Roscotanes and roscoranes: Oxygenated abietane and pimarane diterpenoids from Kaempferia roscoeana

Phytochemistry 143 (2017) 36e44

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Roscotanes and roscoranes: Oxygenated abietane and pimarane diterpenoids from Kaempferia roscoeana Jutatip Boonsombat a, Chulabhorn Mahidol a, b, Pornsuda Chawengrum b, Nanthawan Reuk-Ngam a, Nitirat Chimnoi a, Supanna Techasakul a, Somsak Ruchirawat a, b, c, Sanit Thongnest a, * a b c

Chulabhorn Research Institute, Kamphaeng Phet 6 Road, Bangkok 10210, Thailand Chulabhorn Graduate Institute, Chemical Biology Program, Kamphaeng Phet 6 Road, Bangkok 10210, Thailand Chulabhorn Graduate Institute, Center for Environmental Health and Toxicology (EHT), CHE, Ministry of Education, Thailand

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 May 2017 Received in revised form 17 June 2017 Accepted 21 July 2017

Eight previously undescribed ditepenoids, including four oxygenated abietanes (roscotanes A-D) and four oxygenated pimaranes (roscoranes A-D), along with twelve known diterpenoids were isolated from the whole plants of Kaempferia roscoeana. Their structures were elucidated by extensive spectroscopic analysis, and the structure of roscotane A was further confirmed by single crystal X-ray diffraction analysis. Most isolated compounds were evaluated for their antimicrobial and antimalarial activities. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Kaempferia roscoeana Zingiberaceae Roscotanes A-D Roscoranes A-D Abietane Pimarane Antimalarial activity Antimicrobial activity

1. Introduction Kaempferia L. (Zingiberaceae) contains about 60 species widely distributed in tropical countries including Laos, Myanmar, Cambodia, Indonesia, Malaysia, and China, of which 16 species are found in Thailand (Picheansoonthon and Koonterm, 2008; Sirirugsa, 1992). Quite a number of plants in this genus have been regarded as medicinal plants and they are used as Thai folk medicine against various diseases such as diabetes, cancer, herpes, malaria, wound infection, urticaria and allergy (Pengcharoen, 2002; Thiengsusuk et al., 2013; Tewtrakul and Subhadhirasakul, 2007). Recently, many researchers have indicated the high potential of edible plants in Southeast Asia for cancer chemoprevention and as antimicrobial agents. Zingiberaceae have been found to be one of the desirable sources of effective cancer-preventive agents (Kirana et al., 2003; Win et al., 2015a, 2015b). For antimicrobial activities,

* Corresponding author. E-mail address: [email protected] (S. Thongnest). http://dx.doi.org/10.1016/j.phytochem.2017.07.008 0031-9422/© 2017 Elsevier Ltd. All rights reserved.

reports on the screening of extracts or essential oils of members from Zingiberaceae family against bacterial strains, fungi, and yeast have been published (Chen et al., 2008a; Ficker et al., 2003; Habsah et al., 2000). However, most reports on antimicrobial properties are in Alpinia, Curcuma, and Zingiber species, but a very small number of reports are in Kaempferia species (Thongnest et al., 2005; Vipunngeun et al., 2007). Based on some previous studies on the genus Kaempferia, cyclohexane oxide derivatives, diterpenoids, and flavonoids are found, and more importantly, some of them possess cytotoxic and antimicrobial activities (Panchareon et al., 1996, 1989; Prawat et al., 1993; Tuchinda et al., 1994; Yenjai et al., 2004; Win et al., 2015b). Kaempferia roscoeana Wall, known as “Pro pa”, is used as a spice and food in Thai cuisine. So far, there have been no reports on the chemical constituents of this plant. Preliminary screening of dichloromethane-methanol extracts of the whole plants showed cytotoxic activity against the MOLT-3 cancer cell line (91% cytotoxicity at 30 mg/ml). Additionally, examination of TLC and NMR profiles revealed a number of interesting diterpenoids in the

J. Boonsombat et al. / Phytochemistry 143 (2017) 36e44

dichloromethane-methanol (1:1) extract, we then started a phytochemical investigation on this extract. Here, the structure elucidation of eight previously undescribed diterpenoids named roscotanes A-D (1e4) and roscoranes A-D (5e8) (Fig. 1) along with twelve known compounds (9e20) isolated from the whole plants of K. roscoeana is described. The structure of 1 was further confirmed by single crystal X-ray diffraction analysis. With the exception of compound 4, all diterpenoids were evaluated for antimicrobial activity. Compounds 1e3, 5e7, and 9e17 were also tested for their antimalarial activity. 2. Results and discussion The dichloromethane-methanol (1:1, v/v) extract from the whole plants of K. roscoeana was successively subjected to column chromatography over silica gel or Sephadex LH-20, and preparative HPLC to afford 8 previously undescribed diterpenoids named roscotanes A-D (1e4) and roscoranes A-D (5e8) together with 12 known diterpenoids (9e20), identified as (
)-isopimara-8(14),15 et al., 1997), ar-abietatriene (10) (Miguel del diene (9) (Touche Corral et al., 1994), 7-dehydroabietanone (11) (Su et al., 1994), iso et al., 1997), 1a-hydroxpimara-8(14),15-dien-7-one (12) (Touche yisopimara-8(14),15-diene (13) (Win et al., 2015a), isopimara-8,15dien-7-one (14) (Pinto et al., 1988), abieta-8,11,13-trien-7a-ol (15) (Conner et al., 1980), 7a-hydroxyisopimara-8(14),15-diene (16)  et al., 1997), (1S,5S,9S,10S,11R,13R)-1,11-dihydroxypimara(Touche 8(14),15-diene (17) (Thongnest et al., 2005), sandaracopimaradien1a,2a-diol (18) (Tuchinda et al., 1994), (1R,2S,5S,9S,10S,11R,13R)1,2,11-trihydroxypimara-8(14),15-diene (19) (Thongnest et al., 2005), and (12Z,14R)-labda-8(17),12-dien-14,15,16-triol (20) (Yin et al., 2013) (Scheme S1). Compound 1 was obtained as colorless crystals, and its molecular formula of C21H34O3 was determined by 13C NMR and HRESIMS implying five degrees of unsaturation. The IR spectrum showed absorption bands attributable to hydroxyl (3419 cm
1) and olefinic (1457 cm
1) groups. The 13C NMR (Table 3) and HSQC spectrum of 1 revealed 21 carbon signals corresponding to five methyls [dC 21.4, 21.3, 20.9, 32.9, and 56.1], seven methylenes, four methines including a hemiacetal group (dC 104.4, d) and one olefinic (dC 120.3, d), and five quaternary carbons together with one olefinic carbon (dC 147.7, s) and two oxygenated carbons (dC 79.3 and 75.2, both s). The above data suggested an abietane diterpenoid skeleton (Miguel del Corral et al., 1994). In the 1H NMR spectrum (Table 1), two tertiary methyls [dH 0.93 (H3-18) and 0.92 (H3-19)], two

37

secondary methyls (dH 1.03 and 1.02, both d, J ¼ 2.4 Hz, H3-16 and H3-17), a methoxy group (dH 3.40, br s), an isopropyl group (dH 2.25, m, H-15), an hemiacetal group (dH 5.03, s, H-20), and one olefinic proton (dH 5.23, br s, H-14) were observed. The HMBC correlations of OMe to C-20 (dC 104.4, d), and of H-20, to C-5 (dC 41.8, d), C-8 (dC 79.3, s), C-9 (dC 75.2, s), and C-10 (dC 50.7, s), indicated that one methoxyl was attached to the hemiacetal group which existed between C-10 and C-8. The presence of a trisubstituted D13(14) double bond in 1 was confirmed by cross-peaks between of H-14 to C-8, C-9, C-12, and C-15, and of H-15, H3-16 and H3-17 to C-13 (Scheme S2). Correlations in the NOESY spectrum between H-5 and H3-19 were observed, showing for C-19 and C-18 the ae and beorientation, respectively. Additionally, a correlation between H318 and H-20 suggested that H-20 was in the beorientation and the ring between A and B is trans-fused conformation (Scheme S2). Consequently, the structure of compound 1 was assigned as 8,20epoxy-20a-methoxy-9a-hydroxy-abieta-13-ene and named roscotane A. The structure of 1 was further confirmed by single crystal Xray diffraction (Fig. 2), and this compound represents the first example of a naturally occurring hemiacetal abietane in K. roscoeana. Compound 2 was isolated as an amorphous white powder with the molecular formula C20H32O2 as established by 13C NMR and HRESIMS. The 1H and 13C NMR spectroscopic data of 2 (Tables 1 and 3) closely resemble those of 1 except for the presence of a methylene group at (dH 4.27 and 3.56, both d, J ¼ 8.3 Hz) at C-20 in 2, instead of the methoxy group in 1. Thus, compound 2 was identified as 8,20-epoxy-9a-hydroxy-abieta-13-ene and named roscotane B. Compound 3 was obtained as pale yellow sticky oil. Its molecular formula was established as C20H32O2 determined by 13C NMR and HRESIMS. Detailed analysis of the1H and 13C NMR data of 3 (Tables 1 and 3) were similar to those of 11a-hydroxy-7,13abietadiene (Ashitani et al., 1999) except for the presence of an additional oxygenated carbon at dC 73.8 (C-9). The relative stereochemistry of 3 was obtained using coupling constant data and the NOESY spectrum. Correlations in the NOESY spectrum were observed between H3-20 and H-11/H3-18, as well as between H3-19 and H-5 indicating the a-orientation of both hydroxyl groups at C-9 and C-11 (Scheme S2). The connectivity of oxygen-bearing carbon atoms were confirmed by 2D NMR spectroscopic data (Scheme S2). Compound 3 was thus identified as 9a,10a-dihydroxy-abieta-7,13diene and named roscotane C. Compound 4 was obtained as colorless sticky oil. Its molecular formula C20H32O2 was established by HRESIMS, suggesting five

Fig. 1. Molecular structures of roscotanes A-D (1e4) and roscoranes A-D (5e8).

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J. Boonsombat et al. / Phytochemistry 143 (2017) 36e44

Table 1 1 H NMR Spectroscopic data (600 MHz, CDCl3) for Roscotanes A-D (1e4). positions

1 2 3

1

2a

1.95, ddd (13.1, 4.8, 3.0) 0.99, td (13.4, 4.5) 1.45, m

1.59, 1.32, 1.50, 1.28, 1.36, 1.15, 1.83, 1.63,

13 14 15 16 17 18 19 20

1.38, 1.19, 1.83, 1.64, 1.47, 1.73, 1.49, e 2.22, 1.51, 2.13, 1.98, e 5.23, 2.25, 1.03, 1.02, 0.93, 0.92, 5.03,

OMe

3.40, br s

5 6 7 9 11 12

a

dH (J in Hz)

m td (13.4, 4.0) dd (12.8, 5.1) dt (13.3, 5.4) m m m m m dddd (18.0, 12.5, 5.1, 2.1) dd (18.0, 5.8) br s m d (2.4) d (2.4) br s br s s

m m m m m m m m

1.77, m 1.55, m e 1.63, m 2.15, 2.02, e 5.22, 2.22, 1.02, 1.04, 0.93, 0.93, 4.27, 3.56,

4

2.03, td (13.0, 4.0) 1.64*, m 1.64*, m 1.50, ddd (13.0, 7.0, 4.0) 1.39, ddd (13.3, 4.7, 3.0) 1.25, td (13.3, 3.3) 1.77, dd (9.9, 7.0) 1.96*, t (9.1)

1.66, m 0.80, m 1.50, m 1.40e1.36, m 1.40e1.36, m 1.10, m 1.10, m 2.10, ddd (14.9, 3.9, 1.5) 1.71, ddd (14.9, 12.8, 2.2) 3.24, t (2.0)

5.53, t (3.9) e 4.12, br s

dddd (18.0, 12.1, 5.6, 2.1) dd (17.7, 5.1) br s m d (1.1) d (1.0) s s d (8.3) d (8.3)

Data were recorded at 300 MHz * Overlapped signals within the same column.

3b

b

Fig. 2. Asymmetric unit in the single-crystal X-ray structure of 1.

degrees of unsaturation. The 13C NMR (Table 3) and HSQC spectra of 4 revealed 20 carbon signals corresponding to five methyls, six methylenes, five methines including two oxygenated at dC 62.5 and 59.3, and four quaternary carbons. The 1H NMR spectrum (Table 1) showed the presence of three tertiary methyls (dH 0.89, 0.86, and 0.82, all singlets), two secondary methyls of an isopropyl group (dH 0.99 and 0.95, both d, J ¼ 6.9 Hz), and two oxygenated methines (dH 3.24 and 2.32). The above NMR data of 4 closely resembled those of crotontomentosin B (Song et al., 2015), except for the presence of a

2.71, d (17.0) 1.96 *, dd (17.0, 2.6) e 5.76, d (2.4) 2.08, m 0.93, d (0.9) 0.92, d (0.8) 0.95, s 0.88, s 1.32, s

1.30, 1.45, 1.30, 1.99, 1.78, e 2.32, 1.63, 0.95, 0.99, 0.89, 0.86, 0.82,

m m m dt (14.3, 3.4) ddd (14.3, 12.1, 5.0) br s m d (6.9) d (6.9) s s s

Data were recorded at 600 MHz in C6D6.

methylene group at C-2 in 4 instead of a ketone group in crotontomentosin B. The four high field shifted oxygenated carbons [dC 63.2 (C-13), 62.5 (C-14), 59.3 (C-7), and 55.6 (C-8)] together with the two oxygen atoms were assigned as two epoxides which existed between C-7 and C-8, and between C-13 and C-14, respectively. This assignment was verified by the HMBC correlations of H-7 to C6, of H-14 to C-7, C-8, and C-9, and of H-15 to C-13 and C-14 (Scheme S2). The NOESY correlations between H-5 (dH 1.10, m) and H3-19/H-9 (dH 1.30, m) indicated that these protons were cofacially a
oriented. Likewise, the NOESY correlations between H3-20 and H3-18/H-11b/H-6b indicated that these protons were beoriented. The 1H NMR spectrum of 4, H-7 resonated as a triplet peak (J ¼ 2.0 Hz), implying small coupling constants of H-7 with both H-6a and H-6b; therefore, H-7 was established to be b
oriented. Furthermore, the NOESY correlations between H-14 and H-7/H3-17 also indicated that H-14 and the isopropyl were b
oriented (Scheme S2). Consequently, the structure of 4 was elucidated as 7a,8a:13a,14a-diepoxyabietane and named roscotane D. Compound 5 was isolated as pale yellow oil. Its molecular formula C20H34O was established by HRESIMS, suggesting five degrees of unsaturation. The 13C NMR (Table 3) and HSQC spectra of 5 revealed 20 carbon signals corresponding to four methyls, eight methylenes including one oxygenated carbon at dC 63.5, three methines, and five quaternary carbons together with two olefinic carbons at dC 134.0 and 127.3. The 1H spectrum (Table 2) for 5 was similar to those of acasiane A (Lin et al., 2009) except for a methyl group in 5, instead of the hydroxymethyl group at C-19 in acasiane A. The only one hydroxymethyl group presented at C-15 in 5 was indicated by the signals at dH 3.74 and 3.59 (both dd, J ¼ 11.3, 8.0 Hz, H-16) in the HSQC spectrum. The presence of a tetrasubstituted D8(9) double bond in 5 was confirmed by the HMBC correlations of H3-20 (dH 0.90) and H2-12 (dH 1.89 and 1.28, both m) to C-9 (dC 134.0) and of H2-7 (dH 2.15, m) to C-8 (dC 127.3) and C-9. A high field shifted methine signal at dH 0.62 (d, J ¼ 4.0 Hz, H-14) indicated the presence of a cyclopropane ring, which was confirmed by HMBC

J. Boonsombat et al. / Phytochemistry 143 (2017) 36e44

39

Table 2 1 H NMR Spectroscopic data (600 MHz, CDCl3) for Roscoranes A-D (5e8).

dH (J in Hz)

positions

5 1

1.70, 1.01, 1.70, 1.42, 1.44, 1.12, 1.06, 1.63, 1.58, 2.15,

m m m m m m m m m m

17 18 19

e 1.99, 1.69, 1.89, 1.28, 0.62, 1.36, 3.74, 3.59, 1.23, 0.85, 0.90,

m m m m d (4.0) dd (7.8, 4.3) dd (11.3, 8.0) dd (11.3, 8.0) s s s

20

0.90, s

2 3 5 6 7 9 11 12 14 15 16

a

Data were recorded at 300 MHz

6

7a

8

3.70, t (3.2)

3.62, br s

3.72 br s

1.93, 1.66, 1.80, 1.14, 1.57, 4.39,

tt (13.2, 3.2) ddd (13.1, 6.7, 3.4) td (13.1, 3.1) m m br s

3.99, ddd (12.3, 4.2, 2.6)

3.99, ddd (12.2, 4.3, 2.7)

1.77, 1.41, 1.86, 4.25,

2.32, 2.28, 2.36, 4.17,

dd (14.4, 2.7) dd (14.4, 2.7) d (7.0) ddd (12.0, 7.0, 5.0)

3.92, d (2.7)

1.75, 1.54, 5.49, 5.83, 5.00, 4.95, 1.11, 1.27, 1.05,

dd (12.0, t (11.9) br s dd (17.5, dd (17.5, dd (10.7, s s s

1.91, 1.34, 1.73, 1.49, 1.30, 2.24, 2.06, 2.41, 1.73, 1.56, 1.43,

t (12.6) td (12.6, 4.3) m m m ddd (14.0, 4.2, 2.0) ddd (14.0, 14.0, 4.9) t (7.8) m m m

5.30, 5.78, 4.92, 4.90, 1.06, 0.87, 3.42, 3.15, 0.87,

br s dd (17.5, 10.7) dd (17.5, 1.4) dd (10.7, 1.4) s s d (10.8) d (10.8) s

5.0)

10.7) 1.0) 1.0)

1.14, s b

t (12.5) dd (12.2, 4.1) d (1.8) br s

2.86, 1.72, 1.63, 1.52,

t (7.1) m m m

5.77, 5.81, 5.00, 4.95, 1.12, 1.31, 1.10,

d (2.1) dd (17.8, 10.2) d (1.3) dd (4.7, 1.2) s s s

1.11, s

Data were recorded at 600 MHz in C6D6. *Overlapped signals within the same column.

Table 3 13 C NMR data for Compounds 1e8 (600 MHz, CDCl3). positions

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OMe a

dC mult 1

2a

3b

4

24.8 t 19.4 t 41.6 t 33.5 s 41.8 d 19.2 t 31.6 t 79.3 s 75.2 s 50.7 s 26.8 t 23.0 t 147.7 s 120.3 d 34.7 d 21.3 q 20.9 q 21.4 q 32.9 q 104.4 d 56.1 q

28.6 t 19.9 t 41.6 t 33.4 s 41.9 d 19.6 t 32.4 t 77.7 s 75.9 s 40.2 s 25.8 t 22.9 t 147.3 s 120.5 d 34.6 d 20.9 q 21.3 q 21.4 q 32.8 q 72.5 t

31.6 t 18.8 t 42.2 t 33.0 s 43.3 d 24.9 t 130.1 d 134.5 s 73.8 s 40.7 s 69.1 d 34.0 t 139.7 s 121.1 d 35.1 d 21.3 q 20.9 q 22.5 q 34.2 q 17.6 q

39.3 18.6 42.0 32.5 44.8 22.6 59.3 55.6 49.1 34.4 15.9 23.1 63.2 62.5 33.1 17.9 17.6 22.4 32.7 19.0

Data were recorded at 75 MHz

b

t t t s d t d s d s t t s d d q q q q q

5

6

7a

8

36.3 t 18.9 t 41.9 t 33.3 s 51.8 d 18.9 t 33.2 t 127.3 s 134.0 s 37.5 s 20.4 t 29.2 t 23.4 s 28.47 d 28.54 d 63.5 t 19.8 q 21.5 q 33.1 q 19.0 q

74.2 d 25.3 t 36.1 t 34.2 s 50.1 d 68.6 d 45.4 t 133.2 s 52.7 d 43.3 s 65.4 d 44.5 t 38.5 s 131.8 d 148.1 d 110.5 t 25.3 q 24.4 q 33.5 q 18.6 q

76.4 d 66.7 d 44.2 t 34.8 s 43.2 d 71.8 d 78.1 d 136.4 s 37.3d 43.4 s 17.4 t 33.9 t 37.7 s 137.9 d 147.5 d 111.3 t 26.3 q 26.0 q 33.5 q 18.0 q

75.0 d 66.7 d 36.7 t 38.9 s 40.2 d 22.0 t 35.3 t 136.5 s 43.4 d 42.7 s 18.2 t 34.4 t 37.4 s 129.7 d 148.8 d 110.2 t 26.0, q 19.1 q 71.6 t 15.6 q

Data were recorded at 150 MHz in C6D6 * Assignments may be interchangeable.

correlations of H-14 to C-9, C-16, and C-17 (Scheme S2). Considering the unsaturation with the spectral data described above, a tetracyclic diterpenoid structure possessing a cyclopropane ring at C-13 (dC 23.4, s) and C-14 (dC 28.47, d) and one double bond was suggested. A further correlation between H-14 and H3-17 in the NOESY spectrum suggested the cis-junction between rings C and D. Thus, compound 5 was elucidated as isopimara-8(9)-ene-16b-ol and named roscorane A. Compound 6 was obtained as an off-white amorphous powder. The molecular formula, C20H32O3, was determined by 13C NMR and HRESIMS. The 1H NMR spectrum (Table 2) of 6 displayed signals

characteristic of vinylic protons at dH 5.83 (1H, dd, J ¼ 17.5, 10.7 Hz, H-15), 5.00 (1H, dd, J ¼ 17.5, 1.0 Hz, H-16a), and 4.95 (1H, dd, J ¼ 10.7, 1.0 Hz, H16b), an olefinic proton at dH 5.49, three oxygenated methine groups at dH 4.39 (dC 68.6, d, C-6), 4.17 (dC 65.4, d, C-11), and 3.70 (dC 74.2, d, C-1), and four singlet signals of tertiary methyl groups at dH 1.27 (H3-18), 1.14 (H3-20), 1.11 (H3-17), and 1.05 (H3-19). On the basis of the molecular formula and the presence of two double bonds inferred from the 1H and 13C NMR data, it was apparent that three rings were present in the molecule. The HMBC correlations from a methyl proton at dH 1.11 to the carbon at dC 148.1 (d) as well as 3J correlations from a vinylic proton at dH 5.00 and

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J. Boonsombat et al. / Phytochemistry 143 (2017) 36e44

4.95 to a quaternary carbon at dC 38.5 indicated a pimarane diterpene skeleton with a vinyl group attached to C-13 (Rao et al., 1968). The HMBC correlations of H-1 to C-3, C-5, C-9, and C-20, of H-6 to C4, C-8, C-10, both of H-9 and H-12 to C-11, and of H-11 to C-10 showed the attachment of these hydroxyl groups at C-1, C-6, and C11, respectively (Scheme S2). Correlations in the NOESY spectrum were observed between H-1 and H3-20 (dH 1.14)/H-11 (dH 4.17)/H317 (dH 1.11), and between H-6 and H-5 (dH 1.57)/H-9 (dH 2.36)/H3-19 (dH 1.05) indicated that the hydroxyl groups at C-1 and C-11 to be aeoriented and C-6 to be b-oriented (Scheme S2). Compound 6 was therefore elucidated as 1a,6b,11a-trihydroxyisopimara-8(14),15diene and named roscorane B. Compound 7 was obtained as a white amorphous powder, and its molecular formula was shown as C20H32O4 determined by 13C NMR and HRESIMS. The 1H and 13C NMR spectroscopic data (Tables 2 and 3) of 7 were similar to those of 6 but different in the substituent patterns. Compound 7 differed from 6 due to the absence of the hydroxyl group attached to C-11 and the presence of two additional oxymethine groups [dH 3.99 (ddd, J ¼ 12.3, 4.2, 2.6 Hz), dC 66.7 (C-2) and dH 3.92 (d, J ¼ 2.7 Hz), dC 78.1 (C-7)], instead of methylene groups at C-2 and C-7, respectively. The location of four hydroxyl groups in 7 were verified to be at C-1, C-2, C-6, and C-7 on the basis of the HMBC correlations of H-1 (dH 3.62, br s) to C-2, C-3, C-5, C-9, and C-20, of H-2 to C-1, of H-3 to C-1, C-2, of H-6 (dH 4.25, br s) to C-7, C-8, and C-10, and of H-7 to C-5, C-6, C9, and C-14 (Scheme S2). The NOESY correlations between H-1 and H-2 (dH 3.99, ddd)/H3-20 (dH 1.11, s), and between H-6 and H-5 (dH 1.86, d, J ¼ 1.8 Hz)/H3-19 (dH 1.10, s) suggested H-1 and H-2 to be boriented and H-6 to be a-oriented. The rather small vicinal coupling constant of H-7 [dH 3.92 (d, J ¼ 2.7 Hz)] and the NOESY experiments between H-7 and H-14 suggested that the configuration of H-7 to be beoriented (Scheme S2). Accordingly, compound 7 was established as 1a,2a,6b,7a-tetrahydroxypimara-8(14),15-diene and named roscorane C. Compound 8 was obtained as an off-white amorphous powder. Its molecular formula was established as C20H32O3 by 13C NMR and HRESIMS. The 1H and 13C NMR data (Tables 2 and 3) of 8 were similar to those of sandaracopimaradien-1a,2a-diol (18) except for the hydroxymethyl group at C-19 [dH 3.42 (d, J ¼ 10.8) and 3.15, (d, J ¼ 10.8), dC 71.6 ] in 8, instead of a methyl group in 18. This deduction was supported by HMBC correlations (Scheme S2) from H-19a and H-19b to C-4 (dC 38.9, s), C-5 (dC 40.2, d), and C-18 (dC 19.1, q). The relative configuration of 8 was established by the NOESY experiment. The NOESY correlations between H-5 and H-9/ H2-19 indicated the aeorientation of H-5, H-9, and the hydroxymethyl group; while the correlations between H-1 and H-2/H3-18/ H3-20 suggested the b-orientation of H-1, H-2, H3-18, and H3-20. Consequently, compound 8 was assigned as 1a,2a,19a-trihydroxypimara-8(14),15-diene and named roscorane D. The structures of the known diterpenoids 9e20 (Scheme S1 and Table S4) were identified by comparison of their observed and reported NMR data. On the basis of the present study, the whole plants of K. roscoeana were found to be a rich source of both 9ahydroxy-abietane and 1a-hydroxyisopimarane diterpenoids. Among the twenty isolates, roscotanes A (1) and B (2) isolated from the whole plant of K. roscoeana are the unprecedented entabietane diterpenoids having the unique five-membered ether ring D joining C-8/C-10 bond. Unlike other previously studied Kaempferia species that are rich in isopimarane diterpenoids (Kaewkroek et al., 2013; Sematong et al., 1994; Tang et al., 2011; Thongnest et al., 2005; Tuchinda et al., 1994; Win et al., 2015a,b), abietane diterpenoids were rarely found in Kaempferia species, and the only mention was in K. angustifolia and K. elegans (Tang et al., 2011). A literature survey showed that abietane diterpenoids have been previously obtained from the following genera (family): Ceriops

(Rhizophoraceae) (Wang et al., 2014), Croton (Euphorbiaceae) (Song et al., 2015), Suregada (Euphorbiaceae) (Gondal and Choudhary, 2012; Haba et al., 2009; He et al., 2009; Lee et al., 2008), Euphorbia (Euphorbiaceae) (Haba et al., 2013; Liu et al., 2014, 2016; Reis et al., 2014; Zhang et al., 2009, 2014), Isodon (Labiatae) (Chen et al., 2008b; Huang et al., 2007; Li et al., 2006, 2010, 2011; Liu et al., 2013; Sun et al., 2006; Wang et al., 2013), Goldfussia (Acanthaceae) (Yu et al., 2007), Doellingeria (Bai et al., 2005), Solidago (Compositae) (Lu et al., 1995), Salvia (Lamiaceae) (Jassbi et al., 2015), and Glyptostrobus (Taxodiaceae) (Zhang et al., 2010). Hence, our findings further add to the diversity and complexity of abietane and contribute to the chemotaxonomic significance. It is also worth mentioning that the biogenesis pathway of roscorane A (5) could be derived from sandaracopimaradiene (9). Epoxidation at D15,16 could give intermediate (i) which could undergo cyclopropane ring formation to compound 5 upon protonation at epoxide ring. (Scheme S3). Due to the paucity of compound 4, only nineteen compounds 1e3 and 5e20 were evaluated for antimicrobial activities and compounds 1e3, 5e7, and 9e17 were also tested for their antimalarial activities. According to the results, compound 10 showed the most activity against Gram-positive bacteria strains Staphylococcus epidermidis and Bacillus cereus with MIC (MBC) values of 25 (75) and 25 (50) mg/ml, respectively, implying that this compound had quite specific antimicrobial activity. None of other compounds showed activity against all Gram-negative bacterial and fungal strains. Additionally, none of them exhibited activity against Plasmodium falciparum (Chloroquine resistant) at the highest concentration tested (IC50 > 10
5 M). 3. Concluding remarks Eight previously undescribed diterpenoids including four oxygenated abietanes (1e4) and four oxygenated pimaranes (5e8), and twelve known diterpenoids were isolated from the whole plant of K. rocoeana. The structure of compound 1 was further confirmed by X-ray analysis. On the basis of the present study, this plant was found to be a rich source of both 9a-hydroxy-abietane and 1ahydroxyisopimarane diterpenoids. Additionally, the fact that several abietane diterpenoids were isolated from this plant is quite remarkable; since, abietane diterpenoids are rarely found in Kaempferia species. All tested compounds showed no antimalarial activity and only an aromatic abietane, compound 10 was the most active against Gram-positive bacterial strains Staphylococcus epidermidis and Bacillus cereus. Considering that several other abietane and pimarane diterpenoids have been reported for selective antibacterial activity and the SAR study of these diterpenoids to antibacterial activity is under attention (Dettrakul et al., 2003; Urzúa lez, et al., 2008; Porto et al., 2009; Machumi et al., 2010; Gonza 2015), our antibacterial results from a variation of tested structures thus provide additional information toward the better understanding of the essential structural factors to antibacterial activity. 4. Experimental 4.1. General experimental procedures Melting points were determined on a Büchi 535 apparatus and are uncorrected. Optical rotation values were measured on a JASCO P-1020 polarimeter. IR spectra were recorded on a PerkinElmer Spectrum One Spectrophotometer using a universal attenuated total reflectance (ATR) technique. 1H and 13C NMR spectra were recorded on a Bruker UltraShield Avance 300 NMR (operating at 300 MHz for 1H and 75 MHz for 13C) and a Bruker AVANCE 600

J. Boonsombat et al. / Phytochemistry 143 (2017) 36e44

NMR (operating at 600 MHz for 1H and 150 MHz for 13C) spectrometer using residual signal for CDCl3, CD3OD, and C6D6 as a reference and TMS as an internal standard. Two-dimensional NMR (DEPT, HMQC, HSQC, HMBC, COSY, and NOESY) spectra were obtained using standard Bruker Software. HREIMS and APCI TOF-MS (70 eV) analyses were determined using a Bruker MicroTOFLC spectrometer. Single-crystal X-ray diffractions were recorded on a Bruker X8 APEX diffractometer with APEX II CCD area detector using graphite-monochromatic Mo Ka radiation. All solvents were distilled from commercial grade solvents prior to use, and spectral grade solvents were used for spectroscopic measurements. Analytical and semipreparative HPLC were performed on Waters Delta 600 and 1525 pumps together with Waters 2996 and 2998 photodiode array detectors and on the Thermo Separation Products, San Jose, CA, USA. (Pump P4000; detector, PL-ELS 2100). HiChrom™ 5C18 (250  21.2 mm i.d.) was used for the preparative purposes. Sephadex™ LH-20 (GE Health care Bio-Sciences AB) was used for column gel filtration. RP-C18 silica gel (150e200 mesh, Merck) and silica gel 60 (Merck, 0.063e0.200 mm) were used for column chromatography (CC), while silica gel 60 (Merck, less than 0.063 mm) was used for flash column chromatography. Silica gel 60 PF254 (Merck) was used for preparative thin layer chromatography (prep TLC). TLC was performed on precoated aluminum plates (Merck, silica gel 60 F254). The results were detected by UV absorption at 254 nm and visualized by heating silica gel plates spraying with Godin's reagent. 4.2. Plant material The whole plants of K. roscoeana (Zingiberaceae) were collected from Phetchaburi Province, Thailand, in November 2014. The plant was authenticated by Prof. Dr. Wongsatit Chuakul of the Department of Pharmaceutical Botany, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand. A voucher specimen number BKF 01e00053 (Thongnest No. 3) was deposited at the Department of National Park, Wildlife and Plant Conservation, Ministry of Natural Resources and Environment, Bangkok, Thailand. 4.3. Extraction and isolation The fresh ground whole plants of K. roscoeana (11 kg) were extracted with MeOH (25 L  3) at room temperature, and soaked with the CH2Cl2
MeOH (v/v 1:1, 25  2) mixture for an additional 24 h, in order to maximize extracting capacity. The combined MeOH extract was partitioned in hexane/H2O, and CH2Cl2/H2O, respectively. After removal of the solvent, yielded hexane (22.5 g), CH2Cl2 (4.8 g), and WatereMeOH (102.5 g) extracts, and CH2Cl2
MeOH mixture (66 g). The CH2Cl2 extract and CH2Cl2
MeOH mixture were later combined as the same chemical constituents were observed on TLC and NMR spectroscopy. The combined CH2Cl2
MeOH extracts (66.0 g) were fractionated using silica gel column chromatography (CC) with a gradient of hexane
CH2Cl2 (100:0 to 0:100) and CH2Cl2
MeOH (99:1 to 0:100) to obtain twenty fractions (F1-F20) which were combined according to both TLC results and NMR spectra, and only the interesting fractions were subsequently purified. Fraction 2 (2.9 g) was first subjected to column chromatography on silica gel eluting with a step gradient of hexanes
CH2Cl2 (100:0 to 50:50) to give 9 (1.2 g), 10 (338 mg), and another seven subfractions (2.1e2.7) were collected. Subfraction 2.3 (23 mg) was further purified by preparative TLC using hexane
acetone
CH2Cl2 (10: 0.1: 0.1) to give 11 (12 mg). Subfraction 2.5 (105 mg) was separated by a silica gel CC using hexane
CH2Cl2 (75:25 to 72:28) to give four subfractions (2.5.1e2.5.4). Purification of subfraction 2.5.1 (27 mg) on reversed-phase C18 column eluted with

41

MeOH
H2O (90:10), followed by prep TLC using hexane
acetone
CH2Cl2 (10:0.2:0.2) yielded roscotane D (4, 6 mg) and 12 (3 mg). Roscotane A (1, 5.0 g) was obtained by recrystallization in CH2Cl2 of the filtrate of fraction 10. Fraction 11 (4.4 g) was rechromatographed on silica gel with a step gradient of EtOAc
hexanes (1:99 to 50:50) and then MeOH
EtOAc (2:98) to afford roscotane A (1, 824 mg), 12 (18.2 mg), and fourteen subfractions (11.1e11.14). Subfraction 11.6 (204.2 mg) was purified by flash chromatography with hexanes
CH2Cl2 (100:0 to 0:100) [column: 15 m silica HP 40 g, 20 bar] to give 13 (17.4 mg), and a mixture (92.9 mg). Further purification of the mixture (92.9 mg) by Sephadex LH-20 CC with CH2Cl2
MeOH (20:80) gave 14 (54.6 mg). Subfraction 11.10 (764.9 mg) was separated by Sephadex LH-20 CC with CH2Cl2
MeOH (20:80) to obtain five subfractions (11.10.1e11.10.5). Subfraction 11.10.4 (224 mg) was further purified by reversed-phase C18 HPLC (MeCN
H2O, 70:30 to 100:0, 10 mL min
1, l 210 nm) to furnish roscotane C (3, 12 mg), 15 (16.0 mg), and 16 (30.8 mg). Subfraction 11.12 (500 mg) was subjected to chromatography on silica gel eluted with hexane
CH2Cl2 (80:20 to 50:50) and further purified by silica gel CC with hexane
CH2Cl2 (70:30 to 60:40) to give roscotane B (2, 162.3 mg). Subfraction 11.13 (1.1 g) was separated by Sephadex LH-20 CC with CH2Cl2
MeOH (20:80) to obtain two subfractions (11.13.1e11.13.2). Subfraction 11.13.2 (328 mg) was further subjected to silica gel CC using hexane
CH2Cl2 (80:20) to give roscotane B (2, 58.4 mg) and six subfractions (11.13.2.1e11.13.2.6). Subfractions 11.13.2.4 (43 mg) and 11.13.2.5 (42 mg) were combined and purified by prep TLC using hexane
CH2Cl2 (35:65) yielding roscorane A (5, 65 mg). Fraction 16 (7.8 g) was separated by Sephadex LH-20 CC with 100% MeOH to obtain three subfractions (16.1e16.3). Subfraction 16.3 (1.9 g) was subjected to silica gel CC using hexane
CH2Cl2 (55:45) to give 17 (101.3 mg) and twelve subfractions (16.3.1e16.3.12). Subfraction 16.3.3 (242 mg) was purified by silica gel CC using hexane
EtOAc (99:1 to 82:18) to afford 18 (134 mg) and three subfractions (16.3.3.1e16.3.3). Subfraction 16.3.5 (146 mg) was purified by Sephadex LH-20 CC with CH2Cl2
MeOH (20:80) two times yielded roscorane B (6, 100 mg). Fraction 17 (442 mg) was separated by Sephadex LH-20 CC with CH2Cl2
MeOH (80:20) to obtain eight subfractions (17.1e17.8). Subfraction 17.4 (64 mg) was further purified by reversed-phase C18 HPLC (MeCN
H2O, 30:70 to 100:0, 10 mL min
1, l 210 nm) to furnish roscorane B (6, 1.8 mg), 19 (4.3 mg), and 18 (4.8 mg). Subfraction 17.5 (67 mg) was purified by reversed-phase C18 HPLC (MeCN
H2O, 30:70 to 100:0, 10 mL min
1, l 210 nm) to furnish roscorane C (7, 11.4 mg), roscorane D (8. 33.6 mg), and 20 (2.9 mg). 4.3.1. Roscotane A (1) Colorless crystals (CH2Cl2); mp 180e183  C; [a] 28D
82.7 (c 1.015, CHCl3); IR (neat) nmax 3419, 2960, 2918, 1457, 1441, 1101, 991, 930 cm
1; 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 357.2403 [M þ Na]þ, calcd for C21H34O3Na, 357.2400. 4.3.2. Roscotane B (2) Amorphous, white powder; [a]28D
13.3 (c 0.43, CHCl3); IR (neat) nmax 3392, 2957, 2921, 2865, 1458, 1441, 1111, 996, 909 cm
1; 1 H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 305.2469 [M þ H]þ, calcd for C20H33O2, 305.2475. 4.3.3. Roscotane C (3) Pale yellow sticky oil; [a]27D
49.9 (c 0.59, CHCl3); IR (neat) nmax 3441, 2957, 2921, 1716, 1462, 1384, 11191, 1017, 994 cm
1; 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 327.2305 [M þ Na]þ, calcd for C20H32O2Na, 327.2295.

42

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4.3.4. Roscotane D (4) Colorless sticky oil; [a]27D
11.8 (c 0.58, CHCl3); 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 327.2292 [M þ Na]þ, calcd for C20H32O2Na, 327.2295. 4.3.5. Roscorane A (5) Pale yellow oil; [a]26 D þ 45.9 (c 1.52, CHCl3); IR (neat) nmax 3396, 2926, 2868, 1715, 1662, 1458, 1375, 1263, 1022 cm
1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 289.2540 [M þ H]þ, calcd for C20H33O1, 289.3301. 4.3.6. Roscorane B (6) Amorphous, off-white powder; [a]27D
20.3 (c 0.15, CHCl3); IR (neat) nmax 3367, 2937, 1937, 1772, 1443, 1247, 1048 cm
1; 1H and 13 C NMR data, see Tables 2 and 3; HRESIMS m/z 343.2241 [M þ Na]þ, calcd for C20H32O3Na, 343.2244. 4.3.7. Roscorane C (7) Amorphous, white powder; [a]27D
55.0 (c 0.54, CHCl3); IR (neat) nmax 3378, 2956, 2924, 1464, 1393, 1028 cm
1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 359.2194 [M þ Na]þ, calcd for C20H32O4Na, 359.2193. 4.3.8. Roscorane D (8) Amorphous, off-white powder; [a]27Dþ 37.9 (c 0.34, CHCl3); IR (neat) nmax 3420, 2951, 2927, 2875, 1714, 1456, 1432, 1144, 1025 cm
1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 343.2231 [M þ Na]þ, calcd for C20H32O3Na, 343.2244. 4.4. X-ray diffraction analysis of 1 A suitable crystal was measured by a Bruker APEX-II CCD diffractometer. The crystal was kept at 296.15 K during data collection. Using Olex2 (Dolomanov et al., 2009), the structure was solved with the Superflip (Palatinus and Chapuis, 2007; Palatinus and van der Lee, 2008, 2012) structure solution program using Charge Flipping and refined with the ShelXL (Sheldrick, 2015) refinement package using Least Squares minimization. Software packages used to prepare molecular graphics and materials for publication were Olex2 (Dolomanov et al., 2009) and Mercury (Macrae et al., 2006). Crystallographic data for the structure 1 have been deposited with the Cambridge Crystallographic Data Center as supplementary publication number CCDC 1452789. Copies of these data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/ retriveving.html or on application to CCDC, 12 Union Road, Cambridge CB21EZ, UK (fax (þ44) 1223-336-033 or e-mail: deposit@ ccdc.cam.ac.uk). 4.4.1. Crystal data of 1 C42H68O6; fw 668.96; T ¼ 296.15 K, monoclinic space group P21 (no.4); unit cell dimensions a ¼ 8.7698(9) Å, b ¼ 23.782(3) Å, c ¼ 9.4831(10) Å, V ¼ 1962.2(4) Å3, a ¼ 90 , b ¼ 97.196(4) , g ¼ 90 , Z ¼ 2, rcalc ¼ 1.132 Mg m
3, crystal dimensions 0.44  0.26  0.12 mm, m(MoKa) ¼ 0.073 mm
1, F(000) ¼ 736.0, 17612 reflections measured, (4.682  2Q  50.198 ), 6657 unique [Rint ¼ 0.0354, Rsigma ¼ 0.0501], which were used in all calculations. The final refinement gave R1 ¼ 0.0512 [I > 2s(I)], wR2 ¼ 0.1296 (all data). 4.5. Antimicrobial activity assay Strains of four Gram-positive species: Staphylococcus aureus (S. aureus) TISTR 1466 (ATCC 6538), Staphylococcus epidermidis (S. epidermidis) TISTR 518 (ATCC 14990), Enterococcus faecalis (E. faecalis) TISTR 379 (ATCC 19433), and Bacillus cereus (B. cereus)

TISTR 687 (ATCC 11778), three clinical strains of Gram-negative species: Pseudomonas aeruginosa (P. aeruginosa) TISTR 781 (ATCC 9027), Escherichia coli (E. coli) TISTR 780 (ATCC 8739), and Salmonella typhimunium (S. typhimunium) TISTR 292 (ATCC 13311), three species of Fungi: Penicilium sp. TISTR 3118, Aspergillus flavus TISTR 3366, and Aspergillus niger TISTR 3254, and eight species of Yeasts: Candida albicans (C. albicans) TISTR 5779 (ATCC 10231), Cryptococcus albidus (C. albidus) TISTR 5684 (MUCL 40661), Candida guilliermondii (C. guilliermondii) TISTR 5883, Candida tropicalis (C. tropicalis) TISTR 5043, Candida parapsilosis (C. parapsilosis) TISTR 5924, Candida glabrata (C. glabrata) TISTR 5006, Candida krusei (C. krusei) TISTR 5099, and Candida sake (C. sake) TISTR 5143, obtained from the culture collection center, Thailand Institute of Scientific and Technological Research (TISTR), Thailand, were used for antimicrobial testing. The bacteria were maintained on nutrient agar (NA) at 37  C and fungi were maintained on potato dextrose agar (PDA) at 28  C. The antimicrobial experiments were conducted according to previously reported procedures (Makarasen et al., 2015; French, 2006; Bell and Grundy, 1968). 4.6. Antimalarial activity assay Parasites culture: Human erythrocytes (type O) infected with P. falciparum 94 strain, (Chloroquine resistant) was maintained in continuous culture, according to the method described by Trager and Jensen (1976). RPMI 1640 culture medium (Gibco, USA) supplemented with 25 mM of HEPES (Sigma, USA), 40 mg/L gentamicin sulfate (Government Pharmaceutical Organization, Thailand) and 10 ml of human serum was used in continuous culture. Before starting the experiment, P. falciparum culture was synchronized by using sorbitol-induced hemolysis according to the method of Lambros and Vanderberg (1979) to obtain only ring-infected cells and then incubated for 48 h prior to the drug testing to avoid the effect of sorbitol. The experiments were started with synchronized suspension of 0.5%e1% infected red blood cell during ring stage. Parasites were suspended with culture medium supplemented with 15% human serum to obtain 10% cell suspension. The parasite suspension was put into 96-well microculture plate; 50 ml in each well and then add 50 ml of various test drug concentrations. These parasite suspensions were incubated for 48 h in the atmosphere of 5% CO2 at 37  C. After 48 h incubation, parasites culture was fixed by adding 0.25% (v/v) glutaraldehyde (Sigma) in phosphate buffer saline (PBS) and these were kept for DNA staining. Parasite DNA staining and Flow Cytometric analysis: Before parasites DNA staining, 5  106 red blood cells from each glutaraldehyde-fixed sample were washed once with PBS and resuspended in PBS containing propidium iodide (PI) (Molecular Probe) at a final concentration of 10 mg/ml, and held for at least 1 h in dark. The stained samples were then analyzed with BD FACSCanto™ (Becton-Dickson). The PI stained cells were excited with 488 nm. Red fluorescence was detected at 585 nm. Red blood cells were gated on the basis of their forward scatter and side scatter. For each sample, 30,000 cells were required, stored and analyzed. Percent parasitemia, fluorescence intensity, and any abnormal fluorescence pattern were obtained from an integrated fluorescence histogram between the test and the control sample. Notes The authors declare no competing financial interest. Acknowledgements This work is supported by the Center of Excellence for Environment Health and Toxicology, (EHT-PERDO), the Ministry of

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Education, and the Chulabhorn Research Center. Institute of Molecular Biosciences, Mahidol University is partially funding. We acknowledge to Prof. Dr. Wongsatit Chuakul, Department of Pharmaceutical Botany, Faculty of Pharmacy, Mahidol University, Thailand, for the identification of the plant. We thank Dr. Hunsa Prawat for her assistance with NMR spectroscopy and also thanks to Miss Kamonthip Singbumrung, a student from Kasetsart University Sakonnakhon Campus, for chromatographic analysis during her training course. Appendix B. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.phytochem.2017.07.008. References Ashitani, T., Iwaoka, T., Nagahama, S., 1999. 11a-hydroxy-7,13-abietadiene from Sugi (Cyrptomeria japonica) wood extract. Nat. Prod. Lett. 13, 169e170. Bai, S.-P., Zhu, Z.-F., Yang, L., 2005. An ent-abietane diterpenoid from Doellingeria scaber. Acta Crystallogr. 61, 2853e2855. Bell, S.C., Grundy, W.E., 1968. 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