English

• 1.   Introduction

  Nothotsuga longibracteata (W. C. Cheng) Hu ex C. N. Pagebelongs to Nothotsuga of Pinaceae, one of Chinese rare gymnosperms endemic plant. Nothotsuga contains only one species, N. longibracteata, commonly known as the Bristlecone Hemlock, Tsuga longibracteata, Tsugo-keteleeria longibracteata. It was an archaic plant of the quaternary glacial period, and distributed mainly in Hunan, Guangdong, Guangxi, Fujian and Guizhou provinces of China.^[1] Plants of this genus have long been used as a traditional herbal medicine to treat arthritis and stomach troubles.^[2] Previous chemical investigation of this plant afforded lanostane triterpenoids, lignans, flavonoids, sterols, and phenolic acids.^[3-4] Aiming to identify structurally interesting metabolites from Chinese endemic medical plants, ^[5-8] we conducted an intensive investigation into the constituents of the heartwood of N. longibracteata. In this study, we report the isolation and structure elucidation of a rare benzyl furanone derivate, 3-(4'-hydroxy-3'-methoxy- benzyl)-2(5H)-furanone (1), and two new lignans 2~3, together with nineteen known lignans compounds 4~22 (Figure 1). The structures of these new compounds were elucidated by spectroscopic evidences including HR-ESI- MS, 1D, 2D NMR, and CD spectroscopies. The cytotoxic effects of the isolated new compounds on three human tumour cell lines (A172, SHSY5Y, and Hela) were evaluated by the MTT assay. But their showed no cytotoxic activities at the concentration of 50 μmol/L. Gastrointestinal motility of zebrafish test showed that compound 3 has the function of promoting gastrointestinal motility of zebrafish to excrete Nile red at doses 8 and 24 μmol/L by means of acting on the cholinergic nervous system. We herein present the purification, structural elucidation, and biological evaluation of these isolated and identified lignans compounds.

  Figure 1

  

  Figure 1.  Chemical structures of compounds 1~22

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  2.   Results and discussion

  2.1   Structure identification of compound 1

  Compound 1 was obtained as a brown gum. With the molecular formula, C₁₂H₁₂O₄, which indicated 7 degrees of unsaturation, was established from the ion of protonated molecule peak at m/z 221.0809 [M+H]⁺ (calcd for C₁₂H₁₃O₄⁺: 221.0808) in the HR-ESI-MS. The IR spectrum of compound 1 indicated the presence of hydroxyl and carbonyl groups at 3426 and 1746 cm^－1. ¹H NMR, ¹³C NMR and heteronuclear single quantum correlation (HSQC) spectra (Table 1) of compound 1 showed the presence of a 1, 3, 4-trisubstituted benzyl unit at δ_H 6.72 (dd, J＝8.0, 1.8 Hz, 1H), 6.76 (d, J＝1.8 Hz, 1H), 6.86 (d, J＝8.0 Hz, 1H), 3.52 (br. s, 2H), and δ_C 129.4, 111.7, 146.8, 144.7, 114.7, 121.7, 31.7; a methylene group at δ_H 3.52 (2H, brs) and δ_C 31.7; an oxygenated methylene group at δ_H 4.75 (m, 2H) and δ_C 70.5; an olefinic bond at δ_H 6.95 (m, 1H) and δ_C 134.8, 145.6; a methoxyl group at δ_H 3.87 (3H, s) and δ_C 56.1; a carbonyl group at δ_C 174.2 and a hydroxyl group at δ_H 5.58. These NMR data above indicated that compound 1 might be a benzylbutyrolactone derivative.^[9] The heteronuclear multiple-bond correlations (HMBC) (Figure 2) of OCH₃ (δ_H 3.87) with C-3' (δ_C 146.8), 4'-OH (δ_H 5.58) with C-3', C-4' and C-5', and H-6' with C-4' (δ_C 146.8), indicated that the methoxyl group located at C-3' and hydroxyl group located at C-4'. The HMBC correlations of H-7' with C-1', C-2' and C-6', H-6' with C-7', and H-2' with C-7' confirmed the presence of a benzyl moiety in the molecule. The HMBC correlations of H-4 with C-2 and C-5, H-5 with C-2, C-3 and C-4 established the presence of a furanone ring. Further, the connection of the benzyl moiety and the furanone ring by C-3 and C-7', was established based on the HMBC correlations of H-7' with C-2, C-3 and C-4, and H-4 with C-7'. Therefore, the structure of compound 1 was established as 3-(4'-hydroxy-3'- methoxy-benzyl)-2(5H)-furanone.

  Table 1

  

  Table 1.  ¹H NMR and ¹³C NMR spectral data of compounds 1 and 2 (400/100 MHz)

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  Position	1 (CDCl3)			2 (CD3OD)
  	δH (J in Hz)	δC		δH (J in Hz)	δC
  1					133.3
  2		174.2		6.62 (br. s, 1H)	113.3
  3		134.8			148.9
  4	6.94~6.96 (m, 1H)	145.6			145.7
  5	4.74~4.76 (m, 2H)	70.5		6.69 (overlapped, 1H)	115.9
  6				6.54 (overlapped, 1H)	122.8
  7				2.54~2.68 (m, 2H)	35.9
  8				2.12~2.21 (m, 1H)	40.5
  9				4.24 (dd, 11.1, 5.9, 1H); 4.01 (dd, 11.1, 6.5, 1H)	66.2
  1'		129.4			133.8
  2'	6.76 (d, 1.8, 1H)	111.7		6.56 (overlapped, 1H)	113.5
  3'		146.8			149.0
  4'		144.7			145.8
  5'	6.86 (d, 8.0, 1H)	114.7		6.59 (overlapped, 1H)	116.0
  6'	6.72 (dd, 8.1, 1.8, 1H)	121.7		6.54 (overlapped, 1H)	122.8
  7'	3.52 (br. s, 2H)	31.7		2.54~2.68 (m, 2H),	35.6
  8'				1.89~1.98 (m, 1H)	44.4
  9'				3.68 (dd, 10.8, 6.0, 1H); 4.01 (dd, 10.8, 6.5, 1H)	62.8
  1''					173.1
  2''				2.05 (s, 3H)	21.1
  3-OCH3				3.76 (s, 3H)	56.3
  3'-OCH3	3.87 (s, 1H)	56.1		3.77 (s, 3H)	56.3
  4'-OH	5.58 (s, 1H)

  Figure 2

  

  Figure 2.  Key ¹H-¹H COSY() and HMBC () correlations of compounds 1~3

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  2.2   Structure identification of compound 2

  Compound 2 was isolated as a brown gum, with the molecular formula of C₂₂H₂₈O₇ on the basis of HR-ESI-MS (m/z 427.1714 [M+Na]⁺, calcd for C₂₂H₂₈O₇Na 427.1733, ), which indicated 9 degrees of unsaturation. The IR spectrum of compound 2 indicated the presence of carbonyl group at 1767 cm^－1. The ¹H NMR, ¹³C NMR and HSQC spectra (Table 1) of compound 2 displayed two 1, 3, 4-trisubstituted benzyl units at δ_C 133.3, 113.3, 148.9, 145.7, 115.9, 122.8, and δ_C 133.8, 113.5, 149.0, 145.8, 116.0, 122.8; two aryl methoxyl groups at δ_H 3.76 (3H) and 3.77 (3H); an acetoxy group at δ_C 173.1 and 21.1; four methylene groups at δ_C 66.2, 62.8, 35.9, 35.6; two methine groups at δ_C 44.4, 40.5. These structural moieties would constitute an 8, 8'-lignane skeleton for compound 2, similar to that of secoisolariciresinol^[10] except for the substitution in the C-9. The structure of compound 2 was further supported by HMBC spectra (Figure 2). The HMBC correlations of H-9/C-1'', H-2''/C-1'' revealed the acetoxy group attached to C-9. The HMBC correlations of 3-OCH₃/C-3, H-5/C-1, H-5/C-3, 3'-OCH₃/C-3', H-5'/C-1', H-5'/C-3', showed the two methoxyl groups located at C-3 and C-3', respectively. The (8S, 8'S)-absolute stereochemistry was confirmed by the specific optical rotation and circular dichroic (CD) curve (positive Cotton effects at 228 and 291 nm) as those of 5-demethoxyniranthin.^[11] Therefore, the structure of compound 2 was established to be (8S, 8'S)- 3, 3'-dimethoxyligna-4, 4', 9, 9'-tetraol-9-actate. The plane structure of compound 2 was identified already from MS in the literature, ^[12] but the NMR data were no reported.

  2.3   Structure identification of compound 3

  Compound 3 was isolated as a yellow gum. Its molecular formula, C₂₄H₃₀O₇, which indicated 10 degrees of unsaturation, was established from the quasi-molecular ion peak at m/z 453.1880 [M+Na]⁺ (calcd for C₂₄H₃₀O₇Na 453.1889) in the HR-ESI-MS. The IR spectrum of compound 3 indicated the presence of carbonyl group at 1759 cm^－1. The ¹H NMR spectrum (Table 2) of compound 3 displayed two methoxyl signals at δ_H 3.79 (s, 3H) and 3.83 (s, 3H), an AMX aromatic system at δ_H 6.58 (d, J＝1.9 Hz, 1H), 6.64 (dd, J＝8.0, 1.9 Hz, 1H), 6.81 (d, J＝8.0 Hz, 1H), two aromatic protons at δ_H 6.29 (s, 1H) and 6.54 (s, 1H). The ¹³C NMR and HSQC spectra indicated a carbonyl at δ_C 175.8, 12 aromatic carbons at δ_C 110.1, 111.7, 114.5, 115.8, 122.6, 126.0, 131.6, 132.4, 144.0, 145.2 and 145.5, 146.9, two downfield methylene groups at δ_C 62.4 and 65.0, two methoxyl groups at δ_C 56.1 and 56.1, three methine carbons at δ_C 43.2, 46.7 and 46.9, three upfield methylene carbons at δ_C 19.3, 30.8 and 32.7, and a methyl carbon at δ_C 13.9. These NMR data suggested that compound 3 might be an aryltetrahydronaphthalene-type lignin.^{[13-14] 1}H-¹H correlated spectroscopy (COSY) (Figure 2) showed the presence of OCH₂CH₂CH₂CH₃ moiety. The HMBC correlations (Figure 2) from H-1'' to C-9 (δ_C 175.8), H-7 to C-9, established the location of OCH₂CH₂CH₂CH₃ moiety at C-9. The HMBC correlations from H-9' to C-7', C-8 and C-8' showed an OH attached to C-9'. The HMBC correlations from OCH₃-3' to C-3', H-5' to C-3', H-5' to C-1', confirmed the presence of OCH₃ at C-3'. Similarly, a methoxy group at C-5 was also assigned. The stereochemistry of different substituent groups on the tetrahydronaphthalene ring was designated using a nuclear overhauser enhancement spectroscopy (NOESY) experiment. The cross-peaks of H-7'/H-9', H-7'/H-8 and H-8/H-9' indicated that H-7', H-9' and H-8 were cofacial. The assignments were also buttressed by vicinal coupling constant value of H-7' and H-8' (J＝10.7 Hz) when the dihedral angle of H-7'-H-8' took value close to 180°. The absolute stereochemistry of (7'R, 8S, 8'S)-3 was established by the circular dichroism (CD) spectrum. The positive Cotton effects at 205, 228, 293 nm and the negative Cotton effects at 227, 239, 277 nm were very similar to urinatetralin, ^[11] and it was further confirmed by TD-DFT ECD calculations (Figure 3). In summary, compound 3 was determined to be (7'R, 8S, 8'S)- 3', 5-dimethoxy-2, 7'-cycloligna-4, 4', 9'-triol-9-acid butyl ester.

  Table 2

  

  Table 2.  ¹H NMR and ¹³C NMR spectral data of compound 3 (400/100 MHz, in CDCl₃)

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  Position	δH (J in Hz)	δC		Position	δH (J in Hz)	δC
  1		126.0		4'		144.5
  2		132.4		5'	6.81 (d, 8.0, 1H)	114.5
  3	6.29 (s, 1H)	115.8		6'	6.64 (dd, 8.0, 1.9, 1H)	122.6
  4		144.0		7'	3.91 (d, 10.7, 1H)	46.7
  5		145.2		8'	2.65, 2.92 (overlapped, 1H)	43.2
  6	6.54 (s, 1H)	110.1		9'	3.56 (dd, 11.8, 3.0, 1H) 3.43 (dd, 11.8, 3.5, 1H)	62.7
  7	3.21 (dd, 15.7, 11.5, 1H); 2.86~2.93 (overlapped, 1H)	32.7		1''	4.09~4.15 (m, 2H)	65.0
  8	2.08~2.17 (m, 1H)	46.9		2''	1.59~1.65 (m, 2H	30.8
  9		175.8		3''	1.33~1.42 (m, 2H	19.3
  1'		136.6		4''	0.92 (t, 7.4, 3H)	13.9
  2'	6.58 (d, 1.9, 1H)	111.7		5-OCH3	3.83 (s, 3H)	56.1
  3'		146.9		3'-OCH3	3.79 (s, 3H)	56.1

  Figure 3

  

  Figure 3.  Calculated ECD spectra of 7'R, 8S, 8'S-3

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  The known compounds were identified as matairesinol (4), ^[15] nortrachelogenin (5), ^[16] allo-7'-methoxymatairesi- nol (6), ^[17] 7R-ethoxymatairesinol (7), ^[18] 7'R-hydroxyma- tairesinol (8), ^[19] 7'S-hydroxymatairesinol (9), ^[19] tsugacetal (10), ^[20] 1-ethoxy-4-(4'-hydroxy-3'-methoxy-phenyl)-7-me- thoxy-1, 3, 3a, 4, 9, 9a-hexahydronaphtho[2, 3-c]furan-6-ol (11), ^[21] α-conidendrin (12), ^[22] 8-hydroxy-α-conidendrin (13), ^[23] 4, 4'-dihydroxy-3, 3'-dimethoxy-7-one-lignan-9, 9'- olid (14), ^[24] 8-hydroxyoxomatairesinol (15), ^[25] 8-hydroxy- α-conidendric acid methyl ester (16), ^[23] epinortrachelogenin (17), ^[26] isolariciresinol (18), ^[27] burselignan (19), ^[27] 3-(2, 4-dihydroxy-3-methoxybenzyl)-4-(4-hydroxy-3-meth-oxybenzy1)tetrahydrofuran (20), ^[28] cedrusin (21), ^[29] and 7'-hydroxyisolappaol A (22).^[30]

  All these new compounds were evaluated for their cytotoxicity against A172, SHSY5Y and Hela cells using MTT method. But these compounds are inactive (IC₅₀ > 50 μmol/L) for all cell lines. Compounds 1~3 were evaluated for their activity of promoting gastrointestinal motility of zebrafish treated with Nile red. Compound 3 significantly promoted the Nile red excretion at doses > 8 and > 24 μmol/L. Compound 3 promoted the gastrointestinal motility weaken, at 50 and 5 μmol/L atropine, compared with the test result no atropine at 50 μmol/L. These results showed that compound 3 might promote the gastrointestinal motility of zebrafish via acted on the cholinergic nervous system (Figure 4).

  Figure 4

  

  Figure 4.  (a) Effect of compound 3 on the gastrointestinal motility of zebrafish. (b) and (c) interfering effect of atropine on the gastrointestinal activation of compound 3. Indicates p < 0.001 when compared with the control

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  3.   Conclusions

  The phytochemical investigation of N. longibracteata gave a new benzyl furanone derivate (1), two new lignans 2~3 and 19 known lignans compounds 4~22. Compound 1 was a novel furanone derivate, and it was first isolated from plants as natural product skeleton as well as we know.^[9] Compound 1 maybe the degradation of benzyl product of dibenzylbutyrolactone lignan. Compound 3 has the function of promoting gastrointestinal motility of zeb- rafish to excrete Nile red at doses 8 and 24 μmol/L by means of acting on the cholinergic nervous system.

  4.   Experimental

  4.1   Instruments and reagents

  UV spectra were recorded on a Perkin-Elmer Lambda 35 UV-Vis spectrophotometer. CD spectra were recorded on a Chirascan circular dichroism spectrometer (Applied Photophysics Ltd, Leatherhead, U.K.). HR-ESI-MS spectra were measured on a Bruker MicrO TOF-Q II mass spectrometer. Optical rotations were determined on a Perkin-Elmer-241 polarimeter (Perkin Elmer, Inc., Waltham, MA, USA). IR spectra were measured on a PerkinElmer one FT-IR spectrometer (KBr). 1D NMR and 2D NMR spectra were recorded on a Bruker-AVANCE III-400 or an Agilent DD2400-MR instrument using TMS as the internal reference. Column chromatography was performed using silica gel (300~400 mesh, Qingdao marine Chemical Ltd., Qingdao, P. R. China). Semi-preparative high-pressure liquid chromatography (HPLC) was performed on a LC 3000 system (Beijing ChuangXingTongHeng Science and Technology Co., Ltd.) equipped with ODS column (5 μm, i.d. 10 mm×250 mm, YMC and H & E).

  4.2   Plant materials

  The heartwood of this plant was collected from Huang Sang Nature Reserve in Hunan province, China, in August 2016, and identified as Nothotsuga longibrteata (W. C. Cheng) Hu ex C. N. Page by Prof. Fa-Ming Wu (Zunyi Medical University). A voucher specimen (ZMC No. 20160814) was deposited at School of Pharmacy of Zunyi Medical University.

  4.3   Extraction and separation

  The air-dried and finely ground heartwood of N. longibracteata (5.0 kg) was extracted with 95% ethanol (25 L) for three times at room temperature (5 d each). The ethanol portion was evaporated under reduced pressure to get a crude extract (900 g), which was further suspended in warm distilled water (2.0 L) and extracted successively with petroleum ether, EtOAc, and n-BuOH (repeat each solvent 3 times), respectively. The EtOAc extract (205 g) was subjected to silica gel column chromatography (80 mm×600 mm, 400 g, 300~400 mesh), eluted with a gradient of petroleum ether-EtOAc (V:V＝10:1→1:2) to afford 6 fractions (Fr.1~Fr.6).

  Fr.4 was further separated by silica gel column chromatography to get 3 fractions (Fr.4.1~Fr.4.3). Fr.4.1 was purified by a Sephadex LH-20 column chromatography (MeOH) and divided into 8 subfractionses (Fr.4.1.1~Fr.4.1.8). Fr.4.1.3 was further purified by semi-preparative HPLC with MeOH-H₂O (V:V＝60:40) to yield compound 7 (t_R 10.3 min, 10.5 mg). Fr.4.1.5 was further purified by semi-preparative HPLC with MeOH-H₂O (V:V＝60:40) to get compounds 10 (t_R 9.7 min, 10.9 mg) and 11 (t_R 14.3 min, 14.1 mg). Fr.4.2 was purified by semi-preparative HPLC with MeOH-H₂O (V:V＝50:50) to yield 6 subfractionses (Fr.4.2.1~Fr.4.2.6). Fr.4.2.3 was further purified by semi-preparative HPLC with MeOH-H₂O (V:V＝58:42) to obtain compound 5 (t_R 8.7 min, 17.6 mg). Fr.4.2.5 was further purified by semi-preparative HPLC with MeOH-H₂O (V:V＝65:35) to obtain compound 4 (t_R 17.1 min, 30.6 mg). Fr.4.2.6 was repeated recrystallization to get compound 12 (20.5 mg).

  Fr.5 was further separated by silica gel column chromatography to get 2 fractions (Fr.5.1~Fr.5.2). Fr.5.1 was purified by a Sephadex LH-20 column chromatography (MeOH) and divided into 10 subfractionses (Fr.5.1.1~Fr.5.1.10). Fr.5.1.2 was further purified by semi-preparative HPLC with MeOH-H₂O (V:V＝60:40) to yield compounds 16 (t_R 10.3 min, 10.5 mg) and 6 (t_R 18.3 min, 10.5 mg). Fr.5.1.3 was further purified by semi-preparative HPLC with MeOH-H₂O (V:V＝55:45) to get compound 1 (t_R 9.7 min, 10.9 mg). Fr.5.1.6 was purified by semi- preparative HPLC with MeOH-H₂O (V:V＝40:60) to obtain compound 13 (t_R 24.3 min, 20.3 mg). Fr.5.2 was separated by semi-preparative HPLC with MeOH-H₂O (V:V＝23:77→62:38) to yield 7 subfractionses (Fr.5.2.1~Fr.5.2.7). Fr.5.2.2 was further purified by semi-preparative HPLC with MeOH-H₂O (V:V＝33:67) to obtain compounds 9 (t_R 27.1 min, 16.5 mg) and 8 (t_R 30.0 min, 14.9 mg). Fr.5.2.3 was further purified by semi-pre- parative HPLC with MeOH-H₂O (V:V＝42:58) to obtain compound 14 (t_R 24.1 min, 19.5 mg). Fr.5.2.4 was further purified by semi-preparative HPLC with MeOH-H₂O (V:V＝45:55) to get compound 17 (t_R 32.6 min, 17.5 mg). Fr.5.2.5 was further purified by semi-preparative HPLC with MeOH-H₂O (V:V＝42:58) to obtain compound 15 (t_R 35.4 min, 20.3 mg).

  Fr.6 was further separated by silica gel column chromatography to get 5 fractions (Fr.6.1~Fr.6.5). Fr.6.2 was purified by Sephadex LH-20 column chromatography (MeOH) and divided into 8 subfractionses (Fr.6.2.1~Fr.6.2.8). Fr.6.2.4 was further purified by semi-preparative HPLC with MeOH-H₂O (V:V＝23:77) to yield compound 18 (t_R 18.5 min, 23.5 mg). Fr.6.2.5 was isolated by semi-preparative HPLC with MeOH-H₂O (V:V＝37:63→50:50) to obtain 14 subfractionses (Fr.6.2.5.1~Fr.6.2.5.14). Fr.6.2.5.3 was further purified by semi- preparative HPLC with MeOH-H₂O (V:V＝25:75) to get compounds 21 (t_R 23.9 min, 5.4 mg) and 19 (t_R 25.0 min, 3.7 mg). Fr.6.2.5.6 was purified by semi-preparative HPLC with MeOH-H₂O (V:V＝38:62) to obtain compound 20 (t_R 42.1 min, 12.4 mg). Fr.6.2.5.8 was further purified by semi-preparative HPLC with MeOH-H₂O (V:V＝47:53) to yield compound 22 (t_R 47.7 min, 9.1 mg). Fr.6.4 was separated by semi-preparative HPLC with MeCN-H₂O (V:V＝35:65) to yield 5 subfractionses (Fr.6.4.1~Fr.6.4.5). Fr.6.4.3 was further purified by semi-preparative HPLC with MeCN-H₂O (V:V＝35:65) to obtain compounds 2 (t_R 37.1 min, 10.5 mg) and 3 (t_R 45.9 min, 13.7 mg).

  3-(4'-Hydroxy-3'-methoxybenzyl)-2(5H)-furanone (1): Brown gum; [α]20 D 0 (c 0.01, MeOH); UV (MeOH) λ_max [log ε/(L•mol^－1•cm^－1)]: 282 (3.35) nm; IR (KBr) ν_max: 3426, 1746, 1514, 1070, 1040, 667 cm^－1; ¹H NMR and ¹³C NMR data, Table 1; HR-ESI-MS m/z:221.0809 [M+H]⁺ (calcd for C₁₂H₁₃O₄⁺, 221.0808).

  (8S, 8'S)-3, 3'-Dimethoxyligna-4, 4', 9, 9'-tetraol-9-actate (2): Brown gum; [α]20 D+20 (c 0.01, MeOH); UV (MeOH) λ_max [log ε/(L•mol^－1•cm^－1)]: 280 (2.52) nm; IR (KBr) ν_max: 3432, 1720, 1807, 1516, 1269, 1031 cm^－1; CD (c 6.00× 10^－5 mol/L, MeOH) λ_max [∆ε/(deg•cm²•dmol^－1)] 205 (+4.28), 211 (－9.21), 228 (+2.61), 291 (+0.94) nm; ¹H NMR and ¹³C NMR data see Table 1; HR-ESI-MS calcd for C₂₂H₂₈O₇Na [M+Na]⁺ 427.1733, found 427.1714.

  (7'R, 8S, 8'S)-3', 5-Dimethoxy-2, 7'-cycloligna-4, 4', 9'-triol- 9-acid butyl ester (3): Yellow gum; [α]20 D－24 (c 0.02, MeOH); UV (MeOH) λ_max [log ε/(L•mol^－1•cm^－1)]: 285 (2.42) nm; IR (KBr) ν_max: 3410, 1759, 1516, 1271, 1218, 994 cm^－1; CD (c 6.00×10^－5 mol/L, MeOH) λ_max [∆ε/(deg•cm²•dmol^－1)] 206 (+22.21), 225 (+2.02), 240 (－6.39), 274 (－4.05), 292 (+8.50) nm; ¹H NMR and ¹³C NMR data see Table 2; HR-ESI-MScalcd for C₂₄H₃₀O₇Na [M+Na]⁺ 453.1889, found 453.1880.

  4.4   Cytotoxicity assay

  Three human cancer cell lines, A172, SHSY5Y and Hela cells, were used in the cytotoxicity assay. The cytotoxicity assay was performed using the MTT method as literature.^[31]Using cis-platin and taxol as positive control, all tests were performed in triplicate.

  4.5   Gastrointestinal motility of zebrafish

  Zebrafish culture and experimental procedureof gastrointestinal motility were performed as literature.^[32]

  4.6   Computational details

  The theoretical calculation of compound 7'R, 8S, 8'S-3 was carried out as described previously using Gaussian 16.^[33] Conformational analysis was performed using Conflex 8 (Boltzman Jump, Smart Minimizer using the MMFF). All configurations (relative energy < 41.9 kJ/mol) were used in optimizations at the B3LYP/6-31G(d, p) in MeOH. The theoretical calculation of ECD was performed using TD- DFT at the B3LYP/6-31G(2d, 2p) level in MeOH.^[34] Spec- Dis 1.71 was used to sum up single ECD spectra after a Boltzmann statistical weighting, and for the Gauss curve generation (σ＝0.2 eV) and the comparison with experimental data.

  Supporting Information 1D NMR, 2D NMR, HR-ESI-MS spectra of compounds 1~3, and CD spectra of compounds 2 and 3. The Supporting Information is available free of charge via the Internet at http://sioc-journal.cn.

  
  
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  Figure 1  Chemical structures of compounds 1~22

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  Figure 2  Key ¹H-¹H COSY() and HMBC () correlations of compounds 1~3

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  Figure 3  Calculated ECD spectra of 7'R, 8S, 8'S-3

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  Figure 4  (a) Effect of compound 3 on the gastrointestinal motility of zebrafish. (b) and (c) interfering effect of atropine on the gastrointestinal activation of compound 3. Indicates p < 0.001 when compared with the control

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  Table 1.  ¹H NMR and ¹³C NMR spectral data of compounds 1 and 2 (400/100 MHz)

  Position	1 (CDCl3)			2 (CD3OD)
  	δH (J in Hz)	δC		δH (J in Hz)	δC
  1					133.3
  2		174.2		6.62 (br. s, 1H)	113.3
  3		134.8			148.9
  4	6.94~6.96 (m, 1H)	145.6			145.7
  5	4.74~4.76 (m, 2H)	70.5		6.69 (overlapped, 1H)	115.9
  6				6.54 (overlapped, 1H)	122.8
  7				2.54~2.68 (m, 2H)	35.9
  8				2.12~2.21 (m, 1H)	40.5
  9				4.24 (dd, 11.1, 5.9, 1H); 4.01 (dd, 11.1, 6.5, 1H)	66.2
  1'		129.4			133.8
  2'	6.76 (d, 1.8, 1H)	111.7		6.56 (overlapped, 1H)	113.5
  3'		146.8			149.0
  4'		144.7			145.8
  5'	6.86 (d, 8.0, 1H)	114.7		6.59 (overlapped, 1H)	116.0
  6'	6.72 (dd, 8.1, 1.8, 1H)	121.7		6.54 (overlapped, 1H)	122.8
  7'	3.52 (br. s, 2H)	31.7		2.54~2.68 (m, 2H),	35.6
  8'				1.89~1.98 (m, 1H)	44.4
  9'				3.68 (dd, 10.8, 6.0, 1H); 4.01 (dd, 10.8, 6.5, 1H)	62.8
  1''					173.1
  2''				2.05 (s, 3H)	21.1
  3-OCH3				3.76 (s, 3H)	56.3
  3'-OCH3	3.87 (s, 1H)	56.1		3.77 (s, 3H)	56.3
  4'-OH	5.58 (s, 1H)

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  Table 2.  ¹H NMR and ¹³C NMR spectral data of compound 3 (400/100 MHz, in CDCl₃)

  Position	δH (J in Hz)	δC		Position	δH (J in Hz)	δC
  1		126.0		4'		144.5
  2		132.4		5'	6.81 (d, 8.0, 1H)	114.5
  3	6.29 (s, 1H)	115.8		6'	6.64 (dd, 8.0, 1.9, 1H)	122.6
  4		144.0		7'	3.91 (d, 10.7, 1H)	46.7
  5		145.2		8'	2.65, 2.92 (overlapped, 1H)	43.2
  6	6.54 (s, 1H)	110.1		9'	3.56 (dd, 11.8, 3.0, 1H) 3.43 (dd, 11.8, 3.5, 1H)	62.7
  7	3.21 (dd, 15.7, 11.5, 1H); 2.86~2.93 (overlapped, 1H)	32.7		1''	4.09~4.15 (m, 2H)	65.0
  8	2.08~2.17 (m, 1H)	46.9		2''	1.59~1.65 (m, 2H	30.8
  9		175.8		3''	1.33~1.42 (m, 2H	19.3
  1'		136.6		4''	0.92 (t, 7.4, 3H)	13.9
  2'	6.58 (d, 1.9, 1H)	111.7		5-OCH3	3.83 (s, 3H)	56.1
  3'		146.9		3'-OCH3	3.79 (s, 3H)	56.1

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