Antimicrobial and cytotoxic phenolics and phenolic glycosides from Sargentodoxa cuneata

Fitoterapia 101 (2015) 153–161

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Antimicrobial and cytotoxic phenolics and phenolic glycosides from Sargentodoxa cuneata Xiaobin Zeng a,b,⁎,1, Hai Wang a,c,1, Zhongqing Gong b, Jinghui Huang a, Weijing Pei a, Xueyan Wang a, Jingzhao Zhang a, Xudong Tang a,⁎ a Key Lab for New Drug Research of TCM and Shenzhen Branch, State R&D Centre for Viro-Biotech, Research Institute of Tsinghua University in Shenzhen, Shenzhen 518057, Guangdong, People's Republic of China b Guangdong Key Laboratory for Research and Development of Natural Drugs, Guangdong Medical College, Zhanjiang 524023, Guangdong, People's Republic of China c School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China

a r t i c l e

i n f o

Article history: Received 10 November 2014 Accepted in revised form 7 January 2015 Accepted 8 January 2015 Available online 15 January 2015 Keywords: Sargentodoxa cuneata Phenolic glycosides Dihydronaphthalene lignan Antimicrobial activity Cytotoxic activity

a b s t r a c t Five new phenolic glycosides, Sargentodosides A-E, and two new dihydronaphthalene lignans, Sargentodognans F-G, together with thirty-two known phenolic compounds were isolated from the 60% ethanol extracts of Sargentodoxa cuneata. Their structures including absolute configurations were determined by spectroscopic analysis and electronic circular dichroism experiments. In bioscreening experiments, twelve compounds (22–26, 29, 33–34, 36, 38) exhibited antibacterial activities against S. aureus ATCC 29213 with minimum inhibitory concentration (MIC) values of 2–516 μg/mL. And compound 29 showed the highest antibacterial activity against S. aureus ATCC 29213 with MIC values of 2 μg/mL, while the MIC values of levofloxacin was 8 μg/mL. Three compounds (29, 33, 36) exhibited antibacterial activities against S. aureus ATCC 25923 with MIC values of 256–516 μg/mL. Two compounds (29, 33) exhibited antibacterial activities against A. baumanii ATCC 19606 with MIC values of 128–516 μg/mL. However, no compound exhibited antimicrobial activities against C. albicans ATCC 10231. Moreover, three compounds (10, 25, 36) exhibited significant inhibition of proliferation in the two cell lines Hela and Siha, and showed stronger inhibitive activity of these two selected cell lines than cisplatin in the cytotoxic assay. Thus, S. cuneata is a potential plant source for further research targeting bacteria and cancer diseases. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Sargentodoxa cuneata (Oliv.) Rehd. Et Wils is a Lardizabalaceae plant, as a common Chinese medicinal plant. It is distributed in South, East, Central and Southwest China [1]. The stem of S. cuneata is used in Chinese folk medicine for the treatment of rheumatic arthritis, abdominal pain, acute

⁎ Corresponding authors at: Key Lab for New Drug Research of TCM and Shenzhen Branch, State R&D Centre for Viro-Biotech, Research Institute of Tsinghua University in Shenzhen, Shenzhen 518057, Guangdong, People's Republic of China. Tel.: +86 755 26551399; fax: +86 755 26711832. E-mail addresses: [email protected] (X. Zeng), [email protected] (X. Tang). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.fitote.2015.01.008 0367-326X/© 2015 Elsevier B.V. All rights reserved.

appendicitis, trauma, dysmenorrhea, amenorrhea and painful menstruation [2]. Several kinds of compounds including anthraquinones [3,4], lignans [5–10], triterpenes [6,11], phenylpropanoids [9,10,12], phenolic acids [8–10,13–15], and other compounds [3,6,9] have been previously isolated from S. cuneata. The water and the ethanol extracts of S. cuneata have been reported to inhibit the growth of many kinds of human pathogenic bacilli in vitro [1,16]. Chang and Case (2005) [14] demonstrated that phenolic glycosides, isolated from the water-soluble constituents of the stem of S. cuneata possessed significant activity against two Gram-positive organisms, Staphylococcus aureus and Micrococcus epidermidis. As part of our ongoing search of promising new active compounds from the traditional medicinal plants, we systematically investigated the constituents from the stem of S. cuneata.

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Five new phenolic glycosides, Sargentodoside A-E (1–3, 5, 9), and two new dihydro-naphthalene lignan F-G (30–31), together with thirty-two known phenolic compounds (4, 6–8, 10–29, 32– 39) were isolated and identified from the 60% ethanol extract of S. cuneata. The chemical structures of new compounds were given in Fig. 1. Their potential antimicrobial effects on S. aureus ATCC 29213, S. aureus ATCC 25923, A. baumanii ATCC 19606, and C.albicans ATCC 10231 were evaluated. Additionally, their cytotoxic activities against human tumor cell lines Hela and Siha were also evaluated.

HPLC grade and obtained from Shanghai Chemical Reagents Co., Ltd (Shanghai, China). 2.2. Plant material

2. Experimental

The stems of Sargentodoxa cuneata (Oliv.) Rehd. et Wils. were purchased from Hubei Jingui Chinese Herbal Pieces Company, Hubei Province, China, in March 2011, and were identified by Prof. Naili Wang of Research Institute of Tsinghua University in Shenzhen. A voucher specimen (No. 20110310) was maintained in the key lab for Research and Development of New Drugs, Research Institute of Tsinghua University in Shenzhen (518057), China.

2.1. General

2.3. Extraction and isolation

Infrared radiation (IR) spectra was measured on a Shimadzu FTIR spectrometer on KBr pellets. Ultraviolet (UV) spectra were obtained with a Shimadzu UV2401PC spectrophotometer. Optical rotations were measured using a JASCO P-1020 automatic digital polarimeter. Circular dichroism (CD) measurements were carried out on a JASCO J-805 spectropolarimeter. NMR spectra were recorded on a Bruker DPX-400 spectrometer using standard Bruker pulse programs. Chemical shifts were shown as δ-values with reference to tetramethylsilane (TMS) as an internal standard. ESI-MS data were obtained on a Bruker Esquire LC 200-Ion trap mass spectrometer, and HR-ESIMS were measured on a Bruker microTOF-QIImass spectrometer. Sephadex LH-20 (GE, America), silica gel (Qingdao Ocean Chemical Co., Ltd, Qingdao, China), and ODS (40–63 μm, Merck, Darmstadt, Germany) were used for column chromatography. Thin-Layer Chromatography (TLC) was carried out on preparative Silica gel 60 F254 and RP-18 F254 plates (Merck, Darmstadt, Germany), and spots were visualized by spraying the plates with 15% H2SO4, and heating them at 105 °C. Preparative high performance liquid chromatography (HPLC) was performed using an ODS column (Shim-Pack, 20 × 250 mm, 5 μm, Shimadzu, Kyoto, Japan). All other chemicals were analytical or

The air-dried stems of S. cuneata (5.0 kg) were extracted with 60% EtOH (25 L × 3) and refluxed. Evaporation of the organic solvent under reduced pressure at 55 °C yielded a crude extract (890.0 g). The concentrated brown syrup was resuspended in water and partitioned with ethyl acetate (4.7 L × 3) and watersaturated n-butanol (4.5 L × 3) gradually to afford 90.0 g and 248.0 g of dried organic extracts, respectively. The ethyl acetate fraction (90.0 g) was absorbed on 150 g silica gel and fractionated over a silica gel (763 g, 200 ~ 300 mesh, 5 × 87 cm) column by eluting gradually with CH2Cl2: MeOH [50:1 (3.2 L), 35:1 (5.8 L), 20:1 (4.3 L), 10:1 (17.1 L), 6:1 (10.7 L), 4:1 (8 L)]. This process yielded 8 fractions (FE.): 1 (1.1 g), 2 (1.2 g), 3 (4.2 g), 4 (2.2 g), 5 (1.76 g), 6 (2.4 g), 7 (4.7 g), 8 (5.0 g). Compound 21 (1 mg) was obtained by recrystallizing from FE.1. FE.2 was purified with Sephadex LH20 column [CH2Cl2: MeOH (7:3), 1 L] and preparative HPLC [HPLC condition: 5 μm, 20 × 250 mm, flow rate: 8 mL/min, MeOH: H2O (47:53) containing 0.05% CF3COOH, 0–50 min], yielding compound 29 (23 mg, tR: 43 min). FE.3 was subjected to Sephadex LH-20 column [CH2Cl2: MeOH (7:3), 1 L] and preparative HPLC [HPLC condition: 5 μm, 20 × 250 mm, flow rate: 8 mL/min, MeOH: H2O (43:57) containing 0.05%

Fig. 1. Structures of the Sargentodoside A (1), B (2), C (3), D (5), E (9), and Sargentodognans F (30), G (31).

X. Zeng et al. / Fitoterapia 101 (2015) 153–161

CF3COOH, 0–55 min], yielding compounds 26 (17 mg, tR: 35 min), 27 (6 mg, tR: 41 min) and 28 (10 mg, tR: 48 min), respectively. FE.4 was purified with Sephadex LH-20 column [CH2Cl2: MeOH (7:3), 1 L] and preparative HPLC [HPLC condition: 5 μm, 20 × 250 mm, flow rate: 8 mL/min, MeOH: H2O (38:62) containing 0.05% CF3COOH, 0–60 min], obtaining compound 30 and 31 (8 mg, tR: 56 min), respectively. FE.5 was fractionated by Sephadex LH-20 column [CH2Cl2: MeOH (7:3), 1 L] and preparative HPLC [HPLC condition: 5 μm, 20 × 250 mm, flow rate: 8 mL/min, MeOH: H2O (28:72) containing 0.05% CF3COOH, 0–68 min], yielding compounds 22 (48 mg, tR: 38 min), 23 (210 mg, tR: 43 min) and 25 (6 mg, tR: 51 min), respectively. FE.6 was subjected to Sephadex LH-20 column [CH2Cl2: MeOH (7: 3), 1 L] and preparative HPLC [HPLC condition: 5 μm, 20 × 250 mm, flow rate: 8 mL/min, MeOH: H2O (22: 78) containing 0.05% CF3COOH, 0–78 min], yielding compounds 32 (120 mg, tR: 51 min), 33 (161 mg, tR: 58 min), 34 (60 mg, tR: 66 min) and 35 (30 mg, tR: 72 min), respectively. The n-butanol fraction (248.0 g) was absorbed on 300 g silica gel and fractionated over a silica gel (2350 g, 200 ~ 300 mesh, 9.5 × 81 cm) column by eluting gradually with CH2Cl2: MeOH [15:1 (10.0 L), 85:15 (6.4 L), 80:20 (26.4 L), 70:30 (24 L), 0:1 (8 L)]. This process yielded 6 fractions (FB.): 1 (21.2 g), 2 (27.8 g), 3 (23.0 g), 4 (12.8 g), 5 (24.2 g), 6 (12.4 g). FB.2 was separated on a Sephadex LH-20 column, using MeOH-H2O (10:90, 30:70, 50:50, 70:30, 90:10, 100:0, each 2 L) to afford compound 4 (14 mg, 50:50) and six subfractions (FB.2.1-4). FB.2.1 from MeOH-H2O (10:90) was further purified with preparative HPLC [HPLC condition: 5 μm, 20 × 250 mm, flow rate: 8 mL/min, MeOH: H2O (18:82) containing 0.05% CF3COOH, 0–65 min], obtaining compounds 37 (3 mg, tR: 31 min), 36 (15 mg, tR: 38 min), 12 (25 mg, tR: 48 min), and 17 (54 mg, tR: 53 min), respectively. FB.2.2 from MeOH-H2O (30:70) was further purified with preparative HPLC [HPLC condition: 5 μm, 20 × 250 mm, flow rate: 8 mL/min, MeOH: H2O (23:77) containing 0.05% CF3COOH, 0–78 min], obtaining compounds 18 (15 mg, tR: 23 min), 10 (65 mg, tR: 31 min), 19 (10 mg, tR: 34 min), 9 (2.2 mg, tR: 39 min), 20 (6.5 mg, tR: 46 min), 1 (2.7 mg, tR: 49 min), 2 (15 mg, tR: 61 min), and 3 (116 mg, tR: 68 min), respectively. FB.2.4 from MeOH-H2O (70:30) was further purified with preparative HPLC [HPLC condition: 5 μm, 20 × 250 mm, flow rate: 8 mL/min, MeOH: H2O (43:57) containing 0.05% CF3COOH, 0–68 min], yielding compounds 7 (10 mg, tR: 35 min), 8 (10 mg, tR: 38 min), 15 (60 mg, tR: 45 min), 16 (60 mg, tR: 47 min), 24 (15 mg, tR: 50 min), 38 (45 mg, tR: 55 min) and 5 (15 mg, tR: 62 min), respectively. FB.3 was fractionated by Sephadex LH-20 column [CH2Cl2: MeOH (7:3), 10 L] and preparative HPLC [HPLC condition: 5 μm, 20 × 250 mm, flow rate: 8 mL/min, MeOH: H2O (18:82) containing 0.05% CF3COOH, 0–68 min], obtaining compounds 11 (50 mg, tR: 45 min), 13 (80 mg, tR: 48 min), 14 (59 mg, tR: 53 min), 6 (8 mg, tR: 59 min) and 39 (16 mg, tR: 62 min), respectively. The structures of new compounds 1–3, 5, 9, and 30–31 are showed in Fig. 1. 2.4. Sargentodoside A (1) Yellow amorphous powder; [α]25 D −31.4° (c 0.17, MeOH); UV λmax (CH3OH): 282 nm (logε 3.86); CD (3.42 × 10−4, MeOH) [θ]: −50018 (237 nm), −21053 (274 nm), +5901 (291 nm); For 1H NMR (400 MHz, CD3OD) and 13C NMR (100 MHz, CD3OD) spectroscopic data, see Tables 1 and 2;

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ESI-MS m/z 545 [M + Na]+; HR-ESIMS m/z 521.2032 [M − H]− (calcd for C26H33O11, 521.2023). 2.5. Sargentodoside B (2) Yellow amorphous powder; [α]25 D + 72.5° (c 0.17, MeOH); UV λmax (CH3OH): 282 nm (logε 3.86); CD (3.92 × 10−4, MeOH) [θ]: −14399 (238 nm), −5618 (274 nm), +8273 (291 nm); For 1H NMR (400 MHz, CD3OD) and 13C NMR (100 MHz, CD3OD) spectroscopic data, see Tables 1 and 2; ESIMS m/z 521 [M − H]−; HR-ESIMS m/z 521.2032 [M − H]− (calcd for C26H33O11, 521.2023). 2.6. Sargentodoside C (3) Yellow amorphous powder; [α]25 D −6.9° (c 0.17, MeOH); CD (4.02 × 10−4, MeOH) [θ]: +13606 (239 nm), +6470 (274 nm), −14658 (290 nm); For 1H NMR (400 MHz, CD3OD) and 13C NMR (100 MHz, CD3OD) spectroscopic data, see Tables 1 and 2; ESI-MS m/z 545 [M + Na]+; HR-ESIMS m/z 521.2032 [M − H]− (calcd for C26H33O11, 521.2023). 2.7. Sargentodoside D (5) Colorless amorphous solid; [α]20 D -168.7° (c 0.05, MeOH); For 1H NMR (400 MHz, CD3OD) and 13C NMR (100 MHz, CD3OD) spectroscopic data, see Tables 1 and 2; ESI-MS m/z 547 [M + Na]+; HR-ESIMS m/z 523.2313 [M − H]− (calcd for C26H35O11, 523.2308). 2.8. Sargentodoside E (9) Colorless amorphous solid; [α]25 D + 49.4° (c 0.20, MeOH); UV λmax (CH3OH): 205 nm (logε 4.03), 232 nm (logε 3.94), 279 nm (logε 3.80), 311 nm (logε 3.71); For 1H NMR (400 MHz, CD3OD) and 13C NMR (100 MHz, CD3OD) spectroscopic data, see Table 3; ESI-MS m/z 353 [M + Na]+; HR-ESIMS m/z 329.0882 [M − H]− (calcd for C14H17O9, 329.0873). 2.9. Sargentodognans F and G (30 and 31) Yellow needles; [α]25 D 0° (c 0.20, MeOH); UV λmax (CH3OH): 224 nm (logε 4.49), 283 nm (logε 4.05), and 324(logε 3.99); For 1 H NMR (400 MHz, CD3OD) and 13C NMR (100 MHz, CD3OD) spectroscopic data, see Table 4; ESI-MS m/z 623 [M − H]−; HR-ESIMS m/z 623.24023 [M − H]− (calcd for C36H35N2O8, 623.2393). 2.10. Minimum inhibitory concentration (MIC) assays The assays were performed in a similar manner as previously reported [17] using S. aureus ATCC 29213, S. aureus ATCC 25923, A. baumanii ATCC 19606, and C.albicans ATCC 10231. EBSmedium [18] for bacteria and MYC-medium (1.0% phytone peptone; 1.0% glucose; 50 mM HEPES (11.9 g/L) pH 7) for fungi was used, respectively. Briefly, 200 μL of bacteria suspension were seeded into each well of 96-well cell culture plates containing 106 CFU/mL, exposed to the test compounds and positive control (levofloxacin) at concentrations of 516, 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5 μg/mL in triples for 24 h.

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Table 1 1 H NMR Data for Compounds 1–3 and 5 (CD3OD, 400 MHz, J in Hz, δ in ppm). Positions

1

1

2

3

5

6.71 (s)

6.42 (s)

6.49 (s)

7.01 (d, J = 8.4Hz)

2 3 4

6.80 (s)

6.73 (s)

6.64 (s)

2.99 (dd, J = 17.0, 5.0Hz), 2.68 (dd, J = 17.0, 9.2Hz) 2.08 (m)

2.82 (d, 2.81 (d, J = 7.7Hz) J = 7.6Hz)

6.68 (d, J = 1.9Hz) 2.62 (m)

2.04 (m)

2.04 (m)

7 7a

2.08 (m)

1.81 (m)

1.79 (m)

8

4.26 (d, J = 4.2Hz)

3.81 (overlap)

3.83 (d, J = 11.2Hz)

6 6a

9 10

1.91(m) 3.59 (m), 3.69 (m) 1.91 (m) 3.59 (m), 3.69 (m) 2.68 (m)

6.63 (dd, J = 2.0, 8.4Hz)

1′ 2′

6.69 (d, J = 2.0Hz)

6.69 (d, 6.70(d, J = 2.0Hz) J = 1.9Hz)

6.60 (d, J = 2.0Hz)

3′ 4′ 5′

6.66 (d, J = 8.4Hz)

6.76 (d, J = 8.1Hz) 6.63 (dd, J = 1.9Hz, 8.0Hz) 4.62 (d, J = 7.5Hz)

6.65 (d, J = 8.4Hz) 6.53 (dd, J = 2.0, 8.4Hz) 4.85 (d, J = 7.8Hz)

6′

6.45 (dd, J = 2.0, 8.4Hz)

1″

4.71 (d, J = 6.8Hz)

2″ 3″ 4″ 5″ 6″ 3-OCH3 or 4′OCH3 3′-OCH3

13

Positions

H NMR

1

5

Table 2 13 C NMR Data for Compounds 1–3 and 5 (CD3OD, 100 MHz, J in Hz, δ in ppm).

6.76 (d, J = 8.0Hz) 6.62 (dd, J = 1.9Hz, 8.0Hz) 4.41 (d, J = 7.7Hz)

1 2 3 4 5 6 6a 7 7a 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 3-OCH3 or 4′-OCH3 3′-OCH3

C NMR

1

2

3

5

120.2 146.3 149.8 113.6 33.1 35.7 65.6 44.7 62.2 46.8 132.3 133.5 135.9 115.5 148.5 146.1 115.7 124.2 103.5 75.1 78.0 71.1 78.0 63.6 56.6 56.9

119.2 146.4 146.2 113.5 33.5 40.1 66.0 47.7 62.5 48.3 132.6 134.9 138.4 114.1 149.2 148.8 116.3 123.4 103.6 74.8 78.0 71.0 78.1 62.1 56.6 57.0

118.9 146.3 146.0 113.5 33.8 40.1 66.0 47.8 62.3 48.3 132.4 134.7 138.4 114.1 149.3 148.8 116.3 123.5 102.6 74.8 77.8 70.6 78.0 61.6 56.6 57.0

117.9 146.3 145.7 114.6 36.3 44.5 62.3 44.2 62.2 36.2 137.7 123.0 134.0 113.8 150.7 149.0 116.0 122.9 103.2 75.1 78.0 71.6 78.3 62.7 56.5 56.8

2.12. Statistical analysis All data were expressed as mean ± S.D., From at least three independent experiments, each performed in quintuplicate.

3. Results and discussion 3.85 (3H, s)

3.82 (3H, s)

3.82 (3H, s)

3.75 (3H, s)

3.76 (3H, s)

3.79 (3H, s)

3.79 (3H, s)

3.74 (3H, s)

2.11. Cell proliferation assay

The 60% ethanol extract of the stem of S. cuneata was subjected to separation using various chromatographic techniques, such as liquid-liquid extraction, silica gel column, ODS column, Sephadex LH-20 column and RP-HPLC, to obtain five

Table 3 1 H NMR and 13C NMR Data for Compound 9 (CD3OD, 400 MHz for 1H NMR and 100 MHz for 13C NMR, J in Hz, δ in ppm). Positions

Cell proliferation assay with cell lines Hela and Siha was performed as previously described [19].The Human cervical cancer lines HeLa and Siha were from American Type Culture Collection (ATCC). All the cells were cultured in RPMI-1640 medium (Hyclone, Logan, UT), and supplemented with 10% fetal bovine serum (Hyclone) and antibiotic (100 units/mL penicillin and 100 μg/mL streptomycin) in 5% CO2 at 37 °C. The cytotoxicity assay was performed according to the CCK-8 method in 96-well microplates. Briefly, 200 μL of adherent cells were seeded into each well of 96-well cell culture plates and allowed to adhere for 12 h before drug addition with an initial density of 5 × 104 cells/mL. Each tumor cell line was exposed to the test compound and positivie control (cisplatin) at concentrations of 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5 μg/mL in quadruples for 48 h.

Compound 9 1

H NMR

1 2 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″

5.24 (d, J = 17.2Hz), 4.88 (d, J = 17.2Hz) 7.41 (d, J = 2.0Hz)

6.83 (d, J = 8.4Hz) 7.42 (dd, J = 8.4, 2.0Hz) 4.37 (d, J = 7.6Hz) 3.29 (overlap) 3.36 (m) 3.27 (overlap) 3.28 (overlap) 3.89 (dd, J = 11.9, 1.8Hz), 3.65 (dd, J = 11.9, 5.8Hz)

13

C NMR

197.0 72.2 128.2 116.2 146.9 153.1 115.9 123.0 104.5 75.2 77.9 71.8 78.4 63.0

X. Zeng et al. / Fitoterapia 101 (2015) 153–161 Table 4 1 H NMR, 13C NMR, 1H-1H COSY, HMBC, and NOESY data for Compounds 30 and 31 (CD3OD, 400 MHz for 1H NMR and 100 MHz for 13C NMR, J in Hz, δ in ppm). positions

1

1

4.35 (d, J = 4.0Hz) 3.69 (d, J = 4.0Hz)

2 3 4 5 6 7 8 2a 3a 4a 8a 1′ 2′

H NMR

7.21 (s) 6.88 (s)

6.53 (s)

6.70 (d, J = 2.0Hz)

3′

13 C NMR

1 H-1H COSY

HMBC a

NOESY

47.7

2

8, 2′

51.1

1

2a,3,4a,8,8a, 1′,2′,6′ 2,2a,3,3a, 4,8a 3, 3a,2,5 4,6,7, 8a

5 4

1,4a,6

2 1

127.8 134.8 113.4 149.8 148.3 117.4 174.7 170.6 125.1 132.8 136.1 112.8 149.0

4′ 5′ 6′ 3′-OCH3 4′-OCH3 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 2.70 (t,

6.64 (d, J = 8.1Hz) 6.41 (dd, J = 2.0, 8.1Hz) 3.75 (s) 3.89 (s) 6.82 (d, J = 8.6Hz) 6.65 (d, J = 8.6Hz) 6.65 (d, J = 8.6Hz) 6.82 (d, J = 8.6Hz) 3.24 (m), 3.43 (m) 2.48 (t, J = 7.2Hz) J = 7.2Hz)

3′OCH3

146.4 116.4

6′

1′, 4′

121.6

2′, 5′

1, 2′, 4′

56.5 56.8 131.3 130.9

3′ 4′

2′

3″,5″

1″,3″,4″,7″

7″

116.4

2″,6″

1″,4″,5″

156.9 116.4

2″,6″

1″,3″,4″

130.9

3″,5″

1″,4″,5″,7″

8″

2″,6″,8″

42.5

35.6

7″

2a,1″,7″ 7″

1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 7‴ 8‴ 2.70 (t,

6.98 (d, J = 8.6Hz) 6.68 (d, J = 8.6Hz) 6.68 (d, J = 8.6Hz) 6.98 (d, J = 8.6Hz) 3.24 (m) 3.43 (m) 2.48 (t, J = 7.2Hz) J = 7.2Hz)

131.6 131.0

3‴,5‴

1‴,4‴,7‴

116.4

2‴,6‴

1‴,4‴,5‴

157.0 116.4

2‴,6‴

1‴,3‴,4‴

131.0

3‴,5‴

1‴,4‴,7‴

7‴

8‴

2‴,6‴,8‴

2‴, 6‴

42.8

35.8

7‴

3a,1‴ 7‴

a

HMBC correlations from H to C.

new phenolic glycosides, Sargentodoside A-E (1–3, 5, 9), and two new dihydro-naphthalene lignans, Sargentodognan F-G (30–31), together with 32 known phenolic compounds (4, 6–8, 10–29, 32–39).

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3.1. Structure elucidation of the new compounds (1–3, 5, 9, 30–31) Sargentodoside A (1) was obtained as a yellow amorphous powder, [α]25 D − 31.4° (c 0.17, MeOH). UV λmax (CH3OH): 282 nm (logε 3.86). Its molecular formula was determined to be C26H34O11 by a positive ESI-MS molecule at m/z 545 [M + Na]+ and a negative HR-ESIMS deprotonated molecule at m/ z 521.2032 [M − H]− (calcd for C26H33O11, 521.2023). The 1 H NMR spectrum (Table 1) of 1 showed signals assigned to two methoxyl groups [δH 3.85 (3H, s, 3-OCH3), and 3.76 (3H, s, 3′-OCH3)], an anomeric proton of sugar [δH 4.71(1H, d, J = 6.8 Hz, H-1″)], 1,3,4-trisubstituted benzene ring [δH 6.69 (1H, d, J = 2.0 Hz, H-2′),6.66 (1H, d, J = 8.4 Hz, H-5′), 6.45 (1H, dd, J = 2.0, 8.4 Hz, H-6′)], and 1,3,4,6-tetrasubstituted benzene ring [δH 6.71 (1H, s, H-1), 6.80(1H, s, H-4)]. In 13C NMR spectrum (Table 2), signals arising from a β-glucopyranose moiety, two aromatic nucleus, and six aliphatic carbons along with two methoxyl groups were observed, suggesting that the aglycone of 1 is a lignan. The glucose was determined to be the D-configuration by gas chromatographic (GC) analysis of a chiral derivative of the acid hydrolysate [20]. The NMR and GC data of 1 were almost similar with (−)-isolariciresinol 4-Oβ-D-glucopyranoside reported by Jiang et al. [21]. On the basis of HMBC correlations (Fig. 2), the singlet signals at 3.85 (3H, s, 3-OCH3) and 3.76 (3H, s, 3′-OCH3), which typically represent methoxy protons, showed correlations with 149.8 (C-3) and 148.5 (C-3′), thus confirming the assignment of the methoxy groups at C-3 and C-3′, respectively. The absolute configuration of C-8 of 1 was further established to be R, since a positive Cotton effect at 291 nm [21] was observed in the CD spectrum. A cis configuration of C-7 and C-8 was supported by the coupling constant (J = 4.2 Hz) between H-7 and H-8. So, the absolute configuration of C-7 of 1 was further established to be R. The coupling constant (J = 5.0 Hz) between H-5ax and H-6 and the coupling constant (J = 9.2 Hz) between H-5 eq and H-6 indicate that the corresponding dihedral angle should be ca. 180°. The minimized structure obtained by MM2 calculation [22] was used to generate dihedral angles and an angle of 52° was measured, which is compatible with a 6S configuration. The absolute configuration of C-6, C-7 and C-8 of 1 was finally established to be (6S, 7R, 8R). Thus, compound 1 was determined to be (6S,7R,8R)-5,6,7,8-tetrahydro-2-O-β-Dglucopyranoside-3-methoxy-6,7-bis(hydroxymethyl)-8-(3methoxy-4-hydroxyphenyl)-naphthalene. Sargentodoside B (2) was obtained as a yellow amorphous powder, [α]25 D + 72.5° (c 0.17, MeOH). UV: λmax (CH3OH): 282 nm (logε 3.86). Its molecular formula was determined to be C26H34O11 by a negative HR-ESIMS deprotonated molecule at m/z 521.2032 [M − H]− (calcd for C26H33O11, 521.2023). The 1H and 13C NMR data of 2 were almost similar with those of (−)-isolariciresinol 4-O-β-D-glucopyranoside. On the basis of HMBC correlations (Fig. 2), the singlet signals at 3.79 (3H, s, 3′OCH3) and 3.82 (3H, s, 4′-OCH3), which typically represent methoxy protons, showed correlations with 149.2 (C-3′) and 148.8 (C-4′), thus confirming the assignment of the methoxy groups at C-3′ and C-4′, respectively. The absolute configuration of 2 was further established to be 6S, 7R, 8R, because of a positive cotton effect at 291 nm and two negative Cotton effects at 238 nm and 274 nm was observed in the CD spectrum, which is the same with those of compound 1. Thus, Sargentodoside B

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Fig. 2. Key HMBC correlations of the Sargentodoside A(1), B (2), C (3), D (5), E (9) and Sargentodognans F (30), G (31).

was determined to be (6S,7R,8R)-5,6,7,8-tetrahydro-2-O-βD-gluco pyranoside-3-hydroxy-6,7-bis(hydroxy ethyl)-8-(3,4dimethoxyphenyl)-naphthalene. Sargentodoside C (3) was obtained as a yellow amorphous powder, [α]25 D −6.9° (c 0.17, MeOH). Its molecular formula was determined to be C26H34O11 by a negative HR-ESIMS deprotonated molecule at m/z 521.2032 [M − H]− (calcd for C26H33O11, 521.2023). The 1H and 13C NMR data of 3 were almost similar with those of compound 2. On the basis of HMBC correlations (Fig. 2), the singlet signals at 3.79 (3H, s, 3′-OCH3) and 3.82 (3H, s, 4′-OCH3), which typically represent methoxy protons, showed correlations with 149.3 (C-3′) and 148.8 (C-4′), thus confirming the assignment of the methoxy groups at C-3′ and C-4′, respectively. The absolute configuration of C-8 of 3 was further established to be S, since a negative cotton effect at 291 nm was observed in the CD spectrum. while, the absolute configuration of C-6 and C-7 was concluded to be 6R, 7S by two positive Cotton effects at 239 nm and 274 nm in the CD spectrum, which are different from the compounds 1 and 2. The absolute configuration of C-6 was 6R, which was also conducted by the correlation between H-8 and H-6 in NOESY. Thus, the absolute configuration of C-6, C-7 and C-8 of 3 was further established to be (6R, 7S, 8S). Finally, Sargentodoside C (3) was determined to be (6R,7S,8S)-5,6,7,8-tetrahydro-2-O-βD-glucopyranoside- 3-hydroxy-6,7-bis(hydroxylmethyl)8-(3,4-dimethoxyphenyl)-naphthalene. Sargentodoside D (5) was obtained as colorless amorphous − 168.7° (c 0.05, MeOH). Its molecular formula solid, [α]20 D was determined to be C26H36O11 by a negative HR-ESIMS deprotonated molecule at m/z 523.2313 [M − H]− (calcd for C26H35O11 523.2308), which was supported by 1H NMR and 13C NMR data. The 1H NMR, 13C NMR, DEPT, HMQC and HMBC data for 5 (Tables 1, 2, Fig. 2) indicated the presence of two 1,3,4trisubstituted benzene moieties [δH 7.01 (1H, d, J = 8.4Hz, H-1), 6.68 (1H, d, J = 2.0Hz, H-4), 6.63 (1H, dd, J = 2.0, 8.4Hz, H-10), 6.60 (1H, d, J = 2.0Hz, H-2′), 6.65 (1H, d, J = 8.4Hz, H-5′), 6.53 (1H, dd, J = 2.0, 8.4Hz, H-6′)], two methoxy groups [δH 3.74 (3H, s, 3′-OCH3),3.75 (3H, s, 4′-OCH3)], two oxymethylenes [δH 3.59 (2H, t, H-6a and H-7a), 3.69 (2H, m, H-6a and H-7a)], two methines [δH 1.91 (2H, m, H-6 and H-7)],

two methenes [δH 2.62 (4H, m, H-5 and H-8)], a β-glucose moiety (δH 2.54 - 4.85). The glucose was determined to be the D-configuration by gas chromatographic (GC) analysis of a chiral derivative of the acid hydrolysate [20]. The NMR and GC data of 5 were almost similar with (−)-secoisolariciresinol 4O-β-D-glucopyranoside reported by Zhong et al. [22]. However, on the basis of HMBC correlations (Fig. 2), the singlet signals at 3.74 (3H, s, 3′-OCH3) and 3.75 (3H, s, 4′-OCH3), which typically represent methoxy protons, showed correlations with 150.3 (C-3′) and 149.0 (C-4′), thus confirming the assignment of the methoxy groups at C-3′ and C-4′, respectively. The β-D-glucose, whose anomeric proton [4.85 (1H, d, J = 7.8Hz)] was correlated to C-2 in HMBC (Fig. 2), was concluded to be located at C-2. The aglycon of 5 has a symmetry axis between C-6 and C-7, because C-5, C-6 and C-6a were equivalent to C-8, C-7 and C-7a, respectively. By comparison with the literature values of (−)-secoisolariciresinol 4-O-β-D-glucopyranoside, the obtained optical rotation value of (−)-secoisolariciresinol 4-O-β-Dglucopyranoside {[α]D = −182.1° (c 0.05, MeOH)} indicated an absolute configuration of 8R and 8′R for (−)secoisolariciresinol 4-O-β-D-glucopyranoside [23]. In the same manner, the optical rotation value of 5 {[α]25D = −168.7 (c 0.05, MeOH)} suggested the absolute configuration of C-6 and C-7 to be 6R and 7R. Thus, Sargentodoside D was determined to have the structural formula 5. To our knowledge, this is the first report of this compound. Sargentodoside E (9) was obtained as colorless amorphous solid, [α]25 D + 49.4° (c 0.20, MeOH). UV λmax (CH3OH): 205 nm (logε 4.03), 232 nm (logε 3.94), 279 nm (logε 3.80), 311 nm (logε 3.71). Its molecular formula was determined to be C14H18O9 by a postive ESI-MS deprotonated molecule at m/z 353 [M + Na]+ and a negative HR-ESIMS molecule at m/z 329.0882 [M − H]− (calcd for C14H17O9, 329.0873). The 1 H NMR and 13C NMR data for 9 (Table 3) indicated the presence of a 1,3,4-trisubstituted aromatic moiety [δH 7.41 (1H, d, J = 2.0 Hz, H-2′), 6.83(1H, d, J = 8.4 Hz, H-5′) and 7.42 (1H, dd, J = 8.4, 2.0 Hz, H-6′)], an oxymethylene [δH 4.88 (1H, d, J = 17.2 Hz, H-2 eq), 5.24 (1H, d, J = 17.2 Hz, H-2ax)], and a β-glucose moiety (δH 3.27-4.37). The carbonyl (δC 197.0) was concluded to be located at C-1, which correlated with the

X. Zeng et al. / Fitoterapia 101 (2015) 153–161 Table 5 Antimicrobial activities (MIC) of the compounds (22–26, 29, 33–34, 36, 38–39). MIC (μg/mL) Compounds S. aureus ATCC 29213

S. aureus ATCC 25923

A. baumanii ATCC19606

C. albicans ATCC 10231

22 23 24 25 26 29 33 34 36 38 39 levofloxacin

N512 N512 N512 N512 N512 256 512 N512 512 N512 N512 16

N512 N512 N512 N512 N512 128 512 N512 N512 N512 N512 16

N512 N512 N512 N512 N512 N512 N512 N512 N512 N512 N512 64

256 256 256 64 64 2 64 128 8 32 256 8

hydrogens [δH 7.41(1H, d, J = 2.0 Hz, H-2′) and 7.42 (1H, dd, J = 8.4, 2.0 Hz, H-6′)] on the benzene ring in HMBC. The glucose was determined to be the D-configuration by gas chromatographic (GC) analysis of a chiral derivative of the acid hydrolysate [20]. Thus, Sargentodoside E (9) was determined to be 3′,4′-dihydroxyphenacyl-O-β-D-glucopyranoside. To our knowledge, this is the first report of this compound. Sargentodognan F and G (30 and 31) was obtained as yellow needles, UV λmax (CH3OH): 224(logε 4.49), 283(logε 4.05), and 324(logε 3.99). And the molecular formula was determined to be C36H36N2O8 by a negative ESI-MS molecule at m/z 623 [M − H]− and a negative HR-ESIMS molecule at m/z 623.24023 [M − H]− (calcd for C36H35N2O8, 623.2393), which were supported by 1H NMR and 13C NMR data. The 1H and 13 C NMR of 30 and 31 were assigned by combination of 1H-1H COSY, DEPT, HMQC, HMBC and NOESY experiments (Table 4). The 1H NMR spectrum of 30 and 31 showed the presence of two tyramine moieties, five aromatic and one olefinic protons, two methoxy signals and two methenyl protons signals, which were coupled with each other. The 1H and 13C NMR data of 30 and 31 were almost similar with dihydro-naphthalene lignin: 7-Hydroxy-1-(4-hydroxyl-3-methoxyphenyl)-N2,N3-bis (4hydroxyphenethyl)-6-methoxy-1,2-dihydro-naphthalene2,3-dicarboxamide reported by DellaGreca et al. (2006) [24]. On the basis of HMBC correlations (Table 4), the singlet signals at δH 3.75 (3H, s, 3′-OCH3) and δH 3.89 (3H, s, 4′OCH3), which typically represent methoxy protons, showed correlations with 149.0 (C-3′) and 146.4 (C-4′), thus confirming the assignment of the methoxy groups at C-3′ and C-4′, respectively. A cis configuration of the phenyl group at C-1 and the amide carbonyl at C-2 supported by the coupling constant (J = 4.0 Hz) between H-1 and H-2. Sargentodoside F and G (30 and 31) showed, in the 1H NMR spectrum, the relative protons as two doublets at 4.0 and 4.0 (J = 4.0 Hz). The coupling constant between H-1 and H-2 indicates that the corresponding dihedral angle should be ca. 45°. The minimized structure obtained by MM2 calculation [22] was used to generate dihedral angles and an angle of 52° was measured, which is compatible with a cis configuration [24]. The absolute configuration of C-1 and C-2 of 30 and 31 was further established to be (1R, 2R) and (1S, 2S), since there is no Cotton effect in the CD spectrum. Thus, 30 and 31 are a couple of racemic mixture, determined to be 6,7-dihydroxy-1-(3′,4′-dimethoxyphenyl)-N2,N3-

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bis(4-hydroxyphenethyl)-1R,2R- dihydronaphthalene-2,3dicarboxamide and 6,7-dihydroxy-1-(3′,4′-dimethoxyphenyl) -N2,N3-bis(4-hydroxy phenethyl)-1S,2S-dihydronaphthalene2,3-dicarboxamide. The known compounds were identified, by comparing of their spectroscopic data (such as UV, IR, ESIMS, 1H NMR, 13 C NMR, 1H-1H COSY, HMQC, HMBC, NOESY, et al.) with data previously reported in the literature, as (+)-isolariciresinol9′-O-β-D-glucopyranoside (4) [25], Slvadoraside (6) [26], Glehlinoside C (7) [23], 7-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)-N2,N3-bis(4- hydroxyphenethyl)-6-methoxy-1,2dihydro-naphthalene-2,3-dicarboxamide (8) [24], Sargentol (10) [9], Cuneataside C (11) [27], Osmanthuside H (12) [28,29], Crosatoside B (13) [30], Echipuroside A (14) [31], 6-(β-Dglucopyranosyloxy)-2R,4- dihydroxy-2-[(4-hydroxyphenyl) methyl]-3(2H)-benzofuranone (15) [32], 6-(β-D-glu copyranosyloxy)-2S,4-dihydroxy-2-[(4-hydroxyphenyl)methyl]3(2H)-benzofuranone (16) [32], 1-O-α-rhamnopyranosyl(1″ → 6′)-O-β-D-glucopyranosyl-2-methoxy-4- acetylphenol (17) [33], 1-O-α-L-rhamnosyl(1″-6′)-β-D-glucopyranosyloxy3,4,5- trimethoxybenzene (18) [34], 4-O-β-D-glucopyranosyl3-hydroxylbenzoic acid (19) [35], Protocatecheuic acid 3-O-βD-glucoside (20) [36], Vanillic acid (21) [37], Catechin (22) [38], (−)-Epicatechin (23) [39], Dulcisflavan (24) [40], Cinchonains Ia (25) [41,42], Caffeic acid (26) [43], Trans-N-pCoumaroyltyramine (27) [44], Protocatechuic acid (28) [45], Hydroxytyrosol (29) [46], 3-O-caffeoylquinic acid (32) [47], Calceolarioside B (33) [48], 2-(4-hydroxyphenyl)ethyl[6-O-(E)-caffeoryl]- O-β-D-glucopyranoside (34) [49], Salidroside (35) [50], 2-(3,4-dihydroxyphenyl) ethyl-O-β-Dglucopyranoside (36) [51], Icariside D2 (37) [52], methyl 3-Ocaffeoylquinate (38) [53], Procyanidin B-2 (39) [54].

Table 6 Cytotoxic activities (IC50) of the compounds (7–10, 13–18, 20–26, 28–31, 33–34 and 36–39). IC50 (μg/mL) Compounds

Hela

Siha

7 8 10 13 14 15,16 17 18 20 21 22 23 24 25 26 28 29 30, 31 33 34 36 37 38 39 Cisplatin

46.57 46.57 16.16 128.12 49.87 39.46 138.78 87.45 66.79 67.58 36.53 45.62 29.67 13.79 25.89 38.78 145.98 68.98 21.24 29.01 12.24 81.16 23.33 67.83 16.46

56.66 56.66 16.07 N256 178.98 89.96 148.98 98.52 68.85 72.58 38.12 56.67 31.07 16.67 28.34 41.67 156.16 78.34 78.48 30.12 15.43 105.61 68.90 102.34 20.75

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Fig. 3. Dose–response curve of antiproliferative activity against Hela of compounds 10, 25 and 36 (mean ± SD, n = 3).

3.2. In vitro antimicrobial activity of the compounds (1–39)

4. Conclusion

All the compounds isolated were tested for their potential antimicrobial effects on S. aureus ATCC 29213, S. aureus ATCC 25923, A. baumanii ATCC 19606, and C.albicans ATCC 10231. The antimicrobial abilities of the compounds (22–26, 29, 33– 34, 36, 38–39) isolated from S. cuneata were shown in Table 5. Among the 39 compounds tested, 22–26, 29, 30–31, 33–34, 36, 38–39 showed antibacterial activities against S. aureus ATCC 29213. 29, 33, and 36 showed antibacterial activities against S. aureus ATCC 25923. 29 and 33 showed antibacterial activities against A. baumanii ATCC 19606. However, there are no compounds that showed antimicrobial activities against C.albicans ATCC 10231. 15 and 16 showed no antimicrobial activity which was not compatible with the paper reported by Li et al. (1997) [32]. 25 have been reported to exhibit significantly antimicrobial activity against B. cereus, S. aureus, E. coli and P. aeruginosa [41]. 25 exhibited significantly antibacterial effects on S. aureus ATCC 25913. This finding was in accordance with the former research [41].

Five new phenolic glycosides, Sargentodoside A-E (1–3, 5, 9), and two new dihydro-naphthalene lignins, Sargentodoside F-G (30–31), together with 32 known phenolic compounds (4, 6–8, 10–29, 32–39), were isolated from the 60% ethanolic extract of the stem of S. cuneata. In bioscreening experiments, 22–26, 29, 31, 33–34, 36, 38–39 exhibited antibacterial activities against S. aureus ATCC 29213. 29, 33 and 36 exhibited antibacterial activities against S. aureus ATCC 25923. 29 and 33 exhibited antibacterial activities against A. baumanii ATCC 19606. However, no compound exhibited antimicrobial activities against C.albicans ATCC 10231. In the cytotoxic assay, 10, 25 and 36 exhibited significant inhibition of proliferation in the two cell lines, and showed stronger inhibition of proliferation than cisplatin. Phenolics 10, 25 and 36 were identified as promising lead compounds, especially 25 and 36. Thus, S. cuneata is a potential plant source for further research targeting bacteria and cancer diseases.

3.3. In vitro antiproliferative activity of the compounds (1–39)

Acknowledgements

Meanwhile, their cytotoxic activities against human tumor cell lines Hela and Siha were also evaluated. The cytotoxic activities of the phenolics (7–10, 13–18, 20–26, 28–31, 33–34 and 36–39) against two human tumor cell lines Hela and Siha were shown in Table 6. 10, 25 and 36 exhibited significant inhibition of proliferation in the two cell lines, and showed stronger inhibition of these two selected cell lines than cisplatin. It should be noted that the analogues of compounds 33, 34, 35, 36 and 37 have been reported to exhibit no significant cytotoxic activity against cancer cell lines Hepalclc-7 and MCF-7 [55]. However, 33, 34 and 36 exhibited significant effects on these two cancer cell lines. This finding was not in accordance with the former research. Moreover, 10, 25 and 36 seemed to cause total inhibition of cell viability in two target cell lines, resulting in an antiproliferative rate of about 85% at the concentration 64 μg/ml. From the concentration-response curve of antiproliferative activity against Hela cells among 10, 25 and 36 (Fig. 3), 25 and 36 showed higher concentrationresponse efficacy than that of 10.

This work was supported by the grants from construction supporting project programs of Shenzhen key laboratory (SW201110026), Shenzhen strategic emerging industry development special funds (JCYJ20130401173241464, JCYJ20130401172016183, ZDSY20120616141302982), the Start Fund of Guangdong Medical College (XB1302) and Shenzhen basic research project (JCYJ20140419122040615). The authors deeply appreciated associate professor Zhengshuang Xu and Dr. Delin Zhang for CD analysis (Peking University Shenzhen Graduate School, Shenzhen, China).

Appendix A. Supplementary data The 1D NMR and 2D NMR spectra for new compounds (1-3, 5, 9 and 30−31) are available in the supplementary data. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fitote.2015. 01.008.

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