Heartwood Extract of Neolamarckia Cadamba Ameliorates Oxidative Stress     Linked Type II Diabetes in Wistar Albino Rats

 

Shanti Bhushan Mishra1*, Mukul Maurya2, Vineet Srivastava2

1Department of Pharmacognosy, United Institute of Pharmacy, Prayagraj 211010, India.

2Department of Pharmacology, United Institute of Pharmacy, Prayagraj 211010, India.

*Corresponding Author E-mail: shantipharma15@gmail.com

 

ABSTRACT:

The Neolamarcia cadamba plant has traditionally been claimed for the treatment of a variety of metabolic disorders, including diabetes. Many scientific studies have also proved its effectiveness against various diseases due to the presence of several chemical constituents viz. Quinovic Acid, Cadamabine, Cadambagenic Acid, Cadamine, Beta-Sitosterol, Chlorogenic Acid, etc. Therefore, in this study the effect of 50% ethanolic extract (NCEE) from the heartwood of the plant was evaluated for its potential antidiabetic, hypolipidemic and antioxidant effects in Streptozotocin (STZ) and high fat diet (HFD) induced diabetic rats. Preliminary phytochemical screening of NCEE revealed the presence of phenolic compounds, flavonoids, steroids and alkaloids. NCEE (200 and 400mg/kg body weight) was administered orally to diabetic rats, once daily for 3 weeks. Blood glucose levels, various biochemical parameters, antioxidant enzyme levels and histopathological investigation were performed in the diabetic control group and the extract treated groups. Plant extracts have a dose-dependent antidiabetic effect by normalizing the levels of antioxidant enzyme biomarkers and hematological abnormalities and ameliorating histopathological changes compared to the diabetic control group. The observations confirmed that the 50% ethanolic extract of heartwood of Neolamarckia cadamba (Roxb.) Bosser exhibits anti-diabetic, hypolipidemic effect along with mitigation of oxidative stress.

 

KEYWORDS: Antidiabetic, HFD, Neolamarckia cadamba, Oxidative stress, Streptozotocin.

 

 


INTRODUCTION: 

Chronic hyperglycemia in type II diabetes is accompanied by a sequence of metabolic abnormalities. This is an increasingly common disorder of carbohydrate and lipid metabolism. The disease has two important features, one is the failure of pancreatic beta cells to properly secrete insulin in response to blood glucose levels and the other is insulin resistance, which means a failure of peripheral tissues such as the liver, muscle, and adipose tissue to produce physiological response to dosage of insulin1. Insulin resistance always occurs in the early stages of type II diabetes, followed by prolonged insulin resistance, leading to further weakening of beta cells, resulting in hyperglycemia and altered lipid metabolism.

 

It has been recommended that HFD may be a better way to initiate insulin resistance, which is one of the important features of type II diabetes and low-dose STZ has been known to induce a mild impairment of insulin secretion, which is similar to the feature of the later stage of type II diabetes2- 3.

 

Therefore, in this study, we preferred animal model by feeding the low-dose STZ with a high-fat diet that closely resemblance disease incidence (from insulin resistance to β-cell dysfunction) as well as the metabolic features of type II diabetes in humans.

 

India is the largest producer of medicinal herbs in the world due to which it is often called a botanical garden of the world. Neolamarckia cadamba is one of the medicinal plants traditionally used by the Indian. Various parts of this plant have been traditionally used to treat various diseases4. Scientific exploration of this plant revealed its antimicrobial, antioxidant, analgesic, anti-inflammatory, hepatoprotective, wound healing and antidiabetic properties5-9. This plant phytochemically rich by presence of various chemical constituents like indole alkaloids, terpenoids, sapogenins, saponins, terpenes, steroids, fats and reducing sugars10. Heartwood of plant contain Pinocembrin, Kaempferol, Aromadendrin, Quercetin, Taxifolin, Chlorogenic acid, Chrysin, and Naringenin11-13. The present work establishes antidiabetic activity of N. cadamba in experimental animals.

 

Materials and Methods Plant material:

The Heartwood of Neolamarkia cadamba was collected from the local area of Naini Prayagraj in the month of October 2020 and allow for air-dried at 400°C. Authentication of plant was done by taxonomist from Botanical Survey of India Central region, Prayagraj and voucher specimen has been submitted in the departmental herbarium with Accession No. 104534.

 

Preparation of extract:

The powdered material of heartwood of N. cadamba (1000g) were macerated with petroleum ether to eliminate fatty ingredients; the marc was further extracted with of 50% ethanol for seven days by maceration process and solvent recovery have been performed through distillation assembly at (50-600C) for 18 hrs. The obtained semi solid plant extract have been subjected for drying on water bath under controlled conditions and then store in air tight container in a desiccator. Thus 18.45g of solid residue (yield 1.84% w/w) was obtained in respect of crude  plant material.

 

Experimental animals:

Adult male wistar albino rats (150-210g) were procured from CPCSEA registered Laboratory Animal Supplier m/s Chakraborty Enterprises Kolkata (Regd. No. 1443/PO/Bt/s/11/CPCSEA). All animals were housed in quarantine area for acclimatization for two weeks before the experiment. The animals were kept in polypropylene rat cages under standard conditions, where 12 h light and 12 h dark cycle maintained at temperature (25±20C) and humidity (55±5%). All animals fed with normal chow (Anupam, India) and water ad libitum throughout the experiment. The study was approved by the Institutional Animal Ethics Committee of United Institute of Pharmacy with approval no. UIP/IAEC/Nov.2020/04

 

Acute toxicity study:

To determine the lethal dose (LD50) of NCEE, oral acute toxicity study has been performed in experimental animals as per OECD 423 guidelines14. NCEE was administered orally with the help of oral gavage tube at a dose of 5, 50, 300, 2000 and 5000mg/kg body wt. with regular food and water. No significant toxic response showed in animals, in the first 4 h after the administration of the plant extract. All animals were observed for any type of gross abnormality for next fourteen days. No morbidity and mortality were found in all the animals.

 

Experimental induction of diabetes:

Type II diabetes was induced by low-dose of STZ and high fat diet (HFD) administration as per previously reported method with slight modification15. Briefly, the animals were feed with HFD for a period of two weeks and then injected with low dose of STZ (single dose of 35mg/kg b.w.) by intra-peritoneal route. The composition of HFD was tested from food analysis & research laboratory, Centre of Food Technology, University of Allahabad.

 

Seven days after STZ injection, the fasting blood glucose levels of all rats were estimated; Those whose blood glucose level was more than 200mg/dL were considered diabetic and were selected for further experiments. These animals were continued on the HFD until the end of the study.

 

Experimental design:

The Wistar rats were divided into five groups of six animals in each group:

Group I: Normal control rats administered 1% gum acacia in normal saline daily for 21 days

Group II: Diabetic control rats administered HFD + 1% gum acacia in normal saline daily for 21 days Group III: Diabetic rats administered HFD + NCEE (200mg/ kg, p.o) daily for 21 days

Group IV: Diabetic rats administered HFD + NCEE (400mg/kg, p.o) daily for 21 days

Group V: Diabetic rats were administered HFD + metformin (100mg/kg, p.o) daily 21 days

 

The drugs and vehicles were administered orally through oral gavage tube once daily for the period of 21 days. Blood was drawn by retro orbital puncture on day 0 day, 7 day, 14 and day 21 to measure blood glucose levels. Gluco-One blood glucose monitoring kit (Morepen Laboratories, New Delhi) used for assessing the blood glucose level. Biochemical parameters such as serum insulin, cholesterol, Triglycerides, HDL, LDL and VLDL were estimated by using diagnostic kits (Erba Mannheim, Mumbai). After 21 days of treatment, All the animals were sacrificed by intra-peritoneal injection of thiopentone (120mg/kg). The organs viz. pancreas liver and kidney were dissected out, cleaned and washed in ice-cold phosphate buffer saline and fixed in 10% neutral buffered formalin solution for histopathological observation and antioxidant assessment. The collected blood samples were instantly centrifuged at 2500rpm for 15min. The separated serum was collected in fresh serum tubes and stored in refrigerator (2-4ºC) after tightly capped.

 

In vivo antioxidant activity:

Measurement of Catalase (CAT), Reduced glutathione (GSH), Superoxide dismutase (SOD) and Glutathione peroxidase (GPx) were estimated for ascertaining antioxidant efficacy of NCEE as per previously reported method16.

 

Histopathological Examination:

For the histopathological analysis, the animal from each group has been sacrificed by cervical dislocation under anesthetic condition and pancreas was isolated and preserved by immersion in a 10% formalin solution. The organs were sliced at 5µm in a rotary microtome and stained with Eosin & Hematoxyline stain. The snaps of histopathological slides were captured at 40X with a Nikon E400 microscope (Chiyoda, Tokyo).

 

Statistical analysis:

Statistical analysis was performed using GraphPad Prism software version 9.1.2 Values are represented as the mean±S.D. for six rats in each group. Statistical comparisons were analyzed by one-way ANOVA with post-hoc differences using the Newman–Keuls method. p < 0.001 was considered significant.

 

RESULTS:

Preliminary phytochemical analysis:

Preliminary phytochemical analysis divulges the presence of phenolic compounds, flavonoids, saponins, carbohydrates, steroids and alkaloids in 50% ethanolic extract of heartwood of N. cadamba.

 

Acute oral toxicity study:

It was observed that all the animals that received the extract orally in different doses (5mg/kg, 50mg/kg, 300 mg/kg, 2000mg/kg, and 5000mg/kg b.w.) of N. cadamba showed regular behavior and motility, moreover, no mortality recorded during this toxicity study period. Results indicates that oral administration of NCEE even at a dose of 5000mg/kg b.w. was found to be nontoxic. Two different doses i.e., 200mg/kg and its double strength 400 mg/kg body weight was selected for the anti-diabetic activity.

 

Antihyperglycemic effect of NCEE:

The effect of NCEE on STZ and HFD induced diabetes in animals was shown in Fig.2a. At the end of treatment, there is decrease in blood glucose level in treated rats with NCEE (200mg/kg) was 247.1 - 130.3mg/dL (p< 0.01); after oral delivery of NCEE (400mg/kg), blood glucose level was reduced from 255.3 – 115mg/dL (p< 0.001); while in case of Metformin (100mg/kg) it was found to be 241.8 - 107.8mg/dL (p< 0.001).

Effect of NCEE on biochemical markers:

Fig. 2b showed that triglyceride (TG), total cholesterol (TCh), low density lipoprotein (LDL) and very low-density lipoprotein (VLDL) levels were significantly elevated in diabetic animals compared to those in control group and drug treated groups while high density lipoprotein (HDL) level was significantly decreased in diabetic model group compared to those in control group. Oral administration of NCEE (400mg/kg) showed the significant amelioration in biochemical parameters viz. TG (110.6-68.3; p<0.001), TCh (151.3-75.8; p<0.001) HDL (13.3- 18.0; p<0.001), LDL (72.1-44.6; p<0.01) and VLDL (31.5-19.2; p<0.001) levels in diabetic rats.

 

The fasting serum insulin (FSI) levels were estimated at the end of treatment in all the groups. FSI level has been reduced in the animals of the diabetic control group. NCEE (200 and 400mg/kg) treated rats were exert significant  influence on serum insulin levels from 14.8 ± 1.4 to 24.5±1.0 and 14.8±1.4 to 26.1±1.3 IU in STZ induced diabetic animals.

 

Figure. 1 Effect of NCEE on blood glucose level (1a) and on biochemical parameters (1b)

 

Effect of NCEE on antioxidant enzymes:

The outcome of NCEE on antioxidant level, the activities of enzymatic antioxidants CAT, GPx, SOD, and non- enzymatic antioxidant GSH were determined and results were mentioned in (Table 1). The level of enzymatic and non-enzymatic antioxidants were significantly reduced in diabetic control rats. After treating for 21 days with NCEE 200mg/kg there is a significant increase in GPx and CAT level in liver 5.6 ± 0.5 to 8.1±0.4 (p< 0.001) and 30.8±2.8 to 56.1±0.9, (p< 0.01) respectively. There are ameliorative changes in other parameters viz. GSH and SOD in a dose dependent practice as compared with diabetic control group.


Table 1: Effect of NCEE on various antioxidant enzymes

Effect of NCEE on in vivo anti-oxidant enzymes Parameters

SOD (U min/ (mg/Hb in erythrocytes)

CAT (μmol of H2O2 consumed/ min/mg protein)

GPx (μg glutathione consumed / min/mg protein)

GSH (nM of DTNB conjugated/mg protein)

Control

Liver

8.2 ±0.3

86.2 ± 1.2

10.3 ± 0.5

51.9 ± 0.9

Kidney

13.2 ±0.9

72.5 ± 1.0

13.1 ± 1.1

34.6 ± 1.1

Diabetic Control

Liver

3.9 ±0.3z

30.8 ± 2.8z

5.6 ± 0.5z

23.3 ± 0.3z

Kidney

8.2 ± 0.6z

51.3 ± 1.4z

7.5 ± 4.5

27.6 ± 0.79z

Metformin (100 mg/kg)

Liver

7.1 ±0.7c

83.3 ±1.1c

9.3± 0.4c

47.8 ± 1.2c

Kidney

12.6 ± 1.3c

67.3 ± 1.9c

11.7 ± 0.9

32.8 ± 1.4c

NCEE (200 mg/kg)

Liver

5.3 ±0.4b

56.1 ±0.9b

8.1 ±0.4c

38.6 ±0.87b

Kidney

11.8 ± 3.2b

61.0 ± 1.7

9.6 ± 1.1

30.6 ±1.5

NCEE (400 mg/kg)

Liver

6.3 ±0.4b

77.4 ± 0.8c

8.83 ±1.25c

42.7 ±1.1c

Kidney

12.2 ± 0.9c

64.4± 3.8c

10.5 ± 1.2

31.9 ± 1.3c

The data represent as mean ± standard deviation of six rats in each group. bp <0.01 and cp < 0.001 compared to diabetic control group. zp<

0.01 as compared to control group.

 

 


Histopathological Investigation

Pancreas:

In Fig. 2, microscopically examined pancreas section of normal control group (2a) shows normal architecture that means islets are normal and no fibrosis or any inflammation was seen where as in diabetic control group (2b) architecture is somewhat distorted with lymphocytic infiltration. NCEE (200 and 400mg/kg) treated groups (2c and 2d) shows normal architecture which is partially effaced. The islets are normal, no fibrosis or any inflammation was seen. The acini cells are lined by round two oval cells with moderate cytoplasm. In standard group (2e) i.e., treated with Metformin (100 mg/kg) showed normal architecture same as that of control group with no lymphocytic infiltration. Histopathological observation of pancreas revealed morphological changes in the diabetic groups whereas the NCEE treated groups retain the normal architecture.

 

 

Figure: 2 Histopathology of Pancreas

DISCUSSION:

Type II diabetes, known as adult-onset or non-insulin-dependent diabetes, concerned for more than 90–95% of all diabetes; Therefore, it is imperative to find an excellent and acceptable treatments and new prevention strategies for type II diabetes. To achieve this goal, suitable experimental models are considered as essential tools. It has been suggested that HFD may be a better way to initiate insulin resistance which is one of the important features of type II diabetes17. Additionally, streptozotocin (STZ) is commonly used to reprogram both insulin- dependent and non-insulin dependent diabetes mellitus, low-dose streptozotocin has been known to induce mild impairment of insulin secretion known to be characteristically similar to the later stages of type II diabetes18-19. Therefore, in the present study HFD and low dose of STZ model was selected that closely reflects the metabolic    characteristics of human type II diabetes mellitus, this model is beneficial for testing insulin sensitizing and insulinotropic agents. Moreover, this model is cost-effective, readily available, easy to generate and demonstrates stable and long-lasting hyperglycemia.

 

Acute toxicity study confirmed the absence of any toxicity or mortality of N. cadamba at a higher dose of 5000 mg/kg. According to OECD guidelines, If LD50 is estimated to be above 5,000mg/kg then no further acute toxicity    testing is required. Acute toxicity test serves as a guide in dosage selection for long term toxicity studies as well as other studies that involve the use of animals20. It is therefore concluded that the 50% ethanolic extract of N. cadamba could be used with some degree of safety especially when consumed by the oral route.

 

Experimental studies proved that N. cadamba exhibits an anti-hyperglycemic action on the treatment of diabetic rats. Supplementation for diabetic rats, Cadamba extract has been shown to have a significantly lowering effect on blood glucose levels. In this study, a substantial reduction in blood glucose levels in a dose-dependent manner  that is to say there is no significant reduction in blood glucose levels at the 200mg/kg dose compared with other  doses of NCEE and Metformin.

 

Significant blood glucose lowering effects were reported at the higher dose (400mg/kg) compared to the 200 mg/kg dose. The presence of active components with different mechanisms such as: quercetin, taxifolin, chrysin, naringin, kaempferol, aromadendrine and chlorogenic acid in Cadamba tree is responsible for its hypoglycemic        effects21-22.

 

Increased lipids in the liver and triglycerides impair insulin's ability to regulate gluconeogenesis and glycogen synthesis. This impaired insulin action will lead to insulin resistance which eventually results in pancreatic beta- cell failure and circulating insulin levels become insufficient to control blood glucose levels, leading to hyperglycemia. NCEE had decreased the levels of Cholesterol, triglycerides and increase the level of HDL in dose dependent manner thereby improves the action of insulin23.

 

The free radicals produced during diabetes mellitus lead to oxidative damage, particularly in the liver and kidney, which exert oxidative stress and damage to internal endothelial tissues that ultimately lead to high blood sugar. Oxidative stress in diabetes is accompanied by a decrease in antioxidant status, which can increase the damaging effects of free radicals. Possible causes of oxidative stress in diabetes mellitus include increased production of reactive oxygen species by NADPH oxidase. The decreased activities of antioxidant enzymes such as SOD and CAT are mainly involved in the direct elevation of reactive oxygen species. SOD protects tissues from oxygen free radicals by scavenging superoxide radicle which damage membranes and biological structures24-25.

 

CAT catalyzes the transformation of hydrogen peroxide into harmless products within the cell. Reduced activities of GPx, GST, SOD and CAT have been observed in the liver and kidney of diabetic rats that results the multiple deleterious effects due to the accumulation of reactive oxygen species26-27.

 

Administration of NCEE resulted in the activation of   SOD and CAT and the activities of these enzymes returned to normal levels. The resulting increased activities of these enzymes clearly suggest that the heartwood extract of Cadamba tree has free radical scavenging properties, which exert beneficial action against pathological changes caused by ROS.

 

Histopathological examination of isolated pancreas of diabetic rats revealed morphological changes in the diabetic groups whereas the NCEE treated groups retain the normal architecture of       distinct organ.

 

The findings of the present study suggest that administration of NCEE may be effective in preventing oxidative stress and cell damage by increasing antioxidant enzyme activities, and ultimately contributing to the improvement of tissue dysfunction in diabetic rats.

 

CONCLUSION:

In view of the present study, it is believed that N. cadamba has been observed to exert significant antidiabetic effects in wistar albino rats. This contented research leads to the development of herbal formulations and gives support to reported folkloric usage of N. cadamba in the treatment of oxidative stress linked diabetes.

 

CONFLICT OF INTEREST:

Authors declares no conflict of interest in this study.

 

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Received on 27.09.2021             Modified on 11.05.2022

Accepted on 02.09.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(3):1121-1126.

DOI: 10.52711/0974-360X.2023.00187