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J. M. A. Hannan , Asma Ahammed , P. R. Flatt

Department of Pharmacy, School of Pharmacy and Public Health, Independent University, Bangladesh (IUB), Dhaka, Bangladesh

Diabetes Research Centre, School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland BT52 1SA, UK

Corresponding Author Email: jmahannan@iub.edu.bd

DOI : https://doi.org/10.51470/JPB.2025.4.1.117

Abstract

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1. Introduction

Diabetes mellitus is recognized as a significant health concern worldwide and is a primary determinant of disability, morbidity, and mortality. Nevertheless, progress in knowledge and therapeutic approaches presents significant challenges in managing diabetes [1] [2]. Type 2 diabetes is a widespread and critical chronic condition arising from complex genetic-environment interactions, aggravated by additional risk factors such as obesity and a sedentary lifestyle [3]. There are several different classes of drugs, both oral and injectable, for the management of type 2 diabetes (T2DM) [4]. However, these nonprescription drugs are costly, not freely available to some populations, and often associated with significant drawbacks, let alone the inability to insure against complications of the disease [5].

The World Health Organization (WHO) has documented approximately 21,000 plant species globally that are frequently used for medicinal purposes. Of these, around 2,500 species are utilized in India, with nearly 150 species being commercially available on a large scale. India is recognized as the largest producer of medicinal herbs and is often referred to as the “botanical garden of the world” [6]. According to a WHO survey, traditional medicine systems account for approximately 80% of healthcare treatments in India, 85% in Myanmar (Burma), and 90% in Bangladesh [7]. Medicinal plants play a significant role in the traditional management of type 2 diabetes mellitus (T2DM) across various countries. Many of these plants have demonstrated notable antidiabetic activity, often with minimal or no side effects.

These plants are rich sources of bioactive compounds such as flavonoids, alkaloids, phenolics, and tannins, which are known to enhance pancreatic islet function, stimulate insulin secretion or improve insulin action, and reduce intestinal glucose absorption [8]. Due to these properties, herbal medicines have gained widespread popularity, especially in communities with limited access to conventional pharmaceuticals [5]. Such plant-based therapies are often preferred in economically disadvantaged populations due to their affordability, accessibility, and lower risk of drug-related adverse effects. Consequently, they are increasingly being used as alternatives or adjuncts to synthetic antidiabetic drugs to help manage and reduce the complications associated with diabetes [9].

Several studies have demonstrated the antihyperglycemic benefits of medicinal plants in the management of type 2 diabetes mellitus (T2DM) [10]. One prominent example is Ocimum sanctum L. (syn. Ocimum tenuiflorum L.), commonly known as Holy Basil. This plant holds a distinguished place in traditional and folk medicine systems across Southeast Asia [11]. The leaves of O. sanctum have been widely used in Ayurvedic and Unani medicine for their therapeutic properties and have been traditionally applied in the treatment of various ailments, including diabetes, ulcers, and cancers. Additionally, the plant has been used to manage malaria, diarrhea, dysentery, skin diseases, arthritis, eye infections, insect bites, as well as bacterial and fungal infections [12].

Ocimum sanctum contains a complex profile of chemical constituents, including a variety of nutrients and bioactive compounds that contribute to its pharmacological properties [13]. However, the concentrations and efficacy of these bioactive ingredients can vary significantly depending on cultivation practices, harvesting time, processing techniques, and storage conditions [14]. Various parts of the plant—including leaves, stems, flowers, roots, seeds, and even the whole plant—have been employed in traditional medicine for their therapeutic potential.

Ocimum sanctum leaves have been traditionally utilized in the management of diabetes mellitus for many years. A dietary supplement of fresh leaves at a dosage of 2 g/kg body weight administered to albino rabbits for 30 days resulted in a significant reduction in blood glucose levels [15]. Additionally, tulsi leaf powder has been reported to significantly decrease serum and tissue lipid profiles in both normal and diabetic rats [16]. Oral administration of the extract led to a marked reduction in blood glucose levels in glucose-fed hyperglycemic and streptozotocin-induced diabetic rats [17]. Ethanolic extracts of O. sanctum also significantly reduced glycosylated hemoglobin and urea levels in streptozotocin-induced diabetic rats, while concurrently increasing levels of glycogen, hemoglobin, and total protein. These changes were accompanied by an increase in insulin levels and improved glucose tolerance [18].

It is believed that several bioactive compounds present in O. sanctum leaf extracts exert stimulatory effects on insulin secretion, thereby contributing to its antidiabetic properties [19]. Supporting this, research by Chandra et al. [20] demonstrated that oral treatment with O. sanctum extract at a dose of 500 mg/kg body weight significantly reduced blood glucose levels in insulin-deficient, streptozotocin-induced diabetic rats. The extract also inhibited lipid peroxidation, reactivated key antioxidant enzymes, and helped restore glutathione (GSH) and antioxidant metal levels. Furthermore, it induced a notable increase in liver glycogen content in both normal and alloxan-induced diabetic rats.

To further understand the multiple actions of Ocimum sanctum in type 2 diabetes, the present study aimed to investigate the chronic effects of an ethanol leaf extract on the glycemic, lipidemic, and platelet aggregation status in type 2 diabetic rats.

• Materials and Methods

• Plant material and preparation of the extract

Dried Ocimum sanctum leaves were procured from Ramkrishna Mission, Kolkata, India, and botanically authenticated, with voucher specimens deposited at the National Herbarium, Bangladesh. The leaves were first washed thoroughly with water, and petioles and stems were removed. The cleaned leaves were then oven-dried at 40°C, ground into fine powder (200 mesh) using a Cyclotec grinding machine, and stored in an airtight plastic container.

A total of 2 kg of the powdered O. sanctum leaves was subjected to extraction with 80% ethanol (10 L) in a stainless steel extraction tank over four days at room temperature, with the solvent being replaced daily. The combined extracts were filtered and concentrated to dryness using a rotary evaporator. Residual solvent was removed using a membrane pump. The final extract (275 g) was freeze-dried using a Varian 801-model LY-3-TT freeze-dryer (USA) and stored in a reagent bottle at 4°C in a freezer until further use.

• Animals

Adult male Long-Evans rats (180–220 g body weight) were used in all experiments. The animals were housed under controlled environmental conditions at the animal facility of the Bangladesh Institute of Research and Rehabilitation in Diabetes, Endocrine and Metabolic Disorders (BIRDEM), Dhaka, Bangladesh. They were maintained on a 12-hour light/dark cycle at a temperature of 21 ± 2 °C. The standard laboratory diet provided consisted of 36.2% carbohydrate, 20.9% protein, 4.4% fat, and 38.5% fiber, with a metabolizable energy content of 1.18 MJ/100 g (282 kcal/100 g).

All experimental procedures involving animals were conducted in accordance with local institutional guidelines, the Animals (Scientific Procedures) Act 1986, and the EC Directive 86/609/EEC for animal experimentation.

Induction of type 2 diabetes:

Type 2 diabetes was induced by a single intraperitoneal injection of STZ to 48-hour-old pups of adult Long-Evans [21]. The STZ solution was prepared fresh in 0.5M citrate buffer (pH 4.5) and was injected immediately following dissolution at a dose of 90 mg/kg body weight.  Experiments were conducted in male rats 3 months after injection, when they had gained a weight of approximately 175 – 180 g. Prior to experiments, the diabetic status of the rats was checked by blood glucose estimation. It is understood that no animal model is identical to any human disease; none of the available animal models of Type 2 diabetes mellitus exactly replicate the human Type 2 diabetes mellitus. However, n-STZ rat models have several benefits over the other models and are the appropriate experimental animal models of Type 2 diabetes mellitus [22].

• Chronic effects on glycemic, lipidemic, and body weight:

Type 2 diabetic rats (n = 24) were randomly allocated into two groups (n = 12 per group). The treatment group received Ocimum sanctum extract at a dose of 1.25 g/kg body weight, dissolved in 10 mL water/kg body weight, administered orally via a metallic gavage tube twice daily for 28 days. The control group received an equivalent volume of distilled water (10 mL/kg body weight). The selected extract dose (1.25 g/kg bw) was based on prior studies demonstrating significant hypoglycemic activity without observable toxicity.

All animals were maintained under identical environmental conditions and were given free access to standard laboratory chow and water throughout the study. Body weights were recorded on day 0 and at 7-day intervals thereafter. Blood samples were collected at baseline (day 0) from the tail vein under light ether anesthesia and on day 28 via the abdominal aorta under pentobarbital anesthesia. Serum was separated by centrifugation and used for the analysis of glucose, fructosamine, insulin, total cholesterol, HDL-cholesterol, triglycerides, and non-esterified fatty acids (NEFA). All serum samples were immediately stored at –70 °C until further biochemical analysis.

• Estimation of platelet aggregation:

Platelet aggregation test was conducted by Chrono Log Lumi aggregometer (Chronolog Corp., Havertown, PA, USA) linked to a potentiometric recorder. Platelet-rich plasma (PRP) was prepared using anti-coagulated blood samples (with 3.8% sodium citrate), which were carefully centrifuged at 800 rpm for 15 min, and a portion of the supernatant was transferred into a polypropylene plastic tube. To generate platelet-poor plasma (PPP), the same supernatant was again centrifuged at 3000 rpm for 20 min. This supernatant was then transferred into a polypropylene plastic tube. To estimate platelet aggregation, 500 µl of PRP and PPP were taken into cuvettes (P/N 312) and placed into the aggregometer. Which automatically sets the 100% (PRP) baseline. After 30 seconds, ADP (12 µl) was added to PRP using a micropipette, and the optical curve to run for 5 min. Platelet aggregation was presented as a percentage of the PPP transmission value.

• Acute metabolic studies

The non-diabetic mice housed in Nalgene Cages (USA) were administered with ethanol extract of Ocimum Sanctum (1.25g/kg bw). Food and water intake, as well as urination and defecation, were measured with an interval of one hour over 24 hours.

• Biochemical analysis

Serum glucose was estimated by glucose oxidase (GOD-PAP) based enzymatic colorimetric method using kits from Boehringer Mannheim GmbH (Germany). Serum fructosamine was assessed by a colorimetric method using commercial kits from Boehringer Mannheim GmbH (Germany). Serum triglyceride was estimated by an enzymatic colorimetric method using commercial kits from SERA PAK, USA. Serum total cholesterol was determined by the enzymatic colorimetric (cholesterol oxidase/peroxidase) method using kits from SERA PAK, USA. HDL-cholesterol was determined by enzymatic colorimetric (cholesterol oxidase /peroxidase) method using kits from SERA PAK, USA. Serum NEFA was measured by an enzymatic colorimetric method using kits from SERA PAK, USA.

• Statistical analysis

All statistical analyses were conducted using SPSS version 29 for Windows. Group comparisons were performed using the unpaired Student’s t-test or the Mann–Whitney U test, depending on the normality of the data. For variables measured at multiple time points, repeated measures ANOVA was employed, with Bonferroni correction applied to maintain an overall Type I error rate of 5%. A p-value of less than 0.05 was considered statistically significant.

Results

• Chronic effects on glycemic, lipidemic, and platelet aggregation status

After 28 days of feeding ethanol extract of O sanctum to type 2 diabetic rats,  serum glucose (p<0.01) and fructosamine (p<0.01) levels were substantially reduced by 25% and 14%, respectively, compared to the control group (Fig. 1). In addition, the extract lowered the total cholesterol (p<0.01) and triglyceride (p<0.01) levels by 29% and 26%, respectively, and increased HDL levels by 18% (p<0.05, Fig 2). The extract substantially reduced the NEFA by 50% (Fig. 3a). Platelet aggregation was decreased by 21% compared to the control group (p<0.05, Fig. 3b).

3.2. Effects on water intake, food intake, defecation and urination, and body weight

During 24-hour studies, O sanctum induced no changes in water intake, food intake, defecation, water content of defecation, or urination compared to the control group (Fig. 4 & 5). Over the 28-day study period, administration of ethanol extract of O sanctum did not change body weights as compared to the control group. The body weight increased in all the test groups from the initial to the final day (Fig. 6).

• Discussion

Oral hypoglycemic agents remain the cornerstone of Type 2 diabetes mellitus (T2DM) treatment and are generally effective in managing hyperglycemia. However, they are often associated with significant side effects and typically fail to prevent or delay the progression of diabetes-related complications [23][24]. With the growing understanding of the heterogeneity of T2DM, there is an increasing need for more effective therapies that offer fewer adverse effects. Despite advances in modern pharmacotherapy—such as the development of insulin, biguanides, sulfonylureas, thiazolidinediones, DPP-IV inhibitors, GLP-1 receptor agonists, and SGLT2 inhibitors [25]—many of these treatments remain costly, are not universally accessible, and may still fall short in addressing long-term complications. Consequently, in many developing countries, traditional antidiabetic plants offer a promising and accessible alternative. However, with the exception of a few studies [26][27][28], the therapeutic potential of many of these medicinal plants has not been thoroughly investigated.

There are some scientific reports [29], [16],[30] regarding the antihyperglycemic action of O sanctum, but its metabolic effects and pointers towards effects on diabetes complications have not been evaluated.  In this present study, we examined the effect of the ethanol extract of O sanctum on streptozotocin-induced type 2 diabetes rats in a chronic 28-day feeding experiment. As expected, we found that the extract significantly lowered serum glucose and fructosamine levels in the blood. Indeed, since serum fructosamine reflects the glycemic status over the preceding 2 to 3 weeks, this finding establishes, more conclusively, the anti-hyperglycemic properties of the ethanol extract of O sanctum.

Postprandial hyperglycemia is a well-recognized cardiovascular risk factor in patients with type 2 diabetes mellitus (T2DM) [31]. Studies have shown that elevated blood glucose levels after meals can double the risk of coronary heart disease and fatal cardiovascular events [32]. Furthermore, recent evidence suggests that tight glycemic control is closely linked to a reduced incidence and slower progression of microvascular complications. Similarly, effective management of hyperlipidemia significantly decreases both microvascular and macrovascular risks in individuals with T2DM [33][34].

Administration of ethanol extracts of Ocimum sanctum has been shown to reduce serum triglycerides and total cholesterol while concurrently increasing high-density lipoprotein (HDL) levels. These findings are consistent with previous studies [35][16][36]. Under normal physiological conditions, insulin activates the enzyme lipoprotein lipase, which plays a key role in hydrolyzing triglycerides [37]. However, in insulin-deficient states, this pathway is impaired, leading to hypertriglyceridemia. The ethanol extract of O. sanctum has been reported to enhance insulin secretion in streptozotocin (STZ)-induced hyperglycemic rats, which may help reduce triglyceride levels through the activation of lipoprotein lipase [19].

Patients with type 2 diabetes exhibit an elevated FFA level [38][39]. A high FFA can impair hepatic and peripheral insulin resistance and favour VLDL-triglyceride synthesis [40].  NEFA also influences platelet aggregation and vascular changes by accelerating the rate of prostacyclin in plasma [41][42]. In the present study, O sanctum leaf extract lowered NEFA levels. In an earlier study, it was observed that O sanctum is effective as an analgesic, inflammatory, and antipyretic agent [43]. As O sanctum has anti-inflammatory action, it was assumed that it might have some antiplatelet activity. The extract lowered the platelet aggregation, which might be due to the reduction of NEFA. This reduction in the concentration of serum NEFA may contribute to the improvement in insulin action. A study reveals that metformin significantly decreases serum TG concentrations and NEFA levels in HHTg rats [41].

High levels of triglycerides and total cholesterol in blood are major coronary risk factors [44][45]. Several studies have shown that an increase in HDL cholesterol is associated with a decrease in coronary risk [46]. Hence, the extract not only helps to reduce hyperglycemia but also helps to prevent dyslipidemia and insulin resistance, both of which are important risk factors for the complications of diabetes.

In the 28-day chronic study, the O sanctum-administered group did not exhibit any significant changes in body weight compared with control rats. To check the possible effects of ethanol extract of O sanctum on the normal metabolic processes, food intake, defecation, water content in stool, water intake, and urination were observed during a 24-hour study. It was found that there was no change in any of these parameters in O sanctum-fed mice as compared to the control group. This finding recommends that the extract of O sanctum does not alter behaviour, suggesting also a lack of toxicity.

• Conclusion

This research has exhibited that ethanol extract of O sanctum leaves lowers atherogenic lipids and NEFA in blood whilst also decreasing platelet aggregation. This indicates that O sanctum is beneficial as a source of invention of newer antidiabetic compounds and has promising benefits as an addition to the treatment of diabetes and its complications.

Ethical Approval

Principles of Laboratory Animal Care (NIH publication No. 85-23, revised 1985) were followed, as well as the UK Animals (Scientific Procedures Act 1986 and EU Directive 2010/63 EU) for all types of animal experiments and approved by the ethical committee.

Declaration of competing interests

The authors hereby declare that they have no conflicts of interest.

Acknowledgments

These studies were sponsored by the University of Ulster Research Strategy Funding and the Independent University, Bangladesh (IUB) URC Research Grant. All authors contributed to the idea and design of the experiment. J. M. A. Hannan and Asma Ahammed contributed to the experimental study, analysis, and preparation of the manuscript. Peter R. Flatt to the supervision of the study.

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