INTRODUCTION:

Mikania micrantha kunth belongs to the family Astereraceae. It is also known as climbing hemp fine or Chinese creeper in English, Japanilota in Assamese, and Titaiyabaur in Hindi language¹. This perennial vine grows at lightning speed and is native to tropical America. The plant is also seen in tropical Asia, and Africa along with parts of Papua New Guinea. Traditionally, it has been used to treat wounds and control mild external bleeding². In Bangladesh, it is widely used as an indigenous preservative. Traditional uses for M.micrantha include the treatment of rheumatism, respiratory illnesses, dysentery, and stomach pain.

As an alternative medicine for the treatment of diabetes, hypertension, and hypercholesterolemia, it is also ingested as juice. The juice of M. micrantha is used to lessen rashes and irritation, while plasters prepared from the plant's leaves are used to treat bites from toxic insects. Pharmaceutical research across the world shows that, natural products are potential sources of novel molecules for drug development. Diabetes is the world’s largest endocrine disease involving metabolic disorder of carbohydrate, fat and protein³. Plants have been the basic for medicinal treatment through much of human history and such traditional medicine is still widely practiced today for many diseases and used to treat effectively one of most common and oldest metabolic disorder “DIABETES” around 250 BC across the world many people were suffering from the diabetes⁴ Ancient Egypt and Greece both had a working understanding of diabetic mellitus. The Latin word "Mellitus" means "Sweetened with honey," which relates to the presence of sugar in urine. The word "DIABETES" is derived from the Greek word "DIAB," meaning to pass through; referring to the cycle of intense thirst and frequent urination⁵. Type-1 (Insulin Dependent Diabetes Mellitus) diabetes is an autoimmune disorder, characterized by pancreatic β cell destruction with an absolute deficiency of insulin⁶. Type-2 (Non-Insulin Dependent Diabetes Mellitus) diabetes is due to dysregulated glucose homeostasis, impaired insulin secretion and action⁷. The current approach to treating diabetes focuses on managing and bringing blood glucose levels down to a normal range. Western medications and Chinese traditional treatments both work in the same way to decrease blood sugar:

1. To induce the release of insulin from the beta cell of the pancreatic islet

2. To fend against hormones that raise blood sugar.

3. To enhance the amount of insulin receptor sites' sensitivity and affinity tothe hormone.

4. To reduce glycogen leading out.

5. To reduce free radicals, prevent lipid peroxidation, and enhance the body's microcirculation^5,8.

Free radicals are highly reactive molecules in the body that can damage by destroying the enzymes, protein molecules and entire cells. The oxidative damage caused by free radicals is a pivotal mechanism implicated in the progression of a large number of human diseases⁹. Oxygen free radicals or reactive oxygen species (ROS) are products of normal cellular metabolism¹⁰. Medicinal plant parts are commonly rich in phenolic compounds, such as flavonoids, phenolic acids, stilbenes, tannins, coumarins, lignans and lignins. These compounds have multiple biological effects including antioxidant activity¹¹.

MATERIALS AND METHODS:

Collection and authentication of the plant component:

The leaves of the plant Mikania micrantha kunth (Asteraceae) were collected in the month of November of 2022. A specimen of the plant was deposited in the Central National Herbarium at the Botanical Survey of India, Shibpur, Howrah (CNH/Tech.II/2O23/45) for identification and authentication. The leaves were collected, thoroughly washed with water, and then dried for a month under shade. The dried leaves were then mechanically ground into a powder and placed in an airtight container for later usage.

Fig.1: Mikania micrantha Plant

Preparation of Extracts:

Methanol (boiling point 64⁰C) was used to extract the leaves powder of Mikania micrantha. The extract was concentrated, dried in the open air, and stored in an airtight container.

In-vitro antioxidant studies:

Determination of Total Phenolic content:

The total phenolic content of the sample was determined by the Folin-Ciocalteau method^12,13

Determination of Total Flavonoid content:

The total soluble flavonoid content of the fractions was determined with aluminum chloride using quercetin as a standardwith slight modifications^13,14.

Determination of DPPH radical scavenging activity:

The radical scavenging activities of different extracts were determined by using DPPH assay. The decrease in the absorption of the DPPH solution after the addition of an antioxidant was measured at 517nm¹⁵. Minor adjustments were made to an earlier approachof DPPH activity while using Ascorbic acid as the Standard for comparison¹⁶.

Determination of hydrogen peroxide radical scavenging activity:

Scavenging of H2O2 by extracts may be attributed to their phenolics, which can donate electrons to H₂O_2.¹⁷

The ability of both M. micrantha extracts to scavenge hydrogen peroxide was determined according to the method of Oktay Munir and Oktay et al, 2003^17,18.

In- vitro anti-diabetic activity^19,20

Inhibition of α Amylase:

Inhibition of α-amylase and α-glucosidase enzymes can slow uptake of dietary carbohydrates and suppress postprandial hyperglycemia²¹.The in-vitroanti-diabetic assay was performed by DNSA method using Acarbose as Standard^22–24.

RESULT AND DISCUSSION:

Total Phenolic Content Assay:

The absorbance of the Standard and test solution was represented in table no.1. The total phenolic content in terms of mg Gallic Acid Equivalent (GAE)of Petroleum ether extract was found to be 6.585 mg/g and the methanolic extract was found to be 15.202 mg/g.

Table 1. Observation of absorbance in Total phenolic estimation

Sl No.	Standard (Gallic acid)	Concentration (µg/ml)	Absorbance
1		20	0.238
2		40	0.458
3		60	0.686
4		80	0.815
5		100	0.995
6	Sample (Petroleum ether extract)	100	0.139
7	Sample (Methanolic extract)	100	0.220

Figure 2: Standard curve of Gallic acid

Total Flavonoid Content Assay:

The content of flavonoid compound in both Petroleum ether and methanolic extract of Milania micrantha leaves was measured by aluminum chloride reagent in terms of quercetin equivalent and was found to be 9.0588mg/g and 12.980mg/g.

Table 2: Observation of absorbance in total flavonoid estimation

Sl No.	Standard (Quercetin)	Concentration (µg/ml)	Absorbance
1		20	0.138
2		40	0.250
3		60	0.358
4		80	0.454
5		100	0.547
6	Sample (Petroleum ether extract)	100	0.089
7	Sample(Methanolic extract)	100	0.109

Figure 3: Standard curve of Quercetin

Inhibition of DPPH radicals:

The drop in DPPH's absorbance at 517nm, which is brought on by antioxidants, was used to assess its capacity for reduction. The extract showed maximum hydrogen donating ability in the presence of DPPH free radicals at high concentrations. The extract showed antioxidant activity with an IC₅₀ value of 202.94µg/ml. However, the known antioxidant Ascorbic acid exhibited an IC₅₀ value of 139.66µg/ml on DPPH radical.

Table 3: DPPH Radical Scavenging Assay of Standard Sample and Test Sample

Sl No.	Concentration (µg/ml)	Percentage Inhibition (Mean±SEM)
Sl No.	Concentration (µg/ml)	Test Sample	Standard (Ascorbic acid)
1	20	3.11±0.28	16.92±0.33
2	40	8.24±0.13	18.01±0.08
3	60	12.48±0	28.46±0.5
4	80	17.86±0.1	30.54±0.8
5	100	24.16±0.21	39.90±0.09

Figure 4: DPPH Radical Scavenging Assay

Inhibition of hydrogen peroxide radicals:

The IC₅₀ value of extract and ascorbic acid were 561µg/ml and 481µg/ml respectively.

Table 4: H₂O₂ radical scavenging activity of extract and standard

Sl. No.	Concentration (µg/ml)	Percentage inhibition (Mean±S.E.M)
		Standard (Ascorbic acid)	Extract
1	100	4.21±0.11	2.12±0.10
2	200	14±1.23	10.39±0.62
3	300	27.32±1.15	22.31±1.14
4	400	45.85±0.67	35.25±0.72
5	500	53.12±.011	45.02±.02
6	600	61.04±2.58	52.47±0.61

Figure 5: % Hydrogen peroxide radical scavenging activity

Determination of in-vitro Anti diabetic activity:

α-amylase inhibition assay:

The IC₅₀ of Acarbose and Methanolic extract was calculated and found to be94.66 µg/ml 105.269µg/ml respectively.

Table 5: α -amylase inhibition activity of extract and standard

Sl. No.	Concentration (µg/ml)	% Inhibition of Acarbose, mean±SD (n=3)	*% Inhibition of methanolic extract of M.micrantha,* mean±SD**
1	20	13.2±0.447	21.05±0.615
2	40	25.12±0.510	31.58±0.520
3	60	30.75±0.585	36.84±0.605
4	80	39.38±0.708	42.105±0.796
5	100	55.64±0.517	47.368±0.818

Figure 6: Graph of α-amylase inhibition assay of Acarbose and test sample.

CONCLUSION:

In the area, Mikania micrantha is one of the most prevalent plants. The flavonoids are considered to be responsible for the antioxidant and antidiabetic potential of the leaves extract. The in vitro antioxidant investigations of the methanolic extract of Mikania micrantha leaves were carried out with great care. When compared to other common antioxidants, the methanolic extract of Mikania micrantha leaves shown potent anti-oxidant activity by suppressing DPPH radical scavenging activities. As a result, it may be inferred that the plant Mikania micrantha can be employed as a significant and accessible source of natural antioxidants with subsequent health advantages. These in vitro studies demonstrate that this plant extract is a significant natural source of antioxidants, which may be helpful in reversing the effects of a variety of oxidative stresses. It was seen that there was a dose-dependent increase in percentage inhibitory activity against alpha-amylase enzyme while performing the in vitro ant diabetic evaluation.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

ACKNOWLEDGMENTS:

We sincerely thank Central National Herbarium, Botanical Survey of India, Howrah, West Bengal, India for their expertise and timely support in the identification and authentication of the plant sample.

AUTHORS CONTRIBUTION:

All Authors have contributed equally.

REFERENCES:

1. Deori C, Dutta G, Das S, Phukan D, Gogoi G. To evaluate the anti-inflammatory activity of ethanolic extract of leaves of Mikania micrantha on experimental animal models. J Evol Med Dent Sci. 2017; 6(50): 3818-3822.

2. Facey PC, Pascoe KO, Porter RB, Jones AD. Investigation of plants used in Jamaican folk medicine for anti-bacterial activity. Journal of Pharmacy and Pharmacology. 1999; 51(12): 1455-1460. doi.org/10.1211/0022357991777119

3. Chandel HS, Pathak A, Tailang M. Formulation of Polyherbal and Evaluation for Anti Diabetic Effect. Research Journal of Pharmacognosy and Phytochemistry. 2011; 3(3): 134-136.

4. Raju K, Rose AS, Rohini B, Sahaja P, Shylaja G, Simran S. Formulation and evaluation of anti-diabetic herbal syrup. Research Journal of Pharmacognosy and Phytochemistry. 2020; 12(3): 141-144. doi.org/10.5958/0975-4385.2020.00023.0

5. Tripathi KD. Essentials of Medical Pharmacology. JP Medical Ltd; 2013.

6. Mawlieh BS, Shastry CS, Chand S. Evaluation of anti-diabetic activity of two marketed herbal formulations. Res J Pharm Technol. 2020; 13(2): 664-668. doi.org/10.5958/0974-360X.2020.00127.4

7. Campbell RK. Fate of the beta-cell in the pathophysiology of type 2 diabetes. Journal of the American Pharmacists Association. 2009; 49(5): S10-S15.doi.org/10.1331/JAPhA.2009.09076

8. Li WL, Zheng HC, Bukuru J, De Kimpe N. Natural medicines used in the traditional Chinese medical system for therapy of diabetes mellitus. J Ethnopharmacol. 2004; 92(1): 1-21. doi.org/10.1016/j.jep.2003.12.031

9. Beena P, Purnima S, Kokilavani R. In Vitro Anti-Oxidant Study of Ethanolic Extract of Coldenia procumbens Linn. Asian Journal of Research in Chemistry. 2011; 4(3): 450-451.

10. Samuel DS, Geetha R V. Antioxidant activity of dry fruits: A short review. Res J Pharm Technol. 2014; 7(11): 1319-1322.

11. Stuckey BN, Osborne CE. A review of antioxidant analysis in food products. J Am Oil Chem Soc. 1965; 42(3): 228-232. doi.org/10.1007/BF02541136

12. Kuber RB. In vitro antioxidant potential, total phenolic and flavonoid contents in Justicia gendarussa leaf extracts. Res J Pharm Technol. 2021; 14(5): 2707-2713. doi.org/10.52711/0974-360X.2021.00477

13. Miliauskas G, Venskutonis PR, Van Beek TA. Screening of radical scavenging activity of some medicinal and aromatic plant extracts. Food Chem. 2004; 85(2): 231-237. doi.org/10.1016/j.foodchem.2003.05.007

14. Halliwell B. Reactive oxygen species in living systems: source, biochemistry, and role in human disease. Am J Med. 1991; 91(3): S14-S22. doi.org/10.1016/0002-9343(91)90279-7

15. Gini EJ, Sivakkumar T, Kuppuswami S. Determination of Antioxidant Activity of various extracts of Pajanelia longifolia (Willd.) K. Schum, isolation and characterization of Flavonoid from Ethanol extract by Column Chromatography. Res J Pharm Technol. 2017; 10(10): 3391-3397. doi.org/10.5958/0974-360X.2017.00603.5

16. Krishnaveni R, Rajan S. In vitro Anti-oxidant and Anti-inflammatory Activity of the Aqueous and Ethanol Leaf Extracts of Clerodendrum phlomidis Linn. F. Res J Pharm Technol. 2021; 14(10): 5217-5221.doi.org/10.52711/0974-360X.2021.00908

17. Joshi H, Pagare M, Patil L, Kadam V. In–Vitro Antioxidant Activity of Ethanolic Extract of Leaves of BuchananiaLanzan Spreng. Res J Pharm Technol. 2011; 4(6): 920-924.

18. Oktay M, Gülçin İ, Küfrevioğlu Öİ. Determination of in vitro antioxidant activity of fennel (Foeniculum vulgare) seed extracts. LWT-Food Science and Technology. 2003; 36(2): 263-271. doi.org/10.1016/S0023-6438(02)00226-8

19. Klein SM, Cohen G, Cederbaum AI. Production of formaldehyde during metabolism of dimethyl sulfoxide by hydroxyl radical-generating systems. Biochemistry. 1981; 20(21): 6006-6012. doi.org/10.1021/bi00524a013

20. Helen PA, Bency BJ. Inhibitory potential of Amaranthus viridis on α-amylase and glucose entrapment efficacy In vitro. Res J Pharm Technol. 2019; 12(5): 2089-2092. doi.org/10.5958/0974-360X.2019.00346.9

21. Jebaseelan S. Identification of phytochemical constituents within the rhizome extract of Costusspeciosus (koen) using Gas Chromatography-Mass Spectrometry (GC-MS) analysis and In-vitro anti diabetic activity. Res J Pharm Technol. 2019; 12(2): 812-816. doi.org/10.5958/0974-360X.2019.00141.0

22. Babu SN, Govindarajan S, Vijayalakshmi MA, Noor A. Evaluation of in vitro anti-diabetic and anti-oxidant activities and preliminary phytochemical screening of gel, epidermis and flower extract of Aloe vera. Res J Pharm Technol. 2019; 12(4): 1761-1768. doi.org/10.5958/0974-360X.2019.00295.6

23. Nimbekar T, Jain A, Mohanty PK. Phytochemical screening and In-Vitro Antidiabetic activity of extracts of some Indian medicinal plants. Res J Pharm Technol. 2021; 14(4): 2026-2030.doi.org/10.52711/0974-360X.2021.00359

24. Telagari M, Hullatti K. In-vitro α-amylase and α-glucosidase inhibitory activity of Adiantum caudatum Linn. and Celosia argentea Linn. extracts and fractions. Indian J Pharmacol. 2015; 47(4): 425-429. doi.org/ 10.4103/0253-7613.161270

Received on 12.03.2024 Revised on 06.07.2024

Accepted on 05.09.2024 Published on 27.03.2025

Available online from March 27, 2025

Research J. Pharmacy and Technology. 2025;18(3):1096-1100.

DOI: 10.52711/0974-360X.2025.00157

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Determination of Total Flavonoid content:

Determination of DPPH radical scavenging activity:

Determination of hydrogen peroxide radical scavenging activity:

Scavenging of H2O2 by extracts may be attributed to their phenolics, which can donate electrons to H2O2.17

Scavenging of H2O2 by extracts may be attributed to their phenolics, which can donate electrons to H₂O_2.¹⁷