for major constituents was undertaken using standard qualitative methods. Screening test were performed for carbohydrates, tannins, saponins, flavonoid, alkaloids, quinines, glycosides, cardiac glycosides, terpenoids, phenols, coumarins, phloba-tannins, anthraquinines, phytosteroids in the mentioned Spinach oleracea leaves extract by following procedure mentioned in this study as 7.
Test for Carbohydrates: 2 ml of plant extract was added with 1 ml of Molisch’s reagent and few drops of concentrated sulphuric acid were added. Presence of purple or reddish colour indicates the presence of carbohydrates.
Test for Tannins: 1 ml of plant extract, 2 ml of 0.7 M NaOH and few drops of Folin-Denis reagent was added into to the test tube. Formation of dark blue or greenish black indicates the presence of tannins.
Test for Saponins: 2 ml of plant extract, 2 ml of distilled water was added and shaken in a graduated cylinder for 15 min lengthwise. Formation of 1 cm layer of foam indicates the presence of saponins.
Test for Flavonoids: 2 ml of plant extract, 1 ml of 2N sodium hydroxide was added into to the test tube. Presence of yellow colour indicates the presence of flavonoids.
Test for Alkaloids: 2 ml of plant extract, 2 ml of concentrated hydrochloric acid was added into to the test tube. Then few drops of Mayer’s reagent were added. Presence of green colour or white precipitate indicates the presence of alkaloids.
Test for Quinines: 1 ml of extract and 1 ml of concentrated sulphuric acid was added. Formation of red colour indicates presence of quinines.
Test for Glycosides: 2 ml of plant extract, 3 ml of choloroform and 10% ammonia solution was added. Formation of pink colour indicates presence of glycosides.
Test for Cardiac Glycosides: 0.5 ml of extract, 2 ml of glacial acetic acid and few drops of 5% ferric chloride were added. This was under layered with 1 ml of concentrated sulphuric acid. Formation of brown ring at the interface indicates presence of cardiac glycosides.
Test for Terpenoids: 0.5 ml of extract, 2 ml of chloroform was added and concentrated sulphuric acid was added carefully. Formation of red brown colour at the interface indicates presence of terpenoids.
Test for Phenols: 1 ml of the extract, a few drops of Phenol-Denis reagent was added followed by 2 ml of 15% Sodium carbonate solution. Formation of blue or green color indicates presence of phenols.
Test for Coumarins: 1 ml of extract, 1 ml of 10% NaOH was added. Formation of yellow colour indicates presence of coumarins.
Test for Steroids and Phytosteroids: 1 ml of plant extract equal volume of chloroform is added and subjected with few drops of concentrated sulphuric acid appearance of brown ring indicates the presence of steroids and appearance of bluish brown ring indicates the presence of phytosteroids.
Test for Phlobatannins: 1 ml of plant extract few drops of 2% HCL was added appearance of red colour precipitate indicates the presence of phlobatannins.
Test for Anthraquinones: 1 ml of plant extract and few drops of 10% ammonia solution was added, appearance pink colour precipitate indicates the presence of anthraquinones.
Quantification of total Flavonoids Content: Total Flavanoid content in the extracts was determined using the method described by 8. The flavonoid content was determined by aluminium chloride method using Quercetin as standard. Extracts and Quercetin were prepared in (10 mg/ mL). 0.1 mL of extract was mixed with 0.9 mL of distilled water in test tubes, followed by addition of 75 µL of 5% sodium nitrite solution. After 6 min, 150 µL of 10% aluminium chloride solution was added and the mixture was allowed to stand for further 5 min after which 0.5 mL of 1 M Sodium hydroxide was added to the reaction mixture. Then add 2.5 ml of distilled water and mixed well. The absorbance was measured immediately at 510 nm using a spectro-photometer. A calibration curve was generate during various concentrations of Quercetin 20-100 µg. Blank consist of all the reagents, exceptfor the extract or Quercitin is substituted with 0.1 ml of Results were expressed as the Quercetin equi-valence (QE) of the sample was expressed in mg/g of the extract.
Total Phenolic Content: The amount of phenolic compounds in the extracts was determined by the Folin-Ciocalteu colorimetric method and calculated from a calibration curve obtained with gallic acid as standard (1 mg / 1 ml) 9. From the standard solution 20 to 100 µl was taken and added to different test tubes. Extract was added in a separate test tube at a concentration of 10 mg/ml. To 0.1 ml of each extract, 5 ml of folins – ciocalteu (1:10 dilution) was added and the contents were mixed thoroughly and incubated in dark for three minutes. 5 ml of sodium carbonate (11.25 / 150 ml) was added and the mixture was incubated in dark for 60 min. The absorbance was measured at 760 nm in a UV-Visible Spectrophotometer The results were expressed in Gallic acid equivalence of the samples (GE) µg/mg of the extract.
Anti-oxidant Activity: Free radical scavenging activity was determined by DPPH radical scavenging assay as described by 10. 4mg/100ml of DPPH in methanol was prepared and 1.5 ml of this solution was mixed with 1.5 ml of extractives in methanol at different concentrations (20-100). The reaction mixture was vortexed thoroughly and left in the dark for 20 min. The absorbance of the mixture was measured spectrophotometrically at 517 nm. Ascorbic acid was used as reference standard. Percentage DPPH radical scavenging activity was calculated by the following equation
[(Acontrol – Aextract) / (Acontrol)] × 100
RESULTS AND DISCUSSION:
Phytochemical Analysis: Qualitative analysis performed with Spinach oleracea leaves in three solvents namely Petroleum ether, Ethyl acetate, and Methanol based on the polarity showed positive results for Carbohydrates, Tannins, Saponins, Flavonoids, Alkaloids, Glycosides ,Cardiac glycol-sides, Terpenoids and Phenols. The present study was conducted with an objective to identify the best extraction solvent, which can be used to extract the maximum amount of the phyto-chemicals from the dried Spinach oleraceae plant leaves. The three extracts of Spinach olreaceae leaves commonly showed the presence of phyto-chemical such as Carbohydrate, Tannin, Flavonoid Terpenoid and Phenol Table 1. Spinach oleraceae is a well known medicinal plant known to have health benefits against many diseases.
Qualitative biochemical estimation were conducted to detect the presence of different phytochemicals in the dried Spinach oleraceae plant leaves extract obtained by using different solvents Petroleum ether, ethyl acetate and methanol. Our results highlights that all the extracts formed by using different solvents from Spinach oleraceae plant leaves contain phytochemicals like carbohydrate, tannin, flavonoid, tereponoid and phenol. Previous reports also shown that the preliminary phyto-chemical analysis of Spinach oleraceae revealed that different active constituent present in different extracts such as carbohydrates, proteins, amino-acids, fats, oils, steroids, terepenoids. Glycosides, alkaloids, tannins and other phenolic compound 11.
TABLE 1: PHYTOCHEMICAL ANALYSIS
|Petrolem ether||Ethyl acetate||Methanol|
(+) =Indicates presence (-) =indicates absence
Quantification of Total Flavonoids and Phenol Content: Total flavonoid concentration was quantified by using Quercetin as the standard and measured OD at 510 nm. Using Methanol as an extraction results in the maximum flavonoid extraction with 11.41 mg/g of quercetin equivalent of extract followed by petroleum ether and ethylacetate with 4.21 mg/g, and 3.37 mg/g of quercetin equivalent of extract Table 2. Total phenol content in the plant was estimated using gallic acid as a standard. The OD was taken at 760 nm. Maximum phenol concentration was observed when ethylacetae 4.33 mg/g and Methanol 2.06 mg/g. The ethyl acetate extracts were registered the high amount of phenol content Table 3. A Quantitative study was further conducted to detect the amount of the total flavonoids and total phenols in these plant extracts for direct comparison and have demonstrated that methanolic extract contains the maximum amount of flavonoids and phenols. The quantification of phytochemicals of S. oleraceae showed the presence of the highest amount of phenols and flavonoids justified the potent antioxidant nature of the plant. The study also revealed that flavonoids represent the main group of phenolic compounds in S. oleraceae 12.
TABLE 2: TOTAL FLAVONOID CONTENT
|Extracts||Sample OD at 510 nm (µg)||Total flavonoid content (mg/g) equivalent of Quercetin Content|
TABLE 3: TOTAL PHENOLIC CONTENT
|Extracts||Sample OD at 700 nm (µg)||Total Tannin content mg/g) Equivalent of Quecetin Content|
Antioxidant Activity: The ability of Spinach oleracea extracts to scavenging the DPPH free radical assay by petroleum ether, ethyl acetate and methanol extract the samples are added to the mixture and incubated for 30 min in dark. On completion of 30 min of incubation, colour change is observed in the mixture. The colour changed from violet to pale yellow. The readings are recorded at 517 nm. Colour change represents the scavenging activity of the mixture.
The antioxidant activities of particles were compared with the standard in graphical representation. The mean IC50 values of the leaves and Methanol extract were lower which showed the radical scavenging activity was shown to be effective in this solvent extracts 19.74 mg/g then the ethyl acetate extract 43.42 mg/ml and Petroleum ether extract 49.21 mg/ml. The IC50 value and scavenging activity of Petroleum ether, Ethyl acetatae, methanol extract of Spinach oleracea were 24.31 µg/ml, 23.43 µg/ml, 10.63 µg/ml and scavengimg effect of 55.07% inhibition effect Table 4.
Spinach ranks high among vegetables regarding antioxidant capacity because of an abundance of phenolic compounds 13, 14, suggesting that spinach consumption may afford protection against oxidative stress mitigated by free-radical species. Spinach phenolic compounds exhibit a wide range of biological effects including antioxidative 15.
Flavonoids are the most abundant polyphenols in our diet. The free radicals produced in the body are neutralized by flavonoids which are well known for their antioxidant properties. The results from these studies which highlights that methanolic Spinach oleraceae plant leaves extract possess the maximum concentration of flavonoids, phenols and s also possess the maximum antioxidant activities.
TABLE 4: ANTIOXIDANT ACTIVITY OF SPINACH OLERACEA
|Extract||Concentration (µg/mL)||Control||Test Sample||% of Inhibition|
CONCLUSION: The phytochemical analysis of Spinach oleraceae revealed the presence of phytochemicals such as Carbohydrate, Flavonoid, tannins, terponoids and phenol in three different extracts. Furthermore, the quantification of phyto-chemicals showed the leaves of Spinach oleraceae Flavonoid and Phenolic compound. Hence, the presence of these beneficial secondary metabolites imparts antioxidant potential to Spinach oleraceae. By this present phytochemical scrrening and quantification, we suggest the Spinach oleraceae is a good nutrient rich leafy vegetable with an antioxidant value that can be used as a therapeutic medicine.
CONFLICTS OF INTEREST: Nil
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How to cite this article:
Kaliyaperumal S and Radhika M: Study of phytochemical analysis and antioxidant activity of Spinach oleracea L plant leaves. Int J Pharmacognosy 2020; 7(7): 198-03. doi link: http://dx.doi.org/10.13040/IJPSR.0975-8232.IJP.7(7).198-03.
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