Chalcane–stilbene conjugates and oligomeric flavonoids from Chinese Dragon’s Blood produced from Dracaena cochinchinensis

Phytochemistry xxx (2015) xxx–xxx

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Chalcane–stilbene conjugates and oligomeric flavonoids from Chinese Dragon’s Blood produced from Dracaena cochinchinensis Qian Hao a, Yoshinori Saito a, Yosuke Matsuo a, Hai-Zhou Li b, Takashi Tanaka a,⇑ a b

Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-Machi, Nagasaki 852-8521, Japan Faculty of Life Science and Technology, Kunming University of Science and Technology, Yunnan 650500, People’s Republic of China

a r t i c l e

i n f o

Article history: Received 27 December 2014 Received in revised form 27 September 2015 Accepted 30 September 2015 Available online xxxx Keywords: Dracaena cochinchinensis Asparagaceae Chinese Dragon’s Blood Chalcane Stilbene Flavonoid

a b s t r a c t A detailed chemical investigation of Chinese Dragon’s Blood, which is a traditional medicine produced form the red resin of Dracaena cochinchinensis, yielded two chalcane–stilbene conjugates, named cochinchinenenes G and H, together with 25 known compounds. The structures of these compounds were determined by spectroscopic examination. HPLC analysis of the resin indicated that the major constituents were a complex mixture of oligomeric polyphenols, which were detected as a broad hump on the base line of a HPLC chromatogram. 13C NMR analysis indicated that the oligomers were mainly composed of oxygenated chalcane units. This suggestion was supported by the results of a thiol degradation experiment with mercaptoethanol, which yielded a thioether of 4-[(4-hydroxyphenyl)propyl]3-methoxyphenol. Furthermore, methylation followed by electrospray ionization mass spectroscopic analysis of the resulting fractions established the presence of at least one heptamer of chalcane units. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Dragon’s Blood is the name given to the traditional medicines produced from the red resins of more than 20 different plant species belonging to four distinct genera, including Daemonorops, Dracaena, Croton and Pterocarpus (Gupta et al., 2008; Li et al., 2014), which have been used in many cultures throughout the world for centuries (Gupta et al., 2008). Chinese Dragon’s Blood is the red resin obtained from Dracaena cochinchinensis, and this material has been shown to promote blood circulation and alleviate inflammation, as well as being used to treat stomach ulcers, diarrhea, diabetes and bleeding (Zheng et al., 2006; Gupta et al., 2008; Li et al., 2014). D. cochinchinensis is an evergreen tree or shrub that is native to the tropical regions of southwestern China, Myanmar and Laos (Zheng et al., 2012a), and the resin of this plant is produced by the artificial wounding of its stems (Wang et al., 2011). Previous phytochemical studies of this resin have shown that it mainly contains phenolic compounds, including flavonoids and stilbenoids, as well as several steroids (Zhu et al., 2007). However, the major phenolic constituents of this resin have not yet been fully characterized because of the difficulties associated with the separation of these compounds. Interestingly, the composition of the phenolic substances in this resin was found to be dramati-

⇑ Corresponding author. E-mail address: [email protected] (T. Tanaka).

cally different from that of the fresh plant, which was attributed to the production and conversion of the simple flavonoids with endogenous enzymes and/or the metabolism by exogenous microorganisms during the production of the resin (Wang et al., 2011; Zheng et al., 2004a, 2012a). Most notably, the levels of oligomeric polyphenols increased remarkably during the production of the resin. In this study, isolation and structural elucidation gave two new phenolic metabolites (1 and 2) and 25 known compounds (3–27) from Chinese Dragon’s Blood. Furthermore, this is the first reported chemical and spectroscopic characterization of the oligomeric polyphenols from Chinese Dragon’s Blood, which account for over 50% of the resin by weight.

2. Results and discussion Chromatographic separation of the MeOH soluble part of the red resin of D. cochinchinensis led to isolation of 27 compounds, including two new compounds and 25 known compounds (3–27). The known compounds were identified through a comparison of their spectroscopic data with those reported in the literature (Figs. 1 and S2 in the Supplementary material). Compound 1 was obtained as a yellow amorphous powder, and its molecular formula was determined to be C33H34O6 by highresolution fast-atom-bombardment mass spectrometry (HR-FABMS) in the positive ion mode, which showed the [M+H]+ peak at m/z 527.2437 (calcd for C33H35O6: 527.2434). The 1H NMR and

http://dx.doi.org/10.1016/j.phytochem.2015.09.009 0031-9422/Ó 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Hao, Q., et al. Chalcane–stilbene conjugates and oligomeric flavonoids from Chinese Dragon’s Blood produced from Dracaena cochinchinensis. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.09.009

2

Q. Hao et al. / Phytochemistry xxx (2015) xxx–xxx

H3CO

5

3 2

OCH3 H β'

1

γ'

α'

α

β

4'' 1''

OH

4'

HO

α

1' 2''

3

OCH3

OH H β' γ'

5

1'

4''

α'

OH

4''' 4'

HO

2 1

β

OCH3

1''

H3CO

OH H

OH

2''

O

OCH3

OCH3 4'''

OH

OH

OH

OH 1

2

3 OH

OH

H3CO

H3CO

OH

HO

HO

OH

OCH3

OCH3

OH

SCH2CH2(SCH2CH2)nOH

SCH2CH2OH

H3CO

3b : n=1 3c : n=2

3a

OH HO

OH

OH

OH

HO

OCH3

OCH3

OH SCH2CH2SCH2CH2OH

HO

O

OH

HO OH 4

OH 4a

OH putative structure of oligomeric flavonoids

Fig. 1. Structures of compounds 1–4, thiol degradation products and a plausible partial structure for the oligomer.

1 H–1H COSY spectra of 1 showed two pairs of two-proton doublets [d 7.13 (2H, d, J = 8.2 Hz), 6.73 (2H, d, J = 8.2 Hz), 7.13 (2H, d, J = 8.2 Hz), 6.73 (2H, d, J = 8.2 Hz)], which were consistent with two 1,4-disubstituted aromatic rings (Table 1). Furthermore, a set of ABX coupled signals was observed at d 6.71 (1H, d, J = 8.2 Hz), 6.33 (1H, d, J = 2.3 Hz) and 6.23 (1H, dd, J = 8.2, 2.3 Hz), which were attributed to a 1,2,4-trisubstituted aromatic ring. Two meta-coupled signals were also noted at d 6.64 (1H, d, J = 1.8 Hz) and 6.42 (1H, d, J = 1.8 Hz), which were consistent with the occurrence of a 1,2,3,5-tetrasubstituted aromatic ring. Besides the aromatic protons, signals attributable to an isolated trans-alkene group were observed at d 6.94 (1H, d, J = 16.0 Hz) and 6.70 (1H, d, J = 16.0 Hz). Aliphatic proton signals belonging to two methylene groups [d 2.29 (2H, m), 2.47 (2H, m)], one methine group [d 4.54 (1H, br s)] and four methoxy groups [d 3.77, 3.68, 3.67, 3.61, each (3H, s)] were also observed in the 1H NMR spectrum of 1. The 13C NMR and heteronuclear single-quantum coherence (HSQC) spectra of 1 contained signals consistent with the presence of 11 aromatic quaternary carbons, including six oxygen bearing carbons, 13 aromatic methine carbons, three aliphatic methine carbons, two aliphatic methylene carbons and four methoxy carbons (Table 1). The 1H-detected heteronuclear multiple-bond correlation (HMBC) spectrum of 1 (Fig. 2) had correlations between the trans-alkene sp2 protons and the p-hydroxybenzene and tetrasubstituted phenyl rings, which were consistent with the presence of a stilbene unit. The presence of an aliphatic chain composed of C-a0 , C-b0 and C-c0 moieties was also apparent from 1H–1H COSY correlations, and the connectivity of the 2-methoxy-4-hydroxybenzene group to the C-a0 moiety was established by the observation of HMBC correlations of H-a0 to the aromatic carbons. Although the H-c0 signal at d 4.54 showed no HMBC correlation because of its severe broadening, NOESY correlations from the H-c0 signal to the transalkene unit and p-methoxybenzene ring indicated that the aforementioned stilbene unit was connected to the C-c0 moiety of a chalcane unit. The location of the methoxy groups at the C-3, 5, 200 and 400 0 positions was achieved using HMBC correlations, and these assignments were confirmed by a NOESY experiment. Com-

pound 1 gave a very small specific rotation value ([a] 21 D 1.6°), which suggested that this compound was a racemic mixture. Based on these spectroscopic data, the structure of 1 was elucidated and named cochinchinenene G (Fig. 1). This compound was named in accordance with the system used previously to name similar compounds (Zhu et al., 2007). The severe broadening of the H-c0 and Cc0 signals in the NMR spectra of compound 1 was attributed to the restricted rotation of the bond between the C-2 and C-c0 carbons, which was caused by the presence of large substituents at the neighboring positions. The molecular formula of compound 2 was determined to be C30H28O6 by HR-FAB-MS in the positive ion mode, which gave an [M+H]+ peak at m/z 485.1961 (calcd for C30H29O6: 485.1964). The 1 H NMR spectrum of compound 2 was found to be similar to that of 1, showing two sets of A2B2-type aromatic signals belonging to p-oxygenated phenyl rings, a set of ABX-type aromatic signals consistent with a 1,2,4-trisubstituted phenyl ring and a two-proton singlet attributable to a symmetrical 1,3,4,5-tetrasubstituted phenyl ring (Table 1). The 1H NMR spectrum of compound 2 also contained signals consistent with a pair of trans-alkene protons [d 6.75 and 6.91 (each 1H, d, J = 16.2 Hz)], an aliphatic methine [d 4.57 (1H, t, J = 8.2 Hz, H-c0 )] and two methylene protons [d 2.36 (2H, m, H-a0 ) and 2.27 (2H, m, H-b0 )], which were similar to those found in compound 1. These similarities therefore suggested that compound 2 was also a chalcane–stilbene conjugate. An apparent difference between these compounds, however, was that the 1H NMR spectrum of compound 2 showed the presence of only one methoxy signal [d 3.73 (3H, s)], as well as the symmetric structure of the tetrasubstituted phenyl ring. The 13C NMR spectrum of compound 2 (Table 1) also supported these observations. The HMBC spectrum of compound 2 demonstrated that the aromatic carbons belonging to the symmetrical tetrasubstituted phenyl ring showed crosspeaks with the trans-alkene protons, which also correlated to the carbons of a p-hydroxybenzene ring; confirming the presence of a stilbene unit (Fig. 2). The absence of a correlation from the methoxy group to these two aromatic rings indicated that the stilbene unit was resveratrol (4). The HMBC correlation of the C-3, C-4,

Please cite this article in press as: Hao, Q., et al. Chalcane–stilbene conjugates and oligomeric flavonoids from Chinese Dragon’s Blood produced from Dracaena cochinchinensis. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.09.009

Q. Hao et al. / Phytochemistry xxx (2015) xxx–xxx Table 1 H-(500 MHz) and 13C-(125 MHz) NMR spectroscopic data for compounds 1 and 2 (in CD3OD)a. 1

1

2

1

13

6.42 (1H, d, J = 1.8 Hz)

140.4 125.2 160.5 99.0 160.1 104.2 126.4

H

1 2 3 4 5 6

a b 10 20 , 60 30 , 50 40 100 200 300 400 500 600

a0 b

0

c0

100 0 200 0 , 600 0 300 0 , 500 0 400 0 3-OCH3 5-OCH3 200 -OCH3 400 0 -OCH3

6.64 (1H, d, J = 1.8 Hz) 6.94 (1H, d, J = 16.0 Hz) 6.70 (1H, d, J = 16.0 Hz) 7.13 (2H, d, J = 8.2 Hz) 6.73 (2H, d, J = 8.2 Hz)

6.33 (1H, d, J = 2.3 Hz) 6.23 (1H, 2.3 Hz) 6.71 (1H, 2.47 (2H, 2.29 (2H, 4.54 (1H,

dd, J = 8.2, d, J = 8.2 Hz) m) m) br s)

7.13 (2H, d, J = 8.2 Hz) 6.73 (2H, d, J = 8.2 Hz) 3.67 3.77 3.61 3.68

(3H, (3H, (3H, (3H,

s) s) s) s)

C

131.2 130.6 128.7 116.4 158.0 122.8 159.7 99.7 157.5 107.4 131.1 29.1 34.0 40.1 138.5 129.6 114.1 158.6 55.6 55.7 55.5 55.6

1

13

H

6.44 (1H, s)

6.44 (1H, s) 6.75 (1H, d, J = 16.2 Hz) 6.91 (1H, d, J = 16.2 Hz) 7.32 (2H, d, J = 8.5 Hz) 6.74 (2H, d, J = 8.5 Hz)

6.34 (1H, d, J = 2.3 Hz) 6.26 (1H, 2.3 Hz) 6.84 (1H, 2.36 (2H, 2.27 (2H, 4.47 (1H,

dd, J = 8.2, d, J = 8.2 Hz) m) m) t, 8.2)

7.25 (2H, d, J = 8.2 Hz) 6.62 (2H, d, J = 8.2 Hz)

3.73 (3H, s)

C

130.6 106.1 157.9 119.1 157.9 106.1 127.0 128.3 137.8 128.6 116.4 158.1 123.8 159.6 99.6 157.3 107.4 131.1 29.7 34.5 41.0 138.3 130.2 115.2 155.6

55.6

a Chemical shifts are given d values, multiplicities and coupling constants (J in Hz) in parentheses.

and C-5 positions of the resveratrol unit with the aliphatic methine proton (d 4.47, H-c0 ) indicated that the chalcane unit was attached to C-4 of the resveratrol moiety. The position of the methoxy group at C-200 of the trisubstituted phenyl ring was determined based on its HMBC correlation (Fig. 2). Furthermore, the connection of the Ca0 methylene to C-100 of the trisubstituted phenyl ring and the linkage of C-c to the other p-hydroxybenzene ring was also confirmed by HMBC and NOESY experiments (Fig. 2). This compound was also considered to be a racemic mixture because of its small specific rotation value ([a] 21 D 0.7°). Taken together, these data indicated that the structure of 2 was as shown in the Fig. 1, and the compound was subsequently named cochinchinenene H (Fig. 1). HPLC analysis of Chinese Dragon’s Blood showed that the major constituents of this resin were detected as a large, broad hump on the base line of the chromatogram (Figs. 3A and S1 Supplementary material). The chromatographic properties of this resin were simi-

3

lar to those of the black tea thearubigins (Haslam, 2003; Kusano et al., 2008) and polymeric proanthocyanidins (Kusano et al., 2011), which are both polymers of coexisting monomeric flavan3-ols. The techniques used for characterization of the polymers identified in our previous studies were applied to the oligomeric substance found in Dragon’s Blood. Fraction 5, which was strongly adsorbed on Sephadex LH-20 gel and subsequently eluted with aqueous acetone (Fig. 3A), contained only the oligomeric substances that were detected as a broad hump on the base line of the HPLC chromatogram. The 13C NMR spectrum of this material exhibited broad peaks attributable flavonoid moieties, which were closely related to those of the chalcane unit of 3, a chalcane–chalcone dimer isolated in this study. This observation strongly suggested that this oligomer was mainly composed of chalcane units. Furthermore, the expansion of the spectrum displayed very small signals attributable to a conjugated carbonyl carbon (d 202) and an oxygenated aromatic carbon (d 163) (arrows in Fig. 3A), which were similar to those of the 4-hydroxyphenyl-keto moiety belonging to the chalcone unit of 3. This result therefore indicated that chalcone units bearing carbonyl group were also contained in the oligomers, although their contribution to the oligomerization was apparently very low. Signals characteristic of the stilbene units found in compounds 1 and 2 were not observed in the 13C NMR spectrum of the oligomers. The presence of chalcane units in the oligomers was chemically supported by the thiol degradation reaction of the oligomeric fraction with mercaptoethanol under acidic conditions, which afforded thioethers 3a–c, and therefore confirmed the presence of 4-[(4-hydroxyphenyl)propyl]-3-methoxyphenol units in the oligomers. The yield of 3a–c was very low compared with that expected from the 13C NMR spectrum. This low yield was accounted for in the sense that this degradation method was not effective for the decomposition of the oligomers found in Dragon’s Blood. In addition, thioether 4a of resveratrol (4) was obtained more minor products, suggesting that resveratrol also partly contributes to the oligomerization. This suggestion was consistent with the fact that the thiol degradation method is generally effective for proanthocyanidins with phloroglucinol-type A-rings and ineffective for those with resorcinol-type A-rings (Kusano et al., 2011). An attempt to analyze the oligomers directly by electrospray ionization mass spectroscopy (ESI-MS) failed to provide any meaningful information, and the fractions containing oligomers were subsequently methylated with diazomethane to reduce the variety of structural analogs. ESI-MS analysis of the methylated fractions indicated that they contained at least a heptamer of the flavonoid units (Fig. 4). However, it is likely that only a small number of the components in the methylated fractions possessed molecular weights low enough to be detected in this ESI-MS experiment. Based on these observations, it was possible to propose a plausible partial structure for the oligomeric flavonoid of Chinese Dragon’s Blood, which is shown in Fig. 1. This study therefore represents the first reported account of chemical and spectroscopic evidence

Fig. 2. Selected 1H–1H COSY, NOESY and HMBC correlations for 1 and 2.

Please cite this article in press as: Hao, Q., et al. Chalcane–stilbene conjugates and oligomeric flavonoids from Chinese Dragon’s Blood produced from Dracaena cochinchinensis. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.09.009

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Q. Hao et al. / Phytochemistry xxx (2015) xxx–xxx

solvent

A

HPLC of Fr. 5 CH-09 [MaxABS (200.0--650.0 nm)]

250000

200000

Intensity [µAU]

150000

100000

50000

-0

0.0

5.0

10.0

15.0

20.0

25.0 30.0 Retention Time [min]

35.0

40.0

45.0

50.0

D:\DC-NMR\20140520\Fr223367-Fr223359 ODS 26-44 C.als

PPM 210.0

200.0

190.0

180.0

170.0

160.0

150.0

140.0

130.0

B

120.0

3'/5' 2'/6' 2'''/6'''

110.0

100.0

90.0

80.0

70.0

γ

4'' 2 4''' 2'' 1''' 4 4'

50.0

40.0

30.0

20.0

10.0

20.0

10.0

0.0

3'''/5'''

-OCH3

6''

3

60.0

6,1' 1'' 5 1

5'' 3,3''

γ'

β

β' α' α

PPM 210.0

Fig. 3.

13

200.0

190.0

180.0

170.0

160.0

150.0

140.0

130.0

120.0

110.0

100.0

90.0

80.0

70.0

60.0

50.0

40.0

30.0

0.0

C NMR spectra of fraction 5 containing the oligomeric polyphenols and compound 3. (A) 13C NMR spectrum and HPLC profile of fraction 5. (B) 13C NMR spectrum of 3.

Fig. 4. ESI-MS analysis of fraction 5 containing oligomeric polyphenols and the possible structure of a pentamer.

Please cite this article in press as: Hao, Q., et al. Chalcane–stilbene conjugates and oligomeric flavonoids from Chinese Dragon’s Blood produced from Dracaena cochinchinensis. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.09.009

Q. Hao et al. / Phytochemistry xxx (2015) xxx–xxx

for the structural composition of the oligomeric flavonoids found in Chinese Dragon’s Blood. 3. Conclusion Prior to this investigation, 28 flavonoid and stilbene related compounds had been identified in Chinese Dragon’s Blood (Luo et al., 2011; Zheng et al., 2012a,b; Dai et al., 2012; Guan and Guo, 2012; Mei et al., 2013; Li et al., 2014). In the present study, two additional new chalcane–stilbene conjugates (1 and 2) were isolated together with 25 known compounds. Furthermore, oligomeric flavonoids, which have not been studied in any great detail to date, despite being the main constituents of the resin, were characterized by spectroscopic and chemical methods. The result of 13C NMR spectroscopic analysis and thiol degradation experiments indicated that the oligomer was mainly composed of 4-[(4-hydroxyphenyl)propyl]-3-methoxyphenol units. This study therefore represents the first reported chemical study of the oligomeric substances found in Chinese Dragon’s Blood and is not only important from the perspective of traditional medicine, but could also be important in the context of plant defense systems related to phytoalexin. Further studies towards developing an effective chemical mechanism for the oxidative oligomerization of chalcanes are currently underway in our laboratory. 4. Experimental 4.1. General experimental procedures IR and UV spectra were obtained with Jasco FT/IR-410 and Jasco V-560 UV/Vis spectrophotometers (Jasco Co., Tokyo, Japan). Optical rotations were measured on a Jasco DIP-370 digital polarimeter (Jasco Co.). 1H and 13C NMR spectra were acquired in CD3OD at 27 °C using a Varian Unity plus 500 spectrometer operating at 500 and 125 MHz for the 1H and for 13C spectra, respectively (Varian, Palo Alto, CA, USA) or a JEOL JNM-AL 400 spectrometer operating at 400 and 100 MHz for the 1H and 13C NMR, respectively (JEOL Ltd., Tokyo, Japan). Coupling constants (J) have been expressed in Hz, and chemical shifts (d) are provided on the ppm scale. ESI-MS were obtained using a JEOL JMS-T100TD spectrometer (JEOL Ltd.). HR-FAB-MS were recorded on a JMS 700 N spectrometer (JEOL Ltd.), using m-nitrobenzyl alcohol or glycerol as the matrix. Column chromatography (CC) was conducted over Diaion HP20SS (Mitsubishi Chemical Co., Tokyo, Japan), Sephadex LH-20 (25–100 mm, GE Healthcare UK Ltd., Little Chalfont, UK), Chromatorex ODS (Fuji Silysia Chemical Ltd., Kasugai, Japan) and silica gel 60N (100–210 lm, Kanto Chemical Co., Tokyo, Japan) columns. TLC was performed on precoated Kieselgel 60 F254 plates (0.2 mm thick, Merck, Darmstadt, Germany) with CHCl3–MeOH– H2O (90:10:1 – v/v) mixtures being use as the eluents. The spots on the TLC plates were detected by UV illumination (254 nm) and spraying with a 2% ethanolic FeCl3 and 10% sulfuric acid reagent, followed by heating. Preparative HPLC was performed on a Cosmosil 5C18-PAQ column (250  20 mm, i.d., Nacalai Tesque Inc., Kyoto, Japan) with CH3CN-H2O. Analytical HPLC was performed on a Cosmosil 5C18-AR II column (250  4.6 mm, i.d., Nacalai Tesque Inc.) with a gradient elution of CH3CN in 50 mM H3PO4 from 10% to 30% over 30 min, followed by 30% to 75% over 15 min at a flow rate of 0.8 mL/min. The eluted compounds were detected with a Jasco MD-910 photodiode array detector. 4.2. Plant material Resin of D. cochinchinensis was purchased at a local market in Yunnan Province, China, in 2012, and identified by Prof. Hai-Zhou

5

Li at Kunming University of Science and Technology, China. The resin was obtained by extraction of artificially wounded section of the stem of D. cochinchinensis with EtOH (Yi et al., 2011). A voucher specimen (RDC201301) is deposited in the Laboratory of Natural Product Chemistry, Graduate School of Biomedical Sciences, Nagasaki University, Japan. 4.3. Extraction and isolation Resin (450 g) of D. cochinchinensis was dissolved in MeOH and any insoluble materials were removed by filtration. The MeOH soluble part (432 g) was partitioned between hexane and MeOH. The MeOH layer (414.89 g) was separated by Sephadex LH-20 CC (40  10 cm, i.d.) EtOH: H2O: acetone (1:0:0, 9:1:0, 8:2:0, 6:4:0, 0:1:1, v/v) to give five fractions, Fr. 1 (20.8 g), Fr. 2 (292.0 g), Fr. 3 (70.1 g), Fr. 4 (16.3 g) and Fr. 5 (14.2 g). Fr. 2 was suspended in CHCl3 to give a CHCl3 soluble part (Fr. 2–1, 64.0 g) and insolubles (Fr. 2–2, 228.0 g). The CHCl3 soluble part was separated by silica gel CC (20  8 cm, i.d., CHCl3–MeOH 100:0–0:100, v/v) into five fractions, Frs. 2–1–1 (18.5 g), 2–1–2 (1.24 g), 2–1–3 (8.9 g), 2–1–4 (8.9 g) and 2–1–5 (18.2 g). Fr. 2–1–2 was subjected to further purification by successive rounds of CC over Chromatorex ODS (H2O–MeOH), silica gel (hexane–EtOAc) and preparative HPLC to give 1 (68.8 mg), cochinchinenene A (7) (7.1 mg) (Zhu et al., 2007), 7,40 -dihydroxy8-methylflavane (14) (89.9 mg) (Ioset et al., 2001), 5,40 -dihydroxy7-methoxy-6-methylflavane (15) (69.8 mg) (Zheng et al., 2004b) and 7,40 -homoisoflavane (16) (18.0 mg) (Zheng et al., 2006). The purification of Fr. 2–1–4 by successive rounds of CC over MCI gel (H2O–MeOH), Sephadex LH-20 (H2O–MeOH), silica gel (hexane– EtOAc), Chromatorex ODS (H2O–MeOH) and preparative HPL C gave loureirin C (9) (49.9 mg) (Zheng et al., 2006), 2,6-dimethoxy-4,40 -dihydroxydihydrochalcone (10) (22.1 mg) (Ichikawa et al., 1997), 20 -methoxy-40 ,4-dihydroxychalcone (11) (28.8 mg) (Kajiyama et al., 1992), 30 -methoxy-40 ,4-dihydroxychalcone (12) (30.1 mg) (Arty et al., 2000), 10,11-dihydroxydracaenone C (17) (19.1 mg) (Zheng et al., 2006), 7,40 -dihydroxyflavanone (18) (6.7 mg) (Zhou et al., 2001a), 7,40 -dihydrohomoisoflavanone (19) (9.0 mg) (Zheng et al., 2006), 40 -methoxy-30 ,7-dihydroxyflavone (20) (16.8 mg) (Wei et al., 1998), ()-(70 S,8S,80 R)-4,40 -dihydroxy3,30 ,5,50 -tetramethoxy-70 ,9-epoxylignan-90 -ol-7-one (22) (5.7 mg) (Xiong et al., 2011), secoisolariciresinol (23) (7.3 mg) (Wang et al., 2006), dihydrodehydroconiferyl alcohol (24) (7.5 mg) (Wang et al., 2010) and 5-methoxydihydrodehydroconiferyl alcohol (25) (7.6 mg) (Deyama et al., 1987). A portion (145.0 g) of the CHCl3 insoluble part (Fr. 2–2) was subjected purified by CC over a Sephadex LH-20 column (40  10 cm, i.d.), MeOH: H2O: acetone (6:4:0, 8:2:0, 1:0:0, 0:4:6, 0:2:8, v/v) to give five fractions. Fr. 2–2–1 (26.0 g) and Fr. 2–2–2 (37.1 g) contained steroids, and Fr. 2–2–3 (65.6 g), Fr. 2–2–4 (14.3 g) and Fr. 2–2–5 (0.6 g) contained phenolic substances. Fr. 2–2–1 was purified by silica gel CC eluting with CHCl3–MeOH–H2O, followed by purification of Chromatorex ODS to yield spirosta-5,25(27)-diene-lb,3b-diol (neoruscogenin) 1-O-[O-a-L-rhamnopyranosyl-(1,2)-a-L-arabinopyranoside] (26) (36.7 mg) (Mimaki et al., 1996) and (25R)-spirost-5-en-3-ol-3O-a-L-rhamnopyranosyl-(1,2)-[b-D-glucopyranosyl-(1,3)]-b-D-glucopyranoside (27) (9.8 mg) (Zheng et al., 2004a). Fr. 2–2–3 was purified over a Diaion HP 20SS column (35  7 cm, i.d.), MeOH: H2O (6:4:0, 8:2:0, 1:0:0, v/v) to give 12 subfractions. Subfraction 2–2–3–1 (1.45 g) was purified by silica gel CC (3 cm i.d.  15 cm) with CHCl3–MeOH (20:1–4:1, v/v) to yield resveratrol (4) (660.8 mg) (Hu et al., 2011). Subfraction 2–2–3–2 (3.72 g) was repeatedly subjected to CC using Sephadex LH-20, silica gel and Chromatorex ODS to yield 2 (4.6 mg), cochinchinenene D (6) (136.6 mg) (Zhu et al., 2007), cochinchinenin (5) (6.1 mg) (Zhou et al., 2001b) and 7,40 -dihydroxyflavone (21) (126.4 mg) (Tu et al., 2003). Subfraction 2–2–3–3 (5.31 g) was purified by sequential CC

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Q. Hao et al. / Phytochemistry xxx (2015) xxx–xxx

over Sephadex LH-20, silica gel and Chromatorex ODS columns to yield cochinchinenene C (7) (162.8 mg) (Zhu et al., 2007), 1-[5-(2methoxy-4,40 -dihydroxydihydrochalconyl)]-1-(4-hydroxyphenyl)3-(2-methoxy-4-hydroxyphenyl)propane (3) (53.8 mg) (Tobinaga et al., 2006) and 3-methyl resveratrol (13) (49.7 mg) (Orsini et al., 2004).

1.6 Hz, H-2 and H-6), 5.92 (1H, J = 1.6 Hz, H-4), 3.94 (1H, t, J = 8.4 Hz, –CH), 3.46 (2H, t, J = 6.4 Hz, –CH2OH), 2.77 (2H, m, –CH2), 2.43 (6H, m, –CH2S). 13C NMR (CD3OD, 100 MHz) d: 159.1 (C-3, C-5), 157.5 (C-40 ), 142.7 (C-1), 134.1 (C-10 ), 130.2 (C-20 , C-60 ), 116.1 (C-30 , C-50 ), 108.8 (C-2, C-6), 101.5 (C-4), 62.4 (–CH2OH), 51.9 (–CH), 44.5 (–CH2), 35.2, 33.0, 32.4 (–SCH2).

4.4. Thiol degradation

4.5. Methylation

For this chemical degradation experiment, Fr. 2–2–4 containing the oligomeric polyphenols identical to those in Fr. 5 was used. The identities of these fractions were confirmed by comparison of TLC, HPLC, and 13C NMR spectra. A portion (10.2 g) of Fr. 2–2–4 was dissolved in a solution consisting of 2-mercaptoethanol (50 mL), 0.3% HCl (400 mL) and EtOH (600 mL), and the resulting mixture was heated at 80 °C for 14 h. HPLC analysis of the reaction mixture showed one major peak (tR 17.3 min, Fig. S15), although a large broad hump was observed on the baseline of the chromatogram, which was attributed to the original oligomers remaining in the chromatogram. After concentration, the mixture was purified over a Sephadex LH-20 column eluting with EtOH to give fractions containing a mixture of degradation products (2.46 g) and oligomeric materials (4.79 g). The first fraction was further purified by CC over silica gel eluting with CHCl3–MeOH–H2O (90:10:1, 85:15:1 and 80:20:2 – v/v/v) to give 7 fractions. Fr. 2–2–4–1–3 was subjected to Chromatorex ODS CC (50–100% MeOH) to give 3b (30.3 mg) and 3c (30.1 mg). Separation of Fr. 2–2–4–1–4 with Chromatorex ODS afforded 3a (41.6 mg). Similar separation of Fr. 2–2–4–1–6 gave 4a (7.8 mg). Thioether (3a): Yellow amorphous powder, ESI-MS m/z: 667 [2MH], 333 [MH], 255 [MHSCH2CH2OHH]; 1H NMR (CD3OD, 400 MHz) d: 7.11 (2H, d, J = 8.5 Hz, H-20 and H-60 ), 6.78 (1H, d, J = 8.0 Hz, H-6), 6.73 (2H, d, J = 8.5 Hz, H-30 and H-50 ), 6.35 (1H, d, J = 2.5 Hz, H-3), 6.26 (1H, dd, J = 2.5, 8.5 Hz, H-5), 3.71 (3H, s, OCH3), 3.68 (1H, br s, H-c), 3.46 (2H, t, J = 6.5 Hz, –CH2OH), 2.40 (4H, m, H-a, –CH2S), 2.00 (2H, m, H-b); 13C NMR (CD3OD, 100 MHz) d: 159.7, 157.9, 157.4 (C-2, C-4, C-40 ), 134.7 (C-10 ), 131.2 (C-6), 130.2 (C-20 , C-60 ), 121.9 (C-1), 116.1 (C-30 , C-50 ), 107.5 (C-5), 99.8 (C-3), 62.3 (CH2OH), 55.6 (OCH3), 49.7 (C-c), 38.0 (C-b), 33.9 (CH2S), 28.9 (C-a). Thioether (3b): Yellow amorphous powder, ESI-MS m/z: 787 [2MH], 393 [MH], 255 [MHSCH2CH2SCH2CH2OHH]; 1H NMR (CD3OD, 400 MHz) d: 7.09 (2H, d, J = 8.4 Hz, H-20 and H-60 ), 6.76 (1H, d, J = 8.4 Hz, H-6), 6.71 (2H, d, J = 8.4 Hz, H-30 and H-50 ), 6.32 (1H, d, J = 2.0 Hz, H-3), 6.23 (1H, dd, J = 2.4, 8.0 Hz, H-5), 3.70 (3H, s, OCH3), 3.66 (1H, m, H-c), 3.50 (2H, t, J = 6.4 Hz, –CH2OH), 2.45 (8H, m, H-a, –CH2S), 1.95 (2H, m, H-b). 13C NMR (CD3OD, 100 MHz) d: 159.6 (C-2), 157.9 (C-4), 157.4 (C-40 ), 134.5 (C-10 ), 131.2 (C-6), 130.2 (C-20 , C-60 ), 121.7 (C-1), 116.1 (C-30 , C-50 ), 107.5 (C-5), 99.7 (C-3), 62.3 (–CH2OH), 55.5 (–OCH3), 49.6 (C-c), 37.8 (C-b), 35.1, 33.1, 32.0 (–CH2S), 28.8 (C-a). Thioether (3c): Yellow amorphous powder, ESI-MS m/z: 907 [2MH ], 453 [MH ], 255 [MHSCH2CH2SCH2CH2SCH2CH2 OHH ]; 1H NMR (CD3OD, 400 MHz) d: 7.13 (2H, d, J = 8.4 Hz, H-20 and H-60 ), 6.78 (1H, d, J = 8.0 Hz, H-6), 6.74 (2H, d, J = 8.4 Hz, H-30 and H-50 ), 6.36 (1H, d, J = 2.4 Hz, H-3), 6.27 (1H, dd, J = 2.4, 8.0 Hz, H-5), 3.72 (3H, s, OCH3), 3.69 (1H, m, H-c), 3.65 (2H, t, J = 6.8 Hz, –CH2OH), 2.54 (12H, m, H-a, –CH2S), 1.97 (2H, m, H-b). 13 C NMR (CD3OD, 100 MHz) d: 159.6 (C-2), 157.9 (C-4), 157.5 (C-40 ), 134.5 (C-10 ), 131.3 (C-6), 130.2 (C-20 , C-60 ), 121.7 (C-1), 116.2 (C-30 , C-50 ), 107.5 (C-5), 99.8 (C-3), 62.5 (–CH2OH), 55.6 (–OCH3), 49.6 (C-c), 37.8 (C-b), 35.3, 33.3, 33.1, 33.0, 32.0 (–SCH2), 28.9 (C-a). Thioether (4a): Yellow amorphous powder, FAB-MS m/z: 367 [M +H]+; 1H NMR (CD3OD, 400 MHz) d: 7.01 (2H, d, J = 8.4 Hz, H-20 and H-60 ), 6.61 (2H, d, J = 8.4 Hz, H-30 and H-50 ), 5.94 (2H, dd, J = 7.2,

Fraction 5 (100 mg) was dissolved in MeOH (5 mL) and treated with a solution of CH2N2 in ether at 0 °C for 12 h. The mixture was the concentrated on a rotary evaporator and analyzed by ESI-MS. 4.6. Cochinchinenene G (1) Yellow amorphous powder, [a]21 1.6° (c = 0.55, MeOH), D FAB-MS m/z: 527 [M+H]+; HR-FAB-MS m/z: 527.2437 [M+H]+ (calcd for C33H35O6: 527.2434); IR mmax cm1: 3388, 2999, 2937, 2834, 1599, 1511, 1462; UV kmax (MeOH) nm (log e): 299 (1.98), 287 (2.04), 218 (3.50); for 1H and 13C NMR spectroscopic data, see Table 1. 4.7. Cochinchinenene H (2) Yellow amorphous powder, [a]21 D 0.7° (c = 0.15, MeOH); FABMS m/z: 485 [M+H]+; HR-FAB-MS m/z: 485.1961 [M+H]+ (calcd for C30H29O6: 485.1964); IR mmax cm1: 3360, 2927, 1603, 1509, 1430, 1232, 1195, 1172; UV kmax (MeOH) nm (log e): 325 (1.78), 216 (2.42); for 1H and 13C NMR spectroscopic data, see Table 1. Acknowledgments The authors would like to express their gratitude to Mr K. Inada, Mr N. Yamaguchi and Mr N. Tsuda (Nagasaki University Joint Research Center) for conducting the NMR and MS measurements. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2015. 09.009. References Arty, I.S., Timmerman, H., Samhoedi, M., Sastrohamidjojo, S., Van der Goot, H., 2000. Synthesis of benzylideneacetophenones and their inhibition of lipid peroxidation. Eur. J. Med. Chem. 35, 449–457. Dai, H.F., Wang, H., Liu, J., Wu, J., Mei, W.L., 2012. Two new bioflavonoids from the stem of Dracaena cambodiana. Chem. Nat. Compd. 48, 376–378. Deyama, T., Ikawa, T., Kitagaw, S.A., Nishibe, S., 1987. The constituents of Eucommia ulmoides OLIV. V. Isolation of dihydroxydehydrodiconiferyl alcohol isomers and phenolic compounds. Chem. Pharm. Bull. 35, 1785–1789. Guan, J., Guo, S.X., 2012. Three new bioflavonoids from Chinese dragon’s blood, Dracaena cochinchinensis. Nat. Prod. Commun. 7, 591–594. Gupta, D., Bleakley, B., Gupta, R.K., 2008. Dragon’s blood: botany, chemistry and therapeutic uses. J. Ethnopharmacol. 115, 361–380. Haslam, E., 2003. Thoughts on thearubigins. Phytochemistry 64, 61–73. Hu, Y.Q., Tu, P.F., Li, R.Y., Wan, J., Wang, D.L., 2011. Studies on stilbene derivatives from Dracaena cochinchinensis and their antifungal activities. Chin. Tradit. Herbal Drugs 32, 104–106. Ichikawa, K., Kitaoka, M., Taki, M., Takashi, S., Iijima, Y., Boriboon, M., Akiyama, T., 1997. Retrodihydrochalcones and homoisoflavones isolated from Thai medicinal plant Dracaena loureiri and their estrogen agonist activity. Planta Med. 63, 540–543. Ioset, J.R., Marston, A., Gupta, M.P., Hostettmann, K., 2001. A methylflavan with free radical scavenging properties from Pancratium littorale. Fitoterapia 71, 35–39. Kajiyama, K., Demizu, S., Hiraga, Y., Kinoshita, K., Koyama, K., Takahashi, K., Tamura, Y., Okada, K., Kinoshita, T., 1992. Two prenylated retrochalcones from Glycyrrhiz inflata. Phytochemistry 31, 3229–3232. Kusano, R., Andou, H., Fujieda, M., Tanaka, T., Matsuo, Y., Kouno, I., 2008. Polymerlike polyphenols of black tea and their lipase and amylase inhibitory activities. Chem. Pharm. Bull. 56, 266–272.

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Please cite this article in press as: Hao, Q., et al. Chalcane–stilbene conjugates and oligomeric flavonoids from Chinese Dragon’s Blood produced from Dracaena cochinchinensis. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.09.009