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α-Glucosidase Inhibitory and Glucose Uptake Stimulatory Effects of Phenolic Compounds From Dendrobium christyanum

A methanolic extract from the dried root of Dendrobium christyanum Rchb.f. (Orchidaceae) exhibited α-glucosidase inhibitory activity and glucose uptake stimulatory effect. Chromatographic separation of the extract led to the isolation of 13 phenolic compounds (1-13). Their structures were determined by spectroscopic analysis. The isolates were then evaluated for in vitro α-glucosidase inhibitory and glucose uptake stimulatory activities. Methyl haematommate (1), methyl 2,4-dihydroxy-3,6-dimethylbenzoate (3), n-docosyl 4-hydroxy-trans-cinnamate (4), coniferyl aldehyde (6), 4,5-dihydroxy-2-methoxy-9,10-dihydrophenanthrene (7), gigantol (10), and diorcinolic acid (13) showed higher α-glucosidase inhibitory activity than the drug acarbose. Moreover, n-docosyl 4-hydroxyl-trans-cinnamate (4), vanillin (5), and coniferyl aldehyde (6) could enhance glucose uptake by L6 myotubes. Compounds 4 and 6 appear to be potential hypoglycemic agents since they possess both α-glucosidase inhibitory and glucose uptake stimulatory activities. This study is the first report on the chemical constituents and antidiabetic activity of D. christyanum.

Diabetes mellitus (DM) is a disease of abnormal glucose metabolism caused by impaired insulin secretion and/or inefficient utilization of insulin. The hormone insulin is secreted from the β cells of the islets of Langerhans in the pancreas. It plays an essential role in blood glucose homeostasis by facilitating cellular glucose uptake and regulating the metabolism of protein, lipid, and carbohydrate. Untreated DM can produce several complications, such as diabetic nephropathy, neuropathy, and retinopathy, resulting in premature death. 1 In type II DM, the most common form of diabetes, impaired glucose tolerance leads to insulin resistance, a condition of decreased responsiveness of target tissues (liver, muscles, and adipose tissues) to regular circulating insulin, and results in postprandial hyperglycemia. 2 At present, several types of oral antidiabetic drugs with different mechanisms of action are available. Our study focuses on α-glucosidase inhibiting and glucose-uptake stimulating drugs.
α-Glucosidase inhibitors (AGIs) were discovered to control postprandial hyperglycemia in the 1970s and became a new class of antidiabetic drugs in the 1980s, but so far, only a few are commercially available. 3,4 Examples are voglibose and acarbose. They can decrease the absorption of carbohydrates from the small intestine by competitively inhibiting the α-glucosidase enzyme in the brush border and thus lowering the postprandial blood glucose level. 5 Acarbose is a complex oligosaccharide obtained from the microbial origin, 5 and its recommended doses are 50-200 mg 3 times daily. 6 Studies showed that 56%-76% of patients who took AGIs suffered from the side effects related to the digestive system, such as bloating due to indigestible glucose, flatulence, diarrhea, and nausea. 7 Apart from the above drawbacks, the structures of these AGIs mainly comprise sugar units, and their production processes are complicated. 5 In recent years, researchers have turned their attention to finding AGI drugs from alternative sources, such as medicinal plants. 8,9 There are several classes of compounds from medicinal plants that have been reported as AGIs, such as terpenoids, flavonoids, and phenylpropanoids. 4 The investigation for new AGIs from medicinal plants still remain attractive because of the finding of active compounds would be helpful for the development of dietary supplements.
Insulin maintains the glucose homeostasis after a meal by stimulating glucose uptake into skeletal muscles. In type II DM, peripheral insulin resistance in skeletal muscles or adipose tissues leads to defects in insulin-mediated glucose uptake and utilization and then results in hyperglycemia. 2 Insulin sensitizers can lower the blood glucose level by increasing the glucose uptake in muscles and reducing the hepatic gluconeogenesis and lipogenesis. 10 The most frequently used drug in this class is metformin. Although the side effects of metformin, such as diarrhea and flatulence, are well tolerated, there are serious concerns over the long-term use of this drug due to some side effects, such as the decrease in vitamin B 12 absorption, lactic acidosis, and macrocytic anemia. 11 Thus, alternative glucoseuptake enhancing drugs are still needed.
Dendrobium is one of the large genera in the Orchidaceae family. The genus comprises about 1100 species, and in Thailand, 150 species have been identified. 12 In traditional Chinese medicine, several members of Dendrobium known as "Shihu" have been used to increase body fluid production, relieve indigestion, and improve eyesight. 13 Previous studies have shown that Dendrobium plants produce many classes of secondary metabolites with interesting biological activities, including antioxidant, anticancer, antimicrobial, antifungal, antiherpetic, antidiabetic, anti-inflammatory, antimalarial, neuroprotective, immunomodulatory, and antiplatelet aggregation activities, as well as inhibition of lymphocyte infiltration and apoptosis in Sjogren's syndrome. 14 Over the years, we have been working on several plants in the genus Dendrobium and found that some possess outstanding α-glucosidase inhibitory activity, for instance, loddigesiinols G-J from Dendrobium loddigesii, 15 5-methoxy-7-hydroxy-9,10-dih ydro-1,4-phenanthrenequinone from Dendrobium formosum, 16 and dendrofalconerol A from Dendrobium tortile. 17 Besides, some phenolic compounds with glucose uptake stimulatory activity from D. formosum have been reported. 16 Dendrobium christyanum Rchb.f. is a small epiphytic plant, widely distributed in Thailand, Vietnam, and Southwest China. "Ueang Sae Phukradueng" is the name in Thai. 18 The occurrence of D. christyanum is more abundant than at higher elevations, usually above 5900 ft. 19 Before the present investigation, neither chemical nor biological studies have been reported for this plant.
In the preliminary evaluation, a methanolic extract of D. christyanum at 100 µg/mL exhibited α-glucosidase inhibitory activity by more than 70%. At 10 µg/mL and 100 µg/mL, it enhanced glucose uptake by L6 myotubes by 34.5% ± 51.7% and 56.4% ± 19.7%, respectively. These observations encouraged us to perform a detailed investigation of this plant to find the compounds responsible for the biological activities. The results from this study constitute the first report of glucosidase inhibitory and glucose uptake stimulatory activities of D. christyanum and its active principles.

α-Glucosidase Inhibition Assay
α-Glucosidase inhibition assay was carried out by following the method in the previous report by Inthongkaew et al. 16 α-Glucosidase inhibition was determined by observing the release of pnitrophenol from the substrate pnitrophenol-αdglucopyranoside. Acarbose was used as a positive control.

Glucose Uptake Stimulation Assay
Glucose uptake stimulation assay was performed according to the method by Inthongkaew et al. 16 Insulin (500 nM) and metformin (2 mM) were used as positive controls, and the differentiation medium containing 0.1% dimethyl sulfoxide was used as a negative control.

Cell Viability Assay
Cell viability in each well was evaluated promptly after the determination of glucose uptake in L6 cells. 3-(4,5-Dimethylth iazol-2-yl)-2,5-diphenyltetrazolium bromide assay method was used to determine the cytotoxicity of each test compound. 20

Statistical Analysis
The results of glucose uptake stimulation and cytotoxicity assays were described as the mean ± standard deviation.
Analysis of variance was performed using the GraphPad Prism Version 7.00 for Windows (GraphPad Software, Inc., San Diego, CA, USA). The statistical analysis for evaluating the significance of the difference between means is performed by the incorrect Fisher's least significant difference post hoc test. A P value <0.05 was considered statistically significant.

α-Glucosidase Inhibitory Activity of the Compounds
In the assays for α-glucosidase inhibitory activity, we evaluated each compound at 100 µg/mL. Only 1, 3, 4, 6, 7, 10, and 13 exhibited more than 70% inhibition and, thus, were further analyzed for half-maximal inhibitory concentration (IC 50 ) values. All of them showed stronger activity than acarbose, as evidenced by their lower IC 50 values ( Table 1). The α-glucosidase inhibitory activities of methyl haematommate (1) and diorcinolic acid (13) were reported for the first time in this study whereas those of 3, 4, 6, 7, and 10 were described earlier. 37,38 Compound 4 displayed potent α-glucosidase inhibitory activity, but 2 was devoid of such activity, despite their structural similarity. Other research groups have also reported several esters of transcinnamic acid derivatives as strong AGIs. 39,40 On the other hand, studies revealed that transtetracosylferulate and cisdocosylferulate and cistetracosylferulate from D. tortile and D. scarbrilingue 17,37 did not possess α-glucosidase inhibitory activity. It appears that the presence of the MeO group in the aromatic ring might cause the loss of activity in the ferulic acid esters.

Acknowledgments
HTS is grateful to the Graduate School, Chulalongkorn University for a CU-ASEAN PhD scholarship.

Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.