AIP Conference Proceedings 2092, 030018 (2019); https://doi.org/10.1063/1.5096722 2092, 030018
© 2019 Author(s).
The effect of Acalypha indica Linn. extract on insulin resistance in Sprague-Dawley
rats after induction of high cholesterol-high fructose diet
Cite as: AIP Conference Proceedings 2092, 030018 (2019); https://doi.org/10.1063/1.5096722 Published Online: 09 April 2019
Desak Gede Budi Krisnamurti, Erza Pinayungan, Rani Wardani Hakim, and Erni H. Purwaningsih
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The Effect of Acalypha indica Linn. Extract on Insulin Resistance in Sprague-Dawley Rats after Induction of High
Cholesterol-High Fructose Diet
Desak Gede Budi Krisnamurti
1, Erza Pinayungan
2, Rani Wardani Hakim
1, Erni H.
Purwaningsih
1,a)1Department of Medical Pharmacy, Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6, Central Jakarta 10430 Indonesia
2Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6, Central Jakarta 10430 Indonesia
a)Corresponding author: [email protected]
Abstract. Type 2 diabetes mellitus is a metabolic disorder that is characterized by hyperglycemia, insulin resistance, inadequate insulin secretion, and excessive glucagon secretion. Metformin is the first-line medication prescribed for type 2 diabetes mellitus. Prolonged use of metformin can cause mild and serious side effects. One of the alternative herbal remedies used to treat Type 2 diabetes mellitus is Acalypha indica Linn (AI). The objective of this research is to investigate the effect of ethanol extract AI to insulin resistance. Thirty male Sprague-Dawley rats are divided into six group, four groups (negative control, metformin 100 mg/kgBW/day, AI extract 250 mg/kgBW/day, combination metformin and AI extract) are induced with high fructose and high cholesterol diet; while two other groups (normal and normal treated with AI extract) are given normal diet. After four weeks of treatment, rats were terminated and laboratory test was performed to see plasma insulin level using enzyme-linked immunosorbent assay. We found that AI extract significantly reduced plasma insulin level (p=0,018), similar to metformin (p=0,009), compare with negative control. In addition, we also calculate insulin resistance index using the Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) based on plasma insulin level and blood glucose level. The combination of metformin and AI extract significantly decreased the insulin resistance index compared with the negative control (p=0,034). Therefore, AI extract can be used as a complementary to metformin therapy in type 2 diabetes mellitus and also as a prevention therapy of insulin resistance on normal individual.
Keywords: Acalypha indica Linn., insulin resistance, type 2 diabetes mellitus.
INTRODUCTION
Type 2 diabetes mellitus is a metabolic disorder characterized by long-term hyperglycemia conditions due to pancreatic beta failure in producing adequate amounts of insulin and/or hepatocyte, adipocytes and skeletal muscle cells resistance to insulin [1,2].The pattern of increasing prevalence and high mortality of diabetes mellitus in Indonesia can not be separated from changes in people's lifestyles, including smoking, alcohol consumption, sedentary lifestyle, and high-calorie food consumption [3,4]. One of the high-calorie foods is food contain high-fructose and high-cholesterol level, such as carbonated beverage packaging, cakes, and frozen junk foods [5]. High-fructose and high-fat diet induce disorder such as diabetes risk, hepatic and vascular complication [6]. The study by Motshakeri et al. (2015) [7] showed that administration of high-sucrose and high-fat diet for sixteen weeks could induce hyperinsulinemia, glucose intolerance and insulin resistance in Sprague-Dawley rats. These conditions are the main characteristics of diabetes mellitus [1,2]. Long-term hyperglycemia causes various complications including retinopathy, nephropathy, neuropathy, diabetic ulcers, vascular disease, decreased awareness due to ketoacidosis and death [8].
Lifestyle changes are often unable to lower and control blood glucose levels in patients with type 2 diabetes mellitus. Pharmacological interventions should be given to this group of patients [9]. Metformin is currently the first-
line drug treatment for type 2 diabetes mellitus [8,9]. Metformin belongs to the biguanide class of oral antidiabetic drugs. Metformin frequently described as an insulin sensitizer leading to a reduction in insulin resistance and significant reduction of plasma fasting insulin level. Metformin works in a number of ways to lower blood sugar levels such as increases the sensitivity of muscle cells to insulin, reduces gluconeogenesis in the liver, and also delays the absorption of sugar from the intestines into the bloodstream by increasing the number of glucose transporters on skeletal muscle cell surfaces through activation of the AMP-activated protein kinase (AMPK) enzyme [10]. On the other hand, metformin consumption may cause acute or chronic adverse events, such as gastrointestinal system disorders and vitamin B12 deficiency [10,11].
Based on these problems, herbs can be used as an alternative treatment of type 2 diabetes mellitus, either as a preventive agent or as a complementary chemical drug [12,13].Some herbal remedies are believed to have the ability to improve blood glucose levels, less expensive, less adverse side effects compare to chemical drugs.12 The study by Saha et al. (2011) [14] showed that giving methanol extracts of Acalypha indica Linn. for 21 days was able to decrease both fasting and postprandial blood glucose levels in Wistar rats after streptozotocin-induced. In addition, the study by Reddy et al. (2012) [15] also showed that administration of ethanol extracts of Acalypha indica Linn. for 14 days decreased fasting and postprandial blood glucose levels, and improved the weight of Wistar rats induced by streptozotocin and nicotinamide.
However, there is still no comprehensive study on the effects of ethanol extract of Acalypha indica Linn. on diabetes mellitus in rats after induced high-fructose and high-cholesterol diet. Although some studies have shown that ethanol extract of AI is capable of lowering blood glucose levels in diabetic rats, there is no study that explains the effect of these extracts on insulin resistance states. The results of this study are expected to illustrate the effects of ethanolic extract of Acalypha indica Linn. against insulin resistance and can describe the mechanism of action from Acalypha indica Linn in lowering blood glucose levels.
MATERIALS AND METHODS Acalypha indica Linn Extraction
Acalypha indica Linn (AI) root is obtained from Depok, West Java, Indonesia. AI plant had been identified at the Indonesian Institute of Sciences (LIPI) Bogor. The AI roots are dried and mashed into a dry powder. AI powder is then macerated with 70% ethanol for 3 x 24 hours. The result of the maceration process is then evaporated with a rotary vacuum evaporator to obtain a thickened extract. The dosage of AI extract in this study was 250 mg/kg body weight/day. The ethanol extract of AI was dissolved in 1% CMC solution, then administered orally to SD rats every day.
Animals Preparation for Experiment
Thirty Sprague-Dawley rats, 8-12 weeks old with similar weight were used and obtained from the National Institute of Health Research and Development, Ministry of Health Republic of Indonesia. Before treatment starts, both aged and young Sprague-Dawley rats were given a one-week acclimation, placed in a 24°C room, 12 hours of light-dark cycles. The rats fed with standard rat food and had access to water ad libitum.
Animals Treatment
Thirty male Sprague-Dawley rats, 8-12 weeks old, are divided into six group, four groups (negative control, metformin 100 mg/kg.BW/day, AI extract 250 mg/kg.BW/day, combination metformin and AI extract) are induced with high-fructose and high-cholesterol diet; while two other groups (normal and normal treated with AI extract) are given normal diet. High-fructose diet consists of 1.5 mL of 55% fructose solution dissolved in 0.5 mL of water for a single oral treatment in each rat. High-cholesterol diet is made from mixing quail egg yolks into normal feed. Rats induced with high-fructose and high-cholesterol diet for seven weeks. After seven weeks of induction, treatment for each group starts administered for four weeks, while high-fructose and high-cholesterol diet still continues.
After four weeks of treatment, all Sprague-Dawley rats were terminated under ketamine and xylazine anesthesia.
Each of the blood samples was collected and placed into K3-EDTA tubes and centrifuged at 3000 rpm, 4oC temperature for 10 minutes.
Measurement of Plasma Insulin Level
Blood serum obtained from centrifugation were assessed for plasma insulin level parameters using the enzyme- linked immunosorbent assay (ELISA) method. Laboratory measurements were taken place in the Department of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Indonesia. The results of SD rats plasma insulin level were reported in mean ± standard error of mean pg/mL serum. Kit used in this study is Rat INS (Insulin) ELISA Kit from Elabscience.
Measurement of Insulin Resistance Index (HOMA-IR)
Homeostatic model assessment- insulin resistance (HOMA-IR) is a method for assessing β-cell function and insulin resistance (IR) from basal (fasting) glucose and insulin or C-peptide concentrations. HOMA-IR was calculated according to the formula: fasting insulin (microU/L) x fasting glucose (nmol/L)/22.5.
Statistical Analysis
Laboratory results from blood samples of Sprague-Dawley rats will be analyzed using analysis of variance (ANOVA). Statistical analysis performed with GraphPad Prism 7 for Microsoft Windows and visualized into graphs.
Ethical Consent
Ethical consent regarding the usage of rats as experimental subjects in this study was approved by the Health Research Ethics Committee, Faculty of Medicine, Universitas Indonesia-Cipto Mangunkusumo Hospital in 2016.
RESULTS AND DISCUSSIONS
FIGURE 1. The effects of Acalypha indica Linn on plasma insulin level of Sprague-Dawley rats after induction of high cholesterol – high fructose diet. Results were shown as mean SD (N = 5). (*) Significant difference versus negative control at p
0,05.
The effect of Acalypha indica Linn. on plasma insulin level of Sprague-Dawley rats after induction of high cholesterol – high fructose diet is shown in Figure 1. Our result showed that plasma insulin level in rats treated with
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Acalypha indica Linn. Ethanolic extract was significantly reduced plasma insulin level (P=0,018) compare to negative control. It also observed that Sprague-Dawley rat treated with Metformin as a positive control was significantly reduce plasma insulin level (P=0,09) compare to negative control. Rats treated with a high-fructose and high-cholesterol diet without treatment (negative control) had the highest plasma insulin level.
Induction of high-cholesterol and high-fructose diet will lead to increased blood glucose so pancreatic beta cells will secrete more insulin to reduce blood glucose concentration in the state of insulin resistance, resulting in hyperinsulinemia [16,17]. The study by Raatz et al. (2017) indicates that patients with a high-fructose diet had higher proinflammatory and plasma insulin factors than patients with normal diet [17]. Another study by Park et al. (2015) showed that Sprague-Dawley rats induced a high-fructose diet for eight weeks had higher plasma insulin levels compared with normal diet rats [18]. A study by Chen et al. (2013) also states that Sprague-Dawley rats induced with high-fructose diets for 12 weeks have higher plasma insulin levels compared with normal diet rats [19].
Acalypha indica Linn. is one of the herbs that can reduce fasting and postprandial blood glucose levels in diabetic model rats [12, 14]. No studies have suggested that Acalypha indica Linn. extracts are capable to lowering plasma insulin levels, but studies of Mandal et al. (2013) explains that decreased blood glucose levels in mice may result from the insulinotropic action of Acalypha indica Linn. extract in stimulating insulin secretion by pancreatic beta and / or increasing peripheral tissue sensitivity to insulin [20].
Normal Rats
Negative Control
AI 250 mg/kg BW Metfo
rmin 100 mg/kgBW Metfo
rmin+AI
Normal Rats + AI
0 1 2 3
Treatment Group
Insulin Resistance Index
FIGURE 2. The effects of Acalypha indica Linn on insulin resistance index of Sprague-Dawley rats after induction of high cholesterol – high fructose diet. Results were shown as mean SD (N = 5). (*) Significant difference versus negative control at p
0,05.
The effect of Acalypha indica Linn. on insulin resistance index of Sprague-Dawley rats after induction of high cholesterol – high fructose diet is shown in Figure 2. Our result showed that ethanolic extract combine with metformin was significantly reduced insulin resistance index compare to negative control (P=0,034). Rats treated with a high- fructose and high-cholesterol diet without treatment (negative control) had the highest insulin resistance index.
Insulin resistance index is obtained from HOMA-IR calculation. The study by Wang et al. (2015)showed that Sprague-Dawley rats after induction of high cholesterol – high fructose diet for 13 weeks have higher insulin resistance index compare to rats with normal diet [21].
The study by Garabadu et al. (2016) showed that metformin was able to reduce 50% insulin resistance index in diabetic model rats [22]. Lowest insulin resistance index in Sprague Dawley rats treated with a combination of metformin and Acalypha indica Linn. may occur due to effects combination of metformin and phytochemical components in the Acalypha indica Linn which contain saponin and polyphenol. The other study by Neha et al. (2015), showed that saponin and polyphenol content in grapefruit seed extract have an additive effect on metformin in lowering blood glucose level [23].
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CONCLUSION
In conclusion, this present study showed that Acalypha indica Linn (AI) ethanolic extract can reduce plasma insulin level in Sprague-Dawley (SD) rats after induction of high-fructose and high-cholesterol diet (P=0,018). The result also showed that insulin resistance index in rats treated with Acalypha indica Linn. Ethanolic extract combine with metformin was significantly reduced insulin resistance index compare to negative control (P=0,034). Therefore, AI maybe a potential complementary to metformin therapy in type 2 diabetes mellitus and also as a prevention therapy of insulin resistance on normal individual. We suggest further investigation on human subject to be conducted in the future.
ACKNOWLEDGEMENTS
This study would not be possible without supports from National Institute of Health Research and Development, Ministry of Health Republic of Indonesia and Faculty of Medicine, Universitas Indonesia (Department of Medical Pharmacy, and Department of Biochemistry and Molecular Biology). We also would like to express our gratitude for the financial aid from PITTA Grant, provided by the Directorate of Research and Community Engagement, Universitas Indonesia with contract number 2063/UN2.R3.1/HKP.05.00/2018.
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