Sesquiterpenoids from the herbs of Solanum lyratum and their cytotoxicity on human hepatoma cells

Journal Pre-proof Sesquiterpenoids from the herbs of Solanum lyratum and their cytotoxicity on human hepatoma cells

Shuang-Shuang Li, Zhuo-Yang Cheng, Yang-Yang Zhang, Rui Guo, Xiao-Bo Wang, Xiao-Xiao Huang, Ling-Zhi Li, Shao-Jiang Song PII:

S0367-326X(19)31589-8

DOI:

https://doi.org/10.1016/j.fitote.2019.104411

Reference:

FITOTE 104411

To appear in:

Fitoterapia

Received date:

7 August 2019

Revised date:

29 October 2019

Accepted date:

4 November 2019

Please cite this article as: S.-S. Li, Z.-Y. Cheng, Y.-Y. Zhang, et al., Sesquiterpenoids from the herbs of Solanum lyratum and their cytotoxicity on human hepatoma cells, Fitoterapia (2018), https://doi.org/10.1016/j.fitote.2019.104411

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© 2018 Published by Elsevier.

Journal Pre-proof

Sesquiterpenoids from the herbs of Solanum lyratum and their cytotoxicity on human hepatoma cells Shuang-Shuang Lia, Zhuo-Yang Chenga, Yang-Yang Zhanga, Rui Guoa, Xiao-Bo Wangb,

Xiao-Xiao

Huanga,*

[email protected],

Ling-Zhi

Lia ,*

[email protected], Shao-Jiang Songa,* [email protected] a

Key Laboratory of Computational Chemistry-Based Natural Antitumor Drug

Research & Development, Liaoning Province, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, People′s Republic

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of China b

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Chinese People′s Liberation Army Logistics support force No.967 Hospital, Dalian

116021, People′s Republic of China Corresponding author.

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*

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ABSTRACT

Eleven sesquiterpenoids including four new eudesmane sesquiterpenoids, solanoids

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A-D (1-4), and seven known compounds (5-11) were isolated from the herbs of

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Solanum lyratum. By analyzing the UV, MS and NMR data, the gross structures of all

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isolates were established. The absolute configurations of these new compounds were determined by comparison of the experimental and calculated electronic circular

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dichroism (ECD) spectra. The in vitro cytotoxicity of all isolates against the hepatocellular carcinoma Hep3B and HepG2 cell lines was evaluated. Among them, compounds 7 and 11 exhibited moderate cytotoxicity against two cell lines.

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Keywords: Solanum lyratum; sesquiterpenoids; calculated ECD; cytotoxicity.

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1. Introduction

Solanum lyratum Thunb., belonging to the genus Solanum, is a perennial herbal

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distributed mainly in China, Japan, Korea and Indo-China Peninsula. It is a traditional medicinal species which is known as “Bai- Ying” in Chinese and “Back-Mo-Deung”

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in Korean [1,2]. It has invoked growing attention due to its biologically active

steroidal

alkaloids,

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constituents [3-6]. The predominant isolates from S. lyratum were steroidal saponins, polysaccharide,

and

sesquiterpenoids

[7-9].

Previous

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investigations on the biological of S. lyratum showed that it displayed anti-tumor, anti- inflammatory, anti-anaphylactic, immunomodulatory, and anti-oxidant activities [10-13].

In order to discover more structurally diverse and bioactive metabolites, a phytochemical study on the herbs of S. lyratum led to the isolation of eleven sesquiterpenoids including four new eudesmane sesquiterpenoids (1-4) (Fig. 1). Their structures were unambiguously assigned by the inspection of extensive spectroscopic data and ECD calculations. The cytotoxicity assay of all isolates was undertaken against two human hepatocellular carcinoma Hep3B and HepG2 cell lines. The isolation,

structure elucidation and

sesquiterpenoids were described. 2. Materials and Methods 2.1. General Experimental Procedures

cytotoxic activity evaluation of these

Journal Pre-proof Optical rotations were measured on a JASCO DIP-370 digital polarimeter. UV spectra

were obtained on a Shimadzu UV-1700 spectrophotometer.

ECD

measurements were conducted on a Bio-Logic MOS 450 spectrophotometer. NMR spectra were recorded on Bruker ARX-400 and AVIII-600 spectrometers with CDCl3 as solvent. HRESIMS data were acquired on a Bruker Micro Q-TOF instrument in positive-ion mode. ODS (50 µm, Merck, Germany) and Silica gel (100–200 or 200– 300 mesh; Qingdao Marine Chemical Co. Ltd.) were used

for Column

chromatography. Semipreparative HPLC was performed on a Shimadzu LC-6AD pump system with a Shimadzu SPD-20A detector, using YMC C18 column (250 × 10

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mm, 5 µm). 2.2. Plant Material

The herbs of Solanum lyratum were obtained in September 2018 from Liyang,

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Jiangsu Province, China. It was identified by Professor Jin-cai Lu of department of

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Traditional Chinese Materia Medica, Shenyang Pharmaceutical University. A voucher specimen (No. 20180907) of the plant is stored at the herbarium of the department of

2.3. Extraction and Isolation

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Pharmacognosy, Shenyang Pharmaceutical University.

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The air-dried herbs of S. lyratum (40 kg) were extracted three times with 70% EtOH (50 L × 2 h) under reflux conditions and filtered to obtain a crude methanol

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extract (5.1 kg). This was then partitioned with EtOAc and n-butanol (n-BuOH). The

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EtOAc (293 g) and n-BuOH extracts (1114 g) were fractionated by passage of a column chromatography on silica gel (CH2 Cl2 -MeOH, from 100:1 to 1:1), respectively, to produce six main fractions A-F with TLC analysis. Fraction A (32.5 g) was further chromatographed via open ODS column with EtOH-H2 O (20%-90%) as solvent to afford four fractions (A1-A4). Fraction A1 (4.6 g) was separated by silica gel CC with PE-EtOAc (from 15:1 to 0:1) as gradient solvent to afford subfractions A1a-A1e. Compound 6 (13.7 mg) was isolated from fraction A1e using semipreparative HPLC (CH3 CN-H2 O, 33:67). Fraction A2 (3.6 g) was subjected to silica gel column chromatography with a solvent system of PE-EtOAc, which gave seven subfractions (A2a-A2g). A2a (54.6 mg) was purified by semipreparative HPLC to give compounds 1 (7.2 mg) and 5 (16.5 mg) eluted with CH3 CN-H2O (48:52). Compounds 3 (4.6 mg), 4 (3.8 mg) and 7 (23.5 mg) were acquired by purification of A2b (20 mg) using semipreparative HPLC eluted with CH3 CN-H2 O (42:58). A2c (18.2 mg) was purified by semipreparative HPLC to furnish compound 8 (5.2 mg).

Journal Pre-proof Fraction A3 (7.2 g) was also divided into six subfractions (A3a-A3g) by silica gel CC under identical conditions as described above. Compound 2 (6.3 mg) was finally obtained from subfraction A3a by semipreparative HPLC (CH3 CN-H2 O, 52:48). Subfraction A3b was purified by using the same HPLC conditions as fraction A3a to yield compound 11 (3.9 mg). By a procedure similar to subfraction A3b, A3f yielded 9 (3.4 mg) and 10 (19.7 mg). Solanoids A (1): Yellow powder (MeOH); [α] 20D +189.5 (c 0.1, MeOH); UV (MeOH) λmax (logε): 204 nm (1.4); ECD (MeOH) λmax (Δε) 224 (+10.5), 238 (+8.4) nm; The 1 H (600 MHz, CDCl3 ) and

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C NMR data (150 MHz, CDCl3 ) see Table 1; HRESIMS

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(m/z): 253.1198 [M + Na]+ (calcd for C15 H18 O2 Na, 253.1199).

Solanoids B (2): Yellow powder (MeOH); [α] 20D +17.4 (c 0.1, MeOH); UV (MeOH)

CDCl3 ) and

13

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λmax (logε): 205 nm (1.6); ECD (MeOH) λmax (Δε) 209 (+6.4) nm; The 1 H (400 MHz, C NMR data (100 MHz, CDCl3 ) see Table 1; HRESIMS (m/z):

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217.1597 [M + Na]+ (calcd for C15 H20 ONa, 217.1587).

Solanoids C (3): White amorphous powder (MeOH); [α] 20D –7.0 (c 0.1, MeOH); UV

(600 MHz, CDCl3 ) and

13

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(MeOH) λmax (logε): 201 nm (1.7); ECD (MeOH) λmax (Δε) 233 (+7.9) nm; The 1 H C NMR data (150 MHz, CDCl3 ) see Table 1; HRESIMS

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(m/z): 271.1306 [M + Na]+ (calcd for C15 H20 O3Na, 271.1305). Solanoids D (4): White amorphous powder (MeOH); [α] 20D +42.7 (c 0.1, MeOH);

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UV (MeOH) λmax (logε): 209 nm (1.9); ECD (MeOH) λmax (Δε) 202 (+3.4), 216 (–1.0), 13

C NMR data (150 MHz,

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241 (+4.1), 335 (–0.8) nm; The 1 H (600 MHz, CDCl3 ) and

CDCl3 ) see Table 1; HRESIMS (m/z): 257.1490 [M + Na]+ (calcd for C15 H22O2Na, 257.1512).

2.4. ECD calculations

Conformational searches were performed by means of the MMFF94 force field in CONFLEX software to obtain all the possible conformers of compounds 1-4 [14, 15]. The conformers whose energy was within the window of 10 kcal/mol were further optimized at the B3LYP/6-31G(d) level in the Gaussian 09 software [16]. TDDFT calculations and Boltzmann distributions of all the selected conformers were carried out at the B3LYP/6- 311++G(2d, p) levels with the CPCM model in methanol solution [17]. Finally, the ECD spectra were generated as the sum of Gaussians. 2.5. Cytotoxicity assay Human hepatocellular carcinoma Hep3B and HepG2 cells were cultured in

Journal Pre-proof DMEM medium containing penicillin- streptomycin and 10% fetal bovine serum (FBS), and maintained at 37 °C with 5% CO 2 at a humidified atmosphere. These cells were made into a single-cell suspension and then seeded in 96-well cell culture clusters at a cell density of 5 × 103 cells per well. After incubation for 12 h at 37 °C with 5% CO 2 , the test compounds at different concentrations (3.12-50 μM) and sorafenib (positive control) incubated with the cell [18]. After treatment with 48 h, 20 μL of MTT solution (0.5 mg/mL) was added to each well, and the plates incubated for 4 h at 37 °C. The medium was removed from each well and 150 μL DMSO was added to solubilize the residuum. The absorbance was measured at 490 nm using a

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microplate reader (Thermo, CA, USA). The IC 50 (half maximal inhibitory concentration) values were calculated by GraphPad Prism 6.0 (San Diego, CA, USA). 2.6. Statistical analysis

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All results and data were expressed as mean ± SD at least three independent

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experiments. Statistical significance of the differences was processed through GraphPad Prism 6.0 followed by Student’s t-tests. A p value < 0.05 was taken as a

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significant difference. 3. Results and Discussion

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Solanoids A (1), yellow powder, had the molecular formula C 15 H18O2 , as deduced from its HRESIMS ion at m/z 253.1198 [M + Na]+ (calcd for C 15 H18O 2Na, 253.1199)

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and NMR data, corresponding to seven indices of hydrogen deficiency. The 1 H NMR data (Table 1) contained resonances characteristic of an aromatic proton at δH 6.56

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(1H, s, H-2), two olefinic protons at δH 4.81 (1H, s, H-12a), 4.71 (1H, s, H-12b), three magnetic unequivalent methylene groups, two methines including an oxygenated proton at δH 5.01 (1H, s, H-9), and two methyl singlets attached to the aromatic ring at δH 2.09 (3H, s, H3 -14) and 2.33 (3H, s, H3-15). The

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C NMR data with the aid of the

HSQC spectrum revealed all 15 carbon resonances, which were confirmed as a pentasubstituted aromatic ring, a double bond, three methylenes (one oxygenated), two methines (one oxygenated), and two methyls. The aforementioned NMR data resembled those of rumicifoline A [19], indicating that 1 was also a eudesmane sesquiterpenoid. A comparison of the 1D NMR spectrum of 1 with those of rumicifoline A suggested the presence of a methyl at C-1 in 1, which was confirmed through the HMBC cross-peaks of H3 -14/C-1, C-2 and C-10, H-2/C-9 and C-14. The structure was further confirmed through the HMBC cross peaks of H-2/C-4 and C-10, H-6/C-4, C-7 and C-10, H-8/C-6, C-10 and C-11, H-12/C-7 and C-13, H3 -15/C-3, C-4

Journal Pre-proof and C-5 and the 1 H- 1 H COSY correlations of H2 -6/H-7/H2 -8/H-9 (Fig. 2). In addition, an ether linkage from C-9 to C-13 was observed via the key HMBC correlation of H-9 with C-13, which was in accordance with one index of hydrogen deficiency. Therefore, the gross structure of 1 was defined as an eudesmane sesquiterpenoid featuring a 6/6/6 tricyclic skeleton with an oxygen bridge. The relative configuration of bridgehead ＊

＊

carbons within this bridge-ring could be determined as 7S , 9S

due to the presence

of rigid skeleton. The absolute configuration of 1 was determined by comparison of experimental and calculated ECD spectra based on TDDFT method. The experimental ECD spectrum matched well with the calculated ECD spectra for 7S, 9S as shown in

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Fig. 4. Hence, the absolute configuration of 1 was elucidated as 7S, 9S. Solanoids B (2) possessed a molecular formula of C15 H20 O based on the 1

H

NMR

spectrum

(Table

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HRESIMS (m/z 217.1597 [M + H]+, calcd for C15 H21 O, 217.1587) and NMR data. Its 1)

displayed

signals

for

a

symmetrical

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1,2,3,4-tetrasubstituted benzene ring at δH 6.92 (2H, s, H-2, 6), five methylene groups including one exocyclic methylene at δH 5.15 (1H, s, H-13a), 5.00 (1H, s, H-13b) and

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oxygenated methylene at δH 4.25 (2H, s, H2 -12), one methine at δH 2.41 (1H, m, H-7), and two methyl groups at δH 2.20 (6H, s, H3 -14, 15). The 13 C NMR data (Table 1) and

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HSQC spectrum showed the occurrence of 15 carbon signals, which were confirmed as a benzene ring, two olefinic carbons, four methylenes, one methine and two methyl

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groups. Analyses of its 1D NMR spectra and HMBC spectrum implied that 2 was

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similar structurally to (7R)-14(10→1)-abeoeudesma-1,3,5(10),11(13)-tetraen-12-oic acid [20], which was a rearranged eudesmane with an aromatic ring. This conclusion was further verified via the observed 1 H- 1 H COSY cross peaks of H2 -6/H-7/H2 -8/H2 -9 and H-2/H-3 (Fig. 2). The notable difference was in the presence of a methylol resonance in 2, instead of a carbonyl group at C-12. It was supported by diagnostic chemical shifts at C-12 [δC 65.4; δH 4.25 (2H, s) for 2] and the H2 -12/C-7, C-11, C-13 and H2 -13/C-12 HMBC correlations (Fig. 2). Compound 2 possessed one asymmetric carbon center at C-7, and its absolute configuration was directly confirmed by comparison of experimental and calculated ECD spectra. As shown in Fig. 4, the calculated ECD spectrum of 2 was in accordance with the experimental spectrum in showing a positive Cotton effect at 215 nm. Thus, the absolute configuration of 2 was established as 7R. Solanoids C (3), gave an [M + Na]+ ion at m/z 271.1306 (calcd for C15 H20O3Na,

Journal Pre-proof 271.1305) in the (+)-HRESIMS, which established the molecular formula of C 15 H20O3 . Its 1 H NMR spectrum (Table 1) illustrated three aromatic protons at δH 7.11 (1H, m, H-9), 7.08 (2H, m, H-8, 10), a benzylic methylene group at δH 3.04 (1H, d, J = 14.0 Hz, H-5a) and 2.97 (1H, d, J = 14.0 Hz, H-5b), a methylene group at δH 2.99 (1H, d, J = 16.7 Hz, H-2a) and 2.30 (1H, d, J = 16.7 Hz, H-2b), four methyl signals at δH 1.52 (3H, s), 1.58 (3H, s), 2.37 (6H, s) (two aromatic methyl groups). The

13

C NMR and

HSQC spectra displayed 15 carbon signals attributable to a phenyl group, one carbonyl, two oxygenated methines, two methylenes, and four methyls (two benzyls). The NMR spectra of 3 were similar to solafuranone [21], compound 5 in the present

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study, except for the downfield chemical shift of C-3 (δC-3 90.1 for 3; δC-3 45.9 for 5)

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and the absence of a methine proton at δH 2.45 (1H, m) in 5, which suggested the presence of a hydroxy functionality at C-3 in 3. The HMBC cross peaks of H2 -2/C-1,

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C-3, C-4 and C-5 and H2 -5/C-2, C-3 and C-4 confirmed this deduction (Fig. 2). The

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gross structure of 3 was verified from the additional 2D NMR (HSQC and HMBC) spectroscopic data. Compounds 3 and 5 contained a 2,6-dimethylbenzyl moiety,

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which occurs rarely in nature [22]. The only one stereogenic center at C-3 was present in compound 3. The absolute configuration of 3 was defined by using the same

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method as 2. The calculated ECD curve of 3R matched well with the experimental ECD curve of compound 3 (Fig. 4). Finally, compound 3 was determined as 3R.

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The molecular formula of solanoids D (4) was confirmed as C 15 H22O2 from the HRESIMS ion peak at m/z 257.1490 (calcd for [M + Na]+, 257.1512) and NMR data.

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The 1 H NMR data (Table 1) showed four olefinic protons at δH 6.57 (1H, d, J = 10.1 Hz, H-3), 5.89 (1H, d, J = 10.1 Hz, H-2), 4.75 (2H, m, H2 -12), two methines at δH 1.95 (1H, m, H-7) and 1.81 (1H, m, H-5), three magnetic unequivalent methylenes, and three methyl groups at δH 1.77 (3H, s, H3 -13), 1.38 (3H, s, H3 -14), 1.25 (3H, s, H3 -15), suggesting the occurrence of an eudesmane sesquiterpenoid, with the corresponding 15 carbon signals. The 1 H- 1 H COSY correlations between H-2/H-3 and H-5/H-6/H-7/H-8/H-9 together with the HMBC cross-peaks of H-5/C-4 and H3 -15/C-1 confirmed the A/B ring system of eudesmane sesquiterpenoid. Compound 4

was

elucidated

to

possess

an

identical

(4R,5R,7R,10R)-4-hydroxy-eudesma-2,11-dien-1-one

[23],

planar the

structure known

to

isolated

compound 7, except for the anticipated minor differences in the chemical shifts of C-3 (δC 150.3 for 4; 153.9 for 7), C-4 (δC 68.8 for 4; 70.7 for 7), C-5 (δC 47.9 for 4; 52.2 for 7), and C-14 (δC 29.1 for 4; 22.4 for 7). Among the four stereogenic centers (C-4,

Journal Pre-proof C-5, C-7, and C-10) in 4, the relative configurations were examined initially by NOESY spectrum. The NOESY cross-peaks (Fig. 3) of H-5/H-7, H-5/H-9b, H-5/H3 -14, and H3 -15/H-9a indicated that H-5, H-7 and H3 -14 are oriented in the same direction, indicating that compound 4 was the epimer of 7 at C-4. The absolute configuration of 4 was determined as 4S, 5R, 7R, 10R by the same methods as described above (Fig. 4). The other isolated compounds were identified as solafuranone (5) [21], lycifuranone A (6) [21], (4R,5R,7R,10R)-4-hydroxy-eudesma-2,11-dien-1-one (7) [23], (+)-anhydro-β-rotunol (8) [24], nardoeudesmol A (9) [25], rishitin (10) [26],

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1α-hydroxy-bisabola-2,10-dien-4-one (11) [27], by NMR data and optical rotations compared with the reported spectroscopic data in the references. All isolates 1-11 were tested for their in vitro cytotoxic activities against two

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human hepatocellular carcinoma, HepG2 and Hep3B cells, by MTT assay using

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sorafenib as the positive control. Among the test compounds, six compounds showed effective inhibition activity of more than 40% at 50 μM (Fig. 5). The compounds with

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inhibition ratio > 40% were screened out to treat with HepG2 and Hep3B cells in subsequent experiment. As shown in Fig. 5, compounds 7 and 11 exhibited moderate

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cytotoxicity against two tested hepatocellular carcinoma cells, and 11 had more

4. Conclusion

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cytotoxic effect on Hep3B cells (IC50 = 47.81 μM) than HepG2 cells (IC50 > 100 μM).

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In the present study, eleven sesquiterpenoids including four new eudesmane sesquiterpenoids, solanoids A-D (1-4), and seven known ones (5-11) were isolated from the herbs of Solanum lyratum. Their structures and absolute configurations were determined by UV, MS, NMR data and comparing calculated ECD with the experimental data. Compounds 3 and 5 contained a 2,6-dimethylbenzyl moiety, which occurs rarely in nature. Compound 4 was the epimer of compound 7 at C-4. All isolates were evaluated for their in vitro cytotoxicity against the hepatocellular carcinoma Hep3B and HepG2 cells. The results indicated that compounds 7 and 11 exhibited moderate cytotoxicity against two hepatocellular carcinoma cell lines. Acknowledgments This work was supported by Career Development Support Plan for Young and Middle-aged Teachers in Shenyang Pharmaceutical University (ZQN2018006) and the Project of Innovation Team Foundation (LT2015027).

Journal Pre-proof Conflict of interest The authors declare that there is no conflict of interest.

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Antiproliferative sesquiterpenoids from ligularia rumicifolia with diverse skeletons, J. Nat. Prod.

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[20] X.R. Cheng, S.D. Zhang, C.H. Wang, J. Ren, J.J. Qin, X. Tang, Y.H. Shen, S.K. Yan, H.Z. Jin,

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W.D. Zhang, Bioactive eudesmane and germacrane derivatives from Inula wissmanniana Hand.-Mazz, Phytochemistry. 96 (2013) 214-222. [21] F.J. Guo, Y.C. Li, New sesquiterpenoids from Lycinathes marlipoensis, Helv. Chim. Acta. 88 (2005) 2364-2369.

[22] W.J. Syu, M.J. Don, G.H. Lee, C.M. Sun, Cytotoxic and novel compounds from solanum indicum, J. Nat. Prod. 64 (2001) 1232-1233. [23] L.F. Ding, G.M. Yang, Y.D. Guo, L.D. Song, J. Liu, X.D. Wu, A new sesquiterpenoids from Artemisia lavandulaefolia, Chin. Trad. Herb. Drugs. 49 (2018) 1995-1999. [24] Y.C. Chen, H.Z. Lee, H.C. Chen, C.L. Wen, Y.H. Kuo, G.J. Wang, Anti-inflammatory components from the root of Solanum erianthum, Int. J. Mol. Sci. 14 (2013) 12581-12592. [25] M.L. Liu, Y.H. Duan, J.B. Zhang, Y. Yu, Y. Dai, X.S. Yao, Novel sesquiterpenes from Nardostachys chinensis Batal, Tetrahedron. 69 (2013) 6574-6578. [26] H.W. Gardner, A.E. Desjardins, S.P. Mccormick, D. Weisleder, Detoxification of the potato phytoalexin rishitin by Gibberella pulicaris, Phytochemistry. 37 (1994) 1001-1005. [27] V. Castro, G. Tamayo-Castillo, J. Jakupovic, Sesquiterpene lactones and other constituents

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from Calea prunifolia and C.peckii, Phytochemistry. 28 (1989) 2415-2418.

Journal Pre-proof Figure caption Fig. 1. The structures of compounds 1-11 from the herbs of Solanum lyratum. Fig. 2. Key HMBC and 1 H- 1 H COSY correlations of compounds 1-4. Fig. 3. Key NOESY correlations of compound 4. Fig. 4. Calculated and experimental ECD spectra of 1-4. Fig. 5. Cytotoxic activities of compounds 1-11 against two hepatocellular carcinoma cell lines. (A) Inhibition ratio of all isolates at 50 μM. (B-C) Cell viability of the compounds (3.12-50 μM) with inhibition ratio > 40% against HepG2 and Hep3B cells by MTT assay. Concentration-response curves for the cytotoxicity of compounds in

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HepG2 and Hep3B cells.

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Fig. 1. The structures of compounds 1-11 from the herbs of Solanum lyratum.

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Fig. 2. Key HMBC and 1 H- 1 H COSY correlations of compounds 1-4.

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Fig. 3. Key NOESY correlations of compound 4.

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Fig. 4. Calculated and experimental ECD spectra of 1-4.

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Fig. 5. Cytotoxic activities of compounds 1-11 against two hepatocellular carcinoma cell lines. (A) Inhibition ratio of all isolates at 50 μM. (B-C) Cell viability of the

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compounds (3.12-50 μM) with inhibition ratio > 40% against HepG2 and Hep3B cells

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by MTT assay. Concentration-response curves for the cytotoxicity of compounds in

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HepG2 and Hep3B cells.

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C-NMR data for compounds 1-4 in CDCl3

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Table 1. 1 H-NMR and

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1a δH (J in Hz)

δC 136.4 115.2

6.56, s

3 4 5

9 10 11 12 13 14 15

152.9 118.3 138.4

6.92, s

34.9

2.87, dd (15.8, 3.5) 2.50, dd (15.8, 11.5) 2.41, m 2.08, dd (12.6, 2.7 ) 1.66, dd (12.6, 5.4) 2.80, m 2.64, dd (11.7, 5.8)

35.1 32.7 66.0 125.2 149.4 107.9

4.81, s 4.71, s 3.88, d (13.3) 3.83, d (13.3) 2.33, s 2.09, s

64.4 18.5 10.7

127.0 133.9 134.8

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7 8

2.95, dd (17.1, 6.4) 2.67, d (17.1) 2.99, m 2.29, dt (5.9, 3.6) 1.82, dt (5.9, 3.6) 5.01, s

δC 133.9 126.9

6.92, s

4.25, s 5.15, s 5.00, s 2.20, s 2.20, s

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2b δH (J in Hz)

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1 2

C-NMR data for compounds 1-4 in CDCl3 .

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13

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Table 1. 1 H-NMR and

H NMR (600 MHz),

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C NMR (150 MHz), measured in CDCl3 .

b1

H NMR (400 MHz),

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C NMR (100 MHz), measured in CDCl3 .

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a1

3a δH (J in Hz) 2.99, d (16.7) 2.30, d (16.7)

3.04, d (14.0) 2.97, d (14.0)

33.6

δ 1 4

9 8 3

1

37.4 28.3

7.08, m

1 1

27.7

7.11, m

1

134.9 153.6 65.4

7.08, m 2.37, s

1 1 2

108.6

2.37, s

2

19.6 18.9

1.52, s 1.58, s

2 2

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October 29, 2019 Dear Editor:

The authors declare that there is no conflict of interest.

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incerely yours,

Xiaoxiao Huang, Assistant Professor School of Traditional Chinese Materia Medica

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Shenyang Pharmaceutical University

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Assistant Professor

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Materia Medica

Tel./Fax: +024 43520793

E-mail: [email protected]

Lingzhi

Li,

School of Traditional Chinese

Shenyang

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Pharmaceutical University

Tel./Fax: +024 43520792 E-mail: [email protected]

Shaojiang

Song, Dr., Prof.

School of Traditional Chinese Materia Medica Shenyang Pharmaceutical University Tel./Fax: +024 43520707 E-mail: [email protected]

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Graphical Abstracts