Steroidal glycosides from Reineckia carnea

    Steroidal Glycosides from Reineckia carnea Xiaomei Song, Dongdong Zhang, Hao He, Yuze Li, Xinjie Yang, Chong Deng, Zhishu Tang, Jiucheng Cui, Zhenggang Yue PII: DOI: Reference:

S0367-326X(15)30044-7 doi: 10.1016/j.fitote.2015.07.008 FITOTE 3225

To appear in:

Fitoterapia

Received date: Revised date: Accepted date:

21 May 2015 6 July 2015 7 July 2015

Please cite this article as: Xiaomei Song, Dongdong Zhang, Hao He, Yuze Li, Xinjie Yang, Chong Deng, Zhishu Tang, Jiucheng Cui, Zhenggang Yue, Steroidal Glycosides from Reineckia carnea, Fitoterapia (2015), doi: 10.1016/j.fitote.2015.07.008

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ACCEPTED MANUSCRIPT Steroidal Glycosides from Reineckia carnea Xiaomei Song a, 1, Dongdong Zhang a, 1, Hao He b, Yuze Li a, Xinjie Yang a, Chong Deng a, Zhishu Tang a,

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Jiucheng Cui a, Zhenggang Yue a, *

a Shaanxi Collaborative Innovation Center of Chinese Medicinal Resource Industrialization, Shaanxi Province

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Key Laboratory of New Drugs and Chinese Medicine Foundation Research, Shaanxi Rheumatism and Tumor

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Center of TCM Engineering Technology Research, School of Pharmacy, Shaanxi University of Chinese Medicine, Xianyang 712046, China.

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b School of Pharmaceutical Sciences, Xi’an Medical University, Xi’an 710021, China.

1 Co-first author * Corresponding author Tel: 86-180-9208-6211 E-mail: [email protected] (Z.G. Yue);

ACCEPTED MANUSCRIPT Abstract Three new steroidal glycosides (1-3) and a novel natural product 4 firstly obtained from a plant source, together with two known steroidal glycosides (5-6) have been isolated from the whole plant of Reineckia carnea. Their

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structures were determined by physicochemical properties and spectroscopic methods, and their cytotoxic activities against human 1299 tumor cells were evaluated by MTT method. Compounds 4, 5 and 6 exhibited

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cytotoxicity with IC50 values of 50.3 µmol • L-1, 67.2 µmol • L-1 and 61.8 µmol • L-1, while compounds 1, 2, and 3

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showed no cytotoxicity with the cells.

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Key words: Reineckia carnea; furostanol and spirostanol glycosides; structure identification; cytotoxicity

ACCEPTED MANUSCRIPT 1. Introduction Reineckia carnea (Andr.) Kunth, an evergreen herbaceous perennial plant, is mostly distributed in China and Japan [1]. It is a monotypic plant genera in the Reineckia genus of Liliaceae family, which is widely grown for

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groundcovers or medical herbs in south of China [2], and it is commonly used in Miao Minority of China, as one of the most important ingredients in many medical prescriptions for the treatment of cough, sore throat, traumatic

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injury and bronchial asthma diseases [3, 4]. The previous investigations of bioactive constituents from R. carnea

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had resulted in saponins with the chemical structures as spirostanol [5, 6], furostanol [7], cholestane [5, 8], stigmastane [6], pregnane and ergostane [4, 9] type glycosides. As part of our research project to explore more

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diversity bioactive leading compounds from the medicinal herbs of Qinba mountains [10-14], the chemical constituents and pharmacological studies of R. carnea were studied, and six steroidal glycosides (1-6) were

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acquired, in which 26-O-β-D-glucopyranosyl-(25S)-20-O-methyl-5β-furost-22 (23)-en-1β, 3β, 20α, 26-tetraol 1-O-β-D-fucopyranosyl-(1→2)-[α-L-rhamnopyranosyl]-3-O-α-L-rhamnopyranoside

(1),

26-O-β-D-gluco

-pyranosyl-(25S)-20-O-methyl-5β-furost-22 (23)-en-1β, 3β, 20α, 26-tetraol 1-O-β-D-xylopyranosyl-(1→2)

3β-diol

were

two

new

furostanol

glycosides

and

1-O-β-D-fucopyranosyl-(1→2)-[α-L-rhamnopyranosyl]-3-O-α-L-rhamno

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(25S)-5β-spirostan-1β,

(2)

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-[α-L-rhamnopyranosyl]-3-O-α-L-rhamnopyranoside

-pyranoside (3) was a new spirostanol glycoside, and (25S)-5β-spirostan-1β, 3β-diol 3-O-α-L-rhamnopyranoside

glycosides

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(4) was a spirostanol glycoside firstly isolated from the natural source, together with two known spirostanol (25S)-5β-spirostan-1β, 3β-diol 1-O-α-L-rhamnopyranosyl-(1→2)-β-D-xylopyranoside

(5)

and

(25S)-5β-spirostan-1β, 3β-diol 1-O-β-D-xylopyranoside (6). Their isolation, structural elucidation and cytotoxic evaluation on human 1299 cells were reported in this paper. 2. Results and discussion

Compound 1 was obtained as a white amorphous powder. The positive colorations of Liebermann–Burchard and Ehrlich reactions, suggested that 1 was a furostanol glycoside [15, 16]. The HR-ESI-MS spectrum showed a negative molecular ion peak at m/z 1061.5575 [M _ H] , corresponding to a molecular formula of C52H86O22 . The －

1

H-NMR of 1 showed the presence of an exocyclic olefinic proton at δH 4.35 (1H, t, J = 7.2Hz, H-23), eight

methyls at δH 0.87 (3H, s, H-18), 1.32 (3H, s, H-19), 1.38 (3H, s, H-21), 1.12 (3H, d, J = 6.6 Hz, H-27), 3.20 (3H, s, 20-OMe), 1.54 (3H, d, J = 6.4 Hz, H-fuc-6ʹ ʹ ), 1.74 (3H, d, J = 6.1 Hz, H-rha-6ʹ ʹ ʹ ), 1.66 (3H, d, J = 6.1 Hz, H-rha-6ʹ ʹ ʹ ʹ ), and four anomeric protons at δH 4.86 (1H, d, J = 7.7 Hz, H-glc-1ʹ ), 4.89 (1H, d, J = 7.7 Hz, H-fuc-1ʹ ʹ ), 6.42（1H, brs, H-rha-1ʹ ʹ ʹ ), 5.46 (1H, brs, H-rha-1ʹ ʹ ʹ ʹ ). The 13C-NMR displayed 52 carbon signals, 4 of which were attributed to the anomeric carbons [δC 105.2 (C-glc-1ʹ ), 99.8 (C-fuc-1ʹ ʹ ), 101.7 (C-rha-1ʹ ʹ ʹ ), and 99.6 (C-rha-1ʹ ʹ ʹ ʹ )], and 27 of which were assigned to the aglycone carbons. Among them,

ACCEPTED MANUSCRIPT three methyl carbon signals at δC 14.4, 15.7 and 16.9 were ascribable to C-18, C-19 and C-21; two olefinic carbon signals at δC 157.4 (C-22) and 96.5 (C-23), and the methyl signals at δC 18.7 (C-27), corresponding to their 1

H-NMR characters, indicated the presence of an exocyclic isopentene group, linked to the tetrahydrofuran ring of

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the furostanol saponin with the exocyclic olefinic bond. HMBC correlations (Fig.2) of H-19/C-1, C-5, C-9 and C-10, H-4/C-2, C-3 and C-5, H-6/C-5, C-7, C-8 and C-10, H-8/C-9, C-11 and C-14, H-18/C-12, C-13, C-14 and

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C-17, H-21/C-17, C-20 and C-22, 20-OMe/C-20, H-23/C-22 and C-24, H-27/C-24, C-25 and C-26 revealed the

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aglycone moiety as 20-O-methyl-furost-22(23)-en-1, 3, 20, 26-tetraol. Moreover, HMBC correlations of H-fuc-1ʹ ʹ /C-1; H-rha-1ʹ ʹ ʹ /C-fuc-2ʹ ʹ ; H-rha-1ʹ ʹ ʹ ʹ /C-3; H-glc-1ʹ /C-26, disclosed that the terminal

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rhamnose unit was linked at C-fuc-2ʹ ʹ of the inner fucose unit, and then the fucose unit was linked at C-1 of the aglycone; the other rhamnose unit was linked at C-3 of the aglycone, and the glucose unit was linked at C-26 of

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the aglycone. Acid hydrolysis of 1 resulted in the production of D-glucose, D-fucose and L-rhamnose, which were confirmed by GC analysis of the trimethylsilyl-L-cysteine derivatives of the hydrolysate of 1 and the authentic sugars. Coupling constants of the anomeric proton signals suggested β-configuration of D-glucose and D-fucose,

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and α-configurations of L-rhamnoses, respectively. Then, the planar structure of 1 was deduced as 3,

20,

26-tetraol

1-O-β-D-fucopyranosyl

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26-O-β-D-glucopyranosyl-20-O-methyl-furost-22(23)-en-1,

-(1→2)-[α-L-rhamnopyranosyl]-3-O-α-L-rhamnopyranoside. Meanwhile, in the NOSEY spectrum (Fig.3) of 1,

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the NOE correlations of H-2a/H-5 and Me-19, H-2b/H-1 and H-3, indicated α-axial configurations of H-1 and H-3, and β-orientation of H-5, Me-19, 1-OH and 3-OH, which supported the A/B cis ring junction pattern; the NOE correlations of Me-19/H-8 and H-1/ H-9 supported the B/C trans ring junction pattern; the NOE correlations of Me-18/H-8 and H-15a, H-14/H-15b and H-16, supported the C/D trans ring junction pattern; the correlations of Me-21/Me-18 and H-23 supported α-axial of 20-OMe, β-orientation of Me-21 and, Z configuration of 22 (23)-en.. Finally, the absolute configuration of C-25 was deduced as S by the difference of chemical shifts between H-26 equitorial (δH 3.56) and H-26 axial (δH 4.16), since the difference is usually ∆δ > 0.57 ppm for 25S compounds and ∆δ < 0.48 ppm for 25R compounds [17]. Therefore the structure of 1 was characterized as 26-O-β-D-glucopyranosyl-(25S)-20-O-methyl-5β-furost-(Z)-22

(23)-en-1β,

3β,

20α,

26-tetraol

1-O-β-D-fucopyranosyl-(1→2)-[α-L-rhamnopyranosyl]-3-O-α-L-rhamnopyranoside. Compound 2 was obtained as a white amorphous powder. The positive colorations of Liebermann–Burchard and Ehrlich reactions, suggested that 2 was a furostanol glycoside [15, 16]. The HR-ESI-MS spectrum showed a negative molecular ion peak [M

_

－

H] at m/z 1047.4600, corresponding to a molecular formula of C51H84O22.

Comparison of the HR-ESI-MS and NMR data of 2 and 1, demonstrated almost similar NMR spectroscopic features unless the β-D-fucopyranosyl moiety in 1 was replaced by β-D-xylopyranosyl moiety in 2. This was

ACCEPTED MANUSCRIPT confirmed by acid hydrolysis and subsequent GC analysis of the hydrolysates, according to the same protocol as that described for 1. In the HMBC spectrum (Fig.2) of 2, correlations of H-xyl-1ʹ ʹ /C-1 and H-rha-1ʹ ʹ ʹ /C-xyl-2ʹ ʹ , disclosed the terminal rhamnose unit was linked at C-xyl-2ʹ ʹ of the inner xylose unit,

26-O-β-D-glucopyranosyl-(25S)

-20-O-methyl-5β-furost-(Z)-22

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and then the xylose unit was linked at C-1 of the aglycone. Therefore, the structure of 2 was identified as (23)-en-1β,

3β,

20α,

26-tetraol

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1-O-β-D-xylopyranosyl-(1→2)-[α-L-rhamno -pyranosyl]-3-O-α-L-rhamnopyranoside.

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Compound 3 was obtained as a white amorphous powder. The positive coloration of Liebermann–Burchard reaction and negative coloration of Ehrlich reaction, suggested that 3 was a spirostanol glycoside [15]. The _

－

H] , corresponding to a

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HR-ESI-MS spectrum showed a negative molecular ion peak at m/z 869.4935 [M

molecular formula of C45H74O16. The 1H-NMR of 1 showed the presence of seven methyls at δH 0.82 (3H, s, H-18),

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1.26 (3H, s, H-19), 1.12 (3H, d, J = 6.3 Hz, H-21), 1.04 (3H, d, J = 6.9 Hz, H-27), 1.51 (3H, d, J = 6.4 Hz, H-fuc-6ʹ ), 1.72 (3H, d, J = 6.1 Hz, H-rha-6ʹ ʹ ), 1.64 (3H, d, J = 6.1 Hz, H-rha-6ʹ ʹ ʹ ), and three anomeric protons at δH 4.92 (1H, d, J = 7.7 Hz, H-fuc-1ʹ ), 6.44 (1H, brs, H-rha-1ʹ ʹ ), and 5.43 (1H, brs, H-rha-1ʹ ʹ ʹ ). 13

C-NMR displayed 45 carbon signals, 3 of which were attributed to the anomeric carbons [δC 99.7

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The

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(C-fuc-1ʹ ), 101.4 (C-rha-1ʹ ʹ ), and 99.7 (C-rha-1ʹ ʹ ʹ )], and 27 of which were assigned to the aglycone carbons. Among them, four methyl carbon signals at δC 16.6, 16.7, 14.8 and 16.2 were ascribable to C-18, C-19,

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C-21 and C-27; the quaternary carbon signal at δC 109.6, was identified as an acetal carbon (C-22), a characteristic signal of the spirostanol saponin. The 13C-NMR data of the aglycone moiety was similar to (25S)-5β-spirostan-1β, 3β-diol [18], which was supported by HMBC correlations (Fig.2) of H-19/C-1, C-5, C-9 and C-10, H-4/C-2, C-3 and C-5, H-6/C-5, C-7, C-8 and C-10, H-8/C-6, C-7, C-9, C-11 and C-14, H-18/C-12, C-13, C-14 and C-17, H-21/C-17, C-20 and C-22, H-16/C-15, C-17, C-20 and C-22, H-24/C-23 and C-25, H-27/C-24, C-25 and C-26. Moreover, HMBC correlations of H-fuc-1ʹ /C-1, H-rha-1ʹ ʹ /C-fuc-2ʹ , H-rha-1ʹ ʹ ʹ /C-3, disclosed the terminal rhamnose unit was linked at C-fuc-2ʹ of the inner fucose unit, and then fucose unit was linked at C-1 of the aglycone; and the other rhamnose was linked at C-3 of the aglycone. Meanwhile, the configurations of sugar moieties of 3 were confirmed by acid hydrolysis and subsequent GC analysis of the hydrolysates, according to the same protocol as that described for 1. In the NOSEY spectrum of 3 (Fig.3), the NOE correlations of H-2a/H-5 and H-19; H-2b/H-1 and H-3; H-19/H-8; H-1/ H-9; H-18/H-8 and H-15a; H-14/H-15b; H-15b/H-16 and H-17 indicated the A/B cis, the B/C and C/D trans ring junction pattern, and β-orientation of 1-OH and 3-OH. Finally, the 25S configuration was supported by IR data 987, 916, 896, 849 (916 > 896) cm−1. Thus the structure of 3 was formulated as (25S)-5β-spirostan-1β, 3β-diol 1-O-β-D-fucopyranosyl-(1→2)-[α-L-rhamnopyranosyl]-3-O-α-L -rhamnopyranoside.

ACCEPTED MANUSCRIPT Compound 4 was obtained as a white amorphous powder. The positive coloration of Liebermann–Burchard reaction and negative coloration of Ehrlich reaction, suggested that compound 4 was a spirostanol glycoside. Comparison of the 1H-NMR and

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C-NMR data of 4 and 3, indicated the absence of the fucopyranosyl and

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rhamnopyranosyl moieties in 4, and the chemical shift of C-1 in 4 was transferred to upfield at δC 73.1 (∆δC -5.6 ppm) indicated the disappearance of the sugar chain at C-1. The structure of 4 was finally confirmed by the GC

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analysis, 2D-NMR and IR spectrums as (25S)-5β-spirostan-1β, 3β-diol 3-O-α-L-rhamnopyranoside (4, Fig.1).

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Although compound 4 has been reported as a partial hydrolysate from convallasaponin-D [19], it is the first example of the isolation of 4 from a plant source, and the first time to report the spectrum data.

reported

in

the

literature

[18]

as

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Additionally, the known spirostanol glycosides were identified by comparison of spectroscopic data with those (25S)-5β-spirostan-1β,

3β-diol

1-O-α-L-rhamnopyranosyl

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-(1→2)-β-D-xylopyranoside (5, Fig.1) and (25S)-5β-spirostan-1β, 3β-diol 1-O-β-D-xylopyranoside (6, Fig.1). The cytotoxic activities of compounds 1–6 against the human tumor 1299 cell lines were evaluated by the MTT assay. Compounds 4–6 showed inhibitory activity with IC50 values of 50.3 µmol • L-1, 67.2 µmol • L-1 and 61.8

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µmol • L-1, respectively, and compounds 1–3 exihibited no cytotoxicity (IC50 > 100 µmol • L-1), while the positive

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control 5-Fu gave an IC50 value of 43.3 µmol • L-1. The results showed that the structure of the aglycone was

exhibited none.

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important to the cytotoxic activity, while spirostanol glycosides showed cytotoxicity and furostanol glycosides

3. Experimental

3.1 General experimental procedures

The IR spectra were recorded on a Bruker TENSOR-27 instrument. The HR-ESI-MS spectra was taken on an Agilent Technologies 6550 Q-TOF. 1D and 2D NMR spectra were recorded on a Bruker-AVANCE400 instrument with TMS as an internal standard. The analytical HPLC was performed on a Waters 2695 Separations Module coupled with a 2996 Photodiode Array Detector and a ODS-3 column (4.6 mm × 250 mm, 5 mm particles, COSMOSIL, Tokyo, Japan). Semipreparative HPLC was performed on a system comprising a Shimadzu LC-6AD pump equipped with a SPD-20A UV detector and a Ultimate XB-C18 (10 mm × 250 mm, 5 mm particles) or YMC-Pack-ODS-A (10 mm × 250 mm, 5 mm particles). GC was performed on an Agilent 7890A gas chromatograph (Agilent technologies Inc, Santa Clara, CA, USA) equipped with HP-5 capillary column (30m × 320mm × 0.25 mm). Sephadex LH-20 gel and C-18 (40 –75 mm) silica gel was purchased from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). Silica gel was purchased from Qingdao Haiyang Chemical Group Corporation (Qingdao, China).

ACCEPTED MANUSCRIPT 3.2 Plant material Reineckia carnea (Andr.) Kunth was collected on July in 2013 from Taibai region of Qinba mountains in Shaanxi Province of China, and authenticated by Prof. Benxiang Hu. A voucher specimen (herbarium No.

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20130712) has been deposited in the Medicinal Plants Herbarium (MPH), Shaanxi University of Chinese Medicine, Xianyang, China.

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3.3 Extraction and isolation

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The air-dried and powdered whole plant of R. carnea (4.5 kg) was extracted with 80% EtOH under reflux for three times at 80 . After removing the solvent, the concentrated residue was partitioned with petroleum ether and

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n-BuOH successively. The n-BuOH extract (170 g) was subjected to column chromatography (CC) on silica gel, eluting with gradient solvent system (CHCl3-MeOH-H2O, 100:0:0 − 60:40:10) to give four fractions (Fr.1 − Fr.4).

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Fr.3 (35 g) was subjected to CC on silica gel, eluting with (CHCl3-MeOH-H2O (100:0:0 − 70:30:5) to give six subfractions (Fr.3-1 − Fr.3-6). Fr.3-3 (3.8 g) was subjected to CC on ODS gel, using MeOH-H2O (10:90 − 70:30) as the eluent to afford five fractions (Fr.3-4-1 – Fr.3-4-5). Fr.3-4-2 (130 mg) was purified by HPLC (Ultimate

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XB-C18, 10 mm × 250 mm, 5 µm particles, flow rate: 1.0 mL/min) with MeCN-H2O (28:72) as mobile phase to

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afford compound 1 (12.6 mg; tR = 43 min) and compound 2 (9.8 mg; tR = 47 min), Fr.3-4-1 (195 mg) was purified

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by HPLC with MeCN-H2O (36:64) as mobile phase to afford compound 3 (17.3 mg; tR = 58 min) , Fr.2 (43 g) was subjected to CC on silica gel, eluting with CHCl3-MeOH-H2O (100:0:0 − 80:20:5) to afford compound 4 (6.5 mg), 5 (8.1 mg) and compound 6 (9.4 mg). 3.4

26-O-β-D-glucopyranosyl-(25S)-20-O-methyl-5β-furost-22(23)-en-1β,

3β,

20α,

26-tetraol

1-O-β-D-fucopyranosyl-(1→2)-[α-L-rhamnopyranosyl]-3-O-α-L-rhamnopyranoside A white amorphous power; HRESI-MS m/z 1061.5575 [M _ H] (calcd for C52H85O22, 1061.5532); IR (KBr) －

νmax 3365, 2913, 1650, 1449, 1156, 1060 cm−1; 1H-NMR (400 MHz, in pyridine-d5) and 13C-NMR (100 MHz, in pyridine-d5) spectral data, see Tables 1 and 2. 3.5

26-O-β-D-glucopyranosyl-(25S)-20-O-methyl-5β-furost-22(23)-en-1β,

3β,

20α,

26-tetraol

1-O-β-D-xylopyranosyl-(1→2)-[α-L-rhamnopyranosyl]-3-O-α-L-rhamnopyranoside A white amorphous power; HRESI-MS m/z 1047.4600 [M _ H] (calcd for C51H83O22, 1047.5376); IR (KBr) －

νmax 3363, 2911, 1649, 1450, 1158, 1063 cm−1; 1H-NMR (400 MHz, in pyridine-d5) and 13C-NMR (100 MHz, in pyridine-d5) spectral data, see Tables 1 and 2. 3.6

(25S)-5β-spirostan-1β,

α-L-rhamnopyranoside

3β-diol

1-O-β-D-fucopyranosyl-(1→2)-[α-L-rhamnopyranosyl]-3-O-

ACCEPTED MANUSCRIPT A white amorphous power; HR-ESI-MS m/z 869.4935 [M _ H] (calcd for C45H73O16, 869.4899); IR (KBr) νmax －

3415, 2926, 1449, 1050, 987, 916, 896, 849 cm−1; 1H-NMR (400 MHz, in pyridine-d5) and 13C-NMR (100 MHz, in pyridine-d5) spectral data, see Tables 1 and 2.

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3.7 (25S)-5β-spirostan-1β, 3β-diol 3-O-α-L-rhamnopyranoside

A white amorphous power; IR (KBr) νmax 3416, 2924, 1447, 1051, 985, 916, 894, 844 cm−1; 1H-NMR (400

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MHz, in pyridine-d5) and 13C-NMR (100 MHz, in pyridine-d5) spectral data, see Tables 1 and 2.

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3.8 Acid hydrolysis of compounds 1- 4 and absolute configuration determinations of sugars Compounds 1-4 (3 - 5 mg) were individually hydrolyzed with 1 N HCl-dioxane (1:1, 3 mL) at 60 ℃ for 6 h.

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After dilution with H2O (5 mL), the reaction mixture was extracted EtOAc to yield separate EtOAc and H2O phases. The H2O layer was evaporated under reduced pressure. After addition of H2O (5 mL), the acidic solution

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was evaporated again, and this procedure was repeated until a neutral solution was obtained. The neutral solution was evaporated and dried in vacuo to furnish a monosaccharide residue. The residue was dissolved in pyridine

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(0.5 mL), and 2 mg of L-cysteine methyl ester hydrochloride was added. The mixture was maintained at 60 ℃

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for 2 h, evaporated under a stream of N2, and dried in vacuo. Next, 0.2 mL of N-trimethylsilylimidazole was added, and the resultant reaction mixture was maintained at 60 ℃ for 1 h. The mixture was partitioned between

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n-hexane and H2O (2 mL each), and the n-hexane extract was analyzed by GC under the following conditions: capillary column, HP-5 (30 m × 320 mm × 0.25 µm); detector, FID; detector temperature, 280 ℃; injection temperature, 250 ℃; initial temperature, 100 ℃ for 2 min and subsequent increase to 280 ℃ at a rate of 10 ℃ /min; final temperature, 280 ℃ for 5 min; carrier, N2 gas. The absolute configurations of the sugars isolated from the hydrolysates of 1-4 were determined by comparing by comparing the retention times (tR) of their trimethylsilyl-L-cysteine derivatives with those of authentic sugars prepared by a similar procedure. The tR of the trimethylsily-L-cysteine derivatives of the sugars were follows: D-glucose, 21.35 min, D-fucose, 18.24min; D-xylopyranose, 16.88 min; and L-rhamnopyranose, 18.76 min, respectively. 3.9 Cytotoxicity assay The cytotoxic activity assay toward the human tumor 1299 cell lines was measured by the MTT method in vitro, using 5-fluorouracil (5-Fu) as positive control. Briefly, 1 × 104 ml-1 cells were seeded into 96-well plates and allowed to adhere for 24 h. Compounds 1–6 were dissolved in DMSO and diluted with complete medium to 6 degrees of concentration (from 100 µmol • L-1 to 0.1 µmol • L-1) for inhibition rate determination. After incubation at 37.8℃ for 4 h, the supernatant was removed before adding DMSO (100 µL) to each well. Each test was duplicated for three times, and the inhibition rate (IR) and IC50 were calculated. The positive control 5-Fu gave an

ACCEPTED MANUSCRIPT IC50 value of 43.3µmol• L-1, compounds 4, 5 and 6 exhibited cytotoxicity with IC50 value of 50.3 µmol • L-1, 67.2 µmol • L-1 and 61.8 µmol • L-1, while compounds 1, 2 and 3 showed no cytotoxicity with the cells. Acknowledgments

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This project was financially supported by the National Natural Science Foundations of China (grant No. 81373978), the Open Projects Program of the Key Laboratory of Tibetan Medicine Research, Chinese Academy

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of Sciences (grant No. 2014-TMR-01), the Innovative Research Team in TCM material foundition and key

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preparation technology (grant No. 2012KCT-20), and the Innovation projects of Science and technology in Shaanxi province (grant No. 2013KTCQ03-14). The authors thank Pu Jia in North West University for her kind

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D

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help on HR-ESI-MS spectra test.

ACCEPTED MANUSCRIPT Reference [1] Tsukamoto, Y., The grand dictionary of horticulture, Shogakukan, Tokyo, 1988. [2] Zhang, X.J., The applications of Reineckea carnea in horticulture and pharmacy, Hubei Agricultural Sciences. 48 (2009) 662-3.

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[3] Han, N., Chang, C., Wang, Y., Huang, T., Liu, Z., Yin, J., The in vivo expectorant and antitussive activity of extract and fractions from Reineckia carnea, Journal of ethnopharmacology. 131 (2010) 220-3.

[4] Xing, P.P., Wu, Q., Wu, Z.W., Fu, H.Z., A new pregnane-type glycoside from Reineckia carnea, Journal of Chinese

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Pharmaceutical Sciences. 20 (2011) 347-51.

[5] Iwagoe, K., Konishi, T., Kiyosawa, S., Studies on the constituents of the aerical parts of Reineckia carnea Kunth.,

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YAKUGAKU ZASSHI. 107 (1987) 140-9.

[6] Kanmoto, T., Mimaki, Y., Sashida, Y., Nikaido, T., Koike, K., Ohmoto, T., Steroidal constituents from the underground parts of Reineckea carnea and their inhibitory activity on cAMP phosphodiesterase, Chemical &

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pharmaceutical bulletin. 42 (1994) 926-31.

[7] Wang, Q., Hou, Q., Guo, Z.Y., Zou, K., Xue, Y.H., Huang, N.Y., et al., Three new steroidal glycosides from roots of Reineckia carnea, Natural product research. 27 (2013) 85-92.

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[8] Zhang, Z.Q., Chen, J.C., Zhou, L., Qiu, M.H., Two new cholestane bisdesmosides from Reineckia carnea, Helvetica Chimica Acta. 90 (2007) 616-22.

[9] Li, W.X., Huang, X.Y., Chen, X.M., Deng, L.F., Qi, C., Study on the molluscicidal component isolation and

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structure determinatlon of Reineckia carnea, Agerochemicais. 47 (2008) 97-9. [10] Chai, J., Song, X.M., Wang, X., Mei, Q.B., Li, Z., Cui, J.C., et al., Two new compounds from the roots and

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rhizomes of Trillium tschonoskii, Phytochemistry Letters. 10 (2014) 113-7. [11] Li, Y.H., Fan, L., Sun, Y., Miao, X., Zhang, F., Meng, J., et al., Paris saponin VII from trillium tschonoskii reverses multidrug resistance of adriamycin-resistant MCF-7/ADR cells via P-glycoprotein inhibition and apoptosis

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augmentation, Journal of ethnopharmacology. 154 (2014) 728-34. [12] Li, Y.H., Sun, Y., Fan, L., Zhang, F., Meng, J., Han, J., et al., Paris saponin VII inhibits growth of colorectal cancer cells through Ras signaling pathway, Biochemical pharmacology. 88 (2014) 150-7. [13] Fan, L., Li, Y.H., Sun, Y., Yue, Z.G., Meng, J., Zhang, X.T., et al., Paris saponin VII inhibits metastasis by modulating matrix metalloproteinases in colorectal cancer cells, Molecular medicine reports. 11 (2015) 705-11. [14] Li, Y.H., Liu, C.X., Xiao, D., Han, J., Yue, Z.G., Sun, Y., et al., Trillium tschonoskii steroidal saponins suppress the growth of colorectal Cancer cells in vitro and in vivo, Journal of ethnopharmacology. (2015). [15] Kiyosawa, S.H.U., Hutoh, M., Komori, T., Nohara, T., Hosokawa, I., Kawasaki, T., Detection of proto-type compounds of diosgenin-and other spirostanol-glycosides, Chemical & pharmaceutical bulletin. 16 (1968) 1162-4. [16] Su, L., Feng, S.G., Qiao, L., Zhou, Y.Z., Yang, R.P., Pei, Y.H., Two new steroidal saponins from Tribulus terrestris, Journal of Asian natural products research. 11 (2009) 38-43. [17] Agrawal, P.K., Dependence of 1H NMR chemical shifts of geminal protons of glycosyloxy methylene (H2-26) on the orientation of the 27-methyl group of furostane-type steroidal saponins, Magnetic Resonance in Chemistry. 42 (2004) 990-3. [18] Zhang, Z.Q., Chen, J.C., Zhang, X.M., Li, Z.R., Qiu, M.H., Two new spirostanol saponins from Reineckia carnea, Helvetica Chimica Acta. 91 (2008) 1494-9. [19] Kimura, M., Tohma, M., Yoshizawa, I., Constituents of convallaria. XI. On the structure of convallasaponin-D, Chemical & pharmaceutical bulletin. 16 (1968) 1228-34.

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Fig. 1 Chemical structures of compounds 1-6

Fig. 2 Key HMBC correlations of compounds 1-4

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Fig. 3 Key NOESY correlations of compounds 1-4

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1

H-NMR and 13C-NMR spectrum data* for the aglycones of 1–4

1

2

3

4

No. δC

δH

δC

δH

δC

δH

δC

δH

1

78.6

4.02 (1H, ca.)

78.6

4.04 (1H, ca.)

78.7

4.01 (1H, ca.)

73.1

4.45 (1H, ca.)

2

30.7

3

70.7

4

31.3

5

35.5

2.21 (1H, ca.)

35.7

2.22 (1H, ca.)

34.8

6

27.2

1.28 (2H, ca.)

27.3

1.29 (2H, ca.)

26.7

1.52 (1H, ca.) 1.83 (1H, ca.)

0.91 (1H, ca.)

2.32 (1H, ca.)

70.9

4.23 (1H, ca.) 1.58 (1H, ca.)

31.3

1.81 (1H, ca.)

0.89 (1H, ca.)

70.6 31.2

2.26 (1H, ca.) 2.33 (1H, ca.) 4.23 (1H, ca.)

1.51 (1H, ca.)

1.83 (1H, ca.)

72.6 31.3

1.97 (1H, ca.) 2.11 (1H, ca.) 3.83 (1H, ca.) 1.38 (1H, ca.) 1.84 (1H, ca.)

2.14 (1H, ca.)

35.8

1.54 (1H, ca.)

1.26 (2H, ca.)

26.5

1.27 (2H, ca.)

0.91 (1H, ca.)

8

41.5

2.53 (1H, ca.)

38.9

2.51 (1H, ca.)

40.3

2.53 (1H, ca.)

42.2

1.93 (1H, ca.)

9

46.4

1.25 (1H, ca.)

46.2

1.24 (1H, ca.)

46.6

1.46 (1H, ca.)

42.6

1.44 (1H, ca.)

10

40.1

——

39.7

——

39.6

——

40.5

——

11

22.3

12

40.3

13

40.9

——

40.5

14

57.6

0.96 (1H, ca.)

57.2

15

34

16

84.4

17

67.5

18

14.4

19

16.9

20

82.9

21

1.77 (1H, ca.)

1.45 (1H, ca.) 2.03 (1H, ca.)

9.5 Hz)

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40.5

——

40.7

——

0.96 (1H, ca.)

56.8

1.07 (1H, ca.)

56.4

1.05 (1H, ca.)

1.77 (1H, ca.)

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5.01 (1H, dd, 6.7,

——

1.24 (1H, ca.)

33.9

84.1

1.43 (1H, ca.)

22.3

2.02 (1H, ca.)

39.9

1.42 (1H, ca.)

1.61 (1H, ca.)

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1.24 (1H, ca.)

1.23 (1H, ca.)

23.1

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2.02 (1H, ca.)

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1.23 (1H, ca.)

1.61 (1H, ca.)

26.6

0.91 (1H, ca.)

27.4

1.76 (1H, ca.)

27.1

30.1

7

1.75 (1H, ca.)

27.7

30.8

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4.24 (1H, ca.)

2.27 (1H, ca.)

29.8

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2.32 (1H, ca.)

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2.27 (1H, ca.)

1.45 (1H, ca.) 2.03 (1H, ca.)

5.01 (1H, dd, 6.7, 9.5 Hz)

42.3

32.1

81.1

2.02 (1H, ca.) 1.36 (1H, ca.) 2.13 (1H, ca.)

1.43 (1H, ca.) 2.02 (1H, ca.) 4.54 (1H, ca.) 4.82 (1H, dd, 3.1,

21.1

40.3

32.3

2.04 (1H, ca.) 1.37 (1H, ca.) 2.11 (1H, ca.)

1.45 (1H, ca.) 2.04 (1H, ca.)

81.3

4.52 (1H, ca.)

63

4.60 (1H, ca.)

16.4

0.84 (3H, s)

2.15 (1H, d, 6.0 Hz)

67.1

2.12 (1H, d, 6.0Hz)

62.8

0.87 (3H, s)

14.1

0.87 (3H, s)

16.6

1.32 (3H, s)

15.4

1.32 (3H, s)

16.7

1.26 (3H, s)

18.8

1.22 (3H, s)

——

82.6

——

42.3

1.88 (1H, ca.)

42.2

1.82 (1H, ca.)

15.7

1.38 (3H, s)

17.6

1.38 (3H, s)

14.8

22

157.4

——

157.5

——

109.6

23

96.5

4.35 (1H, t, 7.2Hz)

96.2

4.35 (1H, t, 7.2Hz)

26.3

24

30.1

25

35.4

2.21 (1H, ca.) 2.47 (1H, ca.) 2.12 (1H, ca.)

30.1 35.3

3.56 (1H, dd, 2.4, 26

75.4

7.1Hz)

2.19 (1H, ca.) 2.47 (1H, ca.) 2.13 (1H, ca.)

26.1 27.4

3.55 (1H, dd, 2.4, 75.4

4.16 (1H, ca.)

7.1Hz)

18.7

1.12 (3H, d, 6.6Hz)

18.7

1.12 (3H, d, 6.6Hz)

20-OMe

49.3

3.20 (3H, s)

49.1

3.17 (3H, s)

0.82 (3H, s)

1.12 (3H, d 6.3Hz) —— 1.33 (1H, ca.) 1.42 (1H, ca.) 1.88 (1H, ca.) 2.11 (1H, ca.) 1.54 (1H, ca.)

15.0 109.8 26.5

26.3 27.6

3.34 (1H, d, 64.9

4.16 (1H, ca.)

27

6.2Hz)

10.8Hz)

1.04 (3H, d, 6.9Hz)

6.9Hz) —— 1.35 (1H, ca.) 1.43 (1H, ca.) 1.89 (1H, ca.) 2.10 (1H, ca.) 1.52 (1H, ca.) 3.37 (1H, d,

65.2

4.06 (1H, ca.) 16.2

1.16 (3H, d

10.9Hz) 4.10 (1H, ca.)

16.7

1.08 (3H, d, 7.0Hz)

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H-NMR and 13C-NMR data* for the sugar moieties of 1–4

1

2

No.

3

No. δC

δH

4

No. δC

No.

δH

δC

δH

δC

δH

Glc-1' 105.2 4.86 (1H, d , 7.7Hz) Glc-1' 105.3 4.86 (1H, d, 7.6Hz) Fuc-1' 99.7 4.92 (1H, d, 7.7Hz) Rha-1' 98.9 5.47 (1H, br.s) 75.3

3.96 (1H, ca.)

2'

75.5 3.96 (1H, ca.)

2'

77.0

4.25 (1H, ca.)

2'

72.8 4.48 (1H, ca.)

3'

78.6

4.26 (1H, ca.)

3'

78.7 4.25 (1H, ca.)

3'

74.8

4.57 (1H, ca.)

3'

72.7 4.36 (1H, ca.)

4'

71.8

4.24 (1H, ca.)

4'

71.9

4.24 (1H, ca.)

4'

74.2

4.04 (1H, ca.)

4'

73.9 4.26 (1H, ca.)

5'

78.5

3.98 (1H, ca.)

5'

78.6

3.98 (1H, ca.)

5'

71.2

5'

70.6 4.27 (1H, ca.)

4.47 (1H, ca.)

6'

6'

19.1

62.8

6'

62.9

4.54 (1H, ca.)

4.54 (1H, ca.)

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Rha-1'' 101.4

Fuc-1'' 99.8 4.89 (1H, d, 7.7Hz) Xyl-1'' 100.1 5.08 ( 1H, d, 7.3Hz)

6.4,

12.9Hz)

1.51 (3H, d, 6.4Hz)

2''

72.4

4.64 (1H, ca.)

3''

72.4

4.06 (1H, ca.)

4.24 (1H, ca.)

2''

79.4

4.27 (1H, ca.)

3''

75.1

4.58 (1H, ca.)

3''

77.4

4.26 (1H, ca.)

4''

74.2

4.24 (1H, ca.)

4''

74.4

4.03 (1H, ca.)

4''

71.6

4.15 (1H, ca.)

5''

69.9

4.20 (1H, ca.)

5''

71.4

5''

67.0

6''

18.8 1.72 (3H, d, 6.1Hz)

6''

17.6

6.4Hz)

Rha-1''' 101.9 2'''

72.6

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5.46 (1H, br.s)

10.2Hz)

D

1.54 (3H, d,

3.67 (1H, dd, 11.0,

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12.9Hz)

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77.1

3.72 (1H, dd, 6.4,

6.47 (1H, s)

Rha-1''' 99.7

5.43 (1H, br.s)

4.65 (1H, ca.)

2'''

72.3

4.66 (1H, ca.)

2'''

72.5

4.65 (1H, ca.)

3'''

72.6

4.08 (1H, ca.)

3'''

72.7

4.23 (1H, ca.)

3'''

72.5

4.08 (1H, ca.)

4'''

74.4

4.25(1H, ca.)

4'''

73.4

4.41 (1H, ca.)

4'''

74.2

4.25 (1H, ca.)

5''

70.2

4.22 (1H, ca.)

5'''

69.1

4.75 (1H, ca.)

5'''

70.1

4.22 (1H, ca.)

6'''

19.2 1.74 (3H, d, 6.1Hz)

6'''

18.6 1.64 (3H, d, 6.1Hz)

6'''

19.1

1.76 (3H, d, 6.1Hz)

Rha-1'''' 99.8

5.42 (1H, br.s)

Rha-1'''' 99.6

6.42 (1H, br.s)

2''''

72.6

4.66 (1H, ca.)

2''''

72.5

4.66 (1H, ca.)

3''''

72.9

4.23 (1H, ca.)

3''''

72.8

4.23 (1H, ca.)

4''''

74.3

4.41 (1H, ca.)

4''''

73.5

4.41 (1H, ca.)

5''''

69.7

4.76 (1H, ca.)

5''''

69.2

4.76 (1H, ca.)

6''''

18.8 1.66 (3H, d, 6.1Hz)

6''''

18.8 1.65 (3H, d, 6.1Hz)

H-NMR and

13

1.72 (3H, d, 5.8Hz)

6.44 (1H, br.s)

2''

Rha-1''' 101.7

* 1

17

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6'

3.71 (1H, dd,

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4.48 (1H, ca.)

PT

2'

C-NMR were measured at 400 MHz and 100 MHz in pyridine-d5; and the assignments were based on HSQC,

HMBC and NOESY experiments

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Graphic abstract