Cytotoxic and antimicrobial labdane and clerodane diterpenoids from Kaempferia elegans and Kaempferia pulchra

Phytochemistry Letters 24 (2018) 140–144

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Cytotoxic and antimicrobial labdane and clerodane diterpenoids from Kaempferia elegans and Kaempferia pulchra

T

Pornsuda Chawengruma, Jutatip Boonsombatb, Prasat Kittakoopa,b,c, Chulabhorn Mahidola,b, ⁎ Somsak Ruchirawata,b,c, Sanit Thongnestb, a

Chulabhorn Graduate Institute, Chemical Biology Program, Chulabhorn Royal Academy, Kamphaeng Phet 6 Road, Bangkok 10210, Thailand Chulabhorn Research Institute, Kamphaeng Phet 6 Road, Bangkok 10210, Thailand c Center of Excellence on Environmental Health and Toxicology, Ministry of Education, Bangkok, Thailand b

A R T I C L E I N F O

A B S T R A C T

Keywords: Kaempferia elegans Kaempferia pulchra Labdane Clerodane Cytotoxicity Antimicrobial activity

Three previously undescribed diterpenoids, propadanes A-C (1-3), together with seventeen known compounds (4-20), were isolated from the rhizomes of Kaempferia elegans and Kaempferia pulchra. The structures of the isolated compounds were elucidated through extensive analysis of spectroscopic data. The absolute configuration (12R) of propadane A (1) was established by CD analysis. (−)-Kolavelool (15) and (−)-2β-hydroxykolavelool (18) showed selective cytotoxic activity against the HL-60 cell line with IC50 values of 8.97 ± 0.66 and 9.58 ± 0.88 μg/mL, respectively. Anticopalic acid (5), anticopalol (10), and 8(17)-labden-15-ol (11) showed antimicrobial activity against the Gram positive bacterium, Bacillus cereus, with MIC values of 3.13, 6.25, and 6.25 μg/mL, respectively. Labdane-type diterpenoids were found to be common constituents in K. elegans, while clerodane-type diterpenoids were common in K. pulchra. This information could be used to establish their chemotaxonomy.

1. Introduction The small forest herbs of ginger family belonging to the Zingiberaceae family are important sources of diterpenoids (Thongnest et al., 2005; Win et al., 2016; Boonsombat et al., 2017). Two hundred fifty species of the Zingiberaceae representing twenty-five genera, are endemic to Thailand (Sirirugsa, 1992; Larsen, 1996). In our previous studies, we isolated ten pimarane-type diterpenoids from Kaempferia marginata (Thongnest et al., 2005) and twenty diterpenoids including abietanes, labdanes, and pimaranes from K. roscoeana (Boonsombat et al., 2017). In an ongoing search for bioactive natural compounds from the Kaempferia genus, we have chemically investigated rhizomes of K. elegans and K. pulchra that were collected from Kanchanaburi Province, Thailand. Both species are found in mixed deciduous forest and on limestone boulders. It should be noted that, in the past, botanists regarded K. pulchra as a synonym for K. elegans (Smitinand, 1980; Searle, 1999); however, the differences are evident in morphological comparisons between the leaves, flowers, and anther crests of these two species (Sirirugsa, 1992). Moreover, molecular phylogenetic analyses indicate that these two plants are clearly different species (Techaprasan et al., 2010). So far little is known concerning the chemical constituents of these plants. We therefore performed the isolation and

⁎

characterization of bioactive compounds in K. elegans and K. pulchra. Examination of TLC and NMR profiles of the CH2Cl2-MeOH (1:1) extracts of these two plants revealed the presence of a number of interesting diterpenoids. Consequently, a comprehensive investigation of the chemical constituents was performed. Two new labdanes (1 and 2) and nine previously identified labdanes consisting of anticopalic acid (5) (Zinkel et al., 1971), (+)-15,16-epoxy-8(17),13(16),14-labdatriene (6) (Villamizar et al., 2003), (+)-pumiloxide (7) (Cambie et al., 1990; Vila et al., 2002), methyl anticopalate (8) (Zinkel et al., 1971), 13-oxo14,15-bis-nor-labd-8(17)-ene (9) (Do Khac Manh et al., 1975), anticopalol (10) (Yee and Coates, 1992), 8(17)-labden-15-ol (11) (Bruns, 1968; Maillo et al., 1987), labda-8(17),13(14)-diene-15,16-olide (12) (Nakano and Martín, 1982; Zdero et al., 1991), and (+)-labda8(17),13(Z)-diene-15,16-diol (13) (Villamizar et al., 2003) were isolated from K. elegans (Fig. 1). Additionally, one new clerodane (3), along with eight known compounds consisting of: cleroda-2,4(18),14trien-13-ol (4) (Wuttke et al., 2004), (−)-kolavelool (15) (Misra et al., 1979), dysoxydensin E (16) (Gu et al., 2014), 13-epi-roseostachenone (17) (Fazio et al., 1992), (−)-2β-hydroxykolavelool (18) (Bomm et al., 1999), (+)-13-epi-2α-hydroxykolavelool (13-epi-roseostachenol) (19) (Bomm et al., 1999), one labdane, calcaratarin A (14) (Kong et al., 2000), and the flavone, 2″,2″-dimethylpyrano-[5″,6″:8,7]-flavone (20)

Corresponding author. E-mail address: [email protected] (S. Thongnest).

https://doi.org/10.1016/j.phytol.2018.02.009 Received 25 October 2017; Received in revised form 22 January 2018; Accepted 9 February 2018 1874-3900/ © 2018 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.

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Fig. 1. Structures of compounds (1-4) from K. elegans and K. pulchra.

Table 1 1 H and 13C NMR Spectroscopic Data for Compounds 1-4 (CDCl3, δ in ppm and J in Hz)*. Position

1

2

3

4

δH

δC

δH

δC

δH

δC

δH

δC

1 2 3

1.52 m, 1.13 m 1.52 m, 1.49 m 1.40 m, 1.20 m

38.7 t 19.2 t 41.9 t

39.1 t 19.4 t 42.1 t

1.74 m, 1.52 m 1.95 m, 1.51 m 5.21 m

19.8 t 27.4 t 120.8 d

2.06 m 5.77 dt (9.7, 3.6) 6.02 d (9.7)

23.5 t 128.5 d 128.8 d

4 5 6

33.5 s 55.2 d 24.4 t

33.6 s 55.5 d 24.4 t

144.0 s 41.5 d 38.2 t

– – 1.86 m, 1.46 m

157.2 s 37.7 s 37.2 t

2.39 ddd (12.7, 4.1, 2.5) 2.03 dd (13.0, 5.3) – 1.99 d (10.9) – 1.50 m

38.4 t

– – 1.74 dt (13.0, 3.3) 1.18 td (12.6, 4.0) 2.10 m, 1.46 m

27.5 t

1.51–1.41 m

27.3 t

149.3 s 52.5 d 39.3 s 28.8 t

1.51 m – 1.42 m 1.67 m, 1.52 m

37.1 d 41.5 s 47.1 d 25.9 t

1.41 m – 1.43 m 1.34 m, 1.23 m

36.6 d 38.6 s 43.5 d 31.5 t

12 13 14 15

– 1.20 m 1.78 ddt (12.8, 5.1, 2.7) 1.33 td (13.0, 4.3) 2.43 ddd (12.9, 4.1, 2.4) 2.06 m – 2.25 d (12.5) – 1.92 dd (12.5, 1.6), 1.43 m 4.87 br d (11) – 5.77 br t (1.5) –

1.75 m, 1.07 m 1.57-1.50 m 1.39 dd (13.4, 4.1) 1.20 ddd (13.0, 4.1) – 1.17 dd (12.6, 2.7) 1.72 m, 1.33 m

73.9 d 37.5 d 35.1 t 60.5 t

34.8 t 73.4 s 145.1 d 111.9 t

2.10 br s 4.91 br s, 4.42 br s 0.81 s 0.88 s 0.68 s

1.34 m – 5.86 dd (17.4, 10.8) 5.20 dd (17.4, 1.1) 5.05 dd (10.8, 1.1) 1.26 s 0.79 d (6.6) 4.78 br s, 4.64 br s 0.99 s 0.81 s

35.0 t 73.3 s 145.2 d 111.7 t

16 17 18 19 20

1.40 m – 5.90 dd (17.4, 10.8) 5.23 dd (17.3, 1.1) 5.09 dd (10.8, 1.1) 1.30 s 0.85 d (6.5) 1.58 br s 0.99 s 4.18 d (11.7), 4.03 d (11.7)

7 8 9 10 11

38.1 t 148.7 s 52.4 d 39.4 s 27.9 t 83.5 d 169.3 s 116.5 d 173.0 s 13.8 q 106.0 t 21.6 q 33.5 q 14.5 q

3.48 ddd (9.9, 5.2, 1.6) 1.70 m 1.65 m 3.77 ddd (11.0, 6.7, 5.0) 3.65 ddd (11.3, 6.7, 5.1) 0.98 d (6.7) 4.82 d (1.4), 4.42 d (1.4) 0.88 s 0.81 s 0.68 s

16.4 q 106.3 t 21.7 q 33.6 q 14.6 q

CO AcO * 600 MHz in 1H and 150 MHz in

2.04 s 13

27.9 t 16.6 q 17.9 q 19.4 q 67.8 t

28.0 q 15.8 q 106.8 t 21.9 q 18.3 q

171.2 s 21.1 q

C.

19), 21.6 (C-18), 14.5 (C-20), and 13.8 (C-16), two double bonds of a lactone ring at δC 169.3 (C-13) and 116.5 (C-14) and an exo-methylene at δC 148.7 (C-8) and 106.0 (C-17). The 1H-1H COSY spectrum of 1 included four fragment consisting of H2-1/H2-2, H-5/H2-6/H2-7, H-9/ H2-11, and H2-11/H-12 (Fig. 2). The assignments of the α,β-unsaturated γ-lactone, and H3-16 were confirmed by the HMBC correlations from H-12 as well as H3-16 to C-13; H3-16 to C-12, C-13, and C-14; and H-14 to C-12 and the C-15 lactone carbonyl (Fig. 2). The HMBC correlations from H2-1 to C-2; H2-3 to C-2; both H3-18 and H3-19 to C-3 and C-5; H-5 to C-19 and C-20; H2-7 to C-5, C-6, C-9, and C-17; H2-11 to C-8; H2-17 to C-7 and C-9; and H3-20 to C-1, C-5, and C-9 established the presence of a labdane core structure in 1. Based upon these data, the gross structure of 1 was established. The configuration of 1 was addressed by analysis of NOESY and CD spectra. The NOESY correlation between H3-18 with H3-20 indicated that H3-18 and H3-20 were in the same plane. The NOESY correlations between H-5 and H-9 and H3-19 as well as the correlation between H-11 and H3-20 suggested that C-19 and C-20 were in α- and β- orientations, respectively. Moreover, the ring between A and B had trans-fused conformation. The configuration of C-12 was determined through analysis of CD spectrum, which showed a negative Cotton effect in the 200 to 235 nm region. This indicated a 12R configuration with to a defined definition of left-handed (M) helicity for the ReC(5)eC]C bond system (Gawronski et al., 1996). Therefore, the structure of compound 1 was unambiguously

(Magalhães et al., 1996), were isolated from K. pulchra (Fig. 1). Structures of the isolated compounds were elucidated by analysis of spectroscopic data and by data comparison with those reported in the literature (Supplementary material). This paper reports the isolation, structural elucidation, and cytotoxic and antimicrobial activities of the constituents of K. elegans and K. pulchra. 2. Results and discussion Compound 1 was named as propadane A, and it had the molecular formula of C20H30O2 as revealed by HRESIMS. The 1H NMR (Table 1) spectrum of propadane A (1) revealed signals of four methyl groups [δ 0.68 (H3-20), 0.81 (H3-18), 0.88 (H3-19), 2.10 (H3-16) ppm], one oxygenated methine [δ 4.87 (d, J = 11.0 Hz, H-12)], and an α,β-unsaturated γ-lactone with the diagnostic peaks at δ 4.87 (H-12) and 5.77 (H-14). The presence of an α,β-unsaturated γ-lactone in 1 was also supported by an IR vibration at 1762 cm−1. The 1H NMR signals at δH 4.91 and 4.42 ppm (H2-17, each with br s) were indicative of an exomethylene. Of the required six double bond equivalents, three accounted for the α,β-unsaturated carbonyl group and exo-double bond while the remaining three were satisfied by the bicyclic and lactone rings; suggesting that 1 was a labdane-type diterpenoid. The 13C NMR spectrum of 1 (Table 1) showed signals for 20 carbons, including: a lactone carbonyl at δC 173.0 (C-15), four methyl groups at δC 33.5 (C141

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[δH 5.21, (1H, m, H-3)], three vinylic protons [δH 5.90, (1H, dd, J = 17.4, 10.8 Hz, H-14), δH 5.23 (1H, dd, J = 17.3, 1.1 Hz, H2-15), and 5.09 (1H, dd, J = 10.8, 1.1 Hz, H2-15)] and two methylene protons [δH 4.18 (1H, d, J = 11.7, H2-20) and 4.03 (1H, d, J = 11.7, H2-20)]. In general, NMR data of 3 was similar to that of (−)-kolavelool (15) (Misra et al., 1979). Careful examination of the NMR data suggested that the C-20 methyl in (−)-kolavelool (15) was replaced by an acetoxymethyl group in 3. The HMBC correlations for H-8 and H-10 to C-20 (δC 67.8), H2-20 to C-8 (δC 37.1) and C-11 (δC 25.9) to the carbonyl carbon (δC 171.2) suggested that the acetoxy group in 3 was attached at C-20. Further analysis of the HMBC and 1H-1H COSY spectra (Fig. 2) showed that the proton and carbon resonances in 3 were assigned unambiguously (Table 1). The NOESY spectrum of 3 revealed correlations between H3-17, H2-20, and H3-19, suggesting that these protons were in the same plane, (α) face. The NOESY correlations between H-10 and H2-11 indicated these protons were co-planar. Compound 3 had similar structure to that of kolavelool (15) and like 15 ([α]27 D −25.7, [lit. −40.4]) displayed negative optical rotation ([α]26 D −48.7). Consequently, the configuration of 3 should be the same as that of 15 and was therefore identified as 20-acetoxy-13R-hydroxy-3,14-clerodadiene and named propadane C. Compound 4 was isolated as colorless viscous oil ([α]26 D +45.0) with the molecular formula of C20H32O (by HRESIMS). Of the five degrees of unsaturation of the molecule, correspond to double bonds inside the ring [δH 6.02 (d, J = 9.8 Hz), 5.78 (dt, J = 9.7, 3.6 Hz) and δC 128.5 d, 128.7 d], while the other two are indicative of an exomethylene group [δH 4.78 (br s), 4.64 (br s), and δC 157.2 s, 106.8 t] and a vinyl group [δH 5.86 (dd, J = 17.4, 10.8 Hz), 5.20 (dd, J = 17.4, 1.1 Hz), 5.05 (dd, J = 10.8, 1.1 Hz), and δC 145.2 d, 111.7 t]. Since compound 4 had five degrees of unsaturation with four methyl groups (Table 1), it was suggested to be a bicyclic diterpene. Analyses of the 1H and 13C NMR data indicated that the structure of 4 was similar to that of chelodane (Rudi and Kashman, 1992), except for the presence of a double bond conjugated to an exomethylene group in ring A in 4 (Fig. 2). The presence of a conjugated diene in ring A of 4 was confirmed by the HMBC correlations from H-2 (δH 5.77) to C4 (δC 157.2) and C-10 (δC 43.5); H-3 (δH 6.02) to C-1 (δC 23.5), C4 (δC 157.2) and C18 (δC 106.8); and H2-18 (δH 4.78 and δH 4.64) to C-3 (δC 128.8) and C5 (δC 37.7). The NOESY correlations between H-10 (δH 1.43, m) and H11 (δH 1.34, m) and between H3-17 (δH 0.79, d, J = 6.6 Hz)/H3-19 (δH 0.99, s) and H3-20 (δH 0.81, s) suggested that H-10 and H-11 were βoriented and H3-17, H3-19, and H3-20 were α-oriented. Consequently, the relative configurations of C-5, C-10, 17-Me, and 20-Me in 4 were the same as those in 3. The configuration of C-13 was still unclear; however, 4 had positive optical rotation, ([α]26 D +45.0), while 3 had negative optical rotation, ([α]26 D −48.7) indicating that the C-13 configuration in 4 is likely different from that in 3. Accordingly, compound 4 was identified as cleroda-2,4(18),14-trien-13-ol. A literature survey revealed that 4 had already been patented as an agent for lowering prolactin (Wuttke et al., 2004). However, there has been no report of 1H and 13C NMR data for 4. Assignments for the 1H and 13C NMR resonances of 4 are shown in Table 1. Due to the low yield for some isolated compounds, only compounds 3, 4, 15, and 16-19 were tested for their cytotoxic activity against cell lines for: lung cancer (A-549), acute promyelocytic leukaemia (HL-60), hepatocarcinoma (Hep-G2), human epithelial carcinoma (HeLa), cholangiocarcinoma (HuCCA-1), multidrug resistance small-cell lung cancer (H69AR), acute lymphoblastic leukaemia (MOLT-3), human liver cancer (S102), hormone-dependent breast cancer (T47-D), hormone-independent breast cancer (MDA-MB-231), and normal lung fibroblasts (MRC-5) (see Table S1). Among the compounds tested, compounds 15 and 18 exhibited cytotoxic activity against the HL-60 cell line with the IC50 values of 8.97 ± 0.66 and 9.58 ± 0.88 μg/mL, respectively, while the other compounds (3, 4, 16, 17, and 19) were inactive. Compounds 1-5, and 8-12 were evaluated for antimicrobial activity. Interestingly, compounds 5 and 10 showed antimicrobial

Fig. 2. Selected COSY, HMBC, and NOESY correlations of compounds 1-4.

identified as labda-8(17),13-dien-12R,15-olide. Compound 2 had the molecular formula of C20H36O2 as established by HRESIMS. The IR spectrum of 2 revealed the presence of hydroxyl (3358 cme1) and exo-methylene (885 cm–1) groups in 2. The 1H and 13C NMR spectra (Table 1) of 2 were similar to those of 1 but differed in their side-chains. The 1H and 13C NMR spectra of 2 revealed signals for exo-methylene [δH 4.82 (1H, d, J = 1.4 Hz) and 4.42 (1H, d, J = 1.4 Hz), δC 106.3 (C-17)], hydroxymethyl [δH 3.77 (1H, ddd, J = 11.0, 6.7, 5.0 Hz), 3.65 (1H, ddd, J = 11.3, 6.7, 5.1 Hz), δC 60.5 (C15)], and an oxygenated methine [δH 3.48 (1H, ddd, J = 9.9, 5.2, 1.6 Hz), δC 73.9 (C-12)]. Analyses of the 1H, 13C NMR, and DEPT spectra of 2 indicated the presence of four methyl signals, eight methylenes, four methines, and three quaternary carbons. Comparisons of the 1H and 13C NMR data of 2 with those of (11) [8(17)-labden-15-ol; ([α] 25 +14.4] and its enantiomer, ent-labdenol ([α] 25 −22) D D (Fukuyama et al., 1999) revealed that the C-12 methylene in 11 was replaced by an oxygenated methine in 2. The C-12 oxygenated methine in 2 was assigned by the HMBC correlations from H-9 (δH 1.99, d, J = 10.9 Hz) and H-11 (δH 1.50, m) to C-12 as well as H-12 to C-14 (δC 35.1) and C-16 (δC 16.4); and H3-16 (δH 0.98, d, J = 6.7 Hz) to C-12 and C-14 (Fig. 2). The NOESY spectrum of 2 showed correlations between H3-20/H3-18, H-5/H-9 and H-5/H3-19, which indicated the presence of a trans-fused ring junction for the A/B ring. While the C-12 configuration of 2 could not be determined due to an insufficient amount of the compound, compound 2 did show positive optical rotation ([α]26 D +15.7), which was similar to that seen for 8(17)-labden-15ol (11), ([α]25 D +14.4, [lit. +35]) (Maillo et al., 1987). Given that both compounds share the same biosynthetic origin (Peters, 2010), it was assumed that the stereochemistry of the core structure in 2 was the same as that in 11. Accordingly, the structure of 2 was assigned as 8(17)-labden-12,15-diol and named propadane B. Compound 3 was isolated as an amorphous white powder ([α]26 D −48.7) with a molecular formula of C22H36O3, that was established by HRESIMS. The IR spectrum of 3 revealed the presence of hydroxyl (3432 cm−1), carbonyl (1737 cm−1), and CeO bonds (1236 cm−1). The 13 C NMR and HSQC spectral data (Table 1) of 3 showed 22 carbon resonances consisting of a carbonyl group, four quaternary carbons, including one olefin, in addition to one oxygenated carbon, four methines, two of which are olefinic, seven methylenes and five methyl groups. The 1H spectrum of 3 (Table 1) showed signals for three methyl singlets [δH 0.99 (3H, s, H3-19), 1.30 (3H, s, H3-16), and 1.58 (3H, s, H3-18)], a methyl doublet [δH 0.85, (3H, d, J = 6.5 Hz, H3-17)], an acetoxyl group [δH 2.04, (3H, s, H3-22)], a trisubstituted double bond 142

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MeOH (99:1 to 0:100), to obtain twenty-nine fractions (F1-F29); one of the fractions contained compound 5 (1.9 g), [α]25 D = +44.2 (c 0.40, CHCl3). Fraction 6 (21 mg) was separated by silica gel CC eluting with hexane (100%) to yield seven subfractions (6.1-6.7), and two subfractions contained compounds 6 (3 mg), [α]25 D = +31.6 (c 0.30, CHCl3) and 7 (1.6 mg), [α]25 D = +9.5 (c 0.16, CHCl3). Fraction 11 (87.8 mg) was separated on silica gel CC and eluted with hexane-CH2Cl2 (95:5 to 0:100) to generate twelve subfractions (11.1–11.12). Combined subfractions (11.4-11.8) were further purified by preparative TLC and developed with a mixture of CH2Cl2-MeOH (9:1), followed by a mixture of hexane-EtOAc (9:1), to isolate compound 8 (4.2 mg), [α]25 D = +31.4 (c 0.43, CHCl3). Fraction 14 (55.4 mg) was subjected to a silica gel CC eluted with hexane-EtOAc (97:3) to yield fourteen subfractions (14.1–14.14) and compound 9 (1.4 mg), [α]25 D = +15.6 (c 0.15, CHCl3). Fraction 19 (180 mg) was separated by silica gel CC (hexaneEtOAc, 98:2) into twenty subfractions (19.1–19.20). Subfraction 19.20 (27 mg) was further separated by C18 reversed-phase CC (gradient elution with a mixture of MeOH-H2O, 80:20 to 97:3) resulting in the isolation of compounds 10 (10.2 mg), [α]25 D = +30.2 (c 0.95, CHCl3) and 11 (6.7 mg), [α]25 D = +14.4 (c 0.67, CHCl3). Fraction 20 (77.7 mg) was separated by C18 reversed-phase CC using MeOH-H2O (70:30 to 90:10) to obtain twelve subfractions (20.1–20.12). Subfraction 20.10 (6.6 mg) was subjected to preparative TLC developed with a mixture of hexane-EtOAc (98:2), followed by a mixture of (hexane-CH2Cl2acetone, 4.9:5.0:0.1) to isolate compounds 1 (2.4 mg) and 12 (3.3 mg), [α]25 D = +38.6 (c 0.22, CHCl3). Fractions 21 (1.1 g) was further separated using C18 reversed-phase CC using MeOH-H2O (80:20), which resulted in the isolation of compound 5 (765.9 mg). Fraction 25 (314 mg) was separated by C18 reversed-phase CC using MeOH-H2O (70:30 to 100) to obtain twenty subfractions (25.1–25.20) and compound 5 (80.6 mg). Subfraction 25.16 (14.9 mg) was further subjected to preparative TLC (hexane-EtOAc, 60:40) to yield compounds 2 (1.7 mg) and 13 (2.4 mg), [α]25 D = +15.3 (c 0.22, CHCl3).

activity against Gram-positive bacteria with respective MIC (MBC) values of 12.5 (18.75) and 12.5 (200) μg/mL for Staphylococcus epidermidis; 12.50 (25) and 6.25 (200) μg/mL for Enterococcus faecalis; and 3.13 (6.25) and 6.25 (6.25) μg/mL for Bacillus cereus. Compound 11 displayed selective activity toward B. cereus with an MIC (MBC) values of 6.25 (25) μg/mL. None of the tested compounds showed the activity against either Gram-negative bacterial or fungal strains. In conclusion, we have isolated three new diterpenoids (1-3) namely propadanes A-C, along with 17 known compounds (4-20) from K. elegans and K. pulchra. We found that the majority of the isolated compounds in K. elegans were labdane-type diterpenoids, while those from K. pulchra were clerodane-type diterpenoids, clearly indicating a difference in chemical constituents between the K. elegans and K. pulchra. The results revealed from this study could lend important information that could aid in the elucidation of the chemotaxonomy of K. elegans and K. pulchra. (−)-Kolavelool (15) and (−)-2β-hydroxykolavelool (18) showed cytotoxic activity against the HL-60 cell line, while anticopalic acid (5), anticopalol (10), and 8(17)-labden-15-ol (11) displayed antimicrobial activity against Gram-positive bacteria. 3. Experimental section 3.1. General experimental procedures Optical rotations were measured on a JASCO P-1020 polarimeter. IR spectra were recorded on a PerkinElmer Spectrum One Spectrometer using a universal attenuated reflectance (ATR) technique. CD spectra were recorded on a JASCO J-810 spectropolarimeter. NMR spectra were obtained using Bruker Avance 400 and 600 spectrometers. Chemical shifts are expressed in δ (ppm) and were referenced to the residual solvent signals. HRESIMS analyses were performed using a Bruker Daltonics MicroTOF spectrometer. All solvents were distilled from commercial grade solvents prior to use. Spectral grade solvents were used for spectroscopic measurements. RP-C18 silica gel (150–200 mesh, Merch) and silica gel 60 (Merck, 0.063–0.200 nm) were used for column chromatography (CC), while silica gel 60 (Merck, less than 0.063 nm) was used for flash CC. Silica gel precoated aluminum plates (F254, 0.25 mm) were used for TLC detection. Spots were visualized using UV light (254 and/or 366 nm) and Godin’s reagent.

3.3.2. Extraction and isolation of compounds from Kaempferia pulchra rhizomes The fresh ground rhizomes of K. pulchra (1.4 kg) were soaked in MeOH (3 L × 3) at room temperature and the MeOH extract was partitioned with CH2Cl2-H2O (200 mL × 6), yielding CH2Cl2 (7.75 g) and water-MeOH (13.1 g) extracts. The plant material was further soaked in with a CH2Cl2-MeOH mixture (1:1), which generated 5.1 g of an extract. The crude CH2Cl2 extract and CH2Cl2-MeOH extracts were combined because these extracts had similar chemical profiles, as revealed by TLC and 1H NMR analyses. The combined crude extract (12.9 g) was fractionated by silica gel CC eluted with a gradient elution of hexane-CH2Cl2 (100:0 to 0:100) and CH2Cl2-MeOH (99:1 to 1:1) to obtain ten fractions (F1-F10), as well as compounds 4 (266 mg) and 15 (965 mg), [α]D26 −25.7 (c 0.58, CHCl3). Fraction 8 (250 mg) was subjected to CC on C18 reversed-phase, using a gradient elution of MeOH-H2O (70:30 to 95:5), which yielded thirteen subfractions (6.1–6.13) and compound 16 (131 mg), [α]D26 = −39.6 (c 1.43, CHCl3). Subfraction 6.8 (10.6 mg) was further separated using two rounds of preparative TLC (hexane-EtOAc-CH2Cl2, 4:0.8:5.2), followed by hexane-EtOAc-CH2Cl2; 80:16:104), which resulted in the isolation of compounds 14 (1.8 mg), [α]D26 +7.8 (c 0.20, CHCl3), and 20 (1.7 mg), [α]D24 = +21.0 (c 0.17, CHCl3). Subfraction 6.9 (7.8 mg) was further separated by preparative TLC (hexane-EtOAcCH2Cl2; 4:0.8:5.2) to isolated compound 3 (3.2 mg). Fraction 9 (1.40 g) was separated by Sephadex LH20 CC, eluted with MeOH-CH2Cl2 (80:20), to give subfraction 9.3 (224 mg), which was further purified by silica gel CC using a gradient elution of MeOH-CH2Cl2 (0:100 to 0.1:99.9). This yielded compounds 4 (25 mg) and 17 (37 mg), [α]D26 −16.6 (c 0.56, CHCl3). Fraction 10 (1.35 g) was separated by Sephadex LH 20 CC eluted with MeOH-CH2Cl2 (80:20), followed by silica gel CC with a gradient elution of MeOH-CH2Cl2 (0.5:99.5 to 1:99). Eight subfractions (10.1-10.8) were generated as well as compounds 16

3.2. Plant material The plant materials (K. pulchra and K. elegans) were collected in the mixed deciduous forest and on limestone boulders in Sai Yok District, Kanchanaburi Province, Thailand, in November 2015, and were authenticated by Prof. Dr. Wongsatit Chuakul, Mahidol University. The voucher specimen numbers BKF 192347 (Thongnest No. 1) and BKF 192348 (Thongnest No. 2) were deposited at the Department of National Parks, Wildlife and Plant Conservation, Ministry of Natural Resources and Environment, Bangkok, Thailand. 3.3. Extraction and isolation 3.3.1. Extraction and isolation compounds from Kaempferia elegans rhizomes The freshly ground rhizomes of K. elegans (0.4 kg) were soaked in MeOH (2 L × 3) at room temperature and the MeOH extract was partitioned with CH2Cl2-H2O (200 mL × 6), yielding CH2Cl2 (6.94 g) and water-MeOH (8.29 g) extracts. The plant material was further soaked in a CH2Cl2-MeOH mixture (1:1) and yielded 5.07 g of an extract. The crude CH2Cl2 extract and CH2Cl2-MeOH extract were combined because these extracts shared the same chemical constituents when analyzed by TLC and 1H NMR techniques. The combined crude extracts of CH2Cl2 and CH2Cl2-MeOH (12.0 g) were separated by silica gel column chromatography (CC), eluted with a stepwise gradient of hexane-CH2Cl2 (100:0 to 0:100) and CH2Cl2143

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(129 mg) and 19 (173 mg), [α]26 D = +11.2 (c 1.16, CHCl3). Subfraction 10.2 (226 mg) was further purified by C18 reversed-phase CC using MeOH-H2O (80:20) as the eluent, which yielded four subfractions (10.2.1–10.2.4). Subfraction 10.2.3 (25 mg) was further separated to preparative TLC coated with C18 reversed-phase (100% MeOH) to isolated compound 18 (10 mg), [α]26 D = −26.3 (c 0.95, CHCl3).

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3.3.3. Propadane A (1) A colorless oil; [α]26 D −16.9 (c 0.07, CHCl3); IR (ATR) νmax: 2925, 2853, 1762, 1643, 1459, 1441, 1259, 1020, 959, and 797 cm−1; CD (1.2 × 10−4 μM, MeOH) λmax nm (Δe); 203 (+2.8394) and 216 (−25.6234) nm; 1H and 13C NMR data, see Table 1; HRESIMS m/z 325.2136 [M+Na]+ (calcd for C20H30NaO2, 325.2138, Δ = 0.5 ppm). 3.3.4. Propadane B (2) A yellow powder; [α]26 D +15.8 (c 0.16, CHCl3); IR (ATR) νmax: 3358, 2925, 2852, 1737, 1642, 1459, 1441, 1379, 1060, and 885 cm−1; CD (1.1 × 10−4 μM, MeOH) λmax nm (Δε);195 (+31.3687) and 204 (−13.6530) nm; 1H and 13C NMR data, see Table 1; HRESIMS m/z 331.2605 [M+Na]+ (calcd for C20H36NaO2, 331.2608, Δ = 0.7 ppm). 3.3.5. Propadane C (3) A white amorphous powder; [α]26 D −48.7 (c 0.23, CHCl3); IR (ATR) νmax: 3432, 2924, 2854, 1737, 1657, 1456, 1376, 1236, 1034, and 917 cm−1; CD (6.7 × 10−4 μM, MeOH) λmax nm (Δε); 217 (−32.3989), 233 (+18.9661), and 323 (−5.4963) nm; 1H and 13C NMR data, see Table 1; HRESIMS m/z 371.2555 [M + Na]+ (calcd for C22H36NaO3, 371.2557, Δ = 0.5 ppm). 3.3.6. Cleroda-2,4(18),14-trien-13-ol (4) A colorless sticky oil; [α]26 +45.0 (c 1.2, CDCl3); CD D (4.1 × 10−4 μM, MeOH) λmax nm (Δε);196 (−33.0184) and 234 (+7.7653) nm; 1H and 13C NMR data, see Table 1; HRESIMS m/z 311.2337 [M+Na]+ (calcd for C20H32NaO, 311.2345, Δ = 2.7 ppm). 3.4. Biological assays Antimicrobial activity and cytotoxic activities were evaluated according to previously reported procedures (Boonsombat et al., 2017; Thongnest et al., 2017). Acknowledgements This work was supported by the Center of Excellence on Environmental Health and Toxicology, Science & Technology Postgraduate Education Research Development Office (PERDO), Ministry of Education. Partially supported was also provided by Mahidol University. P. C. acknowledges a grant from His Majesty the King Honour Celebration Scholarships Project for the Scientist Development. The authors thank the CRI staff members, N. Reuk-Ngam, P. Intachote, S. Sengsai, and B. Saimanee for the cytotoxic and antimicrobial activity determinations. We also thank K. Trisuppakant, W. Thamniyom, S. Pisutcharoenpong, and N. Chimnoi, for the IR, NMR, and HRMS data. Prof. W. Chuakul from Mahidol University is gratefully acknowledged for the identification of the plants. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.phytol.2018.02.009.

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