Comparative In Vitro Hypoglycemic Activity of Dolichandrone falcata's Aerial Parts Via α-Amylase and α-Glucosidase Inhibition

 Introduction

Type-2 Diabetes is regarded as the second most common non-communicable disorder, after hypertension, in terms of public health significance. It is a metabolic disorder resulting from the body’s inability to produce sufficient quantities of insulin or properly utilize the same. Dietary phenolics may help regulate hyperglycemia by inhibiting important enzymes (α-Amylase and α-Glucosidase) involved in starch digestion and protecting against free radicals and lipid peroxidation in the pancreas.¹ Dolichandrone falcata Seem (D. falcate) is a deciduous plant from the Bignoniaceae family that is frequently known as ‘Medshingi’ in the districts of Marathwada and Vidarbha in Maharashtra, India.^2,3 The leaves of the plant D. falcata have traditionally been used by the tribes of Maharashtra's Melghat region as a folk treatment for food sickness mostly caused by the ingestion of fish and to induce abortion.

Materials and Methods

Plant material

The leaves and bark of D. falcata were collected from Kunchavaram forest, Gulbarga and local areas of Gulbarga, and supplied by Dr. Vijay Danapur (CEO of Vriksha Vijnan Pvt Ltd). They were identified and authenticated by experts from Central Ayurveda Research Institute, Bangalore, with the identification code RRCBI-19674.

The leaves and bark were shade dried and coarsely powdered. 25 g of powdered leaves and bark were subjected to successive soxhlet extraction with ethyl acetate, methanol and water, respectively. The respective yields of the leaf extracts were 7.96%, 10.16% and 3.84% (w/w), and of the bark extracts were 3.16%, 13.72% and 12.48% (w/w).

Proximate analysis

Total ash (%), loss on drying (LOD) (%), water and alcohol soluble extractive values were calculated for both leaf and bark of D. falcata using appropriate methods

Phytochemical standardization

Preliminary phytochemical screening of the leaf extracts of D. falcata was initially carried out to confirm the presence of various primary and secondary metabolites.

α- Amylase inhibition activity

One mL of 0.02 M sodium phosphate buffer was added to the test extract, along with 0.5 mL of enzyme (0.5 mg/mL), and the mixture was incubated for 10 minutes at 25ºC. 0.5 mL of 1% starch solution in buffer was added. Thereafter, the reaction mixture was incubated at 25oC for 10 minutes and 1 mL of DNSA (3,5-dinitrosalicylic acid) solution was added. The process was then stopped by incubating in a boiling water bath for five minutes before being cooled to room temperature.

The reaction mixture was then diluted to 10 mL by adding distilled water, and absorbance was measured at 540 nm in a spectrophotometer. The reference sample included all other reagents and the enzyme with the exception of the test sample. α-Amylase inhibitory activity was expressed as a percentage inhibition.

Inhibition (%) = [(Absref - Abssample)/Absref] × 100

Where, Absref = absorbance of the reference and Abssample = absorbance of the test sample

Comparative analysis of α-Amylase inhibition by ethyl acetate, methanol and aqueous extracts of both leaf and bark of D. falcata and Acarbose was done by calculating IC75 values using statistical tools. IC75 model was chosen for α-Amylase inhibition because of the data not being compatible with the IC50 model.

α- Glucosidase inhibition activity

To 1 mL of test extract, 1 mL enzyme (1 U/mL) was added and incubated at 25oC for 10 minutes, 0.5 mL of 1 mM p-NPG solution in buffer was added. Thereafter, the reaction mixture was incubated at 25oC for five minutes and 1 mL of 0.1 M Na2CO3 solution was added to terminate the reaction. The reaction mixture was then diluted to 10 mL by adding distilled water, and absorbance was measured at 450 nm in a spectrophotometer. The reference sample included all other reagents and the enzyme with the exception of the test sample.

α-Glucosidase inhibitory activity was measured as % inhibition.

Inhibition (%) = [(Absref - Abssample)/Absref] × 100

Where, Absref = absorbance of the reference and Abssample = absorbance of the test sample

Comparative analysis of α-Glucosidase inhibition by ethyl acetate, methanol and aqueous extracts of both leaf and bark of D. falcata and Acarbose was done by calculating IC50 values using statistical tools

HPTLC Profiling and Rutin Estimation

The ethyl acetate, methanol and aqueous extracts of leaf and bark of D. falcata were dissolved in methanol (2 mg/mL). These six methanolic extracts were subjected to High performance thin-layer chromatography (HPTLC) study for the estimation of the standard Rutin (1 mg/mL).

Results

Proximate Analysis

Leaf: The leaf exhibited high mineral content with a total ash percentage of 8.74±0.21%. It showed a relatively high moisture content as indicated by the loss on drying (LOD) of 11.68±0.15%. The aqueous extractive value was significant at 10.57±0.48%, showing a high presence of water-soluble compounds. Additionally, it had a moderate amount of alcohol-soluble compounds with an extractive value of 5.80±0.41%.

Bark: The bark had a moderate mineral content with a total ash percentage of 6.95±0.55%. Its moisture content was moderate, as indicated by the LOD of 8.38±0.51%. The aqueous extractive value was relatively low at 4.10±0.18%, indicating fewer water-soluble compounds. The alcohol soluble extractive value was also low at 1.09±0.15%, showing minimal organic solvent-soluble compounds.

α-Amylase Inhibition Activity

The enzyme inhibitory activity was carried out for 100, 200, 400 and 1600 μg/mL of all three extracts of leaf and bark, and Acarbose at the dilutions of 50, 100, 150 and 200 μg/mL.

IC75 Data of all Samples for α-Amylase Inhibition

The IC75 values for α-Amylase inhibition of various samples were determined, with standard Acarbose showing an IC75 value of 178.79 μg/mL. Among the leaf extracts, DF L - EtAc exhibited an IC75 value of 1264.09 μg/mL, DF L - MeOH had 1012.17 μg/mL, and DF L - Aq recorded 983.91 μg/mL. For the bark extracts, DF B - EtAc had an IC75 value of 865.65 μg/mL, DF B - MeOH showed 985 μg/mlL, and DF - Aq had the lowest IC75 value among the bark extracts at 764.09 μg/mL. The leaf and bark aqueous extracts showed the most potent α-Amylase inhibitory activity in their respective categories, with the Aq extracts also showing the presence of most phytochemicals during phytochemical screening. Notably, the DF B - Aq extract exhibited the most potent α-Amylase inhibitory activity out of all the plant extracts, with an IC75 value of 764.09 μg/mL, compared to the standard Acarbose's IC75 value of 178.79 μg/mL.

α- Glucosidase Inhibition Activity

The enzyme inhibitory activity was carried out for 20, 40, 60 and 80 μg/mL of all three extracts of leaf and bark, and Acarbose at the dilutions of 10, 20, 30 and 40 μg/mL.The IC50 values for α-Glucosidase inhibition of various samples were determined, with standard Acarbose showing an IC50 value of 23.75 μg/mL. Among the leaf extracts, DF L - EtAc exhibited an IC50 value of 63.56 μg/mL, DF L - MeOH had 63.47 μg/mL, and DF L - Aq recorded 57.89 μg/mL. For the bark extracts, DF B - EtAc had an IC50 value of 48.78 μg/mL, DF B - MeOH showed 57.30 μg/mL, and DF - Aq had the lowest IC50 value among the bark extracts at 44.97 μg/mL. The leaf and bark aqueous extracts demonstrated the most potent α-Glucosidase inhibitory activity in their respective categories, with the Aq extracts also showing the presence of most phytochemicals during phytochemical screening. Notably, the DF B-Aq extract exhibited the most potent α-Glucosidase inhibitory activity out of all the plant extracts, with an IC50 value of 44.97 μg/mL compared to the standard Acarbose's IC50 value of 23.75 μg/mL. Additionally, the DF B - EtAc showed similar α-Glucosidase inhibitory activity to a previous study where the DF Heartwood EtAc extract reported an IC50 value of 32.87 μg/mL.⁴ These results corroborate the claims of D. falcata leaves exhibiting a potent antidiabetic effect in alloxan-induced diabetic rats.⁵

HPTLC Profiling and Rutin Estimation

Rutin peak was observed in all the extracts except for DF L - MeOH

DF L - EtAc extract reported the highest % Rutin ~ 37.3% out of the other extracts. This extract also showed sufficient α-Amylase and α-Glucosidase inhibitory activity.

DF B - Aq which showed the most potent α-Amylase and α-Glucosidase inhibitory activity out of all the other extracts reported the second highest % Rutin ~ 21.24%.

Although Rutin itself has α-Amylase and α-Glucosidase inhibitory potential, presence of other phytoconstituents could account for greater enzyme inhibitory activity of DF B - Aq.⁶

Discussion

Phytochemical screening indicated the existence of many secondary metabolite groups in plant leaf and bark extracts. Comparative analysis of α-Amylase and α-Glucosidase inhibition was carried out through IC75 and IC50 values, respectively by a derivation of the slope-intercept equation for all the extracts. DF B – Aq showed the least IC75 (α-Amylase) and IC50 (α-Glucosidase) values at 764.09 μg/mL and 44.97 μg/mL, respectively. Both the Aq extracts showed the most potent α-Amylase and α-Glucosidase inhibition in their respective categories. Standard drug Acarbose showed IC75 (α-Amylase) and IC50 (α-Glucosidase) values at 178.79 μg/mL and 23.75 μg/mL, respectively. Rutin showed presence in all the extracts except DF L - MeOH. DF L - EtAc showed the highest % Rutin of 37.3% ~18.65 μg. DF B - Aq which showed maximum α-Amylase and α-Glucosidase inhibition showed % Rutin of 21.24% ~ 10.62 μg. There was a justifiable correlation between the % Rutin of plant extracts and digestive enzyme inhibitory activity.

Conclusion

The present study showed significant α-Amylase and α-Glucosidase inhibition potential of various extracts of leaf and bark. The bark extracts have shown higher enzyme inhibition potential than the leaf extracts. Rutin was identified in all the extracts which has also been proven to inhibit α-Amylase and α-Glucosidase. Future studies on this formulation would be of good scope.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgement

The authors are thankful to Government College of Pharmacy for providing all the facilities.