Acute Anti-Hyperglycaemic Activity of Five Traditional Medicinal Plants in High Fat Diet Induced Obese Rats

Background : Diabetes mellitus (DM), a prevalent non-communicable disease, is a metabolic condition involving defective pancreatic β -cells and/or insulin resistance. Researchers are presently exploring traditional medicinal plants to identify alternatives for treating diabetes due to the various disadvantage of current anti-diabetic medicines. Objective : The present study evaluated the anti-hyperglycaemic effects of ethanol extracts of five medicinal plants (EEMPs) ( Gynura nepalensis , Glochidion thomsonii , Clerodendrum splendens , Clero-dendrum infortunatum and Xanthium strumarium ) which are traditionally used as an ethnomedicine to treat diabetes and numerous other health problems. Methods : High-fat fed (HFF) obese rats were used to perform acute in vivo tests, including oral glucose tolerance, feeding test, metabolic studies, and gastrointestinal motility using BaSO4 milk solution. Priliminary phytochemical screening were performed to discover the presence or absence of alkaloids, tannins, saponins, steroids, glycosides, flavonoids, and reducing sugars in extracts. Results : Oral administration of ethanol extracts (250 mg/kg, body weight), along with glucose (18 mmoL/kg body weight), ameliorated glucose tolerance ( p < 0.05–0.01). In addition, the extracts improved gut motility (250 mg/kg; p < 0.05–0.001), as well as reduced food intake during the feeding test (250 mg/kg; p < 0.05–0.001). Phytochemical screening of these medicinal plants depicted the presence of flavonoids, alkaloids, tannins, saponins, steroids and reducing sugars. Conclusions : Phytochemicals such as flavonoids, tannins and saponins may be responsible for the glucose-lowering properties for these plants. Additional research is warranted to fully identify the bioactive phytomolecules and mechanistic pathways that might lead to the development of a viable, cost-effective type 2 diabetes therapy.


Introduction
Mortality rates in diabetes mellitus (DM) are consistently rising across the globe, affected approximately 10% of the adult population worldwide [1]. DM is characterized as a chronic disease resulting from insulin resistance, inadequate insulin production or both. The three main categories of diabetes are: type 1, type 2, and gestational diabetes [2]. Type 1 diabetes mellitus (T1DM) also known as insulin-dependent diabetes, accounts for 10% of all diabetic patients and is distinguished by the almost total loss of pancreatic β-cells [3]. Type 2 diabetes mellitus (T2DM) or non-insulin-dependent diabetes mellitus, accounts for 90% of all diabetic patients and is classified as defective insulin signalling and/or insufficient production of insulin. Obesity, physical inactivity, genetic factors, and chronic hyperglycaemia are the main contributors of T2DM [4]. Chronic hyperglycaemia associated with T2DM results in a number of secondary conditions including both macro and microvascular complications, and without proper treatment these may result in cardiac arrest, stroke, blindness, and renal failure [5,6].
The initial steps in managing T2DM are a proper diet, weight management, as well as regular physical activity [7]. In addition to these, various medication categories, including metformin, sulphonylureas, thiazolidinediones, Glucagon-like peptide 1 (GLP-1) analogues, Glucose-dependent Insulinotropic Peptide (GIP) and GLP-1R co-agonists, dipeptidyl peptidase-IV (DPP-IV) inhibitors, sodium-glucose co-transporter 2 (SGLT2) inhibitors and synthetic insulin, are now used as treatments for T2DM [8]. However, these synthetic medications come with a host of negative side effects, such as gastrointestinal abnormalities, obesity, hepatic and renal disorders, low blood sugar level, and are often expensive and unavailable to rural and economically deprived areas [4,9,10]. As a result, researchers are devoting a great deal of time and energy into studying plants and other natural sources in an effort to discover new treatments to combat diabetes mellitus [11]. Since ancient times, medicinal plants, such as Gynura nepalensis DC., Glochidion thomsonii (Müll.Arg.) Hook.f., Clerodendrum splendens G.Don, Clerodendrum infortunatum L. and Xanthium strumarium L. have been employed in local communities to treat illnesses, such as diabetes, hy-pertension, liver disease, lung problems, microbial infections, cancer, inflammation, and gastrointestinal problems, due to their numerous health benefits [12][13][14][15][16]. These plants contain a variety of potent bioactive phytoconstituents, including rutin, quercetin, linolenic acid, epicatechin, gymnemic acid, kaempferol, berberine, gallic acid and iminosugars which is particularly present in Casuarina equisetifolia L., are capable of reducing high blood sugar levels by increasing insulin secretion, inhibiting glycosidase and glycosyl transferase activity as well as protecting pancreatic β-cells from damage, inhibiting DPP-IV enzyme activity and thus, these compounds may aid researchers to discover and develop alternative therapies to treat T2DM [17][18][19][20][21][22].
The aim of the current study was to evaluate glucose lowering properties of five traditional medicinal plants (X. strumarium, C. infortunatum, C. splendens, G. thomsonii and G. nepalensis) including the effects on food intake and gut motility in vivo.

Collection and Preparation of Plant Extracts
The five plants, X. strumarium (leaves), C. infortunatum (roots), C. splendens (leaves), G. thomsonii (bark) and G. nepalensis (leaves) were obtained from Jahangirnagar University, Dhaka, Bangladesh, and a plant taxonomist from Bangladesh National Herbarium identified and allocated their individual accession numbers DACB87271, DACB87272, DACB87273, DACB87274 and DACB87275 respectively. Following rinsing and air drying of the plant parts, 200 g of the dried plant powder was added to 1 liter of 80 % (v/v) ethanol and shaken at a speed of 900 g for 48 to 72 h at room temperature. The mixture was separated with filter paper (Whatman no. 1), and a rotary evaporator (BibbyRE-200, Sterilin Ltd., Newport, UK) was then used to dehydrate the filtered extract. The final product was lyophilized in a freeze dryer vacuum (Savant Speed vac, New York, NY, USA), and then preserved at 4°C for further experiments [23].

Animals
Six to eight weeks old Long Evan male rats (200-250 g) were fed a high-fat diet (20% of protein, 45% of fat, and 35% of carbohydrate: 26.15 KJ/g total energy percent) for 6 to 8 weeks before the start of the studies. For normal control, same aged rats received a standard rodent diet (30% protein, 60% carbohydrate, and 10% fat, making 12.99 KJ/g total energy, Trouw Nutrition, Cheshire, UK) were used. Before conducting the experiments, fasting blood glucose were measured in high fat fed (HFF) diet rats to separate in individual group. Higher than normal fasting blood glucose levels (5.6 to 7.0 mmol/L) were considered as HFF diet-induced obese rats. The fasting blood glucose of 10 rats were within the normal range which had been excluded from the studies. The groups were divided as follows: Group 1: Lean control (saline) Group 2: HFF diet control (saline) Group 3: HFF diet control + G. nepalensis (250 mg/kg) Group 4: HFF diet control + G. thomsonii (250 mg/kg) Group 5: HFF diet control + C. infortunatum (250 mg/kg) Group 6: HFF diet control + X. strumarium (250 mg/kg) Group 7: HFF diet control + C. splendens (250 mg/kg) Group 8: HFF diet control + Glibenclamide (5 mg/kg)

Oral Glucose Tolerance
Rats given a high-fat diet were starved for 12 h and administered glucose (18 mmol/kg, body weight (b.w.)) alone (control) or in conjunction with ethanolic extracts (250 mg/kg, b.w.) orally. Blood samples were drawn from the tail vein prior to (0 min) and after (30, 60, 120, and 180 min) treatments. The plasma was separated from the blood by centrifugation at 12,000 rpm for 5 min at 4°C and the samples were stored at -20°C for the plasma insulin measurement using Rat Insulin ELISA Kit (Crystal Chem, Elk Grove Village, IL, USA). Ascencia Contour glucose meters (Bayer, Newbury, UK) were used to measure blood glucose levels [24]. Glibenclamide, a sulfonylurea drug, used as a positive control, was dissolved in Dimethyl sulfoxide (0.6% DMSO).

Feeding Test
HFF rats were used to study the impact of extracts on food consumption. Before the experiment, the rats were fasted for 12 h. Oral administration of saline (5 mL/kg, b.w.), extracts (250 mg/kg, b.w.), or glibenclamide (5 mg/kg, b.w.) was followed by measurement of food intake at 0, 30, 60, 90, 120, and 180 min respectively. Glibenclamide, a standard drug, used as the positive control [25].

Metabolic Studies
Metabolic studies were performed in HFF Long Evan male rats using metabolic cages to measure food and fluid consumption. The rats underwent 12-h fasting after a 24-h adaptation period. HFF diet control group received saline (5 mL/kg, b.w.), and the treatment groups received plant extracts (250 and 500 mg/kg, b.w.). The food and fluid intake were observed and recorded every 1 h for the first 6 h, then every 2 h for the next 6 h and finally at 24 h [25].

Gastrointestinal Motility
BaSO4 milk solution was used to assess the gastrointestinal motility. Rats were starved for 20 h and 1 h before administering 10% BaSO4 (W/V of 0.5% Na-CMC) mixture, the treatment groups received extracts (250 mg/kg), bisacodyl (10 mg/kg) and loperamide (5 mg/kg) respectively. After 15 minutes of consuming the BaSO4 milk solution, the animals were killed, and their entire intestines were removed. The distance of BaSO4 travelled was measured and expressed as a fraction of its full length (from the pylorus to ileocecal junction) [25].

Phytochemical Screening
Secondary metabolites such as alkaloids, saponins, steroids, flavonoids, tannins, reducing sugar, and glycosides were identified using previously described techniques [26]. Alkaloids were tested by acidifying 2 mL of the extracts with hydrochloric acid (HCl), to which 1 mL Dragendroff's reagent was added, and the appearance of a red color showed the presence of alkaloids. To test for tannins, a few drops of 10% lead acetate were added to 2 mL of the extracts and the formation of white sediment confirmed the presence of tannins. Testing for the presence of flavonoids involved heating a mixture of 4 mL of the extracts and 1.5 mL methanol; the appearance of a pink color upon the addition of magnesium and a few drops of HCl suggested the presence of flavonoids. To test for saponins, 1 mL of the extracts was mixed with 9 mL distilled water which produced a stable foam suggesting the presence of saponins. To check the presence of steroids, 2 mL of the extracts were mixed with 10 mL chloroform, 1 mL acetic anhydride, and 2 mL sulphuric acid; the formation of a bluish-green color indicated the presence of steroids. Glycosides were tested by combining 1 mL of the extracts with a few drops of glacial acetic acid, ferric chloride, and concentrated sulphuric acid; visualization of bluish-green color suggested the presence of glycosides. Reducing sugars were tested by combining 1 mL of the extracts, 1 mL of distilled water, and a few drops of Fehling's reagent. The mixture was heated, and visualization of a red-brick color indicated the presence of reducing sugars.

Statistical Analysis
To analyse and interpret the data, Graph Pad prism 5 (San Diego, CA, USA) was used. Data analysis was done using an unpaired Student's t-test (nonparametric, with twotailed p values) and a one-way ANOVA with Bonferroni post hoc testing, and the values were represented as Mean ± SEM with a hypothetical significance level of p < 0.05.

Phytochemical Screening and EEMPs
Saponins and flavonoids were detected in all five plants in phytochemical screening of crude extract, as well as tannins in G. nepalensis, G. thomsonii, C. splendens, and X. strumarium (Table 1). Reducing sugar was found in G. nepalensis and G. thomsonii and alkaloids were only present in G. thomsonii (Table 1). Additionally, G. nepalensis also contained steroids (Table 1).

Discussion
Diabetes, one of the most prominent and severe metabolic disorders, has greatly affected many individuals all over the world [27]. Obesity, commonly characterized to be an excess of body fat, is a major risk factor for T2DM. In people who are obese, adipose tissues produce nonesterified fatty acids (NEFAs), which may result in insulin resistance and β-cell dysfunction, and this may ultimately lead to T2DM [28]. Obesity may also potentially raise the risk of cardiovascular disease (CVD) in T2DM patients by deteriorating hypertension and dyslipidaemia [29]. Therefore, we chose HFF rats to conduct in vivo studies to provide an idea on how specific medicinal plants may interact with T2DM and its complications. At this preliminary study, the anti-diabetic potential of ethanol extract of five traditional medicinal herbs G. nepalensis, G. thomsonii, C. splendens, C. infortunatum, and X. strumarium were observed.
In acute in vivo studies, the ethanol extracts of G. nepalensis, G. thomsonii, C. splendens, C. infortunatum and X. strumarium were found to substantially ameliorate glucose tolerance and plasma insulin level in HFF rats, suggesting that all five plants have glucose lowering properties. Recent reports have demonstrated that C. infortunatum improves glucose tolerance and reduces fasting blood glucose in streptozotocin induced diabetic rats [30]. Previous research on G. nepalensis and X. strumarium showed that they possess α-glucosidase inhibitory activities, and they can also reduce oxidative stress damage via free radical scavenging [31,32]. The glucose lowering properties of G. thomsonii and C. splendens may be due to the presence of phytochemicals such as diosgenin, gymnemic acid, epigallocatechin gallate, catechin, proanthocyanidin and ellagic acid, are known to improve glucose homeostasis by reducing blood glucose levels, inhibiting glucose absorption, improving pancreatic β-cell function, and decreasing oxidative stress damage [33][34][35].
Additional in vivo studies on HFF rats comprised feeding tests and metabolic studies to investigate the effect of plant extracts on food and fluid consumption. The plant ex-tracts reduced both fluid and food consumption but was not significant as compared to HFF control. Plant extracts with high dose (500 mg/kg, b.w.) shown more efficacy in decreasing these parameters, particularly at night (between 6 to 8 PM). Rats are known to be more active during nighttime and the blood sugar levels in diabetic rats tend to be at peak at night [36]. Thus, reduction in food intake caused by medicinal plant extracts may be responsible for maintaining healthy blood glucose levels.
Gastrointestinal motility was observed using BaSO4 milk solution. The present studies showed that plant extracts promote gut motility, indicating that the plants may shorten the time available for the digestion and absorption of carbohydrates in the gut, leading to decreased glucose absorption and plasma sugar levels [37]. The presence of saponins in these plants may contribute to improve gut motility, as saponins are known to inhibit disaccharidase activity and intestinal glucose absorption in streptozotocin induced rats [38].
Phytochemical screening of the five plant extracts confirmed the presence of flavonoids and saponins, which is consistent with previous studies on these plants [39][40][41][42][43]. Flavonoids, such as kaempferol, rutin and quercetin, have previously been demonstrated to have glucose lowering, insulin secreting, and β-cell protecting properties in different animal models, including streptozotocin and alloxan induced rats, and HFF rats [44][45][46]. Recent studies on saponins have revealed that these phytochemicals can enhance insulin release and inhibits absorption of carbohydrates by mediating the AMP-activated protein kinase (AMPK) pathway [47]. Tannins were found in four of these plants (G. nepalensis, G. thomsonii, C. splendens, and X. strumarium) is consistent with prior findings [46][47][48][49]. Recent reports indicated that tannins enhance glucose uptake in 3T3L1 adipocyte cells and reduce oxidative stress via the phosphatidylinositol (PI3) and mitogenactivated protein kinases (MAPK) pathways [48,49]. Alkaloids found in G. thomsonii, are also known to be efficacious α-glucosidase inhibitors [50,51]. The anti-diabetic properties of these five plants could be attributed due to the presence of these phytochemicals. However, further studies, including in vitro cell line experiments, and chronic in vivo studies are warranted to fully comprehend the antidiabetic and insulinotropic properties of these plants.

Conclusions
Traditional medicinal plants, G. nepalensis, G. thomsonii, C. splendens, C. infortunatum and X. strumarium, showed improvement in glucose tolerance, plasma insulin level and gastrointestinal motility in HFF rats, suggesting that the extracts may improve diabetes and its complexities by lowering blood sugar levels and inhibiting carbohydrate digestion and absorption in the gut. In addition, the plants were also observed to reduce consumption of food and fluid in HFF rats. The anti-hyperglycaemic properties of these plants may be attributed to the presence of phytochemicals such as flavonoids and saponins. Our findings support the use of these plants as an ethnomedicine for the treatment of T2DM. Additional in-depth research, such as the purification and identification of active components from these plants, could aid in the development of anti-diabetic therapy for T2DM in humans.

Availability of Data and Materials
The information is not available to the public as a result of certain limitations. The corresponding author, however, is willing to provide the information acquired from this study upon request.

Author Contributions
JMAH and PA were equally responsible for the conception and design of the study as well as the supervision of the study; PA, NN, FTT and AT conducted the experiments, analyzed the data, evaluated the results, created the figures, and PA drafted the paper, while PA and JMAH edited the revised manuscript. All authors have participated sufficiently in the work to take public responsibility for appropriate portions of the content and agreed to be accountable for all aspects of the work in ensuring that questions related to its accuracy or integrity. The final manuscript has been read and approved by all authors.

Ethics Approval and Consent to Participate
Independent University, Bangladesh (IUB), Institutional Review Board (IRB) approved protocols on 19th December 2019 for experiments to be performed using animals and the experiments were performed in line with the Animal Welfare Act 2019 of Bangladesh. It was ensured that no animals will be injured over the course of this study.