Evaluation of In-vitro Anti-diabetic activity in Ethanolic Stem Extract of Euphobia hirta

Dr. Sundhararajan R, L K. Dhivya*, Mohan. N, Paul Christober. M,

Surya Prakash. S, Tamil selvan. P

Mohammed Sathak College of Pharmacy, Chennai, Tamilnadu

*Correspondence: lkdhivya97@gmail.com

DOI: https://doi.org/10.71431/IJRPAS.2026.5207

INTRODUCTION

Euphorbia hirta Linn. (E. hirta) belongs to the Euphorbiaceae family. In India, E. hirta is commonly distributed in many provinces of the southern area. Many investigations have demonstrated that this plant has been widely used as traditional medicine to treat many diseases, including wound healing, gastrointestinal disorders, tumors, etc. Additionally, this herb was used as a traditional medicine in the treatment of diabetes for a long time.

In Vitro Antidiabetic activity

Controlling blood glucose levels, particularly postprandial (after-meal) hyperglycemia, is a major therapeutic goal in diabetes management. Among various strategies, the inhibition of digestive enzymes involved in carbohydrate metabolism specifically alpha-amylase and alpha-glucosidase- has emerged as an effective approach.

MATERIALS AND METHODS

COLLECTION OF PLANT MATERIAL

The Euphorbia hirta were obtained from garden and authenticated to verify their botanical identity. Once authenticated, the E.hirta were thoroughly cleaned, shade-dried, and ground into a fine powder.

PREPARATION OF PLANT EXTRACT

A total of 100 grams of the powdered E.hirta material was subjected to extraction using ethanol as the solvent.

The powdered E.hirta were macerated in 500 mL of ethanol acetate for 72 hours at room temperature with periodic stirring. After the extraction, the mixture was filtered using Whatman No. 1 filter paper to remove the plant residues. The filtrate was concentrated using a rotary evaporator under reduced pressure at 40°C until a semi-solid residue was formed. The dried extracts was collected, weighed, and stored in an airtight container at 4°C for further in vitro analysis.

ASSAY AND IVITRO TEST

IN VITRO A-AMYLASE INHIBITION ASSAY

 A total of 250 μL of sample  was placed in a tube and 250 μL of 0.02 M sodium phosphate buffer (pH 6.9) containing α-amylase solution (0.5 mg/mL) was added. This solution was pre- incubated at 25 °C for 10 min, after which 250 μL of 1% starch solution in 0.02 M sodium phosphate buffer (pH 6.9) was added at timed intervals and then further incubated at 25 °C for 10 min. The reaction was terminated by adding 500 μL of dinitrosalicylic acid (DNSA) reagent. The tubes were then incubated in boiling water for 5 min and cooled to room temperature. The reaction mixture was diluted with 5 mL distilled water and the absorbance was measured at 540 nm using spectrophotometer. A control was prepared using the same procedure replacing the sample with distilled water. Acarbose standard was also run in the same manner.

The -amylase inhibitory activity was calculated as percentage inhibition:

The -amylase inhibitory activity was calculated as percentage inhibition:

                           Abs 540 (control) – Abs 540 (sample)

Inhibition (%) = ---------------------------------------------- × 100

                                           Abs 540(control)

Concentrations of sample resulting in 50% inhibition of enzyme activity (IC50) were determined graphically.

IN VITRO Α-GLUCOSIDASE INHIBITION ASSAY

 Yeast α‐glucosidase (0.7 U) dissolved in 100 mM phosphate buffer (pH 7.0) containing 2 g/l bovine serum albumin, and 0.2 g/l NaN3, and 5 mM p-nitrophenyl‐ α‐D‐glucopyranoside in the same buffer (pH 7.0) were used as an enzyme and a substrate solution, respectively. The enzyme solution (2000 μl) and 100 μl of the test sample at various concentrations (20 to 100 μl/mL) were mixed, and absorbance at 405 nm was

measured using a spectrophotometer. After incubation for 5 min, 50 μl of the substrate solution was added and incubated for an additional 5 min. The increase in absorbance from time zero was measured, and inhibitory activity was calculated as a percentage of the blank control. The % inhibition was calculated using the following formula,

                              Abs 405 (Control) – Abs 405 (extract)

Inhibition (%) = -------------------------------------------------- × 100

                                            Abs 405(control)

RESULT AND DISCUSSION

Table 1: In-vitro α-amylase inhibition Assay

                        The values are means of triplicate experiments ± standard deviation.

The inhibitory activity of EEEH against carbohydrate-digesting enzymes was evaluated and compared with the standard drug acarbose. EEEH exhibited moderate α-amylase inhibitory activity, with an IC₅₀ value of 1.16 mg/mL, indicating measurable enzyme suppression. In comparison, acarbose demonstrated significantly stronger inhibition, as reflected by a lower IC₅₀ value of 0.42 mg/mL.

Figure 1: In-vitro α-amylase inhibition assay

Table 2: In-vitro α-glucosidase inhibition Assay

                             The values are means of triplicate experiments ± standard deviation.

The inhibitory activity of EEEH against α-glucosidase was evaluated and compared with the standard drug acarbose. EEEH exhibited moderate α-glucosidase inhibitory activity, with an IC₅₀ value of 1.21 mg/mL, indicating appreciable enzyme suppression. In contrast, acarbose showed stronger inhibitory potential, as evidenced by a lower IC₅₀ value of 0.44 mg/mL.

Figure 2: In-vitro α-glucosidase inhibition assay

CONCLUSION

EEEH exhibited moderate α-amylase and α-glucosidase inhibitory activity these indicating appreciable enzyme suppression, that shows the pharmacological profile of existing antidiabetic drugs but from a natural orign, making it a potential candidate for functional food or phytopharmaceutical intrests.

ACKNOWLEDGEMENT

We thanks to management and staffs of Mohamed Sathak AJ College of Pharmacy Chennai, Tamil Nadu,

India for providing necessary facilities and full support throughout the research in a successful manner.

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