Chemical constituents of Pholidota cantonensis

Phytochemistry xxx (2017) 1e7

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Chemical constituents of Pholidota cantonensis Bin Li a, b, Zulfiqar Ali c, Michael Chan e, Juan Li a, Mei Wang c, Naohito Abe c, Can-Rong Wu a, Ikhlas A. Khan c, d, Wei Wang a, b, *, Shun-Xiang Li a, ** a

Hunan Province Engineering Research Center of Bioactive Substance Discovery of Chinese Medicine, School of Pharmacy, Hunan University of Chinese Medicine, Changsha 410208, China b TCM and Ethnomedicine Innovation & Development Laboratory, Sino-Luxemburg TCM Research Center, School of Pharmacy, Hunan University of Chinese Medicine, Changsha 410208, China c National Center for Natural Products Research, University of Mississippi, Oxford, MS 38677, USA d Department of Pharmacognosy, University of Mississippi, Oxford, MS 38677, USA e Natural Health and Food Products Research Group, British Columbia Institute of Technology, Burnaby V5G3H2, BC, Canada

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 January 2016 Received in revised form 30 January 2017 Accepted 6 February 2017 Available online xxx

Two 9,10-dihydrophenanthrenes trivially named phocantol and phocantone, two diterpenoid glycosidesnamed phocantoside A and phocantoside B were isolated from the ethanol extract of the air-dried whole plant of Pholidota cantonensis Rolfe, together with seventeen known compounds. The structures of the four compounds were identified as 1-hydroxy-2,7-dimethoxy-9,10-dihydrophenanthro-[4,5-bcd] furan, 5-hydroxy-2,7-dimethoxy-9,10-dihydro-1,4-phenanthrenedione, (8R,13E)-ent-labd-13-ene-3a,8,15 -triol 15-O-b-D-gluco-pyranoside and (5S,8R,9S,10R)-cis-cleroda-3,13(E)-diene-15,18-diol 15-O-b-D-glucopyranosyl-18-O-b-D-glucopyranoside by chemical and spectroscopic methods, including 1D and 2D NMR. Twenty compounds were evaluated for their cytotoxic activities against mouse leukemia p388D1 cancer cells, and compound phocantone, phocantoside A, tanshinone IIA and syringate exhibited cytotoxic activity against the mouse leukemia p388D1 cancer cells with IC50 values ranging from 13.37 to 27.5 mM. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Pholidota cantonensis Orchidaceae Diterpenoid glycosides 9,10-Dihydrophenanthrene Cytotoxicity

1. Introduction The genus Pholidota (Orchidaceae) comprises about 30 species with a distribution from tropical Asia to tropical Australia. Among them, about 14 species occur in China (Chen, 1999). Many active functions has been reported, such as cytotoxic, antitumor, DPPH free radical scavenging, nitric oxide inhibiting, and antiinflammatory activities (Bandi and Lee, 2011). Pholidota cantonensis Rolfe, namely ‘Xi-Ye-Shi-Xian-Tao’ and ‘Shuangfei Yan’ in Chinese, is a Tujia ethnomedicine of Hunan province with a long history of use for the treatment of sore throat, tonsillitis, cough phlegm asthma, pneumonia, acute soft tissue trauma and arthritis (Chen, 1999; Yi et al., 2004). Phytochemical studies on P. cantonensis * Corresponding author. TCM and Ethnomedicine Innovation & Development Laboratory, Sino-Luxemburg TCM Research Center, School of Pharmacy, Hunan University of Chinese Medicine, Changsha 410208, China. ** Corresponding author. Hunan Province Engineering Research Center of Bioactive Substance Discovery of Chinese Medicine, School of Pharmacy, Hunan University of Chinese Medicine, Changsha 410208, China. E-mail addresses: [email protected] (W. Wang), lishunxiang@hotmail. com (S.-X. Li).

have reported the existence of several stilbenoids and a 9,10dihydrophenanthraquinone (Li et al., 2008, 2014). In addition, previous phytochemical studies reported stilbenoids, triterpenoids, steroids, lignans, phenanthrene and phenolic compounds in this genus (Bandi and Lee, 2011; Dong et al., 2013; Wang et al., 2012, 2014; Yuan et al., 2013). Keeping in view of its traditional medicinal importance and few phytochemical investigations, thus far a phytochemical study of P. Cantonensis was carried out. The present investigation resulted in isolation and identification of four new compounds (1-4) (Fig. 1) from the ethanol extract of the air dried whole plant, together with seventeen known compounds (5-21). In addition, the cytotoxic activities on mouse leukemia p388D1 cancer line and inhibition of iNOS activity in mouse macrophages (RAW 264.7 cells) for the compounds were evaluated. Compounds 2, 3, 8 and 11 exhibited cytotoxic activities, and no compound showed inhibition of iNOS activity.

2. Results and discussion Twenty-one compounds including four new compounds (Fig. 1)

http://dx.doi.org/10.1016/j.phytochem.2017.02.005 0031-9422/© 2017 Elsevier Ltd. All rights reserved.

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Fig. 1. Chemical structures for compounds 1-4.

were isolated and identified from the ethanol extract of the dried whole plant. The new compounds were elucidated as 1-hydroxy2,7-dimethoxy-9,10-dihydro-phenanthro-[4,5-bcd]furan (1), trivially named phocantol, 5-hydroxy-2,7-dimethoxy-9,10-dihydro-1,4phenanthrenedione (2), trivially named phocantone, (8R,13E)-entlabd-13-ene-3a,8,15-triol 15-O-b-D-glucopyranoside (3), trivially named phocantoside A, (5S,8R,9S,10R)-cis-cleroda-3,13(E)-diene15,18-diol 15-O-b-D-glucopyranosyl-18-O-b-D-glucopyranoside (4), trivially named phocantoside B through chemical methods and spectroscopic techniques. Phocantol (1) was obtained as a colorless powder, and whose molecular formula was determined to be C16H14O4 by HRESIMS (m/ z 271.0999, [MþH]þ) with a degree of unsaturation equal to 10. It's IR spectrum contained strong absorption bands indicative of an aromatic ring (1535 and 1465 cm1). The 13C NMR and DEPT-135 spectra of 1 showed 16 carbons, including two methylenes (dC 22.0 and 25.3), nine quaternary carbons (dC 112.6, 116.3, 117.3, 129.1, 140.9, 146.9, 148.6, 154.6 and 160.4), three methines (dC 95.9, 96.2 and 108.6) and two methoxy carbons (dC 56.1 and 57.1). In the 1H NMR spectrum, the two methylene signals at dH 3.11 (2H, m) and 3.14 (2H, m), together with the two methylene carbons (dC 22.0 and 25.3), in the 13C NMR spectrum, indicated that compound 1 had a typical 9,10-dihydrophenanthrene structure (Majumder et al., 1982; Bi et al., 2008). The three aromatic proton resonaces at dH 7.09 (1H, s), 6.73 (1H, s), 6.95 (1H, s), the two methoxyl signals at dH 3.79 (3H, s), 3.83 (3H, s) and a hydroxyl resonaces at dH 8.49 (1H, br s) were also observed. Long range 1H-13C correlations (Fig. 2, Table 1) suggested methoxyl and hydroxyl groups were located at C-2, C-7 and C-1, respectively. Furthermore, NOE correlations between: H-3 and 2-OCH3; H-6, H-8 and 7-OCH3; and H-8 and H-9 confirmed the connections of the hydroxyl, methoxyls and aromatic protons (Fig. 2). The five oxygenated quaternary carbons, together with the molecular formula and the degree of unsaturation disclosed the presence of a furan ring between C-4 and C-5. Thus, structure 1 was established as 1-hydroxy-2,7-dimethoxy-

9,10-dihydrophenanthro-[4,5-bcd]furan. Phocantone (2) was obtained as a dark purple powder whose HREIMS exhibited a molecular ion peak (m/z 286.0832 [M]þ) indicating a molecular formula of C16H14O5. It's IR spectrum contained strong absorption bands for an aromatic ring (1541 and 1432 cm1) and a conjugated carbonyl group (1635 cm1). The 13C NMR and DEPT-135 spectra of 2 indicated a structure with 16 carbons, including two carbonyls (dC 180.5 and 192.0), two methylenes (dC 21.1 and 29.1), seven quaternary carbons (dC 110.6, 139.1, 140.4, 142.4, 157.6, 158.7 and 162.7), three methines (dC 102.4, 107.9 and 108.4) and two methoxyls (dC 55.3 and 56.6). The 1H NMR spectrum of 2 exhibited three aromatic protons, including a tetra-substituted aromatic ring with meta-coupled protons at dH 6.38 (d, J ¼ 2.7 Hz) and 6.42 (d, J ¼ 2.7 Hz) assigned to H-6 and H-8, and a singlet at dH 6.01 (s) assigned to H-2 or H-3. Two methylenes at dH 2.66 (4H, s) and two methoxyls at dH 3.82 (3H, s), 3.90 (3H, s) were also observed. Based on the above evidence, compound 2 was postulated to be 9,10-dihydrophenanthraquinone (Chen et al., 2013). The 1 H-1H COSY spectrum showed a partial structure indicated by thick lines (Fig. 2). The HMBC (Fig. 2, Table 1) suggested the methoxyls and hydroxyl were connected at C-2, C-7 and C-5 respectively. The connection of 2-OCH3 could also be confirmed by comparing its 13C NMR data with those of known 9,10-dihydrophenanthrene-1,4diones, as when the methoxyl group is attached at C-2, the chemical shifts of C-1 and C-4 were about dC 180 and 192 respectively (Yoshikawa et al., 2012; Cheng et al., 2000), However, when the methoxyl group is attached at C-3, the chemical shifts of C-1 and C4 were about dC 185 (Thangaraja et al., 2011). On the basis of these results, structure 2 was determined to be 5-hydroxy-2,7dimethoxy-9,10-dihydro-phenanthrene-1,4-dione. Phocantoside A (3) was obtained as a light brown gum, [a]20D: 28 (c 0.1, THF). It's HRESIMS showed a pseudo-molecular ion peak (m/z 509.2960, [MþNa]þ), which accounted for a molecular formula of C26H46O8, with a degree of unsaturation equal to 4. It's IR absorption at 3370 cm1 indicated the presence of hydroxyl

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Fig. 2. Key 1He1H COSY, HMBC and NOESY correlations of 1 and 2.

groups. The 1H NMR spectrum showed an olefinic proton at dH 5.68 (t, J ¼ 6.9 Hz, H-14), five tertiary methyls at dH 1.66 (H-16), 1.33 (H17), 1.29 (H-19), 1.26 (H-20) and 1.12 (H-18), together with the anomeric proton of b-glucopyranosyl at dH 4.95 (d, J ¼ 7.7 Hz, H-10 ). The 13C NMR displayed 26 carbon signals, including six resonances assignable to a b-glucopyranosyloxy moiety [dC-10 -C-60 103.9 (d), 75.3 (d), 78.6 (d),71.8 (d), 78.7 (d) and 62.9 (t)], while the remaining 20 signals included: five tertiary methyls dC 15.9, 16.6, 16.8, 29.1 and 31.3; four methines at dC 55.6, 59.4, 78.4 and 121.2; seven methylenes at dC 38.1, 28.1, 18.9, 43.3, 24.6, 43.8 and 66.2, and four quaternary carbons at dC 39.6, 72.0, 39.3 and 140.8. These spectroscopic data suggested that 3 was a diterpenoid glycoside, especially as the carbon chemical shifts of rings A and B were very similar to those of the ent-labdane diterpenoid sylvestin (Wang et al., 2010), except for the position of the b-glucopyranosyloxy unit and the glycosidation shifts of the corresponding carbons. Therefore 3 should be entlabdane diterpenoid glycoside. The overall structure of 3 was confirmed by 1H-1H COSY and HMBC experiments. It's 1H-1H COSY spectrum displayed correlations of H-1, H-2 and H-3, H-6 and H-7, H-9, H-11 and H-12, and H-14 and H-15 (Fig. 3). The anomeric proton signal at dH 4.95 (d, 1H, J ¼ 7.7 Hz) showed an HMBC correlation to C-15 (dC 66.2), which established the b-glucopyranosyl moiety was connected to C-15. Two hydroxyl groups on the diterpene skeleton were deduced by IR absorption and molecular weight analysis. One hydroxyl group was located at C-3 (dC 78.4) by the HMBC observed between H-18, H-19 with C-3, C-4 and C-5. The other hydroxyl was placed on C-8 as indicated through the HMBC between H-17 and C-8, C-7 and C-9 (Fig. 3, Table 2). In addition, H-3 showed as a double doublet with coupling constants of 4.5 and

10.8 Hz, indicating an axial-axial coupling and consequently defining a 3a-OH (Oliveira et al., 2012). ROESY correlations between H-3/H-5 and H-5/H-9; H-18/H-20 and H-20/H-11 further confirmed presence of a 3a-OH. The a-orientation of 8-OH (8R) was determined by the chemical shift of C-17 at dC 31.4 (Wang et al., 2010; Socolsky et al., 2007). The E configuration of the double bond D13 was identified by the chemical shift of H-16 (dH 1.66) (Yang et al., 2004). The D-glucose was identified by HPLC analysis of its acetylated thiazolidine derivative after acid hydrolysis and comparison with an authentic sample (Ali et al., 2013). Thus, the structure of 3 was assigned as (8R,13E)-ent-labd-13-ene-3a,8,15-triol 15-O-b-Dglucopyranoside. Phocantoside B (4), obtained as a colorless gum, was determined to have the molecular formula C32H54O12 based on its HRESIMS (m/ z 653.3499, [MþNa]þ), corresponding to six degrees of unsaturation. Its IR spectrum showed an absorption band at 3365 cm1 which indicated the presence of hydroxyl groups. The 1H, 13C NMR spectroscopic data displayed resonances for two olefinic protons at dH 5.60 (1H, m, H-14), 5.86 (1H, m, H-3), and four olefinic carbons at dC 125.8 (C-3), 140.1 (C-4), 140.7 (C-13), 120.9 (C-14). These data indicated the presence of two trisubstituted double bonds. Resonances for two anomeric protons of b-glucopyranosyl units at dH 4.94 (d, J ¼ 8.0 Hz, H-10 ), 4.93 (d, J ¼ 8.0 Hz, H-100 ) were also observed. The 13C NMR spectrum displayed 32 carbon resonances, including 12 signals assignable to two b-glucopyranosyl units, with the remaining 20 resonances including four tertiary methyls, four methines, eight methylenes and four quaternary carbons. The chemical shifts of 4 were in agreement with those of 13(E)-neocleroda-3,13-diene-15,18-diol (Yang et al., 2004), except for the

Table 1 1 H and 13C NMR spectroscopic data of compounds 1 and 2. Compound 1a

Compound 2b

No.

dC

dH (J in Hz)

1 2 3 4 4a 4b 5 6 7 8 8a 9 10 10a 1-OH 5-OH 2-OCH3 7-OCH3

140.9, s 148.6, s 95.9, d 146.9, s 116.3, s 117.3, s 154.6, s 96.2, d 160.4, s 108.6, d 112.6, s 25.3, t 22.0, t 129.1, s

e e 7.09, e e e e 6.95, e 6.73, e 3.14, 3.11, e 8.49,

57.1, q 56.1, q

a 1

13

b 1

13

H (500 MHz) and H (400 MHz) and

HMBC (H to C)

s

C-1, C-2, C-4, C-4a

s

C-5, C-7, C-8, C-4b

s

C-6, C-7, C-9, C-4b

m m

C-4b, C-8, C-8a, C-10, C-10a C-1, C-4a, C-8a, C-9, C-10a

dC

dH (J in Hz)

180.5, s 158.7, s 107.9, d 192.0, s 139.1, s 110.6, s 157.6, s 102.4, d 162.7, s 108.4, d 142.4, s 29.1, t 21.1, t 140.4, s

e 6.01, s e e e e 6.42,d (2.7) e 6.38, d (2.7) e 2.66, br s 2.66, br s e

56.6, q 55.3, q

10.41, s 3.90, s 3.82, s

HMBC (H to C)

C-1, C-2, C-4, C-4a

C-5, C-7, C-8, C-4b C-9, C-6, C-7, C-4b C-4b, C-8, C-8a, C-10, C-10a C-1, C-4a, C-8a, C-9, C-10a

s

3.83, s 3.79, s

C-2 C-7

C-4b, C-5, C-6 C-2 C-7

C NMR (125 MHz) (in DMSO-d6). C NMR (100 MHz) (in CDCl3).

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Fig. 3. Key 1He1H COSY, HMBC and ROESY correlations of 3 and 4.

resonances attributable to the two glucopyranosyls. Hence, compound 4 was expected to be a diterpenoid glycoside with a structure of a clerodane skeleton. The overall structure of 4 was established by 1H-1H COSY and HMBC experiments. The correlations of H-10, H-1, H-2 and H-3, H-6, H-7, H-8 and H-17, H-11 and H12, and H-14 and H-15 were observed in the 1H-1H COSY spectrum (Fig. 3). Anomeric proton signals at dH 4.94 (H-10 ) and 4.93 (H-100 ) showed HMBC correlations to dC 65.8 and 70.9 respectively, which suggested connections between the b-glucopyranosyl moieties were connected at C-15 and C-18 respectively. The D3,13 double

bonds were deduced from the HMBC between: H-3 and C-1, C-2, C4, C-5, C-18; and H-14 and C-16, C-12 respectively. The HMBC between: H-17 and C-7, C-8, C-9; H-18 and C-3, C-100 ; H-19 and C-4, C5, and C-10; and H-20 and C-8, C-10, C-11 were also observed (Fig. 3, Table 2). The stereochemistry of compound 4 was established from the combined evidence of its spectroscopic data in comparison with those of known clerodane diterpenoids. The chemical shifts of C-19 (dC 35.0) suggested the presence of an A/B ring cis fused clerodane, by comparing the spectroscopic data with literature (Morales et al., 2000; Cifuente et al., 2002; Kalpoutzakis

Table 2 1 H and 13C NMR spectroscopic data of compounds 3 and 4 (Pyridine-d5, 500 MHz/125 MHz). No.

Compound 3

Compound 4

dC

dH, Mult. (J in Hz)

1

38.1 t

2 3 4 5 6 7 8 9 10 11 12 13 14 15

28.1 t 78.4 d 39.6 s 55.6 d 18.9 t 43.3 t 72.0 s 59.4 d 39.3 s 24.6 t 43.8 t 140.8 s 121.2 d 66.2 t

16 17 18

16.8 q 31.3 q 16.6 q

1.75 dt (13.0, 5.5) 1.10 td (13.0, 5.5) 1.95 m, 1.92 m 3.55 dd (10.8, 4.5) e 0.92, br d (11.9) 1.95 m, 1.65 m 2.00 m, 1.54 m e 0.75 br s e 1.99 m, 1.57 m 2.18 m, 2.09 m e 5.68 t (6.9) 4.71 dd (12.0, 6.4) 4.42 dd (12.0, 7.0) 1.66 s 1.34 s 1.12 s

19 20 10 20 30 40 50 60

29.1 q 15.9 q 103.9 d 75.3 d 78.6 d 71.8 d 78.7 d 62.9 t

100 200 300 400 500 600 a

1.29 1.26 4.95 4.09 4.29 4.25 3.99 4.57 4.37

s s d (8.0) t (8.0) t (8.0) t (8.0) m dd (12.0, 2.5) dd (12.0, 5.6)

dC

dH, Mult. (J in Hz)

17.4 t

1.93 m, 1.70 m

C-13

23.8 t 125.8 d 140.1 s 36.2 s 36.9 t 29.0 t 37.4 d 40.0 s 45.2 d 36.5 t 32.6 t 140.7 s 120.9 d 65.8 t

C-13, C-14 C-7, C-8, C-9 C-3, C-4, C-5,C-19

16.6 q 15.8 q 70.9 t

C-3, C-4, C-5,C-18 C-1, C-5, C-9,C-10 C15

34.5 q 17.5 q 103.4 d 75.1 d 78.3 d 71.6 d 78.4 d 62.6 t

2.10 m, 2.02 m 5.86 br t (4.2) e e 2.24 br d (13.8), 1.07 m 1.24 m, 1.13 m 1.34 m e 1.31 m 1.51 m, 1.24 m 1.82 t (8.0) e 5.60 t (6.6) 4.69 dd (11.9, 6.2) 4.45 dd (11.9, 7.2) 1.63 s 0.66 d (6.5) 4.65 d (13.1) 4.37 d (13.1) 1.22 s 0.74 s 4.94 a d (8.0) 4.07 a t (8.0) 4.28 a 4.23 a 3.98 a 4.57 a br d (12.0) 4.37 a dd (12.0, 5.7) 4.93 a d (8.0) 4.07 a t (8.0) 4.26 a 4.22 a 3.98 a 4.57 a br d (12.0) 4.37a dd (12.0, 5.7)

HMBC (H to C)

104.1 d 75.0 d 78.3 d 71.5 d 78.5 d 62.6 t

HMBC (H to C)

C-1, C-2, C-4, C-5, C-18

C-1, C-5, C-9 C-10, C-13, C-14, C-16 C-12, C-16 C-100 , C-14 C-12, C-13, C-14 C-7, C-8, C-9 C-10 , C-3 C-4, C-5, C-10 C-8, C-10, C-11 C15

C18

Signals overlapped.

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et al., 2003; Liu et al., 2009). In cis-clerodanes, the C-19 carbon resonates at about dC 30, whereas in trans-clerodanes it appears at dC 15-20 (Cifuente et al., 2002). The ROESY experiment confirmed the proposed cis-configuration of the decalin moiety, since the correlations between H-10 and H-11, H-11 and H-8 could be observed, which indicated the H-20 was on the other side. For such cis-clerodanes, even the absolute stereochemistry can be estab€s et al., 2004). lished from the 1H NMR (Seaman et al., 1990; Bla 5a,10a-cis- and 5b,10b-cis-clerodanes differ in the orientation of methyl H-20 and therefore in its chemical shifts. In the 5a,10a-cisclerodanes (cis-normal-clerodanes), the methyl H-20 shows an equatorial orientation and produces a chemical shift (dH 1.08) downfield from the axial methyl H-20 (dH 0.84) of the 5b,10b-cisclerodanes (cis-ent-clerodanes). The C(13) ¼ C(14) bond had to be in the (E)-configuration due to the observed correlation between H-14 with H-12 (Fig. 3) and the chemical shift of H-16 (dH 1.63) (Yang et al., 2004). The D-glucose was identified through the same method used with compound 3. Therefore, the structure of 4 was established as (5S,8R,9S,10R)-cis-cleroda-3,13(E)-diene-15,18-diol 15-O-b-D-glucopyranosyl-18-O-b-D-glucopyranoside. A further 15 compounds isolated in this study were identified through comparison of their spectroscopic data (1H and 13C NMR) with those reported in the literature. These known compounds were identified as gigantol (5) (Leong et al., 1997), thunalbene (6) (Majumder et al., 1998), lusianthridin (7) (Majumder and Lahiri, 1990), tanshinone IIA (8) (Lee et al., 2005), neo-tanshinlactone (9) (Wang et al., 2004), syringaldehyde (10) (Kim et al., 2003), syringate (11) (Phadungkit and Luanratana, 2006), syringin (12) (Mizutani et al., 1988), butylcinnamate (13) (Lin et al., 2007), Eferulic acid docosyl ester (14) (Kotowicz et al., 2005), tangshennoside Ⅲ (15) (Yuda et al., 1990), 24-methylenecycloartone (16) (Aves et al., 2000), 24-methylenepollinastanone (17) (Koorbanally et al., 2000), hop-22(29)-ene (18) (Ageta et al., 1993), b-sitostenone (19) (Georges et al., 2006), respectively. Two other compounds isolated in this study were determined to be stigmasterol (20) and daucosterol (21) through comparison of their 1H NMR and 13C NMR spectra to those of authentic samples. 3. Concluding remarks As described in this paper, the phytochemical investigation into P. cantonesis led to the isolation of a new 9,10dihydrophenanthrene, a new 9,10-dihydrophenanthraquinone and two new diterpenoid glycosides. To the best of our knowledge, it's rarely that diterpenoids were isolated from the Orchid family, and these findings provide some further insight into the diversity of natural products found within the Orchid family. Twenty compounds were evaluated for their in vitro cytotoxic activities against mouse leukemia p388D1 cancer cells, with taxol as a positive control by using the MTT assay as described in the literature (Yue et al., 2008). Compounds 2, 3, 8 and 11 exhibited weak cytotoxic activity against the cells with IC50 values ranging from 13.37 to 27.5 mM. No compounds showed iNOS inhibitory activities on RAW 264.7 macrophages (IC50 > 50 mM) (see Table 3) (Zhao et al., 2014). 4. Experimental 4.1. General experimental procedures Optical rotations were measured on a Rudolph Research Analytical AutoPol IV polarimeter. IR spectra were recorded on a Thermo NICOLET 6700 FT-IR spectrometer. HR-ESI-MS and HR-EIMS data were obtained using an Agilent Series 1100 SL mass spectrometer and AEI-MS-50 mass spectrometer, respectively. 1H

5

Table 3 Results of cytotoxic activities against mouse leukemia p388D1 cancer cells. Compound

IC50 (mM)

Compound

IC50 (mM)

1 2 3 4 6 8 9 10 taxol

73.0 27.5 17.7 >200 64.1 13.7 64.5 87.8 0.03

11 12 14 15 16 17 18 20

87.8 >200 81.6 >200 81.4 72.6 94.0 84.3

and 13C NMR spectra were recorded on American Varian INOVA400 (1H 400 MHz, 13C 100 MHz) and American BRUKER-500 (1H 500 MHz, 13C 125 MHz) NMR spectrometers. Column chromatography (CC) was performed using silica gel (32e63 mm, Selecto INC., United States), MCI gel CHP20/P120 (75e150 mm, Mitsubishi Chemical Corporation), reversed-phase RP-C18 silica gel (Polarbond, JTBaker), and Sephadex LH-20 (Sigma). TLC was carried out on aluminum-backed plates pre-coated with silica gel GF254 (20  20 cm, 200 mm, 60 Å, Merck). Visualization was accomplished by spraying with 5% vanillin (Sigma) solution in conc. H2SO4-EtOH (5:95), followed by heating. HPLC analysis was carried out on a Waters Alliance 2695, equipped with a 996 photodiode array detector (Waters Corp., Milford, MA), with Waters Empower-2 software. A Luna C-18 column (150  4.6 mm, 5 mm particle size, Phenomenex Inc., Torrance, CA) was protected with a 2 cm LC-18 guard column (Phenomenex Inc.). Semi-preparative chromatography was carried out on a Agilent 1260, equipped with a G1314F UVeVis detector (Agilent technologies Co. Ltd), with Agilent Open LAB CDS chemstation edition. A Zorbax SB-C18 column (150  9.4 mm, 5 mm particle size, Agilent technologies Co. Ltd.). The solvents (Fisher) used for HPLC and other chromatographic procedures were of HPLC and certified grades, respectively. Sugar standards were purchased from Sigma-Aldrich. 4.2. Plant material The whole plant of P. cantonensis was collected in Suining County Hunan Province, China, in October 2011 and was identified by Prof. Ta-Si Liu, a plant taxonomist within the Department of Pharmacognosy at Hunan University of Chinese Medicine. A voucher specimen (No.20111024 PC) is deposited in the Hunan Province Engineering Research Center of Bioactive Substance Discovery of Chinese Medicine, School of Pharmacy, Hunan University of Chinese Medicine. 4.3. Extraction and isolation Dried whole plant of P. cantonensis (5.0 kg) were ground into a powder, then extracted with EtOH-H2O (3000 mL, 80:20 v/v, 7 days each time) at room temperature for three times. After removal of the combined solvents, a crude extract (504.6 g) was obtained. The crude extract was suspended in H2O and partitioned with petroleum ether, CHCl3, EtOAc and n-BuOH (each 2000 mL) in succession. The resulting four fractions were evaporated to dryness in vacuo, to yield petroleum ether (4.15 g), CHCl3 (32.00 g), EtOAc (26.39 g), n-BuOH (118.21 g) extracts. The CHCl3 (32.00 g) extract was separated into seven fractions (A1-A7) by silica gel CC (1 kg) eluting with gradient of petroleum ether:EtOAc (40:1, 20:1, 10:1, 5:1 and 2:1, each 15 L). Fraction A2 (835 mg) was separated by silica gel CC and eluted with petroleum ether:EtOAc (5:1) followed by Sephadex LH-20 CC (petroleum

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ether:CHCl3:MeOH, 4:5:1) to obtain 1 (3.0 mg). Fraction A3 (560 mg) was subjected to Sephadex LH-20 CC eluting with petroleum ether:CHCl3:MeOH (4:5:1) and recrystallization to give 18 (1.8 mg). Fraction A4 (3.18 g) was repeatedly recrystallized with EtOAc to afford 14 (560 mg). Fraction A5 (6.13 g) was subjected to MCI gel CC and eluted with a gradient mixture of MeOH:H2O (5:5, 6:4, 7:3, 8:2, 9:1 and 10:0) this being followed by Sephadex LH-20 CC (CHCl3:MeOH, 1:1) to obtain 7 (20 mg) and a mixture. The latter was purified by semipreparative HPLC (MeOH:H2O, 60:40) to yield 5 (10 mg) and 6 (15 mg). Fraction A6 (4.20 g) was isolated using silica gel CC (petroleum ether:acetone, 5:1) to afford 10 (15 mg), and 11 (5.0 mg). Fraction A7 (2.60 g) was separated over MCI-gel CC (200 mL, MeOH:H2O, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1 and 10:0) and eight subfractions. Subfraction A7.2 was recrystallized to obtained 21 (20 mg). The EtOAc extract (26.39 g) was applied to a silica gel column (750 g), this being eluted with petroleum ether:EtOAc (20:1, 20:2, 20:4, 20:10, and 20:20) to give 10 fractions (B1-B10). Fraction B7 was applied to a silica gel column eluting with (petroleum ether:EtOAc, 5:1) and further purified by recrystallization to afford 2 (5.0 mg). The n-BuOH extract (80 g) was subjected to silica gel CC (1.2 kg) eluting with a gradient system of CHCl3:MeOH (100:0, 95:5 and 90:10), to yield 6 fractions (C1-C6). Fraction C2 (248 mg) was separated by silica gel CC (hexane:Et2O, 10:0 / 9:1) to obtain 9 (2.0 mg), 13 (7.2 mg), 16 (9.5 mg), 17 (4.5 mg). Fraction C3 (19.37 g) was subjected to sequential chromatographic purification using Sephadex LH-20 CC to yield 7 subfractions C3.1- C3.7. Subfraction C3.4 (2.95 g) was further separated using silica gel CC (CHCl3 and MeOH) into 11 subfractions C3.4.1-C3.4.11. Subfraction C3.4.1 (75.6 mg) was further purified by silica gel CC (hexane:Et2O, 9:1 / 7:3) to obtain 8 (8.3 mg). Subfraction C3.4.5 (41.0 mg) was subjected to silica gel CC (hexane:Et2O, 9.5:0.5 / 8:2) to afford 19 (10.6 mg). Subfraction C3.4.7 (96.0 mg) was further purified by silica gel CC (hexane:acetone, 9.5:0.5 / 8.5:1.5) to obtain 20 (6.2 mg). Fraction C5 (5.0 g) was subjected to RP-C18 CC, then being eluted with a gradient mixture of MeOH:H2O (5:5, 6:4, 7:3 and 10:0), according to differences in composition monitored by TLC yielding 9 subfractions. Subfraction C5.6 (97.2 mg) was isolated by silica gel CC (CHCl3:MeOH:H2O, 8.5:1.5:0.1) to yield 3 (6.3 mg) and 4 (29.7 mg). Fraction C6 (3.20 g) was subjected to silica gel CC eluting with CHCl3:MeOH (80:1, 50:1, 30:1, 20:1 and 10:1) to yield 6 fractions C6.1-C6.6. C6.5 (1.2 g) and further separated using silica gel CC to afford 3 mixtures with (CHCl3:MeOH:H2O, 8.5:1.5:0.1) as the elution solvent. The second mixture was purified by Sephadex LH20 CC (CHCl3:MeOH, 1:1) to yield 12 (780 mg). Fraction C7 (4.02 g) was separated by silica gel CC (CHCl3:MeOH, 7:1, 6:1 and 4:1) into 13 fractions, the tenth fraction was isolated by silica gel CC (CHCl3:MeOH:H2O, 10:3:0.5) to obtain 15 (15 mg). 4.4. Acid hydrolysis Compound 3 (2 mg) was dissolved in 2 N HCl in dioxane-H2O (1:1, 1 mL) and heated at 90  C for 4 h. The reaction mixture was diluted with H2O 1 mL, neutralized with aqueous NH4OH (28%) and extracted with EtOAc (4  2 mL). The aqueous layer was dried and dissolved in pyridine (1 mL). The reaction mixture was heated at 60  C for 1 h after the addition of 0.1 M cysteine methyl ester hydrochloride in pyridine (1 mL). Phenyl isothiocyanate in pyridine (10 mg/mL, 1 mL) was added and heated at 60  C for 1 h. The mixture was filtered and analyzed by reversed-phase HPLC. CH3CN with 0.1% HOAc (A) and H2O with 0.1% HOAc (B) were used as mobile phases at a flow rate of 1 mL/min with the following gradient: 10% A for 20 min and 55% A for 25 min. Chromatographic peaks were detected at 250 nm. Compound 4 was treated similarly.

The standard sugar (Sigma-Aldrich) derivatives were prepared identically and analyzed by HPLC under similar conditions. A pair of isomers (major & minor) was detected in each case and D-glucose in both compounds was identified by comparing the retention times of their derivatives with those of authentic sugar samples [Dglucose: 12.5 min (minor)/15.3 min (major), L-glucose: 13.1 min (minor)/15.1 min (major)]. 4.5. Cytotoxic assay The inhibition ratio reflecting the cytotoxicity of the compounds was assessed through an MTT assay (Yue et al., 2008). Mouse leukemia p388D1 cancer cells (Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum. The cancer cells (1  104 cells/mL, 90 mL each well) were then incubated in 96-well culture plates for 24 h, and treated with certain concentrations of the compounds for an additional 72 h. At the end of the incubation, 20 mL of the dye MTT (5 mg/mL) was added to each well, and the plates were incubated for 4 h at 37  C. Then 100 mL of lysis buffer (20% SDS in 50%, N-dimethylformamide containing 0.5% (v/v) AcOH:H2O (80:20 v/v) and 0.4% (v/v) 1 N HCl) was added to each well and incubated overnight. The optical density was assessed with a Bio-Rad 550 microplate reader at 595 nm. Positive controls (cells with Taxol), negative controls (cells only), and blank controls (0.1% DMSO in culture medium only) were set up under the same conditions. IC50 values were determined graphically for each experiment using the curvefitting routine of the Prism software (Graph Pad Software, Inc., USA). 4.6. Assay for the inhibition of iNOS activity The assay was performed in mouse macrophages (RAW264.7). The cells were cultured in phenol red-free RPMI medium with 10% bovine calf serum, 100 U/mL penicillin G sodium, and 100 mg/mL streptomycin. Cells were seeded in 96-well plates (100 000 cells/ well) and incubated for 24 h for a confluence of 75% or more. Then the test compounds were added 30 min later, LPS (5 mg/mL) was added and further incubated for 24 h. The activity of iNOS was determined in terms of the concentration of NO by measuring the level of nitrite in the cell culture supernatant using Griess reagent (Sigma-Aldrich). The percent inhibition of nitrite production by the test compound was calculated in comparison to the vehicle control. Parthenolide was used as the positive control, with IC50 values obtained from dose-response curves (Zhao et al., 2014). 4.7. Phocantol (1) Colorless powder. IR (THF): 2957, 1535, 1456, 1261, 1095, 1042 cm1. For 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSOd6, 125 MHz) spectroscopic data see Table 1. HRESIMS (positive): m/ z 271.0999, [MþH]þ(calcd. for C16H15O4: 271.0970). 4.8. Phocantone (2) Dark purple powder. IR (THF): 2961, 1635, 1541, 1432, 1260, 1045 cm1. For 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) spectroscopic data see Table 1. HREIMS (positive): m/z 286.0832 [M]þ (calcd. for C16H14O5: 286.0841). 4.9. Phocantoside A (3) Light brown gum. [a]20D: 28 (c 0.1, THF). IR (THF): 3370, 2924, 1457, 1375, 1263, 1092 cm1. For 1H NMR (pyridine-d5, 500 MHz)

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and 13C NMR (pyridine-d5, 125 MHz) spectroscopic data see Table 2. HRESIMS (positive): m/z 509.2960, [MþNa] þ(calcd. for C26H46O8 Na: 509.3090). 4.10. Phocantoside B (4) Colorless gum. [a]20D: 37 (c 0.1, THF). IR (THF): 3365, 2924, 1458, 1261, 1083, 1025 cm1. For 1H NMR (pyridine-d5, 500 MHz) and 13C NMR (pyridine-d5, 125 MHz) spectroscopic data see Table 2. HRESIMS (positive): m/z 653.3499, [MþNa]þ (calcd. for C32H54O12 Na: 653.3512). Acknowledgements This work was supported by the Natural Science Foundation of Hunan province (grant number 2016JJ6118), Scientific Research Fund of Hunan Provincial Education Department (grant number 15C1036), Aid Program for Science and Technology Innovative Research Team in Higher Educational Institutions of Hunan Province (grant number 2010/212), Hunan Department of Science and Technology (grant numbers 2014FJ1007 and 2014SK4037) and State Key Laboratory of Chinese Medicine Powder and Medicine Innovation in Hunan (Incubation) are acknowledged (grant number ZYFT201408). Partial support from the United States Food and Drug Administration (grant number U01-FD004246-01) is acknowledged. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.phytochem.2017.02.005. References Ageta, H., Shiojima, K., Suzuki, H., Nakamura, S., 1993. NMR spectra of triterpenoids. I: conformation of the side chain of hopane and isohopane, and their derivatives. Chem. Pharm. Bull. 41, 1939e1943. Ali, Z., Smillie, T.J., Khan, I.A., 2013. Two spirostan steroid glycoside fatty esters from Dioscorea cayenensis. Nat. Prod. Commun. 8, 323e326. Aves, J.S., Castro, J.C.M., Freire, M.O., Cunha, E.V.L., Barbosa-Filho, J.M., Silva, M.S., 2000. Complete assignment of the 1H and 13C NMR spectra of four triterpenes of the ursane, artane, lupane and friedelane groups. Magn. Reson. Chem. 38, 201e206. Bandi, A.K.R., Lee, D.U., 2011. Chemical constituents and bioactivities of plants from the genus Pholidota. Chem. Biodivers. 8, 1400e1409. Bi, Z.M., Zhou, X.Q., Wang, Z.T., 2008. A new phenanthrene derivative from Pholidota yunnanensis. Chin. Pharm. J. 43, 576e577. Bl€ as, B., Zapp, J., Becker, H., 2004. ent-clerodane diterpenes and other constituents from the liverwort Adelanthus lindenbergianus (lehm.) mitt. Phytochemistry 65, 127e137. Chen, X.Q., 1999. Flora of China (18). Science Press, Beijing, pp. 386e400. Chen, X.J., Mei, W.L., Zuo, W.J., Zeng, Y.B., Guo, Z.K., Song, X.Q., Dai, H.F., 2013. A new antibacterial phenanthrenequinone from Dendrobium sinense. J. Asian Nat. Prod. Res. 15, 67e70. Cheng, Q.F., Wei, M.Z., Guo, W.Q., 2000. New bibenzyl and phenanthrenedione from Dendrobium densiflorum. Chin. Chem. Lett. 11, 705e706. Cifuente, D.A., Borkowski, E.J., Sosa, M.E., Gianello, J.C., Giordano, O.S., Tonn, C.E., 2002. Clerodane diterpenes from Baccharis sagittalis: insect antifeedant activity. Phytochemistry 61, 899e905. Dong, F.W., Fan, W.W., Xu, F.Q., Wan, Q.L., Su, J., Li, Y., Zhou, L., Zhou, J., Hu, J.M., 2013. Inhibitory activities on nitric oxide production of stilbenoids from Pholidota yunnanensis. J. Asian Nat. Prod. Res. 15, 1256e1264. Georges, P., Sylvestre, M., Ruegger, H., Bourgeois, Paul, 2006. Ketosteroids and hydroxyketosteroids, minor metabolites of sugarcane wax. Steroids 71, 647e652. Kalpoutzakis, E., Aligiannis, N., Skaltsounis, A.L., Mitakou, S., 2003. cis-Clerodane type diterpenes from Cistus monspeliensis. J. Nat. Prod. 66, 316e319. Kim, H., Ralph, J., Lu, F., Ralph, S.A., Boudet, A.M., MacKay, J.J., Sederoff, R.R., Ito, T., Kawai, S., Ohashi, H., Higuchi, T., 2003. NMR analysis of lignins in CAD-deficient plants. Part 1. Incorporation of hydroxycinnamaldehydes and hydroxybenzaldehydes into lignins. Org. Biomol. Chem. 1, 268e281.

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Please cite this article in press as: Li, B., et al., Chemical constituents of Pholidota cantonensis, Phytochemistry (2017), http://dx.doi.org/10.1016/ j.phytochem.2017.02.005