Introduction
Diabetes mellitus (DM) type 2, a metabolic disorder is characterized by the abnormal increase of sugar (glucose) in blood due to insufficient production of insulin by pancreas [
1]. Hyperglycemia may affect other body functions and organs, especially kidney, heart, eyes, veins and nerves [
2]. Pathogenesis of diabetes mellitus is directly proportional to the decreased antioxidant status of the body [
3]. The free radicals (superoxide dismutase, hydrogen peroxide etc.) cause devastation of β cells of pancreas and at last diminished insulin production [
4]. The disease may bring about weak digestion of glucose and different biomolecules like lipids, carbohydrates and proteins [
5]. According to some estimations type 2 diabetes mellitus has an impact on 415 million people in 2015 and this will increase up to 642 million by 2040 [
6]. Different drugs like; sulphonylureas, biguanides, thiazolidinediones and insulin are available to treat diabetes and reduce the risk of complications [
7]. Despite remarkable development for treating diabetes by oral hypoglycemic drugs, investigations for new drugs continue, as the existing synthetic drugs have several safety issues [
8].
Herbal medicines derived from plants have been utilized for the therapy of type 2 diabetes mellitus [
9].
Pandanus tectorius Parkinson ex Du Roi is a little tree grown wild in littoral habitats all through the coast of South and Southeast Asia, Australia and Pacific islands of Oceania [
10]. In folk medicine, different parts of this plant have been used for the treatment of bronchitis, leprosy, dermatitis, measles, urinary tract ailments and type 2 diabetes mellitus [
11]. Its leaves have been used for alleviating hepatitis, asthma, cold and cancer, where its roots were found helpful in managing digestive and respiratory disorders [
12]. Ten phenolic compounds isolated from
P. tectorius, out of which vanillin showed strong antioxidant activity [
13]. Various medicinal properties of
P.tectorius have been reported but, scientific evaluation of these properties received little attention. The present research describes the anti-diabetic, nephron-protective and hypolipidemic activity of water and ethanol extracts of various parts of
P. tectorius in alloxan diabetic rats.
Materials and methods
Chemicals and reagents
Solvent (ethanol) of analytical grades was purchased from Merck (France). Alloxan, DPPH and Tris (hydroxymethyl) aminomethane were purchased from Sigma Aldrich, U.S.A. All the kits including ALAT, AST, ALP, LDH, urea, creatinine glucose, triglycerides, cholesterol, HDL cholesterol, LDL cholesterol were purchased from Merck (France) and Ecoline (Germany).
Collection of plant material
Pandanus tectorius Parkinson ex Du Roi was collected from Bali Bagh, Malir, Karachi. Fresh plant part of P. tectorius (root, stem and leaf) were cleaned with the tap water and stored at -20 °C until used. Herbarium sheet was kept in the Department of Botany, University of Karachi, bearing herbarium No. 14/2/2014, G.H: 86550.
Different parts of P. tectorius, leaf, root and stem (500 g each) were cut into small pieces with a knife and then chopped in an electric grinder. The minced material of each part was soaked for 2 weeks in distilled ethanol (96%) and filtered over cotton wool. The filtrate was concentrated to a gummy mass on a rotary vacuum evaporator (Büchi R-200, New Castle, DE) that give the yield of 2.7% from stem, 9.5% from root and 7.5% from leaves respectively. Extracts were stored at a room temperature until used.
The Leaves, root and stem (100 g each) of P.tectorius were cut separately and grinded, soaked in distilled water for 1 h and homogenized using a homogenizer (Polytron (Kinematica) PT-MR 2100), then filtered over through Whatman no. 1 filter paper. The filtrate was lyophilized on a freeze dryer (Eyela, FD-1) that gave the yield of 14.5% from stem, 11.6% from root and 2.6% from leaves and stored at -20 °C until used.
Animals
Male (Wistar albino) rats, of body weight 150–200 g were bought from Dow University of Health Science, Karachi. Rats were then kept in cages under standard laboratory conditions (23 ± 2 °C and 12 h light/dark cycle) for 2 weeks prior to experimentation. All the animals were fed with a standard pellet diet and water (ad libitum).
Induction of diabetes mellitus
Diabetes in rats was produced by using method of Zhang et al. [
14], where intraperitoneal injection of alloxan monohydrate (Sigma aldrich, St. Louis, MO) at 100 mg/kg BW in saline was given to rats for 3 days continuously. The sugar level was evaluated on the third day by using a glucometer (Gluco trend Roche). The level of blood sugar 250 mg/dl or above was considered as diabetic.
The rats were separated into 3 major groups, where the third group was further divided into 6 sub-groups with each consisting of 6 rats.
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➢ Normal control group: This group of rats was fed on standard diet and water with no treatment.
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➢ Alloxan diabetic control group: This group of rats was intraperitoneally injected with alloxan (100 mg/kg) for 3 days.
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➢
Ethanol extract of P.tectorius treated group rats: This group of rats was further separated into 6 sub-groups:
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Ethyl alcohol extract of leaves of P.tectorius treated group: This group of rats was orally dosed with ethanol extract of leaves of P.tectorius (200 mg/kg b.w.,) at day 3.
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Ethyl alcohol extract of stem of P.tectorius treated group: This group of rats was orally dosed with ethanol extract of stem of P.tectorius (200 mg/kg b.w.,) at day 3.
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Ethyl alcohol extract of roots of P.tectorius treated group: This group of rats was orally dosed with ethanol extract of roots of P.tectorius (200 mg/kg b.w.,) at day 3.
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Ethyl alcohol extract of leaves of P.tectorius + alloxan dosed group: This group of rats were intraperitoneally injected with alloxan (100 mg/kg) for 3 days. On day 3 they were fed with ethanol extract of leaves of P.tectorius (200 mg/kg b.w.,).
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Ethyl alcohol extract of stem of P. tectorius + alloxan dosed group: This group of rats were intraperitoneally injected with alloxan (100 mg/kg) for 3 days. On day 3 they were fed with ethanol extract from the stem of P.tectorius (200 mg/kg b.w.,).
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Ethyl alcohol extract of roots of P. tectorius + alloxan dosed group: This group of rats were intraperitoneally injected with alloxan (100 mg/kg) for 3 days. At day 3 they were fed with ethanol extract of roots of P. tectorius (200 mg/kg b.w).
On the 4th day, rats from each group were decapitated after 12 h fasting [
14]. After collection of Blood, serum was obtained by centrifugation to do biochemical analysis.
Assessment of anti-diabetic, nephron-protective and cardio-protective effect of P. tectorius
Anti-diabetic effect was evaluated by estimating blood glucose level; while nephro-protective effect was determined by using kidney profile (urea and creatinine); whereas cardio-protective effect was examined using hepatic enzyme including i) alanine aminotransferases (ALAT), ii) aspartate aminotransferases (AST), iii) alkaline phosphatase (ALP), iv) lactate dehydrogenase (LDH). Lipid profile i) total cholesterol, ii) triglycerides, iii) HDL cholesterol iv) LDL cholesterol was also determined and coronary artery risk index (CRI) and atherogenic index (AI) were calculated [
15]. All estimations were performed on a blood chemistry analyzer {Micro lab 300 (Merck)}using kits (Merck / Ecoline).
Antioxidant activity of ethanol and water extracts of P. tectorius
Free radical capturing capacity of extracts of water and ethanol of different parts of
P. tectorius were estimated using 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) assay [
16]. Where 200 μL of each extract having 0.002, 0.05, and 0.01 mg/ml concentration was mixed with 800 μl of 100 mM Tris-HCl buffer (pH 7.4). DPPH (30 μM dissolved in DMSO), 1 mL was added to the mixture and vortex. The absorbance was recorded at 517 nm against blank by using UV-visible spectrophotometer. One ml ethanol in 1 ml DPPH was used as control. Below mentioned formula was used to calculate the activity.
$$ \left(\%\right)\kern0.5em \mathrm{Antioxidant}\kern0.5em \mathrm{activity}=\frac{\mathrm{Absorbance}\ \mathrm{of}\ \mathrm{control}-\mathrm{Absorbance}\ \mathrm{of}\ \mathrm{sample}}{\mathrm{Absorbance}\ \mathrm{of}\ \mathrm{control}}\kern0.5em \mathrm{X}\kern0.5em 100 $$
Estimation the phytochemical constituents
For the determination of different phytochemicals in leaves, stem and roots of
P. tectorius standard methods were used. Polyphenol were extracted from water and ethanol of different parts (root, stem, leaves) of
P. tectorius by using the method of Jimenez-Escrig et al. [
17] and estimated by Chandini et al. [
18]. Alkaloids were estimated according to the method of Harborne [
19]. Obdoni & Ochuko [
20] method was used to estimate saponin. Total tannin was estimated by using the method of Julkunen-Tiitto [
21]. Whereas, method of Chang et al. [
22] was used for the determination of flavonoid.
Statistical analysis
To analyze the data “Analysis of Variance” (ANOVA) was used and means were compared at [
23]. Duncan’s Multiple Range Test was also performed and means ± SD was calculated.
Discussion
In the present study root, stem and leaf of plant
Pandanus tectorius Parkinson ex Du Roi were tested for their hypoglycemic potential in healthy and alloxan induced diabetic rats. The results showed that both water and ethanol extracts exhibited anti-hyperglycemia in normal rats, as well as in diabetic rat models. Medicinal plants have been reported to possess hypoglycemic potential which can be used to treat diabetes mellitus [
24]. Many mechanisms of action have been proposed by which extracts of medicinal plants produced hypoglycemia. The proposed mechanisms include the effect of plant material on β-cells of pancreas, which may diminish synthesis of insulin, or release, or cell regeneration. The plants also produce anti-diabetic effects by improving peripheral glucose utilization or enhancing the synthesis of liver glycogen or may inhibit absorption of glucose from intestine [
25]. For anti-diabetic activity, plants are responsible via their capability to reestablish the function of pancreas either by increasing the secretion of hormone or by decreasing the absorption of glucose from intestine [
26,
27]. In this study water extract of root, stem (65.98%) and leave displayed a marked reduction in serum glucose concentration in alloxan diabetic rats. Similarly, ethanol extract of root, stem and leave also significantly decreased the level of glucose in comparison with their respective diabetic control (untreated) rats, this indicates stimulation of insulin release from the pancreas [
28]. Alloxan causes heavy destruction of pancreatic β-cells, which results in reduced synthesis and release of hormones from the pancreas. The normal functioning of insulin is impaired which causes hyperglycemia [
29]. In our study alloxan induced hyperglycemia, but treatment with ethanol and water extract significantly reduced glucose level. The water extract of
Euonymus alatus decreased the streptozotocin induced elevated level of serum glucose level [
30]. Alloxan causes permanent damage to β-cells [
31]. In our study alloxan treated rats after treatment with ethanol and water extract of root stem and leave demonstrated variable but significant reduction in glucose level, so we concluded that these extracts produced anti-diabetic effects by a mechanism other than the stimulation of insulin release from β- cells of pancreas.
Hyperlipidemia is characterized by enhanced levels of triglycerides, cholesterol, low density lipoprotein cholesterol (LDL-c). The results of the in vivo study revealed that both ethyl alcohol and water extracts of root, leave and stem significantly reduced the level of cholesterol, triglycerides, and LDL-c with subsequent increase in HDL-c in normal and diabetic rats in comparison with their respective control. All three extracts corrected hyperlipidemia. Hyperlipidemia is the leading endangerment for the premature development of heart disease like coronary artery disease, atherosclerosis and hypertension. Many studies on hyperlipidemia suggested that decrease in lipid profile: cholesterol, triglycerides and LDL-c and increase in HDL-c reduces risk of developing ischemic heart diseases [
32]. Prolonged hyperglycemia produced more oxidative stress. Oxidation is one of the destructive activities in which various molecules are broken down. Oxidative stress is the main factor which relates hyperlipidemia with pathogenesis of atherosclerosis [
33]. These reactive oxygen species react with lipids and cause lipid peroxidation, which results in membrane necrosis. Lipid peroxidation is declined which leads to curtailment in arterial wall cholesterol and deduction in atherosclerosis caused by hyperlipidemia [
34]. In the study the coronary risk index (CRI) and atherogenic index (AI) increased in alloxan induced diabetic rats, whereas indexes reduced or came towards normal range after treatment with extract, this indicates reduction in risk for development of coronary disease. Several indices have been derived from lipid profile to establish an index for the prediction of cardiac risk, among these AI is considered as a strong index for predicting risk of ischemic diseases [
35].
Free radicals also play a vital role in the production of secondary entanglement in diabetes mellitus and affect or impair the function of kidney, blood vessels, eye and nerves [
36]. The elevated level of oxidative stress increases the lipid profile [
37]. In this study different parts of
P.tectorius showed significant antioxidant activity. There is a report that destruction of βcells of pancreas is prevented by antioxidants [
38]. The anti-diabetic activity of ethyl acetate
fraction of E. alatus has been reported in alloxan treated mice evidenced by reduced plasma glucose, triglyceride and total cholesterol level with elevated SOD. This study also revealed that the ethyl acetate fraction contains flavonoids and polyphenols, including quercetin and kaempferol as main components with strong antioxidant activities [
39].
Plasma urea and creatinine is widely accepted as a measurement of renal function [
40]. In the study it was observed that urea and creatinine increased significantly in the alloxan diabetic control group. Administration of ethanol and water extract significantly decreased the serum level of both. The reduction in the level of these metabolites by the extract indicates the protective role of extracts against renal disorder due to diabetes [
41,
42]. Anti-diabetic, anti-hyperlipidemia and nephron-protective (via reducing urea and creatinine level in serum of diabetic rats) has been reported [
43].
Determination of cardiac and hepatic enzymes activity in serum or plasma is significantly important in order to diagnose the progression of disease. In this study the anti-diabetic activity and decrease in the level of ALP, ALAT, ASAT and LDH in alloxan induced diabetic rats might be due to the presence of antioxidant molecules in different parts of the plant. Certain enzymes are confined to certain tissues, and they enter in the blood only when these tissues are damaged. The presence of a significant increased amount of these specific enzymes in the blood reveals cell damage or destruction [
44]. In present study enzyme activity of alanine aminotransferase (ALAT)/ serum glutamic pyruvic transaminase (SGPT), aspartate aminotransferase (ASAT)/ serum glutamic oxaloacetic acid (SGOT) alkaline phosphatase, and lactate dehydrogenase (LDH) in serum were reduced by the treatment of water and ethanol extracts of root, stem and leave as compared to alloxan induced diabetic rats. Attenuation of increased activities of all tested enzymes as compared to control rats indicate the protective and nontoxic nature of plant extract [
45]. ALP is a liver marker enzyme and high level of enzyme in diabetic control indicates damage to structural integrity of liver. However diabetic rats treated with plant extract showed decreased in ALP level indicating healing effect of the extract. The transaminases ALAT and ASAT are also liver markers and used to detect toxicity to the liver [
46]. Increased in activity of both transaminases in diabetic rats suggest damage of liver cells [
47]. In this study LDH increased significantly in diabetic rats, and treatment with water and ethanol extract reduced the level towards normal. The increase in LDH activity after in diabetic rats may result from loss of membrane integrity [
48].
Phytochemical estimation revealed the highest phenol content in water extract of stem. Extracts from different solvents showed a variable amount of phenol. Phenols are responsible for producing multiple biological effects including antioxidant and anti-diabetic [
49,
50]. Our study also demonstrated the presence of a variable number of flavonoids, tannin, alkaloid and saponin in different parts of the plant. Many reports are available suggesting antioxidant and anti-diabetic and hypolipidemic potential of plants due to the presence of these constituents [
29,
30,
49].
Conclusion
Diabetes is a serious metabolic disorder affecting peoples worldwide. Long term hyperglycemia results in many complications like irregular functions of liver, heart and kidney. It also damages nerves, blood vessels and eyes. Treatment with oral hypoglycemic agents and insulin are not only expensive but also produce many adverse effects. We studied the anti-diabetic potential of water and ethanol extract of leaves, stem and root of
P. tectorius in alloxan diabetic rats. This study demonstrated that all parts of plants have tremendous effect in lowering the blood glucose level and as well as attenuating the diabetes related complications like heart related problems and kidney dysfunction. In this study, different parts of
P. tectorius showed good antioxidant activities and also showed presence of polyphenols. Dietary polyphenols have been reported to possess antidiabetic activity and inhibit α-amylase and α-glucosidase activities which may inhibit glucose absorption in the intestine and stimulate insulin secretion [
51]. Polyphenols from
P. tectorius could be a dietary therapy for the prevention and management of Type 2 diabetes. However, to confirm these effects and to make dietary recommendations for patients with type 2 diabetes, further studies are necessary.
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