Components with Anti-Diabetic Activity Isolated from the Leaves and Twigs of Glycosmis pentaphylla Collected in Vietnam

A phytochemical investigation of the leaves and twigs of Glycosmis pentaphylla (Rutaceae), collected in Vietnam, yielded three new compounds named glyfuran (1), glyphyllamide (2), and glyphyllazole (3), along with twenty-five known compounds (4–28). The structures of isolates were determined by IR, MS, NMR, and UV data analyses. In the anti-diabetic activity screening, (+)-isoaltholacton (4), glycoborinine (17), 2′,4′-dihydroxy-4,6′-dimethoxychalcone (24), and flavokawain A (25) simultaneously exhibited inhibition of dipeptidyl peptidase-4 (DPP4) and stimulation of the glucagon-like peptide-1 (GLP-1) secretion on the murine intestinal secretin tumor cell line (STC-1).


Introduction
Diabetes mellitus is a metabolic disease characterized by excessive amounts of blood sugar, leading to a family of diseases that includes hypertension, osteoporosis, retinopathy, and urethritis. Along with the increasing popularity of a sedentary lifestyle, diabetes has gradually become a global epidemic. According to the World Health Organization, approximately 422 million people worldwide suffer from this disease, with up to 1.5 million dying yearly [1]. Owing to the eating habits of East Asians (rice is the staple food), the prevalence of diabetes is much higher than in other areas of the world. It is estimated that 8.5% of adults in Taiwan are afflicted with this disease, and the number of patients is predicted to increase in the future [2]. Anyway, the treatment of many diabetes complications is very costly and has become an economic burden for governments everywhere. Moreover, people with type 2 diabetes are twice as likely to develop liver or pancreatic cancer [3,4].
The leading causes of diabetes are the pancreas being unable to produce enough insulin (juvenile diabetes) or when the body becomes resistant to insulin (adult-onset diabetes). There is no cure for diabetes, but it can be treated and controlled, with treatment being divided into two kinds: subcutaneous insulin injection and oral hypoglycemic drugs. Oral hypoglycemic drugs induce effects that include the promotion of insulin secretion (Diamicron) with an increase in insulin receptor sensitivity (Metformin), α-glucosidase inhibitors (Acarbose), and dipeptidase-4 (DDP-4) inhibitors (Galvus). These medications often cause unpleasant side effects such as flatulence, diarrhea, hypoglycemia, weight gain, and even liver toxicity [1]. As a result, many people are turning to herbal medicines to treat diabetes [5]. So far, many Traditional Chinese Medicines or folk herbs have shown significant effects on lowering blood sugar or reducing the side effects of western medicines. For example, Gastrodia elata water extract can increase the quality of islet β cells and reduce cell apoptosis [6]; polyacetylene from Bidens pilosa var. radiata can decrease blood sugar and increase insulin release [7], while the polysaccharides in the flower buds of Lonicera japonica can lower blood sugar [8]. In terms of pure compounds, iminosugars and sugar derivatives were regarded as important antidiabetic agents [9][10][11][12][13].
Glucagon-like peptide-1 (GLP-1) is an incretin that can decrease blood sugar levels in a glucose-dependent manner by enhancing insulin secretion; moreover, the action of GLP-1 is preserved in patients with type 2 diabetes, and substantial pharmaceutical research has therefore been directed towards developing GLP-1-based treatment. However, endogenous GLP-1 is rapidly degraded primarily by dipeptidyl peptidase-4 (DPP-4) [14]. In our pre-screenings for anti-diabetes, the extract of Glycosmis pentaphylla (Retz.) DC. was able to simultaneously inhibit DPP-4 and stimulate the secretion of GLP-1 ( Figure S1) in a dose-dependent manner (Figure S2), so G. pentaphylla has been proven to have the potential for development of an anti-diabetes drug and was accordingly selected for the following studies.
G. pentaphylla, a species of plant belonging to the family Rutaceae, is a shrub or small tree, 1.5-5.0 m high, and is widely distributed over India, Malaysia, Southern China, and the Philippine Islands to Vietnam. This plant is a Traditional Chinese Medicine (TCM) that can strengthen the stomach and relieve pain. In Vietnam, the roots, leaves, and branches of G. pentaphylla are collected year-round to relieve pain, to treat rheumatism, body aches, boils, impetigo, and snakebite. It is also used for postpartum women to cure uterine bleeding, eating indigestion, and abdominal distention [15,16]. Herein, the details of extraction, isolation, structural elucidation, anti-diabetes, and cytotoxicity of isolated compounds are described.
The anti-diabetic activity of isolated secondary metabolites was evaluated by stimulating the secretion of GLP-1 and inhibiting DPP-4. As shown in Figure 4A, compounds 1, 4,  6, 7, 17

General Experimental Procedures
The optical rotations, UV, and IR spectra were recorded on a Jasco P-2000 digital polarimeter, a Jasco V-530 UV/VIS spectrophotometer, and a Jasco FT-IR 4600 spectrometer, respectively. NMR spectra data were corrected in CDCl3 (δH 7.26 and δC 77.0) using solvent peaks as the internal standard on Bruker AVIII HD 700X NMR and Varian VNMRS 600

Plant Material
The leaves and twigs of G. pentaphylla were purchased in July 2018 from Cu Lao Cham Island (Hoi An City, Quang Nam province, Vietnam) by Associate Professor Quang Vinh Nguyen (Institute of Biotechnology and Environment, Tay Nguyen University, Vietnam) and Associate Professor Chia-Hung Yen (Graduate Institute of Natural Products, Kaohsiung Medical University, Taiwan). A voucher specimen (code No. KMU-VN016) was deposited at Kaohsiung Medical University.

DPP-IV Activity Assay
According to the manufacturer's instructions, the measurement of the activity and potential inhibition of DPP-IV, a type II membrane glycoprotein, was performed using the DPP-IV GloTM Protease Assay (Promega).