Three Novel Biphenanthrene Derivatives and a New Phenylpropanoid Ester from Aerides multiflora and Their α-Glucosidase Inhibitory Activity

A phytochemical investigation on the whole plants of Aerides multiflora revealed the presence of three new biphenanthrene derivatives named aerimultins A–C (1–3) and a new natural phenylpropanoid ester dihydrosinapyl dihydroferulate (4), together with six known compounds (5–10). The structures of the new compounds were elucidated by analysis of their spectroscopic data. All of the isolates were evaluated for their α-glucosidase inhibitory activity. Aerimultin C (3) showed the most potent activity. The other compounds, except for compound 4, also exhibited stronger activity than the positive control acarbose. Compound 3 showed non-competitive inhibition of the enzyme as determined from a Lineweaver–Burk plot. This study is the first phytochemical and biological investigation of A. multiflora.


Introduction
Diabetes mellitus (DM) is one of the main causes of global morbidity and mortality [1]. The disease is caused by insufficient insulin secretion and/or action. DM is associated with high blood glucose levels, and type 2 DM is the most common form, covering 90-95% of all diabetes cases [2]. Drugs currently used for treating DM can be classified into several classes following their chemical structures and modes of action, and some have limitations due to their adverse reactions or unpleasant effects [3]. α-Glucosidase is an enzyme located in the small intestine. It is responsible for converting starch and disaccharides into monosaccharides (glucose). Inhibition of this enzyme can significantly reduce postprandial hyperglycemia [4]. α-Glucosidase inhibitors (AGIs) have been widely used in combination with other anti-DM drugs in the management of type 2 DM [5][6][7][8].
However, AGIs can cause liver injuries and gastrointestinal side effects [9,10]. There has been a growing interest in developing antidiabetic drugs of botanical origin because they are perceived as possessing fewer undesired effects [11,12]. Several promising AGIs have been reported from some members of the family Orchidaceae, such as Dendrobium tortile [13], Bulbophyllum retusiusculum [14], and Arundina graminifolia [15].
Aerides is a small genus of epiphytes in the family Orchidaceae. It consists of approximately 21 species that are native to South and South-East Asia [16]. Some Aerides species have been used in traditional medicine. For example, Aerides falcata has been used for boosting the immune system, whereas Aerides odoratum has been known for its antibacterial properties [17]. Phytochemical screening of Aerides odoratum suggested the presence of alkaloids, glycosides, flavonoids, saponins, tannins, terpenoids, steroids, and anthroquinones [18]. Several phenanthrene derivatives have been identified from Aerides rosea [19] and Aerides crispum [20].
Aerides multiflora Roxb. (Figure 1) is commonly known as "The Multi-flowered Aerides" [21] and called "Malai Dang" in Thai [22]. It has several synonyms, including Aerides affinis, Aerides godefroyana, Aerides lobbii, Cleisostoma vacherotiana, and Epidendrum geniculatum [23]. The plant is indigenous to Bangladesh, India, Nepal, Myanmar, Thailand, Malaysia, Philippines, Laos, Cambodia, and Vietnam. A. multiflora has been traditionally used as a tonic [24]. It has also been used to treat cuts and wounds [17,25] and fractured and dislocated bones [26]. In an earlier study, its tubers showed an antibacterial effect in vitro [27]. As a continuation of our investigation of orchids for α-glucosidase inhibitors [28][29][30], a MeOH extract obtained from the whole plants of Aerides multiflora was evaluated and found to possess strong inhibitory property against the enzyme (82.4 ± 9.5% inhibition at 100 µg/mL). In this communication, we describe our findings on the chemical constituents of this plant and their α-glucosidase inhibitory activity.
Compound 3 was obtained as a brown amorphous solid. Its UV absorptions and IR absorption bands were similar to those of compound 2, indicating a phenanthrene derivative. The HR-ESI-MS exhibited [M+Na] + at m/z 533.1218 (calculated for C 30 Table 2) revealed their structural similarity, except for the presence of a hydroxyl group at C-6/C-6 in 3, instead of a methoxy group. Moreover, the two phenanthrene units were symmetrically linked to each other through a C−C bond between C-1 and C-1 as supported by the HMBC correlations from C-1/C-1 to H-3/H-3 , H-10/H-10 and HO-2/HO-2 [39]. On the basis of above spectral evidence, the structure of compound 3 was established as shown, and the trivial name aerimultin C was given to the compound.
Compound 4 was obtained as a yellow amorphous solid. The molecular formula was determined as C 21    The NOESY cross-peak between MeO-3 /MeO-5 protons and H-2 /H-6 confirmed the locations of the methoxy groups at C-3 /C-5 (δ 147.7). The two phenylpropanoid units were connected by an ester bond at C-9 and C-9 , as determined by HMBC correlation of C-9 (δ 172.2) with H 2 -9 . Based on the above spectroscopic evidence, compound 4 was determined as dihydrosinapyl dihydroferulate. Prior to this study, the natural occurrence of 4 was unknown. However, the compound was earlier synthesized by acylation of the lignins from Arabidopsis thaliana [42].
It should also be noted that the genus Aerides belongs to the same clade as Rhynchostylis [43]. Both genera have been called "the foxtail orchid" due to the erect or pendent inflorescences of closely packed flowers, and this has sometimes led to confusion. Up to the present, no reports on the secondary metabolites of the latter genus have appeared. Comparative chemical studies, in combination with detailed genetic analysis, may help shed light on the distinction between these two sister genera.

α-Glucosidase Inhibitory Activity
Yeast α-glucosidase enzyme was used in this study. In general, α-glucosidase enzymes can be obtained from several sources, for example, Saccharomyces cerevisiae, Rattus norvegicus, and GANC-human [65]. The enzyme derived from the yeast shows approximately 55% sequence homology with that obtained from mammalian sources [66], and therefore is widely employed in the investigations of natural compounds for α-glucosidase inhibitory potential [67,68].
Overall, the dimeric phenanthrenes (1, 2, 3, and 8) demonstrated higher activity than the monomers (5, 7, and 10), as indicated by their IC 50 values (Table 4). Aerimultin C (3) was the most potent α-glucosidase inhibitor, with an IC 50 value of 5.2 ± 0.7 µM. Replacing the phenolic groups at C-6 and C-6 of this compound with methoxy groups reduced the activity by about seven-fold, as can be seen from the increased IC 50 value (37.2 ± 4.5 µM) for agrostonin (8). The importance of free OH groups is also supported by the potent activity (IC 50 2.08 ± 0.19 µM) earlier observed for a biphenanthrene (from Dioscorea bulbifera, Dioscoreaceae), the structure of which contains four free phenolic groups [69]. A molecular docking study on flavones with α-glucosidase inhibitory activity has also revealed that replacement of the hydroxyl groups with methoxy groups could lead to loss of activity [70].
A kinetics study was then performed on compound 3 to analyze the mode of enzyme inhibition using various substrate concentrations (0.25-2.0 mM). From the Lineweaver-Burk plot in Figure 3A, the drug acarbose showed the intersection of the lines on the y-axis, indicating competitive type of inhibition. The K i value of acarbose (190.57 µM) was obtained from the secondary plot by replotting the slopes of the lines against inhibitor concentrations. For compound 3, the increase in concentration (4 and 8 µM) decreased the V max from 0.10 to 0.035 but did not affect the K m value ( Figure 3B). The results suggested non-competitive inhibition of the enzyme by 3. The K i value of compound 3 (4.18 µM) was obtained from the secondary plot, as shown in Table 5.  Generally, non-competitive inhibitors have some advantages over competitive inhibitors [72]. Non-competitive inhibitors bind to the allosteric site of the enzyme, and thus do not depend upon the substrate concentration. Moreover, they require lower concentrations than competitive inhibitors to produce the same effect [73]. Compound 3, as a potent non-competitive inhibitor of α-glucosidase, provides a lead structure for the further design and development of AGI drugs.

Plant Material
The plant materials, whole plants of Aerides multiflora, were purchased from Chatuchak market in May 2019. Plant identification was performed by Mr. Yanyong Punpreuk, Department of Agriculture, Bangkok, Thailand. A voucher specimen BS-AM-052562 has been deposited at the Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University.

Extraction and Isolation
The dried powder from the whole plants of Aerides multiflora (6.1 kg) was macerated with MeOH (4 × 18 L). The MeOH extract, at a concentration of 100 µg/mL, showed 82.4 ± 9.5% inhibition of α-glucosidase. This MeOH extract (550 g) was then suspended in water and partitioned with EtOAc and butanol to give an EtOAc extract (201.1 g), a butanol extract (80.8 g), and an aqueous extract (150 g), respectively. The EtOAc extract exhibited 92.9 ± 3.2 inhibition at 100 µg/mL, whereas the others were devoid of activity (<50% inhibition). Therefore, the EtOAc extract was subjected to further investigation.

α-Glucosidase Inhibition Assay
The assays were performed following previous protocols [74]. The liberation of pnitrophenol from the substrate p-nitrophenol-α-D-glucopyranoside (PNPG) was observed to determine the inhibition of the α-glucosidase enzyme. Each sample was initially dissolved in 50% DMSO. Then, 0.1 U/mL of α-glucosidase (40 µL) in phosphate buffer (pH 6.8) was added to each well of a 96-well plate which contained the sample solution (10 µL). The plate was pre-incubated at 37 • C for 10 min. Then, 2 mM p-nitrophenol-α-Dglucopyranoside (50 µL) was added, and the mixture was incubated again at 37 • C for 20 min. Finally, 1 M Na 2 CO 3 solution (100 µL) was added to terminate the reaction. The absorbance of the mixture was measured at 405 nm using a microplate reader. Two-fold serial dilution was performed for IC 50 determination. The drug acarbose was used as the positive control.
The mode of enzyme inhibition of the test compound was determined using the double reciprocal Lineweaver-Burk plot (1/V vs. 1/[S]). The experiment was performed by varying the PNPG concentrations (0.25, 0.5, 1.0, and 2.0 mM) in the absence or presence of compound 3 (4 µM and 8 µM) or acarbose (930 µM and 465 µM). The secondary plot was constructed by replotting the slopes of the lines against inhibitor concentrations, and the K i was calculated from the line equation of the plot.

Conclusions
In this communication, ten compounds were isolated from Aerides multiflora, including three new compounds, namely, aerimultins A-C (1-3), the new natural product dihydrosinapyl dihydroferulate (4), and six known compounds (5)(6)(7)(8)(9)(10). The structures of the new compounds were established by spectroscopic methods. The findings in this study suggested that biphenanthrenes might be taken as a chemotaxonomic marker for the subfamilies Epidendroideae and Orchidoideae within the family Orchidaceae. For the first time, the dimeric phenanthrenes obtained from this plant family were investigated for an α-glucosidase inhibitory activity. Among the isolates, the biphenathrene aerimultin (3) emerged as the most potent inhibitor, showing much higher potency than the drug acarbose. An enzyme kinetic study on this compound revealed a non-competitive type of inhibition and suggested that it could be a candidate structure for α-glucosidase inhibitor drug development.