2. Results and Discussion
The 95% EtOH and 50% EtOH extract of the roots and rhizomes of
D. versipellis were adsorbed by silicious earth, and then fractioned by CH
2Cl
2, EtOAc, and MeOH, respectively. The MeOH extract was isolated and purified by repeated column chromatography, allowing the isolation of one new dibenzyltyrolactone lignan dysoslignan A (
1), three new arylnaphthalide lignans dysoslignan B–C (
2–
4), along with fourteen known metabolites (
5–
18). By comparing their physical and spectroscopic data with literature values, the known metabolites were identified as sinolignan B (
5) [
12], 4-demethylpicropodophyllotoxin 7′-
O-β-
d-glucopyranoside (
6) [
13], 4-demethylpicropodophyllotoxin (
7) [
13], picropodophyllotoxin (
8) [
13], 4-demethyldehydropodophyllotoxin (
9) [
12], dehydropodophyllotoxin (
10) [
12], taiwanin H (
11) [
14], cleistanthin B (
12) [
15], arabelline (
13) [
16], podophyllotoxin (
14) [
12], 4-demethylpodophyllotoxin (
15) [
12], 4-demethylpodophyllotoxin 7′-
O-β-
d-glucopyranoside (
16) [
12], podophyllotoxin 7′-
O-β-
d-glucopyranoside (
17) [
12], and aegineoside (
18) [
17].
Compound
1 was obtained as a white amorphous powder and its molecular formula was determined as C
34H
44O
18 on the basis of its HR-ESI-MS (
m/
z 763.2419 [M + Na]
+, calcd for 763.2425). The
1H NMR spectrum (
Table 1 and
Figure S1) showed three methoxy groups at δ 3.73 (6H, s), 3.63 (3H, s); one 1,3,4-tri-substituted benzene ring at δ 6.40 (1H, d,
J = 1.0 Hz), 6.64 (1H, d,
J = 7.9 Hz), 6.35 (1H, dd,
J = 7.9, 1.0 Hz); one 1,3,4,5-tetra-substituted benzene ring at δ 6.76 (2H, s); and one methylenedioxy group at δ 5.92 (1H, s), 5.94 (1H, s). The
13C NMR spectrum (
Table 1 and
Figure S2) exhibited one carbonyl group at δ 176.7; twelve aromatic carbons and five aliphatic carbons at δ 76.8, 51.7, 38.6, 36.0, 72.1; as well as three methoxy groups at δ 55.5 (×2), 59.9; one methylenedioxy group at δ 100.7; one set of glucopyranosyl group at δ 99.8, 73.5, 76.5, 81.0, 76.3, 61.0; and one set of galactopyranosyl group at δ 103.2, 73.3, 74.6, 70.8, 74.7, 61.1. The aglycone was identified as poporhizol by comparison of its NMR and ECD data with those reported in the literature [
18], combined with data observed in the HSQC, HMBC, DEPT,
1H-
1H COSY, NOESY, and HR-ESI-MS spectra (
Figures S3–S9). The
13C NMR chemical shifts δ 99.8, 103.2, and spin-spin coupling constants (7.8, 7.9 Hz) of two anomeric protons allowed the identification of β-glucopyranosyl and β-galactopyranosyl moieties. The absolute configurations of glucose and galactose were determined by a microhydrolysis method and HPLC analysis [
19]. The HMBC cross peaks (
Figure 2) of the anomeric proton at δ 4.14 (1H, d,
J = 7.8 Hz, H-1″) with C-7 (δ 76.8) and the other anomeric proton at δ 4.26 (1H, d,
J = 7.9 Hz, H-1‴) with C-4″ (δ 81.0), respectively, indicated that the sugar sequence was β-
d-glucopyranosyl-(1→4)-β-
d-galactopyranosyl group and was attached at C-7 of the aglycone.
Establishment of the relative configuration was based on the chemical shift of H-9′ and NOESY experiment (
Figure S7). NOE correlation of H-7 (δ 5.23) with H-8′ (δ 2.85) indicated that the relationship for H-8/H-8′ was trans. This was also supported by the Δδ Hα-9′-Hβ-9′ value of 0.35 (this value ≥ 0.2 for trans, and ≈ 0 for cis) [
20]. The ECD spectrum of
1 (
Figure S8) was in good agreement with the ECD spectrum of the 7R,8S,8′R-isomer cleistonkiside B [
20]. So the 7R, 8S, and 8′R-configurations were assigned for
1. Thus, compound
1 was identified as poporhizol 7-
O-β-
d-glucopyranosyl-(1→4)-β-
d-galactopyranoside, and named dysoslignan A.
Compound
2 was obtained as a white amorphous powder and its molecular formula was determined as C
39H
50O
23 on the basis of its HR-ESI-MS (
m/
z 909.2637 [M + Na]
+, calcd for 909.2641). The
1H NMR spectrum (
Table 1 and
Figure S10) showed two methoxy groups at δ 3.73 (6H, s), four aromatic protons at δ 7.23 (1H, s), 5.95 (1H, s), 6.57 (2H, s); and one methylenedioxy group at δ 5.94 (1H, s), 5.86 (1H, s). The
13C NMR spectrum (
Table 1 and
Figure S11) exhibited one carbonyl group at δ 178.1, twelve aromatic carbons and five aliphatic carbons, as well as two methoxy groups at δ 56.2 (×2), one methylenedioxy group at δ 100.9, and three sets of glucopyranosyl groups at δ 103.6, 73.9, 77.0, 70.4, 76.9, 68.4, 103.4, 73.6, 76.8, 70.1, 76.8, 68.3, 103.2, 73.5, 76.4, 69.8, 75.0, 61.1. The aglycone was identified as picropodophyllotoxin by comparison of its NMR data with those reported in the literature [
21], combined with data observed in the HSQC, HMBC, DEPT,
1H-
1H COSY, NOESY, and HR-ESI-MS spectra (
Figures S12–S18). The
13C NMR chemical shifts δ 103.6, 103.4, 103.2 and spin-spin coupling constants (7.4, 8.3, 8.0 Hz) of three anomeric protons allowed the identification of three β-glucopyranosyl moieties. The absolute configuration of glucose was determined by the same method as compound
2. The HMBC cross peaks (
Figure 2) of the anomeric proton at δ 4.51 (1H, d,
J = 7.4 Hz, H-1″) with C-7′ (δ 76.6), and the other two anomeric protons at δ 4.12 (1H, d,
J = 8.3 Hz, H-1‴) and δ 4.10 (1H, d,
J = 8.0 Hz, H-1⁗) with C-6″ (δ 68.4) and C-6‴ (δ 68.3), respectively, indicated that the sugar sequence was β-
d-glucopyranosyl-(1→6)-β-
d-glucopyranosyl (1→6)-β-
d-glucopyranosyl group and was attached at C-7′ of the aglycone.
Establishment of the relative configuration was based on the chemical shift of C-9, the
1H coupling constants (
J values) and NOESY experiment (
Figure S16). For a cis-orientation of lactone at C-8′ and C-8, the signal of C-9 was at around δ 178.0 ppm, while for a trans-orientation, the signal of C-9 upfield shifted to around δ 175.0 ppm [
21]. According to a signal of C-9 at δ 178.1, the orientation of H-8′/H-8 of compound
2 was determined to be
cis. The
JH-7/H-8 (8.1 Hz) and
JH-7′/H-8′ (10.0 Hz) values indicated the trans-forms of H-7/H-8 and H-7′/H-8′. The NOE correlation of H-7/H-7′ and H-8/H-8′ also supported the relative configuration of 7,8-trans-7′,8′-trans-8,8′-cis. Studies on the ECD curves of 7-aryltetralin lignans showed that all 7β (
S)-aryl compounds gave negative Cotton effects at around 280–290 nm, while all 7α (
R)-aryl compounds gave a positive Cotton effect [
12]. The ECD spectrum (
Figure S17) of compound
2 exhibited a positive Cotton effect at 290 nm. Consequently, the absolute configuration of C-7 was determined to be
R. Thus, compound
2 was established as 4-demethylpicropodophyllotoxin 7′-
O-β-
d-glucopyranosyl-(1→6)-β-
d-glucopyranosyl-(1→6)-β-
d-glucopyranoside, and named dysoslignan B.
Compound
3 was obtained as a white amorphous powder and possessed a molecular formula C
20H
16O
8, as revealed by its HR-ESI-MS analysis (
m/
z 407.0737 [M + Na]
+, calcd for 407.0743). The
1H NMR spectrum (
Table 2 and
Figure S19) showed two methoxy group at δ 3.72 (6H, s); four aromatic protons at δ 7.54 (1H, s), 7.03 (1H, s), 6.48 (2H, s); and one methylene group at δ 5.30 (2H, s). The
13C NMR spectrum (
Table 2 and
Figure S20) revealed a skeleton of arylnaphthalide lactone lignan including one carbonyl group at δ 169.9, sixteen aromatic carbons and one aliphatic carbon at δ 66.5, as well as two methoxy groups at δ 56.0 (×2). A careful comparison of the NMR spectra of
3 with 4-demethyl-dehydropodophyllotoxin, combined with data observed in the HSQC, HMBC, and HR-ESI-MS spectra (
Figures S21–S23), indicated that compound
3 was a demethylene derivative of 4-demethyl-dehydropodophyllotoxin [
12]. The HMBC correlation (
Figure 2 and
Figure S22) between two methoxy groups at δ 3.72 (6H, s) and δ 147.5 (C-3, 5) indicated that they were located at C-3 and C-5. Thus, compound
3 was identified as 6′,7′-demethylene-4-demethyldehydropodophyllotoxin, and named dysoslignan C.
Compound
4 was obtained as a white amorphous powder and possessed a molecular formula C
20H
14O
8, as revealed by its HR-ESI-MS analysis (
m/
z 383.0768 [M + H]
+, calcd for 383.0767). The
1H NMR spectrum (
Table 2 and
Figure S24) showed one methoxy group at δ 3.70 (3H, s); four aromatic protons at δ 7.60 (1H, s), 6.89 (1H, s), 6.30 (1H, d,
J = 1.9 Hz), and 6.28 (1H, d,
J = 1.9 Hz); one methylenedioxy group at δ 6.151 (1H, s), 6.152 (1H, s); and one methylene group at δ 5.30 (2H, s). The
13C NMR spectrum (
Table 2 and
Figure S25) revealed a skeleton of arylnaphthalene lactone lignan including one carbonyl group at δ 169.5, sixteen aromatic carbons and one aliphatic carbon at δ 66.5, as well as one methoxyl group at δ 55.9, and one methylenedioxy group at δ 101.9. A careful comparison of the NMR spectra of
4 with 4-demethyl-dehydropodophyllotoxin, combined with data observed in the HSQC, HMBC, and HR-ESI-MS spectra (
Figures S26–S28), suggested compound
4 to be a demethylation derivative of 4-demethyldehydropodophyllotoxin [
12]. The HMBC correlation (
Figure 2 and
Figure S27) between the methoxy group at δ 3.70 (3H, s) and δ 148.0 (C-3), indicated that it was located at C-3. Thus, compound
4 was identified as 3,4-di-demethyldehydropodophyllotoxin, and named dysoslignan D.
All isolated compounds were evaluated for their in vitro cytotoxic activities against the A-549 and SMMC-7721 cell lines using the MTS assay [
22] with cisplatin and paclitaxel as positive controls, and the IC
50 values are summarized in
Table 3. Compounds
7–
12 and
14–
17 showed more potent cytotoxicities against the SMMC-7721 cell line than the A549 cell line. Compounds
7–
10 and
14–
16 exhibited more potent activities than cisplatin in two tumor cell lines. Compound
14 showed the highest cytotoxicity against the A-549 and SMMC-7721 cell lines, with IC
50 values of 0.130 and 0.0088 μM, respectively. The glycosylation of 7′-hydroxy group strongly reduced the activity; for example, comparing
16 to
15,
17 to
14, and
2,
5, and
6 to
7. The cis-fusion compounds (
6,
7 and
8) between the tetraline and lactone were more cytotoxic than those corresponding trans-fusion analogues (
16,
15, and
14). Compounds
7,
15 and
8,
14 containing a non-aromatized ring C exhibited more cytotoxic activity than aromatized compounds
9 and
10, indicating that the non-aromatized ring C played an important role in the cytotoxicity against A-549 and SMMC-7721 cells lines. The methylenedioxy-bearing compound (
9) was found to be more potent than the ring A-opened analogue (
3). The preliminary structure-activity relationship investigation suggested that the trans-fusion between the tetraline and lactone, non-aromatized ring C, and a methylenedioxy at ring A, were structurally required for maintaining cytotoxicity for related podophyllotoxin analogues.
3. Experimental Section
3.1. General Experimental Procedures
Optical rotations and ECD spectra were determined by a Rudolph AP-IV polarimeter (Rudolph, Hackettstown, NJ, USA) and an Applied Photophysics Chirascanq CD spectropolarimeter (AppliedPhotophysics, Leatherhead, Surrey, UK), respectively. UV and IR spectra were obtained using a Thermo EVO 300 spectrometer (Thermo, Waltham, MA, USA) and a Thermo Nicolet IS 10 spectrometer (Thermo, Waltham, MA, USA), respectively. NMR and mass spectra were performed on a Bruker Avance III 500 spectrometer (Bruker, Rheinstetten, Germany) and a Bruker maXisHD mass spectrometer (Bruker, Bremen, Germany), respectively. Preparative HPLC separations were run on a SEP system (Beijing Sepuruisi scientific Co., Ltd., Beijing, China) equipped with a variable-wavelength UV detector, using a YMC-Pack ODS-A column (250 × 20 mm, 5 μm). ODS (50 μm), sephadex LH-20 (40–70 μm), and silica gel (160–200 mesh) were acquired from YMC Co. Ltd. (Kyoto, Japan), Amersham Pharmacia Biotech AB, (Uppsala, Sweden), and Marine Chemical Industry, (Qingdao, China), respectively. MCI gel CHP-20 and Diaion HP-20 were obtained from Mitsubishi Chemical Corp. (Tokyo, Japan). Chemical reagents for isolation were of analytical grade and purchased from Tianjin Siyou Co., Ltd., Tianjin, China. Biological reagents were from Sigma Company.
3.2. Plant Material
The roots and rhizomes of D. versipellis were collected in Qingzhen, Guizhou Province, China, in July 2019, and identified by Prof. Cheng-Ming Dong at School of Pharmacy, Henan University of Chinese Medicine, where a voucher specimen (DV 20190706) was deposited.
3.3. Extraction and Isolation
The powered roots and rhizomes of D. versipellis (40 kg) were refluxed with 95% EtOH (v/v 120 L × 3, 1.5 h each) and 50% EtOH (v/v 120 L × 1, 1.5 h each) at 95 ℃, respectively. The filtrate was evaporated under reduced pressure to give a dark brown residue (5.4 kg). The residue was adsorbed by silicious earth and eluted by CH2Cl2, EtOAc, and MeOH. The MeOH extract (3.4 kg) was fractioned by silica gel column chromatography (CC), eluting with a gradient of CH2Cl2–MeOH (v/v 100:0, 100:1, 100:3, 100:5, 100:7, 100:10, 100:30, 100:50, 0:100). Nine fractions M1~M9 were obtained on the basis of TLC monitoring results. The white precipitates (3.5 g) from fraction M4 was isolated by preparative HPLC (MeOH:H2O, 66:34) at a flow rate of 3 mL min−1 to give compounds 7 (tR 13.7 min, 3.6 mg), 15 (tR 16.0 min, 12.4 mg), 8 (tR 18.3 min, 2.7 mg), 14 (tR 21.2 min, 2.7 mg), 9 (tR 28.1 min, 5.2 mg), and 10 (tR 44.5 min, 3.0 mg). Fraction M4 (90.2 g) was subjected to sephadex LH-20 CC eluted by methanol to yield subfractions M4–1~M4–3. Subfraction M4–1 (23.7 g) was submitted to ODS CC eluted by MeOH–H2O (10:90, 30:70, 50:50, 70:30, 90:10, 100:0) to afford subfractions M4–1–1~M4–1–6. Subfraction M4–1–2 (4.8 g) was separated by sephadex LH-20 CC eluted by methanol to yield subfractions M4–1–2–1~M4–1–2–6. Subfraction M4–1–2–3 (1.6 g) was isolated by preparative HPLC (MeOH:H2O, 52:48) at a flow rate of 3 mL min−1 to give subfractions M4–1–2–3–1 (tR 7.9 min), M4–1–2–3–2 (tR 8.3 min), M4–1–2–3–3 (tR 10.8 min), M4–1–2–3–4 (tR 12.8 min), M4–1–2–3–5 (tR 16.4 min), and M4–1–2–3–6 (tR 25.1 min). Subfraction M4–1–2–3–2 (22.7 mg) was purified by preparative HPLC (MeOH:H2O, 48:52) at a flow rate of 3 mL min−1 to afford 3 (tR 11.9 min, 2.5 mg). Subfraction M4–1–2–3–5 (19.2 mg) was isolated by preparative HPLC (MeOH:H2O, 42:58) at a flow rate of 3 mL min−1 to afford 11 (tR 54.1 min, 2.1 mg). Subfraction M4–1–2–3–6 (25.3 mg) was purified by preparative HPLC (MeOH:H2O, 50:50) at a flow rate of 3 mL min−1 to afford 4 (tR 32.5 min, 2.2 mg). Fraction M5 (110.0 g) was subjected to sephadex LH-20 CC eluted by methanol to yield subfractions M5–1~M5–8. Subfractions M5–1~M5–5 (22.9 g) were combined and submitted to MCI CC eluted by MeOH–H2O (0:100, 10:90, 30:70, 50:50, 70:30, 90:10, 100:0) to afford subfractions M5–1–1~M5–1–4. Subfraction M5–1–2 (3.7 g) was applied to silica gel CC with a CHCl3-MeOH (100:0, 100:1, 100:3, 100:5 100:7, 100:10, 7:1, 3:1) gradient to give subfractions M5–1–2–1~M5–1–2–8. Subfraction M5–1–2–6 (50 mg) was isolated by preparative HPLC (MeOH:H2O, 54:46) at a flow rate of 3 mL min–1 to give compound 16 (tR 18.5 min, 5.6 mg). Subfraction M5–1–3 (0.25 g) was separated by silica gel CC with a CHCl3-MeOH (100:0, 100:1, 100:3, 100:5 100:7, 100:10, 7:1, 3:1) gradient to give subfractions M5–1–3–1~M5–1–3–9. Subfraction M5–1–3–5 (2.5 g) was purified by preparative HPLC (MeOH:H2O, 55:45) at a flow rate of 3 mL min–1 to give compounds 12 (tR 49.0 min, 4.3 mg) and 17 (tR 32.2 min, 3.3 mg). The precipitates from subfraction M5 were washed repeatedly by MeOH, and then the white powder (compound 6) was obtained. Fraction M6 (130.0 g) was subjected to sephadex LH-20 CC eluted by methanol to yield subfractions M6–1 and M6–2. The subfraction M6–1 (57.3 g) was applied to ODS CC with a MeOH-H2O (10:90, 30:70, 50:50, 70:30, 90:10, 100:0) gradient to give subfractions M6–1–1~M6–1–4. Subfraction M6–1–2 (5.8 g) was separated by silica gel CC with a CH2Cl2-MeOH (100:0, 100:1, 100:3, 100:5, 100:7, 100:10, 100:30) gradient to give subfractions M6–1–2–1~M6–1–2–7. Subfraction M6–1–2–4 (1.03 g) was isolated by preparative HPLC (MeOH:H2O, 45:55) at a flow rate of 3 mL min–1 to give subfraction M6–1–2–4–1 (tR 11.0 min) and 2 (tR 39.1 min, 6.6 mg). Subfraction M6–1–2–4–1 (15.2 mg) was applied to preparative HPLC (MeOH:H2O, 38:62) at a flow rate of 3 mL min−1 to give 18 (tR 17.0 min, 3.2 mg). The subfraction M6–1 (57.3 g) was applied to ODS CC with a MeOH-H2O (10:90, 30:70, 50:50, 70:30, 90:10, 100:0) gradient to give subfractions M6–1–1~M6–1–4. Subfraction M6–2 (65.9 g) was separated by silica gel CC with a CH2Cl2-MeOH (100:1, 100:3, 100:5, 100:7, 100:10, 100:30) gradient to give subfractions M6–2–1~M6–2–6. Subfraction M6–2–5 (1.7 g) was submitted to preparative HPLC (MeOH:H2O, 60:40) at a flow rate of 3 mL min−1 to give 13 (tR 22.9 min, 4.0 mg). The subfractions M7 and M8 were combined and then applied to Diaion HP-20 CC with an EtOH-H2O (10:90, 30:70, 50:50, 70:30, 90:10, 100:0) gradient to give subfractions M7–1~M7–6. The white sticky gum from subfraction M7–2 was washed repeatedly by MeOH and then separated by preparative HPLC (MeOH:H2O, 35:65) at a flow rate of 3 mL min−1 to give compounds 1 (tR 16.5 min, 10.0 mg) and 5 (tR 21.7 min, 3.7 mg).
3.4. Spectroscopic and Physical Data
Dysoslignan A (
1): white, amorphous powder; [α
–24.6 (c 0.28, MeOH); ECD (MeOH) λmax (Δε) 206 (–15.0), 222 (+0.5), 237 (–2.0), 285 (–0.3) nm; UV (MeOH) λmax (log ε) 204 (4.81), 275 (3.79), 285 (3.66) nm; IR (iTR)
νmax 3386, 2931, 2905, 2832, 1759, 1653, 1594, 1506, 1462, 1447, 1422, 1389, 1334, 1244, 1192, 1169, 1127, 1074, 1037 cm
−1; HR-ESI-MS (positive):
m/
z 763.2419 [M + Na]
+ (calcd for C
34H
44O
18Na, 763.2425); NMR data (DMSO-
d6), see
Table 1.
Dysoslignan B (
2): white, amorphous powder; [α
–39.7 (c 0.25, MeOH); ECD (MeOH) λmax (Δε) 208 (+3.75), 238 (+0.31), 290 (+0.42) nm; UV (MeOH) λmax (log ε) 204 (4.45), 242 (3.62), 284 (3.36) nm; IR (iTR)
νmax 3381, 2361, 1764, 1616, 1523, 1475, 1375, 1335, 1264, 1219, 1168, 1121, 1033 cm
−1; HR-ESI-MS (positive):
m/
z 909.2637 [M + Na]
+ (calcd for C
39H
50O
23Na, 909.2641); NMR data (DMSO-
d6), see
Table 1.
Dysoslignan C (
3): white, amorphous powder; UV (MeOH) λmax (log ε) 204 (4.49), 225 (4.19), 264 (4.35), 326 (3.76), 363 (3.60) nm; IR (iTR)
νmax 3367, 2989, 2946, 2833, 1741, 1608, 1520, 1467, 1420, 1348, 1274, 1213, 1186, 1115, 1090, 1027 cm
−1; HR-ESI-MS (positive):
m/
z 385.0920 [M + H]
+ (calcd for C
20H
17O
8, 385.0923),
m/
z 407.0737 [M + Na]
+ (calcd for C
20H
16O
8Na, 407.0743); NMR data (DMSO-
d6), see
Table 2.
Dysoslignan D (
4): white, amorphous powder; UV (MeOH) λmax (log ε) 202 (4.48), 225 (4.29), 263 (4.38), 312 (3.82), 355 (3.60) nm; IR (iTR)
νmax 3410, 2939, 2839, 1745, 1605, 1535, 1465, 1352, 1244, 1131, 1094, 1030 cm
−1; HR-ESI-MS (positive):
m/z 383.0768 [M + H]
+ (calcd for C
20H
15O
8, 383.0767); NMR data (DMSO-
d6), see
Table 2.
3.5. Acid Hydrolysis and Sugar Determination
The absolute configurations of the galatose and glucose moieties were determined by the previously reported method [
19]. Compounds
1 (1.0 mg) and
2 (1.0 mg) were dissolved in 1.0 mL of 2M HCl, and then hydrolyzed at 90 °C for 3 h. The HCl in the reaction mixture was removed under reduced pressure. The remaining reaction mixture was extracted with CH
2Cl
2. The water layers were directly analyzed by HPLC [column: Asahipak NH
2P-50 4E (4.6 mm × 250 mm); mobile phase: CH
3CN-H
2O (17:3), flow rate: 0.7 mL/min]. The peaks at 13.15 and 14.27 min were coincided with D-glucose and D-galatose.
3.6. Cytotoxicity Asssay
By the previously reported MTS method [
22], the cytotoxic activities of compounds
1–
18 were evaluated against human lung cancer A-549, hepatocellular carcinoma SMMC-7721 cell lines. The cells were cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS) at 37 ℃ under 5% CO
2 in a humidified atmosphere. Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). To be brief, 100 µL of cells were seeded into each well in a 96-well cell culture plate in advance. After 24 h, various concentrations of all test compounds were added. After the incubation for 48 h, MTS (20 μL) was added to each well, and the incubation continued for 4 h at 37 ℃. The optical density at 492 nm was determined using a 96-well microtiter plate reader. The IC
50 values were calculated by the Reed–Muench method. Statistical analysis were performed by SPSS 20.0 software (SPSS Inc., Chicago, IL, USA). All experiments were performed in triplicate.