New Fluorene Derivatives from Dendrobium gibsonii and Their α-Glucosidase Inhibitory Activity

Two new compounds, dihydrodengibsinin (1) and dendrogibsol (2), were isolated from the whole plant of Dendrobium gibsonii, together with seven known compounds (3–9). The structures of the new compounds were elucidated by their spectroscopic data. All these isolates were evaluated for their α-glucosidase inhibitory activities. Dendrogibsol (2) and lusianthridin (7) showed strong α-glucosidase inhibitory activity when compared with acarbose. An enzyme kinetic study revealed that dendrogibsol (2) is a noncompetitive inhibitor of α-glucosidase.


Introduction
Diabetes is a metabolic disease associated with chronic hyperglycemia due to deficiency in insulin secretion or action [1]. The prevalence of diabetes has been increasing all over the world. Around 8.8% of the world's adult population suffered from diabetes in 2017, and it is estimated that the number will rise to 9.9% by 2045 [2]. Many diabetic patients suffer from chronic complications such as nephropathy, neuropathy, retinopathy and macrovascular problems, which are the major causes of morbidity and mortality. About 90% of all diabetic patients are caused by type II diabetes [3].
α-Glucosidase is one of the key enzymes involved in carbohydrate metabolism and is essential for maintaining normal physiological functions [4]. It has been considered a suitable model for observing the action of nutraceuticals on type II diabetes [5]. α-Glucosidase inhibitor (α-GI) drugs, given alone or combination with other oral antidiabetic agents, have been used for the treatment of type II diabetes [6]. Acarbose and miglitol are examples of α-GIs; they decrease postprandial hyperglycemia by retarding the absorption of glucose in the intestine [7]. However, these drugs have several side effects, including diarrhea, flatulence, abdominal pain and liver damage [8]. Thus, new α-GI drugs with less adverse effects are still needed. α-Glucosidase enzymes obtained from yeast, rat intestine and mouse intestine have been used as screening tools for identifying potential α-GI agents [9].
A large number of α-GIs have been reported from natural sources [10]. Several α-GIs of plant origin appear to be more potent and safer than their synthetic counterparts [11]. Dendrobium, a major
Compound 1 was obtained as a brownish-white amorphous solid. The molecular formula C 15 H 14 O 5 was analyzed from its [M − H] − at m/z 273.0764 (calcd. for C 15 H 13 O 5 273.0763). The IR spectrum showed absorption bands for hydroxyl (3420 cm −1 ) and aromatic (2925, 1618 cm −1 ) functionalities. The UV spectrum exhibited absorption peaks at 220, 255 and 300 nm, indicating a fluorene structure [30]. This was supported by the presence of twelve aromatic carbons and one oxygenated methine carbon of C-9 (δ 74.5), which correlated to the proton at δ 5.38 (1H, d, J = 7.8 Hz, H-9) in the HSQC spectrum ( Table 1). The HO-9 proton at δ 4.57 (d, J = 7.8 Hz) displayed two-bond HMBC correlation with C-9. The 1 H-NMR spectrum of 1 showed four aromatic proton signals at δ 6.77-7.13 and signals for two methoxyl groups at δ 3.93 (3H, s, MeO-2) and δ 4.12 (3H, s, MeO-4). On ring A, the 1 H-NMR spectrum exhibited three aromatic protons with ortho-coupling at δ 6.77 (1H, d, J = 7.5 Hz, H-6), 7.05 (1H, d, J = 7.5 Hz, H-8) and 7.13 (1H, t, J = 7.5 Hz, H-7). The assignment of H-8 was based on its HMBC correlations with C-9. The HO-5 proton at δ 9.44 (s) showed correlation with C-5 (δ 151.1) and C-6 (δ 116.1) in the HMBC spectrum. On ring B, the singlet proton signal δ 7.10 was assigned to H-1 from its HMBC correlation with C-9. The first methoxyl (δ 3.93) was located at C-2 and the second methoxyl (δ 4.12) was at C-4, as supported by their NOESY correlations with H-1 and HO-5, respectively. Based on the above spectral data, compound 1 was characterized as 2,4-dimethoxy-9H-fluorene-3,5,9-triol and given the trivial name dihydrodengibsinin. Prior to this study, the natural occurrence of 1 was not known. This compound, however, was earlier synthesized by reduction of the corresponding fluorenone dengibsinin [20,21]. Molecules 2020, 25, 3 of 9 Compound 1 was obtained as a brownish-white amorphous solid. The molecular formula C15H14O5 was analyzed from its [M − H] − at m/z 273.0764 (calcd. for C15H13O5 273.0763). The IR spectrum showed absorption bands for hydroxyl (3420 cm −1 ) and aromatic (2925, 1618 cm −1 ) functionalities. The UV spectrum exhibited absorption peaks at 220, 255 and 300 nm, indicating a fluorene structure [30]. This was supported by the presence of twelve aromatic carbons and one oxygenated methine carbon of C-9 (δ 74.5), which correlated to the proton at δ 5.38 (1H, d, J = 7.8 Hz, H-9) in the HSQC spectrum ( Table 1). The HO-9 proton at δ 4.57 (d, J = 7.8 Hz) displayed two-bond HMBC correlation with C-9. The 1 H-NMR spectrum of 1 showed four aromatic proton signals at δ 6.77-7.13 and signals for two methoxyl groups at δ 3.93 (3H, s, MeO-2) and δ 4.12 (3H, s, MeO-4). On ring A, the 1 H-NMR spectrum exhibited three aromatic protons with ortho-coupling at δ 6.77 (1H, d, J = 7.5 Hz, H-6), 7.05 (1H, d, J = 7.5 Hz, H-8) and 7.13 (1H, t, J = 7.5 Hz, H-7). The assignment of H-8 was based on its HMBC correlations with C-9. The HO-5 proton at δ 9.44 (s) showed correlation with C-5 (δ 151.1) and C-6 (δ 116.1) in the HMBC spectrum. On ring B, the singlet proton signal δ 7.10 was assigned to H-1 from its HMBC correlation with C-9. The first methoxyl (δ 3.93) was located at C-2 and the second methoxyl (δ 4.12) was at C-4, as supported by their NOESY correlations with H-1 and HO-5, respectively. Based on the above spectral data, compound 1 was characterized as 2,4-dimethoxy-9H-fluorene-3,5,9-triol and given the trivial name dihydrodengibsinin. Prior to this study, the natural occurrence of 1 was not known. This compound, however, was earlier synthesized by reduction of the corresponding fluorenone dengibsinin [20,21].   6.85 (1H, s, H-1) and 6.93 (1H, t, J = 8.0 Hz, H-7) and two methoxyl groups at C-2 (δ 3.77, 3H, s,) and C-4 (δ 4.18, 3H, s). The presence of a dihydrophenanthrene unit in 2 was deduced from the characteristic signals for 2 methylene carbons at δ 20.9 (C-9 ) and 26.9 (C-10 ) in addition to 12 aromatic carbon resonances. In the 1 H-NMR spectrum, the dihydrophenanthrene unit displayed two aromatic proton singlets at δ 6.04 (1H, s, H-6 ) and 6.61 (1H, s, H-1 ), and three methoxyl groups at δ 3.37 (3H, s, MeO-3 ), 3.54 (3H, s, MeO-7 ) and 3.82 (3H, s, MeO-2 ). The assignment of H-6 of ring C was supported by its HBMC correlations with C-4b (δ 120.6) and C-8 (δ 143.4). On ring C, the first methoxy group should be placed at C-7 according to its NOESY correlation with H-6 . On ring D, the assignment of H-1 was deduced from its HMBC correlations with C-10 . The NOESY cross-peak between H-1 and H 2 -10 was also observed. The second methoxy group was located at C-2 , as supported by its NOESY correlation with H-1 . The HMBC correlations of C-3 (δ 137.3) with H-1 and MeO-3 indicated the location of the third methoxy group at C-3 . Compound 2 had the fluorene moiety connected to the dihydrophenanthrene unit through a C-C linkage between C-5 (δ 123.4) and C-9 (87.4) and ether bond between C-9 and the oxygen atom at C-4 (δ 145.3), forming a spiro structure. This was supported by the HMBC correlations of C-9 with H-1, H-8 and H-6 . Thus, it was concluded that 2 was a fluorene-dihydrophenanthrene adduct, with the structure as shown in Figure 1, and it was given the trivial name dendrogibsol. It is the first representative of this class of dimeric compounds. The biogenesis of the unprecedented fluorene-dihydrophenanthrene adduct (2) is proposed to occur as shown in Figure 2. The coupling reaction is initiated by the nucleophilic attack from C-5 of the dihydrophenanthrene unit (II) onto the keto carbon (C-9) of the fluorenone (I) to give a quinone-like structure (III). This structure subsequently isomerizes to form intermediate IV. Finally, the nucleophilic attack by the oxygen of the OH-4 group of the dihydrophenanthrene unit on the carbinol carbon (C-9) of the fluorene part, with concomitant loss of H 2 O, generates compound 2.
The biogenesis of the unprecedented fluorene-dihydrophenanthrene adduct (2) is proposed to occur as shown in Figure 2. The coupling reaction is initiated by the nucleophilic attack from C-5′ of the dihydrophenanthrene unit (II) onto the keto carbon (C-9) of the fluorenone (I) to give a quinone-like structure (III). This structure subsequently isomerizes to form intermediate IV. Finally, the nucleophilic attack by the oxygen of the OH-4′ group of the dihydrophenanthrene unit on the carbinol carbon (C-9) of the fluorene part, with concomitant loss of H2O, generates compound 2.
Further investigation was conducted on compound 2 to study its kinetic properties with regard to the enzyme α-glucosidase using varying concentrations of the substrate. From Lineweaver-Burk plots in Figure 3A, it can be seen that acarbose inhibited α-glucosidase in a competitive manner.

α-Glucosidase Inhibitory Activity
All the isolated compounds (1-9) were evaluated for their α-glucosidase inhibitory activities. In this study, each compound was initially tested at 100 µg/mL. Half-maximal inhibitory concentration (IC 50 ) was determined if the compound showed more than 50% inhibition of the enzyme. Acarbose was used as the positive control. Dendrogibsol (2) and lusianthridin (7) showed potent α-glucosidase inhibitory activities with IC 50 values of 19.8 ± 0.9 µM and 185.4 ± 6.9 µM, respectively, when compared with acarbose (IC 50 514.4 ± 9.2 µM). The other compounds were devoid of activity.
Further investigation was conducted on compound 2 to study its kinetic properties with regard to the enzyme α-glucosidase using varying concentrations of the substrate. From Lineweaver-Burk plots in Figure 3A, it can be seen that acarbose inhibited α-glucosidase in a competitive manner. When the acarbose concentration was increased, the K m decreased from 6.74 to 1.55 mM while the V max value (0.11 ∆OD/min) was unaffected. On the other hand, compound 2 was found to be a noncompetitive inhibitor of α-glucosidase, with decreasing V max from 0.12 to 0.052 ∆OD/min and unchanging K m (1.55 mM), as illustrated in Figure 3B. The generated secondary plots for compound 2 and acarbose revealed that the K i value of 2 (20.38 µM) was much less than that of acarbose (190.57 µM), as shown in Figure 3 and summarized in Table 2.

Plant Material
The whole plant of D. gibsonii was purchased from Chatuchak market, Bangkok, in February 2018. Plant identification was performed by B. Sritularak. A voucher specimen (BS-DG-022561) has been deposited at the Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University (Bangkok, Thailand).

Extraction and Isolation
The dried powder of whole-plant D. gibsonii (4.2 kg) was macerated with methanol (MeOH) (5 × 15 L), and a MeOH extract (371 g) was obtained. This extract was dissolved in water and then partitioned with EtOAc and BuOH to give an EtOAc extract (100 g), a BuOH extract (72 g) and an aqueous extract (95.5 g) after evaporation of the solvent. These extracts were then evaluated for their α-glucosidase inhibitory activity. Only EtOAc extract exhibited strong α-glucosidase, with 77.7 ± 1.8% inhibition at concentration 100 µg/mL, and therefore was further investigated The BuOH and aqueous extracts were devoid of activity (<50% inhibition at concentration 100 µg/mL).

Assay for α-Glucosidase Inhibitory Activity
The α-glucosidase inhibition assay was performed according to previous protocols [31]. The assay was based on the release of p-nitrophenol from p-nitrophenol-α-d-glucopyranoside (substrate). The test samples were prepared by dissolving in 50% DMSO. Two-fold serial dilution was done for IC 50 determination of active compounds. The sample solution (10 µL) and 0.1 U/mL α-glucosidase (40 µL) in phosphate buffer (pH 6.8) were added to a 96-well plate. The mixture was preincubated at 37 • C for 10 min before adding 2 mM p-nitrophenol-α-d-glucopyranoside (50 µL). Then, the reaction was incubated again at 37 • C for 20 min. Finally, 1 M Na 2 CO 3 solution (100 µL) was added to stop the reaction. The absorbance of the mixture was determined using a microplate reader at 405 nm. In this assay, acarbose was used as the positive control.
An enzyme kinetic study was conducted based on the α-glucosidase assay as mentioned above. The PNPG concentrations were varied from 0.25 to 2 mM in the absence or presence of compound 2 (11 and 22 µM) or acarbose (930 and 465 µM). The inhibition mode was determined by double-reciprocal Lineweaver-Burk plot (1/V vs. 1/[S]). In order to estimate the K i value, slopes of double-reciprocal lines were used to construct a secondary plot, and the K i was calculated from the line equation of the plot [32].