Research Article |
Corresponding author: Asri Sulfianti ( asri.sulfianti@brin.go.id ) Academic editor: Rumiana Simeonova
© 2023 Asri Sulfianti, Nisrina Firdausi, Nurhadi Nurhadi, Ngatinem Ngatinem, Kurnia Agustini, Sri Ningsih.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Sulfianti A, Firdausi N, Nurhadi N, Ngatinem N, Agustini K, Ningsih S (2023) Antidiabetic activity of Anredera cordifolia (Ten.) Stennis extracts with different ethanol percentages: an evaluation based on in vitro, in vivo, and molecular studies. Pharmacia 70(1): 39-47. https://doi.org/10.3897/pharmacia.70.e94899
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Anredera cordifolia (Ten.) Stennis, also known as Binahong (B), is an Indonesian plant used to treat diabetes. The purpose of this study was to determine the best extragent for preparing Binahong extract as an antidiabetic agent using different concentrations of ethanol (50%, 70%, and 96%), labelled as BE50%, BE70%, and BE96%. An alpha-glucosidase inhibiting assay was used to assess the activity. The most active extract was tested in vivo assay using an oral glucose tolerance test (OGTT) and alloxan-high feed diet (alloxan-HFD)-induced diabetes in rats, with glucose level and beta cell Langerhans repair as parameters. A molecular assay was also performed to look into the expression of homeostasis regulator genes on 3T3-L1 adipose cells. The results showed that 96% ethanol extract (BE96%) inhibited alpha-glucosidase the most effectively (IC50 119.78± 11.14 μg/mL). The in vivo assay revealed that the treatment BE96% at 250 mg/kg BW for 21 consecutive days significantly reduced plasma glucose levels in Type 2 DM rats compared to the control group (p ≤ .05) with improved of Langerhans beta cells. BE96% also significantly reduced postprandial glucose levels. At the cellular level, Oil-Red-O staining revealed that differentiated adipocytes treated with BE96% had the highest lipid absorbance (p ≤ .05), compared to the control. BE96% significantly increased the expression of Glucose Transporter Isoform 4 (GLUT4) at the molecular level. It could be concluded that BE96% exhibited the best antidiabetic properties.
Alloxan-HFD-induced diabetic rat, alpha-glucosidase inhibition activity, Binahong, glucose tolerance test, GLUT4 expression
A metabolic condition known as diabetes mellitus (DM) is characterized by chronically high blood sugar levels (hyperglycemia) and blood insulin levels (hyperinsulinemia) (
One of the additional resources for treating diabetes is medicinal plants (
Numerous studies have been conducted to identify an active sugar-lowering candidate agent in A. cordifolia. In general, the antidiabetic effect of a new compound is usually evaluated in vivo on a diabetogenic-compound-induced DM animal model using alloxan or streptozotocin (
However, evaluation at molecular levels is also critical to support the pharmacodynamic of A. cordifolia. One gene that critically regulates T2DM is GLUT4, which is expressed mainly in insulin-sensitive tissues, such as the adipose tissue, liver tissue, and skeletal muscle tissue. Increased GLUT4 expression has been shown to reduce blood glucose and improve glucose transport at a cellular level (
In this study, we determined the best ethanol concentration for preparing A. cordifolia extract to be used as an antidiabetic agent. A. cordifolia extracts with 50, 70, and 96% ethanol (BE50%, BE70%, and BE96%) were evaluated for in vitro and in vivo activity, as well as their capability to regulate GLUT4 expression. To begin, all extracts (BE50%, BE70%, and BE96%) were tested in vitro to determine which extract was the most active using an alpha-glucosidase enzyme inhibition assay. The selected A. cordifolia extract was then tested for hypoglycemic activity using the OGTT and long-term treatment with alloxan-HFD-induced diabetic rats. The extracts were also administered during 3T3-L1 adipose cell differentiation to investigate the effect of plant on GLUT4 expression. The choice of solvent type in this study became one of the most critical points in extraction to obtain an appropriate extract containing the desired metabolites and suitable for the intended purpose. The application of solvents in the industrial-scale extraction process is regulated by the Indonesian Food and Drug Administration (BPOM). Due to safety concerns, ethanol or a mixture of ethanol and water is preferred over other organic solvents (
Plant collection and authentication
Fresh A. cordifolia leaves were collected from Yogyakarta, Indonesia, and authenticated by Biology Research Centre, LIPI (Indonesian Institute of Science) (No. B-104/ IV/ DI.01/I/2021).
A. cordifolia leaves were cleaned, washed, and dried in a 50 °C oven before being milled into coarse powder. One hundred grams of powder were extracted with 1 L 96% food grade ethanol in a shaking incubator at room temperature for 24 hours. The extraction process was repeated twice after filtering. Furthermore, the filtrate was collected and evaporated using a rotary evaporator at 50 °C. The obtained semisolid mass then stored in an amber bottle at -80 °C for future experiments. The same extraction was carried out with 50% and 70% ethanol. Finally, the extracts were labelled with the percentages BE96%, BE70%, and BE50%. The yield of each extract was 11,72%, 10,54%, and 9.77%, respectively.
The inhibitory activity of alpha-glucosidase was conducted based on a previous study (
The IC50 value was derived from a linear regression equation of the curve that plotted each concentration toward the % inhibition value using Microsoft Excel. The sample that demonstrated the lowest IC50 was then chosen for further experiments.
The experiment was conducted at the animal laboratory of the National Research and Innovation Agency (BRIN), Indonesia. 31 male Sprague Dawley rats (10–12 weeks of age, 150–200 BW) were used for efficacy test. 15 male and 15 female rats (6–8 weeks, 80–100 BW) were used for toxicity test. All animals were procured from the Central Animal House of BPOM. The animals were placed in polycarbonate cages (32×24×16 cm) with husk as bedding material and kept at a controlled temperature (21–25 °C), humidity (55±5%), and a 12-light/dark circle. Animals were regularly fed and watered ad libitum. All animals were acclimatized for seven days before the experiment. The Health Research Ethical Committee, Faculty of Medicine, the University of Indonesia had approved all these in vivo experiments (Ethical approval: No. KET- 411/UN2.F1/ETIK/PPM.00.02/2021).
The acute toxicity test of selected A. cordifolia extract followed WHO protocol guideline (
Oral glucose tolerance activity of BE96% was determined based on a previous study (
In addition, as other lowering glucose parameter, Area Under the Curve (AUC) was calculated from the glucose-time graphic using a trapezoidal mathematic equation (
The hypoglycemic activity of the selected extract was evaluated in alloxan-HFD-induced Type 2 diabetic rat was based on the method of
The 3T3-L1 pre-adipocyte cells were obtained from the collection of LAPTIAB National Research and Innovation Agency (BRIN), Indonesia. The cells were maintained in DMEM High Glucose (Sigma-Aldrich, USA), supplemented with 10% Fetal Bovine Serum (FBS) (Gibco, USA), 100 U/mL penicillin (Gibco, USA), 100 g/mL streptomycin (Sigma-Aldrich, USA), 0.5 percent amphotericin (Sigma-Aldrich, USA) (DMEM Hg + FBS Complete) and incubated at 37 °C in a humidified incubator with 5% CO2. The adipocyte differentiation procedure followed a previous protocol (
Total RNA of adipocytes treated was isolated using Genezol (Geneaid, Taiwan) reagent and quantified using a Nano-Drop UV-vis spectrophotometer (SensiFAST SYBR No-ROX). One-Step Kit (Bioline, USA) was used to perform a direct quantitative real-time PCR according to the manufacturer’s instructions. The primer sequences were seen on Table
Primer sequences for Glucose Transporter Isoform 4 (GLUT4) and Beta-actin genes.
No | Gene | Primer Sequences |
---|---|---|
1 | GLUT4 | Forward: 5’ -GATTCTGCTGCCCTTC TGTC-3’ |
Reverse: 5’-ATTGGACGCTCTCTCTCCAA-3’ | ||
2 | Beta-actin | Forward: 5’-CTCTGGCTCCTAGCACCATGAAGA-3’ |
Reverse: 5’-GTAAAACGCAGCTC AGTAACAGTCCG-3’ |
ΔCT: CT (target gene) – CT (house keeping gene)
ΔΔCT: ΔCT (target gene) – Δct (reference gene)
Statistical analysis was performed by GraphPad Prism 8.0. The difference between groups was compared using a One-way analysis of variance (ANOVA), followed by Tukey’s Multiple Comparison Test post hoc. Statistical significance was defined as a p value of less than 0.05 (p ≤ .05).
As a preliminary study, A. cordifolia extracts were assessed for their activity by alpha-glucosidase inhibitory assay. Our experiment showed that among three extracts, BE96% demonstrated the highest inhibitory activity (IC50 119.78 ± 11.14 μg/mL). Thus, this extract (BE96%) was selected as sample for in vivo experiments. However, its activity was lower than that of acarbose (IC50 3.34 ± 0.95 μg/mL) (Table
The acute toxicity test of BE96% showed no mortality in both sexes until the dose of 4.25 g/kg BW. Additionally, no symptoms such as drowsiness, convulsion, tremor, diarrheal, constipation, or polyuria were observed in all groups. A closer examination of the animal body weights revealed that all rats gained weight during the research.
Based on OGTT (Fig.
Percent of plasma glucose level (left) and AUC value of each group (right) during Oral glucose tolerance test (OGTT). *: Significantly different from Normal (p ≤ .05). Different letters (a, b) indicated significant differences (p ≤ .05). Glucose (% toward M0) = (glucose level Mi/glucose level M0) × 100%.
Following that, the potency of BE96% was also examined in an alloxan-HFD-induced diabetic rats (Table
Groups | FBGL (mg/dL) at day of measurement | ||
---|---|---|---|
D0 | D14 | D21 | |
Normal | 122.93±9.84a | 119.54±6.95c | 92.05±4.83e |
BE96% | 277.65±23.41b | 135.94±7.29c | 104.20±6.51e |
Glibenclamide | 266.15±29.66 a | 113.61±24.09c | 85.56±11.98e |
Negative | 233.98±27.11 a | 245.89±16.48d | 221.18±27.56f |
Based on the cellular study, A. cordifolia expedited the differentiation of 3T3-L1 fibroblast cells into an adipocyte-like phenotype. It can be seen microscopically by changes in pre-adipocyte cell shape, clonal growth, and an increase in the storage of lipids (
The ability of A. cordifolia extract to inhibit alpha-glucosidase enzyme was demonstrated in vitro. It was seen by the highest inhibition of BE96% compared to the two other A. cordifolia extracts. It was shown by the IC50 value of BE96% which was the lowest among the other extracts. Theoretically, the lower the level of IC50 demonstrated, the higher the quality of enzymatic inhibition (
Furthermore, the polarity of solvent that used in extraction process influenced the chemical compounds in the extract. The polarity of solvent is inversely proportional to ethanol content, the higher the concentration of ethanol, the lower the polarity of the solvent. Our results showed that extract produced using the higher percentage ethanol solvent demonstrated a more potent inhibitory activity of the alpha-glucosidase enzyme. It was most likely due to the higher ethanol content of the solvent, increasing the active compounds concentration of inhibitor alpha-glucosidase enzyme, although it remained to be measured.
It was reported that several compounds in A. cordifolia that be claimed to play a role in antidiabetic activity were Boussingoside A1 Saponin (
Further evaluation on BE96% also was carried out by in vivo assay. The acute toxicity assay was firstly performed to investigate the extract safety profile used in the efficacy trials. According to the results, the BE96% was categorized as practically non-toxic. It was indicated that until the dose of 4.25 g/kg BW, there was no mortality in both sexes, and no acute symptoms were observed in all groups. Additionally, all rats gained weight during the research, indicating that the dose bellowed of the LD50 threshold was safe to be used in further in vivo research.
The efficacy study indicated that BE96% (250 mg/kg BW) reduced glucose levels in both glucose-loaded rats and alloxan-HFD-induced diabetic animals. Activity BE96% reducing the postprandial glucose (PBG) after exposure to glucose solution supported the in vitro experiment on the alpha-glucosidase inhibitory assay. The potency BE96% as a hypoglycemic agent was also demonstrated in an alloxan-HFD-induced diabetic rats. The lowering FBGL after BE96% treatment was due to its ability to repair the damaged pancreatic beta cells generated by alloxan induction. Previous research have shown that A. cordifolia extract and its isolated compounds have hypoglycemic activity. The 96% ethanolic extract of A. cordifolia at the dose of 25–100 mg/kg BW lowered BGL in HFD-induced diabetic rats after 21 days of treatment compared to the negative group significantly (p ≤ .05) (
Induction of the combined alloxan i.p. and long-term HFD p.o. raised FBGL. The combination of HFD and alloxan could likely hinder the occurrence of auto-reversion to normal circumstances, as described if alloxan was administered in the ranges of 90–140 mg/kg BW, i.p., without HFD (
Further research showed that the bioactive ingredient in A. cordifolia engaged in controlling the balance of lipid and glucose in the 3T3-L1 adipocyte cell culture. Treatment with three different A. cordifolia extracts (BE50%, BE70%, and BE96%) on 3T3-L1 cells showed that A. cordifolia enhanced the differentiation of 3T3-L1 fibroblast cells into an adipocyte-like phenotype. It can be seen microscopically by changes in pre-adipocyte cell shape, clonal growth, and an increase in the storage of lipids (
A. cordifolia functioned as an insulin mimic, controlling the anabolic response in adipose tissue by increasing glucose uptake. As a result, fatty acid synthesis from glucose was generated in adipocytes. This activity is most likely due to bioactive compounds in A. cordifolia, such as flavonoids, which can stimulate glucose uptake. The other study showed that flavonoids influence peripheral insulin sensitivity (
To summarize, the results of this study demonstrated that the higher the ethanol content of the solvent in A. cordifolia extracts, the greater the efficacy of plant extract as an anti-diabetic agent. It was demonstrated by in vitro, in vivo, and molecular studies that A. cordifolia has a promising inhibitory activity against alpha-glucosidase enzyme, significantly reduced postprandial hyperglycemia (p ≤ .05), reduced fasting blood glucose level (p ≤ .05), and potentially promote the improvement of damaged beta cells, and finally regulated glucose level in cell and molecular level by upregulating the GLUT4 mRNA level. However, further studies needed to be conducted to elaborate on the compound in BE96% that played in lowering blood glucose.
Animal experiment was conducted at the animal laboratory, National Research, and Innovation Agency (BRIN), Indonesia. Male and female Sprague Dawley rats were procured from the Central Animal House of BPOM (Indonesian FDA). The animals were placed in polycarbonate cages (32×24×16 cm) three animals each with husk as bedding material and kept at a controlled temperature (21–25 °C), humidity (55±5%), and a 12-light/dark circle. Animals were regularly fed and watered ad libitum. All animals were acclimatized for seven days before the experiment. At the end of the experiments, all animals were euthanized for pancreas collection. The Health Research Ethics Committee, Faculty of Medicine, the University of Indonesia had approved all these in vivo experiments (Ethical approval: No. KET- 411/UN2.F1/ETIK/PPM.00.02/2021).
All authors declared that they have no known conflict of financial interest or personal relationships that could have appeared to influence the outcome of this research.
This work was supported by Insinas Research Grant, First Term of 2019, from the Ministry of Research, Technology and Higher Education, Republic of Indonesia (No: 4/E/KPT/2019). The authors thank all technicians in mammalian cell culture and animal laboratory, National Research and Innovation Agency (BRIN) Republic of Indonesia, for their support in this study.