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. 2022 Jul 6;7(28):24511–24521. doi: 10.1021/acsomega.2c02163

Antidiabetic and Cytotoxic Activities of Rotenoids and Isoflavonoids Isolated from Millettia pachycarpa Benth

Virayu Suthiphasilp §, Narawadee Rujanapun ‡, Pakit Kumboonma #, Boonyanoot Chaiyosang †, Sarawut Tontapha ⊥, Tharakorn Maneerat §,‡, Brian O Patrick ∥, Raymond J Andersen ∥, Thidarat Duangyod ‡,○, Rawiwan Charoensup ‡,○, Surat Laphookhieo §,‡,*
PMCID: PMC9301698  PMID: 35874225

Abstract

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A phytochemical investigation of the root and leaf extracts of Millettia pachycarpa Benth resulted in the isolation and identification of 16 compounds, including six rotenoids (1–6) and 10 prenylated isoflavonoids (7–16). Compound 4 was isolated as a scalemic mixture, which was resolved by chiral HPLC to afford (−)-(6aS,12aS)-12a-hydroxy-α-toxicarol (4) and (+)–(6aR,12aR)-12a-hydroxy-α-toxicarol (4). (+)-(6aR,12aR)-Millettiapachycarpin (3) and (−)-(6aS,12aS)-12a-hydroxy-α-toxicarol (4) were isolated as new compounds. The absolute configuration of (−)-(6R)-pachycarotenoid (2), (+)–(6aR,12aR)-millettiapachycarpin (3), (−)-(6aS,12aS)-4 and (+)–(6aR,12aR)-12a-hydroxy-α-toxicarol (4), (+)-(6aS,12aS)-(5), and (−)-(6aS,12aS,2″R)-sumatrol (6) were identified by electronic circular dichroism (ECD) data. (−)-(6aS,12aS,2″R)-Sumatrol (6) was also confirmed by X-ray diffraction analysis using Cu–Kα radiation. Antidiabetic activities, including α-glucosidase and α-amylase inhibitory activities, and cytotoxicities against lung cancer A549, colorectal cancer SW480, and leukemic K562 cells of some isolated compounds were evaluated. Of these, isolupalbigenin (11) exhibited the highest α-glucosidase inhibitory activity, with an IC50 value of 11.3 ± 0.2 μM, whereas the scalemic mixture of 12a-hydroxy-α-toxicarol (4) displayed the best α-amylase inhibitory activity, with an IC50 value of 106.9 ± 0.2 μM. Euchrenone b10 (15) exhibited the highest cytotoxicity against lung cancer A549, colorectal cancer SW480, and leukemic K562 cells, with IC50 values of 40.3, 39.1, and 15.1 μM, respectively. In addition, molecular docking simulations of α-glucosidase inhibition of the active compounds were studied.

Introduction

Millettia pachycarpa Benth. (Fabaceae) is a perennial climbing tree, which is distributed throughout Southeast Asian countries. Some parts of the plant have been used in traditional medicines. For example, the seeds have been used for the treatment of worm infestations, skin diseases, bruising, and as anthelmintics in the Southeast Asia region,1,2 whereas the root barks have been used to treat intestinal infections by direct consumption.3,4 Moreover, the root peels have been used as insecticides and fish poison.5 In previous phytochemical investigations, rotenoids and flavonoids have been identified as major phytochemicals.6−9 These types of compounds display interesting biological activities, including cytotoxicity, α-glucosidase inhibition, NF-κB inhibition, anti-inflammation, and antibacterial behavior.6−9 For example, berectones A and B showed inhibitory activities toward α-glucosidase with IC50 values of 144 and 172 μM, respectively.10 (−)-Hydroxyrotenone and (−)-rotenone showed significant cytotoxicity against HT-29 with IC50 values of 0.1 and 0.3 μM, respectively,11 whereas barbigerone showed significant cytotoxicity against HepG2 cell lines with the IC50 value of 0.61 μM.12 In addition, 4′,5′-dimethoxy-6,6-dimethylpyranoisoflavone and deguelin showed butyrylcholinesterase inhibitory activity with an IC50 value of 2.34 and 14.25 μM, respectively,1 whereas tephrosin had an anti-inflammatory activity with an IC50 value of <10 μM without cell toxicity at a concentration of 3.125 μM.13 In our continuing studies of biologically active natural products from medicinal plants,14−19 we report here the isolation and structure elucidation of two new rotenoids, (+)-(6aR,12aR)-millettiapachycarpin (3) and (−)-(6aS,12aS)-12a-hydroxy-α-toxicarol (4), together with 14 known compounds (Figure 1) from the root and leaf extracts of M. pachycarpa. Antidiabetic activities, including α-glucosidase and α-amylase inhibitory activities, and cytotoxicities against three human cancer cell lines (lung cancer A549, colorectal cancer SW480, and leukemic K562 cells) of some isolated compounds were evaluated. In addition, molecular docking simulations of α-glucosidase inhibition of the selected active compounds are reported.

Figure 1.

Figure 1

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Compounds isolated from root and leave extracts of Millettia pachycarpa Benth.

Results and Discussion

Structure Elucidation of Isolated Compounds

The EtOAc extracts of the roots and leaves of M. pachycarpa were individually separated and purified by various column chromatographic techniques to yield 16 compounds, including two new rotenoids, (+)-(6aR,12aR)-3 and (−)-(6aS,12aS)-4, and 14 known compounds (Figure 1). Compounds 1–7 were isolated from the root extract, whereas compounds 8–16 were isolated from the leaf extract. The known compounds were identified as 6a,12a-dehydro-α-toxicarol (1),20 (−)-6-hydroxy-6a,12a-dehydro-α-toxicarol (2),21 (+)-(6aR,12aR)-12a-hydroxy-α-toxicarol (4),20,22 (+)-α-toxicarol (5),20,23 (−)-sumatrol (6),24,25 toxicurol isoflavone (7),26 6,8-diprenylorobol (8),27 6,8-di-C-prenylpratensein (9),28 6,8-diprenylgenistein (10),27 isolupalbigenin (11),29,30 lupalbigenin (12),30 isoerysenegalensein E (13),31,32 erysenegalensein E (14),32 euchrenone b10 (15),33 and senegalensin (16)33 by analysis of their spectroscopic data and by comparisons of these data with those reported.

Compound 2 ((−)-6-hydroxy-6a,12a-dehydro-α-toxicarol) was first isolated and identified from the fruit extract of Amorpha fruticosa by Somleva and Ognyanov in 198521 without the absolute configuration identification. In this study, the absolute configuration at C-6 of compound 2 was identified from the experimental and calculated electronic circular dichroism (ECD) as shown in Figure 2. The experimental ECD spectrum of (−)-2 [[α]D25 −66, c 0.5 (MeOH)] was similar to that of the computed ECD spectra of (6R)-2 (Figure 2). Accordingly, the absolute configuration of compound (−)-2 was identified as (−)-(6R)-6-hydroxy-6a,12a-dehydro-α-toxicarol.

Figure 2.

Figure 2

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Experimental and calculated ECD spectra of (−)-(2).

Compound 3 was obtained as a yellow solid. Its molecular formula, C22H20O8, was established on the basis of its HRESIMS data, which showed an ion peak at m/z 413.1230 [M + H]+ (calcd for 413.1231). Compound 3 was a tephrosin-like rotenoid,31 which showed the key resonances of diastereotopic oxymethylene protons [δH/δC 4.56 (dd, 12.1, 2.4) and 4.46 (dd, 12.1, 1.1)/63.7 (C-6)] and an oxymethine proton of the cis-B/C ring junction [δH/δC 4.51 (dd, 2.4, 1.1)/75.6 (C-6a)] in the 1H and 13C NMR spectroscopic data (Table 1). The 1H and 13C NMR spectroscopic data also revealed the resonances for a hydrogen-bonded hydroxy proton [δH 11.59 (s, 11-OH)], three singlet aromatic protons [δH/δC 6.78 (s)/112.2 (C-1), 6.43 (s)/100.5 (C-4), and 5.94 (s)/98.0 (C-10)], a methoxy group [δH 3.79 (s)/ δC 56.0], and a chromene moiety [δH/δC 6.48 (d, 10.3)/115.2, 5.44 (d, 10.3)/126.6, 1.41 (s)/28.7, 1.33 (s)/28.4, and δc 78.6]. The methoxy group was located at C-3 because of the HMBC correlation between δH 3.79 (3-OMe) and δc 148.7 (C-3). The HMBC correlations between δH 6.48 (H-1′) to 155.5 (C-7a), 102.0 (C-8), 163.4 (C-9), and δH 5.44 (H-2′) to 102.0 (C-8) supported the location of the chromene moiety at C-8/C-9 (Figure 3). The (6aR,12aR) absolute configuration of compound 3 was confirmed by the comparison of its experimental ECD spectrum and specific rotation ([α]D25 +57 (c 1, MeOH)) to those of (+)-(6aS,12aS)-α-toxicarol (5, ([α]D25 +32 (c 1, MeOH)) (Figure 4). The ECD spectrum of compound 3 (Figure 4) showed positive Cotton effects at 367 nm and negative Cotton effects at 235 and 318 nm, similar to that of (+)-α-toxicarol (5) (Figure 4), suggesting the (6aR,12aR) absolute configuration of compound 3. Thus, the structure of compound 3 was assigned as (+)-(6aR,12aR)-millettiapachycarpin.

Table 1. NMR Spectroscopic Data (1H-NMR (δ in ppm, J in Hz, 500 MHz) and 13C (125 MHz)) of (+)-(6aR,12aR)-3 in CDCl3 and (−)-(6aS,12aS)-4 in Acetone-d6.

  (+)-(6aR,12aR)-3
 (−)-(6aS,12aS)-4
position 1H 13C, type 1H 13C, type
1a   109.5, C   109.4, C
1 6.78 (s) 112.2, CH 6.82 (s) 112.2, CH
2   140.2, C   144.7, C
3   148.7, C   152.6, C
4 6.43 (s) 100.5, CH 6.49 (s) 101.9, CH
4a   147.6, C   149.6, C
6 4.56 (dd, 12.1, 2.4); 4.46 (dd, 12.1, 1.1) 63.7, CH2 4.64 (dd, 12.2, 2.5); 4.48 (dd, 12.2, 1.2) 64.4, CH2
6a 4.51 (dd, 2.4, 1.1) 75.6, CH 4.72 (dd, 2.5, 1.2) 76.9, CH
7a   155.5, C   156.5, C
8   102.0, C   102.4, C
9   163.4, C   163.5, C
10 5.94 (s) 98.0, CH 5.88 (m) 98.0, CH
11   164.0, C   165.0, C
11a   100.0, C   101.1, C
12   194.8, C   197.4, C
12a   66.8, C   67.9, C
1′ 6.48 (d, 10.3) 115.2, CH 6.52 (d, 10.2) 115.6, CH
2′ 5.44 (d, 10.3) 126.6, CH 5.62 (d, 10.2) 127.7, CH
3′   78.6, C   79.2, C
4′ 1.41 (s) 28.7, CH3 1.42 (s) 28.7, CH3
5′ 1.33 (s) 28.4, CH3 1.34 (s) 28.4, CH3
2-OMe     3.77 (s) 55.9, CH3
3-OMe 3.79 (s) 56.0, CH3 3.64 (s) 56.7, CH3
11-OH 11.59 (s)   12.05 (s)  
12a–OH     5.88 (m)  
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Figure 3.

Figure 3

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Selected HMBC (1H→13C) and COSY (1H—1H) correlation of compounds (+)-3 and (−)-4.

Figure 4.

Figure 4

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ECD spectra for compounds (+)-3 and (+)-5.

The mixture of compound 4 (12a-hydroxy-α-toxicarol) was first isolated from the root extract of Tephrosia candida (Roxb.) DC in 1997.20 In 2016, Yulin Ren and co-workers5 isolated (+)-(6aR,12aR)-12a-hydroxy-α-toxicarol ([α]D20 +5.0 (c 0.2, CHCl3) and its EDC spectrum displayed positive Cotton effects at 202 (+16.5), 248 (+4.9), and 367 (+5.2) nm and negative Cotton effects at 234 (−13.9), 270 (−3.7), and 310 (−9.8) nm. In this study, we isolated the scalemic mixture of compound 4 as a light-yellow powder and further resolved it by semipreparative chiral-phase HPLC to provide compounds (+)-4 [(tR 14.6 min), [α]D25 +22 (c 0.2, MeOH)] and (−)-4 [(tR 17.2 min), [α]D25 −25 (c 0.15, MeOH)] in a ratio of ca. 1:0.9 (Figure 5), with an ee of 1.15% (Figure S27). The ECD spectrum (Figure 6) of (+)-(6aR,12aR)-12a-hydroxy-α-toxicarol ((+)-4) was similar to that previously reported.5,34 However, the ECD spectrum of (−)-4 [213 (−2.32), 236 (−2.37), 272 (−2.43), and 316 (+2.49) nm] was the mirror image of that of (+)-(6aR,12aR)-12a-hydroxy-α-toxicarol ((+)-4), indicating the absolute configuration of (−)-4 was opposite to that of (+)-(6aR,12aR)-12a-hydroxy-α-toxicarol ((+)-4). To confirm the absolute configuration of (−)-4 and (+)-4, we calculated the ECD spectra for (−)-(6aS,12aS)-4 and (+)-(6aR,12aR)-4. The experimental and computed ECD spectra of (+)-4 were similar to those of a rotenoid, oblarotenoid A, previously reported by Tsegaye Deyou and co-workers in 2017,34 whereas (−)-4 experimental and computed ECD spectra were mirror. Therefore, compound (−)-4 was identified as a new enantiomer of (+)-(6aR,12aR)-12a-hydroxy-α-toxicarol ((+)-4) and assigned as (−)-(6aS,12aS)-12a-hydroxy-α-toxicarol. Full assignments of the 1H and 13C NMR as well as selected HMBC correlations are shown in Table 1 and Figure 3, respectively.

Figure 5.

Figure 5

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Experimental and calculated ECD spectra of (+)-(6aR,12aR)-4 and (−)-(6aS,12aS)-4.

Figure 6.

Figure 6

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ECD spectrum for compound (−)-(6aS,12aS,2″R)-6.

(−)-Sumatrol (6) was first isolated and identified from the resin of Derris malaccenis var. sarawakenis by Cahn and Boam in 1935.35 In 1961, Crombie and Peace established the stereochemistry of (−)-sumatrol by angular orientation and specific rotation of ([α]D20 −182 (c 2.84, benzene).36 In addition, (−)-sumatrol was also isolated from the roots of Dahlstedtia pinnata in 1988 by Garcez and co-workers37 and its relative configuration of (6aS,12aS,2″R) was proposed. To date, the absolute configuration has been not identified. In this study, (−)-sumatrol (6) ([α]D25 −65 (c 1, MeOH); ECD spectrum [215 (−2.54), 241 (+2.01), 287 (−2.14), and 346 (+2.55) nm] (Figure 6)) was isolated as a single enantiomer. Single colorless crystals of (−)-6 were recrystallized from CH2Cl2 and analyzed by X-ray crystallography using Cu–Kα radiation. The (6aS,12aS,2″R) absolute configuration of (−)-sumatrol (Figure 7, CCDC 2155505) was established with a Flack parameter of 0.04(8).

Figure 7.

Figure 7

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ORTEP diagram for (−)-(6aS,12aS,2′′R)-sumatrol (6).

α-Glucosidase and α-Amylase Inhibitory Activities

A preliminary screening of α-glucosidase inhibitory activity of the root (EtOAc) and leaf (EtOAc) extracts displayed promising α-glucosidase inhibitory activities with 99.2 ± 0.2 and 65.1 ± 1.7% inhibition (IC50 values of 19.2 ± 0.4 and 42.0 ± 0.8 μg/mL), respectively. As a consequence, compounds 1, 3–6, and 9–11 and the standard drug acarbose at various concentrations were screened for α-glucosidase inhibitory activity (Table 2). Of these, (+)-(6aR,12aR)-millettiapachycarpin (3), (+)-α-toxicarol (5), 6,8-di-C-prenylpratensein (9), 6,8-diprenylgenistein (10), and isolupalbigenin (11) showed strong α-glucosidase inhibitory with IC50 values of 65.2 ± 0.4, 70.8 ± 1.1, 18.6 ± 1.2, 14.8 ± 1.8, and 11.3 ± 0.2 μM, respectively, which was stronger than that of the acarbose (IC50 of 77.2 ± 0.4 μM). 6a,12a-Dehydro-α-toxicarol (1), the scalemic mixture of 12a-hydroxy-α-toxicarol (4), and (−)-(6aS,12aS,2″R)-sumatrol (6) displayed α-glucosidase inhibitory activities less than the standard control with IC50 values of 95.8 ± 0.3, 77.4 ± 0.9, and 251.9 ± 0.6 μM, respectively (Table 2). It is interesting to note that the structural difference between 6,8-diprenylgenistein (10) and isolupalbigenin (11) is only the location of the isoprenyl group at C-6 and C-3′. The two isoprenyl groups of 6,8-diprenylgenistein (10) are located on C-6 and C-8, whereas in isolupalbigenin (11), they are located on C-8 and C-3′, which plays an important role in the α-glucosidase inhibitory activity. The structural variation between 6,8-di-C-prenylpratensein (9) and 6,8-diprenylgenistein (10) also corresponds to the remarkably different α-glucosidase inhibitory activity. The differences between 6,8-di-C-prenylpratensein (9) and 6,8-diprenylgenistein (10) are the substituents at C-3′ and C-4’. 6,8-Di-C-prenylpratensein (9) possesses hydroxy (3′–OH) and methoxy (4′-OMe) groups, whereas 6,8-diprenylgenistein (10) contains a hydrogen atom (H-3′) and a hydroxy group (4′–OH). The presence of additional hydroxy and methoxy groups in 6,8-di-C-prenylpratensein (9) appears to reduce the α-glucosidase inhibitory activity. It should be noted that α-glucosidase inhibitory activity of 6,8-diprenylgenistein (10) in this study was well agreed with the previous report (IC50 values of and 16.338 and 19.48 μM), whereas compounds 1, 3–6, 9, and 11 were reported here for the first time. In the case of α-amylase inhibitory activity, only four compounds, including (+)-(6aR,12aR)-millettiapachycarpin (3), the scalemic mixture of 12a-hydroxy-α-toxicarol (4), 6,8-diprenylgenistein (10), and isolupalbigenin (11), showed α-amylase inhibitory activity with IC50 values ranging from 106.9 ± 0.2 to 126.9 ± 0.5 μM, and less active than the standard control (acarbose, IC50 of 103.4 ± 0.9 μM).

Table 2. α-Glucosidase and α-Amylase Inhibitory Activities of Compounds 1, 3–6, and 9–11 from M. pachycarpa Leaves and Rootsa.

  α-glucosidase inhibition
 α-amylase inhibition
compds inhibition rate (%) IC50 (μM) inhibition rate (%) IC50 (μM)b
1 95.4 ± 1.3 95.8 ± 0.3 36.6 ± 0.3 inactive
3 99.4 ± 0.3 65.2 ± 0.4 76.4 ± 0.7 116.9 ± 0.8
4 87.8 ± 0.2 77.4 ± 0.9 76.0 ± 2.2 106.9 ± 0.2
5 99.2 ± 0.2 70.8 ± 1.1 41.6 ± 0.4 inactive
6 50.7 ± 0.3 251.9 ± 0.6 25.3 ± 0.8 inactive
9 98.9 ± 0.5 18.6 ± 1.2 24.2 ± 0.4 inactive
10 98.4 ± 0.3 14.8 ± 1.8 77.2 ± 0.9 126.2 ± 0.8
11 99.9 ± 0.8 11.3 ± 0.2 74.1 ± 1.0 126.9 ± 0.5
acarbose 57.2 ± 0.8 77.2 ± 0.4 58.5 ± 1.9 103.4 ± 0.9
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a

Data expressed as mean ± SD.

b

Inactive = not determined because α-amylase inhibition at a concentration of 100 μg/mL was less than 50%.

Molecular Docking Simulation of α-Glucosidase Inhibition

In silico molecular docking was used to investigate the potential interaction of isolated compounds (1, 3–6, and 9–11) with the α-glucosidase enzyme (PDB ID: 2QMJ) (Figures 8, Figures S30–S33, and Table 3) applying previously published methodologies.39 According to the best docking conformations, 6,8-di-C-prenylpratensein (9) (−9.25 kcal/mol) has the lowest free binding energy when compared to acarbose (−8.98 kcal/mol) (Table 3). Therefore, the results showed that the 6,8-di-C-prenylpratensein (9) binds to the α-glucosidase enzyme more readily than the positive control (acarbose). In the case of hydrophilic interactions, it was observed that the hydroxy group (C-11) of 6a,12a-dehydro-α-toxicarol (1) and (+)-α-toxicarol (5) showed hydrogen bonding with the carbonyl oxygen of Asp474 (2.0) and Asp542 (3.9) in the active site residue, respectively, whereas the hydroxy group (C-12a) of (+)-(6aR,12aR)-millettiapachycarpin (3) showed a single hydrogen bond with the carbonyl oxygen of Met444 (2.7). In addition, the methoxy group (C-2) of 6a,12a-dehydro-α-toxicarol (1) displayed strong hydrogen bonding with the carbonyl oxygen of Asn449 (2.0) in the active site, whereas the methoxy group (C-3) of 6a,12a-dehydro-α-toxicarol (1), the scalemic mixture of 12a-hydroxy-α-toxicarol (4), and (+)-α-toxicarol (5) showed hydrogen bonding with the carbonyl oxygen of Asp203 (2.1), Asp474 (2.8), and Arg202 (2.7), respectively. Moreover, the methylene protons at C-6 of the scalemic mixture of 12a-hydroxy-α-toxicarol (4) and (+)-α-toxicarol (5) showed hydrogen bonding with Asp203 (2.9) and Asp203 (3.2), respectively. The remaining hydrogen bonding interactions between the hydrogen atom at C-1 and CH3-4′ of the scalemic mixture of 12a-hydroxy-α-toxicarol (4) and (+)-α-toxicarol (5) were with Thr205 (1.9) and Tyr299 (3.3), respectively (Figures S30 and S31). 6a,12a-Dehydro-α-toxicarol (1), (+)-(6aR,12aR)-millettiapachycarpin (3), the scalemic mixture of 12a-hydroxy-α-toxicarol (4), (+)-α-toxicarol (5), 6,8-di-C-prenylpratensein (9), 6,8-diprenylgenistein (10), and isolupalbigenin (11) did not show the Zn chelation upon docking to the α-glucosidase enzyme except for (−)-sumatrol (6) with a bond length of 3.1 Å (Figure S31E). This could explain why (−)-sumatrol (6) is less inhibitory than other active compounds. Undoubtedly, the docking score (−7.75 kcal/mol) and the lack of significant interactions between the binding site and ligand afford weak binding (Figure S31E). In the case of isoflavonoids, the hydroxy groups on the ring A and B at C-5, C-7, C-3′, and C-4′ are the important functional groups for inhibiting α-glucosidase enzymes. The hydroxy groups on the ring A at C-5 of 6,8-diprenylgenistein (10) and isolupalbigenin (11) interacted with the Asp203 (2.2) and Thr205 (2.0) active site residues, whereas 6,8-di-C-prenylpratensein (9) and isolupalbigenin (11) showed hydrogen bonding with the same active site residue (Asp203 (1.7) and Asp203 (2.8)) at C-7.

Figure 8.

Figure 8

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2D binding mode of the active isolated (A) 6a,12a-dehydro-α-toxicarol (1), (B) (+)-(6aR,12aR)-pachycarotenoid (3), (C) (±)-12a-hydroxy-α-toxicarol (4), (D) (+)-α-toxicarol (5), (E) (−)-sumatrol (6), (F) 6,8-di-C-prenylpratensein (9), (G) 6,8-diprenylgenistein (10), and (H) isolupalbigenin (11) in the binding pocket of the α-glucosidase enzyme (light green, van der Waals; Green, conventional hydrogen bond; purple, carbon hydrogen bond; and pink, π-alkyl interactions.

Table 3. In Silico α-Glucosidase Inhibitory Activities of Active Compounds 1, 3–6, 9–11, and Acarbose.

compds binding affinity ΔG (kcal/mol) hydrophilic interactions (hydrogen bonding) hydrophobic interactions
1 –7.77 Asp203, Asp474, Ser448, Asn449 Phe575, Tyr299, Trp406, Met444, Asp203, Asp474
3 –8.64 Met444 Trp406, Gln603, Tyr299, Gly602, Phe575, His600, Trp441, Asp327
4 –8.35 Asp203, Asp474, Thr205 Phe575, Tyr299, Trp406, Met444
5 –8.21 Asp203, Asp542, Arg202, Tyr299 Phe575, His600, Try299, Ile328, Trp441, Met444, Trp406
6 –7.75 non-interaction Asp203, Arg526, Phe575, His600, Tyr299, Ile364, Trp441, Met444
9 –9.25 Asp203, Gln603 Tyr299, Trp406, Lys480
10 –8.80 Asp203, Phe450 Trp406, Asp542, Phe575, Tyr299
11 –7.92 Asp203, Asp474, Thr205 Leu473, Lys480, Phe450
acarbose –8.98 Asp203, Asp542, Gln603 Phe450
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The hydroxy groups on the ring B at C-3′ of 6,8-di-C-prenylpratensein (9) and C-4′ of 6,8-diprenylgenistein (10) and isolupalbigenin (11) showed hydrogen bonding with Gln603 (2.6), Phe450 (2.0), and Asp474 (2.1), respectively. Two hydrogen bonding interactions between the hydroxy and carbonyl groups at C-5 and C-4 of 6,8-diprenylgenistein (10) are formed with Asp203, whereas isolupalbigenin (11) showed three hydrogen bonding interactions with Thr205 (Figure 8G, H and Figure S32B, C). These data further confirm that the α-glucosidase inhibitory activity of 6,8-diprenylgenistein (10) and isolupalbigenin (11) is likely to be greater than other active compounds. Alkyl interactions of 6,8-di-C-prenylpratensein (9), 6,8-diprenylgenistein (10), and isolupalbi genin (11) also form. For example, the 4′- and 4′′-methyls of isoprenyl units from 6,8-diprenylgenistein (10) engage in π-alkyl interactions with Phe575 and Tyr299, whereas isolupalbigenin (11) was also formed when the same interactions occur with Phe450, Lys480, and Leu473. Similarly, the 4′-methyl of the isoprenyl unit in 6,8-di-C-prenylpratensein (9) forms π-alkyl interactions, this time with Lys480 (Figure 8F). These results show that compounds 9–11, an isoflavonoid with two linear isoprenyl groups, could be introduced into the active site of the α-glucosidase enzyme and efficiently attach to the catalytic amino acid residues via alkyl interactions, demonstrating the relevance of linear isoprenyl groups. These findings suggest that the active rotenoids and isoflavonoids might fill the active site of α-glucosidase and securely bind to the critical residues via a variety of interactions, resulting in the inhibitory activity for α-glucosidase.

Cytotoxicity against Lung Cancer (A549), Colorectal Cancer (SW480), Leukemia (K562), and Mammalian Cells (RAW264.7)

All isolated compounds, except compounds 2 and 14, were evaluated for their cytotoxicities against A549, SW480, K562, and, RAW 264.7 cells (Table S1). Of these, compounds 3, 9, 11, 12, 15, and 16 were cytotoxic against all cell lines with cell viability in the range of 4.53–48.19%. Compound 4 displayed cytotoxicity against all cell lines except against A549 cells with the cell viability in the range of 7.52 to 40.99%, whereas all remaining compounds (8, 10, and 13) were cytotoxic against RAW 264.7 and SW480 cells with the cell viability ranging from 5.66 to 77.04%. The cytotoxicities of compounds 3, 4, 8-13, 15-17 were further evaluated using the MTT assay at concentrations of 6.25, 12.5, 25, 50, and 100 μg/mL (Table 4). Euchrenone b10 (15), showed the strongest cytotoxicity against A549, SW480, K562 cells, and RAW 264.7 cells with IC50 values of 40.3, 39.1, 15.1, and 31.4 μM, respectively. (+)-(6aR,12aR)-Millettiapachycarpin (3) exhibited the highest cytotoxicity against A549, SW480, and K562 cells, and RAW 264.7 cells with IC50 values of 81.0, 60.8, 25.8, and 45.3 μM, respectively, whereas the scalemic mixture of 12a-hydroxy-α-toxicarol (4) displayed the weakest cytotoxicity with IC50 values of >100 μM.

Table 4. Cytotoxicities of Isolated Compounds 3, 4, 8–13, 15, 16, and Doxorubicina.

    cell viability (%)
compds concentration RAW 264.7 A549 (lung cancer) SW480 (Colorectal cancer) K562 (leukemia)
3 6.25 109.34 ± 4.62 116.95 ± 2.56 124.45 ± 3.87 67.77 ± 0.73
12.5 95.70 ± 7.74 108.28 ± 4.38 158.77 ± 8.13 34.90 ± 1.26
25 27.04 ± 3.25 99.22 ± 3.45 83.31 ± 2.29 7.64 ± 0.10
50 6.52 ± 0.71 11.44 ± 1.39 7.72 ± 0.50 7.19 ± 0.15
100 5.53 ± 0.07 4.85 ± 0.08 6.03 ± 0.07 7.09 ± 0.24
IC50 (μM) 45.3 81.0 60.8 25.8
4 6.25 78.44 ± 9.98 not detected 85.51 ± 3.64 96.87 ± 2.53
12.5 88.71 ± 8.08 79.22 ± 0.58 92.94 ± 2.37
25 74.52 ± 5.30 73.57 ± 1.17 91.81 ± 3.96
50 65.31 ± 1.16 61.55 ± 1.26 46.90 ± 1.70
100 7.52 ± 0.41 26.87 ± 2.57 40.99 ± 0.33
IC50 (μM) 156.7 >100 154.7 108.9
8 6.25 113.06 ± 7.41 not detected not detected not detected
12.5 90.28 ± 1.33
25 34.46 ± 2.49
50 5.49 ± 0.46
100 5.66 ± 0.23
IC50 (μM) 43.3 >100 >100 >100
9 6.25 97.22 ± 5.23 114.31 ± 3.98 92.32 ± 1.58 88.04 ± 2.23
12.5 132.32 ± 11.09 113.05 ± 1.29 83.20 ± 3.12 55.01 ± 2.60
25 63.87 ± 1.32 113.71 ± 0.69 71.96 ± 2.03 19.30 ± 1.17
50 5.01 ± 0.10 27.09 ± 3.02 12.60 ± 0.50 13.18 ± 0.37
100 5.12 ± 0.20 9.21 ± 0.62 7.83 ± 0.22 11.63 ± 0.20
IC50 (μM) 57.6 104.9 72.7 29.4
10 6.25 not detected not detected 117.51 ± 0.31 not detected
12.5 118.82 ± 0.92
25 117.40 ± 3.08
50 94.68 ± 3.85
100 77.04 ± 3.95
IC50 (μM) >100 >100 >100 >100
11 6.25 67.79 ± 3.80 236.35 ± 5.44 168.48 ± 3.95 93.20 ± 1.55
12.5 65.97 ± 3.70 224.79 ± 7.80 159.39 ± 8.08 68.47 ± 1.42
25 50.56 ± 2.63 59.73 ± 4.32 30.09 ± 1.45 8.68 ± 0.11
50 3.81 ± 0.11 11.03 ± 0.08 7.64 ± 0.08 7.15 ± 0.09
100 3.96 ± 0.06 13.09 ± 0.53 7.67 ± 0.31 7.08 ± 0.14
IC50 (μM) 67.5 49.7 46.8 34.9
12 6.25 124.12 ± 5.80 107.04 ± 0.37 126.72 ± 6.25 98.45 ± 2.07
12.5 150.93 ± 4.12 117.29 ± 2.47 134.35 ± 6.64 95.82 ± 2.42
25 159.20 ± 1.73 107.51 ± 1.16 152.12 ± 0.87 35.58 ± 2.47
50 8.67 ± 0.50 55.78 ± 1.90 33.83 ± 3.08 9.22 ± 0.44
100 5.55 ± 0.23 5.09 ± 0.04 6.42 ± 0.22 8.19 ± 0.06
IC50 (μM) 110.1 128.9 116.2 53.7
13 6.25 102.52 ± 8.01 not detected 94.01 ± 4.18 not detected
12.5 100.08 ± 2.13 92.77 ± 1.57
25 61.85 ± 3.12 90.43 ± 1.91
50 47.31 ± 2.94 89.53 ± 3.29
100 29.42 ± 3.72 59.45 ± 1.30
IC50 (μM) 56.1 >100 >100 >100
15 6.25 138.44 ± 4.64 141.71 ± 4.57 143.44 ± 4.69 53.82 ± 0.06
12.5 107.31 ± 5.20 112.08 ± 7.52 139.87 ± 4.32 14.15 ± 0.22
25 5.34 ± 0.18 9.75 ± 0.60 9.03 ± 0.44 13.34 ± 0.10
50 5.62 ± 0.07 5.57 ± 0.01 8.07 ± 0.32 11.57 ± 0.24
100 7.78 ± 0.55 7.44 ± 0.33 8.78 ± 0.48 10.60 ± 0.06
IC50 (μM) 31.4 40.3 39.1 15.1
16 6.25 154.04 ± 1.59 126.40 ± 1.36 165.98 ± 6.12 76.60 ± 3.38
12.5 138.95 ± 6.94 115.72 ± 2.68 142.31 ± 1.71 28.75 ± 0.39
25 58.72 ± 2.23 97.01 ± 9.76 122.25 ± 6.64 11.54 ± 0.06
50 6.00 ± 0.65 6.25 ± 0.33 6.60 ± 0.14 10.18 ± 0.15
100 5.30 ± 0.01 4.53 ± 0.07 6.09 ± 0.11 9.70 ± 0.15
IC50 (μM) 51.5 70.9 69.8 21.5
doxorubicin 6.25 4.62 ± 0.17 79.61 ± 1.89 79.99 ± 2.48 74.4 3 ± 1.49
12.5 5.35 ± 0.27 74.02 ± 0.75 73.89 ± 1.19 70.29 ± 0.89
25 4.59 ± 0.01 57.66 ± 1.71 60.17 ± 1.51 59.45 ± 1.80
50 4.44 ± 0.08 52.84 ± 1.51 17.83 ± 1.49 49.64 ± 2.20
100 5.96 ± 0.42 10.11 ± 3.11 16.79 ± 1.29 32.37 ± 0.89
IC50 (μM) <11.5 133.6 53.4 86.8
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a

Data expressed as mean ± SD.

Conclusions

The phytochemical investigation of the root and leaf extracts of M. pachycarpa led to the identification of six rotenoids and ten isoflavonoids. Of these, two rotenoids, (+)-(6aR,12aR)-millettiapachycarpin (3) and (−)-(6aS,12aS)-12a-hydroxy-α-toxicarol (4), were isolated as new compounds. In addition, the absolute configurations of (−)-(6R)-6-hydroxy-6a,12a-dehydro-α-toxicarol (2) and (−)-(6aS,12aS,2″R)-sumatrol (6) were reported for the first time. 6a,12a-Dehydro-α-toxicarol (1) was a major compound found in the extract of the roots, whereas 6,8-diprenylgenistein (10) and isolupalbigenin (11) were major compounds found in the leaf extract. Isolupalbigenin (11) showed the greatest α-glucosidase inhibitory activity. Meanwhile, the scalemic mixture of 12a-hydroxy-α-toxicarol (4) showed moderate α-amylase inhibitory activity. In addition, the important role of the hydroxy, methoxy, and linear type of isoprenyl moieties for α-glucosidase inhibition was suggested by molecular docking analysis. Moreover, the cytotoxic activity evaluation demonstrated that euchrenone b10 (15) had the highest cytotoxicity against A549, SW480, and K562 cells, whereas isolupalbigenin (11) and senegalensin (16) showed moderate cytotoxicity against three cancer cell lines. The current discovery of bioactive components from this plant could be beneficial for future studies into the use of medicinal plants for the treatment of diabetes and cancer.

Experimental Section

General Experimental Procedures

A Buchi M-560 melting point apparatus was used to determine the melting point. A Varian Cary 5000 UV–vis–NIR spectrophotometer was used to record UV–vis spectra. A PerkinElmer FTS FT-IR spectrometer was used to record IR spectra as KBr discs. The specific rotations were measured with a JASCO P-2000 polarimeter. A JASCO J-1500 spectrometer was used to record electronic circular dichroism spectra. High-performance liquid chromatography (HPLC) analysis was performed on a Finnigan Surveyor LC pump plus, coupled to a Finnigan Surveyor PDA plus detector and equipped with a C-18 column. CHIRALCEL OD-H @ a 5 μm column was used for chiral HPLC. Flash column chromatography was performed with the BUCHI Pure C-815 Flash using the following column: FP SELECT Si 25g and SELECT C18 30 μm spherical. The NMR spectra were recorded on a 500 MHz Bruker FT-NMR Ultra Shield. The ESIHRTOFMS were carried out on a 1290 infinity II/6545B QTOF/MS mass spectrometer. Silica gel C60 (0–20 μm), silica gel G60 (60–200 μm), and Sephadex LH-20 (25–100 μm) were performed for column chromatography (CC). Precoated TLC plates of silica gel 60F254 were used for analytical purposes.

Plant Material

The roots and leaves of M. pachycarpa were collected in March 2018 from Doi Tung, Chiang Rai, Thailand (N: 20.3256°, E: 99.3332°). The plant was identified by Mr. Martin van de Bult (Doi Tung Development Project, Chiang Rai, Thailand), and a voucher specimen (MFU-NPR0207) was deposited at the Natural Products Research Laboratory, School of Science, Mae Fah Luang University.

Extraction and Isolation

Air-dried roots of M. pachycarpa (629.5 g) were macerated in EtOAc (3 × 10 L) at room temperature. The root extract (54.5 g) was subjected to column chromatography (CC) over silica gel, eluting with a gradient system of EtOAc in hexanes to afford eight fractions (MPR1–MPR8). Fraction MPR8 (1.8321 g) was purified by silica gel CC eluting with EtOAc–CH2Cl2 (1:20, v/v, 2000 mL) to yield compound 1 (104.4 mg) together with four fractions (MPR8A–MPR8D). Fraction MPR8C (32.6 mg) was further isolated by flash column chromatography eluting with EtOAc–CH2Cl2 (1:20, v/v, 500 mL) to give compound 2 (4.6 mg, tR 9.32 min) and a fraction MPR8C–1 (12.6 mg, tR 7.35 min). Compound 7 (2.1 mg) was obtained from fraction MPR8C–1 by preparative thin-layer chromatography (1:20, v/v, EtOAc-CH2Cl2). Fraction MPR2 (1.3116 g) was chromatographed by silica gel CC (1:5, v/v, EtOAc-hexanes, 1000 mL) to give five fractions (MPR2A–MPR2E). Fraction MPR2E (62.7 mg) was further subjected to C18 reverse-phase silica gel CC (9:10, v/v, MeOH–H2O, 500 mL) to provide two fractions (MPR2E–A and MPR2E–B). Subfraction MPR2E–A (56.7 mg) was purified by silica gel CC (1:50, v/v, EtOAc-CH2Cl2, 1000 mL) to yield compounds 4 (25.6 mg), 5 (4.8 mg), and 6 (3.5 mg). MPR7 (6.9 g) was separated by silica gel CC (1:5, v/v, EtOAc–hexanes, 1500 mL) to give eight fractions (MPR7A–MPR7H). MPR7H (832.6 mg) was further purified by silica gel CC (1:20, v/v, EtOAc-CH2Cl2, 1000 mL) to provide 13 subfractions (MPR7H-A to MPR7H-M). Subfractions MPR7H-G and MPR7H–H were combined (97.6 mg) and further purified by silica gel CC (1:20, v/v, MeOH–CH2Cl2, 1500 mL) to afford four subfractions (MPR7H–GH1 to MPR7H–GH4). Compound 3 (7.9 mg) was obtained from subfraction MPR7H–GH3 (35.7 mg) by silica gel CC (1:20, v/v, acetone-CH2Cl2, 500 mL).

Air-dried leaves of M. pachycarpa (856.7 g) were extracted with EtOAc (3 × 20 L) at room temperature. The leaf extract (45.7 g) was subjected to quick column chromatography (QCC, 11 × 27 cm) over silica gel (0–20 μm) eluting with a gradient of EtOAc-hexanes to afford nine fractions (MPL1–MPL9). Fraction MPL5 (892.7 mg) was further isolated by C18 reverse-phase silica gel CC (8:2, v/v, MeOH–H2O, 500 mL) to give five fractions (MPL5A–MPL5E). Fraction MPL5A (72.1 mg) was separated by Sephadex-LH20 CC (100% MeOH) to provide five fractions (MPL5AA–MPL5AE). Fraction MPL5AC (52.8 mg) was further purified by silica gel CC (1:20, v/v, EtOAc–CH2Cl2, 1000 mL) to give compounds 9 (6.2 mg), 10 (16.7 mg), 15 (7.2 mg), 16 (3.1 mg), and a fraction MPL5ACD (12.7 mg). Compounds 13 (1.3 mg, tR 25.8 min) and 14 (1.5 mg, tR 29.0 min) were obtained from fraction MPL5ACD (12.7 mg) by semipreparative HPLC eluted with ACN–H2O (2 mL/min, 7:3, v/v). Fraction MPL6 (2.9231 g) was purified by C18 reverse-phase silica gel CC (7:3, v/v, MeOH–H2O, 500 mL) to afford three fractions (MPL6A–MPL6C). Fraction MPL6A (1.6213 g) was separated by Sephadex-LH20 CC (100% MeOH) to provide two fractions (MPL6A1 and MPL6A2). The fraction MPL6A1 (62.7 mg) was further purified by silica gel CC (2:8, v/v, EtOAc–hexanes, 1000 mL) yielding compounds 11 (13.9 mg), 8 (5.7 mg), and a fraction MPL6A1C (35 mg). Compound 12 (3.1 mg, tR 27.5 min) was obtained from fraction MPL6A1C (35 mg) by semipreparative HPLC and eluted with ACN–H2O (2 mL/min, 7:3, v/v).

(+)-(6aR,12aR)-Millettiapachycarpin (3)

A yellow powder; mp 163–165 °C; [α]D25 +57 (c 1, MeOH); UV (MeOH) λmax (log ε) 214 (2.70), 229 (2.58), 268 (2.73); ECD (c 1.05, MeOH) λmax (Δε) 235 (−2.37), 318 (−2.50), 367 (+2.56) nm; IR (neat) νmax 3430, 2933, 1636, 1589, 1507, 1455, 1338, 1272, 1162, 1116, 1023, 758, 541 cm–1; 1H NMR (500 MHz, CDCl3) δH 11.59 (1H, s, 11-OH), 6.78 (1H, s, H-1), 6.48 (1H, d, J = 10.3 Hz, H-1′), 6.43 (1H, s, H-4), 5.94 (1H, s, H-10), 5.44 (1H, d, J = 10.3 Hz, H-2′), 4.56 (1H, dd, J = 12.1, 2.4 Hz, H-6), 4.51 (1H, dd, J = 2.4, 1.1 Hz, H-6a), 4.46 (1H, dd, J = 12.1, 1.1 Hz, H-6), 3.79 (3H, s, OMe-3), 1.41 (3H, s, CH3-4′), and 1.33 (3H, s, CH3-5′); 13C NMR (125 MHz, CDCl3) δC 194.8 (C-12), 164.0 (C-11), 163.4 (C-9), 155.5 (C-7a), 148.7 (C-3), 147.6 (C-4a), 140.2 (C-2), 126.6 (C-2′), 115.2 (C-1′), 112.2 (C-1), 109.5 (C-1a), 102.0 (C-8), 100.5 (C-4), 100.0 (C-11a), 98.0 (C-10), 78.6 (C-3′), 75.6 (C-6a), 66.8 (C-12a), 63.7 (C-6), 56.0 (MeO-3), 28.7 (C-4′), and 28.4 (C-5′); (+)-ESIHRTOFMS m/z 413.1230 [M + H]+ (calcd for C22H21O8, 413.1231).

12a-Hydroxy-α-toxicarol (4)

A light-yellow powder; mp 134–135 °C; [α]D25 0 (c 1, MeOH); UV (MeOH) λmax (log ε) 207 (3.09), 273 (3.25); IR (neat) νmax 3456, 2923, 1636, 1586, 1509, 1456, 1271, 1162, 1119, 1043, 756 cm–1.

Resolution of the scalemic mixture of compound 4 (5.7 mg) was performed by semipreparative HPLC on a chiral column (CHIRALCEL OD-H @ 5 μm (4.6 mm φ × 250 mmL)), isocratic elution with n-hexane/isopropanol (1:9, v/v), a flow rate of 1.0 mL/min, and detection at UV 254 nm (Figure 5).

(+)-(6aR,12aR)-12a-Hydroxy-α-toxicarol (4)

tR 14.629 min; 0.7 mg; [α]D25 +22 (c 0.2, MeOH); ECD (c 0.17, MeOH) λmax (Δε) 213 (+2.32), 236 (+2.37), 272 (+2.43), 316 (−2.49) nm.

(−)-(6aS,12aS)-12a-Hydroxy-α-toxicarol (4)

tR 17.164 min; 1.2 mg; [α]D25 −25 (c 0.15, MeOH); ECD (c 0.3, MeOH) λmax (Δε) 213 (−2.32), 236 (−2.37), 272 (−2.43), 316 (+2.49) nm; 1H NMR (500 MHz, acetone-d6) δH 12.05 (1H, s, 11-OH), 6.82 (1H, s, H-1), 6.52 (1H, d, J = 10.2 Hz, H-1′), 6.49 (1H, s, H-4), 5.88 (1H, m, H-10), 5.88 (1H, m, 12a–OH), 5.62 (1H, d, J = 10.2 Hz, H-2’), 4.64 (1H, dd, J = 12.2, 2.5 Hz, H-6), 4.72 (1H, dd, J = 2.5, 1.2 Hz, H-6a), 4.48 (1H, dd, J = 12.2, 1.2 Hz, H-6), 3.77 (3H, s, OMe-2), 3.64 (3H, s, OMe-3), 1.42 (3H, s, CH3-4′), and 1.34 (3H, s, CH3-5′); 13C NMR (125 MHz, acetone-d6) δC 197.4 (C-12), 165.0 (C-11), 163.5 (C-9), 156.5 (C-7a), 152.6 (C-3), 149.6 (C-4a), 144.7 (C-2), 127.7 (C-2′), 115.6 (C-1′), 112.2 (C-1), 109.4 (C-1a), 102.4 (C-8), 101.9 (C-4), 101.1 (C-11a), 98.0 (C-10), 79.2 (C-3′), 76.9 (C-6a), 67.9 (C-12a), 64.4 (C-6), 56.7 (MeO-3), 55.9 (MeO-2), 28.7 (C-4′), and 28.4 (C-5′); (+)-ESIHRTOFMS m/z 427.1384 [M + H]+ (calcd for C23H23O8, 427.1387).

Single-Crystal X-ray Diffraction Analysis of (−)-(6aS,12aS,2″R)-sumatrol (6)

A single colorless irregular-shaped crystal of (−)-(6aS,12aS,2″R)-sumatrol (6) (0.66 × 0.15 × 0.11 mm3) was mounted on a mylar loop in oil on a Bruker APEX-II CCD diffractometer. The structure was solved with SHELXT40,41 by the Intrinsic Phasing solution method and by using Olex2(42) as a graphical interface. The model was refined with version 2017/1 of SHELXL40,41 using Least Squares minimization.

X-ray Crystallographic Data for (−)-(6aS,12aS,2″R)-sumatrol (6)

C23H22O7, Mr = 410.40, triclinic, P1 (No. 1), a = 4.5145(4) Å, b = 7.5184(6) Å, c = 14.5090(13) Å, α = 81.136(4)°, β = 86.813(4)°, γ = 83.240(4)°, V = 482.85(7), T = 189.60 K, Z = 1, Z′ = 1, μ(CuKα) = 0.872, 20 858 reflections measured, 3068 unique (Rint = 0.0400) which were used in all calculations. The final wR2 was 0.0857 (all data) and R1 was 0.0318 {I ≥ 2σ(I)}. The Flack parameter was refined to 0.04(8). The crystallographic data of (−)-6 have been deposited in the Cambridge Crystallographic Data Centre as CCDC 2155505.

α-Glucosidase Inhibitory Activity

The previously reported approach was used to perform a colorimetric α-glucosidase assay.14 Briefly, the tested samples (50 μL) were combined with 100 μL of the α-glucosidase enzyme solution (0.35 U/mL) and preincubated at 37 °C for 10 min. The substrate (100 μL, c 1.5 mM), p-nitrophenyl α-d-glucoside, was added and incubated at 37 °C for 20 min. Then, 1 mL of 1 M Na2CO3 was added. The absorption of the mixture was measured at 405 nm. Acarbose was used as a positive control (77.2 ± 0.4 μM).

α-Amylase Inhibitory Activity

The α-amylase inhibitory activity was carried out by the same procedure as in the previous report.43 In brief, the mixture of the sample (20 μL) and the starch solution (40 μL) was incubated at 37 °C for 20 min. Amylase solution (20 μL, 50 μg/mL) was added and incubated for 15 min. Then, 1 mL of HCl (0.1 M) and 100 μL of iodine solution (1 mM) were added. The absorbance of the mixture was measured at 650 nm. Acarbose was used as a positive control (103.4 ± 0.9 μM).

Cytotoxicity Assay in Mammalian Cells (RAW264.7 Cells)

This assay was performed as previously described.44 Cell viability was measured by an MTT assay. RAW 264.7 cells were seeded at 4 × 104 cells/well in 96-well plates and incubated at 37 ◦C and 5% CO2 overnight. Cells were treated with different concentrations of samples for 24 h. Then, cells were washed with PBS and incubated with 0.5 mM MTT reagent for 4 h. The detection of formazan was measured at 570 nm. The data were calculated as IC50 values with GraphPad Prism 6.0 software.

Cytotoxicity Assay against Lung Cancer (A549), Colorectal Cancer (SW480), and Leukemia (K562 cells)

These assays were performed as previously described.44 The detection of formazan was measured at 570 nm. The standard control was doxorubicin, and the IC50 values were summarized in Table 4.

In Silico Molecular Docking Studies

The crystal structure of α-glucosidase [PDB entry code: 2QMJ] was obtained from the Protein Data Bank (http://www.rcsb.org/pdb). The molecular modeling program Gaussview was used for ligand set up to build the ligands. The ligands were optimized at the AM1 level by using Gaussian 03W. Molecular docking studies were performed using AutoDockTools 1.5.4 (ADT), AutoDock 4.2, and the Lamarckian genetic algorithm (LGA). A grid box size of 60 × 60 × 60 points with a spacing of 0.375 Å between the grid points was implemented and covered almost the entire α-glucosidase protein surface.39

Computational Method

The Gaussian09 program package45 was used for ECD calculations of compounds (−)-2, (+)(6aR,12aR)-4, and (−)-(6aS,12aS)-4. ECD spectra were simulated using a Gaussian band shape with a bandwidth of 0.25 eV. SpecDis 1.64 was used to create the ECD curves (University of Wurzburg, Wurzburg, Germany).

Acknowledgments

This work was supported by the Thailand Science Research and Innovation Fund (DBG6280007). The Postdoctoral Fellowship from Mae Fah Luang University to Dr. Virayu Suthiphasilp was also acknowledged. We thank Mr. Martin van de Bult (Doi Tung Development Project, Chiang Rai, Thailand) for plant collection and identification. The University of British Columbia, Canada is also acknowledged for its laboratory facilities.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.2c02163.

  • 1D and 2D NMR, HRESITOFMS, IR spectra of compounds 3, 4, and 2D NMR spectra of compound 6, chiral HPLC-UV chromatogram of compounds 3–6, hydrophilic interaction with α-glucosidase enzyme of active compounds 1, 3–6, and 9–11, and Table S1: cell viability of isolated compounds 1, 3–13, 15, and 16 from M. pachycarpa leaves and roots. (PDF)

Accession Codes

CCDC 2155505 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_request@ccdc.cam.ac.uk or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: + 44 1223 336033.

This research was supported by a Direct Basic Research Grant (DBG6280007) and a Postdoctoral Fellowship from Mae Fah Luang University to Dr. Virayu Suthiphasilp.

The authors declare no competing financial interest.

Supplementary Material

ao2c02163_si_002.pdf (4.3MB, pdf)

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