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Verma et al                                                                                                                                   Asian Journal of Dental and Health Sciences. 2023; 3(1):11-14 

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Copyright  © 2023 The  Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 
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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


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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 

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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 

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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. 

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