Amelioration of diabetes and its complications by Manilkara zapota (L) P. Royen fruit peel extract and its fractions in alloxan and STZ-NA induced diabetes in Wistar rats

Abstract

Purpose

This study aims to evaluate the effects of Manilkara zapota (L) P. Royen fruit peel extract (EMZFP) and its fractions in ameliorating diabetes and its complications in alloxan and STZ-NA induced diabetes in Wistar rats.

Methods

Antidiabetic effects of EMZFP were assessed in alloxan (150 mg kg^-1) induced diabetes in differently grouped rats (n=6). Diabetic rats were treated with EMZFP 150, 300, and 600 mg kg^-1 while, glimepiride (0.09 mg kg^-1) was used as a reference standard. Treated animals were assessed for various biological parameters i.e. blood glucose, serum lipids, nephroprotective markers, cardiovascular risk indices, liver glycogen, neuropathy, body weight, and histopathology of kidneys. However, for evaluating antidiabetic effects of fractions (chloroform, acetone, ethyl acetate, and remaining ethanol fraction) of EMZFP, diabetes was induced by streptozotocin (60 mg kg^-1)–nicotinamide (120 mg kg^-1/ml) in differently grouped male rats (n=6). Diabetic rats were treated with EMZFP fractions 200 mg kg^-1 however; glibenclamide (10 mg kg^-1) was a reference standard and evaluated for blood glucose, serum lipids, cardiovascular risk indices, and diabetic neuropathy.

Results

EMZFP 300 and 600 mg kg^-1/day demonstrated significant antihyperglycemic effects with augmentation in glycogen content, perfection in serum lipid profile, cardiovascular risk indices, body weight enhancement, nephroprotective effects, beneficial in peripheral neuropathy, and histopathological evidence of reversal of glomerulosclerosis. EMZFP-Et and EMZFP-EA fractions depicted a significant improvement in blood glucose, serum lipid profile, cardiovascular risk indices, and peripheral neuropathy.

Conclusion

EMZFP and its Et and EA fractions ameliorated diabetes and its complications by improving glycemic control and associated biochemical alteration.

Highlights

• Manilkara Zapota (L.) P. Royen fruit peel 70% ethanolic extract exert antidiabetic effects

• EMZFP significantly ameliorated diabetic biochemical parameters and its complications.

• EMZFP-Et and EMZFP-EA fractions exert potential antihyperglycemic, hypolipidemic effects and significantly improved cardiovascular risk indices, and peripheral neuropathy.

• Studied MZFP can be used as promising natural herbal source of antidiabetic principles.

Introduction

Diabetes mellitus (DM) is a chronic metabolic dysregulation with several causes and is manifested by elevated blood glucose levels with altered carbohydrate, lipid, and protein metabolism as a consequence of pancreatic β-cell dysfunction, distortion of the insulin signaling pathway and deprived of insulin resistance [1, 2]. All over the world Type 2 diabetes mellitus (T2DM) is the most common chronic ailment due to the practice of fast food habits and sedentary lifestyles. T2DM is specifically a consequence of insulin resistance and/or inadequate insulin synthesis [3], in chronic stage manifested by severe complications, such as retinopathy, cardiomyopathy, neuropathy, nephropathy, diabetic foot syndrome, and dyslipidemia [4].

Normally, feedback control of the antioxidant system keeps free radicals at a control level in the body. Persistent hyperglycemia resulted in oxidative stress and creates the key and collective events in the pathogenesis of elevated vascular complications such as advanced glycation end product, polyol pathway flux, cytokines, sorbitol, hexamine pathway flux, and activation of protein kinase C isoforms [5].

Accordingly, the current drug regimen focuses on refining the glycemic load to counteract the complications accompanying DM. The management of DM is done by three classes of drug interventions. The first one increases endogenous insulin availability- sulphonylureas, insulin analogs, glinides, dipeptidyl peptidase IV inhibitors, and glucagon-like peptide-1 agonists [6]. Second, drugs increasing the sensitivity of insulin- thiazolidinedione and biguanide [7] and the third one is the carbohydrate hydrolyzing enzyme inhibitors- α-amylase and α-glycosidase inhibitors [8]. Amongst these, sulphonylureas and glitazone may induce obesity, osteoporosis, and inhibition of hepatic regeneration [9]. While, a thiazolidinedione, may induce risk reduction of IGT conversion to T2DM and prolonged use is associated with noticeable weight gain and edema increasing cardiac threats [10]. Owing to these limits of available drug interventions phytotherapy or use of bioactive phytochemicals from traditionally known antidiabetic herbal resources are courtesy of diabetic management and treatment.

Realizing this, herbal extracts of food industry byproducts rich in a nutritionally valuable mixture of components, which may act additively or in synergy in the management of DM, attracted many researchers to investigate and evaluate their antidiabetic potentials [11]. The previous literature findings for extraction of herbal food byproducts were so far up to single-step extraction, which may limit the pulling out of bioactive phytoconstituents. Further fractionation with a specific type of solvent can enrich the extract consequently improving its bioactivity considerably [12].

Manilkara zapota (L) P. Royen fruit from the Sapotaceae family is widely consumed and cultivated nutritional fruit across the tropical reasons of the world. This fruit’s nonedible part like peel constitutes a higher amount of various biologically active phytochemicals as compared to its edible pulp portion [13]. Manilkara zapota (L) P. Royen fruit peel (MZFP) has been reported to the presence of antioxidant principles, enriched variety of phenolic compounds and flavonoids with phenolic hydroxyl groups which effectively scavenges reactive oxygen species [14, 15] and most of the flavonoids had already reported as antidiabetic, helps to minimize cardio-vascular complications [16–19]. 5-caffeoyl quinic acid conjugates, a phenolic compound identified in MZFP reported for treating different diseases and disorders [20, 21]. Lycopene- a carotenoid found in MZFP capable to eradicate the singlet oxygen and protects low-density lipoproteins, proteins, DNA, and lipid molecules from free radicals and performs a key role in shielding from many diseases [22–24]. Furthermore, other various bioactive phytoconstituents were also reported in MZFP, like hydroxybenzoic acids (gallic, ellagic, and p-hydroxybenzoic), flavanols (catechin, epicatechin, and quercetin), hydroxycinnamic acids (chlorogenic, ferulic, and trans-cinnamic), and kaempferol. All these phytochemicals highlight their role in antioxidant activity, antidiabetic effects, and in various other diseases where free radicals scavenging perform a key role in eradicating the disease and its complications [25, 26].

In hyperglycemic state glucose oxidation and non-enzymatic protein glycation causes the excessive free radical formation and this worsened free radical level is concurrently suppressed by an antioxidant defense mechanism, leading to critical impairment of cellular organelles and enzymes, rise in lipid peroxidation, and advances in insulin resistance; this concerned of oxidative stress can stimulate the advances and progression of DM complications [27]. Antioxidants of natural plant sources are capable of scavenging free radicals and considerably diminish the chance of progression of DM complications [28].

In our preliminary studies, we observed that MZFP possesses significant in-vitro free radical scavenging and α-glucosidase enzyme inhibition potentials when ascorbic acid and acarbose were taken as reference standards respectively [29]. Still, no studies were carried out regarding the in-vivo antidiabetic potentials of this fruit peel. Hence, in this presented investigation we further assessed EMZFP and its various fractions, to unfold the hindrance abilities for antidiabetic potentials with its complications like nephropathy, neuropathy, and dyslipidemia in alloxan and STZ-NA induced diabetic rat models respectively.

Materials and methods

Chemicals

All the chemicals used were of analytical grade. Pet ether, n-hexane, ether, chloroform, acetone, ethyl acetate, ethanol, Na-citrate buffer, formalin, Xylene (Merck Life Sci. Pvt. Ltd., Ind.). Alloxan monohydrate (Fine chem., Pvt. Ltd., Mumbai, Ind). Streptozotocin and nicotinamide (SRL diagnostics, Pvt. Ltd., Mumbai, Ind.). Glucose oxidase/peroxidase standard diagnostic kit (Reckon diagnostics, Pvt. ltd., Ind.). Hematoxylin and eosin-stains, urethane, trichloroacetic acid, anthrone reagent (Sigma Aldrich Pvt. Ltd., Ind.).

Collection and authentication of plant sample

Fresh and just ripened Manilkara zapota (L) P. Royen fruits were collected from the local farm area of Nanded, MS, India (latitude 19.13⁰ N & longitude 77.32⁰ E). For taxonomic identity and authentication, the plant herbarium sheet was prepared and deposited at the herbarium of Botanical Survey of India, Pune, India and voucher specimen number issued was BSI/WRC/100-1/TECH./2019/68/ specimen no. 1; dated 24 Dec 2019.

Sample preparation, extraction, and fractionation

The fruits peels were removed with the steel spoon and shade dried. Later it was pulverized to make it in coarse powder. The 1000 ml of 70% ethanol was added to 100 g of dried peel powder (pet ether defatted) and kept for shaking at 28°C for 2 h with 150 rpm on an orbital incubator shaker (Remi RIS 24+). After cooling to room temperature, centrifuged at 2500 rpm for 15 min and filtered by Whatman filter paper No 1. The filtrate was then evaporated for 3 days and obtained extract was put in storage in airtight dark bottles at 4°C before further analysis. [30, 31].

Further; EMZFP was dissolved in ethanol before mounting for column chromatography to separate it into fractions with chloroform, acetone, and ethyl acetate. Slurry of 150 g silica gel G (60-120 mesh size) mixed with 350 ml of n-hexane was prepared. A vertical borosilicate glass column (20 mm width × 500 mm length) was rinsed with acetone and well dried before packing, to the bottom a piece of glass wool was placed, and to its top sea sand (particle size 60) added up to 1 cm height. The prepared slurry was poured into this column up to 2/3^rd of the column height and to the top 1 cm height level of sea sand was placed. This whole assembly was rinsed with solvent to aid the proper packing of the column. The dry powder sample for fractionation was prepared by evaporating a mixture of 10 g of EMZFP and 20 g of silica gel G in ethanol on the hot plate. The top of the prepared column was filled with this dry powder, to its top 1 cm height level sea sand was added, and the whole assembly was eluted with various solvents (chloroform, acetone, and ethyl acetate) by using the gradient elution method to get respective soluble fractions along with remaining ethanol fraction. A solvent level of 6 cm height above the extract was maintained to prevent drying of the column. At a flow rate of 5 ml/min, 50 ml of solvent was collected for each fraction. The obtained each fraction was stored in amber vials and was kept in a refrigerator at 4°C before further usage [32].

Experimental animals

Albino rats of Wistar inbred, 2-3 months old, weighing 200-220 g were selected and kept in ventilated cages with a standard pellet diet and water ad libitum for seven days before initiating the study. Animal care and all the animal experiments were performed and approved by the Institutional animal ethics committee (SNIOP/CPCSEA/IAEC/CP-PL/01-2021) following the CPCSEA guidelines for care and use of laboratory experimental animals in India. The blood samples (3 ml) for analysis were collected using 23 gauze needles from the lateral tail vein following restraint. All the dosing and collection of the biological sample were done between 9 am to 10 am to avoid circadian rhythm-induced changes [33].

Acute oral toxicity study

The acute oral toxicity studies were performed individually for EMZFP and its fractions in two individual stages in nulliparous and non-pregnant, 8-12 weeks old female mice, as per the OECD 423 guidelines. For the first stage, mice were grouped into three groups (n= 03) and given p.o. 05, 50, 300, and 2000 mg kg^-1 b.w. doses of test extract. Then mice were witnessed for the next 24 h for any marks of toxicity, moribund status, and or death. The outcomes from the first stage recommended the doses for the second stage were 3000 and 5000 mg kg^-1 b.w. doses of test extract were administered p.o. to the other three groups (n=03) of mice. However, for the each fraction in the second stage, the doses of 2000 mg kg^-1 b.w were given p.o. Any sign of toxicity, moribund status, and or mortality was noted in the next 72 h for both EMZFP and its fractions.

Experimental induction of diabetes in Wistar rats

Before induction of diabetes, rats were kept for overnight fasting and prior injection of diabetogenic agent animals were anesthetized by light ether. For evaluation of antidiabetic effects of EMZFP, to induce and establish diabetic condition, a single i.p. dose of alloxan monohydrate (150 mg kg^-1 b.w.) was given to the rats [34, 35].

While evaluating the antidiabetic effects of fractions of EMZFP, diabetes was induced by streptozotocin (STZ) –nicotinamide (NA) rat model. Due to the less sensitivity of female rats to STZ, male rats were preferred in this study. STZ causes pancreatic β-cell damage, whereas NA partially protects the β-cell against STZ, while the combination of these two makes the rats’ insulin-deficient but not insulin-resistance [36]. For induction of diabetes a single i.p. dose of NA (120 mg kg^-1 b.w./ml) in saline was given 15 min before the inoculation of a single i.p. dose of 60 mg kg^-1 b.w. of STZ to each rat [37, 38].

The alloxan monohydrate was dissolved in 0.9 % saline [39], while STZ was freshly prepared by solubilizing in 0.1 ml of cold Na-citrate buffer (0.1 M, pH 4.5) [40]. 1 ml syringe with 23 G needle was used for inoculations of all freshly prepared (alloxan, STZ, and NA) volumes of 1 ml kg^-1 b.w., and inoculated within 5 min after its preparation to avoid degradation. Normal water fed was replaced with 5% glucose water for up to the next 48 h to counteract severe acute hypoglycemia in all induced rats. On day 3^rd after inoculations, rats with fasting blood glucose level ≥ 200 mg/dl for alloxan and fasting blood glucose level ≥ 250 mg/dl for STZ- NA induced diabetes were well-thought-out as diabetic rats and utilized for further experiments [41, 42].

Experimental design

For evaluation of antidiabetic effects of EMZFP

After induction of diabetes, survived rats of either sex were randomly divided into different groups (n = 6), while for the control normal rats were selected, and fasting blood glucose levels of all rats were recorded. The treatment group animals were administered with EMZFP and blood glucose levels were recorded at 0^th, 7^th, 14^th, and 21^st days [33, 43, 44].

• Normal control (Gr. I): Normal rats were received p.o. 1 ml kg^-1 b.w. suspension of 1% gum acacia.

• Diabetic control (Gr. II): Alloxan-induced diabetic untreated rats were received p.o. 1 ml kg^-1 b.w. suspension of 1% gum acacia.

• Glimepiride (Gr. III): Alloxan-induced diabetic rats were received p.o. glimepiride 0.09 mg kg^-1 b.w. in suspension of 1% gum acacia.

• EMZFP 150 (Gr. IV): Alloxan-induced diabetic rats were received p.o. EMZFP 150 mg kg^-1 b.w., in suspension of 1% gum acacia.

• EMZFP 300 (Gr. V): Alloxan-induced diabetic rats were received p.o. EMZFP 300 mg kg^-1 b.w., in suspension of 1% gum acacia.

• EMZFP 600 (Gr. VI): Alloxan-induced diabetic rats were received p.o. EMZFP 600 mg kg^-1 b.w., in suspension of 1% gum acacia.

For evaluation of antidiabetic effects of fractions of EMZFP

STZ-NA induced diabetic, survived rats (male) were randomly divided into different groups (n = 6), while for the control group, normoglycemic rats were selected, and fasting blood glucose levels of all grouped animals were recorded. The treatment groups animals were administered p.o. fractions of EMZFP for 21 days [45, 46].

• Normal control (Gr-I): Normal rats were received p.o. 1 ml kg^-1 b.w. suspension of 1% gum acacia.

• Diabetic control (Gr-II): STZ-NA induced diabetic untreated rats were received p.o. 1 ml kg^-1 b.w. suspension of 1% gum acacia.

• Glibenclamide (Gr-III): Diabetic rats were received p.o. glibenclamide 10 mg kg^-1 b.w.

• Treatment group (Gr-IV to Gr-VII): Diabetic rats were received a suspension of various fractions (chloroform, acetone, ethyl acetate, and remaining ethanol fraction) of EMZFP at the doses of 200 mg kg^-1 b.w., prepared in 1% gum acacia solution.

Blood glucose level estimation

Antihyperglycemic effect of EMZFP extract was assessed by estimating blood glucose level on 1^st, 7^th, 14^th, and 21^st day, while, for fractions of EMZFP on 1^st, and 21^st day, by using Reckon diagnostic’s GOD/POD kits.

Body weight

Animals were weighed on the 7^th, 14^th, and 21^st days to estimate any weight changes on treatment with EMZFP extract.

Estimation of Serum levels of Total Cholesterol, HDL, LDL, and Triglyceride

Standard diagnostic kits from Reckon diagnostics, Pvt., ltd., India were used to estimate serum levels of total cholesterol, HDL, LDL, and triglyceride on the 21^st day of treatment with EMZFP and its fractions.

Assessment of the nephroprotective effect

a) Serum biochemical analysis

On the 21^st day, serum levels of total protein, albumin, and creatinine were assessed for any possible effects of EMZFP on diabetic renal conditions. Standard diagnostic kits from Reckon diagnostics, Pvt., ltd., India were used.

b) Histological study

On the 21^st day, any possible nephroprotective effect of EMZFP was studied by performing a histological study. Isolated kidneys from each rat belonging to each group were cleaned and kept in a 10% formalin solution. After embedding in paraffin wax, sliced with a rotary microtome in to about 5 μm thickness. Xylene and ethanol were used for deparaffinization. Further, hematoxylin and eosin-stained sections were microscopically examined for evaluation of histoarchitecture, hyalinization, and glomerulosclerosis [47].

Evaluation of liver glycogen content

On the 21^st day, liver glycogen content was determined by the standard method described [48]. Animal livers were isolated after scarifying with a urethane overdose, cleaned with saline, and homogenized (4 min.) with 40 ml of 5% trichloroacetic acid in the homogenizer vessel. After centrifuging the homogenate for 10 min at 3500 rpm the supernatant was filtered by filter paper and the residue was again extracted with the same procedure to extract 98% of glycogen. To the 1 ml of filtrate 5 ml of 95% ethanol was added and kept overnight at room temperature for precipitation in a test tube. On the next day, this precipitated content was centrifuged for 15 min at 3000 rpm and supernatant clear content was decanted. The test tube containing the remaining pellet was kept in an inverted state (15 min) until getting a dry pellet. This pellet was then added with 10 ml of anthrone reagent, 2 ml of distilled water and shaken vigorously for 5 min. The tube was then kept in boiling water for 15 min, later on, cooled to room temperature.

The optical density was colorimetrically determined at 620 nm. 2 ml of standard glucose (0.1 mg) solution and anthrone reagent (10 mL) was used as a standard solution while, for blank solution 2 ml water with 10 ml anthrone reagent was used. Glycogen content was estimated by the formula;

Mg of glycogen in 100 g of tissue = DU/DS×0.1 ×Volume of extract/weight of tissue ×100×0.9

Where, DU is the optical density of unknown; DS is the optical density of standard.

Assessment of cardiovascular risk indices [49]

Various cardiovascular risk indices were calculated as;

$$\begin{array}{c}\mathrm{Atherogenic}\mathrm{index}=\left(\mathrm{Total}\mathrm{cholesterol},/,\mathrm{HDL}\mathrm{cholesterol}\right)-\mathrm{HDL}\mathrm{cholesterol}\hfill \\ \mathrm{Cardiac}\mathrm{index}=\mathrm{Total}\mathrm{cholesterol}/\mathrm{HDL}\mathrm{cholesterol}\hfill \\ \mathrm{Coronary}\mathrm{artery}\mathrm{index}=\mathrm{LDL}\mathrm{cholesterol}/\mathrm{HDL}\mathrm{cholesterol}\hfill \end{array}$$

Assessment of neuropathic pain

Post-treatment, on the 21^st day hot plate-induced pain sensation ability of diabetic rats treated with EMZFP and its fractions was assessed. Paw licking responses of these diabetic rats were observed by placing them on a hot and cold plate apparatus set at the temperature of 50 °C with thermostat connected heating element of stainless steel (V J Enterprise). To prevent tissue damage a 10 s cutoff time was kept. The average of two times, for hot plate response latency for ipsilateral (uninjured) paw licking was measured, with 15 min time intervals [50].

Statistical analysis

The values presented were mean ± SEM (n=6). The presence of a significant difference between the mean values was calculated by two-way ANOVA followed by tukey's multiple comparison tests at 95% confidence interval with GraphPad Prism software version 9.2.0. While, the p-values < 0.05 were considered statistically significant.

Results

Acute oral toxicity study

During acute oral toxicity studies, in all cases when mice treated with EMZFP and its fractions behaved normally with no signs of any toxicity or moribund status. No death was observed at doses of 2000, 3000, and 5000 mg kg^-1 b.w. with EMZFP and at 2000 mg kg^-1 b.w. with fractions of EMZFP. In both the cases, no problematic clinical marks were detected in surviving mice (Table 1). As such, the minimal lethal dose (LD₅₀) could be more than 5000 mg kg^-1 b.w. for EMZFP; the therapeutic dose preferred was the 1/10^th (300 mg kg^-1 b.w.) of the safe dose (3000 mg kg^-1 b.w.) for this extract. However, for fractions of EMZFP the therapeutic dose preferred was the 1/10^th (200 mg kg^-1 b.w.) of safe dose (2000 mg kg^-1 b.w.).

Table 1.

The time course of signs of toxicity in mice treated with EMZFP-2000, 3000, and 5000 mg kg^-1 b.w. and 2000 mg kg^-1 b.w. for fractions of EMZFP.

Observations	At 3 h	At 24 h	At 72 h
Physical activity	N	N	N
Skin and fur	N	N	N
Eyes and mucous membranes	N	N	N
Behavioral pattern	N	N	N
Respiratory	N	N	N
Circulatory	N	N	N
Autonomic profile	N	N	N
Neurologic profile	N	N	N
Sleep	N	N	N
Diarrhea	N	N	N
Tremors	N. O.*	N. O.*	N. O.*
Mortality	N. O.*	N. O.*	N. O.*

N normal, N.O.* not observed

Effect of EMZFP on blood glucose

As predicted, rats from the untreated diabetic control group showed a continued increase in blood glucose levels from the 1^st to 21^st day. The glimepiride-treated diabetic animals exhibited a reduction in blood glucose level from the 14^th day onwards when compared with diabetic control group animals (p < 0.001) (Table 2). The treatment of diabetic animals with EMZFP 150 mg kg^-1 b.w. was exhibited a reduction in blood glucose level significantly on the 21^st day (p<0.05), and treatment with EMZFP 300 mg kg^-1 b.w. it was significantly reduced on the 14^th day (p<0.05), and the 21^st day (p<0.001). However, a highly significant reduction in blood glucose level was elucidated on treatment with EMZFP 600 mg kg^-1 b.w. on the 14^th day (p<0.01), and on the 21^st day (p<0.001).

Table 2.

Effect of EMZFP on blood glucose level in alloxan-induced diabetic rats

Groups	Blood glucose level (mg/dl) at
	1st day	7th day	14th day	21st day
Normal control	72.45 ± 4.56	65.15 ± 3.87	78.89 ± 2.47	71.47 ± 2.73
Diabetic control	266.43 ± 12.87 $	273.36 ± 11.15 $	279.62 ± 10.28 $	285.92 ± 9.45 $
Diabetic + Glimepiride	271.67 ± 11.42	244.76 ± 10.74	192.91 ± 14.36 ***	161.32 ± 10.28 ***
Diabetic + EMZFP 150	272.67 ± 8.96	253.58 ± 10.28	247.74 ± 11.24	228.58 ± 9.34 *
Diabetic + EMZFP 300	260.35 ± 10.12	248.64 ± 13.78	221.52 ± 14.82 *	198.02 ± 11.29 ***
Diabetic + EMZFP 600	265.34 ± 14.47	229.56 ± 15.24	215.36 ± 9.68 **	173.83 ± 12.87 ***

Data was represented as mean ± SEM (n=6), $p < 0.001 when compared with normal control; *p < 0.05, **p < 0.01, and ***p < 0.001 when compared with diabetic control. Diabetic rats treated with EMZFP 600 mg kg-1 b.w. elucidated significant blood glucose lowering effects on the day 14th and 21st.

Effect of fractions of EMZFP on blood glucose

Treatment with the doses of 200 mg kg-1 b.w. of EMZFP fractions for 21 days in STZ-NA induced diabetic rats were elucidated notable blood glucose-lowering effects (Table 3). A fall in blood glucose level was recorded when treated with EMZFP-Et (230.57 ± 10.48 mg/dl) and EMZFP-EA (238.83 ± 8.87 mg/dl) at a significant level of p<0.01 and p<0.05 respectively, reveling the marked antihyperglycemic effects.

Table 3.

Effect of fractions of EMZFP on blood glucose level in STZ-NA induced diabetic rats

Groups	Blood glucose level (mg/dl) at
	Day 1st	Day 21st
Normal control	66.35 ± 5.46	67.78 ± 4.12
Diabetic control	268.33 ± 14.24 $	290.48 ± 12.37 $
Diabetic + Glibenclamide	273.47 ± 12.65	158.44 ± 8.25 ***
Diabetic + EMZFP-C 200	285.76 ± 10.87	272.88 ± 9.18
Diabetic + EMZFP-A 200	288.16 ± 14.39	264.95 ± 8.54
Diabetic + EMZFP-EA 200	289.26 ± 9.52	238.83 ± 8.87 *
Diabetic + EMZFP-Et 200	286.18 ± 11.76	230.57 ± 10.48 **

(EMZFP-C: chloroform fraction; EMZFP-A: acetone fraction; EMZFP-EA: ethyl acetate fraction; EMZFP-Et: remaining ethanol fraction)

Data represented as mean ± SEM (n=6), $p < 0.001 when compared with normal control; *p < 0.05, **p < 0.01, and ***p < 0.001 when compared with diabetic control.

Diabetic animals treated with EMZFP-Et 200 showed a significant reduction in the blood glucose levels (p < 0.01) on 21st day.

Body weight changes

Untreated diabetic rats showed a notable reduction in body weight. While the rats treated with EMZFP for 21 days showed a gain in body weight at the doses of 150 mg kg^-1 b.w. (6.00 ± 3.92), 300 mg kg^-1 b.w. (7.65 ± 1.92), and 600 mg kg^-1 b.w. (7.71 ± 2.13), when compared with untreated diabetic rats (Table 4).

Table 4.

Effect of EMZFP on body weight changes in alloxan-induced diabetic rats

Groups	Body weight changes (g)
	7th day	14th day	21st day
Normal control	05.12 ± 1.45	6.25 ± 2.14	7.34 ± 2.34
Diabetic control	-9.12 ± 2.15 $	-7.82 ± 2.78 $	-6.72 ± 5.88 $
Diabetic + Glimepiride	1.56 ± 1.24	5.00 ± 2.28 *	7.56 ± 1.88 **
Diabetic + EMZFP 150	-7.57 ± 1.82	-6.64 ± 1.25	6.00 ± 3.92 *
Diabetic + EMZFP 300	-5.45 ± 2.10	5.10 ± 1.23*	7.65 ± 1.92 **
Diabetic + EMZFP 600	-2.65 ± 2.73	6.50 ± 1.16 **	7.71 ± 2.13 **

Values were presented as mean ± SEM (n=6), $p < 0.01 when compared with normal control; *p < 0.05, **p < 0.01 when compared with diabetic control. A Significant change in body weight was observed in diabetic rats treated with EMZFP 300 and 600 mg kg-1 b. w.

Effect of EMZFP on serum levels of total cholesterol, HDL, LDL, and triglyceride

Diabetic rats treated with EMZFP showed some protective effects, dosing with EMZFP-300 mg kg^-1 b.w. showed a significant reduction in serum LDL, and triglyceride (p<0.05) and a notable rise in serum HDL levels when compared with diabetic control rats. However, rats treated with EMZFP-600 mg kg^-1 b.w. indicated more decline in serum cholesterol (p<0.05), LDL and triglyceride levels (p<0.01), and perfection in the serum HDL level (p<0.05) when compared with diabetic control rats (Table 5).

Table 5.

Effect of EMZFP on serum lipids in alloxan-induced diabetic rats

Groups	Serum cholesterol (mg/dl)	Serum HDL (mg/dl)	Serum LDL (mg/dl)	Serum Triglyceride (mg/dl)
Normal control	83.45 ± 5.47	72.24 ± 3.41	43.54 ± 3.14	108. 28 ± 6.94
Diabetic control	176.72 ± 8.28 $	40.28 ± 2.14 $	118.45 ± 5.47 $	164.48 ± 6.82 $
Diabetic + Glimepiride	142.57 ± 5.26 ***	72.94 ± 2.42 ***	85.78 ± 3.98 ***	120.67 ± 5.29 ***
Diabetic + EMZFP 150	170.65 ± 3.12	62.45 ± 2.45	98.62 ± 2.72	158.72 ± 8.27
Diabetic + EMZFP 300	161.49 ± 2.45	63.72 ± 1.87	93.48 ± 3.38 *	139.40 ± 2.86 *
Diabetic + EMZFP 600	151.90 ± 3.57 *	65.28 ± 2.65 *	89.43 ± 2.69 **	136.28 ± 7.62 **

Values were expressed as mean ± SEM (n=6), $p < 0.001 when compared with normal control; *p < 0.05, **p < 0.01, and **p < 0.001 when compared with diabetic control. EMZFP 600 mg kg-1 b.w. elucidated a significant decline in serum cholesterol, triglyceride, and LDL level, furthermore a significant perfection in serum HDL level.

Effect of fractions of EMZFP on serum levels of total cholesterol, HDL, LDL, and triglyceride

Untreated STZ-NA induced diabetic rats exhibited a substantial upsurge in serum cholesterol, LDL, and triglyceride whereas, a decrease in HDL level. Diabetic rats treated with EMZFP-Et elucidated a significant (p<0.01) decline in serum level of cholesterol (140.90 ± 3.55), LDL (85.52 ± 3.92), and triglyceride (160.54 ± 1.75) but an increase in serum HDL (60.15 ± 2.24) when compared with diabetic control group rats. However, the treatment with EMZFP-EA has recorded a significant decrease in serum cholesterol (144.94 ± 2.42) at p<0.05 while, for LDL (87.04 ± 2.86), and triglyceride (161.98 ± 2.18) at p<0.01, and for increment in serum HDL (58.24 ± 2.65) at p<0.05 (Table 6).

Table 6.

Effect of fractions of EMZFP on serum lipids in STZ-NA induced diabetic rats.

Groups	Serum cholesterol (mg/dl)	Serum HDL (mg/dl)	Serum LDL (mg/dl)	Serum Triglyceride (mg/dl)
Normal control	92.62 ± 1.42	71.32 ± 2.36	45.38 ± 2.78	107.09 ± 4.72
Diabetic control	161.54 ± 3.57 $	41.58 ± 2.48 $	105.75 ± 3.48 $	180.75 ± 3.37 $
Diabetic + Glibenclamide	130.82 ± 2.52 ***	64.15 ± 1.87 ***	79.37 ± 4.58 ***	145.42 ± 3.67 ***
Diabetic + EMZFP-C 200	158.52 ± 2.89	55.82 ± 3.25	95.67 ± 2.57	170.58 ± 3.48
Diabetic + EMZFP-A 200	152.35 ± 3.24	57.65 ± 1.24	89.28 ± 4.22 *	164.20 ± 2.65 *
Diabetic + EMZFP-EA 200	144.94 ± 2.42 *	58.24 ± 2.65 *	87.04 ± 2.86 **	161.98 ± 2.18 **
Diabetic + EMZFP-Et 200	140.90 ± 3.55 **	60.15 ± 2.24 **	85.52 ± 3.92 **	160.54 ± 1.75 **

(EMZFP-C: chloroform fraction; EMZFP-A: acetone fraction; EMZFP-EA: ethyl acetate fraction; EMZFP-Et: remaining ethanol fraction)

Values were presented as mean ± SEM (n=6), $p < 0.001 when compared with normal control; *p < 0.05, **p < 0.01, and **p < 0.001 when compared with diabetic control. Diabetic rats treated with Et and EA fractions demonstrated a significant reduction in serum levels of cholesterol, LDL. triglyceride and increment in serum HDL level.

Effect of EMZFP on cardiovascular risk indices

Convinced cardiovascular protective effects were recorded in diabetic rats when treated with EMZFP (Table 7). A noteworthy decrease in cardiovascular risk indices viz. atherogenic, cardiac and coronary artery indices were demonstrated at 150, 300, and 600 mg kg^-1 b.w. doses of EMZFP extracts when compared with diabetic control group animals.

Table 7.

Effect of EMZFP on cardiovascular risk indices in alloxan-induced diabetic rats

Groups	Cardio-vascular risk indices (mg/dl)
	Atherogenic Index	Cardiac Index	Coronary Artery Index
Normal control	0.472 ± 0.34	1.485 ± 0.72	0.646 ± 0.58
Diabetic control	5.276 ± 0.74 $	6.247 ± 0.62 $	3.981 ± 0.47 #
Diabetic + Glimepiride	1.245 ± 0.47 **	2.254 ± 0.87 **	0.849 ± 0.76 *
Diabetic + EMZFP 150	2.314 ± 0.78	3.824 ± 0.35	1.476 ± 0.34
Diabetic + EMZFP 300	2.200 ± 0.27 *	3.110 ± 0.58 *	1.143 ± 0.79
Diabetic + EMZFP 600	1.746 ± 0.26 **	2.700 ± 0.76 **	0.901 ± 0.610 *

Data represented as mean ± SEM (n=6), $p < 0.001, #p< 0.05 when compared with normal control; *p < 0.05, **p < 0.01 when compared with diabetic control.

A significant reduction in cardiovascular risk indices viz. atherogenic, cardiac and coronary artery indices were demonstrated by EMZFP 600 mg kg-1 b.w. doses.

Effect of fractions of EMZFP on cardiovascular risk indices

Treatments of diabetic rats with the fractions of EMZFP were demonstrated cardiovascular protective effects when assessed for cardiovascular risk indices viz. atherogenic, cardiac, and coronary artery indices. EMZFP-EA fraction showed a notable (p < 0.05) decrease in cardiovascular risk indices compared to diabetic control rats. EMZFP-Et fraction demonstrated a significant (p < 0.05) reduction in cardiac and coronary artery index while, highly significant (p < 0.01) reduction in the atherogenic index when compared with diabetic control group animals (Table 8).

Table 8.

Effect of fractions of EMZFP on cardiovascular risk indices in STZ-NA induced diabetic rats.

Groups	Cardio-vascular risk indices (mg/dl)
	Atherogenic Index	Cardiac Index	Coronary Artery Index
Normal control	0.742 ± 0.65	1.511 ± 0.48	0.752 ± 0.37
Diabetic control	4.772 ± 0.46 $	5.010 ± 0.27 #	4.343 ± 0.82 $
Diabetic + Glibenclamide	1.000 ± 0.64 **	1.612 ± 0.64 *	0.912 ± 0.62 *
Diabetic + EMZFP-C 200	2.558 ± 0.38	3.388 ± 0.94	1.627 ± 0.78
Diabetic + EMZFP-A 200	2.272 ± 0.75	2.268 ± 0.25	1.375 ± 0.61
Diabetic + EMZFP-EA 200	1.601 ± 0.87 *	1.813 ± 0.37 *	1.200 ± 0.58 *
Diabetic + EMZFP-Et 200	1.201 ± 0.59 **	1.715 ± 0.72 *	1.127 ± 0.23 *

(EMZFP-C: chloroform fraction; EMZFP-A: acetone fraction; EMZFP-EA: ethyl acetate fraction; EMZFP-Et: remaining ethanol fraction)

All data represented as mean ± SEM (n=6), $p < 0.01, #p< 0.05 when compared with normal control; *p < 0.05, **p < 0.01 when compared with diabetic control. Diabetic rats treated with Et and EA fractions elucidated a significant reduction in cardiovascular risk indices.

Assessment of the nephroprotective effect

After induction of diabetes in diabetes control group animals, a significant (p<0.001) decrease in serum total protein and albumin, while a noticeable (p<0.01) increase in serum creatinine was observed. However, diabetic animals when treated with EMZFP-600 mg kg^-1 b.w., a significant (p<0.05) rise in serum total protein and albumin, and a decrease in serum creatinine was observed. Whereas, EMZFP-300 mg kg^-1 b.w., administration recorded a considerable (p<0.05) decrease in serum creatinine, when compared with diabetic control group animals (Table 9).

Table 9.

Effect of EMZFP on kidney parameters in alloxan-induced diabetic rats

Groups	Serum Total Protein (g/dl)	Serum Albumin (g/dl)	Serum Creatinine (mg/dl)
Normal control	5.64 ± 0.87	6.15 ± 0.48	0.94 ± 0.36
Diabetic control	1.41 ± 0.55 $	2.14 ± 0.26 $	4.52 ± 0.39 #
Diabetic + Glimepiride	5.10 ± 0.34 **	5.98 ± 0.37 **	1.28 ± 0.64 *
Diabetic + EMZFP 150	3.82 ± 0.28	3.18 ± 0.72	1.77 ± 0.87
Diabetic + EMZFP 300	4.24 ± 0.72	4.85 ± 0.82	1.52 ± 0.72 *
Diabetic + EMZFP 600	4.62 ± 0.18 *	5.33 ± 0.67 *	1.40 ± 0.58 *

Data represented as mean ± SEM (n=6), $p < 0.001, #p< 0.01 when compared with normal control; *p < 0.05, **p < 0.01 when compared with diabetic control. Diabetic rats treated with EMZFP 600 mg kg-1 b.w. demonstrated a significant increment in serum total protein, albumin, and reduction in serum creatinine.

Histopathological findings of kidneys for control and EMZFP treated rats

Earlier histological investigations of kidneys reveal that prolonged DM may encourage severe pathological alterations, such as changes in kidney tubule segments, hyalinization, changes in the mesangial matrix, and nephritis (glomerulosclerosis). The untreated diabetic animals showed significant scripts of disruptions of kidney tubule segments with hyalinization and nephritis. However, histopathological findings from kidneys of diabetic animals treated with glimepiride and EMZFP-300, and EMZFP-600 showed the well-conserved segments of kidney tubules with a clear picture of interspersed blood capillaries. All the architecture of kidneys appears to be normal, with no signs of any histological findings for hyalinization, changes in the mesangial matrix, and nephritis (Photomicrograph 1).

Fig. 12.

Photomicrographs (100×) showing effects on 21 days administration (p.o.) of EMZFP on histoarchitecture of kidney in alloxan-induced diabetic rats. A) Control group: depicted normal glomerulus architecture. B) Diabetic control group: abnormal glomerulus appearance. C) Diabetic + Glimepiride group: glomerular capillaries appear to be normal, with no signs of hyalinization, changes in the mesangial matrix, and nephritis. D) Diabetic + EMZFP-150 group: shows few signs of hyalinization and glomerulosclerosis. E) Diabetic + EMZFP-300 and F) Diabetic + EMZFP-600 group: picture suggests normal histoarchitecture with an absence of matrix expansion and no marks of hyalinization and nephritis.

Analysis of liver glycogen

Untreated diabetic rats were observed with a significant (p<0.01) low liver glycogen content as compared to normal control group rats. While rats treated with EMZFP-600 mg kg^-1 b.w. showed a significant (p<0.05) increase (14. 65 ± 0.47mg/100g) in liver glycogen content when compared with diabetic control group rats (Table 10).

Table 10.

Analysis of liver glycogen content in alloxan-induced diabetic rats post-treatment with EMZFP

Groups	Liver Glycogen (mg/100 g)
Normal control	16.25 ± 0.57
Diabetic control	11.18 ± 0.67 $
Diabetic + Glimepiride	15.17 ± 1.13*
Diabetic + EMZFP 150	12.64 ± 0.68
Diabetic + EMZFP 300	13.92 ± 1.08
Diabetic + EMZFP 600	14. 65 ± 0.47 *

Diabetic rats when treated with EMZFP 600 mg kg-1 b.w. elucidated a significant increment in liver glycogen content. Values were presented as mean ± SEM (n=6), $ p < 0.01 when compared with normal control; *p < 0.05 when compared with diabetic control.

Effect of EMZFP extracts and its fractions on hot plate induced neuropathic pain in diabetic rats

Latency to foot licking was recorded and statistically analyzed for all grouped animals. A decreased sensitivity towards the thermal stimuli was observed with untreated diabetic control group rats. Alloxan-induced diabetic rats when treated with EMZFP-600 mg kg^-1b.w. demonstrated a significant (p<0.01) improvement in pain sensitivity by licking the foot at an average time of 6.7 s from individual two readings (Table 11). However, STZ-NA induced diabetic rat’s treatment with EMZFP-EA and EMZFP-Et elucidate a significant (p<0.05) improvement in pain sensitivity by licking the foot at an average time of 6.8 s and 6.6 s from individual two readings respectively (Table 12).

Table 11.

Effect of EMZFP extracts on hot plate induced neuropathic pain in alloxan induced diabetic rats

Groups	Foot licking response(s)
Normal control	4.2 ± 0.5
Diabetic control	8.9 ± 0.2 $
Diabetic + Glimepiride	6.3 ± 0.4 **
Diabetic + EMZFP 150	8.1 ± 0.5
Diabetic + EMZFP 300	7.1 ± 0.3 *
Diabetic + EMZFP 600	6.7 ± 0.5 **

Values were presented as mean ± SEM (n=6), $ p < 0.001 when compared with normal control; *p < 0.05, **p < 0.01 when compared with diabetic control.

Diabetic rats when treated with EMZFP 600 mg kg-1 b.w. were demonstrated a significant improvement in pain sensitivity.

Table 12.

Effect of fractions of EMZFP extracts on hot plate induced neuropathic pain in STZ-NA induced diabetic rats

Groups	Foot licking response(s)
Normal control	4.5 ± 0.4
Diabetic control	8.7 ± 0.3 $
Diabetic + Glibenclamide	6.9 ± 0.5 *
Diabetic + EMZFP-C 200	8.5 ± 0.3
Diabetic + EMZFP-A 200	7.8 ± 0.3
Diabetic + EMZFP-EA 200	6.8 ± 0.5 *
Diabetic + EMZFP-Et 200	6.6 ± 0.4 *

(EMZFP-C: chloroform fraction; EMZFP-A: acetone fraction; EMZFP-EA: ethyl acetate fraction; EMZFP-Et: remaining ethanol fraction)

Diabetic rats when treated with Et and EA fractions were revealed a significant improvement in pain sensitivity. Values were presented as mean ± SEM (n=6), $ p < 0.001 when compared with normal control; *p < 0.05 when compared with diabetic control.

Discussion

The folkloric treatment with the use of ethnobotanicals has an extensive history for treating diabetes and thus it is continued to be an area of research for finding efficacious natural antidiabetic agents. Owing to enriched variety of phytocontents and antioxidant principles in EMZFP [51] it was selected to investigate its in-vivo antidiabetic potentials by evaluating various biochemical diabetic parameters and complications.

Insulin receptor or post-receptor defects with insulin resistance are a characteristic of T2DM. Various researchers reported that STZ-NA induced T2DM model is more suitable for evaluating the antidiabetic potentials of both pharmacological and natural plant sources on the course of DM [52]. Furthermore, this model has certain features like stable moderate hyperglycemia without any exogenous insulin to survive, glucose intolerance, and impairment in insulin secretion by glucose stimulation, reduction in pancreatic insulin stores, pancreatic β-cell reduction, receptiveness to sulphonylureas- glibenclamide [53].

Treatment with EMZFP for 21 days in alloxan-induced diabetic rats demonstrated significant dose-dependent serum glucose-lowering effects by EMZFP 600 mg kg^-1 b.w. p.o. when compared with diabetic untreated rats (Fig. 1). Similarly, with 200 mg kg^-1 b.w. p.o. doses of fractions EMZFP-Et and EMZFP-EA were also elucidated significant antihyperglycemic effects (Fig. 2). However, the blood glucose level in glimepiride and glibenclamide treated rats was lower than all EMZFP and its fractions treated groups. The gallic and ellagic acid [25, 26] had been accountable for enhanced uptake of glucose, α-glucosidase enzyme inhibition, lipid peroxidation, reduction of oxidative stress [54] and regeneration of β-cells and GLUT4 mediated glucose uptake [55]. Also, much literature had documented that gallic acid on oral administration in both the STZ and alloxan-induced diabetic animals had significantly reduced the blood glucose level, creatinine, and an increment in protein content [56, 57]. As MZFP possesses gallic and ellagic acid together with other rich variety of phytoconstituents like p-hydroxybenzoic acid, catechin, epicatechin, quercetin, etc., which might have enhanced the glucose tolerance, regulated carbohydrate metabolism key pathways, or insulin secretion. Thus, perfection in glycemic control and improvement in insulin secretion mechanism of remnant β-cells in diabetic animals might have lowered blood glucose level.

Fig. 1.

Effect of EMZFP on blood glucose level in alloxan-induced diabetic rats. Values were presented as mean ± SEM (n=6), $p < 0.001 when compared with normal control; *p < 0.05, **p < 0.01, and ***p < 0.001 when compared with diabetic control. Diabetic animals treated with EMZFP 600 showed a significant reduction in the blood glucose levels (p < 0.01) from14th day onwards.

Fig. 2.

Effect of fractions of EMZFP on blood glucose level in STZ-NA induced diabetic rats. Values were presented as mean ± SEM (n=6), $p < 0.001 when compared with normal control; *p < 0.05, **p < 0.01, and ***p < 0.001 when compared with diabetic control. Diabetic animals treated with EMZFP-Et 200 showed a significant reduction in the blood glucose levels (p < 0.01) on 21st day.

Hyperglycemia in diabetic animals corresponds to failure in weight gain during 21 days study period. While, EMZFP 300 and 600 mg kg^-1 b.w. treated diabetic rats demonstrated a significant weight gain slightly more than normal control rats (Fig. 3) which might be due to improvement in the synthesis of a structural protein and glycemic control [58].

Fig. 3.

Effect of EMZFP on body weight changes in alloxan-induced diabetic rats. Bars showed with different superscripts for every profile are significantly different. Values were expressed as mean ± SEM (n=6), $p < 0.01 when compared with normal control; *p < 0.05, **p < 0.01 when compared with diabetic control. Diabetic rats treated with EMZFP 300 and 600 mg kg-1 b.w. showed a significant change in body weight.

Hypertriglyceridemia and hypercholesterolemia are some of the etiological factors in DM complications like coronary heart diseases, atherosclerosis, and acute pancreatitis [59]. Depletion in insulin, grounds for insufficiency in lipoprotein lipase activity which considerably contributes to the triglycerides increment in DM [45]. While, hyperlipidemia contributes to a rise in cholesterol, triglycerides, LDL, VLDL, and a fall in HDL levels [60]. However, assessment of EMZFP extract and its fraction for dyslipidemia had demonstrated a significant fall in serum levels of cholesterol, triglycerides, LDL, and rise in HDL when treated with EMZFP-600 mg kg^-1 b.w. (Fig. 4) and EMZFP-Et, EMZFP-EA respectively (Fig. 5). It is well postulated and has been reported by many researchers that, owing to impaired glucose metabolizing pathways leads to the generation of free radicals and progression in DM, furthermore; which can be well repealed by phytochemicals having worthy antioxidant stuff [61]. Thus, it is sensible to accomplish that the antioxidants [62] or active phytoconstituents in the EMZFP, EMZFP-EA, and remaining EMZFP-Et fractions normalized the serum lipids possibly by perfecting hydrolysis, selective uptake, and metabolism of certain lipoprotein, and attenuating lipid peroxidation [63]. Literature findings also suggested that within the intestine or in the liver a possibility of merging of flavonoids into lipoprotein and later ward it is conceded within lipoprotein particle. Thus flavonoids may perhaps preferably shield LDL from oxidation [64].

Fig. 4.

Effect of EMZFP on serum lipids in alloxan-induced diabetic rats. Bars showing different superscripts for every profile are significantly different. EMZFP 600 mg kg-1 b.w. elucidated a significant reduction in serum cholesterol, triglyceride, and LDL level, furthermore a significant enhancement in serum HDL level. Values were presented as mean ± SEM (n=6), $p < 0.001 when compared with normal control; *p< 0.05, **p< 0.01, and ***p< 0.001 when compared with diabetic control.

Fig. 5.

Effect of fractions of EMZFP on serum lipids in STZ-NA induced diabetic rats. Bars showing different superscripts for every profile are significantly different. Diabetic rats treated with Et and EA fractions demonstrated a significant reduction in serum levels of cholesterol, LDL. triglyceride and increment in serum HDL level. Values were presented as mean ± SEM (n=6), $p < 0.001 when compared with normal control; *p < 0.05, **p < 0.01, and **p < 0.001 when compared with diabetic control.

Hyperlipidemia is one of the etiological factors for the progression of atherosclerosis under lipid peroxidation [65] however; prolonged hyperglycemia accelerates glycation and oxidation of small dense LDL particles, which leads to increases in the possibility of cardiovascular complications [66]. Treatment with EMZFP-600 mg kg^-1 b.w (Fig. 6) and its fractions EMZFP-EA and EMZFP-Et have elucidated a more significant reduction in atherogenic, cardiac and coronary artery indices (Fig. 7). The direct possible mechanism for these observations may account for the enriched antioxidants and above-discussed phytoconstituents liable for repletion of hyperglycemia and hyperlipidemia, along with phenolic compounds, such as quercetin and kaempferol [67] present in MZFP.

Fig. 6.

Effect of EMZFP on cardio vascular risk indices in alloxan-induced diabetic rats. Bars showing different superscripts for every profile are significantly different. Diabetic rats treated with EMZFP 600 mg kg-1 b. w. demonstrated a significant reduction in cardiovascular risk indices viz. atherogenic, cardiac and coronary artery indices. Values were presented as mean ± SEM (n=6), $p < 0.001, #p< 0.05 when compared with normal control; *p < 0.05, **p < 0.01 when compared with diabetic control

Fig. 7.

Effect of fractions of EMZFP on cardio vascular risk indices in STZ-NA induced diabetic rats. Effect of fractions of EMZFP on cardio vascular risk indices in STZ-NA induced diabetic rats treated for 21 days. Bars showing different superscripts for every profile are significantly different. Diabetic rats treated with Et and EA fractions demonstrated a significant reduction in cardiovascular risk indices. Values were presented as mean ± SEM (n=6), $p < 0.01, #p< 0.05 when compared with normal control; *p < 0.05, **p < 0.01 when compared with diabetic control.

Progressive proteinuria signifies the reduction in total protein content in diabetic untreated rats [68]. In diabetic nephropathy, accumulation of urea nitrogen and an increment in uric acid is responsible for renal dysfunction with glomerulosclerosis [69]. Diabetic rats when treated with EMZFP 600 mg kg^-1 b.w. had demonstrated a significant improvement in kidney parameters like serum protein, albumin, and decline in the serum creatinine level (Fig. 8), which was supported by histological findings from the kidneys showing evidence of reversal of glomerulosclerosis (Photomicrograph 1).

Fig. 8.

Effect of EMZFP on kidney parameters in alloxan-induced diabetic rats. Bars showing different superscripts for every profile are significantly different. Diabetic rats treated with EMZFP 600 mg kg-1 b.w. demonstrated a significant increment in serum total protein, albumin, and reduction in serum creatinine. Values were expressed as mean ± SEM (n=6), $p < 0.001, #p< 0.01 when compared with normal control; *p < 0.05, **p < 0.01 when compared with diabetic control.

In diabetic state defects in activation of synthase, phosphatase is responsible for depletion in glycogen content of liver and skeleton muscles [70]. After treatment with EMZFP-600 mg kg^-1 b.w. to the diabetic rats showed a marked increase in liver glycogen elucidating that imperfect glycogen storage of diabetic state was moderately improved (Fig. 9). This effect may be attributable to the perfection in the activation of synthase phosphatase due to an increment in insulin response promoting glycogenesis and/or decline in hepatic glycogenolysis [71].

Fig. 9.

Analysis of liver glycogen content in alloxan-induced diabetic rats post-treatment of EMZFP. Analysis of liver glycogen content in alloxan-induced diabetic rats post-treatment of EMZFP extracts for 21 days. Bar superscripts indicates a significance difference. Values were presented as mean ± SEM (n=6), $ p < 0.01 when compared with normal control, *p < 0.05 when compared with diabetic control. A significant rise in liver glycogen content was observed when diabetic rats treated with EMZFP-600 were compared with diabetic control group rats.

Prolonged hyperglycemia increases the possibilities of diabetic neuropathy and is clinically intervened by nontraumatic lower-limb amputations in diabetic patients. Numerous data from preclinical and clinical studies stated the pathogenesis of diabetic peripheral neuropathy signifying the key role of hyperglycemia persuaded advanced glycation end products (AGEs) [72]. The non-enzymatic reaction of methylglyoxal and interaction of elevated glucose with lipids, nucleic acids, and proteins is responsible for the generation of AGEs [73]. Decreased antioxidant defenses, increase in AGEs, altered glucose metabolism, disruption of the polyol pathway, and oxidative stress are some illustrative associated contrivances in the pathogenesis of peripheral neuropathy [74]. Assessment of EMZFP and its fractions for diabetic peripheral neuropathic complications by the hot plate method demonstrated that, EMZFP-600 mg kg^-1 b. w., (Fig. 10) EMZFP-EA and EMZFP-Et (Fig. 11) significantly lowered the foot licking response time and elucidated peripheral nerve protective property. EMZFP had a considerable antioxidant effect and radical-scavenging properties [29] which may account for protection against oxidative cell damage [75]. Thus, the amelioration in peripheral diabetic neuropathic pain by this extract and its fraction might be due to a part or large extent of polyphenolic and flavonoid contents and to the free radical scavenging or antioxidant potentials of Manilkara zapota peel.

Fig. 10.

Effect of EMZFP extracts on hot plate induced neuropathic pain in alloxan-induced diabetic rats. Effect of EMZFP extracts on hot plate induced neuropathic pain in alloxan-induced diabetic rats. Bar superscripts indicates a significance difference; Values were presented as mean ± SEM (n=6), $p < 0.001 when compared with normal control, *p < 0.05, **p < 0.01 when compared with diabetic control. A significant improvement in pain sensation was observed when diabetic rats treated with EMZFP-300 and EMZFP-600 was compared with diabetic control group rats.

Fig. 11.

Effect of fractions of EMZFP extracts on hot plate induced neuropathic pain in STZ-NA induced diabetic rats. Effect of fractions of EMZFP extracts on hot plate induced neuropathic pain in STZ-NA induced diabetic rats. Bar superscripts indicates a significance difference; Values represented as mean ± SEM (n=6), $p < 0.001 when compared with normal control, *p < 0.05 when compared with diabetic control. A significant improvement in pain sensation was observed, when diabetic rats treated with EMZFP-EA 200 and EMZFP- Et 200 were compared with diabetic control group rats.

Conclusions

Rats when treated with EMZFP, a noteworthy antihyperglycemic effects with augmentation in liver glycogen content, perfection in lipid profile and cardiovascular risk indices, enhancement in body weight, nephroprotective effects, beneficial effects in peripheral neuropathy, and histopathological evidence of reversal of glomerulosclerosis were observed. Although these results were not similar to that glimepiride effect, gradual positive augmentation in diabetic parameters is indicative of antidiabetic potentials of this peel extract when evaluated in the alloxan-induced diabetic rat model. However, during the antidiabetic evaluation of the EMZFP fractions in the STZ-NA induced diabetic rat model, EMZFP-EA and EMZFP-Et fractions contributed significant antihyperglycemic effects with perfection in lipid profile, cardiovascular risk indices, and positive peripheral neuropathic effects.

These preliminary study findings provide a basis for future research, where isolated phytoconstituents from this fruit peel should further be comprehensively evaluated pharmacologically and biochemically in exploring mechanism(s) for the antidiabetic outcome of this fruit peel. Further preclinical findings with isolated compounds could be supporting the traditional herbal medicine system for counting Manilkara zapota fruit peels in the dietary management of DM and likely prevention of its impediments.

Declaration of interest

The authors report no declarations of interest.

Consent for publication

Not applicable

Abbreviations

i.p.

Intraperitoneal

p.o.

Administered orally

b.w.

Body weight

STZ

Streptozotocin

NA

Nicotinamide

MZFP

Manilkara zapota (L.) P. Royen fruit peel

EMZFP

70% ethanolic extract of Manilkara zapota (L.) P. Royen fruit peel

EMZFP-C

Chloroform fraction of EMZFP

EMZFP-A

Acetone fraction of EMZFP

EMZFP-EA

Ethyl acetate fraction of EMZFP

EMZFP-Et

Remaining ethanol fraction of EMZFP

Author contribution

KPP: Conceptualization, methodology, investigation, data interpretation, editing & writing original draft. DSC: Methodology supervision. NVV: Formal analysis. All authors have read and approved the final manuscript.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability

Availability of data on reasonable request to corresponding author

Declaration