Extraction and phytochemical analysis Verma et al Asian Journal of Dental and Health Sciences. 2023; 3(1):11-14 [11] AJDHS.COM Available online at ajdhs.com Asian Journal of Dental and Health Sciences Open Access to Dental and Medical Research Copyright © 2023 The Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited Evaluation of Antioxidant Activity of Trigonella foenum graecum Seeds Extract Neha Verma*, Vivekanand Katare, Nisha Kalme, Abhilasha Delouri, Shivam Nema Vivekanand College of Pharmacy, Shikandrabad, Bhopal, MP, 462044, India Article Info: _______________________________________ Article History: Received 09 Dec 2022 Reviewed 11 Jan 2023 Accepted 26 Jan 2023 Published 15 March 2023 _______________________________________ Cite this article as: Verma N, Katare V, Kalme N, Delouri A, Nema S, Evaluation of Antioxidant Activity of Trigonella foenum graecum Seeds Extract, Asian Journal of Dental and Health Sciences. 2023; 3(1):11-14 DOI: http://dx.doi.org/10.22270/ajdhs.v3i1.33 _______________________________________ *Address for Correspondence: Neha Verma, Vivekanand College of Pharmacy, Shikandrabad, Bhopal, MP, 462044, India Abstract ___________________________________________________________________________________________________________________ In the current work, we completed a comprehensive assessment of the relative antioxidant activity in extracts from a few different medicinal plant species.The extracts' effectiveness at scavenging 1, 1- diphenyl-2-picryl hydrazyl (DPPH) radicals was assessed spectrophotometrically. The highest degree of radical scavenging was 52.54%, while the IC50 value was 93.54.Trigonella foenum graecum extract demonstrates how the greater concentration of phytoconstituents chemicals leads to more powerful radical scavenging results. Keywords: Trigonella foenum graecum, Antioxidant, DPPH, Phytochemical analysis Mail Id: nv132611@gmail.com INTRODUCTION Free radicals donate to more than one hundred disorders in humans counting atherosclerosis, arthritis, ischemia and reperfusion damage of numerous tissues, central nervous system injury, gastritis, cancer and AIDS. Free radicals due to ecological pollutants, radiation, chemicals, toxins, profound fried and spicy foods as well as corporeal stress, cause exhaustion of immune system antioxidants, modify in gene expression and persuade abnormal proteins. Oxidation development is one of the most imperative routs for producing free radicals in food, drugs and still living systems. Catalase and hydroperoxides enzymes change hydrogen peroxide and hydroperoxides to nonradical forms and purpose as natural antioxidants in human body. Owing to depletion of immune system natural antioxidants in dissimilar maladies, overwhelming antioxidants as free radical scavengers may be essential. At present available synthetic antioxidants similar to butylated hydroxy anisole (BHA), butylated hydroxy toluene (BHT), tertiary butylated hydroquinone and gallic acid esters, have been supposed to cause or punctual negative health effects. Consequently, strong restrictions have been placed on their application and there is a trend to substitute them with naturally occurring antioxidants. Furthermore, these synthetic antioxidants also show low solubility and reasonable antioxidant activity1,2. Recently there has been an increase of interest in the therapeutic potentials of medicinal plants as antioxidants in dropping such free radical induced tissue injury. Polyphenolic compounds with known properties which include free radical scavenging, inhibition of hydrolytic and oxidative enzymes and anti-inflammatory action3. A number of confirmations suggest that the biological actions of these compounds are related to their antioxidant activity4. An easy, rapid and sensitive method for the antioxidant screening of plant extracts is free radical scavenging assay using 1,1- diphenyl-2-picryl hydrazyl (DPPH) stable radical spectrophotometrically. In the occurrence of an antioxidant, DPPH radical obtains one more electron and the absorbance decreases5. In particular, despite extensive use of wild plants as medicines in Iran, the prose contains few reports of antioxidant activity and chemical composition of these plants. In current study, we carried out a systematic record of the relative free radical scavenging activity in selected medicinal plant species, which are being used traditionally: The seeds Trigonella foenum graecum. In the longer term, plant species (or their active constituents) recognized as having high levels of antioxidant activity in vitro may be of value in the design of additional studies to unravel novel treatment strategy for disorders connected with free radicals induced tissue damage. Besides well-known and traditionally used natural antioxidants from tea, wine, fruits, vegetables and spices, some natural antioxidant (e.g. rosemary and sage) are already exploited commercially either as antioxidant additives or a nutritional supplements6. Also many other plant species have been investigated in the search for novel antioxidants7,8 but generally there is still a demand to find more information concerning the antioxidant potential of plant species. It has been mentioned the antioxidant activity of plants might be due to their phenolic compounds9. Open Access Research Article http://jddtonline.info/ http://dx.doi.org/10.22270/ajdhs.v3i1.33 Verma et al Asian Journal of Dental and Health Sciences. 2023; 3(1):1-4 [12] AJDHS.COM MATERIALS AND METHODS Selection, collection and processing of plant Seeds of fenugreek were purchased from local retail market. The seeds were cleaned before drying it in oven at 50 °C for 24 h. Then, the dried seeds were ground using a mill with ultra-centrifugal equipped with ring sieve owning trapezoid holes sized 0.5 mm. The moisture content of the seed was (5.51 ± 0.14% d.w basis). The powdered seeds were kept in dark airtight container before extraction. Reagents and chemicals 2,2-diphenyl-1-picrylhydrazyl (DPPH), methanol , ascorbic acid, potassium phosphate buffer-ph6.6 (dihydrogen phosphate , sodium hydroxide ), potassium ferricyanide , trichloroacetic acid , ferric chloride . Extraction All the seeds are initially blended into coarse then into powder form. The powdered plant seeds were packed inside the Soxhlet extractor and were successively extracted with petroleum ether, and methanol for 8-10 hours and 40-60oC temperature of the heating mantle were adjusted. The extracts so collected were distilled on a water bath at atmospheric pressure and the last traces of solvent were removed using vacuum. Extracts were collected in air tight container10. Qualitative phytochemical analysis of plant extract Following standard methods by Rani et al, 202111 the Trigonella foenum graecum extract obtained was subjected to the preliminary phytochemical analysis. The extract was screened to spot the presence or absence of many active constituents like carbohydrates, glycosides, phenolic compounds, alkaloids, flavonoids, saponins, fats or fixed oils, protein, amino acid and tannins. Activity (In-vitro anti-oxidant activity) DPPH radical scavenging activity a) Preparation of DPPH reagent 0.1mM solution of 2,2-Diphenyl-1-picrylhydrazyl (DPPH) in methanol was prepared. b) Preparation of Sample/Standard Freshly 1 mg/ml methanol solution of extracts of Trigonella foenum graecum standard was prepared. 1 mg of extracts/standard was taken with methanol to make 1mg/ml stock solution. Different volume of extracts/standard (20 – 100μl) was taken from stock solution in a set of test tubes and methanol was added to make the volume to 1 ml. To this, 2 ml of 0.1mM DPPH reagent was added and mixed thoroughly and absorbance was recorded at 517 nm after 30 minutes incubation in dark at room temperature. C) Preparation of control For control, Take 3 ml of 0.1mM DPPH solution and incubated for 30 min at room temperature in dark condition. Absorbance of the control was taken against methanol (as blank) at 517 nm12 .Percentage antioxidant activity of sample/standard was calculated by using formula: % Inhibition = [(Ab of control- Ab of sample/ Ab of control x 100] Reducing power assay Preparation of standard solution 3 mg of ascorbic acid was dissolved in 3 ml of distilled water/solvent. Dilutions of this solution with distilled water were prepared to give the concentrations of 20, 40, 60, 80 and 100μg/ml. Preparation of extracts of Stock solutions of extracts of Trigonella foenum graecum was prepared by dissolving 10 mg of dried extracts in 10 ml of methanol to give a concentration of 1mg/ml. Then sample concentrations of 20, 40, 60, 80 and 100 μg/ml were prepared. Protocol for reducing power According to this method, the aliquots of various concentrations of the standard and extracts of Trigonella foenum graecum (20 to 100μg/ml) in 1.0 ml of deionized water was mixed with 2.5 ml of (pH 6.6) phosphate buffer and 2.5 ml of (1%) potassium ferricyanide. The mixture was incubated at 50°C in water bath for 20 min after cooling. Aliquots of 2.5 ml of (10%) trichloroacetic acid were added to the mixture, which was then centrifuged at 3000 rpm for 10 min. The upper layer of solution 2.5 ml was mixed with 2.5 ml distilled water and a freshly prepared 0.5 ml of (0.1%) ferric chloride solution. The absorbance was measured at 700 nm in UV spectrometer (Systronic double beam-UV-2201). A blank was prepared without adding extract. Ascorbic acid at various concentrations (20 to 100μg/ml) was used as standard13. RESULTS Table 1: Percentage yield of Trigonella foenum graecum extract S. No Solvent Color of extract Weight of Plant Material (gms) Weight of extract (gms) % Yield 1 Petroleum ether Dark Green 180 4.32 2.4 2 Methanol Green 164.71 10.68 6.484 Verma et al Asian Journal of Dental and Health Sciences. 2023; 3(1):1-4 [13] AJDHS.COM Table 2: Qualitative phytochemical analysis Phytoconstituents Extracts Pet. ether Methanol Alkaloids Mayer +++ + Dragendoff +++ + Flavonoids Lead acetate - - Ferric chloride +++ +++ Carbohydrates Molish +++ +++ Bendict - + Glycosides Killer-lini ++ ++++ Steroids Salkowskis - +++ Libermanburchard +++ +++ Protein and amino acids Ninhydrin ++++ +++ Biuret - - Phenolic compounds and tannins Ferric chloride - - Gelatin +++ + Lead acetate ++++ + Terpenoids Trim hill - - Libermann-burchard +++ +++ Saponins Foam test +++ - Mercuric chloride +++ ++ Note :-+++ Very large quantity, +++ large quantity, +small quantity, - absent Table 3: DPPH activity of Ascorbic acid Ascorbic acid Concentration (µg/ml) Absorbance (nm) % Inhibition 20 0.447 46.97 40 0.353 58.12 60 0.235 72.12 80 0.137 83.74 100 0.069 91.81 Control 0.843 IC50 24.34 Figure 1: Graph represents the percentage inhibition vs concentration of ascorbic acid Table 4: DPPH activity of Trigonella foenum graecum extract Trigonella foenum graecum Concentration (µg/ml) Absorbance (nm) % Inhibition 20 0.49 16.9492 40 0.43 27.1186 60 0.38 35.5932 80 0.33 44.0678 100 0.28 52.5424 Control 0.59 IC50 93.4568 Figure 2: Graph represents the percentage inhibition Vs concentration of extract y = 0.5765x + 35.967 R² = 0.9929 0 10 20 30 40 50 60 70 80 90 100 0 20 40 60 80 100 120 y = 0.4407x + 8.8136 R² = 0.9985 0 10 20 30 40 50 60 0 20 40 60 80 100 120 Verma et al Asian Journal of Dental and Health Sciences. 2023; 3(1):1-4 [14] AJDHS.COM Table 5: Reducing power activity of Ascorbic acid Concentration (µg/ml) Absorbance (nm) 20 0.14 40 0.244 60 0.367 80 0.43 100 0.505 Figure 3: Graph represents the percentage inhibition Vs concentration of ascorbic acid Table 6: Reducing power activity of Trigonella foenum graecum extract Concentration (µg/ml) Absorbance (nm) 20 0.02 40 0.03 60 0.05 80 0.06 100 0.08 Figure 4: Graph represents the percentage inhibition Vs concentration of extract CONCLUSION The current study concluded that a Trigonella foenum graecum seed which was purchased from local market of Bhopal, Madhya Pradesh is rich source of phytochemicals. Results showed the presence of alkaloids, amino acids, carbohydrates, flavonoids, phloba tannins and tannins in the seeds of Trigonella foenum graecum. Results of DPPH assay showed that Trigonella foenum graecum (100µg/ml) has about 52.54% scavenging activity. Therefore, it is concluded from this preliminary study that Trigonella foenum graecum can be used for isolation of important compounds with medicinal and pharmacological importance. REFERENCES 1. Barlow SM. Toxicological aspects of antioxidants used as food additives. In Food Antioxidants, Hudson BJF (ed.) Elsevier, London, 1990; pp 253-307. https://doi.org/10.1007/978-94-009- 0753-9_7 2. Branen AL. Toxicology and biochemistry of butylatedhydroxyanisol and butylatedhydroxytoluene. J. American Oil Chemists Society, 1975; 5:59- 63. https://doi.org/10.1007/BF02901825 3. Frankel E. Nutritional benefits of flavonoids. International conference on food factors: Chemistry and Cancer Prevention, Hamamatsu, Japan. Abstracts, 1995; C6- 2. 4. Gryglewski RJ, Korbut R, Robak J. On the mechanism of antithrombotic action of flavonoids. Biochemical Pharmacol, 1987; 36:317- 321. https://doi.org/10.1016/0006- 2952(87)90288-7 5. Koleva II, Van Beek TA, Linssen JPH, De Groot A, Evstatieva LN. Screening of plant extracts for antioxidant activity: a comparative study on three testing methods. Phytochemical Analysis, 2002; 13:8-17. https://doi.org/10.1002/pca.611 6. Schuler P. Natural antioxidants exploited commercially, In Food Antioxidants, Hudson BJF (ed.). Elsevier, London, 1990; pp 99- 170. https://doi.org/10.1007/978-94-009-0753-9_4 7. Mantle D, Eddeb F, Pickering AT. Comparison of relative antioxidant activities of British medicinal plant species in vitro, J. Ethnopharmacol. 2000; 72:47- 51. https://doi.org/10.1016/S0378-8741(00)00199-9 8. Merghem M, Dahamna S, In-Vitro Antioxidant Activity and Total Phenolic Content of Ruta montana L. Extracts, Journal of Drug Delivery and Therapeutics 2010; 10(2):69-75 https://doi.org/10.22270/jddt.v10i2.3919 9. Cook NC, Samman S. Flavonoids- chemistry, metabolism, cardioprotective effects, and dietary sources. Nutritional Biochemistry, 1996; 7:66-76. https://doi.org/10.1016/0955- 2863(95)00168-9 10. Dutta R, Sharma MK, Khan A, Jha M. Phytochemical and in vitro antioxidant assay of Fumaria officinalis leaf extract. Journal of Advanced Scientific Research. 2020; 11(03):176-82. 11. Rani D, Kharkwal H, Jha M, Rai N. Assessment of the total flavonoid, phenol, alkaloid content and sun protection factor in Grewia abutilifolia leaf extract. Journal of Pharmaceutical Research International.2021; 33(49A):42-51. https://doi.org/10.9734/jpri/2021/v33i49A33300 12. Jha M, Ganesh N, Sharma V In vitro evaluation of free radical scavenging activity of Pistia stratiotes. International Journal of Chemtech Research 2010; 2(1):180-184 13. Jha M, Sharma V, Nema N, Hussain T. Effect of the topical application of the extract of Glycosmis pentaphylla on excisional wound model in mice. Pharmacologyonline. 2009; 3:356-60. 0 0.1 0.2 0.3 0.4 0.5 0.6 0 20 40 60 80 100 120 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0 20 40 60 80 100 120 https://doi.org/10.1007/978-94-009-0753-9_7 https://doi.org/10.1007/978-94-009-0753-9_7 https://doi.org/10.1007/BF02901825 https://doi.org/10.1016/0006-2952(87)90288-7 https://doi.org/10.1016/0006-2952(87)90288-7 https://doi.org/10.1002/pca.611 https://doi.org/10.1007/978-94-009-0753-9_4 https://doi.org/10.1016/S0378-8741(00)00199-9 https://doi.org/10.22270/jddt.v10i2.3919 https://doi.org/10.1016/0955-2863(95)00168-9 https://doi.org/10.1016/0955-2863(95)00168-9 https://doi.org/10.9734/jpri/2021/v33i49A33300