INTRODUCTION:

Diabetes mellitus (DM) is a metabolic condition characterized by impaired glucose homeostasis (chronic hyperglycemia) with disruptions of carbohydrates, fats, and proteins metabolism resulting from defects in insulin secretion, insulin action, or both^1,2. Chronic hyperglycemia during diabetes is interconnected to cardiovascular, kidney, nerve, and ocular dysfunctions. Moreover, its effects are mostly related to dyslipidemia, increased oxidative stress, and changes in the body's antioxidant defense mechanism³. Over the past few decades, diabetes cases and prevalence have gradually risen worldwide (over 690 million people by 2045)⁴, but it is a severe health issue and societal burden in developing nations⁵.

In countries like Jordan, medicinal plants account for around 20% of Jordan's flora^6,7. Due to the relatively high cost of allopathic medicines, it is advantageous to use local plant remedies. For to their efficiency, lack of clinically significant side effects, and affordable price, herbal medicines are gaining popularity^8,9.

In Jordan, several studies reported using indigenous medicinal plants to manage diabetes mellitus. Al-Aboudi and Afifi (2011) reviewed that Achillea santolina L., Ajuga iva L., Allium sativum L., Aloe vera L., Artemisia herba-alba Asso., and Capparis spinosa L. showed in vitro/ in vivo hypoglycemic activities¹⁰.

Issa et al. (2019) also reviewed other plants with an antidiabetic effect¹¹ such as Artemisia vulgari, Fucus vesicolosus, Lepidium sativum, Olea europae, Teucrium polium, Urtic apilulifera, Salvia triloba, and Cinnamomum ceylanicum.

Further, in the review reported by Abu-Odeh and Talib. (2021) some medicinal plants treat diabetes mellitus in the Middle East and Jordan. Achillea santolina, Geranium graveolens, Eryngium creticum, Pistaci aatlantica, and Varthemia iphionoides have an antidiabetic effect¹².

Ajuga orientalis L. was used by traditional healers to manage diabetes mellitus in Jordan. A. orientalis is an annual herbaceous flowering plant belonging to the family Lamiaceae, also known as the Eastern bugle. Ajuga species has been reported to have antibacterial¹³, antitumor¹⁴, antioxidant¹⁴, and anti-inflammatory properties¹³.

Most in vitro studies focus on the inhibitory effects of medicinal plants on α-glucosidase and α-amylase. Alpha-amylase digests dietary starch into maltose, which is digested by glucose in the intestine with alpha-glucosidase. Inhibition of these two enzymes delays the digestion of carbohydrates, lowering postprandial blood glucose levels^2,3. Most of the plants studied inhibit these two enzymes that regulate carbohydrate metabolism. However, different activity levels may be detected for a single plant, depending on extract preparation and study conditions¹⁵.

On the other hand, there are no prior investigations on A. orientalis antidiabetic effects. Therefore, the present study desired to assess the in vitro antidiabetic activity of aqueous and ethanolic extracts of A. orientalis aerial part.

MATERIALS AND METHODS:

Chemicals and Reagents:

All the chemicals and reagents were of AR grade and were procured from Sigma Chemical Co, USA.

Plant Material and Extract Preparation:

The fresh aerial parts of A. orientalis were collected from Ajloun county-Northern of Jordan (31.9494964, 35.9342189) in April 2022 and authentically identified by Prof. Dr. Sawsan Oran, University of Jordan, Amman- Jordan. A. orientalis aerial parts were air-dried at room temperature in the dark for about six weeks before being ground to a fine powder. Ten grams of powder were soaked in 100ml of solvents (absolute ethanol, distilled water) (1:10 w/v sample-to-solvent ratio) for 72 h at room temperature with frequent agitation to make ethanolic and aqueous extracts. The extracts were then filtered with Whitman filter paper (No.1), and the solvents were then evaporated using a rotary evaporator under reduced pressure. The crude extract was then collected and kept at 20°C¹⁴.

Alpha-amylase Inhibitory Activity:

The a-amylase inhibitory activity was conducted using the method reported by Ibrahim et al. (2017) with slight modifications¹⁶. The reaction mixture contained 1ml of (sample) A. orientalis extracts at concentration ranges of 100-500μg/ml and 1ml of a-amylase solution (0.5 mg/ml prepared in 0.20mM phosphate buffer (pH 6.9). The mixture was pre-incubated for 30 min, and then 1 ml of starch solution (1%) in 0.02mol/L sodium phosphate buffer (pH 6.9) was added to the reaction and incubated at 37°C for 10 min. The reaction mixture was terminated by adding 1ml of 3, 5-dinitrosalicylic acid reagent (DNS), and the mixture was boiled for 5 min. Acarbose(100-500μg/ml) was used as a standard (positive control). The absorbance of the reaction mixture was measured at 540nm using a UV- vis spectrophotometer. All assays were carried out in triplicate. The percentage of inhibition was determined using the following equation:

% Inhibition = [(Absorbance _Control - Absorbance _Sample) / Absorbance _Control] x 100

Alpha-glucosidase Inhibitory Activity:

The α-glucosidases inhibitory activity of A. orientalis extracts was determined by incubating 1ml of starch solution (2% w/v maltose) with 0.2M tris buffer (pH 8.0) and various concentrations of extracts (100-500 mg/ml). The reaction mixture was incubated at 37°C for 10min. The reaction was started by adding 1ml of the α-glucosidase enzyme (1U/ml) to the mixture and incubated at 35°C for 40 min. Then the reaction was terminated by adding 2ml of 6 N HCl. Acarbose was used as a positive control. All assays were done in triplicate. The absorbance of the mixture was measured at 400 nm via a spectrophotometer¹⁷. The inhibitory effect was calculated using the following equation.

% Inhibition = [(Absorbance _Control - Absorbance _Sample) / Absorbance _Control] x 100

Statistical Analysis:

All assays were conducted in triplicate, and values are expressed as the mean ± standard error of the mean (SEM) using GraphPad Prism9.0.2 (GraphPad Software, San Diego, USA). Furthermore, the IC₅₀ value was calculated to determine the concentration of the plant extract needed to inhibit 50% of a-amylase or a-glucosidase activity under assayed conditions. The differences between extracts were determined using one-way ANOVA and Tukey's post hoc test, where a p-value < 0.05 was considered statistically significant.

RESULTS:

α- amylase and α- glucosidase inhibition assay:

In the current study, A. orientalis extracts were evaluated for their inhibitory effect on α-amylase and α-glucosidase enzymes by the in-vitro method.

Both extracts significantly demonstrated inhibition to α-amylase and α-glucosidase enzymes in a dose-dependent manner (p-value< 0.05) (Figures 1 and 2). The aqueous and ethanolic extracts of A. orientalis (at a concentration of 500 mg/ml) exhibited 67.21% and 77.9% α-amylase inhibitory activity (Figure 1) and 72.23%, and 83.44% α-glucosidase inhibitory activity (Figure 2), respectively. However, an ethanolic extract inhibited α-amylase and α-glucosidase more than an aqueous extract. Acarbose was used as a positive control (standard), which showed the best α-amylase and α-glucosidase inhibitory activity of 89.18% and 93.35%, respectively, at a concentration of 500 μg/ml.

Moreover, the ethanolic extract of A. orientalis has shown higher enzyme inhibitory activity than the aqueous extract, with an IC₅₀ value of 226.79±3.22 and 209.81±1.33mg/ml (α-amylase and α-glucosidase) (Table 1) compared with that of acarbose, which showed α-amylase inhibitory activity with an IC₅₀ value of 94.96 ± 4.25mg/ml and α-glucosidase inhibitory activity with an IC₅₀ value of 64.15±4.22mg/ml (Table 1).

Table 1. Alpha-amylase and alpha-glucosidase inhibitory effects of aqueous and ethanolic extracts and of Ajuga orientalis, and acarbose.

	IC₅₀ values of α-amylase (mg/ml)	IC₅₀ values of α- glucosidase (mg/ml)
Aqueous extract of A. *orientalis* (AEAO)	346.41±2.10	306.21±2.22
Ethanolic extract of A. *orientalis* (EEAO)	226.79±3.22	209.81±1.33
Acarbose (Standard)	94.96±4.25	64.15±4.22

Values were expressed as Mean ± SEM (N=3).

Figure 1. In vitro α-amylase inhibitory activity of AEAO: Aqueous Extract of Ajuga orientalis and EEAO: Ethanolic Extract of Ajuga orientalis. Acarbose was used as a standard. Values are represented as Mean±SEM (n=3). *p<0.05, **p<0.01, ***p<0.001 vs. Acarbose (ANOVA, Tukey post hoc test).

Figure 2. In vitro α-glucosidase inhibitory activity of AEAO: Aqueous Extract of Ajuga orientalis and EEAO: Ethanolic Extract of Ajuga orientalis. Acarbose was used as a standard. Values are represented as Mean±SEM (n=3). *p<0.05, **p<0.01, ***p<0.001 vs. Acarbose (ANOVA, Tukey post hoc test)

DISCUSSION:

Chronic hyperglycemia in diabetes is associated with long-term damage and failure of various organ systems, most notably the eyes, nerves, kidneys, and heart, while macrovascular complications refer to increased atherosclerosis-related events such as myocardial infarction and stroke¹⁸.

Traditional diabetes medications are effective, but they also have unavoidable side effects. On the other hand, medicinal plants may serve as an alternative source of anti-diabetic agents^19,20. Many plants have been considered a primary source of powerful anti-diabetic drugs for centuries. Medicinal plants are used to treat diabetes in developing countries, mainly to alleviate the financial burden of conventional medicines on the population^21,22. Currently, medicinal plants treat diseases such as diabetes²³; because they contain diverse bioactive constituents such as flavonoids, saponins, terpenoids, carotenoids, alkaloids, and glycosides that may have anti-diabetic properties^24,25,26,27.

Postprandial hyperglycemia is primarily caused by two carbohydrate hydrolyzing enzymes (a-amylase and a -glucosidase). Alpha-amylase initiates carbohydrate digestion by hydrolyzing 1, 4-glycosidic linkages of polysaccharides (starch, glycogen) to disaccharides, and a-glucosidase catalyzes disaccharides to monosaccharides, resulting in postprandial hyperglycemia²⁸. As a result, a-amylase and a-glucosidase inhibitors help manage hyperglycemia because they delay carbohydrate digestion, lowering postprandial plasma glucose levels^29,30.

Many bioactive compounds derived from various plants may be responsible for enzyme inhibition (hypoglycemic effect). Most phenolics and triterpenoids positively correlate as anti-diabetic agents³¹. These bioactive constituents had a hypoglycemic effect via a variety of mechanisms. Polyphenolic compounds may interact with or inhibit specific enzyme positions, reducing the potency of α-amylase and α-glucosidase³². Flavonoid compounds may protect against diabetes by preventing glucose absorption or improving glucose tolerance through competitive inhibition of sodium-dependent glucose transporter-1³³. Moreover, flavonoid compounds such as luteolin, kaempferol, chrysin, and galangin reduced blood glucose levels by inhibiting α-amylase and α-glucosidase activity in the intestine³⁴.

In a study on the aqueous and ethanolic extracts of Ajuga remota, the results showed that the aqueous extract caused more reduction in blood glucose levels than ethanolic extract in diabetic mice ³⁵.

Furthermore, the methanolic and aqueous extracts of Ajuga iva showed inhibition of the vital digestive enzymes linked to type 2 diabetes, with a more potent inhibitory effect against a-glucosidase and a significant inhibition against a-amylase³⁶.

In 2020, Alene and his colleagues conducted an in vivo and in vitro study. They revealed that hydro-methanolic crude extract and its aqueous fraction of Ajuga integrifolia root possess significant anti-diabetic activity³⁷.

In Jordan, there was no available data about the in-vitro (α-amylase and α-glucosidase inhibitory activity) anti-diabetic studies of A. orientalis. As a result, the current study sought to determine the inhibitory activity of ethanolic and aqueous extracts of A. orientalis against α -amylase and α -glucosidase.

Previously, the phytochemical constituents of methanolic extract of A. orientalis were determined by Oran et al. (2022), where the major components were 9-octadecenoic acid, methyl ester, (E)- (27.2%), hexadecanoic acid, methyl ester (12.8%), and methyl stearate (9.6%). Also, Oran et al. (2022) reported that the ethanolic and aqueous extracts of A. orientalis have antioxidant properties and anti-cancer activity¹⁴.

The current study showed that both extracts significantly inhibited the α-amylase and α-glucosidase (p-value<0.05). Ethanolic extract of A. orientalis had more inhibitory activity to α-amylase and α-glucosidase than aqueous extract. Hence, it may be helpful in the management of diabetes mellitus.

CONCLUSION:

The results of this study show that A. orientalis ethanolic and aqueous extracts are effective α-amylase and α-glucosidase inhibitors, which may help reduce of postprandial glucose levels. However, the main compounds responsible for α-amylase and α-glucosidase inhibitory action must be identified and characterized further. Moreover, in-vivo studies are needed to confirm the obtained results further, and it could help develop new anti-diabetic agents based on native plant resources.

CONFLICT OF INTEREST:

The author has no conflicts of interest regarding this investigation.

REFERENCES:

1. Dilworth L, Facey A, Omoruyi F. Diabetes mellitus and its metabolic complications: the role of adipose tissues. Int. J. Mol. Sci. 2021;22(14):7644.doi: 10.3390/ijms22147644.

2. Anarthe S, Chaudhari S. Hypoglycemic Activity of Leucaslinifolia (Lamiaceae) Spreng. Res. J. Pharmacogn. Phytochem. 2011; 3:34–7.

3. Pereira ASP, Banegas-Luna AJ, Peña-Garcia J, Pérez-Sánchez H, Apostolides Z. Evaluation of the anti-diabetic activity of some common herbs and spices: providing new insights with inverse virtual screening. Molecules. 2019; 24:4030. doi: 10.3390/molecules24224030

4. Khalaf RA, Jarad HA, Al-Qirim T, Sabbah D. Synthesis, Biological evaluation, and QPLD studies of piperazine derivatives as potential DPP-IV inhibitors. Med. Chem. 2021; 17:937–44. DOI: 10.2174/1573406416666200917105401

5. Lin X, Xu Y, Pan X, Xu J, Ding Y, Sun X, et al. Global, regional, and national burden and trend of diabetes in 195 countries and territories: an analysis from 1990 to 2025. Sci. Rep. 2020; 10:1–11. doi: 10.1038/s41598-020-71908-9

6. Oran S. The status of medicinal plants in Jordan. J AgricSciTechnol A. 2014;4(6A):461–7

7. Oran S, Al-Eisawi D. Ethnobotanical Survey of the medicinal plants in the central mountains (North-South) in Jordan. JBES. 2015;6(3):381–400.

8. El-Dahiyat F, Rashrash M, Abuhamdah S, Abu Farha R, Babar Z-U-D. Herbal medicines: a cross-sectional study to evaluate the prevalence and predictors of use among Jordanian adults. J. Pharm. Policy Pract. 2020;13 (1):1–9. https://doi.org/10.1186/s40545-019-0200-3

9. Padmaja M, Praveen B, Reddy BR, Madhav PV. Therapeutic Efficiency of Leucasaspera (Lamiaceae) on Lead Acetate Induced Nephrotoxicity and Oxidative Stress in Male Wistar Rat. Asian J. Res. Chem 2011; 4:185–6.

10. Al-Aboudi A, Afifi FU. Plants used for the treatment of diabetes in Jordan: A review of scientific evidence. Pharm. Biol. 2011;49 (3):221–39. https://doi.org/10.3109/13880209.2010.501802

11. Issa R, Khattabi A, Alkarem TA, Altameemi O. The Use of antidiabetic herbal remedies by Jordanian herbalist: a comparison of folkloric practice vs. evidence-based pharmacology. Jordan J. Pharm. Sci. 2019;12 (3):23-37.

12. Abu-Odeh AM, Talib WH. Middle East medicinal plants in the treatment of diabetes: a review. Molecules 2021;26(3):742. https://doi.org/10.3390/molecules26030742

13. Purohit MC, Kandwal A, Purohit R, Semwal AR, Parveen S, Khajuria AK. Antimicrobial Activity of Synthesized Zinc Oxide Nanoparticles using Ajuga bracteosa Leaf Extract. Asian J. Pharm. Anal. 202; 11.

14. Oran SA, Althaher AR, Al Shhab MA. Chemical composition, in vitro assessment of antioxidant properties and cytotoxicity activity of ethanolic and aqueous extracts of Ajuga orientalis L. (Lamiaceae). J. Pharm. Pharmacogn. Res. 2022;10(3):486–95.

15. Seetaloo AD, Aumeeruddy MZ, Kannan RRR, Mahomoodally MF. Potential of traditionally consumed medicinal herbs, spices, and food plants to inhibit key digestive enzymes geared towards diabetes mellitus management—A systematic review. South African J. Bot. 2019; 120:3–24. https://doi.org/10.1016/j.sajb.2018.05.015

16. Ibrahim MA, Habila JD, Koorbanally NA, Islam MS. α-Glucosidase and α-amylase inhibitory compounds from three African medicinal plants: an enzyme inhibition kinetics approach. Nat. Prod. Commun. 2017;12 (7):1934578X1701200731. https://doi.org/10.1177/1934578X1701200731

17. SLDV RMK, Das MC, Vijayaraghavan R, Shanmukha I. In vitro evaluation of antidiabetic activity of aqueous and ethanolic leaves extracts of Chloroxylonswietenia. Natl. J. Physiol. Pharm. Pharmacol. 2017;7(5):486. DOI: 10.5455/njppp.2017.7.1235104012017

18. Mota RI, Morgan SE, Bahnson EM. Diabetic vasculopathy: macro and microvascular injury. Curr. Pathobiol. Rep. 2020;8(1):1–14. doi: 10.1007/s40139-020-00205-x.

19. Dirir AM, Daou M, Yousef AF, Yousef LF. A review of alpha-glucosidase inhibitors from plants as potential candidates for the treatment of type-2 diabetes. Phytochem. Rev. 2021;1–31. https://doi.org/10.1007/s11101-021-09773-1

20. Khamis M, Talib F, Rosli NS, Dharmaraj S, Mohd KS, Srenivasan S, et al. In vitro α-amylase and α-glucosidase inhibition and increased glucose uptake of Morindacitrifolia fruit and scopoletin. Res J Pharm Tech 2015; 8:189–93.

21. Verma S, Gupta M, Popli H, Aggarwal G. Diabetes mellitus treatment using herbal drugs. Int. J. Phytomedicine. 2018;10 (1):1–10. DOI: 10.5138/09750185.2181

22. Tiwari VJ. Ethnopharmacology of Leonotisnepetifolia (L.) R. Br., Lamiaceae, used to cure Jaundice and Liver Disorders by Baiga Tribe of Mandla District of Madhya Pradesh. Res. J. Pharmacogn. Phytochem. 2019; 11:1–7. DOI: 10.5958/0975-4385.2019.00001.3

23. Mamun-or-Rashid ANM, Hossain MS, Hassan N, Dash BK, Sapon MA, Sen MK. A review on medicinal plants with antidiabetic activity. J. Pharmacogn. Phytochem. 2014;3 (4):149–59.

24. Sewidan N, Khalaf RA, Mohammad H, others. In-Vitro Studies on Selected Jordanian Plants as Dipeptidyl Peptidase-IV Inhibitors for Management of Diabetes Mellitus. Iran. J. Pharm. Res. IJPR. 2020;19 (4):95.doi: 10.22037/ijpr.2020.1101232

25. Dongare P, Dhande S, Kadam V. A Review on Pogostemon patchouli. Res. J. Pharmacogn. Phytochem 2014; 691:41–7.

26. Shanaida M, Pryshlyak A, Golembiovska O. Determination of triterpenoids in some Lamiaceae species. Res. J. Pharm. Technol. 2018; 11:3113–8. DOI: 10.5958/0974-360X.2018.00571.1

27. Shagana S, Navenaa S, Vijay J, Vishali S, Rajendran V. Investigation of in vitro Anthelmintic activity of Ocimumbasilicum Linn .(Lamiaceae). Res. J. Pharm. Technol. 2021; 14:52–4. DOI: 10.5958/0974-360X.2021. 00010.X

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

29. Sathish M, Nandhini V, Suresh R. In vitro Alpha amylase and Alpha glucosidase enzyme inhibition of leaf extracts of JatrophaglanduliferaRoxb. Res. J. Pharm. Technol. 2022; 15:2493–7. DOI: 10.52711/0974-360X.2022.00416

30. Helen PA, Bency BJ. Inhibitory potential of Amaranthusviridis on α-amylase and glucose entrapment efficacy In vitro. Res. J. Pharm. Technol. 2019; 12:2089–92.DOI: 10.5958/0974-360X.2019.00346.9

31. Tran N, Pham B, Le L. Bioactive compounds in anti-diabetic plants: From herbal medicine to modern drug discovery. Biology (Basel). 2020;9(9):252. doi: 10.3390/biology9090252

32. Gong L, Feng D, Wang T, Ren Y, Liu Y, Wang J. Inhibitors of α-amylase and α-glucosidase: Potential linkage for whole cereal foods on prevention of hyperglycemia. Food Sci. Nutr. 2020;8(12):6320–37. doi: 10.1002/fsn3.1987

33. Alkhalidy H, Wang Y, Liu D. Dietary flavonoids in the prevention of T2D: An overview. Nutrients. 2018;10(4):438. doi: 10.3390/nu10040438

34. Gu C, Zhang H, Putri CY, Ng K. Evaluation of α-amylase and α-glucosidase inhibitory activity of flavonoids. Int J Food Nutr Sci. 2015; 2(2):174–9. DOI: 10.15436/2377-0619.15.042

35. Tafesse TB, Hymete A, Mekonnen Y, Tadesse M. Antidiabetic activity and phytochemical screening of extracts of the leaves of Ajuga remotaBenth on alloxan-induced diabetic mice. BMC Complement. Altern. Med. 2017;17(1):1–9. https://doi.org/10.1186/s12906-017-1757-5

36. Fettach S, Mrabti HN, Sayah K, Bouyahya A, Salhi N, Cherrah Y, et al. Phenolic content, acute toxicity of Ajuga iva extracts and assessment of their antioxidant and carbohydrate digestive enzyme inhibitory effects. South African J. Bot. 2019; 125:381–5. DOI : 10.1016/j.sajb.2019.08.010

37. Alene M, Abdelwuhab M, Belay A, Yazie TS. Evaluation of antidiabetic activity of Ajuga integrifolia (Lamiaceae) root extract and solvent fractions in mice. Evidence-Based Complement. Altern. Med. 2020; 2020. https://doi.org/10.1155/2020/6642588

Received on 15.07.2022 Modified on 30.08.2022

Research J. Pharm. and Tech 2023; 16(4):1828-1832.

DOI: 10.52711/0974-360X.2023.00300