Binahong (Anredera cordifolia (Tenore) Steen.) Leaf Extract Modulates Fatty Acids and Amino Acids to Lower Blood Glucose in High-Fat Diet-Induced Diabetes Mellitus Rats

Patients with diabetes are 1.6 times more likely to use complementary alternative medicine than nondiabetic patients. Previous studies have shown that Anredera cordifolia (Tenore) Steen. (A. cordifolia) leaf extract has the capacity to lower blood glucose, but the actual mechanisms are unclear. Therefore, in this study, we explored the effect of A. cordifolia leaf extract on the metabolism of fatty acids and amino acids. Six-week-old male Wistar rats were randomly divided into six experimental groups (n = 5 per group). Two groups were fed with a regular diet or a high-fat diet (HFD) for six weeks. The regular diet and HFD groups were administered with 0.5% carboxymethylcellulose as a vehicle, and HFD rats were also fed with a suspension of glibenclamide (0.51 mg/kg body weight (BW)) or A. cordifolia leaf extract (25, 50, and 100 mg/kg BW). During the whole treatment, BW and food intake were recorded weekly. The rats were euthanized seven weeks after treatment. Blood glucose was evaluated by spectrophotometry, while fatty acids and amino acids were evaluated using a gas chromatography/flame ionization detector (GC/FID). All doses of A. cordifolia administration reduced blood glucose significantly, and 50 mg/kg BW was most effective in lowering blood glucose, similar to the effects of glibenclamide. A. cordifolia leaf extract affected the levels of medium-chain fatty acids, especially at 50 mg/kg BW. In contrast, glibenclamide affected long-chain fatty acids (LCFAs) to lower blood glucose. Based on the analysis conducted, we conclude that administration of A. cordifolia leaf extract can decrease blood glucose levels by regulating fatty acid metabolism and that a dose of 50 mg/kg BW in rats was the optimal dose.


Introduction
Diabetes mellitus is characterized by chronic hyperglycemia resulting from defects in insulin secretion, insulin action, or both. Diabetes mellitus is also associated with an increased risk of microvascular and macrovascular diseases [1]. e metabolism of foodstuffs is an altered type I and type II diabetes mellitus. e efficiency of glucose uptake and the utilization by most body cells is prevented by the lack of insulin or insulin resistance. erefore, blood glucose concentration increases, the cell utilization of glucose decreases, and the utilization of fats and proteins also increases. e concentrations of branched-chain amino acids (BCAAs) in plasma are elevated in human and animal models of obesity [2]. BCAAs are hypothesized to be responsible for some beneficial effects of high-protein diets, including improving body weight (BW) control and adiposity. BCAAs improve muscle glucose uptake, whole-body glucose metabolism, and oxidation. BCAAs in plasma stop increasing due to a block in mitochondrial branched-chain amino acid aminotransferase, which has also been associated with improvements in glucose tolerance and resistance to diet-induced obesity [2].
Free fatty acids (FFAs) are essential for the normal function of pancreatic ß cells. ese cells have the capacity to reverse insulin resistance, and their failure results in type II diabetes [3]. Elevated FFA can lead to the accumulation of fat depots in muscle, liver, and pancreatic ß cells, and accumulated triglycerides might promote a lipid environment that could interfere with metabolic signaling or action or both in these different tissues [4].
Recently, hyperlipidemia has been shown using metabolomics to be induced by high-fat diet-(HFD-) related metabolites, such as fatty acids, amino acids, phospholipids, and bile acids involved in fatty acid biosynthesis, beta-oxidation, amino acid metabolism, glycolysis, the TCA cycle, purine metabolism, energy metabolism, and bile secretion. ese molecules have been demonstrated to be disturbed in metabolism in both animal models and humans [5]. e ideal treatment of type II diabetes should reverse insulin resistance and ß-cell dysfunction in treated patients and prevent, delay, or reverse long-term complications. Current treatment strategies are aimed at the amelioration of insulin resistance (diet, exercise, weight loss, and metformin and troglitazone therapy), augmentation of insulin supply (sulfonylurea and insulin therapy), or limitation of postprandial hyperglycemia (acarbose therapy). Future therapies may target (1) insulin resistance, using a multifaceted approach; (2) hepatic glucose production, using gluconeogenesis inhibitors; (3) excess nonesterified fatty acid production, using lipolysis inhibitors; and (4) fat oxidation, using carnitine palmitoyltransferase I and II inhibitors [6].
Many patients commonly use complementary alternative medicine as an alternative or in addition to their current medication regimen to prevent or treat diseases (7). Patients with diabetes have been found to be 1.6 times more likely to use complementary alternative medicine than other patients [7].
Some native tribes in Indonesia use the leaves of the A. cordifolia (Anredera cordifolia (Tenore) Steen.) plant to empirically reduce blood sugar, even as vegetables [8].
Other studies report that A. cordifolia leaf extract can inhibit α-glucosidase with an IC 50 value of 54.24 μg/ml. A. cordifolia leaf methanol extract at a dose of 50 mg/kg body weight (BW) and 200 mg/kg BW reduced blood glucose levels of alloxan-treated mice after seven days by 61.02% and 60.68%, respectively, while after 14 days, the decrease in glucose levels reached 75.64% and 66.61%, respectively, and was histologically shown to repair β-pancreatic cell damage.
ere is, to our knowledge, no data concerning the effect of A. cordifolia leaf extract on the fatty acid and amino acid profiles in serum to evaluate alterations in metabolism. erefore, it is necessary to research the role of A. cordifolia leaf extracts on fatty acid and amino acid metabolism to elucidate the mechanism by which it lowers the glucose concentration in plasma. e powder obtained was extracted by maceration using 96% ethanol and then filtered using filter paper. e maceration results were evaporated using a rotary evaporator to obtain a concentrated extract that could still be poured [9].

Animals and Experimental
Design. All animals in this experiment were used following the animal care guidelines issued by Muhammadiyah University, HAMKA (certificate no. 02/19.04/0183). irty male Wistar rats were purchased from an animal breeding company in Solo, Indonesia, and were acclimated for one week prior to the initiation of the experiment. Rats were caged in an air-conditioned room (relative humidity 45%-65%) under a 12 h light-dark cycle at 30°C ± 2°C and were given free access to food and tap water [11].
Six-week-old male Wistar rats, weighing 150-200 grams, were randomly divided into six experimental groups (n � 5 per group). Two groups were fed with a regular diet or HFD ad libitum for six weeks and were administered 0.5% carboxymethylcellulose as a vehicle. HFD-induced type II diabetes mellitus (HFD T2DM) rats in the remaining groups were administered a suspension of glibenclamide (0.51 mg/ kg BW) or A. cordifolia leaf extract (25, 50, or 100 mg/kg BW). During the treatment, BW and food intake were recorded weekly. e rats were euthanized seven weeks after the beginning of the treatment [8].

Induction of the Hyperglycemia of Diabetes Mellitus by HFD Feeding.
Hyperglycemia was induced by providing an HFD of 20 g/day/200 g rat BW. e HFD feeding of this study was performed for 49 days; the composition of HFD is shown in Table 1 [12].

Biochemical Analysis.
Plasma levels of glucose, fatty acids, and amino acids were determined as previously described [13]. Blood samples were taken on day 0 (before treatment), day 28 (28 days after induction of HFD), and day 49 (21 days after drug administration). Blood samples were taken from the retroorbital sinus and collected in a microtube. e blood was centrifuged at 7,000 rpm for 15 min, and the supernatant was used to obtain plasma samples. Plasma samples were analyzed to determine the levels of glucose and FFA.

Measurement of Glucose Levels.
Glucose levels were measured by enzymatic colorimetry on a UV-VIS spectrophotometer at 500 nm using a glucose liquicolor kit. e absorbance obtained was compared with the absorbance of the blank control and then multiplied by the standard concentration [14].

Oral Glucose Tolerance Test (OGTT).
e OGTT was performed during the last week of the experiment. Rats have fasted for 12 h before OGTT. Subsequently, a 20% glucose solution (2 g/kg BW) was administered to the rats, blood samples were collected after 30, 60, and 120 min with heparinized capillary tubes, and blood samples were used to determine the glucose concentrations as described above [16].

Statistical
Analysis. Data were expressed as means ± standard deviation. Data were statistically processed using SPSS version 16.

Effects of HFD and A. cordifolia Leaf Extract on BW.
Hyperglycemia was induced by providing an HFD of 20 g/ day/200 g rat BW.
e HFD induction of this study was performed for 49 days. e BW gain was the highest in the negative control group (negative), as shown in Table 2.
Obesity is associated with nonalcoholic fatty liver disease, which leads to excessive lipid accumulation in hepatocytes, also called steatosis [17]. Our study showed a significant reduction in the HFD-increased liver weight of rats treated with glibenclamide or A. cordifolia leaf extract. is result agrees with previous studies that showed that HFD-increased liver weight [18,19].

Effects of HFD and A. cordifolia Leaf Extract on Blood
Glucose and OGTT. Increased serum glucose was observed in HFD rats compared with the normal diet group (Table 3). Generally, rats displaying glucose levels more than 200 mg/ dl were considered hyperglycemic [20]. In this experiment, glucose levels typically reached above 200 mg/dl after 21 days of HFD and were stable after 28 days HFD for an additional 28 days. erefore, we administered glibenclamide and A. cordifolia leaf extract after 28 days of HFD treatment. Glucose levels declined below 200 mg/dl shortly after glibenclamide or A. cordifolia leaf extract administration and became insignificant from the normal group after 28 days of treatment. A. cordifolia leaf extract and glibenclamide showed significantly reduced serum glucose levels compared with the HFD group (p < 0.05).
Treatment of HFD rats with A. cordifolia leaf extract significantly improved glucose tolerance (Figure 1). In the HFD group, A. cordifolia leaf extract-treated rats had reduced blood glucose area under the curve compared with the controls during the seven weeks on HFD (Figure 1). e blood glucose responses of rats on the HFD + A. cordifolia leaf extract were similar to those of the glibenclamidetreated rats (Figure 2). Similar results were observed when the rats were treated with oral glucose in other studies [21,22]. e World Health Organization (WHO) recommends that GTT be used as a diagnostic method to classify people with reduced glucose tolerance. Nayak et al. showed a similar result: when normal Wistar rats were given oral glucose (2 g/kg), they showed no signs of intolerance compared to diabetic rats AUC after a 120-minute glucose challenge, and both test compounds and standards showed a reduction in blood glucose levels [23].

Effects of HFD and A. cordifolia Leaf Extracts on Fatty Acid Concentration.
e profiles of fatty acids in HFD rats were detected by GC-FID. Sixteen fatty acids were detected, as shown in Figure 2. e HFD-increased almost all fatty  Advances in Pharmacological and Pharmaceutical Sciences 3 acids, while A. cordifolia leaf extract reduced the increases in medium-chain fatty acids such as oleic acid (C16 : 0) and palmitic acid (C18 : 0), whereas glibenclamide reduced the increases in long-chain fatty acids, especially behenic acid (C22 : 0).

Effects of HFD and A. cordifolia Leaf Extract on Amino
Acid Concentration. From the profiles of amino acids following HFD, all 20 amino acids were evaluated (Figure 3). HFD reduced the levels of almost all amino acids, while A. cordifolia administration, especially dose II (50 mg/kg BW), increased amino acid concentrations of L-arginine, L-isoleucine, L-valine, and L-glycine. Glibenclamide administration did not alter the levels of these amino acids, indicating that these compounds worked via different mechanisms to reduce plasma glucose.

Discussion
e current research was intended to investigate the effect of A. cordifolia leaf extract on HFD-induced diabetic Wistar rats. e administration of HFD in Wistar rats increased BW by approximately 25.60 g, similar to results reported in previous studies [23][24][25]. e composition of HFD was mainly fructose and butter as sources of saturated fatty acids (SFAs). Fructose entry into the liver is broken down into dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. Dihydroxyacetone phosphate ultimately becomes the glycerol backbone of triglycerides, and glyceraldehyde-3-phosphate becomes the acetyl-CoA moiety required for de novo lipogenesis and the synthesis of fatty acids [26]. e postprandial insulin levels are more significant following the addition of saturated fat (butter) [27]; therefore, in this study, high glucose concentrations were used as a marker for diabetes mellitus.
HFD can induce nonalcoholic fatty acid liver disease (NAFLD) due to an imbalance between energy intake and expenditure. Leptin and its receptor LepR regulate BW by balancing food intake and energy expenditure by activating AMPK. AMPK is a major energy sensor of the cell and regulates hepatic and adipose lipid metabolism by modulating lipogenesis, lipolysis, gluconeogenesis, and adipogenesis [28].
HFD destroy the equilibrium between the formation and degradation of lipids and lead to excessive lipid deposition in hepatocytes, resulting in hepatic steatosis, NAFLD, or even more harmful conditions such as fibrosis or cirrhosis [29]. Elevated serum levels of total cholesterol (TC) and low-density lipoprotein (LDL)-c are important risk factors for developing atherosclerosis [30]. In our study, A. cordifolia leaf extract significantly reduced the serum lipid profile, indicating that it may be helpful in suppressing the formation of atherosclerosis.
Monounsaturated and polyunsaturated fatty acids showed positive correlations with high-density lipoprotein  e glibenclamide-treated rat (positive groups) and three doses of A. cordifolia leaf extract-treated rats could decrease the concentration of blood glucose after 30 minutes of glucose administration. e glucose level in the blood is expressed in mg/dl. e values indicate means ± SD of five rats per group. and negative correlations with TC, triglycerides, and LDL levels. In HFD, various tested SFAs and the total SFA increased due to the high-fat concentration, which contains abundant SFAs, especially C16 : 0 and C18 : 0 [31]. Studies that focused on lipid peroxidation in pathological conditions have shown that high levels of unsaturated fatty acids in cells produce reactive oxygen species and result in membrane and mitochondrial permeability, leading to cell apoptosis and death [32,33]. C18 : 1 and C20 : 1 are monounsaturated fatty acids (MUFA), the accumulation of which induces the expression of Bim and FasL, molecules involved in apoptosis, as well as enhancing endoplasmic reticulum stress, resulting in hepatocellular damage [34]. Oleic acid (C18 : 1) is the predominant MUFA induced by HFD. Its elevation is speculated to be caused by the increase in Δ9 desaturases [35]. e activity of Δ9 desaturases is estimated using the ratio of C18 : 1/C18 : 0 and has been shown to be increased in both the serum and liver following HFD. Δ9 desaturase is also known as stearyl-coenzyme desaturase 1 (SCD1). is enzyme can catalyze the transformation from stearyl-coenzyme A (C18 : 0) to oleoyl-coenzyme A (C18 : 1). When comparing normal and SCD1-knockout mice fed HFD, insulin sensitivity was enhanced, and lipid accumulation in the liver was reduced in the SCD1-knockout group [32], indicating that the deletion of Δ9 desaturase could help reduce insulin resistance and hepatic steatosis. Furthermore, HFD has also been shown to contribute to the activation of Δ9 desaturase and results in the generation of C18 : 1. e correlation between serum C18 : 1 and liver C18 : 1 concentrations and hepatic steatosis, inflammation, and cell ballooning scores in our previous experiments suggested that the metabolic change in C18 : 1 in mouse sera might reflect liver damage related to HFD [33].
In this study, decreased levels of alanine and certain glucogenic amino acids (e.g., isoleucine and valine) and elevated levels of glucose in HFD-fed rats implied that glycolysis and aerobic oxidation of glucose were inhibited and that energy consumption might be shifted toward lipid oxidation in response to hyperlipidemia.
is was also observed in apolipoprotein E knockout mice suffering similar energy metabolism impairment [34,35]. e concentration of serum lipids is elevated in diabetes, which is a high-risk factor for coronary heart disease. Under reasonable conditions, insulin activates the enzyme  lipoprotein lipase, which hydrolyzes triglycerides. However, in a diabetic state, lipoprotein lipase is not activated or is insufficiently expressed due to insulin deficiency, resulting in hypertriglyceridemia [36].
Insulin deficiency produced complex alterations in the concentrations of amino acids in the plasma and heart muscle; the concentrations of some amino acids (alanine, valine, leucine, and isoleucine) increased, while others were decreased and a small number were unchanged [38].

Conclusions
Based on the analyses conducted, it can be concluded that the administration of A. cordifolia leaf extract decreased glucose levels in HFD T2DM rats by regulating fatty acid and amino acid metabolism. A dose of 50 mg/kg BW A. cordifolia leaf extract elicited a better effect than other dosages or glibenclamide.
Data Availability e data that support the findings of this study are available from the corresponding author (AB) upon reasonable request. : e level of plasma-amino acids lowers in diabetic rats as compared to normal rats. HFD lowered most of the amino acids, oral administration of A. cordifolia leaves extract doses 25, 50, and 100 mg/kg body weight, and glibenclamide 0.51 mg/kg BW increased the plasma-amino acid as compared to untreated diabetic rats. All values are described as mean ± SD for five rats in each group. 6 Advances in Pharmacological and Pharmaceutical Sciences