Identification and Isolation of Active Compounds from Astragalus membranaceus that Improve Insulin Secretion by Regulating Pancreatic Β-Cell Metabolism

In type 2 diabetes (T2D), insufficient secretion of insulin from the pancreatic β-cells contributes to high blood glucose levels, associated with metabolic dysregulation. Interest in natural products to complement or replace existing antidiabetic medications has increased. In this study, we examined the effect of Astragalus membranaceus extract (ASME) and its compounds 1–9 on glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells. ASME and compounds 1–9 isolated from A. membranaceus stimulated insulin secretion in INS-1 cells without inducing cytotoxicity. A further experiment showed that compounds 2, 3, and 5 enhanced the phosphorylation of total insulin receptor substrate-2 (IRS-2), phosphatidylinositol 3-kinase (PI3K), and Akt, and activated pancreatic and duodenal homeobox-1 (PDX-1) and peroxisome proliferator-activated receptor-γ (PPAR-γ), which are associated with β-cell function and insulin secretion. The data suggest that two isoflavonoids (2 and 3) and a nucleoside (compound 5), isolated from the roots of A. membranaceus, have the potential to improve insulin secretion in β-cells, representing the first step towards the development of potent antidiabetic drugs.


Introduction
Diabetes is characterized by high blood glucose levels (hyperglycemia) and is a common health problem that affects 387 million people worldwide [1]. It is an important risk factor for eye, kidney, nerve, and cardiovascular damage [2]. Type 2 diabetes (T2D) accounts for approximately 90% of all diabetes cases, in which insulin resistance is the primary pathogenic condition. It results in the failure of insulin action in metabolic target tissues, such as muscle, liver, and adipose tissues, due to the insufficient secretion of insulin from the pancreatic β-cells located within the islets of Langerhans [3,4]. A decline in the mass of the pancreatic β-cells, rate of insulin secretion from these cells, or a combination of both, leads to insufficient insulin secretion [5]. Thus, a study focused on preserving the secretory function and mass of the pancreatic β-cells is a strategic approach for the treatment of diabetes.
Interest in natural products to complement or replace existing antidiabetic medications has increased [6]. Approximately 50% of the drugs approved by the US Food and Drug Administration (FDA) are natural compounds or their derivatives, because currently available antidiabetic drugs, including insulin, metformin, and sulfonylureas, are often associated with adverse effects [7]. Some antidiabetic drugs, such as pycnogenol, a trademarked supplement for diabetes derived

General Experimental Procedures
Column chromatography was performed on silica gel (70-230 and 230-400 mesh ASTM, Merck, Kenilworth, NJ, USA) and Diaion HP-20 (Mitsubishi Chemical Co., Tokyo, Japan). Flash chromatography was performed using the flash purification system (Combi Flash Rf, Teledyne Isco, Lincoln, NE, USA) with Redi Sep-C18 (26 g, 43 g Teledyne Isco). High-performance liquid chromatography (HPLC) was performed using the Gilson purification system, with a J'sphere ODS column (250 × 20.0 mm i.d., 4.0 µm, YMC Co., Tokyo, Japan). Thin-layer chromatography (TLC) analysis was performed on silica gel 60 F 254 and RP-18 F 254S plates (Merck). Nuclear magnetic resonance (NMR) spectra were obtained using a JEOL 500 MHz, using tetramethylsilane as an internal standard, and chemical shifts were expressed as δ values. Organic solvents for the chromatographic separations and extractions were distilled before use.

Plant Material
The roots of A. membranaceus (Leguminosae) were purchased from Hyunjin Pharmaceutical Co. (Seoul, Korea), in January 2015. The origin of the plant material was identified by one of authors D.S.J., and a representative specimen (ASME1-2015) was deposited at the Laboratory of Natural Product Medicine, College of Pharmacy, Kyung Hee University, Seoul, Korea.

Cell Viability
An Ez-Cytox cell viability assay kit (Daeil Lab Service Co., Seoul, Korea) was used to measure cell viability [24]. To determine non-toxic dose ranges of ASME and compounds 1-9 isolated from A. membranaceus, INS-1 cells were cultured in 96-well plates for 24 h and, subsequently, treated with ASME and compounds 1-9 for 24 h. Ez-Cytox reagent (10 µL) was added and incubated for 2 h. Absorbance values at 450 nm were measured using a microplate reader (PowerWave XS, Bio-Tek Instruments, Winooski, VT, USA).

GSIS Assay
An insulin secretion assay was used to determine the effect of ASME and compounds 1-9 on GSIS in INS-1 cells, using gliclazide as the positive control. To assess GSIS after treatment with ASME and compounds 1-9, INS-1 cells were cultured in 12-well plates for 24 h, washed twice with Krebs-Ringer bicarbonate HEPES buffer and 2.8 mM glucose, and starved in fresh KRBB. After starvation for 2 h, the cells were treated with ASME, compounds 1-9, and gliclazide. After 2 h, glucose (2.8 and 16.7 mM as basal and stimulant, respectively) was added to each well and incubated for 1 h. GSIS was measured using a rat insulin ELISA kit, as reported previously.

Western Blot Analysis
INS-1 cells were cultured in 6-well plates for 24 h and treated with compounds 2, 3, and 5 for 24 h to assess the levels of protein expression of PPAR-γ, PI3K, Akt, P-IRS-2 (Ser731), IRS-2, P-PI3K, P-Akt (Ser473), and PDX-1 after treatment. Western blot analysis was carried out to evaluate the expression of proteins related to pancreatic β-cell metabolism, as reported previously [25].

Statistical Analysis
Statistical significance was assessed using one-way analysis of variance (ANOVA) and multiple comparisons with a Bonferroni correction. P values less than 0.05 were considered statistically significant. All analyses were determined using SPSS Statistics ver. 19.0 (SPSS Inc., Chicago, IL, USA).

Western Blot Analysis
INS-1 cells were cultured in 6-well plates for 24 h and treated with compounds 2, 3, and 5 for 24 h to assess the levels of protein expression of PPAR-γ, PI3K, Akt, P-IRS-2 (Ser731), IRS-2, P-PI3K, P-Akt (Ser473), and PDX-1 after treatment. Western blot analysis was carried out to evaluate the expression of proteins related to pancreatic β-cell metabolism, as reported previously [25].

Statistical Analysis
Statistical significance was assessed using one-way analysis of variance (ANOVA) and multiple comparisons with a Bonferroni correction. P values less than 0.05 were considered statistically significant. All analyses were determined using SPSS Statistics ver. 19.0 (SPSS Inc., Chicago, IL, USA).

Effect of ASME and Compounds 1-9 on GSIS
The non-toxic dose of ASME and compounds 1-9 was determined using a cell viability assay on INS-1 cells. Although ASME did not show any toxic effect at 12.5, 25, and 50 μM (Figure 2A), some compounds isolated from ASME were slightly cytotoxic at concentrations of 25 μM and above, as cell viability decreased to below 80% ( Figure 2B, C, D, E, and J). ASME at 2.5, 5, and 10 μg/mL, and

Discussion
This study demonstrated that ASME and the compounds identified in this extract exerted insulin secretory effects. ASME and some active compounds isolated from ASME have been reported for their antidiabetic properties [14]. In an STZ-induced rat model of T1D, after treatment with astragalus polysaccharides [34][35][36] and soy isoflavones [36] isolated from ASME, glucose homeostasis was improved, but not by enhancing the capacity of insulin secretion levels [34,35]. In our study, ASME enhanced GSIS without inducing cytotoxicity in INS-1 cells. From this extract, nine compounds (1-9) including calycosin, calycosin-7-O-β-d-glucoside, formononetin, formononetin-7-O-β-d-glucoside, adenosine, 3-(β-d-ribofuranosyl)-2,3-dihydro-6H-1,3-oxazine-2,6-dione, acetylastragaloside I, astragaloside I, and astragaloside II, were isolated and identified. Among the isolated compounds, calycosin-7-O-βd-glucoside (2), formononetin (3), and adenosine (5) led to a significant increase in GSI in a dose-dependent manner, without inducing cytotoxicity in INS-1 cells. The effects of these three compounds were similar to the effect of gliclazide, a medicine used to treat T2D and classified in the sulfonylurea class of insulin secretagogues [37], which improved pancreatic β-cell sensitivity to glucose and enhanced insulin secretion in clinical studies [38]. These results suggest that sufficient secretion of insulin after treatment with ASME and its bioactive compounds, in response to an increase of glucose, may inhibit characteristic diabetic hyperglycemia and improve the sensitivity of pancreatic β-cells to glucose.
The most effective isoflavonoid identified in our study, formononetin (3), has been reported for its antidiabetic effect. In an experimental model of T2D, treatment with formononetin improves insulin sensitivity and reduces hyperglycemia by activating sirtuin 1 (SIRT1), an important regulator of energy metabolism that is involved in the regulation of insulin production and sensitivity and controls co-regulators, such as nuclear factor-kappa B (NF-κB), FOXO proteins, and PPAR-γ in pancreatic β cells [39]. In our previous study, the mechanisms of action mediating insulin secretion by compounds 2, 3, and 5 were evaluated. PPAR-γ is an important regulator of glucose metabolism by regulating gene expression [40], and is activated by compounds 2, 3, and 5. Thiazolidinediones (TZDs), PPARγ agonists, are widely used antidiabetic drugs, but have side effects, including weight gain and hepatic dysfunction. Because of these side effects, a variety of natural compounds, including stilbenes, flavonoids, neolignans, sesquiterpenes, amorfrutins, and coumarins have been identified as PPAR-γ agonists in an attempt to increase the effectiveness of PPAR-γ, while limiting its side effects [41].
PPAR-γ binds to the PDX-1 promoter to upregulate PDX-1 expression, which is associated with pancreatic development and the capacity of β-cells [42,43]. In human pancreatic β-cells, PDX-1 mRNA levels are increased in the presence of gliclazide [44], which has a similar insulin secretory capacity to compounds 2, 3, and 5. These three active compounds also increase the protein expression of PDX-1. Pancreatic β-cell malfunction is characterized by the lack of insulin production and secretion to regulate glucose metabolism, which results in hyperglycemia in PDX-1 knockout mice [45] and IRS-2 knockout mice [46]. IRS-2 is a member of a family of large adaptor proteins, linking insulin receptors to the activation of the PI3K/Akt pathway, which plays an important role in β-cell function [47,48]. Upregulation of IRS-2, PI3K, and Akt leads to the proliferation of these proteins in pancreatic β-cells, maintaining functional β-cell mass and enhancing insulin secretion [49]. In pancreatic islets isolated from patients with T2D, these expression levels are reduced [50,51]. We investigated the role of IRS-2 in the presence of compounds 2, 3, and 5. IRS-2 phosphorylation at Ser731 was increased by compounds 2, 3, and 5. In addition, PI3K-dependent Akt phosphorylation at Ser473 was observed after treatment with compounds 2, 3, and 5.
Consequently, the three active compounds (2, 3, and 5) facilitated insulin secretion by enhancing the expression of IRS-2, PI3K, Akt, PPAR-γ, and PDX-1. Despite these findings, further investigation is needed to determine how inhibitors of IRS-2, PI3K, Akt, PPAR-γ, and PDX-1 affect GSIS. Studies on the solubility, membrane permeability, absorption, distribution, and metabolism of the active compounds in vivo are also required, because these factors limit the oral bioavailability of the compounds [52,53]. In addition, in vivo studies of T2D are required to assess the antidiabetic potential of the active compounds, because their effects on insulin secretion from the pancreatic β cells may not be as significant upon oral administration. Clarifying the underlying mechanisms of action through further investigation may lead to the development of new drugs to prevent or delay the development of diabetes in patients who do not adequately respond to currently available antidiabetic drugs.