Comparative Evaluation of Alpha-Amylase and Alpha- Glucosidase Inhibitory Potential of Aqueous Seed Extract of Trigonella Foenum-Graecum and Moringa Oleifera-An In vitro Study

Background: Trigonella foenum-graecum is one of the commonly used herbs in food. Moringa oleifera is a source of food, accommodation and conventional medicine for many peoples in the developing countriesThe seeds of both the plants were explored for antidiabetic potential. Methods: The current work was designed to probe the in vitro anti diabetic potential of the aqueous seed elicit of trigonella foenumgraceus and Moringa oleifera using the enzymes alpha amylase and alpha glucosidase.Both the extracts were screened for their phytochemicals and antioxidant potential was also analysed.The data were examine statistically by a one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test was used to see the statistical significance in conjunction with groups. The results with the p<0.05 level were considered to be statistically significant. Result: Due to its chemical ingredient and active compounds like amino acids, alkaloids, flavonoids, it proceeds as a good antioxidant. Trigonella foenum-graecum seed extract exhibited comparatively higher antidiabetic potential with an IC50 of 300μg/ml than the Moringa oleifera extract. Results of the work designate that both extracts of the plant possessed by forbidding alpha amylase, alpha glucosidase show maximum inhibition. Hence concluded that, Trigonella foenum Original Research Article Prithiksha et al.; JPRI, 34(3B): 46-54, 2022; Article no.JPRI.79883 47 graecum leaves might be considered as herbal remedies for diabetes. Conclusion: In vitro antioxidant and antidiabetic potential of seed elicit of Trigonella foenumgraecum and Moringa oleifera were analyzed and compared. This study shows that Trigonella foenum-graecum has higher antidiabetic potential than the Moringa oleifera extract.


INTRODUCTION
Trigonella foenum-graecum is an herb extensively cultivated in India and some parts of China.In India,seeds are used as condiments [1]. It is an annual plant, which belongs to the family leguminosae 1 [2]. Seeds place a predominant role in the herbal therapeutics and are acknowledged for tonic,carminative and aphrodisiac potential [3]. Trigonella foenumgraecum is a self pollinating crop and it is a vernacular plant used to make maple syrup and the steroid saponin are used widely in industries.Trigonella foenum-graecum is rich in calcium,iron,beta carotene,vitamins,lipids [4] and the seeds are rich in carbohydrates and proteins.It is also used as a folk treatment for non insulin dependent diabetes [5]. Trigonella foenum-graecum is also used to control Hyperglycemia and hyperlipidemia [6]. It is also called fenugreek.It is used to control the glucose,cholesterol,triglyceride and acts as a lowering compounds.It exhibits a long time antidiabetic action [4].
In the current framework, phytochemicals have obtained much attention in the treatment of diabetes. For various reasons, many researchers have concentrated on isolation of hypoglycemic agents from the medicinal plant [7]. Plant polyphenols and the flavonoids are some of the commonly occurring antidiabetic agents [8]. Trigonella foenum-graecum contributes to lower postprandial hyperglycemia in diabetes the mechanisms of action involves provocation of glucose dependent insulin secretion from beta cells of pancreas along with embarrassment of activity of enzymes like amylase and glucosidase [9,10].
Moringa oleifera is a famous consumable plant having therapeutic and the nutritive values.Moringa oleifera is also known as Horseradish tree and also called as drumsticktree.It is rich in phytochemicals and minerals [9][10][11]. It is a constituent of folk medicine and helps to cure the diabetes.It is commonly called as Miracle tree.It also helps to control cancer [12][13][14]. This medicinal plant is used as a food additive to get relieved from malnutrition. It acts as an antioxidant,anti inflammatory,antidiabetic,antimicrobial agent and also takes measures as a natural coagulant. Moringa oleifera is also used in the treatment of anemia [15][16][17]. It has being widely accepted by major population as nutrition and therapeutic agents [18]. It can be suggested as a potential herbal source to treat diabetes mellitus and as being commonly accepted by major population. Our team has extensive knowledge and research experience that has interpret into high quality publications [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34]. The aim of the study is to compare and assess the alpha amylase and alpha glucosidase inhibitory potential of aqueous seed extracts of Trigonella foenum gracus and Moringa oleifera through an in vitro study.

Plant Extract
Trigonella foenum-graecum and Moringa oleifera seed were collected from a farm in Chennai. Washed, crushed and made into powder. Powder was utilized to prepare an 80% aqueous extract. The extract was prepared by a hot percolation method.

Test for phlobatannin
1ml of the extract was treated with 1ml of 1% HCl and boiled for 10 mins. The formation of red color precipitate indicates the presence of phlobatannin.

Test for Carbohydrates
Three to five drops of Molisch reagent was added with 1 mL of the extract and then 1 mL of concentrated sulphuric acid was added carefully through the side of the test tube. The mixture was then allowed to stand for two minutes and diluted with 5 mL of distilled water. The development of red or dull violet rings at the junction of the liquids showed the presence of carbohydrates.

Test for Flavonoids
Few drops of 1% liquid ammonia were taken in a test tube and along with it 1ml of the extract was added resulting in the formation of yellow color thereby indicating the presence of flavonoids.

Test for Alkaloids
2ml of sample was mixed with 2ml of HCl. Then 6 drops of HCN was added and further 2 drops of picric acid was added that resulted in a creamish pale yellow ppt indicating the presence of alkaloids.

Test for Terpenoids
2 ml of sample along with 2ml of chloroform and 3ml of con. H 2 SO 4 was added. Red color ppt obtained indicates the presence of terpenoids.

Test for proteins
One milliliter of ninhydrin was dissolved in 1 mL of acetone and then a small amount of extract was added with ninhydrin. The formation of purple colour revealed the presence of protein.

Detection of saponins
Foam test: A fraction of the extract was vigorously shaken with water and observed for persistent foam.

Test for steroids
One milliliter of chloroform was mixed with 1 mL of extract and then ten drops of acetic anhydride and five drops of concentrated sulphuric acid were added and mixed. The formation of dark red colour or dark pink color indicates the presence of steroids.

DPPH free radical scavenging activity
Scavenging of 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) radicals was assessed by the method of [35]. DPPH solution (1.0 ml) was added to 1.0 ml of extract at different concentrations (0.1 to 0. 5mg/ml). The mixture was kept at room temperature for 50 minutes and the activity was measured at 517 nm. Ascorbic acid at the same concentrations was used as standard. The capability to scavenge the DPPH radical was calculated and expressed in percentage (%) using following formula: DPPH radical scavenged (%) = Control OD-Sample OD X100 Control OD

Alpha amylase inhibitory activity of aqueous seed extract of Trigonella foenum-graecum and Moringa oleifera
Alpha amylase inhibitory activity of extract was carried out according to the standard method of Ademiluyi et al 2013, In a test tube a reaction mixture containing 500 mu/l phosphate buffer ( 100mM ; pH=6.8). 100 mu alpha amylase (2 mu/l) and varying concentration of extract (0.1 -0.5 mg/ml) was Incubated at 37degree Celsius for 20 minutes. Then the 200 mu/l of 1% soluble starch(100 MM phosphate buffer 6.8) was added as a substrate and incubated further at 37 degree Celsius for 30 minutes, 1000 mu/l of the 3,5 Dinitrosalicylic acid [DNS], DNS colour reagent was then added and boiled for 10 minutes. The absorbance of the resulting mixture was measured at 540 nm using a multi plate reader. Acarbose at various concentrations (0.1-0.5 mg/ml) was used as a standard.

Alpha glucosidase inhibitory activity of aqueous seed extract of Trigonella foenum-graecum and Moringa oleifera
Alpha glucosidase inhibitory activity of extract was carried out according to the method of Ademiluyi et al (2013) Reaction mixture containing 500 mu/l phosphate buffer(100mM pH 6.8), 100mu/l glucosidase (10 ml) and varying concentration of extract (0.1 to 0.5 mg /ml) was pre incubated at 37 degree Celsius for 15 minutes. Then 200 mu/l p-NPG(5mM) was added as a substrate and incubated further at 37degree Celsius for 30 minutes. The reaction was stopped by adding 50 mu/l sodium carbonate (0.1M). The absorbance of the released pnitrophenol was measured at 405 nm using multiple readers. Acarbose at various concentrations (0.1-0.5mg/ml)was used as a standard.

Statistical Analysis
The data were subjected to statistical analysis using two-way analysis of variance (ANOVA) and Tukey's multiple range test to assess the significance of individual variations between the groups. In Tukey's test, significance was considered at the level of p<0.05 .

RESULTS
Trigonella foenum-graecum shows the presence of Amino Acids, Flavonoids, Alkaloids which have higher antidiabetic potential than Terpenoids and carbohydrates. In Moringa oleifera , the presence of flavonoids, steroids indicates the higher antidiabetic potential than Alkaloids and carbohydrates.

DISCUSSION
Phytochemicals are secondary metabolites only present in plants. Plants possess various biologically active compounds that protects and helps human body [36]. Phytochemical analysis has revealed (Table 1)a energetic presence of alkaloids, flavonoids and terpenoids in trigonella foenum-graecum and Moringa oleifera seed elicit [3]. The presence of phytochemicals like alkaloids, glycoside ,tannins, carbohydrates, phenols indicates that the extract is potential for further in vitro analysis like antioxidant activity and antidiabetic activity.

Phytochemicals
Fenugreek Moringa oleifera Amino Acids represents Antioxidant potential of aqueous seed extracts of Trigonella foenumgraecum and Moringa oleifera compared with standard Ascorbic acid. X axis represents the concentration in µg/ml and Y axis represents the inhibitory potential of the extracts.Blue bar represents standard Acarbose, orange bar represents Trigonella foenum-graecum and green bar represents Moringa oleifera Each bar represents Mean ± SEM of 3 independent observations . Significance at p < 0.05

Graph 2. Represents Alpha Amylase Inhibitory potential of aqueous seed extracts of Trigonella foenum-graecum and Moringa oleifera.X axis represents the concentration in µg/ml and Y axis represents the Alpha amylase inhibitory potential of the extracts.Green bar represents standard Acarbose, orange bar represents Trigonella foenum-graecum and purple bar represents Moringa oleifera Each bar represents Mean ± SEM of 3 independent observations .
Significance at p <0.05 Graph 3. Represents Alpha Glucosidase Inhibitory potential of aqueous seed extract of Trigonella foenum-graecum and Moringa oleifera. X axis represents the concentration in µg/ml and Y axis represents the alpha glucosidase inhibitory potential of the extracts.Green bar represents standard Acarbose, red bar represents Trigonella foenum-graecum and purple bar represents Moringa oleifera Each bar represents Mean ± SEM of 3 independent observations . Significance at p < 0.05 Antioxidant activity of aqueous seed extracts of Trigonella foenum-graecum and Moringa oleifera was determined by performing DPPH free radical scavenging assay.Free radicals are molecules influencing an unpaired electron [37]. Herbal elicit which are rich in phenolic compounds have great importance in free radical scavenging.The effect of antioxidants on DPPH free radical scavenging was considered to be due to their hydrogen donating ability.The extracts were analyzed for their antioxidant potential. Both the extract exhibited a significant antioxidant potential, when compared Trigonella foenumgraecum exhibited a strong antioxidant potential with Ic 50 of 320 µg/ml then Moringa oleifera ( Ic 50 of 360 µg/ml) (Graph 1) as compared with the standard vitamin C.
In vitro Antidiabetic activity was analyzed for the extract and compared with the standard drug metformin Both Alpha amylase and Alpha glucosidase inhibitory potential was estimated to analyze antidiabetic potential [38]. The ethanolic seed extract of Trigonella foenum-graecum exhibited significantly more inhibition for Alpha amylase and Alpha glucosidase (Graph 2and 3) with Ic 50 of 280 and 390 µg/ml respectively.Previous studies on antidiabetic potential of Trigonella foenum-graecum has also exhibited a significant inhibitory potential, where aqueous seed extract of Trigonella foenumgraecum a dose of 25mg/kg body weight given to experimental animals for 60days,significantly decreased blood glucose and liver enzymes level.However,no histological liver protective role was reported [3].
The extracts are tested for its role as a starch blocker [39]. Since,it prevents or slows the absorption of starch mainly by blocking the hydrolysis of glycosidic linkage in starch [40]. There is a positive relationship between phytoconstituents contents and its ability to inhibit alpha glucosidase and alpha amylase.From the obtained results,it was evident that both the aqueous seed extract of trigonella foenum and Moringa oleifera possess antioxidant and antidiabetic potential and escalated in a dose dependent manner as compared to standard [41]. The extracts are capable of inhibiting the absorption of glucose [42]. Further research is needed to assess the in vivo antidiabetic potential of the extracts. Further validation of the drug's ability to decrease glucose concentration in serum can help in the drug formulation.

CONCLUSION
Even though there are many drugs available for diabetes, a complete cure remains a dream. Current In vitro findings on the antioxidant and antidiabetic potential of seed elicit of Trigonella foenum-graecum and Moringa oleifera showed that both the plant extracts have potential antidiabetic activity due the existence of the bioactive compounds.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.

SOURCE OF FUNDING
The present study was supported by the following agencies.