A metabolic condition known as diabetes mellitus is characterized by the presence of hyperglycemia as a result of impaired insulin production, impaired insulin action, or both (Rosa Martha et al., 2011). Numerous pathogenic mechanisms, such as the autoimmune death of pancreatic beta cells brought on by insulin shortage, can lead to the development of diabetes (Pettitt et al., 2014). Another cause of diabetes is a metabolic disease or abnormality. Anabolic peptide hormone insulin performs this action. Proteins, lipids, and carbs all exhibit aberrant metabolism when there is insufficient insulin in the body. One of the most prevalent metabolic illnesses, DM is spreading alarmingly around the globe (Hamidreza Ardalani et al., 2021). In just 34 years, the number of DM patients has quadrupled (from 108 million in 1980 to 422 million in 2014), while the global incidence of diabetes among individuals over the age of 18 has increased from 4.7% in 1980 to 8.5% in 2014 (Saruar Alam et al., 2021). By 2030, diabetes is expected to rank as the seventh leading cause of death, according to the WHO (Cristina Rey-Reñones et al., 2022). The International Diabetic Federation (IDF) anticipated that in 2021, the prevalence of diabetes was greater in urban (12.1%) than in rural (8.3%) places and in high-income (11.1%) than in low-income countries (5.5%). Between 2021 and 2045, middle-income countries are predicted to experience the largest relative increase in the prevalence of diabetes (21.1%), followed by high-income (12.2%) and low-income (11.9%) nations (Hong Sun et al., 2022). In addition, it is predicted that 536.6 million people globally have diabetes, with a projected increase to 783.2 million by 2045 if the prevalence of diabetes is not adequately controlled (Saruar Alam, 2021). The actual onset of the disease involves several risk factors. The main risk factors for prediabetes and DM are genetics, environment, loss of the very first phase of insulin release, sedentary lifestyle, lack of exercise, smoking, alcohol consumption, dyslipidemia, reduced-cell sensitivity, hyperinsulinemia, and improved glucagon activity (Lee et al., 2012; Knott et al., 2015; Akter et al., 2017; Danaei et al., 2011; Cornier et al., 2005; Chen et al., 2015; Maj et al., 2011; Schulze et al., 2005).  These elements seem to be crucial in the development of disease-causing insulin resistance or insulin nonfunctionality (Sharif IH et al., 2019; Saruar Alam et al., 2021). 

According to WHO (2011), T2DM affects 90% of patients, primarily due to increased body weight. Obstructive sleep and sleep disorders are frequent risk factors for insulin resistance and glucose sensitivity, two conditions that together lead to prediabetes and subsequently T2DM in overweight adult patients (Saruar Alam et al., 2021). The onset of diabetes is assumed to be favorably correlated with diets with low fiber but high glycemic indexes (GI) (Saruar Alam et al., 2021). South American and South-East Asian nations often have high or intermediate incidences. The region-stratified diabetes prevalence is estimated for a number of nations and shows which nations have the greatest proportion of diabetic individuals worldwide as of 2019 (Saruar Alam et al., 2021). With almost 116 million diabetes patients, China has the greatest percentage of diabetic patients among these nations. India comes in second on the list with 77 million people, followed by the US with 31 million people, indicating that the US will have one of the highest rates of diabetes in the ensuing ten years. According to projections, high-end diabetes populations include Pakistan, Brazil, and Mexico, with 19 million, 16 million, and 12 million diabetic people, respectively (Saruar Alam et al., 2021). IDF standing with 8.4 million cases, Bangladesh has the tenth-highest proportion of adults (20–79 years) with diabetes worldwide. By 2030, it is predicted to move up to ninth place, and by 2045, it will almost double to 15.0 million cases. In Bangladesh in 2019, an additional 3.8 million persons are projected to have had prediabetes. Given the high number of cases in Bangladesh, which places it among the Southeast Asian nations with the highest diabetes burden, policies promoting the introduction of diabetes prevention programs in this nation are urgently required (Islam et al., 2021; Atlas, D, 2015). Type 1, Type 2, and gestational diabetes mellitus (GDM) are the traditional categories for diabetes, according to the American Diabetes Association (ADA) (Saruar Alam et al., 2021).

Type 1 diabetes is one of the most prevalent endocrine and metabolic conditions in kids. People with type 1 diabetes can receive life-saving and ongoing care from insulin therapy. The self-management plan for a person with type 1 diabetes must include insulin delivery, blood glucose monitoring, physical activity, and a healthy diet (Bjornstad P et al., 2018; Ramachandran, A. 2014). Hyperglycemia in type 2 diabetes is caused by the bodys cells failure to respond adequately to insulin, a condition known as insulin resistance. Insulin resistance causes the hormone to be inefficient, which eventually leads to an increase in insulin production (Cristina Rey-Reñones et al., 2022). Inadequate insulin production can develop over time as a result of the pancreatic beta cells inability to keep up with demand (Rosa Martha et al., 2011). Type 2 diabetes is most frequent in older persons, although it is becoming more common in children and younger adults as obesity, physical inactivity, and poor nutrition become more prevalent. Furthermore, pinpointing the precise period of the start of type 2 diabetes is very hard (WHO, 2024). As a result, there may be a long delay between diagnosis and treatment, and up to one-third to half of people with type 2 diabetes may not even know they have the condition. If the illness has gone undiagnosed for a long time, complications including retinopathy or a non-healing lower-limb ulcer may be present at the time of diagnosis (Saruar Alam et al., 2021). Although there is no known cause of type 2 diabetes, there is a strong association between being overweight or obese, being older, and having a family history of the condition. Similar to type 1 diabetes, type 2 diabetes is brought on by a combination of multi-gene pre-disposition and environmental variables (World Health Organization, 2024). The promotion of a healthy lifestyle, which includes a nutritious diet, frequent physical activity, quitting smoking, and maintaining healthy body weight, is the cornerstone of type 2 diabetes management (Health Di-rect, 2024). Oral therapy is generally started with metformin as the first-line drug if lifestyle changes arent enough to regulate blood glucose levels. If a single anti-diabetic medicine isnt enough, a variety of combination therapy alternatives (such as sulphonylureas, DPP4 inhibitors, and GLP-1 analogs) are now available. Insulin injections may be required if oral drugs fail to control hyperglycemia to acceptable levels. However, the benefits of early screening, more effective treatment, and longer longevity, as a result, are also contributing to the growth in prevalence (Uraka-mi et al., 2018; Shahen MZ et al., 2019).

Gestational diabetes mellitus is described as glucose intolerance that develops during pregnancy; the prevalence of gestational diabetes and type 2 diabetes is increasing globally, resulting in significant healthcare and economic consequences (Health Direct, 2024). Insulin resistance in the mother is common throughout pregnancy and is critical for maintaining the maternal fuel supply to sustain the developing fetus. There are a few risk factors for gestational diabetes. The following risk factor has the potential of causing gestational diabetes in a pregnant woman (Bellamy et al., 2009; Khanolkar et al., 2020).

The risk factors include i) Strong familial history of diabetes, ii) Body mass index >30 kg/m2, iii) Having given birth to large infants (4 kg or more), iv) Previous gestational diabetes, v) Previous polyhydramnios, vi) Over the age of 30, and, vii) Previous unexplained fetal losses. Obesity is harmful to ones health. Obesity is characterized as an excess of adipose tissue that compromises both physical and mental health and well-being. Obesity has been linked to both type 1 and type 2 diabetes (Saruar Alam et al., 2021). The quantity of glycerol, hormones, cytokines, proinflammatory chemicals, and other compounds involved in the development of insulin resistance is increased in obese individuals. Another risk is obesity, which causes cell dysfunction, which plays a critical part in the development of diabetes. The flow chart below shows how fat leads to type 2 diabetes (Abdullah S. et al., 2014).

Obesity has already been deemed a global pandemic by the World Health Organization (WHO), making it one of the most pressing public health issues of our day. Overweight and obesity cause 80% of type 2 diabetes, 35% of ischemic heart disease, and 55% of hypertensive illness in people in Europe (Hamidreza Ardalani et al., 2021). Obesity is linked to the development of diabetes, as well as hypertension, lipotoxicity, and other diseases (Abdullah S et al., 2014). Traditional medicine in Bangladesh is a unique amalgamation of ethnomedical elements. It involves traditionally rooted features inspired by local indigenous people and close-by Indian Ayurveda and Unani medicine due to the countrys geographic location and social traits (Mohiuddin AK, 2019). Traditional medicine is a vital aspect of public health care in Bangladesh, with its practitioners strongly ingrained within the local community due to its low cost, easy accessibility, and well-established health services (Ashraf et al., 1982; Ahmed et al., 2009; Rahman et al., 2012).

Recent studies have shown that traditional medicine health services are widely used in Bangladesh (Amonkar et al., 1986) and are crucial in providing health care to the underprivileged, those residing in rural areas, and tribal people (Cristina Rey-Reñones et al., 2022). In Ayurveda and other Indian literature, it is suggested that plants can be used to heal a variety of human illnesses (Negin Mehdinezha et al., 2016). In India, there are approximately 45000 plant species, and many of them are said to have medicinal properties. Over 100 medicinal plants are included in the Indian system of medicine, including folk remedies for the treatment of diabetes that can be used singly or in combination (Hamidreza Ardalani et al., 2021).

Betel leaf is mostly used as betel leaf or in paan, with areca nut and/or tobacco, throughout Asia and by certain Asian emigrants elsewhere in the world. Piper betle Linn. (Piperaceae) is a perennial semi-woody climber that is dioecious. Strongly inflated at nodes, papillose when young, glabrous shortly after. Simple and yellowish green to brilliant green in hue, the leaves alternate (L.S.R. et al., 2008). Fertile branch leaves are 1–2 cm long, 1.2–1.8 mm thick when dry, and glabrous when mature. Berries form a fleshy spadix when they are produced seldom. Bangladesh, Sri Lanka, India, the Malay Peninsula, the Philippines, and East Africa are all home to the Piper betle (Rimando et al., 1986).

Taxonomy of the plant (Sarkar et al., 2008)

• Kingdom: Plantae
• Division: Magnoliphyta
• Class: Magnolipsida
• Order: Piperales
• Family: Piperaceae
• Genus: Piper
• Species: betle

In Bangladesh, Betel leaves are traditionally used for various purposes. Traditionally, green betel leaf is believed to be used to overcome bad breath and body odor, constipation, headache, nervous disorders, itching, rash, boils, rheumatism, blisters, and others (A Malik et al., 2017) Many Researchers already claimed that betel leaves possess antimutagenic, anticarcinogenic, antiplaque, antidiabetic, anti-inflammatory, and antibacterial bioactivities (Gori et al., 1998; Ponnusamy et al., 2010; Amonkar et al., 1986; Padma et al., 1989; Arambewela et al., 2005). 

Ethics approval

Ethical approval was taken from the Ethical Review Board of Bangladesh University of Health Sciences (BUHS) and ethical guidelines of BUHS were followed throughout the study (Rosa Martha, 2011).  All surgical procedures were carried out under anesthesia, and pain was kept to a minimum. Sodium pentobarbital was injected intraperitoneally to anesthetize the rats.

Collection and extraction of Piper betle leaves

Fresh betel leaves were collected from the local market in the Demra region. Botanically authenticated voucher specimens were deposited in the National Herbarium (DACB 49727), Bangladesh. The betel leaves were sun-dried completely. The P. betle was dried and prepared in small pieces by convenient methods. The dried sample was extracted with methanol according to the maceration technique. Obtaining soluble part was concentrated by using Rota-evaporator and finally dried by a Freeze dryer. The dried extract was stored in an airtight container and kept in a freezer until use. The crude extract was used for preliminary phytochemical screening according to standard protocol and bioassay on type 2 diabetic model rats (Yazachew Engida Yismaw et al., 2020).

Phytochemical screening of Piper betle

3g of Piper betle with 100% methanolic extract was boiled with 30 ml distilled water for 5 minutes in a water bath and was filtered while hot. The extract sample or filtrate was being taken for the experiments wherever applicable using standard protocols to test the presence of bioactive compounds. The following protocols were used in the screening test: (Gayathri, V. and Kiruba, D, 2014).

i) Test for Alkaloids (Hagers Test)

1 ml of filtrate was taken and 3 ml of Hagers reagent was mixed in it and observed for the formation of a yellow precipitate. 

ii) Test for Tannins 

1 ml of cool filtrate was mixed with 5 ml distilled water and a few drops (2-3) of 10% Ferric chloride were added and observed for any formation of bluish-black or brownish-green color. This color indicated the presence of tannins. 

iii) Test for Saponins (Forth test)

2.5 ml of cool extract was diluted with 10 ml of distilled water and shaken vigorously for 2 minutes. Frothing indicated the presence of saponins (Negin Mehdinezhad et al., 2016). 

iv) Test for Flavonoids 

1 ml of cool filtrate was mixed with a few fragments of Magnesium ribbon and concentrated HCL was added dropwise (Jahnovi Brahma and Dhananjoy Narzary, 2015). Pink scarlet color indicated the presence of flavonoids. 

v) Test for Phenol 

2 ml of filtrate was taken then freshly prepared 1% Ferric chloride and 1 ml of potassium-Ferro-cyanide was added to it. The formation of bluish-green color indicated the presence of phenol (Negin Mehdinezhad et al., 2016). 

vi) Test for Steroids and Terpenoids (Salkowski Test) 

1 ml of filtrate was mixed with chloroform and a few drops of concentrated sulphuric acid then shaken and allowed to stand for some time (Negin Mehdinezhad et al., 2016). Red color appearance in the upper layer indicated steroid. The yellow color in the lower layer indicated terpenoids. 

vii) Test for Quinine

1 ml of conc.H2SO4 was added to the plant extract sample (1 ml) (Negin Mehdinezhad et al., 2016). Red color appearance designated the existence of quinones in the test samples.

viii) Glycosides 

25ml of dilute sulphuric acid was added to 5ml extract in a test tube and boiled for 15 minutes, cooled, and neutralized with 10% NaOH, then 5ml of Fehling solution was added (Jennifer Adline and Anchana Devi, 2014).

Biological analysis 

The current study was analyzed to determine the biochemical status of type-2 diabetic modal rats and considers blood glucose level, lipid profile, liver glycogen, and insulin level as a parameter to investigate the anti-diabetic activity of Piper betle.

Experimental Animals

Long Evans rats, bred at BUHS animal house and weighing between 222-232g were used in the study (Yazachew Engida Yismaw et al., 2020). The rat was housed in six rats per cage and the temperature was maintained at 22±2˚C with a 12hr light-dark cycle with access to feeding with a standard pellet diet and water ad libitum (Rosa Martha et al., 2011). Type 2 diabetic model of rats was induced with an intraperitoneal injection of streptozotocin (STZ) with a dose of 90mg/kg to 48 hr old pups. After three months, an OGTT test was performed and rats with serum glucose levels 7-8 mmol/L or more were considered for the experiment.

Experimental groups

A total of 24 rats 6 normal rats and 18 type 2 diabetic model rats were used within 28 days experimental period. They were divided into 4 groups as shown in Table 1. Every group contains six rats (n=6) (Pidaran Murugan and Leelavinothan Pari, 2006). 

Table 1: Name of the experimental groups.

Statistical Analysis

Statistical analysis was performed using Statistical Package for Social Sciences (SPSS, version 12, Chicago, IL, USA). Results were expressed as mean ± SD (Negin Mehdinezhad et al., 2016). Statistical evaluation of data was performed by using one-way analysis of variance (ANOVA) and paired t-test. The level of significance was considered at p=0.05 (Rosa Martha et al., 2011).

Preliminary phytochemical screening

In the present study, preliminary phytochemical analysis was carried out on Piper betle extract to identify the presence of phytochemical constituents (Table 2) (Negin Mehdinezhad et al., 2016).

Table 2: Screening of phytochemicals in the methanol extract of Piper betle (Abdulelah H Al-Adhroey et al., 2011).

(+): present, (-): absent, (+++): strongly present

The phytochemical screening revealed the presence of a number of phytochemicals in P. betle like tannins, saponins, phenols, flavonoids, terpenoids, quinine, and glycosides.

Effect on the body weight of rats after different treatments 

In the study it was observed that there is a slight tendency of decrease in body weight in the entire group compared with initial day expect normal water control group (Rosa Martha et al., 2011). Changes in body weight in different groups of rats have been depicted in Table 3. Initial body weight (g) were226±1.60, 222±1.63, 228± 4.02, and 232±3.26(M±SD) in normal water control, diabetic water control rats, gliclazide treated and P. betle treated diabetic model rats respectively. No significant changes were observed during the experimental period compared to the baseline values as well as among different treated groups.

Table 3: Effect of Piper betle methanolic extract treated type-2 diabetic model rats body weight (Rosa Martha et al., 2011).

Group NDWC, DWC, GT, & P. betle represent non diabetic water control rat, diabetic water control rat, Gliclazide treated diabetic rat, Piper betle treated diabetic rat respectively. Statistical comparison between groups was performed using one-way ANOVA and paired sample t-test.

Effect of P. betle  on serum glucose level of type 2 diabetic model rats

The fasting serum glucose level at the initial and end day of the experiment was demonstrated in Fig. 1. The baseline value of serum glucose level was (M+SD, mmol/L) 7.95±0.13, 8.59±0.08, 9.49±1.49, 9.14±0.94 and the 28th-day serum glucose level was 7.91±0.32, 8.74±1.75, 7.25±0.69, and 6.03±1.12 in NDWC, DWC, GT, and P. betle treated groups respectively. As expected, there was no change in serum glucose level in the normal water control group (Saruar Alam et al., 2021). On the contrary, serum glucose level of DWC group increased by 1% compared to baseline value. It was observed that there was a significant (p≤0.01) decrease in serum glucose level in P. betle treated and GT treated groups when compared to the baseline value (0 day vs 28day) (Rosa Martha et al., 2011). The results showed that P. betle had a significant (p≤0.001) antidiabetic effect in relation to fasting serum glucose level when compared to the DWC control groups (Hamidreza Ardalani et al., 2021).

Fig. 1: Effect of methanolic extract of Piper betle on fasting blood sugar levels at the initial day of the experiment (0th day) and the final experimental day (28th day) of diabetic rats. NDWC: non-diabetic water control; DWC: diabetic water control GT: Glyclazide treated and Piper betle treated diabetic rats (Md. Shamim Gazi et al., 2022).Data presented as mean±standard deviation (M±SD). Statistical comparison between groups was performed using one-way ANOVA and paired sample t-test.p*≤0.01, p**≤0.001.

Effects of Piper betle extract on lipid profile of type-2 diabetic model rats

The Chronic effect of Piper betle on lipidemic status of type 2 diabetic rats was also observed (Table 4). There were significant (P≤0.001; P≤0.02) reduction in serum triglyceride (TG) and cholesterol level shown in gliclazide and Piper betle treated group on 28th day respectively. Moreover, Piper betle treated group also significantly (p≤0.001) decreased serum TG and cholesterol level when compared to the DWC (Rodríguez-Correa et al., 2020). However, HDL cholesterol level of was significantly (p≤0.01) increased when comparing with initial day value by Piper betle treated group. Also 28days feeding of Piper betle extract LDL cholesterol 10% level was decreased when compares to the base line value. Whereas GT group reduces 35% and 9% in serum TG, cholesterol and LDL cholesterol respectively and also increases significantly (p≤0.001) HDL level.

Table 4: Effect of Piper betle extract on lipid profile (Rosa Martha et al., 2011). 

Results were expressed as Mean ±SD (Negin Mehdinezhad et al., 2016). Statistical analysis between groups; comparison was done by using one-way ANOVA with post hoc Bonferroni test and within-group comparison was done by paired samples t-test. NDWC= Nondiabetic water control; DWC = Diabetic Water Control; GT = Glicazide treated group. M.B= Musa balbisiana treated group; TG= Triglyceride, HDL= High-Density Lipoprotein, LDL= Low-Density Lipoprotein. p*≤0.02, p**≤0.001.

Effect of Piper betle extract on hepatic glycogen Content of  Type 2 Diabetic model rats

The effect of methanolic extract of Piper betle on hepatic glycogen level in type-2 diabetic model rats has been represented in (Fig. 6). At the end of the experiment, the glycogen content of NDWC, DWC GT and P. betle treated groups were 20.49±0.68, 16.10±1.33, 18.75±0.21 (16%↑), 20.02±0.74 (24%↑) (Mean ±SD) respectively (Rosa Martha et al., 2011). Piper betle GT treated group showed 24% increase of hepatic glycogen level compared to DWC group. Moreover, GT treated group increased 16% glycogen content compared to DWC.

Fig. 2: Effect of hepatic glycogen content groups NWC, DWC, GT, Piper betle, represent Normal diabetic water control rat, gliclazide treated diabetic rat treated rat respectively (Shinu Pottathil et al., 2020) Data presented as mean±standard deviation (M±SD). Statistical comparison between groups was performed using one-way ANOVA.

Chronic effects of Piper betle on insulinemic status of type-2 diabetic model rats

A significant (p≤0.001) increment of insulin level was observed in Piper betle treated group compared with the baseline value (Table 5). Piper betle also significantly (p≤0.006) increase serum insulin levels when compared to the positive control GT group treated rats (Yazachew Engida Yismaw et al., 2020) In case of other groups, the gliclazide treated group and normal water control group, the serum insulin level was found to be increased by 29% whereas DWC group, the serum insulin level decreased by 4% (Mohammad Mahedi Hassan Tusher et al., 2021). 

Table 5: Effects of Piper betle on insulinemic status of type-2 diabetic model rats (Mohammad Mahedi Hassan Tushe et al., 2021). 

Group NDWC, DWC, GT & P. betle represent non diabetic water control rat, diabetic water control rat, Gliclazide-treated diabetic rat, Piper betle treated diabetic rat respectively. Statistical comparison between groups was performed using one way ANOVA and paired sample t test. P*≤0.001,p**≤0.006.

The demand for more effective treatments is increasing as scientists get a greater knowledge of the heterogeneity of diabetes, which is possibly the metabolic ailment with the highest global growth (Baily, C.J. and Flatt, P.R., 1986). Since reactive oxygen species (ROS) (T2DM), a by-product of oxygen stress, are thought to be the root cause of -cell dysfunction, insulin resistance, poor glucose tolerance, and type 2 diabetes mellitus (Saruar Alam et al., 2021). Micro- and macrovascular dysfunctions have also been mentioned as long-term effects of diabetes (Wright Jr. et al., 2006). The need for alternative diabetic treatment options arises from the high cost of complex medications. All across the world, medicinal herbs have long been used to treat diabetes mellitus and other diabetic problems (Negin Mehdinezhad et al., 2016). The future potential of a diabetologists pharmacist may be unlocked by analysing such drugs (Hassan et al., 2018).

P. betel is a well-known traditional medicinal plant with numerous biological and pharmacological benefits, including the capacity to reduce blood sugar and serum cholesterol levels. Additionally, it has a long history of usage in the management of hyperlipidemia and diabetes (Hamidreza Ardalani et al., 2021). The current study sought to determine the impact of a 28-day chronic dose of a methanol extract of Piper betle leaves on type 2 diabetic model rat blood glucose, lipids, insulin, and liver glycogen levels (Jihye Seo et al., 2022). By administering STZ, a pancreatic cell toxin that splits the -cell into glucose and methylnitrogenase, to 48-hour-old pups, type 2 diabetes was created (Mohiuddin AK, 2019). The latter kills cells, breaks down DNA, and alkylates and alters macromolecules, leading to diabetes (Hassan et al., 2018; Rao et al., 2006) Early cell damage led in partial cell regeneration, resulting in type 2 diabetes due to insulin resistance in target tissues and reduced insulin production, as well as increased adiposity (Abdullah S et al., 2014). When at the age of 3 months these rats have been checked with a glucose load all of them could not cope with the glucose load. Although their fasting glucose levels were slightly higher (ranging from 7.38 to 7.56), they did have some cells. These rats had considerably greater postprandial glucose levels, indicating that they had developed type 2 diabetes. 

The phytochemical screening revealed the presence of a number of phytochemicals in P. betle like tannins, saponins, phenols, flavonoids, terpenoids, quinine, and glycosides (Table 2) (Hiwa M Ahmed. 2018). Existing scientific data supports the present findings that P. betle contains essential phytochemicals (tannins, phenols, flavonoids,  saponins, terpenoids, quinine, and glycosides) that are crucial for glucose homeostasis (Unusan, 2020). Therefore, the obtained hypoglycemic effect may be due to the presence of phytochemicals in P. betle  (Yazachew Engida Yismaw et al., 2020). In 28 days experimental period consecutive feeding of the methanol extract Piper betle (1.25mg/kg) didnt show any significant change in the body weight of the experimental rats. All of the groups had a tendency to decrease body weight when compared with the baseline group (Rodríguez-Correa et al., 2020). Previous report claimed that treatment of young adult rats with streptozotocin (STZ) produces a diabetic state that is characterized by loss of weight, polydipsia, and polyuria (F. C. Howarth et al., 2005) A highly significant reduction of fasting serum glucose level was observed after 28 days of consecutive oral feeding of Piper betle leaves when compared to the baseline value and type 2 diabetic control group DWC (p≤0.01& p≤0.001) (figure-5) (Rosa Martha, 2011). Previous researchers found the hypoglycemic effect of ethanol extract of Piper betle leaves in STZ-induced diabetic rats. So, this finding potentiates our finding (Hamidreza Ardalani et al, 2021). It is now well established that relentless hyperglycemia in type 2 diabetic patients is an important cardiovascular risk factors (Saruar Alam et al., 2021). The affiliation of hyperglycemia with a modification of lipid parameters acquaints a major risk for cardiovascular complications in diabetes. Along with hyperglycemia, hyperlipidemia also leads to significant micro- and macrovascular complications in individuals with type 2 diabetes (Abdullah S et al., 2014). 

Hence, the effects of Piper betle on lipid profiles were also examined after 28 days of experiment and found that methanolic extract of Piper betle has significant (p≤0.001,p≤0.02) serum TG and cholesterol-lowering effect on 28th day compared to initial day of the experiment. Phytochemical screening showed the presence of tannins (Table 4) (Negin Mehdinezhad et al., 2016). those have anti hyperlipidemic activity as researchers that tannins have the capacity to reduce cholesterol by decreasing dietary absorption of cholesterol (Negin Mehdinezhad et al., 2016). Moreover HDL cholesterol level was significantly (p≤0.01) increased when compared to the base line value (Rodríguez-Correa et al., 2020). Beside these methanolic extract of Piper betle have an anti-atherogenic effect as it lowered LDL-cholesterol by 10% on the final day compared to the initial day of the experiment (Badrul Alam et al., 2013). Thirunavukkarasu Thirumala and his colleagues also revealed that methanolic leaf extract of P. betel exhibited a significant hypolipidemic activity on high-fat diet-induced hyperlipidemic rat (Thirunavukkarasu Thirumalai et al., 2014). Another researcher found that Piper betle leaves ethanol extract has a lowering capacity of total cholesterol and LDL-cholesterol (Saravanan, R. and Pugalendi, K.V., 2004). Therefore, the obtained hypolipidemic activity by methanol extract of  Piperbetle is in accordance with other investigators.

Current investigation also evaluated that Piper betle significantly (p≤0.006) (Table 5) increased the serum insulin level when compared to the positive control GT group rats (Yazachew Engida Yismaw et al., 2020). According to Arambewela et al. Piper betle extract has the capacity to boost insulinomic activity for decreasing blood glucose levels in Streptozocin (STZ) induced rats (Rosa Martha, 2011). The effects of Piper betle after 28 days of chronic treatment on hepatic glycogen content of type 2 diabetic model rats is presented in (Fig. 6) (Rosa Martha, 2011). It is clear from the figure that Piper betle 24% increase the hepatic glycogen level. Thus, this finding strongly suggests that Piper betle methanolic extract possesses hypoglycemic and lipid-lowering properties.

Moreover, liver glycogen estimation revealed that a stimulatory effect on hepatic glycogenesis which could be the possible reason to decrease the blood glucose level (Hamidreza Ardalani, 2021). Author Santhakumari and their colleagues demonstrated that insulin increases hepatic glycolysis by increasing the activity and amount of several key enzymes, including glucokinase, phosphofructokinase, and pyruvate kinase (Muthukumaran Jayachandran et al., 2018) Hexokinase is universally present in cells of all types. Hepatocytes also contain a form of hexokinase called hexokinase D or glucokinase, which is more specific for glucose and differs from other forms of hexokinase in kinetic and regulatory properties (Arambewela et al., 2005). Glucokinase (also called hexokinase IV) catalyzes the conversion of glucose to glucose6-phosphate and plays a central role in the maintenance of glucose homeostasis. In liver, the enzyme is an important regulator of glucose storage and disposal (Howarth et al., 2005). The treatment of P. Betle elevated the activity of glucokinase in the liver (Saruar Alam et al., 2021). P. betle may stimulate insulin secretion, which may activate glucokinase, thereby increasing utilization of glucose, and thus the increased utilization leads to a decreased blood sugar level that was found in our type 2 diabetic rats treated with P. betle methanol extract.

The findings of the present study suggest that the methanolic extract of Piper betle leaves was effective in reducing blood glucose levels in type-2 diabetic model rats and therefore possesses significant hypoglycemic properties (Badrul Alam et al., 2013). It also decreased cholesterol, Triglyceride (TG), and Low-Density Lipoprotein (LDL) levels and increased High-Density Lipoprotein (HDL) levels. Therefore, Piper betle has hypolipidemic activity on type-2 diabetic model rats. Thus, Piper betle leaves may represent a potential functional food for the prevention and management of type-2 diabetes and its attendant complications.

M.S.A.R.: Conceptualization, visualization and contribution to investigation; B.R.: Investigation, writing and checking the manuscript, editing and funding acquisition; M.M.H.T.: Writing and checking the manuscript along with editing.

The authors are thankful to the Department of Pharmacology and the Department of Chemistry at Bangladesh University of Health Sciences (BUHS) for granting the authors permission to use their lab facilities throughout the study. We also acknowledge the National Herbarium in Dhaka, Bangladesh, for helping us identify the plant.

The author(s) declared that there is no conflict of interest.