Four Novel Phenanthrene Derivatives with α-Glucosidase Inhibitory Activity from Gastrochilus bellinus

Four new phenanthrene derivatives, gastrobellinols A-D (1–4), were isolated from the methanolic extract of Gastrochilus bellinus (Rchb.f.) Kuntze, along with eleven known phenolic compounds including agrostophyllin (5), agrostophyllidin (6), coniferyl aldehyde (7), 4-hydroxybenzaldehyde (8), agrostophyllone (9), gigantol (10), 4-(methoxylmethyl)phenol (11), syringaldehyde (12), 1-(4′-hydroxybenzyl)-imbricartin (13), 6-methoxycoelonin (14), and imbricatin (15). Their structures were determined by spectroscopic methods. Each isolate was evaluated for α-glucosidase inhibitory activity. Compounds 1, 2, 3, 7, 9, 13, and 15 showed higher activity than the drug acarbose. Gastrobellinol C (3) exhibited the strongest α-glucosidase inhibition with an IC50 value of 45.92 μM. A kinetic study of 3 showed competitive inhibition on the α-glucosidase enzyme. This is the first report on the phytochemical constituents and α-glucosidase inhibitory activity of G. bellinus.


Introduction
Diabetes mellitus (DM), one of the most common metabolic diseases, is characterized by high blood glucose levels due to a deficiency of insulin in the body or insufficient insulin sensitivity [1]. α-Glucosidase inhibitors (AGIs), for example, acarbose and voglibose, are the drugs of choice for treating type II DM patients owing to their minimal side effects. However, these AGI drugs still have shortcomings, involving the high cost of production and the need for sugar-like unit(s) in their core structure, and this has motivated researchers to find alternative sources of AGIs [2]. Recently, several plant secondary metabolites of different classes have been investigated as potential candidates for AGI drug development. Interestingly, a great number of phytochemicals obtained from the Orchidaceae family have been found to possess strong α-glucosidase inhibitory activity [3][4][5]. Orchidaceae is one of the largest families in the plant kingdom and consists of about 735 genera and more than 20,000 species [6]. Many have been widely used as principal components in traditional Chinese medicine (TCM) and thus have been extensively investigated and found to be a rich source of unusual secondary metabolites [7]. Gastrochilus, a small genus of monopodial herbs in Orchidaceae, comprises approximately 62 species, mainly found in Southeast Asia [8]. Gastrochilus bellinus (Rchb.f.) Kuntze ("Suea Dam" in Thai and "Wat-Won-Thit-Khwa" in Myanmar) has stems enclosed in basal sheaths of leaves. Its sub-umbellate inflorescence contains small flowers (2-3 cm in diameter), which are distinct morphological features of this genus. The key characteristics of G. bellinus are flowers having pale yellow sepals, petals with brownish-purple markings, and a central cushion on white lip epichile with a groove or cavity at the base ( Figure 1) [9]. Prior to this study, there were no records on the phytochemical and biological investigation of this plant. As part of our continuing studies on bioactive compounds from orchids [10][11][12], we investigated the chemical constituents of G. bellinus and their α-glucosidase inhibitory potential. In this study, a dried MeOH extract prepared from G. bellinus was suspended in water and partitioned with EtOAc and butanol to give EtOAc, butanol, and aqueous extracts, which were then evaluated for their α-glucosidase inhibitory effect. The EtOAc extract exhibited the strongest activity with 80% inhibition at a concentration of 100 µg/mL and, therefore, was selected for further study. The butanol and aqueous extracts were found to be devoid of activity (less than 10% inhibition).

α-Glucosidase Inhibitory Activity
NA means no inhibitory activity.
Due to its high potency and availability, compound 3 was subjected to a kinetic study to determine the mode of enzyme inhibition. Lineweaver-Burk plots of the inverted values of velocity (1/V) versus the inverted values of substrate concentration (1/[S]) were prepared and analyzed, in comparison with that of acarbose, by varying the concentration (0.25-2.0 mM) of the substrate (pNPG) in the presence or absence of compound 3 at two different concentrations (20 and 40 µM).
The drug acarbose, as expected, showed competitive inhibition, as determined from the Lineweaver-Burk plot (Figure 4a). The secondary plot of acarbose, generated by replotting the slopes of the lines against the inhibitor concentration, gave a K i value of 143.6 µM ( Table 4). The obtained kinetic parameters of compound 3 are listed in Table 4. The maximum velocity (V max ) value was determined as 0.1 A 405/min , and the Michaelis-Menten constant (K m ) as 0.8, 0.9, and 1.1 µM (Figure 4b). The presence of compound 3 at different concentrations (20 µM and 40 µM) did not change the V max , but the K m of the enzyme was increased. These results suggest that 3 is a competitive inhibitor of this enzyme. A secondary plot of 3 gave a K i value of 87.3 µM. Several phenolic compounds from plants have been earlier reported as competitive inhibitors of α-glucosidase, for example, dihydrobenzoxanthones from Artocarpus elasticus [25] and flavonoids from Agrimonia pilosa [26].

General Experimental Procedures
UV spectroscopic data were determined using an Agilent Cary 60 Spectrophotometer (Penang, PG, Malaysia), and the IR data were obtained via a Perkin-Elmer FT-IR 1760x spectrophotometer (Boston, MA, USA). High-resolution Atmospheric Pressure Chemical Ionization mass spectra (HR-APCI-MS) were recorded with a Bruker micro TOF-QII mass spectrometer (Billerica, MA, USA). 1 H and 13 C NMR spectra were recorded with a Bruker Avance DPX-300 (Billerica, MA, USA).

Plant Material
The whole plants of Gastrochilus bellinus were purchased from Chatuchak market, Bangkok, in March 2018. Plant identification was done by one of the authors (B. Sritularak) and compared with the database of the Botanical Garden Organization. A voucher specimen (BS-GBel-032561) has been deposited at the herbarium of the Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University.

α-Glucosidase Inhibitory Assay
The α-glucosidase enzyme inhibition assay was carried out according to the method in our previous report [27]. The inhibitory activity was determined by measuring the p-nitrophenol, a yellow color substance that can be monitored at 405 nm, released from pnitrophenyl-α-D-glucopyranoside (pNPG) by the α-glucosidase enzyme. The test samples were initially dissolved in 50% DMSO, and then, 10 µL of the sample solution and 40 µL of 0.1 unit/mL α-glucosidase were incubated at 37 • C for 10 min. After that, to start the reaction, 50 µL of 2 mM pNPG was added to the mixture and incubated at 37 • C for 20 min. One hundred microliters of 1 M Na 2 CO 3 was added to stop the reaction, and then, the absorbance was measured at 405 nm. Acarbose was used as a positive control, and 5% DMSO was used as a negative control. Each experiment was performed in triplicate. Data were displayed as mean ± SD.
The enzyme kinetics parameters (K m and V max ) were determined by analyzing the double reciprocal Lineweaver-Burk plot (1/V vs. 1/[S]). Each experiment was carried out by varying the concentration of pNPG (2.0, 1.0, 0.5, and 0.25 mM) in the absence and presence of different concentrations of the test sample. The reaction was monitored at 405 nm by a microplate reader every 5 min for a total time of 25 min. Each experiment was performed in triplicate. Acarbose and 5% DMSO served as the positive and negative controls, respectively. A secondary plot for acarbose was generated by plotting the slopes of the double-reciprocal lines versus inhibitor concentration [28]. For compound 3, secondary plots were plots of the inverted values of K m (1/K m ) as a function of inhibitor concentration. The inhibition constant (K i ) was then calculated from the intersection point.

Conclusions
This study is the first report on the secondary metabolites of Gastrochilus bellinus. In summary, the phytochemical investigation of Gastrochilus bellinus led to the isolation of four new compounds gastrobellinols A-D (1-4), along with eleven known compounds. When isolates were determined for α-glucosidase inhibitory activity, compounds 1, 2, 3, 7, 8, 13, and 15 showed higher α-glucosidase inhibitory activity than acarbose. The potent α-glucosidase inhibitor, compound 3, revealed its competitive inhibition on the α-glucosidase enzyme.