ANTIHYPERGLYCAEMIC AND ANTIOXIDANT ACTIVITY OF METHANOLIC EXTRACT OF CAPPARIS DECIDUA (FORSSK.) EDGEW. IN STREPTOZOTOCIN-INDUCED DIABETIC RATSHTML Full Text
ANTIHYPERGLYCAEMIC AND ANTIOXIDANT ACTIVITY OF METHANOLIC EXTRACT OF CAPPARIS DECIDUA (FORSSK.) EDGEW. IN STREPTOZOTOCIN-INDUCED DIABETIC RATS
C. Mathur * 1, M. Soni 2 and R. S. Gupta 1
Reproductive Physiology Section 1, Department of Zoology, University of Rajasthan, Jaipur - 302004, Rajasthan, India.
School of Engineering and Technology 2, Department of Biotechnology, SADTM Campus, Jaipur National University, Jagatpura, Jaipur - 302017, Rajasthan, India.
ABSTRACT: The present study evaluates the antihyperglycaemic and antioxidant activity of the methanolic extract of Capparis decidua (CDMtE) fruits in streptozotocin (STZ) induced diabetic rats. Streptozotocin (50mg/kg body weight/) was injected intraperitoneally in rats to induce diabetes. The STZ-induced diabetic rats were treated with CDMtE (200 mg/kg body weight /day) for seven days. Biochemical analysis, toxicity studies, and histopathological studies were carried out to assess the antihyperglycaemic and antioxidant effects. Treatment with CDMtE (200 mg.kg b.wt/day) caused a significant decrease in fasting blood glucose and improvement in the serum insulin level of the diabetic rats. Antioxidant enzymes SOD and GSH activity was increased, and LPO levels of diabetic rats decreased after oral administration of CDMtE (200 mg/kg body weight/day). Histopathological studies showed significant recovery in liver architecture after CDMtE administration. Histopathological results suggest the antihyperglycaemic and antioxidant ability of CDMtE. This study supports the therapeutic potential of C.decidua fruits in the treatment of diabetes.
Capparis decidua, Antidiabetic, Antioxidant, Streptozotocin
INTRODUCTION: Diabetes mellitus is a complex metabolic disease in which there may be either a defect in insulin secretion or the body is not able to respond to insulin, resulting in hyper-glycemia 1. According to IDF (International Diabetes Federation) currently, there are 415 million people suffering from diabetes mellitus globally and are estimated to rise by 225 million by 2040 2.
Hyperglycaemia is the major factor for the onset and progress of diabetic complications, mainly by producing oxidative stress 3. In diabetes, besides hyperglycaemia, abnormalities in lipid level, which is directly correlated with accelerated athero-sclerosis and subsequent cardiovascular diseases are one of the major causes of death in the world. It has been found that chronic hyperglycaemia is associated with the increased production of free radicals. Several hypotheses have been proposed for this genesis that includes oxidation of glucose, a constant increase in the formation of glucose-derived AGEs, and degradation of glycated protein.
In low-income countries where the resources are limited, traditional medicines are the best source for the management of diabetes mellitus 4. Despite the availability of novel antidiabetic drugs, diabetes continues to be one of the major medical problems. All oral hyperglycaemic agents are accompanied by several serious, undesirable side effects like hypoglycaemia, gain in body weight, gastro-intestinal disorders, lactic acidosis, and body fluid retention 5. Therefore, management and treatment of diabetes remain a big challenge for both scientific fraternity and society. Hyperglycaemia-induced oxidative stress can be treated and managed by natural products of medicinal plants 6.
Capparis decidua (family Capparaceae) commonly known as Kair, folk plant is cultivated abundantly in the “Thar desert” of India 7. Flavanoids, alkaloids, phenols, sterols, and glycosides are abundant in this folk plant. This plant has been used in the treatment of several diseases, as reported in the Unani medicine system. The plant finds applications in the treatment of various conditions like flatulence, tumor, respiratory disorders, inflammation, skin diseases, and as a diuretic, and antihelminthic, antifungal 8. Though the effect of Capparis decidua on blood sugar, hemoglobin, and lipids are well-known among traditional healers, there is a lack of well-conducted study to weigh up of its medicinal properties. Therefore, this research study was designed to assess the effect, efficacy, and potential benefits of the methanolic extract of the plant Capparis decidua as a potent anti-hyperglycaemic agent.
MATERIALS AND METHODS:
Drugs and Chemicals: Streptozotocin (STZ) was purchased from Sigma-Aldrich, St Louis, MO. Glucose kits based on the glucose oxidase method were purchased from Ecopak, Accurex Biomedical Pvt. Ltd, Mumbai. Other chemicals and kits were obtained from HiMedia (Mumbai, India) and SD Fine Chemicals Limited (Mumbai, India).
Preparation of Plant Extract: Fruits of C. decidua were collected freshly from the local market of Jaipur, India. The authentication of the species was done at the Department of Botany, University of Rajasthan, Jaipur, Rajasthan. A voucher specimen has been provided by the Department of Botany, the University of Rajasthan with reference no. RUBL 211632. The collected fruits were shed dried for four weeks at room temperature. The dried sample was pulverized using a manual grinder. Plant powder (250 g) was soaked in 500 ml of 100% methanol and kept at room temperature for 24 h. The mixture was extracted in a soxhlet apparatus with methanol for 72 h 9. The extract was filtered, and methanol was distilled off to obtain a dark reddish mass.
Experimental Model: Healthy adult male albino Wistar rats (Rattus norvegicus) were used as a model organism for all the experiments conducted. Animals weighed 160g to 180g were used in the experiment and housed in polypropylene cages in 60-70% relative humidity and 12 h light/12 h dark condition, temperature maintained at 24-28 oC. Water ad-libitum and standard pellet diet were provided to the animals. The experiments were performed as per the ethical guidelines proposed by the Committee for the Purpose of Control and Supervision of experiments on animals (CPCSEA), Ministry of Environment and Forest, Government of India (1678/GO/a/12/CPCSEA Dated 09-01-2013).
Experimental Design: Rats used for the experiments were divided into four groups. Dose 200 mg/kg body weight/day was given for 7 days.
Group I: served as normal control orally received 0.5ml distilled water.
Group II (Diabetic control): STZ-induced untreated diabetic rats (50 mg/kg body weight).
Group III: This group included STZ-induced diabetic rats that were administered with C. deciduas fruits methanolic extract at 200mg/kg body weight /day.
Group IV: This group included STZ-induced diabetic rats that were administered with glibenclamide at 0.3 mg/kg body weight/day.
Experimental Diabetes Induction: Streptozotocin (50mg/kg. body weight) was prepared in citrate buffer (0.1M, pH 4.5) to induce diabetes in an overnight fasted rat by injecting it intraperitoneally 10. Glucose solution (5%) was supplied for 24 h to avoid early drug-induced hypoglycaemic mortality to STZ-induced rats 11. Fasting blood glucose was measured to confirm diabetes in STZ-injected rats 72 h after injection with STZ.
Administration of Crude Plant Extract: Methanolic extract of C. decidua (CDMtE) and standard drug glibenclamide were suspended in-vehicle solution (distilled water, 0.5ml) and administered via oral route by force-feeding for 7 days daily to the rats belonging to respective groups. Feeding to the rats wasstopped30 min before and after the treatment. Only vehicle solution was provided to control (group I) and diabetic control groups (group II). Glibenclamide (dissolved in 0.5ml distilled water) at a dose of 0.3 mg/kg per day was used as a standard drug based on reports of a previous study12.
Biochemical Analysis: An autopsy was performed at the end of the experiment by giving mild ether anesthesia to overnight fasted rats. Blood was collected from the heart via cardiac puncture and allowed to clot at room temperature, further centrifuged at 4 °C at 3000 rpm for separation of serum. The liver was dissected, rinsed with ice-cold saline and stored at −20 °C for further studies. The activities of all the antioxidant enzymes were calculated in liver tissue homogenates. Reduced glutathione (GSH) was estimated using standard method 12. Alteration in the activity of Superoxide Dismutase (SOD) was measured by the procedure given by Marklund and Marklund 13. Liver glycogen was calculated by Montogmery’s method 14. Lipid peroxides were estimated in liver tissue homogenates using standard method 15. The concentrations of serum insulin were assessed according to the standard protocol using radioimmunoassay (RIA).
Acute Oral Toxicity Study: Acute oral toxicity test was performed for C. decidua fruit methanolic extract 16. The male Wistar rats were treated with graded dose of methanolic extracts of fruit of C. decidua (5, 50, 300, 2000 mg/kg b.wt./rats/day) were kept under close observation to find out any possible toxic effects or changes in behavioural pattern. On the basis of the observations, the biological dose was fixed at a maximum of 200 mg/kg body weight for the extract for the treatment.
Histopathological Studies: Hepatic tissue was cut into small pieces and fixed in Bouin’s solution. Ethanol-xylene series was used to process the fixed tissues. Harris hematoxylin and eosin staining were preceded by section cutting and paraffin embedding of the processed tissues. Microscopy was performed using Leitz-diaplan microscope (Leica, Germany) for micrographs of H&E stained sections Fig. 3a-3d.
Statistical Analysis: Values are mentioned as mean ± SEM (standard error of the mean). One-way ANOVA with Tukey-Kramer multiple comparison tests was used to calculate variations in groups 17. Values of p<0.05 were considered statistically significant.
RESULTS: The fruits of C. decidua were extracted with methanol, and different identification tests were conducted for evaluation of chemical constituents present in the extract as per the standard methods 18. The phytochemical screening indicated presence of proteins, alkaloids, carbohydrates, saponins phenols, flavonoids, and glycosides, in methanolic extract of fruits of Capparis decidua (Forssk.) Edgew Table 1.
TABLE 1: PHYTOCHEMICAL INVESTIGATION OF METHANOLIC EXTRACT OF FRUITS OF CAPPARIS DECIDUA (FORSSK.) EDGEW.
|Chemical category||Name of test||Methanolic extract|
|Proteins and Amino acids||Biuret test||+|
|Phenolics/Tannins||Ferric chloride test||+|
|Saponins||Drug + water + shaking||+|
(+ = present)
FIG. 1: EFFECT OF CDMTE ON BLOOD GLUCOSE. Values are expressed as mean ± SEM (n=6). Level of significance: - #-non significant, *p<0.05, ** p<0.01, ***p<0.001 when compared with Group I (Normal Control), ap<0.01; bp<0.01 when compared with Group II (Diabetic Control)
FIG. 2: EFFECT OF CDMtE ON SERUM INSULIN. Values are expressed as mean ± SEM (n=6). Level of significance:- #-non significant, *p<0.05, ** p<0.01,***p<0.001 when compared with Group I (Normal Control), ap<0.01; bp<0.01 when compared with Group II (Diabetic Control)
A significant increase (p<0.001) in blood glucose level was observed in STZ treated rats. A significant antihyperglycaemic effect was marked after oral treatment with CDMtE for 7 days Fig. 1. The results of the current study revealed that the CDMtE exhibited significant antihyperglycaemic activity in STZ-diabetic rats.
A significant decrease (p<0.01) in serum insulin level was also noticed in STZ treated rats. Oral treatment with CDMtE at a dosage of 200 mg/kg for 7 days boosted insulin level in diabetic rats Fig. 2.
Table 2 shows that there is a significant elevation in LPO levels and reduction in GSH, SOD, and Glycogen levels in the liver of diabetic rats compared to control rats. A significant change in biochemical parameters- LPO, SOD, GSH, and liver glycogen was observed after administration of Capparis decidua fruits methanolic extract at a dosage of 200 mg/kgin comparison to diabetic rats of group II Table 2.
TABLE 2: EFFECT OF CDMtE ON LIVER BIOCHEMISTRY
|Group I - Control (Vehicle Treated)||5.83±0.17||2.02±0.19||4.79±0.12||9.08±0.20|
|Group II - Diabetic Control||3.10±0.28**||8.27±0.22***||2.40±0.50**||3.21±0.51***|
|Group III - Diabetic + C. decidua
|Group IV - Diabetic + Glibenclamide (0.3mg/kg b.wt/day)||5.02±0.26#b||4.09±0.29 #a||4.11±0.31#a||7.05±0.30 #b|
Values are expressed as mean ± SEM (n=6). Level of significance: - #-non significant, *p<0.05, ** p<0.01, ***p<0.001 when compared with Group I (Normal Control), ap<0.05; bp<0.01 when compared with Group II (Diabetic Control)
FIG. 3: MICROSCOPIC IMAGES OF LIVER OF CONTROL, DIABETIC AND CDMtE TREATED DIABETIC RATS. Fig. 3a shows the normal architecture of liver tissue, normal portal triad, and sinusoids of normal control rats. (Hematoxylin and eosin stain; original magnification X 200). Fig. 3b is showing degeneration in the normal structure of the liver showing dilation of sinusoids of STZ-diabetic untreated rats. (Hematoxylin and eosin stain; original magnification X 200).In Fig. 3c, CDMtE (200 mg/kg b.wt./day) treatment to diabetic control rats showing the reversion of ruptured liver to near-normal structure. (Hematoxylin and eosin stain; original magnification X 200). In Fig. 3d, glibenclamide (0.3 mg/kg b.wt./day) treatment to diabetic control rats showing the normal structure of liver. (Hematoxylin and eosin stain; original magnification X 200).
Fig. 3 shows the histological results of hepatic tissues. The structure of hepatic tissue, portal triad and blood sinusoids were normal in the control rats Fig. 3a. Induction of diabetes in animals caused severe pathological changes, including dilation of hepatic sinusoids and degeneration of hepatic tissue Fig. 3b. However, liver of rats treated with CDMtE extract significantly reduced the histological alterations and reversed the structure near to normal Fig. 3c and 3d.
DISCUSSION: Oxidative stress plays a vital role in the development of complications of diabetes such as dysfunction of β-cells, insulin resistance, and impaired glucose tolerance. Oxidative stress refers to overproduction of reactive oxygen species or ROS. Reactive oxygen species (ROS) are oxygen-containing chemically reactive species such as peroxides, superoxide, hydroxyl radical. Under the diabetic state, excessive glucose reacts with plasma protein and forms advanced glycation end products (AGEs). These AGEs trigger the over-production of ROS, resulting in the generation of oxidative stress. ROS are responsible for hepato-cyte cytotoxicity 19. Streptozotocin is also responsible for the increase of ROS/RNS generation 20.
To balance the increased ROS, cells have their own defense system- the antioxidant protection system. This system includes enzymatic and non-enzymatic components. Antioxidants are stabilizing agents that regulate the over-production of ROS by scavenging activity. Antioxidants donate their electrons to stabilize free radicals or ROS and minimize their harmful effects. Organ damage due to oxidative stress can be prevented by various antioxidants (both enzymatic and non-enzymatic) 21.
SOD is an enzymatic antioxidant that removes the superoxide radicals and repairs cells from the damage caused by these superoxides 22. In contrast, GSH is a non-enzymatic antioxidant and also an intracellular non-protein thiol which reduced H2O2 and acts against oxidative stress by removing over produced free radicals.
Many plants also contain compounds that possess antioxidant activity. Capparis decidua is one of them. Phytochemical investigations of the plant also confirmed the presence of flavonoids, tannins, phenols, saponins, and sterols. These phytochemicals exhibit antioxidant activities 23.
In this study, hyperglycaemia was noticed in STZ–induced diabetic rats due to overproduction of ROS. Administration of C. decidua fruits methanolic extract to diabetic rats significantly improved the blood sugar level. The possible mechanism by which CDMtE at a dose of 200 mg/kg significantly decreased the blood glucose in diabetic rats is probably due to antioxidants present in the extract. These antioxidants stimulate surviving β-cells or regenerate β-cells, resulting in improved insulin secretion. It has been reported that antioxidants play a major role in β-cells regeneration 24.
In the present study, a reduction in the activity of antioxidants such as SOD and GSH in hepatic tissue of diabetic control rats was observed. This finding was in accordance with a study conducted on male albino wistar rats 25. This study explained that a reduction in SOD indicates free radical-induced damage. Additionally, in diabetes, glycosylation of SOD and loss of copper, which act as an essential cofactor in SOD activity, can be another factor responsible for a reduction in SOD activity 26.
In CDMtE treated diabetic rats, an elevation in GSH and SOD level was observed. Antioxidants present in Capparis decidua extract reduce oxidative stress, resulting in an increase in GSH levels and SOD activity. The increased activity of SOD shows the potential effect of CDMtE on the scavenging of free radicals. The activity of many enzymes, glutathione S‐transferase, γ‐glutamyl transferase, glucose 6‐phosphate dehydrogenase, etc. decide the levels of GSH 27. The CDMtE extract might be responsible for the activation of these enzymes, which further increase the glutathione pool in hyperglycaemic rats.
Overproduction of free radicals is also associated with an increase in Malondialdehyde production (MDA). MDA is a byproduct of polyunsaturated fatty acid peroxidation 28. An elevation in the level of MDA in diabetic rats has been reported in previous researches 29, 30. The finding of our study confirms these observations. A low amount of insulin in diabetes also increases fatty acyl coenzyme-A oxidase enzyme activity which is responsible for the initiation of beta-oxidation, resulting in peroxidation of lipids 31.
Treatment of diabetic rats with CDMtE reduced peroxidation of lipids which is probably due to the presence of active components in C. decidua methanolic extract (CDMtE). The antioxidants present in C. decidua are responsible for free radicals scavenging activity and hepatoprotective properties of the extract.
In our study, a decrease in glycogen content in hepatic tissue was noticed in STZ-induced diabetic rats. These results are in agreement with the effect of Calanthe fimbriata on streptozotocin-induced diabetic mice 32. STZ is a toxic compound that induces diabetes by destroying pancreatic β-cells, which results in a decrease in insulin level 33. When CDMtE at a dosage of 200 mg/kg was given orally to diabetic rats, glycogen content in hepatic tissue increased significantly which may be due to an increase in insulin level. Insulin stimulates glucose uptake and activates glycogen synthase in hepatocytes 34. Insulin mediates activation of phosphoinositide-dependent serine-threonine protein kinase (AKT), which plays a major role in glucose homeostasis 34. Another serine-threonine protein kinase GSK-3, acts as a key component in the regulation of glucose metabolism 35. In insulin-stimulated conditions, AKT phosphorylates and inactivate GSK-3. Inactivation of GSK-3 inhibits phosphorylation of enzyme glycogen synthase (GS) 34. Hence, glycogen synthesis increases. This mechanism is responsible for the conversion of glucose into glycogen.
The observations of this study clearly indicate that the CDMtE decreases oxidative stress and effective in preventing oxidative damage, which is considered as one of the major factors responsible for liver damage in the diabetic state.
CONCLUSION: It can be concluded that CDMtE is potentially effective in protecting the liver tissue from the damage caused by ROS. Phytochemical investigations suggest that CDMtE consists of many phytoconstituents, which elevate cellular antioxidant defense activities and reduction of hyperglycemia in STZ‐treated diabetic control rats. Hence, the findings of this research can be used to develop a potent antidiabetic drug from the plant extract used. Though further research is required to isolate and characterize the potent molecules, which probably play an important role in the treatment of diabetic mellitus and associated hepatotoxicity and more pharmacological investigations are also required to elucidate the exact mechanism and to identify and isolate active principle(s).
ACKNOWLEDGEMENT: The authors are very thankful to the Head and Coordinator (CAS) of the Department of Zoology, University of Rajasthan, Jaipur, India, for providing the necessary facilities for the above investigation.
CONFLICTS OF INTEREST: The authors declare that they have no conflict of interest.
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How to cite this article:
Mathur C, Soni M and Gupta RS: Antihyperglycaemic and antioxidant activity of methanolic extract of Capparis decidua (Forssk.) Edgew. in streptozotocin-induced diabetic rats. Int J Pharm Sci & Res 2021; 12(5): 2700-06. doi: 10.13040/IJPSR.0975-8232.12(5).2700-06.
All © 2013 are reserved by the International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
C. Mathur *, M. Soni and R. S. Gupta
Department of Zoology, University of Rajasthan, Jaipur, Rajasthan, India.
30 April 2020
05 September 2020
15 September 2020
01 May 2021