Evaluation of the anti-hyperglycemic and anti-dyslipidemic potential of the hydroethanolic extract of Graptophyllum pictum (Acanthaceae) aerial parts in streptozotocin-nicotinamide-induced diabetic Wistar rats

Abstract

Context

Diabetes is a chronic metabolic disorder that affects approximately 537 million people worldwide. Modern diabetic therapies include adverse effects and are expensive. Scientists are often exploring affordable natural treatments with little or no side effects. Graptophyllum pictum was chosen for the study due to its traditional claim of reducing blood glucose levels and established glucosidase inhibitory action.

Objective

In this experimental work, the hydroethanolic extract of G. pictum (L.) Griff aerial part was evaluated for hypoglycemic, antihyperglycemic, antidyslipidemic, and protective effects on liver enzymes in streptozotocin (STZ)-nicotinamide (NA)-induced diabetic Wistar rats.

Methods

Diabetes was induced in Wistar rats using an intraperitoneal injection of STZ (60 mg/kg) just 15 min after injecting NA (120 mg/kg). Glibenclamide (10 mg/kg) as a standard and hydroethanolic extract (250 and 500 mg/kg) were used for the treatment of diabetic rats. Throughout the study (21 days), normal and diabetic control animals were given only normal saline (5 mL/kg, p.o).

Results

The findings revealed that hydroethanolic extract did not have any significant hypoglycemic activity but had a significant effect (P < 0.001) when compared to the diabetic control group on lowering blood glucose levels (106 ± 1.41 versus 322.16 ± 2.08) and body weight of diabetic rats, as well as significantly restoring lipid profile (105.83 ± 1.77 versus 195.33 ± 1.49, P < 0.001, total cholesterol; 128.33 ± 1.38 versus 204.5 ± 1.94, P < 0.01, triglycerides; 42.66 ± 1.32 versus 94.83 ± 1.77, P < 0.01, low-density lipoprotein cholesterol; 29.5 ± 0.42 versus 41.83 ± 0.99, P < 0.01, very low-density lipoprotein cholesterol; 29 ± 2.13 versus 15.5 ± 0.84, P < 0.01, high-density lipoprotein cholesterol) and liver enzymes (166.5 ± 1.72 versus 314.83 ± 1.71, P < 0.001, aspartate aminotransferase; 114 ± 1.98 versus 173.5 ± 1.52, P < 0.01, alanine aminotransferase).

Conclusion

Conclusively, results from our study on G. pictum hydroethanolic extract validates its use as an anti-diabetic agent and may offer a new perspective on type 2 diabetic therapy.

Highlights

• •Graptophyllum pictum, known as Justicia picta, is an Acanthaceae plant used to reduce blood glucose.
• •Streptozotocin (STZ)-nicotinamide (NA)-induced diabetes in Wistar rats is used to evaluate the hypoglycemic, antihyperglycemic, and antidyslipidemic effects of the hydroethanolic extract of G. pictum aerial parts.
• •The STZ-NA-induced diabetic rat model is more suitable for acute and chronic antidiabetic research because it has long-term diabetic induction and insulin response to glucose like human type 2 DM.
• •Graptophyllum pictum hydroethanolic extract at 250 and 500 mg/kg demonstrated outstanding antihyperglycemic and anti-dyslipidemic actions.
• •The research suggests that extract may be as effective as glibenclamide in meal-related hyperglycemia and other problems of non-insulin-dependent diabetes.

1. Introduction

Diabetes mellitus (DM) is a group of complex metabolic disorders caused by impaired insulin release and resistance, with approximately 537 million (10.5% of the global adult population aged 20 to 79) diabetic patients worldwide [[1], [2], [3]]. It is characterized by chronic hyperglycemia, which may further trigger other associated complications like nephropathy, neuropathy, retinopathy, and higher chances of cardiovascular disorders [4,5]. Diabetes pathogenesis may involve anything from autoimmune destruction of beta cells of Langerhans distributed throughout the pancreas to some abnormalities that lead to insulin resistance, and both may coexist in patients most of the time [5].

According to the American Diabetes Association (ADA), DM can be classified into two etiopathogenetic groups: type 1 DM and type 2 DM. Type 1 DM, also called insulin-dependent diabetes (IDM) or juvenile-onset diabetes, accounts for 5-10% of diabetic patients. It occurs due to the autoimmune destruction of beta cells in the pancreas, leading to an absolute lack of insulin. Type 2 DM, also known as non-insulin-dependent diabetes mellitus (NIDDM), accounts for 90-95% of diabetic patients [5]. It is caused by insulin resistance or relative insulin deficiency due to a decrease in glucose-stimulated insulin secretion from beta cells or both. Ageing, obesity, reduced physical activity, hypertension, dyslipidemia, and strong genetic predisposition are the factors increasing the probable risk of type 2 diabetes [[5], [6], [7]]. Streptozotocin (STZ)-nicotinamide (NA)-induced diabetes in the Wistar rat model is used to mimic exactly similar conditions as type-2 diabetes in humans [8].

STZ is a nitrosourea analogue (linkage between the N-methyl-N-nitroso urea and carbon-2 hexose) that has been identified as the most potent diabetogenic agent in diabetes research so far. It serves as a glucose analogue and enters through glucose transporters (GLUT2) in the pancreatic beta cells. As an alkylating agent, STZ causes toxicity by selectively transferring its methyl group to the DNA of beta cells and insulinoma cells, resulting in DNA fragmentation [4,9].

Nicotinamide (NA; pyridine-3-carboxamide), an amide form of vitamin B₃ (niacin), inhibits the DNA alkylation effect of STZ and thus reduces the damage of pancreatic islet cells. This results in moderate and stable diabetes and a 40% increase in pancreatic insulin reserve [7,10]. Therefore, this model appears to be the most appropriate for testing the hyperglycemic activity of various drugs [11,12].

Because modern diabetic therapy has many side effects and costs a lot of money, the globe is exploring new antidiabetic medicines from natural sources [13] because of the belief that they have minimal or no side effects and are affordable [14]. Around the globe, more than 1200 medicinal plant species have been explored as remedies for diabetes by various ethnic populations in their traditional medicine systems [15]. This encourages more research into medicinal plants for anti-diabetic activity.

Graptophyllum pictum (L.) Griff., belonging to the Acanthaceae family, is a tropical plant species, also known as Justicia picta. The plant is native to New Guinea and neighboring islands in the Oceania continent. It is well distributed in the United States, India, Indonesia, Ghana, Nigeria, and Ethiopia [[16], [17], [18]]. The plant is traditionally used to treat a variety of ailments, including constipation, hemorrhoids, rheumatism, urinary infection, hepatomegaly, and anuria, as well as to lower blood glucose levels [16,[19], [20], [21], [22]]. Previous research has shown that various extracts, including hydroethanolic extract of G. pictum, demonstrated remarkable antioxidant potential and antidiabetic activity, as evidenced by a significant α-glucosidase inhibitory activity in vitro [21,23,24] and a positive alloxan-induced antidiabetic potential [[25], [26], [27], [28]]. Therefore, the present study aimed to assess the hypoglycemic, antihyperglycemic, and antidyslipidemic activities of the hydroethanolic extract of G. pictum aerial parts in STZ-NA-induced diabetic Wistar rat model.

2. Materials and methods

2.1. Plant material

The fresh aerial parts of the plant were collected from Addis Ababa, Ethiopia. The plant material was taxonomically identified by a taxonomist at the Department of Biology, Debre Tabor University, Debre Tabor, Amhara, Ethiopia, and a voucher specimen (GP01/2022) was deposited in the herbarium for future reference.

2.2. Drugs, chemicals, reagents and instruments

The following drugs, chemicals, reagents, and instruments were used for the study: ammonia solution, ethanol, ferric chloride, gelatin, glacial acetic acid, hydrochloric acid, lead acetate, nitric acid, sulphuric acid, petroleum ether, sodium hydroxide, and sodium chloride (Rankem, India); Dragendorff's reagent, Hager reagent, Mayer's reagent, and Wagner's reagents (Himedia, India); iodine, citric acid, and sodium citrate (Loba Chemie, Mumbai, India); Gluco-one (glucose determination kit, Dr. Morepen, Delhi, India); glibenclamide (Cadila Pharmaceuticals, India); glucose (Reyoung Pharmaceuticals, China); STZ and NA (Sigma-Aldrich, USA); Whatman filter paper no. 1, rotary vacuum evaporator, deep freezer, centrifuge, orbital shaker, and refrigerator. All the drugs, chemicals, and reagents used were of an analytical standard.

2.3. Experimental animals

Healthy Wistar rats (male and female) of body weight 180 g-200 g and aged 8-10 weeks were used for the studies, except for the acute toxicity study, where healthy young adult nulliparous and non-pregnant female Wistar rats, aged 8-10 weeks and weighing 120-140 g, were used. The Wistar rats were purchased from the Ethiopian Health and Nutrition Research Institute. All the Wistar rats were kept in the animal house of the Department of Pharmacy, Debre Tabor University, in ventilated cages maintained at 25 ± 2 °C with a 12-h day and night cycle with water and food (standard pellets, Alema Farms PLC, Bishoftu, Ethiopia) ad libitum. All the animals were allowed to acclimatize to the environment for 2 weeks before the initiation of the experimental work. All of the experimental protocols adhered to internationally accepted guidelines for laboratory animal research [[29], [30], [31]].

2.4. Ethical consideration

All the experimental animals were handled as per international care welfare [[30], [31], [32]]. This study was approved by ethical clearance and certified by the Research and Ethics Review Committee of the College of Health Sciences, Debre Tabor University, Ethiopia, with reference no. SOP12/109/22.

2.5. Extract preparation

A predetermined amount of coarsely powdered drug was defatted with petroleum ether at 40 °C. The defatted and air-dried plant material was then cold macerated with aqueous ethyl alcohol (80% ethanol) and was kept in a rotary shaker at 120 rpm for 72 h for periodic shaking.

The obtained menstruum was filtered by the use of doubled-layered muslin cloth and then vacuum filtered with Whatman filter paper (no. 1) fitted on the Buchner funnel. To maximize the yield, the remaining mark was repeated with a similar process with fresh solvent. All of the menstruum was mixed together and concentrated in a rotary vacuum evaporator at 45 °C to remove the alcohol at a reduced temperature and pressure. The remaining aqueous extract was then lyophilized at −50 °C in order to remove water and to obtain a free-flowing powdered extract. The percentage yield of the obtained extract was calculated and stored at −4 °C for further studies [[33], [34], [35], [36], [37], [38]].

2.6. Phytochemical screening

The hydroalcoholic extract was subjected to phytochemical analysis to detect the presence of various groups of phytoconstituents, such as alkaloids, glycosides, saponins, tannins, resins, flavonoids, steroids, and other phenolic constituents, by using standard methods [33,34].

2.7. Acute oral toxicity studies

The acute oral toxicity study was performed according to the Organization for Economic Cooperation and Development (OECD) test guidelines-425 with a slight modification [29]. Five healthy young adult female nulliparous and non-pregnant Wistar rats aged between 8 and 10 weeks and weighing 120–140 g were selected for the study. All of the animals were housed in individual cages in the experimental animal room, maintained at 25 ± 2 °C with a relative humidity of 40% and a 12-h day and night cycle. All the animals received feed and water ad libitum. All five animals were acclimatized for the first five days before the start of the experiments. Prior to dosing, animals had fasted overnight from food, but water was provided ad libitum.

The first rat orally received 2000 mg/kg ethanolic extract with the use of an oral gavage and was kept under observation for the next 24 h. When no mortality occurred, four other rats were treated in a similar manner and observed for 4 h at a 30 min time interval and then for fourteen consecutive days with an interval of 24 h for general signs and symptoms of toxicity, food and water intake behavior, any change in the skin, eyes, or respiratory pattern, and death [29].

2.8. Hypoglycemic effect on normoglycemic Wistar rats

The Wistar rats were fasted overnight but given water ad libitum. The fasting blood glucose level of each rat was measured. Twenty-four animals with normal blood glucose levels were placed into four groups of six each. The rats in the control group were given normal saline (5 mL/kg, p. o.), the positive control group received the standard drug glibenclamide (10 mg/kg, p. o.), and the third and fourth test groups were administered low (250 mg/kg, p. o.) and high (500 mg/kg, p. o.) doses of hydro-ethanolic extract of G. pictum (HEEGP) aerial parts suspended in normal saline, respectively. A glucometer was used to measure blood glucose levels from blood withdrawn from the tail vein just prior to and at 1, 2, 4 and 8 h after administration [[39], [40], [41]].

2.9. Antihyperglycemic effect on oral glucose tolerance test in normal Wistar rats

Healthy Wistar rats were selected for the assessment of oral glucose tolerance. All the animals were kept fasting overnight with water available ad libitum. Animals were divided into four groups with six animals each. Rats from all four groups were orally administered normal saline, glibenclamide (10 mg/kg), and the hydroethanolic extract of G. pictum aerial parts at 250 mg/kg (HEEGP250) and 500 mg/kg (HEEGP500) according to their respective groupings. After 30 min of the treatment, glucose (2000 mg/kg) was given orally to all the rats. Each animal's blood glucose was tested just before the treatment at 0 h as a baseline and then again at 30, 60, and 120 min after the glucose administration [36,37,42,43].

2.10. Induction of experimental Type-2 diabetes, dosage and treatment

2.10.1. Preparation of citrate buffer

The citric acid (1.09 g) and sodium citrate (1.48 g) were carefully weighed and dissolved in sufficient distilled water to make 100 mL, which was then adjusted to pH 4.5 using hydrochloric acid [44].

2.10.2. Preparation of streptozotocin and nicotinamide solution

STZ is soluble in both water and saline, although both are unstable. In 0.1 M citrate buffer, it is stable. STZ was prepared fresh in ice-cold citrate buffer and given i. p. in 2 mL/kg doses (dose: 60 mg/kg). To make a strength suitable for administration, NA was dissolved in normal saline [45,46].

2.10.3. Induction of diabetes in Wistar rats

Type-2 experimental DM was induced in overnight-fasted Wistar rats (weight, 180-200 g) by injecting STZ (60 mg/kg) intraperitoneally exactly 15 min after the intraperitoneal administration of NA (120 mg/kg). Confirmation of rats becoming diabetic was assessed by measuring the blood glucose levels 72 h following STZ-NA injection using a glucometer, and rats with blood glucose levels of 250 mg/dl or more were considered diabetic and were chosen for the study [45].

2.10.4. Antidiabetic effect on blood glucose, body weight, and biochemical parameters

A total of twenty-four diabetic and six non-diabetic Wistar rats were selected for the study and divided into five groups. Group 1, considered the normal control, consisted of six non-diabetic rats and received only normal saline for 21 days. Groups 2–5 consisted of six diabetic rats each. Group 2, considered a diabetic control group, received normal saline only orally throughout the study. Group-3 rats orally received glibenclamide 10 mg/kg bodyweight (GB10) for 21 days. HEEGP250 and HEEGP500 are 250 and 500 mg/kg of the hydro-ethanolic extracts of G. pictum aerial parts that were given orally to groups 4 and 5 for 21 days. All the animals under study were regularly monitored for food intake and water consumption [42,45].

The fasting blood glucose levels of all the groups were measured using a glucometer on the first, seventh, fourteenth, and twenty-first days, while body weight was measured on days one and twenty-one of the study.

At the end of the study on day 21, all the animals were deprived of food, but the water was available ad libitum. Following mild anesthesia using ether, blood samples were collected via cardiac puncture, transferred to plain bottles, and centrifuged at 3500 rpm for 20 min in order to obtain the serum. The obtained serum samples were used for the estimation of the lipid profile [total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL-c), very low-density lipoprotein cholesterol (VLDL-c)] and serum enzymes like aspartate aminotransferase or aspartate transaminase (AST) and alanine aminotransferase or alanine transaminase (ALT) [45,47].

2.11. Statistical analysis

The statistical analysis was carried out with SPSS software, version 24 (IBM Corp., Armonk, NY, USA), using ANOVA followed by Dunnett's multiple comparison tests. The results are presented as mean ± standard error of the mean (SEM), with levels of significance set at p < 0.05, p < 0.01 and p < 0.001.

3. Results

3.1. Extract yield

The extractive value of the powdered plant material with aqueous ethyl alcohol (80% ethanol) was recorded as 24.0% w/w, as shown in Table 1.

Table 1.

Qualitative and quantitative estimation of HEEGP.

Solvent	Color and texture of extract	Yield (g)	Percentage yield (w/w)
80% ethanol	Light brown free flowing powder	240 g	24%

Abbreviations: HEEGP, hydroethanolic extract of Graptophyllum pictum aerial parts; g, gram; w/w, weight by weight.

3.2. Phytochemical screening results

Preliminary phytochemical screening of the hydroalcoholic extract showed the presence of alkaloids, flavonoids, tannins, glycosides, and saponins as organic phytoconstituents (Table 2).

Table 2.

Qualitative phytochemical profiling of HEEGP.

Phytochemical constituents	Results
	Hydroethanolic extract
Alkaloids	+
Glycoside	+
Flavonoids	+
Saponins	+
Tannins	+
Steroids	_

Abbreviations: HEEGP, hydroethanolic extract of Graptophyllum pictum aerial parts; +, present; -, absent.

3.3. Acute oral toxicity test

At 2000 mg/kg during observation, the hydroethanolic extract of G. pictum (HEEGP) aerial parts lacked signs and symptoms of toxicity, behavioural alteration, and morbidity. Thus, the extract was relatively safe and falls under GSH category 5/unclassified toxicity. Based on these safety observations, two doses were selected for pharmacological evaluation as fractions of the maximum non-toxic dose (2000 mg/kg): 1/8th of the limit dose (250 mg/kg) as the low experimental dose and 1/4th of the limit dose (500 mg/kg) as the high experimental dose, following a standard and widely accepted approach in preclinical studies.

3.4. Hypoglycemic effect of HEEGP on normoglycemic Wistar rats

Table 3 and Fig. 1 show the results of the effects of G. pictum aerial parts hydroethanolic extract and the standard drug glibenclamide (10 mg/kg) on blood glucose levels in normal, healthy Wistar rats. In normoglycemic Wistar rats, the plant extract at 250 and 500 mg/kg doses had no effect on blood glucose levels, but the standard drug glibenclamide had a significant effect (∗∗∗P < 0.001) on blood glucose levels compared to the control group.

Table 3.

Effect of HEEGP on normoglycemic rats.

Group	Blood glucose level (mg/dl)
	0 h	1 h	2 h	4 h	8 h
NC	80.33 ± 5.57	87.83 ± 3.36	85.44 ± 5.83	86.16 ± 5.35	82.66 ± 4.88
GB10	81.83 ± 4.63	75.66 ± 5.33	62.33 ± 5.21	57.13 ± 6.56∗∗∗	56.16 ± 4.38∗∗∗
HEEGP250	81.32 ± 4.92	83.56 ± 5.71	86.32 ± 5.73	85.16 ± 4.42	82.22 ± 3.96
HEEGP500	80.66 ± 2.92	84.22 ± 3.33	85.32 ± 4.72	79.92 ± 4.57	79.11 ± 4.28

Note: Results are expressed in mean ± SEM, (n = 6 rats per group) and analyzed by one-way ANOVA followed by Dunnet tests; ∗∗∗P < 0.001 as compared to control.

Abbreviation: NC, normal control; GB10, glibenclamide 10 mg/kg, HEEGP250 & 500, hydroethanolic extract of Graptophyllim pictum aerial parts at 250 & 500 mg/kg, respectively.

Fig. 1.

Effect of HEEGP on normoglycemic rats.

3.5. Antihyperglycemic effect of HEEGP in glucose-loaded Wistar rats on oral glucose tolerance test

Findings of the oral glucose tolerance study on Wistar rats presented in Table 4 and Fig. 2 showed no significant difference in baseline blood glucose levels among the groups just prior to administration of normal saline, glibenclamide (10 mg/kg) and hydroethanolic extract of G. pictum aerial parts (250 & 500 mg/kg). The blood glucose level was at its peak 30 min after the administration of 2000 mg/kg of glucose in all four groups, in comparison with their baseline values. When compared to the control group, glibenclamide significantly reduced the blood glucose level (∗∗∗P < 0.001) at 30, 60, and 120 min. However, the hydroethanolic extract of G. pictum aerial parts significantly lowered the blood glucose level with doses of 250 mg/kg (∗P < 0.05) and 500 mg/kg (∗∗∗P < 0.001) at 60 and 120 min compared to control.

Table 4.

Effect of HEEGP in glucose loaded Wistar rats on oral glucose tolerance test.

Group	Blood glucose level (mg/dl)
	0 min	30 min	60 min	120 min
NC	78.833 ± 1.13	172.83 ± 2.94	152.32 ± 1.183	122.66 ± 0.88
GB10	80.833 ± 0.60	125.66 ± 1.33∗∗∗	81.33 ± 1.20∗∗∗	65.66 ± 1.49∗∗∗
HEEGP250	81.333 ± 1.60	147.833 ± 1.24###	114.33 ± 1.28∗###	84.43 ± 0.96∗##
HEEGP500	75.166 ± 1.30	140.5 ± 1.66##	95.16 ± 1.57∗∗∗#	71.22 ± 1.28∗∗∗

Note: Results are expressed in mean ± SEM, (n = 6 rats per group) and analyzed by one-way ANOVA followed by Dunnet tests; ∗P < 0.05 & ∗∗∗P < 0.001 as compared with control; ^#P < 0.05, ^##P < 0.01 & ^###P < 0.001 as compared with glibenclamide.

Abbreviation: NC, normal control; GB10, glibenclamide 10 mg/kg, HEEGP250 & 500, hydroethanolic extract of Graptophyllim pictum aerial parts at 250 & 500 mg/kg, respectively.

Fig. 2.

Effect of HEEGP in glucose loaded Wistar rats on oral glucose tolerance test.

3.6. Antidiabetic effect of HEEGP on blood glucose level of diabetic Wistar rats

The fasting blood glucose level was measured for all the rats on day one and then on days 7, 14, and 21 of the study. In all the diabetic rats (Table 5 and Fig. 3), the baseline blood glucose level increased significantly (^###P < 0.001) in STZ-NA administration. On the 7th, 14th, and 21st days, glibenclamide (10 mg/kg) exerted a significant (∗∗∗P < 0.001) control over the elevation of blood glucose levels in diabetic rats. The study outcome showed that the hydroethanolic extract at doses of 250 mg/kg (∗P < 0.05 on the 14th and ∗∗∗P < 0.001 on the 21st day) and 500 mg/kg (∗P < 0.05 on the 7th, ∗∗P < 0.01 on the 14th & ∗∗∗P < 0.001 on the 21st day) significantly reduced the blood glucose level compared to the diabetic control. On the 21st day, the hydroethanolic extract of the plant at both doses (250 & 500 mg/kg) was found to be as effective as the standard drug glibenclamide.

Table 5.

Antidiabetic effect of HEEGP on blood glucose level of diabetic Wistar rats.

Groups	Days
	1	7	14	21
NC	79.333 ± 1.22	83.33 ± 1.42	84.83 ± 1.53	84.16 ± 1.53
DC	314.5 ± 1.94###	333.5 ± 2.23###	377.5 ± 1.94###	361.83 ± 2.08###
GB10	319.83 ± 1.35###	190.16 ± 1.42∗∗∗#	144.5 ± 1.68∗∗∗	91.5 ± 1.56∗∗∗
HEEGP250	319.83 ± 1.19###	275 ± 1.78##$$	218.333 ± 1.52∗#$$	147 ± 1.63∗∗∗$
HEEGP500	322.16 ± 2.08###	237 ± 1.91∗∗##$	185.25 ± 1.23∗∗#$	106 ± 1.41∗∗∗

Note: Results are expressed in mean ± SEM, (n = 6 rats per group) and analyzed by one-way ANOVA followed by Dunnet tests; ∗P < 0.05, ∗∗P < 0.01 & ∗∗∗P < 0.001 as compared with diabetic control; ^#P < 0.05, ^##P < 0.01 & ^###P < 0.001 as compared to normal control; ^$P < 0.05 & ^$$P < 0.01 as compared to glibenclamide.

Abbreviation: NC, normal control; DC, diabetic control; GB10, glibenclamide 10 mg/kg, HEEGP250 & 500, hydroethanolic extract of Graptophyllim pictum aerial parts at 250 & 500 mg/kg, respectively.

Fig. 3.

Antidiabetic effect of HEEGP on blood glucose level of diabetic Wistar rats.

3.7. Effect of HEEGP on body weight of diabetic Wistar rats

The experimental findings revealed that there was no significant weight loss on day one of the study when weight was measured after three days of the STZ-NA treatment. However, the diabetic control group showed a significant (^###P < 0.001) weight loss on day 21st compared to the normal control. Furthermore, the standard drug glibenclamide and the hydroethanolic extract of G. pictum aerial parts at doses of 250 and 500 mg/kg had a significant (∗∗∗P < 0.001) control on the weight loss of diabetic rats at the end of the study when compared to the diabetic control (Table 6 and Fig. 4).

Table 6.

Effects of HEEGP on body weight of diabetic Wistar rats.

Group	Body weight
	1st Day	21st Day
NC	197.16 ± 1.04	203.35 ± 2.23
DC	195.16 ± 1.44	131.46 ± 1.25###
GB10	196.33 ± 1.76	191.52 ± 1.25∗∗∗
HEEGP250	189.24 ± 1.4	170.33 ± 1.54∗∗∗$
HEEGP500	194.66 ± 1.64	177.55 ± 1.29∗∗∗

Note: Results are expressed in mean ± SEM, (n = 6 rats per group) and analyzed by one-way ANOVA followed by Dunnet tests; ∗∗∗P < 0.001 as compared with diabetic control; ^###P < 0.001 as compared to normal control; ^$P < 0.05 as compared to glibenclamide.

Abbreviation: NC, normal control; DC, diabetic control; GB10, glibenclamide 10 mg/kg, HEEGP250 & 500, hydroethanolic extract of Graptophyllim pictum aerial parts at 250 & 500 mg/kg, respectively.

Fig. 4.

Effects of HEEGP on body weight of diabetic Wistar rats.

3.8. Effect of HEEGP on biochemical parameters of diabetic rats

It has been shown that on induction of diabetes by administration of STZ-NA, there was a significant elevation of TC (^###P < 0.001), TG (^###P < 0.001), LDL-c (^###P < 0.001), VLDL-c (^##P < 0.01) and liver enzymes, AST (^###P < 0.001) and ALT (^###P < 0.001), whereas a significant decrease in the level of HDL-c (^###P < 0.001) was observed when compared with the normal control. In addition, glibenclamide significantly controlled the elevation of TC, TG, VLDL-c, AST, ALT (∗∗∗P < 0.001), LDL-c (∗∗P < 0.01) and the reduction of HDL-c (∗∗∗P < 0.001), when compared with the diabetic control group. On the other hand, the hydroethanolic extract of G. pictum aerial parts showed a marked reduction in the levels of TC, TG (∗P < 0.05 for HEEGP250, ∗∗∗P < 0.001 for HEEGP500), VLDL-c (∗P < 0.05 for HEEGP250, ∗∗P < 0.01 for HEEGP500), LDL-c (∗∗P < 0.01 for HEEGP500), serum enzymes AST (∗∗P < 0.01 for HEEGP250, ∗∗∗P < 0.001 for HEEGP500) and ALT (∗P < 0.05 for HEEGP250, ∗∗P < 0.01 for HEEGP500) as compared to diabetic rats. A substantial increase in HDL-c has been noticed in diabetic rats treated with HEEGP500 (∗∗P < 0.01) when compared with the diabetic control group. Nevertheless, no significant change (P > 0.05) in HDL-c and LDL-c for HEEGP250 was shown in comparison with the diabetic control group. All the outcomes of the study are collated in Table 7, Table 8 (Fig. 5, Fig. 6).

Table 7.

Effects of HEEGP on serum lipids of diabetic Wistar rats.

Group	Biochemical parameter
	TC (mg/dl)	TG (mg/dl)	HDL (mg/dl)	LDL (mg/dl)	VLDL (mg/dl)
NC	82.83 ± 1.57	103.83 ± 1.77	40.83 ± 0.87	21.33 ± 1.11	21.16 ± 1.07
DC	195.33 ± 1.49###	204.5 ± 1.94###	15.5 ± 0.84###	94.83 ± 1.77###	41.83 ± 0.99##
GB10	77 ± 1.67∗∗∗	107.5 ± 1.43∗∗∗	36.33 ± 1.46∗∗∗	32.33 ± 1.21∗∗	23 ± 0.68∗∗∗
HEEGP250	140.66 ± 1.11∗$$	160.5 ± 1.23∗$$	19 ± 0.44$$$	73.66 ± 1.15$$$	33.5 ± 0.71∗$$
HEEGP500	105.83 ± 1.77∗∗∗$	128.33 ± 1.38∗∗$	29 ± 2.13∗∗$	42.66 ± 1.32∗∗$	29.5 ± 0.42∗∗$

Note: Results are expressed in mean ± SEM, (n = 6 rats per group) and analyzed by one-way ANOVA followed by Dunnet tests; ∗P < 0.05, ∗∗P < 0.01 & ∗∗∗P < 0.001 as compared with diabetic control; ^###P < 0.001 as compared to normal control; ^$P < 0.05, ^$$P < 0.01 & ^$$$P < 0.001 as compared to glibenclamide.

Abbreviation: NC, normal control; DC, diabetic control; GB10, glibenclamide 10 mg/kg, HEEGP250 & 500, hydroethanolic extract of Graptophyllim pictum aerial parts at 250 & 500 mg/kg, respectively.

Table 8.

Effects of HEEGP on liver function enzymes of diabetic Wistar rats.

Group	Biochemical parameter
	SGOT (U/L)	SGPT (U/L)
NC	155 ± 1.63	69.5 ± 1.35
DC	314.83 ± 1.71###	173.5 ± 1.52###
GB10	146.66 ± 2.19∗∗∗	82.5 ± 1.33∗∗∗
HEEGP250	216 ± 1.57∗∗$$	137.83 ± 1.61∗$$
HEEGP500	166.5 ± 1.72∗∗∗$	114 ± 1.98∗∗$

Note: Results are expressed in mean ± SEM, (n = 6 rats per group) and analyzed by one-way ANOVA followed by Dunnet tests; ∗P < 0.05, ∗∗P < 0.01 & ∗∗∗P < 0.001 as compared with diabetic control; ^###P < 0.001 as compared to normal control; ^$P < 0.05 & ^$$P < 0.01 as compared to glibenclamide.

Abbreviation: NC, normal control; DC, diabetic control; GB10, glibenclamide 10 mg/kg, HEEGP250 & 500, hydroethanolic extract of Graptophyllim pictum aerial parts at 250 & 500 mg/kg, respectively.

Fig. 5.

Effects of HEEGP on serum lipids of diabetic Wistar rats.

Fig. 6.

Effects of HEEGP on liver function enzymes of diabetic Wistar rats.

4. Discussion

DM is a complex metabolic disorder affecting around 5% of the global population [5]. Among these diabetic patients, 90–95% suffer from type-2 DM, which is characterized by insulin resistance and impaired insulin release [7,48]. In patients with type-2 diabetes, the functioning of the beta cells of the islets of Langerhans in the pancreas is reduced and this progression results in beta cell failure, which leads to severe hyperglycemia [48].

For testing the new antidiabetic drugs against type-2 DM from herbal sources or synthesized molecules, various experimental animal models are used, like Goto-Kakizaki (GK) rats (genetic model), partially pancreatectomized rats, alloxan-induced diabetes, STZ-induced diabetes, and STZ-NA-induced diabetic rats. The STZ-NA-induced diabetic rat model is a well-established experimental model by mimicking important features of type-2 diabetes, including partial pancreatic beta-cell dysfunction and insulin resistance. This model exhibits a satisfactory insulin response to glucose, which is a key feature of type-2 diabetes in humans [10,49,50]. The STZ-NA-induced diabetic rat model is more like human type 2 DM in terms of insulin responsiveness to glucose as well as long-term diabetic induction, making it more suitable for acute and chronic antidiabetic pharmacological studies [7,10].

The present investigation highlights the hypoglycemic, antihyperglycemic and anti-dyslipidemic activities of the hydroethanolic extract of G. pictum aerial parts using STZ-NA-induced diabetes in the Wistar rat. Glibenclamide (10 mg/kg) was used as a standard drug for comparative study, as has been done in many previous pharmacological studies [42]. For evaluation of the antidiabetic and other related effectiveness, the selected plant parts were extracted first with petroleum for the purpose of defatting and then with hydro-ethanol (80% ethanol) using the cold maceration method. After lyophilized drying, a free-flowing light brown-colored extract powder was obtained with a 24% percentage yield. The aforementioned findings of the phytochemical screening of the extract revealed the presence of alkaloids, glycosides, saponins, flavonoids, and tannins, which may significantly contribute to its antidiabetic and antidyslipidemic properties [35,46,51]. The glycosides present in the extract have sugar moieties structurally resembling glucose that may lead to stimulus through glucose transporters (GLUT) for insulin release [6,51]. Through their antioxidant mechanisms, flavonoids, tannins, and other phenolic compounds present in the extract may ameliorate diabetes and dyslipidemia by reducing the reactive oxygen species produced by STZ [47,52].

An acute toxicity test for the extract was done by using a modification of the standard method described by the OECD [29]. The extract showed no signs and symptoms of toxicity, behavioural alteration, morbidity, and mortality up to 2000 mg/kg, and then 250 (1/8th) and 500 mg/kg (1/4th) doses of the extract were decided for the study, similarly utilized by other researchers [35,42,53].

The antihyperglycemic evaluation of HEEGP was done by evaluating its effects on normoglycemic rats, followed by the oral glucose tolerance test on nondiabetic rats, and finally via an STZ-NA-induced diabetic model. The normoglycemic study demonstrated that HEEGP has no hypoglycemic effect on normoglycemic rats, whereas glibenclamide seems to have a promising hypoglycemic effect (∗∗∗P < 0.001, 4h and 8h) [54]. This analysis shows that HEEGP has a distinct mechanism of action different from glibenclamide, pointing to the fact that the extract has a beneficial effect in the treatment of diabetes without causing hypoglycemia, which is a serious complication in diabetes management [38,40]. These findings are in accordance with the work done by Ref. [55].

An oral glucose tolerance test was performed to determine the efficacy of HEEGP against dysglycemia and glucose utilization abilities in glucose-loaded normal rats. The study found that HEEGP had an excellent postprandial antihyperglycemic effect after 60 min (∗P < 0.05, 60 and 120 min, HEEGP250; ∗∗∗P < 0.001, 60 and 120 min, HEEGP500), whereas glibenclamide (10 mg/kg) had a significant antihyperglycemic action after 30 min (∗∗∗P < 0.001, 30, 60 and 120 min) compared with the normal control. HEEGP at 500 mg/kg was found to be almost equipotent when compared with glibenclamide. The dose-dependent antihyperglycemic effect of the extract could be attributed to the strength of phytoconstituents in the extract and/or the elimination rate of the extract from the animals [38]. The underlying mechanisms implicated in postprandial antihyperglycemic effects may be attributed to higher glucose utilization for energy synthesis, stimulation of peripheral glucose utilization, inhibition of glucose absorption, insulin secretion, insulin sensitivity, and suppressed glycogenolysis and gluconeogenesis [56,57]. The research findings suggest that the efficacy of HEEGP in treating meal-related hyperglycemia and other complications of NIDDM is nearly equivalent to the effect of glibenclamide.

In the present study, diabetes was induced in the Wistar rats by STZ-NA, an experimental model for type-2 DM, which is used by many researchers for the determination of the antihyperglycemic effects of many drugs derived from synthetic and natural origins [42,45,47,55,[58], [59], [60], [61], [62], [63], [64]]. Glibenclamide exerted a significant effect (∗∗∗P < 0.001) on the blood glucose levels of diabetic rats on the 7th day of the study protocol, and this effect lasted till the 21st day. However, the effect of the extract, which was in a dose-dependent manner, spanned from the 14th day for HEEGP250 (∗P < 0.05, 14th and 21st days) and the 7th day for HEEGP500 (∗∗P < 0.01, 7th and 14th days; ∗∗∗P < 0.001, 21st day) compared with the negative control. All the findings aligned with another study [35]. HEEGP did not lower blood glucose levels in normoglycemic rats, but it did lower blood glucose levels in glucose-loaded rats in oral glucose tolerance and STZ-NA-induced diabetic rats. The findings suggest that HEEGP may be a beneficial antihyperglycemic agent rather than a hypoglycemic agent [55].

One of the complications of diabetes is weight loss. During insulin deficiency, decreased ATP synthesis from carbohydrates, increased fatty acid synthesis, and protein degradation begin to provide amino acids to initiate gluconeogenesis, which may be responsible for muscle mass loss and weight loss [57,58]. In the present study, a significant weight loss (^###P < 0.001) was observed in all the animals treated with STZ-NA. On treatment with HEEGP (250 and 500 mg/kg) and glibenclamide (10 mg/kg), a significant improvement (∗∗∗P < 0.001) in weight for all the diabetic rats in the respective groups was noticed when compared with the negative control. This restored body weight in rats may be associated with increased insulin secretion, better utilization of glucose as an energy source, and enhanced structural protein synthesis. The weight loss caused by STZ-NA treatment and then restored by the extract is similar to previous findings [35,42].

Diabetic dyslipidemia is a disorder of lipoprotein metabolism that occurs in type 2 DM and is a major contributor to cardiovascular severity and morbidity in diabetic patients [65]. The disorder may lead to an elevated level of TC, TG, LDL-c, VLDL-c and a decrease in HDL-c levels in the serum. Similar dyslipidemia was observed in diabetic rats during the experiment. In diabetic conditions, a higher concentration of smaller and denser LDL particles highly susceptible to oxidation due to hyperglycemia increases the risk of coronary heart disease (CHD) [65,66]. Insulin deficiency and resistance inhibited lipase activity suppression, resulting in an increase in free fatty acids in the circulation. These free fatty acids promote the formation and release of VLDL-c into circulation from the liver. VLDL-c is a major lipoprotein that transports TGs, resulting in an elevated level of TGs in the blood [67]. Moreover, VLDL causes the formation of LDL-c via a phenomenon known as beta shift, and LDL-c on lipolysis by hepatic lipase present in the hepatic endothelium produces smaller and denser LDL-c particles. VLDL, in conjunction with cholesteryl ester transfer protein (CETP), also stimulates a decrease in HDL-c levels, which may be reduced as a result of decreased production and increased clearance [65]. The experimental findings revealed that treatment with glibenclamide and HEEGP (250 and 500 mg/kg) resulted in a significant restoration of the lipid profile (TC, TG, LDL, VLDL, and HDL), which is in agreement with other previously reported work [45,60,64,68].

The liver plays a pivotal role in maintaining blood glucose levels during fasting and the postprandial period. Insulin deficit or resistance stimulates the liver to start glycogenolysis and enhance the production of hepatic glucose. Furthermore, hyperlipidemic conditions during diabetes, particularly elevated VLDL and free fatty acids, cause hepatocyte toxicity, resulting in an increase in the levels of hepatic enzymes, AST and ALT [69,70]. Similar observations were recorded in our study, such that treatment with glibenclamide and HEEGP (250 and 500 mg/kg) significantly ameliorated the levels of the liver enzymes AST and ALT. Hence, based on the foregoing, HEEGP may protect diabetic rats from hepatotoxicity by increasing insulin production and sensitivity.

5. Conclusion

The present study elaborated the hypoglycemic, antihyperglycemic, and antidyslipidemic activities of hydroethanolic extract of Graptophyllum pictum aerial parts in STZ-NA-induced diabetes in Wistar rats. The findings of the study indicated that the extract at doses of 250 and 500 mg/kg had remarkable antihyperglycemic and anti-dyslipidemic effects. Nevertheless, no hypoglycemic effect was observed in normoglycemic rats. Furthermore, the plant extract has shown the presence of several phytoconstituents, such as alkaloids, glycosides, saponins, tannins and other phenolic compounds, and flavonoids, which might contribute to its antihyperglycemic and anti-dyslipidemic potential based on biological plausibility and supportive literature, rather than direct causal evidence. Future research into the isolation of active constituents as well as the underlying mechanism of action of bioactive constituents is warranted.

CRediT authorship contribution statement

Pradeep Singh: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Garima Mishra: Writing – review & editing, Methodology, Formal analysis, Data curation, Conceptualization.

Limitations

While the study shows important effects on lowering blood sugar and improving fat levels, it does not clearly explain how these happen, such as by stimulating insulin release, moving GLUT4, blocking alpha-glucosidase, or possibly affecting antioxidant pathways. It may affect the translation of the findings into targeted drug development.

Practical implications and future research

This study scientifically validates the traditional claims of Graptophyllum pictum for lowering blood glucose and supports the integration of the plant into complementary medicine for type 2 diabetes management.

The results show that G. pictum effectively lowers blood sugar and improves fat levels in the blood without causing hypoglycemia in normal rats. It can be safely used alongside standard treatments to help manage blood sugar and fat levels. These results may reduce the dosage requirement of modern drugs and their possible side effects.

Given that acute toxicity was absent at high doses (up to 2000 mg/kg) and that the safe profile observed during the 21-day study provided preliminary evidence of safety, the data supports further preclinical studies on long-term toxicity and efficacy. Additionally, it paves the way for investigating antioxidant, anti-inflammatory, and beta cell regenerative mechanisms, as well as translating these findings into drug development in future research.

Data sharing statement

Upon reasonable request, the corresponding author will provide all of the data that has been used to support the findings of this study available to anyone.

Disclosure

The authors report no conflict of interest in this work.

Funding statement

The authors did not receive any funding from any source to carry out this research work.