Evaluation of antidiabetic and hypolipidemic activity of Barleria cristata Linn. leaves in alloxan-induced diabetic rats

Abstract

The present study was undertaken to evaluate the antidiabetic and hypolipidemic action of leaf extract of Barleria cristata Linn in rats. Diabetes was induced in the rats by a single intraperitoneal (IP) injection of alloxan (150 mg/kg) and randomly divided into 7 groups. Animals were treated with low (250 mg/kg) and high (500 mg/kg) doses of ethyl acetate leaf extract (EALE) and hydro-alcoholic leaf extract (HALE) up to 21 days. The body weight and blood glucose level (BGL) were measured on weekly basis. The rats were killed under mild ether anesthesia on 21st day, blood and the vital organ were collected to estimate biochemical parameters and to study histopathological changes. A single-dose administration of alloxan induced hyperglycemia in all the groups. A regular increase in BGL was observed in toxic control groups when compared with the normal control. Daily oral administration of rats with extracts (HALE and EALE) and standard drug (Glimepiride, 5 mg/kg), reduced elevated BGL significantly (p < 0.001), and body weight was regained in diabetic rats. The extract treatment also improved the normal functioning of the liver and kidneys as evidenced by the restoration of the biochemical profile. The study revealed that B. cristata possesses promising antidiabetic and hypolipidemic activity.

Introduction

Diabetes mellitus (DM), a disease affecting the metabolism of carbohydrates, fats, and proteins occurs when the pancreatic β-cell does not produce a sufficient amount of insulin that the body needs for proper dispersal of blood glucose in different tissue cells (Sun et al. 2020). Polydipsia, polyuria, polyphagia, and weight loss are some of the common symptoms of DM (Kipasika et al. 2020). It has been reported in various studies that diabetics are more vulnerable to papillary necrosis and glomerular lesions, urinary tract infection, and renal atherosclerosis (Turrent-Carriles et al. 2018). The disease slowly progresses towards severe complications such as diabetic neuropathy, diabetic retinopathy, diabetic nephropathy, and diabetic foot disease if neglected. About 50% of people diagnosed with diabetes complain about one or more above-mentioned complications (Zheng et al. 2018).

Presently, due to modernization, diabetes has become a major threat to the World. The number of diabetic patients is increasing rapidly worldwide especially in underdeveloped countries such as India and China. Recent studies have reported that at present 463 million people are suffering from type 2 diabetes and 578 million people (10.2% of the population) will have diabetes by 2030 and 700 million (10.9% of the adult population) by the end of 2045, a big reason to worry (IDF 2019). Among these, a major part would be contributed by India and China due to their high population density (Wang et al. 2013).

Undoubtedly, the disease has been managed successfully by several available allopathic and Ayurveda drugs, however, perfect glycemic control is rarely achieved by these drugs. In addition to very high cost, most of the allopathic drugs causing very serious toxicities in the patients (Haghighatpanah et al. 2016). Therefore, in this situation, use of herbal medicines is the best solution left for diabetes. Both cost and toxicity can be reduced up to large extent using the traditional medicinal plants (Uniyal et al. 2006; Zambrana et al. 2014). The use of traditional medicinal plants for diabetes is from ancient times in countries such as India and China. Garlic, turmeric, and ginger are some of the most effective herbal medicines for diabetes. Several studies have scientifically proved the antidiabetic potential of these herbal drugs (Chan et al. 2012; Patel et al. 2012; Preethi 2013).

Barleria cristata (B. cristata) commonly known as Kala Bansa, belonging to the family Acanthaceae, is one such plant traditionally used in skin diseases, bronchitis, asthma, microbial infections, ulcer, snakebite, bee bite, and toothache (Kumar et al. 2018). From phytochemical screening, the plant leaves and seeds were found to contain alkaloids, flavonoids, glycosides, saponins, and phenols which are responsible for its antioxidant potential (Hemalatha et al. 2012). As we know, alloxan affects the normal functioning of the pancreas by generating free radicals which damage the deoxyribonucleic acid (DNA) of pancreatic β-cells and produces hyperglycemia (Rohilla and Ali 2012). Since plant B. cristata possesses very good antioxidant activity (Charoenchai et al. 2010), the same principle could be used to protect the β-cells of the pancreas and thus to cure hyperglycemia caused by alloxan. Therefore, the present study was ventured to evaluate the antidiabetic and hypolipidemic action of B. cristata extract possibly based on its antioxidant principle.

Materials and methods

Chemicals, reagents, and diagnostic kits

Alloxan for induction of diabetes was purchased from Loba Chemicals Pvt. Ltd Mumbai, India. Glimepiride was purchased from Oster Lab, Ambala (Haryana, India). Ethylene diamine tetraacetic acid (EDTA), Tween80, Ascorbic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and Hydrogen peroxide (H₂O₂) for antioxidant activity estimation was purchased from Hi-Media Laboratories Pvt. Ltd. Mumbai, India. Reagents such as ether, hexane, alcohol, and ethyl acetate were procured from SD-fine Chemicals Ltd, Mumbai, India. Kits for biochemical parameters were purchased from ERBA Diagnostics, Germany. All the chemicals used in this study were of analytical grade.

Collection of plant material

The fresh green leaves of B. cristata were collected from the campus of Kurukshetra University, Kurukshetra, India, and were authenticated by Dr. B. D. Vashishta, Director, Department of Botany, Kurukshetra University, Kurukshetra, India. A specimen of the same plant (No.26/IPS/2013) has been preserved in the herbarium of Botany Department, Kurukshetra University, Kurukshetra, India.

Extract preparation

The leaves dried in shadow (5 kg) were crushed to a coarse powder and then defatted with petroleum ether, then, powdered leaves were extracted in 70% methanol in Soxhlet apparatus at a temperature not exceeding 60ºC. The hydro-alcoholic leaf extract (HALE) was thus obtained, concentrated under reduced pressure in a rotary evaporator to obtain a semi-solid mass (350 g, yield—7%). The marc (semi-solid mass) was successively extracted with ethyl acetate and hexane to prepare ethyl acetate leaf extract (EALE) and hexane leaf extract (HLE). The percentage yield of EALE was found to be 2% (90 g) and that of HLE was found to be 8.57% (300 g). The marc was completely dried before employing the solvent of lower polarity for each subsequent extraction.

In vitro evaluation of the antioxidant activity of B. cristata extracts

DPPH radical-scavenging activity

A 1 mM DPPH solution in ethanol was added to various concentrations of plant extracts (0.25, 0.50, 0.75, and 1 mg/ml) followed by incubation in dark for 30 min at room temperature, the intensity of yellow color chromophore formed was measured at 517 nm. The percentage DPPH-scavenging activity of extracts was calculated using the given formula (Singh and Rajini 2004):

$$\%\text{DPPH scavenging}=\left[\left({\text{A}}_{\text{control}}-{\text{A}}_{\text{sample}}\right)/{\text{A}}_{\text{control}}\right]\times 100$$

H₂O₂-scavenging assay

To determine the H₂O₂-scavenging ability of the B. cristata, Ruch et al. method was followed (1989). As described, a 40 mM solution of H₂O₂ was prepared in phosphate buffer of pH 7.4. Then, different concentrations of semi-solid extracts (0.25, 0.50, 0.75, and 1 mg/ml) were prepared in methanol. Afterward, an equal volume of extract solution was added to H₂O₂ solution (0.6 ml, 40 mM). The absorbance of the resultant solution was read at 560 nm on a UV–visible spectrophotometer (Ahn et al. 2007). The percentage of H₂O₂-scavenging activity was calculated as follows:

$$\%\text{Scavenge of}\left[{\text{H}}_2{\text{O}}_2\right]=\left(\text{AB}-\text{AE}\right)/\text{AB}\times 100,$$

where AB is the absorbance of blank (without extract) and AE is the absorbance of extract samples.

Experimental animals

Male Sprague–Dawley (SD) rats in the weight range of 300–350 were purchased from National Institute of Pharmaceutical Education and Research (NIPER), Mohali after the approval of protocol (protocol no. IPS/AH/224). The animals were kept in the University animal house of Kurukshetra University, Kurukshetra, India, and had free access to standard animal feed, water ad libitum. Animals were acclimatized to the new laboratory conditions for 1 week before the commencement of the experiment. All the experiments were performed according to Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines.

Acute toxicity study

The OECD-423 guidelines were followed for an acute toxicity test (OECD 2001). Briefly, overnight fasted rats were randomly divided into three groups (n = 6). Groups 1 and 2 were exposed for oral administration of HALE and EALE (2 g/kg), while group 3 received only normal saline (10 ml/kg). All animals were maintained at similar housing conditions and continuously observed for the first 4 h at every 15 min intervals, for any sign of toxicity/or deaths, then every 30 min for the next 6 h, and then daily for 2 days. Since no mortality was observed at 2 g/kg, the dose of extracts was increased to 4 g/kg and the treated rats were further observed for the next 2 days.

Selection of dose

Doses of the extracts were selected based on the earlier study (Gambhire et al. 2009).

Assessment of hypoglycemic activity in normal rats by OGTT

To assess the blood glucose level (BGL) by oral glucose-tolerance test (OGTT), animals on 12-h fasting were grouped as: Group 1—normal control, Group 2—glucose loaded (2% glucose solution, 2 ml/kg, PO), Group 3—EALE low dose (250 mg/kg, PO), Group 4—EALE high dose (500 mg/kg, PO), Group 5—HALE low dose (250 mg/kg, PO), Group 6—HALE (500 mg/kg, PO), and Group 7—Standard (Glimepiride 5 mg/kg, PO). To all groups (except normal control), a 2% glucose solution was administered and blood glucose was measured at intervals of 0, 30, 60 and after 90 min (Mari et al. 2001).

Assessment of antidiabetic activity in alloxan-induced diabetic rats

Experimental design and induction of diabetes

A freshly prepared solution of alloxan monohydrate (150 mg/kg, IP) in 5% tween80 was administered to induce diabetes in experimental animals. The BGL was measured on the 4th day after alloxan injection (El-Demerdash et al. 2005; Diniz et al. 2008). Only those animals in which the development of hyperglycemia was confirmed (BGL > 200 mg/dl), were selected for the study (Emiru et al. 2020) and randomly divided into control and treatment groups as shown below:

• Group 1: Normal control treated with normal saline (2 ml/kg, PO).

• Group 2: Diabetic control (Alloxan 150 mg/kg, IP), normal saline (2 ml/kg, PO).

• Group 3: Diabetic rats treated with a low dose of EALE (250 mg/kg, PO).

• Group 4: Diabetic rats treated with a high dose of EALE (500 mg/kg, PO).

• Group 5: Diabetic rats treated with a low dose of HALE (250 mg/kg, PO).

• Group 6: Diabetic rats treated with a high dose of HALE (500 mg/kg, PO).

• Group 7: Diabetic rats treated with Glimepiride (5 mg/kg, PO).

Treatment once started continued up to 21 days. Both, vehicle and the drug solution were administered daily through gastric intubation at a specified time. The BGL was measured on 0, 7, 14, and 21 days to evaluate the effect of extract and standard drug. At the end of the study, maximum blood was collected from the retro-orbital plexus, and animals were sacrificed by cervical dislocation under light ether anesthesia. The abdominal cavity was opened and liver, pancreas, and kidney tissues were extracted to examine the effect of hyperglycemia at the cellular level by histopathology.

Biochemical profile analysis

Various biochemical parameters such as blood lipid profile, creatinine, urea, serum glutamate oxaloacetate transaminase (SGOT), and serum glutamate pyruvate transaminase (SGPT) were evaluated in alloxan-treated groups to assess the normal functioning of the vital organs such as liver and kidney (Primarianti and Sujono 2015).

In vivo antioxidant activity assay

Pancreatic tissue samples from each group were precisely weighed and homogenized in 0.15 M KCl. The resultant mixture was centrifuged at 10,000 rpm for 15 min and supernatants were collected and stored in ice-cold water until further analysis. At the time of analysis, a fraction of the supernatant was taken and used for the determination of superoxide dismutase (SOD), catalase, thiobarbituric acid reactive substances (TBARs), protein carbonyl (PC), and glutathione (GSH) according to the previous method described elsewhere spectrophotometrically (Singh et al., 2019).

Statistical analysis

Statistical analysis of the results was carried out by one-way analysis of variance (ANOVA) followed by Bonferroni test. Values are represented as Mean ± SD. Values p < 0.05 were considered statistically significant.

Results

Acute toxicity

Both extracts were well tolerated by rats as showed no mortality and none of them showed any symptoms of toxicity.

In vitro evaluation of antioxidant activity by DPPH and H₂O₂ radical-scavenging assay

The antioxidant potential of three leaf extracts (HALE, EALE, and HLE) of B. cristata was assessed by DPPH and H₂O₂ radical-scavenging assay. Results show that all three extracts of B. cristata possess good antioxidant properties when compared to standard antioxidant (ascorbic acid). It would be pertinent to mention that HALE (DPPH-scavenging assay-IC50: 92 mg/ml and H₂O₂-scavenging assay IC50: 464.83 mg/ml) and EALE (DPPH-scavenging assay IC50:332 mg/ml and H₂O₂-scavenging assay IC50:544.19 mg/ml) showed the highest antioxidant activity, hence, used further for in vivo animal studies. Whereas HLE extract showed minimum antioxidant activity, hence not used for animal studies (Fig. 1a, b).

Fig. 1.

In vitro antioxidant potential of different extracts of Barleria cristata by (a) DPPH assay and (b) H₂O₂ radical-scavenging assay

Effect of EALE and HALE of B. cristata on blood glucose level of normal rats

When tested for oral glucose-tolerance test (OGTT), a constant rise in BGL was observed in the diabetic control group (70.16 ± 5.63, 133.35 ± 3.13, 121.50 ± 1.40 and 96.06 ± 4.72) with time (Table 1). However, in the extract-treated groups, a significant reduction in BGL was noted. A dose-dependent reduction was observed in extract-treated groups. In EALE low dose treated, initially rose from 65.83 ± 2.99 to 121.13 ± 2.09 (p < 0.001) but after 60th min, BGL level started decreasing and came to normal. The higher dose of EALE also brought the BGL back to normal after an initial increase to 119.45 ± 2.61 (p < 0.001) at 30th min and the deceased to 85.87 ± 4.24 (p < 0.01) after 90th min. A similar pattern of hypoglycemic activity was also observed in HALE extract-treated groups. HALE low dose reduced the BGL from 117.21 ± 2.65 (p < 0.001) to 73.63 ± 4.51 (p < 0.001) A better reduction of BGL was observed with a higher dose of HALE [114.57 ± 3.09 (p < 0.001), 84.31 ± 1.88 (p < 0.001), 65.58 ± 1.91(p < 0.001)] at 30th, 60th and 90th min. A consistent and significant fall in glucose level was also observed in rats treated with Glimepiride (5 mg/kg) at 0, 30, 60, and 90 min after glucose administration.

Table 1.

Effect of EALE and HALE on BGL in normal rats by OGTT models

Group	Blood glucose (mg/dl)
	0 min	30 min	60 min	90 min
G1—normal control (2 ml/kg, PO)	70.96 ± 3.37	67.50 ± 3.45	65.22 ± 1.98	65.98 ± 2.17
G2—glucose loaded (GL) (2 ml/kg, PO)	70.16 ± 5.63	133.35 ± 3.13***	121.50 ± 1.40***	96.06 ± 4.72***
G3—EALE (250 mg/kg, PO)	65.83 ± 2.99	121.13 ± 2.09**	118.73 ± 1.18	86.46 ± 5.45**
G4—EALE (500 mg/kg, PO)	68.51 ± 8.40	119.45 ± 2.61***	108.48 ± 1.11***	85.87 ± 4.24**
G5—HALE (250 mg/kg, PO)	72.51 ± 5.31	117.21 ± 2.65***	98.58 ± 1.20***	73.63 ± 4.51***
G6—HALE (500 mg/kg, PO)	69.26 ± 5.25	114.57 ± 3.09***	84.31 ± 1.88***	65.58 ± 1.91***
G7—glimepiride (5 mg/kg, PO)	66.50 ± 6.44	87.65 ± 11.16***	75.88 ± 3.60***	59.58 ± 2.86***

Values are represented as Mean ± SD, each group contains 6 animals. Comparisons were made on the basis of the one-way ANOVA followed by Bonferroni test. The glucose-loaded group was compared with normal control group and all treated groups were compared to the glucose-loaded group **p < 0.01, ***p < 0.001

Effect of EALE and HALE on blood glucose level in alloxan-induced diabetic rats

Experimental animals developed hyperglycemia on treatment with alloxan (150 mg/kg, IP). Alloxan significantly (p < 0.001) increased the BGL in diabetic control rats (BGL on the initial day was 391.66 ± 0.21 mg/dl and BGL on 21st day was 475.49 ± 0.24 mg/dl) (Table 2). There was no reduction in BGL was observed when given the only vehicle for 21 days. Both extract preparations significantly reduced BGL in dose-dependent manner. EALE low dose, when administered up to 21 days, brought back BGL from 417.533 ± 0.19 mg/dl to 267.18 ± 0.24 mg/dl (p < 0.001). A higher dose of EALE extract reduced BGL from 387.55 ± 0.31 to 223.31 ± 0.26 mg/dl (p < 0.001) when administered daily up to the end of the study. Similarly, HALE low dose (250 mg/kg) decreased the raised BGL from 427.31 ± 0.24 to 207.02 ± 0.03 mg/dl (p < 0.001). However, HALE higher dose provided better hypoglycemia (BGL on initial day 430.37 ± 0.14 mg/dl, BGL at the end of study 129.72 ± 0.52 mg/dl) when compared to the normal group proving its better hypoglycemic potential.

Table 2.

Effect of EALE and HALE on BGL of alloxan-induced diabetic rats

Group	Blood glucose (mg/dl)
	0th Day	7th day	14th day	21st day
G1—normal control (2 ml/kg, PO)	67.38 ± 0.34⁎⁎⁎	77.35 ± 0.27	88.34 ± 0.27	89.19 ± 0.16
G2-diabetic control (150 mg/kg, IP)	391.66 ± 0.21	405.10 ± 0.11***	471.78 ± 0.13***	475.49 ± 0.24***
G3-EALE (250 mg/kg, PO)	417.53 ± 0.19	307.44 ± 0.29***	301.19 ± 0.15***	267.18 ± 0.24***
G4—EALE (500 mg/kg, PO)	387.55 ± 0.31	326.55 ± 0.0***	289.41 ± 0.06***	223.31 ± 0.26***
G5—HALE (250 mg/kg, PO)	427.31 ± 0.24	335.37 ± 0.36***	277.04 ± 0.03***	207.02 ± 0.03***
G6—HALE (500 mg/kg, PO)	430.37 ± 0.14	314.13 ± 0.18***	219.53 ± 0.44***	129.72 ± 0.52***
G7—glimepiride (5 mg/kg, PO)	417.55 ± 0.11	237.41 ± 0.38***	125.32 ± 0.49***	113.58 ± 0.15***

Values are represented as Mean ± SD, each group contains 6 animals. Comparisons were made on the basis of the one-way ANOVA followed by Bonferroni test. Diabetic control group was compared with normal control group and all treated groups were compared with diabetic control group ***p < 0.001

In the standard control (Glimepiride) group, BGL decreased from 417.55 ± 0.11 mg/dl to 113.58 ± 0.15 mg/dl on 21st day (p < 0.001). This shows the presence of a significant glucose lowering potential of Glimepiride.

Effect of B. Cristata leaf extract on body weight of alloxan-induced diabetic rats

A continuous decrease in body weight of experimental animals was observed after alloxan administration up to day 21 in the diabetic control group (Table 3). The diabetic rats treated with Glimepiride (5 mg/kg) as well as the extract (HALE and EALE)-treated groups showed significant (p < 0.001) improvement in body weight compared to the normal control rats.

Table 3.

Effect of EALE and HALE on body weight of alloxan-induced diabetic rats

Group	Body weight (g)
	0th Day	7th day	14th day	21st day
G1—normal control (2 ml/kg, PO)	300.19 ± 0.32	300.29 ± 0.43	307.04 ± 0.95	311.33 ± 0.47
G2—diabetic control (150 mg/kg, IP)	351.49 ± 0.65	288.41 ± 0.45***	246.34 ± 0.29***	151.46 ± 0.23***
G3—EALE (250 mg/kg, PO)	300.49 ± 0.44	237.14 ± 0.34***	234.22 ± 0.38***	234.27 ± 0.24***
G4—EALE (500 mg/kg, PO)	300.77 ± 1.06	242.34 ± 0.33***	254.36 ± 0.35***	245.33 ± 0.57***
G5—HALE (250 mg/kg, PO)	300.38 ± 0.23	246.85 ± 0.13***	244.74 ± 0.36***	267.44 ± 0.40***
G6—HALE (500 mg/kg, PO)	300.37 ± 0.38	251.27 ± 0.46***	281.41 ± 0.06***	276.54 ± 0.47***
G7—glimepiride (5 mg/kg, PO)	350.56 ± 0.69	295.67 ± 0.58***	287.06 ± 0.05***	288.33 ± 0.39***

Values are represented as Mean ± SD, each group contains 6 animals. Comparisons were made on the basis of the one-way ANOVA followed by Bonferroni test. Diabetic control group was compared with normal control group and all treated groups were compared with diabetic control group ***p < 0.001

Effect of B. cristata leaf extracts on liver and kidney function of alloxan-induced diabetic rats

Results show a significant (p < 0.001) increase in the level of liver enzymes such as SGOT and SGPT in diabetic control rats when compared to the normal control rats (Table 4). An elevated level of SGOT and SGPT was restored to normal in extract and standard drug treatment groups (p < 0.001). A similar trend was also observed in the level of protein, urea, and creatinine on extract treatment indicating a significant (p < 0.001) improvement in kidney function.

Table 4.

Effect of EALE and HALE on kidney and liver parameters of alloxan-induced diabetic rats

Group	Kidney parameters			Liver parameters
	Urea (mg/dl)	Creatinine (mg/dl)	Protein (mg/dl)	SGPT (mg/dl)	SGOT (mg/dl)
G1—normal control (2 ml/kg, PO)	39.45 ± 0.78	0.91 ± 0.01	7.96 ± 0.18	45.89 ± 0.80	30.4 ± 0.47
G2—diabetic control (150 mg/kg, IP)	66.37 ± 0.90***	2.11 ± 0.09***	5.28 ± 0.27***	65.19 ± 0.75***	50.31 ± 1.08***
G3—EALE (250 mg/kg, PO)	56.00 ± 1.65**	1.08 ± 0.03***	6.27 ± 0.22***	61.99 ± 0.79	43.72 ± 1.11***
G4—EALE (500 mg/kg, PO)	49.98 ± 0.46***	0.99 ± 0.01***	6.89 ± 0.19***	58.56 ± 5.95***	39.33 ± 0.89***
G5—HALE (250 mg/kg, PO)	48.96 ± 0.83***	0.95 ± 0.06***	6.92 ± 0.29***	50.63 ± 0.42***	35.35 ± 0.85***
G6—HALE (500 mg/kg, PO)	45.22 ± 1.14***	0.92 ± 0.02***	7.01 ± 0.23***	46.86 ± 0.71***	35.49 ± 0.26***
G7—glimepiride (5 mg/kg, PO)	39.84 ± 0.34***	0.83 ± 0.01***	7.86 ± 0.17***	44.98 ± 1.23***	32.48 ± 0.55***

Values are represented as Mean ± SD, each group contains 6 animals. Comparisons were made on the basis of the one-way ANOVA followed by Bonferroni test. Diabetic control group was compared with normal control group and all treated groups were compared with diabetic control group **p < 0.01, ***p < 0.001

Effect of B. cristata leaf extract on blood lipid level of alloxan-induced diabetic rats

Sustain hyperglycemia disturbs the lipid profile in diabetic patients and the same was observed in diabetic control animals. Significant (p < 0.001) increase in cholesterol, low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL) was observed after alloxan administration while high-density lipoprotein (HDL) cholesterol level was reduced. Treatment with HALE and EALE significantly (p < 0.001) diminished the level of cholesterol, LDL, VLDL and restored to normal. In addition, extract treatment up to 21 days significantly raised the level of HDL cholesterol (Table 5).

Table 5.

Effect of EALE and HALE on blood lipid level in alloxan-induced diabetic rats

Group	Cholesterol (mg/dl)	HDL (mg/dl)	LDL (mg/dl)	VLDL (mg/dl)
G1—normal control (2 ml/kg, PO)	165.48 ± 0.45	42.74 ± 0.13	89.48 ± 0.34	67.43 ± 0.80
G2—diabetic control (150 mg/kg, IP)	230.52 ± 0.26***	31.49 ± 0.35***	160.49 ± 0.22***	91.42 ± 0.85***
G3—EALE (250 mg/kg, PO)	209.31 ± 0.22***	35.78 ± 0.02***	148.42 ± 2.51***	86.77 ± 0.51***
G4—EALE (500 mg/kg, PO)	199.28 ± 0.05***	38.11 ± 0.11***	144.93 ± 2.25***	82.68 ± 0.29***
G5—HALE (250 mg/kg, PO)	189.26 ± 0.18***	38.02 ± 0.82***	123.56 ± 1.58***	83.61 ± 1.03***
G6—HALE (500 mg/kg, PO)	180.78 ± 0.94***	41.47 ± 0.27***	109.99 ± 0.91***	73.16 ± 0.55***
G7—glimepiride (5 mg/kg, PO)	167.14 ± 0.32***	43.43 ± 0.42***	100.07 ± 0.73***	70.38 ± 0.84***

Values are represented as Mean ± SD, each group contains 6 animals. Comparisons were made on the basis of the one-way ANOVA followed by Bonferroni test. Diabetic control group was compared with normal control group and all treated groups were compared with diabetic control group ***p < 0.001

In vivo evaluation of antioxidant activity

Antioxidant marker such as TBARS, Protein carbonyl, SOD, catalase, and GSH plays an important role by neutralizing free radical generated in the body (Table 6). A significant (p < 0.001) increase in TBARS and protein carbonyl level was observed in diabetic control when compared with the normal control. When therapy was started with lose dose of EALE, a significant ((p < 0.001)) decrease in TBARS and Protein carbonyl levels was observed while at high dose, a significant (p < 0.001) decrease in TBARS and moderate (p < 0.01) decrease in Protein carbonyl levels were observed.

Table 6.

Effect of EALE and HALE on in vivo antioxidant markers

Groups	TBARs (nm of MDA/µg of protein	GSH (mg %)	SOD (units of SOD/mg of protein)	Catalase (nm of H2O2/min/mg of protein)	Protein carbonyl (nmol/mg of protein)
G1—normal control (2 ml/kg, PO)	489.70 ± 56.4	0.71 ± 0.30	0.03 ± 0.01	0.43 ± 0.20	21.30 ± 6.40
G2—diabetic control (150 mg/kg, IP)	868.60 ± 67.31***	0.63 ± 0.03	0.02 ± 0.00***	0.19 ± 0.04*	57.08 ± 7.34***
G3—EALE (250 mg/kg, PO)	304.50 ± 54.90***	1.4 ± 0.09***	0.04 ± 0.00***	0.34 ± 0.02*	24.40 ± 3.40***
G4—EALE (500 mg/kg, PO)	698.56 ± 90.00***	0.72 ± 0.09	0.02 ± 0.00	0.28 ± 0.06	45.30 ± 0.40**
G5—HALE (250 mg/kg, PO)	592.40 ± 25.80***	0.80 ± 0.09	0.03 ± 0.00	0.34 ± 0.12	35.60 ± 0.60***
G6—HALE (500 mg/kg, PO)	207.50 ± 54.90***	1.90 ± 0.09***	0.07 ± 0.00***	0.56 ± 0.06***	27.30 ± 0.40***
G7—glimepiride (5 mg/kg, PO)	379.70 ± 56.40***	0.71 ± 0.34	0.06 ± 0.01***	0.45 ± 0.20*	23.30 ± 6.40***

Values are (Mean ± SD), each group contains 6 animals. Comparisons were made on the basis of the one-way ANOVA followed by Bonferroni test. Diabetic control group was compared with normal control group and all treated groups were compared with diabetic control group *p < 0.05, **p < 0.01, ***p < 0.001

GSH, SOD, and catalase levels were significantly reduced in the toxic control group (0.63 ± 0.00, 0.02 ± 0.00, and 0.19 ± 0.01) after alloxan administration. In animals treated with HALE at a low dose, an upraised level of GSH (0.80 ± 0.09), SOD (0.034 ± 0.001), and catalase (0.34 ± 0.12) was observed. Treatment with high dose of HALE significantly (p < 0.001) increased the levels of GSH, SOD, and catalase (1.90 ± 0.00, 0.07 ± 0.00, and 0.56 ± 0.06). Both, low and high doses of EALE and HALE worked significantly to restore the antioxidant parameters but EALE treatment at low dose showed better effect than the high dose. Standard therapy with Glimepiride significantly raised the level of GSH, SOD, and catalase (0.71 ± 0.34, 0.06 ± 0.01, and 0.45 ± 0.20) like the normal control animals.

Effect of B. cristata leaf extract on histology of liver, kidney, and pancreas

Histopathological examination of pancreatic tissue of normal control (G1) rats shows a normal cellular architecture of islets of Langerhans (ISL), normal arrangement of the pancreatic lobule (PL), and connective tissue septa (CTS) (Fig. 2a1). However, an excessive cellular disturbance in the cellular arrangement in ISL and distortion of PL was observed in the histology of diabetic control (G2) rats (Fig. 2b1) when compared with normal control animals. Treatment with a high dose (500 mg/kg, PO) of EALE (Fig. 2c1) and HALE (Fig. 2d1) prevented further damage to the pancreatic tissue by alloxan evidenced by the normal architecture of ISL and PL. Glimepiride (5 mg/kg) a well-known antidiabetic drug prevented further damage to the pancreas (Fig. 2e1).

Fig. 2.

Effect of EALE and HALE extract on histology of pancreas, liver and kidney (magnification 4X, H and E). a Section of normal group animals; b section of diabetic control rats; c–e sections of rats treated with EALE (500 mg), HALE (500 mg) and Glimepiride (5 mg/kg), respectively. PL Pancreatic lobules, ISL Islets of Langerhans, CTS connective tissue septa, He hepatocytes, PT portal triad, CV central vein, dCV dilated central vein, dPT distorted portal triad, BC Bowman’s capsule, G Glomerulus, PCT proximal convoluted tubules and CD collecting ducts

Liver histology of the normal control (G1) shows the presence of healthy hepatic cells with the normal architecture of cytoplasm, nucleus, portal triad (PT), and that of central vein (CV) (Fig. 2a2). In diabetic control rats (G2), the normal lobular structure significantly found altered as evidenced by damaged PT (dPT) and the dilated CV (dPT) was prominently found dilated (Fig. 2b2) when compared with normal control animals. No significant damage was observed in the liver tissue of rats treated with extracts (Fig. 2c2–d2) and standard drug (Fig. 2e2). Both high dose extract-treated groups (G4 and G6) significantly used their antioxidant potential to prevent the destruction induced by alloxan.

The kidney of the normal control (G1) rats (Fig. 2a3) showed the normal appearance of renal parenchymal cells, normal proximal convoluted tubule (PCT), collecting duct (CD), glomeruli (G), and Bowman’s capsule (BC) whereas, the kidney histology of diabetic control (G2) rats presented congestion of blood vessels, swelling of tubules and damaged glomeruli (dG) (Fig. 2b3). However, on treatment with a high dose of the extracts (EALE and HALE), the changes induced by alloxan were reversed back to normal as evident by the normal appearing PCT, G, and regeneration of BC (Fig. 2c3–d3).

Discussion

Alloxan being diabetogenic, generates free radicals, which damage the DNA of pancreatic β-cells produce hyperglycemia by reducing insulin synthesis and secretion (Rohilla and Ali 2012). Insulin deficiency disrupts the metabolism of carbohydrates, fats, and proteins mainly in the liver, adipose, and muscle tissues thereby raising the blood level of glucose, cholesterol, urea, creatinine, altering the function of liver enzymes (SGOT, SGPT), negative nitrogen balance, muscle wasting, and weight loss (Nguyen et al. 2011; Mathur et al. 2016). B. cristata, a well-known plant, possessing potent antioxidant activity reverted back all the above-mentioned negative effects produced by alloxan-induced diabetes in rats. Before the commencement of the animal study, in vitro antioxidant (Fig. 1) potential of plant extracts was evaluated by DPPH and H₂O₂ assay (Lalitha et al. 2013). The DPPH and H₂O₂ free radical-scavenging assays are commonly used model to test in vitro antioxidant activity of plant extracts. A lower IC50 value suggests better antioxidant activity (Ekin et al. 2017). In the present study, the HALE and EALE showed good antioxidant potential, probably containing the higher flavonoid content (Hemalatha et al. 2012), henceforth used further to investigate their antidiabetic potential in alloxan-induced diabetic rats.

The glucose-tolerance test is used to measure the body’s ability to metabolize glucose and it measures changes in the concentration of postprandial glucose that typically occur before changes in fasting glucose. OGTT can be used to detect diabetes mellitus or insulin resistance (Kim et al. 2016). In the present study, the preliminary hypoglycemic effect of the extracts was evaluated in normal rats by OGTT. The results of the OGTT test with EALE and HALE documented good hypoglycemic potential of B. cristata as no further increase in BGL level was observed after 30 min of glucose solution administration like glucose-loaded (GL) animals (Table 1). From these findings, EALE and HALE seem to be able to minimize postprandial hyperglycemia through an oral antidiabetic agent mimetic action.

Experimentally induced diabetes has provided great insight into the physiological and biochemical disruptions of the diabetic state. Several studies used alloxan-induced rat model to evaluate the antidiabetic potential of plant extracts and compounds (Etuk 2010). In the present study also, alloxan (150 mg/kg, IP) was used to induce diabetes in rats. Alloxan causes over-production of reactive oxygen species which leads to the cytotoxicity in β-cells of the islets of Langerhans in the pancreas and diminishes synthesis and release of insulin (Fröde and Medeiros 2008). The same result was observed in the current research in alloxan-induced diabetic rats with elevated BGL relative to normal rats. Daily administration of extracts (HALE and EALE) up to 21 days brought the BGL level to normal in alloxan-induced diabetic rats. All treatment groups showed dose-dependent reduction in BGL. In addition, a greater BGL reduction was observed in the HALE high dose-treated group. Results of this study reveal that the leaf extract of B. cristata possesses significant hypoglycemic activity. Similarly, treatment with standard drug Glimepiride significantly reduced the BGL comparable to normal rats. Glimepiride, a well-known oral hypoglycemic drug, acts by stimulating the insulin secretion from pancreatic β-cell (Mowla et al. 2009). Extract treatment could have produced the same effect either through insulin-secreting mechanism or by regenerating the β-cells.

Continuous reduction in body weight is the most peculiar characteristic of diabetes due to glycosuria, reduced peripheral uptake of glucose, reduced glycogen synthesis, negative nitrogen balance, and muscle wasting (Pi-Sunyer 2005). A reduction in average body weight was arrested on administration of HALE (250 and 500 mg/kg) and EALE (500 mg/kg) extract (Table 3). The body weight of treated animals controlled in the treated groups due to the protective effect of the extract and the standard drug. A decrease in average body weight reduction indicates control over polyphagia and muscle wasting resulted due to hyperglycemic condition (Mowla et al. 2009).

To assess the normal functioning of the liver, SGOT and SGPT level in normal and extract-treated grouped animals were estimated. It is well known that the elevation of liver enzymes (SGOT and SGPT) is characteristically associated with the glycemic condition in type 2 diabetic patients when there is no insulin in the blood (Teshome et al. 2019). In this situation, the elevated SGOT and SGPT might contribute to the development of ketogenesis and gluconeogenesis (Qian et al. 2015). Increased SGOT and SGPT activity was also observed in diabetic control rats which indicate a severe deficiency of insulin and a high level of amino acids and fatty acids as well (Table 4). It is evidenced from the results of the current study that the levels of SGOT and SGPT were brought to normal in the respective treated groups. In diabetic nephropathy, the serum urea and creatinine levels are markedly elevated (Yu and Bonventre 2018). Similarly, the levels of urea and creatinine were elevated in diabetic rats and restored towards normal in extract-treated rats. This suggests the well functioning of the vital organs such as liver and kidney.

Hypercholesterolemia is the most frequent lipid abnormality found in diabetic patients due to metabolic disturbance and characterized by increased TG and LDL-C levels and decreased levels of HDL-C (Matheus et al. 2013). It further increases the risk of heart attack and thus necessitates control over hypercholesterolemia. In the present study also, increased levels of cholesterol, LDL, and VLDL and decreased levels of HDL were observed in diabetic rats. However, repeated administration of HALE and EALE for 21 days significantly decreased hypercholesterolemia in alloxan-induced diabetic animals and the levels of LDL, VLDL, and cholesterol restored back to normal (Table 5). This indicates extract treatment might have interfered somewhere in the cholesterol and fatty acid synthesis pathway due to which hypolipidemic action was resulted (Singh et al. 2014). Increased lipid levels cause decreased antioxidant capacity and increased oxidative stress, resulting in multiple organ damage (Suryawanshi et al. 2006).

Diabetes mellitus and oxidative stress have a direct link with each other and the same has been reported in various studies. SOD, catalase, and GSH are family enzymes that work in tandem to fight against this oxidative stress. Excess of H₂O₂ produced in pancreatic tissue may be due to the increased production of superoxide anions (Rohilla and Ali 2012). Alloxan treatment decreased the enzymatic activity of SOD, GSH, and catalase which indicates the development of oxidative stress and EALE and HALE restored the enzymatic activity back to normal (Table 6). Increased activity of TBARS and Protein carbonyl is described as the indicator oxidation of lipid membrane which was also restored back to normal upon initiation of therapy with EALE and HALE (Sharma et al. 2006).

These findings were also confirmed by histopathological studies. Histology of pancreas, liver, and kidney tissues showed greater cellular damage in diabetic control groups (Fig. 2). The extract treatment healed the cellular damage caused by alloxan in experimental animals and the same was evidenced by the histology of the pancreas, liver, and kidney.

Conclusion

It is obvious from the discussion section that leaf extract of B. cristata possesses very good antioxidant, antidiabetic, and hypolipidemic activity proved through in vitro and in vivo experiments. The HALE and EALE extracts of plant have potential to reduce the blood glucose and lipid levels level in experimental animals. All in all, B. cristata could be a better herbal alternative for diabetic patients. Therefore, further work should be carried out to explore its active constituent responsible for hypoglycemic action.

Authors contributions

We declare that this work was done by the author(s) named in this article. MNA, ASS, SB and LS designed the study. SB and LS did experimental work. MNA and ASS collected and analyzed the data. MNA wrote the manuscript with support from ASS, SB and LS. All the authors read and approved the manuscript for publication.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest in the publication.