Antidiabetic and Cardioprotective effect of Terminalia phillyreifolia

(Van Heurck and Mull. Arg.) Gere and Boatwr. bark extract in Experimental animals

Archana Navale¹, Devanshu Patel², Archana Paranjape³

¹Department of Pharmacology, Parul Institute of Pharmacy, Parul University, Limda, Vadodara, India.

²Department of Pharmacology, Parul Institute of Medical Science and Research,

Parul University, Limda, Vadodara, India.

³Edutech Learning Solutions Pvt. Ltd., Vadodara, India.

*Corresponding Author E-mail: archanachavan_83@yahoo.co.in, archana.navale@paruluniversity.ac.in

ABSTRACT:

Objective: Objective of the current study was to evaluate the effect of methanolic extract of Terminalia phillyreifolia (Van Heurck and Müll. Arg.) Gere and Boatwr. (TP) bark in Type 2 diabetes mellitus and cardiovascular complications. Methods: Diabetes mellitus was induced in Wistar rats by administration of fructose and streptozotocin (40mg/kg, i.p.). TP bark extract was administered to diabetic animals at doses of 100mg/kg and 300mg/kg for 12 weeks. Glibenclamide (5mg/kg/day, p.o.) was administered as standard treatment. Various biochemical and functional parameters were evaluated at appropriate time intervals. Results: Methanolic extract of TP bark produced statistically significant (p< 0.05) hypoglycemic and antioxidant effect. Treatment with extract resulted in significant reduction in plasma glucose, HbA1C, lipid levels and oxidative stress parameters. TP treated rats demonstrated significantly lower insulin resistance and improved β cell function as compared to untreated diabetic rats. TP treatment resulted in improvement in cardiac and left ventricular hypertrophy as reflected by lower cardiac and left ventricular hypertrophy index. It could successfully improve the levels of cardiac enzyme markers such as LDH and CK-MB levels in a dose dependent manner. TP treatment could also efficiently prevent abnormalities of haemodynamic parameters such as mean blood pressure and heart rate. Conclusion: The results suggest that methanolic extract of TP bark exerted hypoglycemic effect in diabetic rats, comparable to that of standard. TP bark extract treatment was also able to inhibit development of cardiomyopathy in diabetic animals.

KEYWORDS: Anogeissus acuminata, Terminalia phillyreifolia, diabetic cardiomyopathy, type 2 diabetes mellitus, Insulin resistance, pancreatic beta cell function, Cardiac hypertrophy index, Glycated haemoglobin.

1. INTRODUCTION:

Type 2 diabetes mellitus (T2DM) is a disorder with direhealth outcomes characterized by various long term complications such as, neuropathy, nephropathy, retinopathy and cardiomyopathy¹. It has been proposed by various scientists from time to time that protection from macrovascular complications requires a multifactorial approach with tight management of blood glucose, blood pressure and lipid levels².

Evidence from other studies shows the importance of reducing oxidative stress, insulin resistance³ and inflammationfor prevention of diabetic complications^4-6. This makes it evident that an ideal treatment should embody anamalgamation of different pharmacological actions for prevention ofdevelopment of diabetic complications. Herbal drugs have more than one active constituents with varied mechanisms making them suitable for management of multifactorial diseases such as DM. Terminalia phillyreifolia (Van Heurck and Müll. Arg.) Gere and Boatwr. (TP) (Synonym Anogeissus acuminata) is known to be rich in tannins and flavonoids. The same wasalso substantiated by our previous study with bark extract of the plant⁷. Methanolic extract of bark has also demonstrated antidiabetic action in streptozotocin (STZ) induced diabetes mellitus in our previous study. It also exhibited protective effect on development of nephropathy in diabetic animals⁸. The plethora of beneficial effects displayed by TP treatment in previous studies encouraged us to study its possible beneficial effects in T2DM and diabetic cardiomyopathy.

2. MATERIALS AND METHODS:

2.1 Plant material and extraction:

Barks of plant were collected from Khedbrahma, Gujarat. Herbarium was submitted at NISCAIR, New Delhi (reference no. NISCAIR/RHMD/consult/2013/2290/70). Bark material was shade dried, powdered and extracted using methanol by Soxhlet extraction method (% yield 14.2% w/w).

2.2 Animal tests:

Male wistar rats (160-200gm body weight) were used for the experiment approved by Institutional Animal Ethics Committee (Protocol No. PIPH 35/13). Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines were followed for care and maintenance of animals.

2.3 Induction of DM and experimental design:

DM was induced by Fructose + STZ administration⁹. The rats were provided 10% fructose in place of drinking water for three weeks. After three weeks STZ (40mg/kg, i.p.) was injected in 6 hour fasted rats. Rats with fasting blood sugar level >250mg/dl after 48hours were considered hyperglycaemic. The animals were randomly divided in groups as below for a 12 weeks experiment.

Group I: Normal Control (NC): Acacia suspension, 1.0 ml, p.o.

Group II: Diabetic Control (DC): (DM induced with fructose+STZ) Acacia suspension, 1.0ml, p.o.

Group III: Standard treatment (Std): (DM induced with fructose+STZ) Glibenclamide (5mg/kg b.w.) in acacia suspension, p.o.

Group IV: (DM induced with fructose+STZ) BE100: 100mg/kg b.w. of acacia suspension of TP bark extract, p.o.

Group V: (DM induced with fructose+STZ) BE300: 300mg/kg b.w. of acacia suspension of TP bark extract, p.o.

2.4 Intravenous insulin tolerance test for assessing insulin sensitivity:

This test was carried out to confirm the type of diabetes mellitus and to validate the used model for type 2 DM induction. After two weeks of diabetes induction, Insulin was administered at a dose of 0.1 IU/kg i.v. Plasma glucose levels were determined at 3, 10, 15 and 20 minutes. A graph of % reduction in plasma glucose level Vs time was plotted. Slope (K_ITT) of the regression line was determined¹⁰.

2.5 Evaluation of antidiabetic activity:

Plasma glucose level was determined at 0, 2, 4, 8 and 11 weeks. HbA1C levels were determined by immunoturbidimetry method at 11 weeks.

2.6. Evaluation in diabetic cardiomyopathy

2.6.1. Measurement of haemodynamic parameters

The animals were anaesthetized by Ketamine (100 mg/kg, i.p.) + Xylazine (7mg/kg, i.m.). Mean blood pressure was determined by invasive method using Student physiograph and calibrated manometer¹¹. After euthanasia the heart was isolated and mounted as per the Langendorff heart technique. Chenoweth-Keolle buffer was used for perfusion of heart using air as aeration¹². The responses were recorded employing a force transducer and strain gauge coupler using to the Student’s physiograph.

2.6.2. Determination of cardiac hypertrophy index and left ventricular hypertrophy index:

After recording haemodynamic parameters, the weight of the heart and left ventricle was noted. Index of hypertrophy was calculated using following formula¹³.

Hert weight (mg)

Cardiac Hypertrophy Index = ------------------------------

Body weight (gm)

Left ventricular weight (mg)

Left Ventricular = -------------------------------------------

Hypertrophy Index Body weight (gm)

2.6.3. Assessment of biochemical markers:

Serum lipid levels were measured at 2 and 12 weeks post DM induction. LDH and CK-MB levels were determined using autoanalyzer at 12 weeks.

2.6.4. Assessment of oxidative stress parameters:

Oxidative stress parameters were determined from blood at 2^nd and 12^th week. Method described by Ohkawa et al. was used for Malondialdehyde (MDA)¹⁴, that by Aeibi and Bergmeyer¹⁵ for catalase and that by Beutler et al¹⁶forReduced glutathione (GSH).

2.6.5. Determination of insulin level, HOMA-IR and HOMA-β:

Eleven weeks after starting the treatment fasting blood samples for determination of insulin. HOMA-IR (Homeostatic Model Assessment for Insulin Resistance) and HOMA- β (HOMA to quantify β-cell function) scores were calculated using following formula and conversion factors¹⁷:

Blood glucose (1 mmol/l = 18 mg/dl).

[Insulin (U/1) × Blood Glucose (mmol/i)]

HOMA-IR = --------------------------------------------------

22.5

[20 × Insulin (U/l)]

HOMA- β = ---------------------------------------------------

[Blood glucose (mmol/l)-3.5

2.6.6. Statistical Analysis:

The observations were expressed as mean ± SEM (Standard Error of Mean). Further, statistical analysis was performed by either one-way ANOVA or two way ANOVA, which was followed by appropriate post hoc test, using GraphPad Prism version 5.03 for Windows, GraphPad Software, San Diego California USA. P values <0.05 were considered as significant.

3. RESULTS:

3.1. Insulin sensitivity using intravenous insulin tolerance test:

The % reduction in plasma glucose level Vs time plot (figure 1) represented a line with slope (K_ITT) of 0.644 ±0.23 %/min. The K_ITT value_,if more than 2.0 %/min represents normal insulin sensitivity while that less than 1.5 %/min indicates reduced insulin sensitivity. This indicates that diabetic animals in present study had IR.

Figure 1: Insulin sensitivity using intravenous insulin tolerance test

Analyzed by using GraphPad Prism 5.03. Linear regression analysis. Each point represents Mean ± SEM of 6 observations, D-FrSTZ: Animals with diabetes induced by 10% fructose for 3 weeks + 40 mg/kg streptozotocin, i.p. once

3.2. Effect of TP treatment on plasma glucose levels:

Methanolic extract of TP produced a significant hypoglycemic effect as seen by plasma glucose levels of 346.6±16.86 Vs 116.3±9.93mg/dl for DC and BE300 respectively at 11 weeks as shown in figure 2.

Figure 2: Effect of TP bark extract on Plasma glucose levels in diabetic rats

Analysed using two way ANOVA followed by Bonferroni post test, *** Values differ significantly from Diabetic Control (DC) group (p< 0.001), $ Value differs significantly from Normal Control (NC) group (p<0.001), Each point represents Mean ± SEM of 6 observations, NC: Normal control animals, DC: Diabetic control animals, Glibenclamide: Diabetic animals treated with glibenclamide ( 5 mg/kg, p.o./day), BE100: Diabetic animals treated with bark extract (100 mg/kg, p.o./day), BE300: Diabetic animals treated with bark extract (300 mg/kg, p.o./day)

3.3. Effect on glycated haemoglobin levels:

A consistent reduction in PGL was evident from the values of HbA1C measured at 11 weeks (11.6±0.44 Vs 7.5±0.61 % for DC and BE300 respectively) as shown in figure 3.

Figure 3: Effect of TP extract on glycated haemoglobin levels in diabetic rats at 11 weeks

Analysed by one way ANOVA followed by Tukey’stest, **Values differ significantly from Diabetic Control (DC) group (p< 0.01), * (p< 0.05) $Value differs significantly from Normal Control (NC) group (p< 0.001), Each point represents Mean ± SEM of 6 experiments, Glib: Diabetic animals treated with glibenclamide ( 5 mg/kg, p.o./day), BE100: Diabetic animals treated with bark extract (100 mg/kg, p.o./day), BE300: Diabetic animals treated with bark extract (300 mg/kg, p.o./day)

3.4. Evaluation in Diabetic Cardiomyopathy:

3.4.1. Effect on haemodynamic parameters:

DC rats had significantly higher mean blood pressure (MBP) (118.5±2.8mmHg) as compared to NC (88.6±3.1 mmHg) rats. Animals treated with glibenclamide and BE300 had significantly lower MBP (98.0±4.6 and 96.6±5.5mmHg) as compared to DC rats (figure 4A). It was observed that DC rats had significantly lower heart rate (255±14.0beats/min) as compared to NC rats (350±8.1beats/min), while that in standard (285±20.7 beats/min) and BE300 (321.67±10.14beats/min) treated rats was significantly higher compared to DC rats (figure 4B).

Figure 4 A: Effect on mean blood pressure, 4B: Effect on heart rate, 4C: Effect on cardiac hypertrophy, 4D: Effect on left ventricular hypertrophy, Analysed by one way ANOVA followed by Tukey’s test, ***Values differ significantly from Diabetic Control (DC) group (p< 0.001), **(p< 0.01), *(p< 0.05), $$$Value differs significantly from Normal Control (NC) group (p< 0.001), $Value differs significantly from corresponding normal control group (P<0.05), Each point represents Mean±SEM of 6 observations, Glib: Diabetic animals treated with glibenclamide ( 5mg/kg, p.o./day), BE100: Diabetic animals treated with TP bark extract (100 mg/kg, p.o./day), BE300: Diabetic animals treated with TP bark extract (300mg/kg, p.o./day)

3.4.2. Determination of cardiac hypertrophy index and left ventricular hypertrophy index:

DC rats had significantly higher cardiac and left ventricular hypertrophy index (4.29±0.04 and 0.795±0.013) indicating hypertrophy of heart and left ventricle as compared to NC (3.10±0.07 and 0.623± 0.017 respectively). TP treatment could prevent both cardiac and LV hypertrophy in a dose dependent manner (3.65±0.19 and 0.733±0.016 for BE100, 3.18±0.10 and 0.700±0.022 for BE300).

3.4.3. Effect on biochemical markers of cardiac damage:

LDH and CK-MB levels in serum were significantly elevated in DC rats (182.16±12.26U/L and 28.71±3.14U/L respectively), while these levels were significantly lower in standard (100.9±6.93 and 17.53±2.46U/L respectively). As depicted in figure 5 animals treated with higher dose of TP had significantly low levels of LDH and CK-MB (112.83±12.3 U/L LDH and 18.15±1.9U/L CK-MB). 100mg/kg dose of TP demonstrated significantly lower LDH (120.66±21.0 U/L) but not CK-MB levels (21.21±3.4 U/L).

Figure 5: Effect of TP extract on Serum LDH (A) and Creatine Kinase levels (B) in diabetic rats at 12 weeks

Analysed by one way ANOVA followed by Tukey’s test, ***Values differ significantly from Diabetic Control (DC) group (p< 0.001), **(p< 0.01), *(p<0.05), $$$Value differs significantly from Normal Control (NC) group (p<0.001), $$Value differs significantly from corresponding normal control group (P<0.01), Each point represents Mean±SEM of 6 observations, Glib: Diabetic animals treated with glibenclamide ( 5 mg/kg, p.o./day), BE100: Diabetic animals treated with TP bark extract (100mg/kg, p.o./day), BE300: Diabetic animals treated with TP bark extract (300mg/kg, p.o./day)

3.4.4. Effect on lipid levels:

TP extract treatment at both doses also elicited a significant reduction in total cholesterol, triglyceride and LDL levels, and increase in HDL levels as compared to DC rats (Table 1).

Table 1: Effect of TP treatment on lipid levels of type 2 diabetic rats

	Weeks	NC	DC	Glibenclamide	BE100	BE300
TC (mg/dl)	2 weeks	80.42 ±3.97	123.98 ±3.99^$	98.56 ±3.18**	111.22 ±8.74	100.91 ±5.24*
TC (mg/dl)	12 weeks	78.21 ±3.67	134.88 ±5.62^$	106.89 ±3.03***	113.89 ±5.98*	102.96 ±7.62***
TG (mg/dl)	2 weeks	78.67 ±6.14	195.83 ±35.22^$	111.25 ±16.41**	99.98 ±10.37**	111.69 ±22.36**
TG (mg/dl)	12 weeks	82.65 ±10.56	204.03 ±40.32^$	125.94 ±20.43*	120.87 ±13.91**	114.18 ±16.33**
LDL (mg/dl)	2 weeks	34.83 ±1.05	50.61 ±4.33^$	46.45 ±4.29	39.67 ±2.69*	41.32 ±2.09*
LDL (mg/dl)	12 weeks	37.8 ±1.71	54.82 ±1.97^$	40.05 ±3.69***	43.98 ±3.43*	41.23 ±3.39**
HDL (mg/dl)	2 weeks	27.72 ±3.18	15.99 ±1.73^$	22.6 ±2.45	25.96 ±4.4	26.41 ±2.24*
HDL (mg/dl)	12 weeks	28.49 ±3.6	14.29 ±1.18^$	21.83 ±1.68	25.48 ±3.25*	25.81 ±0.9*

Values are expressed as Mean±SEM, N=6. $: Value differs significantly from corresponding normal control group (P<0.001), * significantly different from diabetic control (P < 0.05), ** (P < 0.01), *** (P < 0.001)

Table 2.Effect of TP treatment on oxidative stress parameters of type 2 diabetic rats at 2 and 12 weeks

Values are expressed as Mean ± SEM, N=6. $: Value differs significantly from corresponding normal control group (P<0.001), * Significantly different from diabetic control (P < 0.05), ** (P < 0.01), *** (P < 0.001)

	Time	NC	DC	Glibenclamide	BE100	BE300
MDA levels (nmol/ml)	2 weeks	0.193 ± 0.011	0.533 ± 0.03^$	0.397 ± 0.03*	0.368 ± 0.035**	0.277 ± 0.035***
MDA levels (nmol/ml)	12 weeks	0.207 ± 0.019	0.608 ± 0.028^$	0.478 ± 0.038*	0.417 ± 0.059***	0.352 ± 0.049***
GSH levels (mg/ml)	2 weeks	12.35 ± 0.48	8.03 ± 0.27^$	10.65 ± 0.62**	10.77 ± 0.45**	10.87 ± 0.62**
GSH levels (mg/ml)	12 weeks	12.53 ± 0.45	7.23 ± 0.16^$	10.42 ± 0.51***	10.92 ± 0.49***	11.4 ± 1.17***
Catalase (U/ml)	2 weeks	0.882 ± 0.03	0.447 ± 0.019^$	0.635 ± 0.058	0.643 ± 0.074*	0.722 ± 0.089**
Catalase (U/ml)	12 weeks	0.86 ± 0.034	0.445 ± 0.032^$	0.632 ± 0.058	0.652 ± 0.082*	0.688 ± 0.064*

Analysed by one way ANOVA followed by Tukey's Test, Values differ significantly from Diabetic Control (DC) group ***(p< 0.001), **(p< 0.01), * (p<0.05), $ Value differs significantly from Normal Control (NC) group (p< 0.001), Each point represents Mean ± SEM of 6 observations, Glib: Diabetic animals treated with glibenclamide (5mg/kg, p.o./day), BE100: Diabetic animals treated with TP bark extract (100mg/kg, p.o./day), BE300: Diabetic animals treated with TP bark extract (300mg/kg, p.o./day).

4. DISCUSSION:

Insulin resistance is an important attribute that differentiates T1DM from T2DM. We assessed the insulin sensitivity in fructose + STZ induced diabetes model to confirm the type of disease developed. The insulin sensitivity index (K_ITT) inthese animals was found to be < 1.5%/min indicating reduced insulin sensitivity or insulin resistance. Similar characteristic was also confirmed by HOMA IR values in disease control animals¹⁸. Development of T2DM in experimental animals caused an elevation in plasma glucose levels and HbA1C levels. TP bark extract treatment could reduce plasma glucose levels as well as HbA1C levels in a dose dependent manner. This corroborates a glucose lowering effect of test agent. Dyslipidaemia is known to be the major predictor of cardiovascular adverse events in patients of T2DM¹⁹. Disturbances in lipid levels were also developed in our experimental animals. TP treatment was able to significantly mitigate the disturbances in lipid levels, indicating a favorable effect on cardiovascular risk of diabetic animals.Treatment with TP bark extract also demonstrated a highly significant reduction in oxidative stress in the diabetic animals. This action of TP bark extractis in accordance with our previous experiments in which it has shown potent in-vitro anti-oxidant activity²⁰. Oxidative stress is an important factor contributing towards development of diabetic cardiomyopathy²¹. Therefore, anti-oxidant mechanism of TP extract may be one of the several factors responsible for its protective effect against cardiomyopathy in DM.

Previous research studies have also demonstrated the anti-diabetic effect of methanolic and aqueous extracts of the plant in alloxan induced DM ^22-24. However, our study is the first to demonstrate the effectiveness of TP bark extract in animal model of T2DM. The reduction in insulin resistance was also confirmed by results of HOMA assessment. TP treatment could significantly reduce the insulin resistance in diabetic animals supporting its effectiveness in T2DM. Quercetin, a flavonoid present in TP has shown β cell protective effect and improved insulin sensitivity in earlier studies^25-26. Our previous study has also shown PTP1B inhibitory effect of TP bark extract²¹. Thus, it can be deduced that probable mechanism of insulin resistance reduction by TP may be via PTP1B inhibition. Apart from HOMA IR, TP extract was also able to improve HOMA β score. HOMA β represents the pancreatic β cell function. Study by Zaruva et al., suggests insulin secretogogue activity of one of the constituent of TP- castalagin²⁴. Other compounds present in TP such as, pterostilbene, gallic acid and quercetin have also demonstrated insulin secretogogue activity in earlier studies^27-29. LDH and CK-MB levels were elevated in diabetic animals at 12 weeks indicating considerable cardiac damage ^30,31. However, their levels were significantly lower in TP treated animals, indicating its cardioprotective effect. T2DM is also known to promote deposition of protein and collagen fibers in heart wall, reducing the functional efficiency of myocardium³². TP treatment in our study animals prevented the development of such hypertrophy, indicating a protective effect against cardiac remodeling. Castalagin present in TP has demonstrated hypotensive effect in spontaneously hypertensive rats³³.

5. CONCLUSION:

The methanolic extract of TP bark demonstrated antidiabetic effect. It could also protect against development of diabetic cardiomyopathy in experimental animals. Further investigation is required to identify active constituent/s responsible for antidiabetic and cardioprotective effects and verifying their mechanisms.

6. ACKNOWLEDGEMENTS:

We heartily acknowledge the infrastructural support received from Parul University. We are grateful to Sun Pharma Advanced Research Centre, Tandalja, Vadodara for generous gift of experimental animals. We are thankful to Mr. Suresh Purohit, Cadila Pharmaceuticals Ltd. for the gift sample of glibenclamide.

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Received on 03.02.2022 Modified on 09.09.2022

Research J. Pharm. and Tech 2023; 16(9):4009-4015.

DOI: 10.52711/0974-360X.2023.00657