α-Glucosidase Inhibitory Activity and Anti-Adipogenic Effect of Compounds from Dendrobium delacourii

Chemical investigation of Dendrobium delacourii revealed 11 phenolic compounds, and the structures of these compounds were determined by analysis of their NMR and HR-ESI-MS data. All compounds were investigated for their α-glucosidase inhibitory activity and anti-adipogenic properties. Phoyunnanin E (10) and phoyunnanin C (11) showed the most potent α-glucosidase inhibition by comparing with acarbose, which was used as a positive control. Kinetic study revealed the non-competitive inhibitors against the enzyme. For anti-adipogenic activity, densifloral B (3) showed the strongest inhibition when compared with oxyresveratrol (positive control). In addition, densifloral B might be responsible for the inhibition of adipocyte differentiation via downregulating the expression of peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT enhancer-binding protein alpha (C/EBPα), which are major transcription factors in adipogenesis.


Introduction
Diabetes mellitus (DM) is a chronic metabolic disorder characterized by a high level of blood glucose resulting from a relative or absolute deficiency of insulin action. Type II is the most common type of diabetes, caused by β-cell dysfunction and insulin resistance [1]. The inhibition of α-glucosidase is effective for the treatment of type II diabetes [2]. α-Glucosidase is a membrane-bound enzyme produced from the epithelial cells of the small intestine. This enzyme is capable of converting starch and disaccharides into monosaccharides (glucose). Thus, glucose absorption can be reduced by inhibition of this enzyme, and postprandial blood glucose levels can also be decreased [3,4].
The relation between diabetes and obesity is well established in both traditional and modern therapy. The World Health Organization (WHO) estimates that 44% of diabetes cases are associated with overweightness and obesity [5]. Obesity is a risk factor of type 2 diabetes, coronary heart disease, and hypertension and is becoming a major health problem [6]. As a complex multifactorial chronic disease, obesity is characterized by an excessive adipocyte tissue mass. Adipogenesis is the process of cell differentiation during which fibroblast-like preadipocytes develop into mature adipocytes. Recently, the inhibition
For further investigation of the mechanism of enzyme inhibition, a kinetic study was carried out on the most potent compounds, phoyunnanin E (10) and phoyunnanin C (11). The experiment was performed by using Lineweaver-Burk plots of the reciprocal of velocity (1/V) against the reciprocal of substrate concentration (1/[S]) ( Figure 2). The substrate p-nitrophenol-α-D-glucopyranoside concentration was varied from 0.25 to 2.0 mM in the absence or presence of compound 10 at 12 µM and 22 µM and compound 11 at 12 µM and 24 µM. As summarized in Table 2, the different concentrations of 10 and 11 reduced the V max but did not affect the K m value, indicating that 10 and 11 are non-competitive types of enzyme inhibitors. On the other hand, the drug acarbose showed an intersection of the lines on the y-axis, indicating a competitive type of inhibition. A secondary plot of each compound was then constructed to evaluate the inhibition constant (K i ). We found that the K i value of acarbose 190.57 µM was obtained, and both 10 (K i 5.89 µM) and 11 (K i 5.97 µM) showed much greater affinity to the enzyme than acarbose. Compounds 10 and 11 as non-competitive inhibitors have some benefit over competitive inhibitors according to their binding to the allosteric site of the enzyme; therefore, they do not depend on the substrate concentration [38]. Furthermore, non-competitive inhibitors demand lower concentrations than competitive inhibitors to generate the same result [39]. Table 2. Kinetic parameters of α-glucosidase inhibition in the presence of phoyunnanin E (10) and phoyunnanin C (11).   To investigate their anti-adipogenic effect, the cytotoxic profile of D. delacourii extracts in preadipocytes was determined using MTT (methyl-thiazolyl-diphenyl-tetrazolium bromide) and nuclear staining assays. After culturing with 5 µg/mL of the methanolic, ethyl acetate, or butanolic extracts for 48 h, there were no significant alterations in the viability percentage observed via MTT assay in mouse embryonic preadipocyte 3T3-L1 cells (Figure 3a), compared with the untreated control. It is worth noting that treatment with all extracts at 10-20 µg/mL reduced viability in 3T3-L1 cells to lower than 90% (data not shown). Figure 3b depicts neither apoptosis nor necrosis, which were respectively observed as bright blue fluorescence of Hoechst33342 and propidium iodide red fluorescence in all treated 3T3-L1 cells. Thus, the extracts at 5 µg/mL, which were considered as non-toxic concentration, were chosen for the investigation of anti-adipogenic activity. Notably, treatment with oxyresveratrol (positive control), an anti-adipogenic natural compound, at 20 µM for 48 h also caused no change in cell viability percentage and cell death in preadipocyte 3T3-L1 cells.
For determination of their anti-adipogenic effect, preadipocyte 3T3-L1 cells were incubated with the differentiation medium with or without D. delacourii extracts, as mentioned in Materials and Methods. After the differentiation period was complete, the accumulated intracellular lipid droplets were analyzed by oil red O staining. As shown in Figure 3c, the lower level of oil red O percentage was indicated in 3T3-L1 cells cultured either with 5 µg/mL methanolic extract or 5 µg/mL ethyl acetate extract, compared with the control group. Meanwhile the significant decrease in oil red O staining percentage was not demonstrated in the treatment of the butanolic extract. A comparable inhibition of adipocyte differentiation between the methanolic extract, the ethyl acetate extract, and oxyresveratrol (20 µM) was evidenced with not only the reduction in the oil red O percentage but also the diminution of cellular lipid droplets presented in differentiated 3T3-L1 cells stained with oil red O (Figure 3d). Taken together, the ethyl acetate extract that indicated anti-adipogenic potential was selected for further investigation.

Screening for Anti-Adipogenic Activity of Compounds (1-11)
According to previous report about anti-adipogenic activity of phenolic compound, batatasin I, at 20 µM [40], the suppressive effect of compounds (1-11) isolated from ethyl acetate extract of D. delacourii at the same concentration (20 µM) was preliminarily demonstrated by the remarkable reduction in both oil red O staining percentage ( Figure 4a) and oil red O staining cells (Figure 4b) in differentiated 3T3-L1 cells. Although ephemeranthoquinone (2) and densifloral B (3) showed the highest anti-adipogenic activity among various isolates as well as positive control (oxyresveratrol), with approximately 64.7% and 65.2% reduction in oil red O staining, respectively, compound 2 at 20 µM significantly decreased viability in 3T3-L1 cells (data not shown). Therefore, compound 3, which caused no cytotoxicity in 3T3-L1 cells, was selected to further investigate the related anti-adipogenic mechanisms. β-actin was used as an internal control. Data are presented as means ± SD from three independent experiments. * p < 0.05 versus non-treated control cells at the same time point.
To compare the potency with another natural compound, preadipocyte 3T3-L1 cells were cultured with differentiation medium containing either various concentrations (0-50 µM) of compound 3 or oxyresveratrol. Figure 4c presents the relationship between concentration and oil red O staining percentage in response to compound 3 and oxyresveratrol treatment.
The results indicate that compound 3 and oxyresveratrol clearly restrained the adipocyte differentiation in 3T3-L1 cells in a dose-dependent manner. In addition, the effect of a 50% inhibitory concentration (IC 50 ) of compound 3 and oxyresveratrol on adipocyte differentiation was about 14.8 ± 1.6 µM and 21.1 ± 1.5 µM, respectively (Figure 4d). According to IC 50 data, compound 3 showed higher potency in anti-adipogenesis compared with oxyresveratrol.

Densifloral B (3) Suppresses Adipocyte Differentiation-Related Proteins
Adipogenesis involves a network of transcription factors that contribute to adipocyte differentiation and lipid accumulation [41]. Although the adipogenic gene CCAAT-enhancerbinding protein beta (C/EBPβ) is expressed soon after exposure to the adipogenic inducers, the upregulation of PPARγ and C/EBPα is acquired after 36-48 h of the induction [42]. In this regard, the translational level of PPARγ and C/EBPα was examined to elucidate the anti-adipogenic mechanism in preadipocyte 3T3-L1 cells cultured with 20 µM densifloral B (3) at both early (38 h) and late (48 h) stage. Western blot analysis indicated the prolong expression of PPARγ and C/EBPα, the adipogenic transcription factors, in 3T3-L1 cells from early until late stage of adipogenic process. Moreover, the expression level of PPARγ and C/EBPα was restrained in densifloral B-treated 3T3-L1 cells compared with untreated control cells (Figure 4e). Interestingly, the diminution in PPARγ and C/EBPα relative protein levels, which was promptly detected at 38 h, was sustained until 48 h of adipocyte differentiation in the presence of 20 µM densifloral B (Figure 4f,g, respectively).
Recent reports indicate that the downregulation of PPARγ and C/EBPα are correlated with the activation of AMPKα/β and ACC [43]. As presented in Figure 5a (Figure 4a). The results presented in this study correspond with the evidence of the stimulation of AMPK and ACC protein being associated with the inhibition of 3T3-L1 adipocyte differentiation [44]. Interestingly, the densifloral B (3) also suppressed the activated Akt (p-Akt), an upstream regulator of the AMPK-ACC signal [45] to reduce the adipocyte formation ( Figure 5d).
Due to the expression of adipogenic transcription factors also modulated via Akt/ GSK3β [46][47][48], the alteration of the p-GSK3β/GSK3β level was additionally examined in densifloral B-treated 3T3-L1 cells. The phosphorylation by p-Akt results in the inactivation of the GSK3β degradation complex following the initiation of targeted gene expression [49]. Surprisingly, the presence of 20 µM densifloral B (3) in differentiation medium significantly lessened p-GSK3β/GSK3β expression level in preadipocyte 3T3-L1 cells at 38-48 h of differentiation time (Figure 5e). Taken together, the present results suggest that densifloral B (3) isolated from D. delacourii might inhibit adipocyte differentiation via suppression of the Akt-mediating GSK3β and AMPK-ACC signals ( Figure 6).

Plant Material
The whole plant of Dendrobium delacourii was purchased from a Chatuchak market in May 2018. Plant identification was performed by Dr. Boonchoo Sritularak. A voucher specimen (BS-Ddela-052561) was deposited at the Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University.

Extraction and Isolation
The dried powder of whole plant D. delacourii (3.5 kg) was macerated with MeOH (4 × 15 L), and a methanolic extract (300.6 g) was obtained. This extract was dissolved in water and then partitioned with ethyl acetate (EtOAc) and butanol to give an EtOAc extract (159.7 g), a butanol extract (98.2 g), and an aqueous extract (42.1 g), respectively, after evaporation of the solvent. The EtOAc extract exhibited 85.5% inhibition of α-glucosidase enzyme at 100 µg/mL and also showed 49.0% inhibition of adipocyte differentiation of 3T3-L1 cells at 5 µg/mL, whereas the other extracts were devoid of both activities. Therefore, the EtOAc extract was subjected to further investigation.

Assay for α-Glucosidase Inhibitory Activity
The assay was based on the inhibition in the sample of α-glucosidase enzyme, which can release p-nitrophenol (PNP) from p-nitrophenyl-α-D-glucoside (PNPG) by hydrolysis [48]. In this assay, acarbose was used as the positive control. For IC 50 determination, twofold serial dilution was performed for each sample. Each experiment was accomplished in triplicate. Data are expressed as mean ± SD.
The kinetic study of enzyme inhibition was analyzed by the double reciprocal Lineweaver-Burk plot (1/V versus 1/[S]). The experiment was carried out by performing various concentrations of substrate p-nitrophenol-α-D-glucopyranoside (0.25, 0.5, 1.0, 2.0 mM) in the absence or presence of compound (10) (12 and 22 µM) and compound (11) (12 and 24 µM). The reaction was monitored every 5 min for a total time of 25 min and measured at 405 nm by a microplate reader. Each experiment was performed in triplicate. The K i value was estimated by constructing a secondary plot which is plotted by the slopes of the double-reciprocal lines versus inhibitor concentration.
3.5. Assay for Anti-Adipogenic Activity 3.5.1. Cell Culture and Adipocyte Differentiation Mouse embryonic preadipocyte 3T3-L1 cells were cultured in DMEM containing 10% FBS, 100 units/mL of penicillin/streptomycin, and 2 mmol/L of L-glutamine under humidified conditions of 5% CO 2 at 37 • C until 70-80% confluence was reached. For differentiation into adipocyte, preadipocyte 3T3-L1 cells were incubated with differentiation media composed of 10% FBS, 0.5 mM isobutylmethylxanthine, 1 µM dexamethasone, and 5 µg/mL insulin in DMEM with or without test compound for 2 days. Then, the differentiation media was replaced with culture media containing 5 µg/mL of insulin. After further incubation for 2 days, the cells were maintained in complete DMEM, which was changed every 2 days until adipocytes containing lipid droplets were observed under microscope [40].

Determination of Cytotoxicity
To evaluate the effect of D. delacourii extracts on cell viability, 3T3-L1 preadipocytes were seeded into 96-well plates at density of 2 × 10 3 cells/well and allowed to attach overnight at 37 • C. Then, the cells were further cultured with extracts (5 µg/mL), compounds (20 µM), or left untreated for 48 h before adding of 0.45 mg/mL MTT solution to assess cell viability. After incubation for 3 h at 37 • C and kept from light, the optical density (OD) of the purple formazan product dissolved in DMSO was measured at 570 nm using a microplate reader (Anthros, Durham, NC, USA). The relative OD ratio of treated to non-treated cells was presented as percentage cell viability [50].
The cytotoxicity of D. delacourii extracts was confirmed via cell death detection using costaining of Hoechst33342 and propidium iodide. After 48 h incubation with indicated treatment, the cells were further incubated with nuclear staining solution containing 2 µg/mL of Hoechst33342 and 1 µg/mL of propidium iodide for 30 min. The mode of cell death was observed under a fluorescence microscope (Olympus IX51 with DP70, Olympus Corp., Shinjuku-ku, Tokyo, Japan).

Quantification of Cellular Lipid Content Using Oil Red O Staining
The lipid droplets presenting in differentiated adipocytes were detected via oil red O staining. After the differentiation process, 3T3-L1 cells were fixed with 10% formalin for 45 min and further incubated with oil red O solution at room temperature for 1 h. After washing with 60% isopropanol for three times, oil red O-stained cells were captured using a Nikon Ts2 inverted optical microscope (Tokyo, Japan). For quantification, cellular oil red O was extracted using absolute isopropanol for measurement of OD at 570 nm by microplate reader (Anthros, Durham, NC, USA) [51]. The percentage of oil red O staining was calculated relative to the total protein content determined by BCA assay [52].

Western Blot Analysis
After the indicated treatment, 3T3-L1 cells were washed with phosphate-buffered saline (PBS, pH 7.4), then the cell membranes were broken using RIPA buffer supplemented with a protease inhibitor cocktail. After incubation on ice for 45 min, the cell lysates were centrifuged at 12,000 rpm at 4 • C for 15 min to collect the clear supernatant containing cellular protein, which was measured for total protein content using a BCA assay kit. The total protein (30 µg) from each sample was loaded and separated onto 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, the separated proteins were transferred onto nitrocellulose membranes, which were blocked with 5% skim milk in TBST buffer (Tris-buffered saline with Tween 20, pH 7.2) and further immunoblotted with primary antibodies against p-Akt (Thr308), Akt, p-GSK3β (Ser9), GSK3β, p-AMPKα (Thr172), AMPKα, p-AMPKβ1 (Ser128), AMPKβ1/2, p-ACC (Ser79), ACC, PPARγ, C/EBPα, and β-actin at 4 • C overnight. Before immersion in horseradish peroxidase (HRP)-linked secondary antibody at room temperature for 2 h, the membranes were washed with TBST for 7 min, three times. The reactive protein signals exposed with chemiluminescent substrates were captured and quantified using Chemiluminescent ImageQuant LAS 4000 (GE Healthcare Bio-Sciences AB, Björkgatan, Uppsala, Sweden).

Statistical Analysis
All data are expressed as means ± standard deviation (SD) obtained from three independent experiments. Statistical analysis was performed using GraphPad Prism 8.0.2 (GraphPad Software Inc., San Diego, CA, USA) with one-way ANOVA. Differences with p value < 0.05 were considered to be statistically significant.

Conclusions
In this study, 11 compounds were isolated from the ethyl acetate extract of D. delacourii. Two dimeric phenanthrene derivatives, phoyunnanin E (10) and phoyunnanin C (11), revealed the most potent α-glucosidase inhibition when compared with a positive control, acarbose. An enzyme kinetic study performed on them indicated non-competitive inhibitors. Regarding anti-adipogenic activity, densifloral B (3) showed the most potent activity when compared with a positive control, oxyresveratrol. The anti-adipogenic properties of densifloral B (3) were attributed to the downregulation of PPARγ and C/EBPα expression through the modulation of Akt-related pathways including the Akt/GSK3β and Akt/AMPK-ACC signals. The findings obtained from this study demonstrate the evaluation of α-glucosidase inhibitory activity and anti-adipogenic effect of the Dendrobium delacourii plant, which can be used for the management of diabetes and obesity.