Further New Gypenosides from Jiaogulan (Gynostemma pentaphyllum

Article pubs.acs.org/JAFC

Further New Gypenosides from Jiaogulan (Gynostemma pentaphyllum) Jun Wang,†,‡ Jun-Li Yang,*,† Pan-Pan Zhou,§ Xian-Hua Meng,† and Yan-Ping Shi† †

CAS Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Lanzhou 730000, People’s Republic of China ‡ University of Chinese Academy of Sciences, Beijing 100039, People’s Republic of China § College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, Gansu 730000, People’s Republic of China S Supporting Information *

ABSTRACT: Jiaogulan (Gynostemma pentaphyllum) has been widely used as a herbal tea, dietary supplement, and vegetable in Asian countries. In this study, six new gypenosides (1−6) were isolated from the aerial parts of G. pentaphyllum. Their molecular structures were elucidated through spectroscopic analysis and acid hydrolysis. Gypenosides 1 and 2 represented the first example of a dammar-21-O- glucopyranoside without any unsaturated functional group and a dammar-3-O-glucopyranosyl-25-Oglucopyranoside without any cyclization in the side chain, respectively. In addition, gypenosides 5 and 6 exhibited the first example of a 24-hydroperoxy-19-oxo-dammarane triterpenoid and 19-oxo-dammar-21-O-glucopyranoside with a saturated side chain, respectively. Gypenoside 5 was found to possess protein tyrosine phosphatase 1B inhibitory activity, with an IC50 value of 8.2 ± 0.9 μM, and moderate cytotoxicity against human breast cancer cells MCF7, MCF7/ADR, and MDA-MB-231, with IC50 values ranging from 10.5 ± 1.4 to 14.2 ± 2.6 μM. The outcome of the study provided crucial information regarding the structural diversity and health benefits of gypenosides. KEYWORDS: jiaogulan, Gynostemma pentaphyllum, new gypenosides, PTP1B inhibitory activity, cytotoxicity

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INTRODUCTION

In this present study, we have undertaken a phytochemical analysis of an aqueous ethanol extract of the aerial parts of G. pentaphyllum to understand the structural diversity and bioactivity of gypenosides, and the investigation has led to the isolation of six new gypenosides 1−6. The structure elucidation of these isolates was performed, followed by protein tyrosine phosphatase 1B (PTP1B) inhibitory activity and cytotoxicity assessment. The detailed procedures of isolation, structure elucidation, PTP1B inhibitory activity, and cytotoxicity study have been described in the following sections.

Gynostemma pentaphyllum (Thunb.) Makino (family: Cucurbitaceae), also known as jiaogulan in Chinese, is a perennial creeping plant widely distributed in Shaanxi, Gansu, Hubei, and Guangxi provinces in China as well as Korea, Japan, and Southeast Asian countries.1,2 This plant has wide usage as a herbal tea, dietary supplement, and vegetable. The plethora of utilities of this plant was recorded in the book “Herbs for Famine” during the Chinese Ming Dynasty (1368−1644 A.D.).3,4 The plant possesses various therapeutic efficacies that include immunity-enhancing effect, cholesterol-lowering effect, anti-inflammatory properties, and blood-pressure-regulating activities. For this reason, the plant was included in the list of health products, approved by the Chinese government.5 Previously reported phytochemical investigations indicated the presence of dammarane glycosides and flavonoid glycosides as the major chemical constituents in this plant.6 The dammarane glycosides, also named as gypenosides, are the bioactive saponins, which are structurally similar to ginsenosides, the bioactive principles of Panax ginseng C. A. Meyer (Araliaceae).7 Approximately, 180 gypenosides have been isolated from G. pentaphyllum to date,8 with various bioactivities, such as hepatoprotective, anti-inflammatory, antilipidermic, cardiovascular, and antioxidant effects,9−12 and eight of those ginsenosides were identified as ginsenosides Rc, Rd, Rb1, F2, Rg3, Rb3, malonyl-Rd, and malonyl-Rb1.13 Since the 1990s, many health products and beverages based on G. pentaphyllum have been developed and sold in Chinese markets, for example, total jiaogulan saponin tablets and jiaogulan tea.14 © 2017 American Chemical Society

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MATERIALS AND METHODS

General Experimental Procedures. Optical rotation values and infrared (IR) spectroscopic data were acquired from a PerkinElmer 341 polarimeter and an IFS120HR 670 Fourier transform infrared (FTIR) spectrometer, respectively. Nuclear magnetic resonance (NMR) spectra at 25 °C were recorded through a Bruker Avance III 400 or Varian INOVA 600 spectrometer, and the recorded NMR spectra were processed through processing software MestReNova (version 5.3.1) using the solvent residual signal (CD3OD, δH 3.31 and δC 49.0) as a chemical shift reference. High-resolution electrospray ionization mass spectrometry (HRESIMS) was performed with a Bruker microTOF-Q II mass spectrometer. The D101 macroporous resin was purchased from Xi’an Lan Xiao New Material Company of China. The silica gel of 200−300 mesh was obtained from the Qingdao Marine Chemical Factory of China. The C18 reversed-phase silica gel of 40−60 mesh and Sephadex LH-20 were purchased from Received: Revised: Accepted: Published: 5926

April 1, 2017 June 28, 2017 June 29, 2017 June 29, 2017 DOI: 10.1021/acs.jafc.7b01477 J. Agric. Food Chem. 2017, 65, 5926−5934

Article

Journal of Agricultural and Food Chemistry Table 1. 1H and 13C NMR Data (400 MHz) for Gypenosides 1−4 in CD3OD 1 number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 sugar 1 2 3 4 5 6 sugar 1 2 3 4 5 6 sugar 1 2 3 4 5 6

1

H (J in Hz)

1.67 m, 0.93 m 1.67 m, 1.58 m 3.08 dd (11.3, 5.0) 0.71 m 1.49 m, 1.45 m 1.53 m, 1.24 m 1.33, m 1.34 (2H) m 1.55 m, 1.32 m 1.76, m 1.47 1.56 1.87 0.97 0.85

m, 1.02 m m, 1.00 m m s s

3.85 3.45 1.57 1.38 1.31

d (10.0) d (10.0) m, 1.47 m (2H) m (2H) m

1.14 1.14 0.92 0.73 0.86

s s s s s

Glu-O-C21 4.23 d (7.7) 3.17 m 3.33 m 3.23 m 3.23 m 3.85 m, 3.62 m

2 13

C

40.5 25.2 79.8 40.2 57.5 19.6 36.6 41.8 52.2 38.4 22.8 28.2 42.7 51.5 32.4 28.8 46.8 16.3 17.0 78.0 75.9 37.6 19.9 45.7 71.7 29.4 29.4 28.8 16.3 17.0

1

H (J in Hz)

1.69 m, 0.97 m 1.67 m, 1.40 m 3.15 dd (10.8, 4.8) 0.79 d (11.0) 1.53 m, 1.43 m 1.53 m, 1.24 m 1.37 m 1.50 m, 1.23 m 1.24 m, 1.01 m 1.82 m 1.44 1.70 1.87 1.00 0.85

m m, 1.51 m m s s

4.09 3.30 1.70 1.73 1.55

d (10.9) overlap m, 1.51 m m, 1.57 m m, 1.38 m

1.26 1.18 1.04 0.85 0.90

s s s s s Glu-O-C3 4.32 d (7.8) 3.18 m 3.23 m 3.25 m 3.34 m 3.86 m, 3.67 m

3 13

C

40.5 26.3 91.0 40.5 57.9 19.4 36.7 41.9 52.4 38.2 22.9 28.9 42.8 51.5 32.4 27.4 46.9 16.2 17.0 79.5 73.5 27.4 17.4 38.0 73.2 28.4 33.3 28.6 17.3 17.0 106.9 75.4 77.9 71.9 78.1 63.0

105.1 75.5 78.1 71.9 78.1 63.0 Glu-O-C25 4.19 d (7.8) 3.18 m 3.34 m 3.25 m 3.23 m 3.86 m, 3.67 m

1

H (J in Hz)

1.69 m, 0.98 m 1.70 m, 1.62 m 3.15 dd (11.4, 4.3) 0.79 d (10.2) 1.54 m 1.30 m, 1.27 m 1.37 m 1.52 m, 1.28 m 1.26 m, 1.04 m 1.79 m 1.51 1.94 1.88 1.00 0.90

m, 1.06 m m, 1.70 m m s s

3.87 3.47 1.54 2.00 5.10

d (10.0) d (10.0) m, 1.51 m m, 1.98 m brs

1.67 1.62 1.04 0.84 0.89

s s s s s Glu-O-C3 4.32 d (7.8) 3.18 m 3.34 m 3.25 m 3.23 m 3.86 m, 3.65 m Glu-O-C21 4.27 d (7.8) 3.18 m 3.33 m 3.25 m 3.23 m 3.86 m, 3.68 m

4 13

C

40.5 25.2 91.0 40.5 57.8 19.4 36.6 41.9 52.3 38.2 22.8 28.2 42.7 51.5 32.4 27.4 46.7 16.3 17.0 78.5 75.8 37.0 23.9 126.1 132.2 26.0 18.0 28.6 17.0 16.9 106.9 75.8 77.8 71.9 77.9 63.1 105.2 75.5 78.1 72.0 78.2 63.0

1

H (J in Hz)

2.49 m, 0.82 m 1.04 (2H) m 3.18 dd (10.6, 4.1) 1.24 m 1.99 m, 1.76 m 1.73 m, 1.46 m 1.73 m 1.65 m, 0.97 m 1.72 m 1.71 m 1.49 1.82 1.89 0.87 10.16

m, 1.12 m m, 1.25 m m s s

3.88 3.44 1.60 2.00 5.09

d (10.3) d (10.3) m, 1.49 m m t (6.8)

1.65 s 1.62 s 1.10 s 0.80 s 0.86 s Ara-O-C3 4.24 d (7.6) 3.54 m 3.49 m 3.79 m 3.81 m, 3.51 m Glu-O-C21 4.26 d (7.8) 3.21 m 3.27 m 3.27 m 3.36 m 3.87 m, 3.65 m

13

C

34.6 28.6 89.3 40.9 56.0 18.6 35.6 41.5 54.2 54.0 23.2 25.2 42.5 51.3 32.8 28.2 46.6 17.7 208.3 77.8 75.7 37.0 23.9 126.1 132.2 26.0 18.0 27.1 16.2 16.8 107.3 73.0 74.5 69.8 66.8

105.1 75.5 78.1 72.0 78.1 63.1

105.9 75.8 78.4 72.1 77.9 63.1

YMC of Japan. Prep-high-performance liquid chromatography (HPLC) separation was performed on a prep-HPLC manufactured by Hanbon Sci & Tech of China using a Megres C18 column (250 × 20 mm) or a Hedea ODS-2 column (250 × 20 mm). Thin-layer chromatography (TLC) detections were performed by spraying 5% H2SO4 in EtOH (v/v) on the developed TLC plates, followed by heating. p-Nitrophenyl phosphate (p-NPP), 3-(4,5-dimethylthiazol-2-

yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), Dulbecco’s modified Eagle’s medium (DMEM), ethylenediaminetetraacetic acid (EDTA), and dithiothreitol (DTT) were purchased from SigmaAldrich Corporation. The breast cancer cell lines MCF7, MDA-MB231, and MCF7/ADR were obtained from Shanghai Baili Biotechnology Co., Ltd. D-Glucose, D-xylopyranose, and L-arabinopyranose were 5927

DOI: 10.1021/acs.jafc.7b01477 J. Agric. Food Chem. 2017, 65, 5926−5934

Article

Journal of Agricultural and Food Chemistry Table 2. 1H and 13C NMR Data (400 MHz) for Gypenosides 5 and 6 in CD3OD 5 number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

1

H (J in Hz)

2.50 0.83 1.77 3.16

d (13.7) m m, 1.30 m dd (11.0, 4.2)

1.23 m 1.99 m, 1.75 m 1.73 m, 1.45 m 1.73 m 1.63 m, 0.93 m 1.72 m, 1.60 m 1.79 m 1.57 1.80 1.85 0.88 10.18

m, 1.11 m m, 1.22 m m s s

3.80 2.15 1.54 4.16

m, 3.43 m m, 1.28 m m m

4.96 1.72 1.08 0.80 0.92

(2H) s s s s s

6 13

C

34.6 28.3 88.6 41.0 56.2 18.6 35.6 41.6 54.2 54.1 23.1 25.2 42.6 51.3 32.7 28.5 47.0 17.8 208.1 79.6 75.5 30.8 26.4 91.2 146.0 114.3 17.4 27.1 16.4 16.8

1

H (J in Hz)

2.49 0.83 1.78 3.18

d (13.1) m m, 1.27 m dd (10.5, 4.9)

1.24 m 1.99 m, 1.76 m 1.73 m, 1.47 m 1.73 m 1.63 m, 0.96 m 1.63 m 1.72 m 1.49 1.82 1.89 0.88 10.18

m, 1.12 m m, 1.22 m m s s

3.86 1.58 1.40 1.48

m, 3.46 m m, 1.48 m m,1.34 m m, 1.39 m

1.17 1.17 1.08 0.80 0.92

s s s s s

5 13

1

C

34.6 28.6 88.6 41.0 56.2 18.6 35.6 41.6 54.1 54.1 23.2 25.2 42.6 51.3 32.8 28.3 46.7 17.7 208.1 78.1 75.8 37.6 19.9 45.7 71.9 29.5 29.4 27.1 16.4 16.9

6

H (J in Hz)

13

C

1

H (J in Hz)

13

C

1′ 2′ 3′ 4′ 5′

4.46 3.85 3.80 3.95 3.83

d (5.4) m m m m, 3.48 m

105.2 75.5 81.8 68.8 64.9

4.46 3.80 3.76 3.94 3.83

d (4.0) m m m m, 3.48 m

105.2 74.8 81.7 69.0 64.5

1″ 2″ 3″ 4″ 5″ 6″

5.19 3.70 3.90 3.39 3.84 1.21

s m m m m d (6.1)

102.2 72.3 72.3 74.0 70.5 18.1

5.19 3.70 3.90 3.39 3.85 1.21

s m m m m d (6.2)

102.2 72.3 72.3 74.0 70.4 18.1

1‴ 2‴ 3‴ 4‴ 5‴

4.45 3.20 3.51 3.25 3.89

d (6.8) m m m m, 3.24 m

105.1 75.5 77.5 71.2 66.9

4.46 3.20 3.32 3.51 3.89

d (6.4) m m m m, 3.22 m

105.1 75.4 77.6 71.2 66.9

sugar at C21 1 2 3 4 5 6

4.25 3.28 3.34 3.25 3.26 3.81

d (7.7) m m m m m, 3.64 m

105.0 75.5 77.6 72.1 78.1 63.1

4.25 3.87 3.26 3.25 3.35 3.86

d (7.7) m m m m m, 3.65 m

105.1 75.4 78.1 71.7 77.9 63.0

at a flow rate of 2 mL/min) equipped with a Hedea ODS-2 column (250 × 20 mm) to yield saponin 4 (4.1 mg, with tR = 40 min). Fraction F4 (102 mg) was separated over Sephadex LH-20 using a gradient of CHCl3−MeOH (from 1:1 to 1:2, v/v) to obtain two subfractions, as F4-1 (37 mg) and F4-2 (51 mg). F4-1 was further separated by prep-HPLC (eluted with 85% MeOH, at a flow rate of 2 mL/min) equipped with a Megres C18 column (250 × 20 mm) to afford two saponins 1 (3.5 mg, with tR = 14 min) and 2 (2.9 mg, with tR = 23 min). Saponin 3 (3.5 mg, with tR = 25 min) was isolated from F4-2 by prep-HPLC (eluted with 84% MeOH, at a flow rate of 2 mL/ min) equipped with a Hedea ODS-2 column (250 × 20 mm). 3β,20(S*),21,25-Tetrahydroxydammar-21-O-β-D-glucopyranoside (1): white amorphous power; [α]20 D , +10 (c 0.1, methanol); IR (film) νmax, 3420, 2936, and 1023 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z, 663.4443 [M + Na]+ (calcd for C36H64O9Na, 663.4443). 3β,20(S*),21,25-Tetrahydroxydammar-3-O-β-D-glucopyranosyl25-O-β-D-glucopyranoside (2): white amorphous power; [α]20 D , −20 (c 0.1, methanol); IR (film) νmax, 3368, 2936, 2859, 1074, and 1017 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z, 807.4865 [M − H2O + Na]+ (calcd for C42H72O13Na, 807.4865). 3β,20(S*),21-Trihydroxydammar-24-en-3-O-β-D-glucopyranosyl21-O-β-D-glucopyranoside (3): white amorphous power; [α]20 D , −10 (c 0.1, methanol); IR (film) νmax, 3368, 2942, 2852, 1653, 1087, and 1023 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z, 807.4856 [M + Na]+ (calcd for C42H72O13Na, 807.4865). 3β,20(S*),21-Trihydroxydammar-19-oxo-24-en-3-O-α-L-arabinopyranosyl-21-O-β-D-glucopyranoside (4): white amorphous power; [α]20 D , +10 (c 0.1, methanol); IR (film) νmax, 3375, 2935, 2871, 1700,

purchased from Sigma-Aldrich Corporation. L-Rhamnopyranose was obtained from J&K Chemical Co., Ltd. (Shanghai, China). Plant Material. The aerial parts of G. pentaphyllum were collected from Ping-li county of Shaanxi province of China in 2015 and were authenticated by Prof. Qi of Lanzhou Institute of Chemical Physics (LICP). A voucher specimen (JGL-PL-201501) of that plant was deposited at the Key Laboratory of Chemistry of Northwestern Plant Resources, LICP, CAS. Extraction and Isolation. The aerial parts of G. pentaphyllum (200 g) were air-dried and extracted with 70% ethanol (3 × 1 L, 1 day for each time) at room temperature. The concentrated crude residue (7.6 g) of the extract was chromatographed through a D101 macroporous resin column and eluted with an ethanol−water gradient system (0:100, 30:70, 60:40, 90:10, and 100:0, v/v). The faction obtained from 60:40 ethanol−water elute was evaporated to obtain 1.4 g of dried residue, which is further fractionated by a C18 reversed-phase silica gel column with a methanol−water gradient from 50:50 to 100:0 (v/v) to yield four fractions (F1−F4) according to their TLC profiles. Fraction F1 (184 mg) was purified through a prep-HPLC (eluted with 70% methanol, at a flow rate of 2 mL/min) equipped with a Megres C18 column (250 × 20 mm) to yield compound 6 (3.2 mg, with tR = 11.4 min). Fraction F2 (206 mg) was chromatographed over silica gel using ethyl acetate−methanol gradient (from 10:1 to 1:1, v/v) to obtain a crude subfraction (32 mg), which was further separated by prep-HPLC (eluted with 70% methanol, at a flow rate of 2 mL/min) equipped with a Megres C18 column (250 × 20 mm) to obtain compound 5 (3.6 mg, with tR = 9 min). Fraction F3 (161 mg) was chromatographed through a silica gel column using a gradient of CHCl3−MeOH (from 20:1 to 2:1, v/v) to yield a subfraction (42 mg), which was further purified by prep-HPLC (eluted with 78% methanol, 5928


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Journal of Agricultural and Food Chemistry

Figure 1. Chemical structures of the new gypenosides 1−6. 1640, and 1061 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/ z, 791.4563 [M + Na]+ (calcd for C41H68O13Na, 791.4552). 3β,20(S*),21-Trihydroxy-24-hydroperoxydammar-19-oxo-25-en3-O-[α-L-rhamnopyranosyl-(1 → 2)]-[β-D-xylopyranosyl-(1 → 3)]-[αL-arabinopyranosyl]-21-O-β-D-glucopyranoside (5): white amorphous power; [α]20 D , −5 (c 0.1, methanol); IR (film) νmax, 3445, 2916, 1720, 1670, 1074, and 1036 cm−1; 1H and 13C NMR data, Table 2; HRESIMS m/z, 1101.5431 [M + Na]+ (calcd for C52H86O23Na, 1101.5452). 3β,20(S*),21,25-Tetrahydroxydammar-19-oxo-3-O-[α-L-rhamnopyranosyl-(1 → 2)]-[β-D-xylopyranosyl-(1 → 3)]-[α-L-arabinopyranosyl]-21-O-β-D-glucopyranoside (6): white amorphous power; [α]20 D , −20 (c 0.1, methanol); IR (film) νmax, 3375, 2942, 2878, 1701, 1067, and 1036 cm−1; 1H and 13C NMR data, Table 2; HRESIMS m/z, 1087.5668 [M + Na]+ (calcd for C52H88O22Na, 1087.5659). Acid Hydrolysis of Saponins 1−6. The saponins 1−6 (1 mg each) were hydrolyzed by refluxing with 1 M HCl (1:1 H2O/ethylene oxide, 2 mL) for 2 h. The dried residues thus obtained were partitioned between ethyl acetate and water. The residue obtained from the water part was dissolved in pyridine (1 mL) and mixed with L-cysteine methyl ester hydrochloride (2 mg). The mixture was warmed at 60 °C for 2 h, and 0.2 mL of trimethylsilylimidazole was added thereafter and again maintained at 60 °C for another 2 h. The residue thus obtained was dried and partitioned between water (1.5 mL) and n-hexane (1.5 mL). The n-hexane layer was analyzed through a gas chromatographic system [detector, flame ionization detector (FID); detector temperature, 280 °C; injection temperature, 250 °C; DB-5 capillary column, 30 m × 0.25 mm × 0.25 μm; column temperature, 100 °C for 2 min and then increase to 280 °C at a rate of 10 °C/min; final temperature, 280 °C for 5 min; and carrier gas, N2]. By comparison of the retention time of the trimethylsilyl-L-cysteine

derivatives of the samples with derivatives of authentic sugars, the absolute configurations of sugar components were determined (Dglucose, 19.50 min; L-rhamnopyranose, 18.32 min; D-xylopyranose, 17.59 min; and L-arabinopyranose, 17.72 min). PTP1B Inhibition Assay.15 Human recombinant PTP1B (BIOMOL International, LP, Plymouth Meeting, PA, U.S.A.) and pnitrophenyl phosphate (p-NPP) (as a substrate) were employed to determine the PTP1B inhibitory activity of the saponins 1−6. p-NPP (4 mM) and PTP1B (0.05−0.1 μg) with (at concentrations ranging from 1 to 40 μM) or without saponins in a buffer containing 1 mM EDTA, 0.1 M NaCl, 50 mM citrate (pH 6.0), and 1 mM DTT were applied in each of 96 wells of a microtiter plate with the final volume as 100 μL. After incubation at 37 °C for 30 min, the reaction was quenched by adding 10 M NaOH. The concentration of p-nitrophenol (reaction product) was calculated by measuring the ultraviolet (UV) absorbance measured at 405 nm using an Infinite M200 PRO Nanoquant microplate reader (Tecan Group, Ltd., Mannedorf, Switzerland). The non-enzymatic hydrolysis of p-NPP was estimated through a blank analysis, which was used to correct the concentration of p-nitrophenol. Cell Culture and MTT Cytotoxicity Assay. Breast cancer cell lines MCF7, MDA-MB-231, and MCF7/ADR were maintained at 37 °C in a humidified atmosphere with 5% CO2. We followed the standard trypsinization procedure for the cell subculture every 3 days with DMEM as the cell culture medium. The MTT assay was employed to assess the cytotoxicity of the saponins 1−6. In brief, 1 × 104 MCF7, MDA-MB-231, and MCF7/ADR cells were seeded in 96well plates with the final volume of 100 μL of the culture medium per well. After 24 h, the cells were treated with saponins at concentrations ranging from 1 to 40 μM and incubated for 48 h. A total of 20 μL of MTT solution (2 mg/mL) was added in each well and incubated for 5929


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Figure 2. Key HMBC correlations (from H to C) for the new gypenosides 1−6. another 4 h. UV absorbance at 550 nm (Infinite M200 PRO Nanoquant microplate reader, Tecan Group, Ltd., Mannedorf, Switzerland) was used to determine the cell viability percentage and cytotoxicity of the saponins 1−6. Statistical Analysis. All results were expressed as the mean ± standard deviation (SD) based on triplicate experiments and evaluated using SigmaPlot software.

and C-25, as analyzed by heteronuclear multiple-bond correlation (HMBC) cross peaks from H-3 (δH 3.08) to C-4 (δC 40.2), C-28 (δC 28.8), and C-29 (δC 16.3), from H-21 (δH 3.45,3.85) to C-17 (δC 46.8), C-20 (δC 78.0), and C-22 (δC 37.6), and from H-26/27 (δH 1.14, 6H) to C-24 (δC 45.7) and C-25 (δC 71.7) (Figure 2). The HMBC cross peak from the anomeric proton (δH 4.23) to C-21 (δC 75.9) suggested the location of the glucose unit at C-21. The coupling pattern of H3 (dd, J = 11.3 and 5.0 Hz) suggested the β orientation of OH3,18 which was further supported by the nuclear Overhauser effect (NOE) correlation from H-3 (δH 3.08) to H-5 (δH 0.71) (Figure 3). The NOE correlations from H-17 (δH 1.87) to H21 (δH 3.45) and H3-30 (δH 0.86) (Figure 3) were used to determine the assignments of α-oriented H-17 and the C20(S*) configuration. Finally, the structure of gypenoside 1 was elucidated as 3β,20(S*),21,25-tetrahydroxydammar-21-O-β-Dglucopyranoside, and this compound represented the first example of a dammar-21-O-glucopyranoside without any unsaturated functional group. Gypenoside 2, another white amorphous powder, with an optical rotation value of [α]20 D of −20 (c 0.1, methanol) had a molecular formula of C42H74O14 based on a HRESIMS ion at m/z 807.4865 [M − H2O + Na]+ (calcd 807.4871). The IR absorption at 3368 cm−1 suggested the presence of a hydroxy group. The occurrence of a β-D-glucopyranosyl unit in compound 2 was confirmed by the same procedure as described for gypenoside 1.16,17 The overall NMR pattern (Table 1) and the established molecular formula of the compound indicated that compound 2 could be another gypenoside with two glucopyranosyl units. The two hydroxy

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RESULTS AND DISCUSSION The hydroalcoholic extract of the aerial parts of G. pentaphyllum was chromatographed through D101 macroporous resin, C18 reversed-phase silica gel, silica gel, Sephadex LH-20, and prepHPLC to afford six new gypenosides 1−6 (Figure 1). Gypenoside 1 was obtained as a white amorphous powder with an optical rotation value of [α]20 D of +10 (c 0.1, methanol). Its molecular formula was determined as C36H64O9 based on a HRESIMS ion at m/z 663.4443 [M + Na]+ (calcd 663.4443). The IR absorption at 3420 cm−1 indicated the presence of a hydroxy group. The presence of a D-glucopyranosyl unit in compound 1 was identified by acid hydrolysis and gas chromatography analysis,16 and the configuration of the glucopyranosyl unit was determined by the coupling pattern of the anomeric proton (d, J = 7.7 Hz) and was found to be β configuration.17 Moreover, its 1H and 13C NMR data (Table 1) of compound 1 showed seven methyl singlets [δH 1.14 (6H), 0.97, 0.92, 0.85, 0.86, and 0.73], an oxymethylene [δH 3.85, 3.45 (ABq, J = 10.0 Hz); δC 75.9], an oxymethine [δH 3.08 (dd, J = 11.3 and 5.0 Hz); δC 79.8], and two oxygenated tertiary carbons (δC 78.0 and 71.7). These observations indicated compound 1 to be a dammarane triterpenoid possessing a Dglucose unit. Three hydroxy groups were assigned at C-3, C-20, 5930


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Figure 3. Key NOE correlations for the new gypenosides 1−6.

similar NMR pattern (Table 1) between compounds 3 and 2, in conjunction with the established molecular formula, suggested compound 3 being another gypenoside possessing two glucopyranosyl units. The planar structure of compound 3 was constructed on the basis of the following HMBC cross peaks from H-21 (δH 3.87,3.47) to C-17 (δC 46.7), C-20 (δC 78.5), and C-22 (δC 37.0) and from both H-26 (δH 1.67) and H-27 (δH 1.62) to C-24 (δC 126.1) and C-25 (δC 132.2) as well as from one anomeric proton at δH 4.32 to C-3 (δC 91.0) and from another anomeric proton at δH 4.27 to C-21 (δC 75.8). The coupling pattern of H-3 (dd, J = 11.4 and 4.3 Hz) and the NOE correlation from H-3 (δH 3.15) to H-5 (δH 0.79) were used to determine α-oriented H-3. The NOE correlations from H-17 (δH 1.88) to H-21 (δH 3.47) and H3-30 (δH 0.89) supported the α-oriented assignment of H-17 and the C-20(S*) configuration. Hence, the structure of gypenoside 3 was assigned as 3β,20(S*),21-trihydroxydammar-24-en-3-O-β-Dglucopyranosyl-21-O-β-D-glucopyranoside. Gypenoside 4, a white amorphous power with [α]20 D of +10 (c 0.1, methanol), had a molecular formula of C41H68O13 based on a HRESIMS ion at m/z 791.4563 [M + Na]+ (calcd 791.4552). The IR spectrum of this compound indicated the presence of hydroxy (3375 cm−1), carbonyl (1700 cm−1), and olefinic (1640 cm−1) functionalities. The presence of α-Larabinopyranosyl and β-D-glucopyranosyl units in compound 4 was verified by acid hydrolysis and gas chromatography,16 and they were deduced as α and β configurations, respectively,

groups were located at C-20 (δC 79.5) and C-21 (δC 73.5) based on the HMBC cross peaks from H-21 (δH 4.09 and 3.30) to C-17 (δC 46.9), C-20 (δC 79.5), and C-22 (δC 27.4) and from both H-26 (δH 1.26) and H-27 (δH 1.18) to C-24 (δC 38.0) and C-25 (δC 73.2) (Figure 2). The two glucopyranosyl units were connected to C-3 (δC 91.0) and C-25 (δC 73.2), as confirmed by the HMBC cross peaks from the anomeric proton at δH 4.32 to C-3 (δC 91.0) and the other anomeric proton at δH 4.19 to C-25 (δC 73.2). The coupling pattern of H-3 (dd, J = 10.8 and 4.8 Hz) and the NOE correlation from H-3 (δH 3.15) to H-5 (δH 0.79) determined the α orientation of H-3.18 The NOE correlations from H-17 (δH 1.87) to H-21 (δH 3.30) and H3-30 (δH 0.90) were used to identify the assignments of αoriented H-17 and the C-20(S*) configuration. Consequently, the structure of gypenoside 2 was assigned as 3β,20(S*),21,25tetrahydroxydammar-3-O-β-D-glucopyranosyl-25-O-β-D-glucopyranoside. Gypenoside 2 exhibited the first example of a dammar-3-O-glucopyranosyl-25-O-glucopyranoside without any cyclization in the side chain. Gypenoside 3, a white amorphous power with an optical rotation value of [α]20 D of −10 (c 0.1, methanol), had a molecular formula of C42H72O13 from a HRESIMS ion at m/z 807.4856 [M + Na]+ (calcd 807.4865). The IR peaks at 3368 and 1653 cm−1 indicated the presence of hydroxy and olefinic functionalities, respectively. A β-D-glucopyranosyl unit was identified in gypenoside 3 following similar analytical procedures as described for gypenoside 1.16,17 The closely 5931


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Table 3. Inhibitory Effects of Gypenosides 1−6 on the PTP1B Enzyme and Their Cytotoxicity against Breast Cancer Cell Lines cytotoxicity against breast cancer cell lines (IC50, μM) compound

PTP1B inhibitory activity (IC50, μM)

1 2 3 4 5 6 ursolic acid 4-hydroxytamoxifen

>30 18.2 ± 1.3 23.5 ± 1.8 28.6 ± 2.8 8.2 ± 0.9 12.5 ± 1.8 4.01 ± 0.2

MCF7 >30 21.0 28.1 26.9 12.4 20.6

± ± ± ± ±

0.8 2.6 1.9 2.3 1.3

5.32 ± 0.4

based on coupling constants of anomeric protons with J = 7.6 and 7.8 Hz.16,17 Except for the signals of sugar units, the 1H and 13 C NMR data (Table 1) showed the presence of one oxymethylene (δH 3.88 and 3.44; δC 75.7), one oxymethine (δH 3.18; δC 89.3), one formyl group (δH 10.16; δC 208.3), one oxygenated tertiary carbon (δC 77.8), and one trisubstituted olefinic system (δH 5.09; δC 132.2 and 126.1). These observations indicated that gypenoside 4 possessed one Lrhamnopyranosyl unit and one D-glucopyranoyl unit as well as one formyl group, one hydroxy group, and one trisubstituted olefinic system. The positions of all functional groups were assigned on the basis of the following HMBC cross peaks from the anomeric proton at δH 4.24 to C-3 (δC 89.3), from another anomeric proton at δH 4.26 to C-21 (δC 75.7), from the proton for the formyl group at δH 10.16 to C-1 (δC 34.6), C-9 (δC 54.2), and C-10 (δC 54.0), from H-21 (δH 3.88,3.44) to C-17 (δC 46.6), C-20 (δC 77.8), and C-22 (δC 37.0), from H2-22 (δH 1.60,1.49) to C-20 (δC 77.8), and from both H-26 (δH 1.65) and H-27 (δH 1.62) to C-24 (δC 126.1) and C-25 (δC 132.2). The coupling pattern of H-3 (dd, J = 10.6 and 4.1 Hz) and the NOE correlation from H-3 (δH 3.18) to H-5 (δH 1.24) (Figure 3) supported the assignment of α-oriented H-3.18 The NOE correlation from H-19 (δH 10.16) to H3-18 (δH 0.87) indicated the presence of a β-oriented formyl group (Figure 3). The NOE correlations from H-17 (δH 1.89) to H-21 (δH 3.44) and H3-30 (δH 0.86) (Figure 3) suggested α-oriented H-17 and the C-20(S*) configuration. Finally, the structure of gypenoside 4 was concluded as 3β,20(S*),21-trihydroxydammar-19-oxo-24en-3-O-α-L-arabinopyranosyl-21-O-β-D-glucopyranoside. The molecular formula of gypenoside 5, a white amorphous power with an optical rotation value of [α]20 D of −5 (c 0.1, methanol), was deduced to be C52H86O23 based on a HRESIMS ion at m/z 1101.5431 [M + Na] + (calcd 1101.5452). The IR absorptions indicated the presence of hydroxy (3445 cm−1), carbonyl (1720 cm−1), and olefinic (1620 cm−1) functionalities. The occurrence of α-L-rhamnopyranosyl, β-D-xylopyranosyl, α-L-arabinopyranosyl, and β-Dglucopyranosyl units in compound 5 was determined by acid hydrolysis and gas chromatography analysis16 as well as the coupling constants of anomeric protons (Table 2). Besides, the 1 H and 13C NMR data of compound 5 (Table 2) showed signals for one oxymethylene (δH 3.80 and 3.43; δC 75.5), two oxymethines (δH 3.16; δC 88.6 and δH 4.16; δC 91.2), one formyl group (δH 10.18; δC 208.1), one oxygenated tertiary carbon at δC 79.6, and one terminal double bond (δH 4.96, 2H; δC 146.0 and 114.3). These observations indicated that this gypenoside possessed four sugar units, one formyl group, one hydroxy group, hydroperoxy group, and one terminal double bond. The planar structure of compound 5 was derived on the basis of HMBC cross peaks, as shown in Figure 2, specifically,

MCF7/ADR

MDA-MB-231

>30 16.7 21.6 >30 14.2 25.6

29.1 23.2 15.3 >30 10.5 24.1

± 1.5 ± 1.2 ± 2.6 ± 1.0

2.58 ± 0.3

± 2.5 ± 2.9 ± 1.0 ± 1.4 ± 2.5

2.80 ± 0.2

from H-1′ (δH 4.46) to C-3 (δC 88.6), from H-1″ (δH 5.19) to C-2′ (δC 75.5), from H-1‴ (δH 4.45) to C-3′ (δC 81.8), from the anomeric proton at δH 4.25 to C-21 (δC 75.5), from H-19 (δH 10.18) to C-1 (δC 34.6), C-5 (δC 56.2), C-9 (δC 54.2), and C-10 (δC 54.1), from H2-21 (δH 3.80 and 3.43) to C-17 (δC 47.0), C-20 (δC 79.6), and C-22 (δC 30.8), and from H-26 (δH 4.96) to C-24 (δC 91.2), C-25 (δC 146.0), and C-27 (δC 17.4). The coupling pattern of H-3 (dd, J = 11.0 and 4.2 Hz) and the NOE correlation from H-3 (δH 3.16) to H-5 (δH 1.23) suggested H-3 as α-oriented. The β-oriented formyl group was supported by the NOE correlation from H-19 (δH 10.18) to H3-18 (δH 0.88). The NOE correlations from H-17 (δH 1.85) to H-21 (δH 3.43) and H3-30 (δH 0.92) were used to determine α-oriented H-17 and the C-20(S*) configuration. The configuration of the hydroperoxy group at C-24 remained undetermined. Therefore, the structure of gypenoside 5 was assigned as 3β,20(S*),21-trihydroxy-24-hydroperoxydammar19-oxo-25-en-3-O-[α-L-rhamnopyranosyl-(1 → 2)]-[β-D-xylopyranosyl-(1 → 3)]-[α-L-arabinopyranosyl]-21-O-β-D-glucopyranoside. Gypenoside 5 represented the first example of a 24hydroperoxy-19-oxo-dammarane triterpenoid. Gypenoside 6, a white amorphous power with [α]20 D of −20 (c 0.1, methanol), was assigned a molecular formula of C52H88O22 based on a HRESIMS sodium adduct ion at m/z 1087.5668 [M + Na]+ (calcd 1087.5659). The IR absorptions indicated the presence of hydroxy (3375 cm−1) and carbonyl (1701 cm−1) functionalities. Gypenoside 6 was found to possess α-L-rhamnopyranosyl, β-D-xylopyranosyl, α-L-arabinopyranosyl, and β-D-glucopyranosyl units.16 Its 1H and 13C NMR data (Table 2) were closely similar to those of gypenoside 5. The major differences of them were the presence of signals for one oxygenated tertiary carbon at δC 71.9 in compound 6, and the disappearance of signals for the hydroperoxy group (δH 4.16; δC 91.2) and the terminal double bond [δH 4.96 (2H); δC 146.0 and 114.3] in compound 5. Finally, the planar structure of compound 6 was furnished on the basis of the HMBC cross peaks from H-1′ (δH 4.46) to C-3 (δC 88.6), from H-1″ (δH 5.19) to C-2′ (δC 74.8), from H-1‴ (δH 4.46) to C-3′ (δC 81.7), from the anomeric proton at δH 4.25 to C-21 (δC 75.8), from H-19 (δH 10.18) to C-1 (δC 34.6), C-9 (δC 54.1), and C-10 (δC 54.1), from H2-21 (δH 3.86 and 3.46) to C-17 (δC 46.7), C-20 (δC 78.1), and C-22 (δC 37.6), and from H2-24 (δH 1.48 and 1.39) and H-26/27 (δH 1.17, 6H) to C-25 (δC 71.9). The coupling pattern of H-3 (dd, J = 10.5, 4.9 Hz) and the NOE correlation from H-3 (δH 3.18) to H-5 (δH 1.24) suggested H-3 as α-oriented. The NOE correlation from H-19 (δH 10.18) to H3-18 (δH 0.88) was employed to identify the β-oriented formyl group. The configurations of α-oriented H-17 and C20(S*) were deduced from the NOE correlations from H-17 (δH 1.89) to H-21 (δH 3.46) and H3-30 (δH 0.92). Finally, the 5932


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Hundred Talents Program, and the Top Priority Program of “One-Three-Five” Strategic Planning of the Lanzhou Institute of Chemical Physics of CAS.

structure of gypenoside 6 was assigned as 3β,20(S*),21,25tetrahydroxydammar-19-oxo-3-O-[α-L-rhamnopyranosyl-(1 → 2)]-[β-D-xylopyranosyl-(1 → 3)]-[α-L-arabinopyranosyl]-21O-β-D-glucopyranoside. Gypenoside 6 showed the first example of a 19-oxo-dammar-21-O-glucopyranoside with a saturated side chain. Inhibitors of PTP1B have been recognized as new avenues for the treatment of breast cancers.19,20 The overexpression of PTP1B in the mammary gland could lead to spontaneous breast cancer.21 Even mammary tumors could be induced by the deletion mutation in the region of ErbB2 in transgenic mice; this pathological process could be delayed by deletion of PTP1B activity via mitogen-activated protein kinase (MAPK) and Akt pathways.21 In this present study, gypenosides 1−6 were evaluated for their inhibitory activity against PTP1B enzymes, with ursolic acid as the positive control (Table 3). Of them, gypenosides 2−6 showed PTP1B inhibitory activity, with IC50 values ranging from 8.2 ± 0.9 to 28.6 ± 2.8 μM, whereas gypenoside 1 did not show any activity, even at the concentration of 30 μM. The cytotoxicity of 1−6 was measured against MCF7, MCF7/ADR, and MDA-MB-231 breast cancer cell lines, with 4-hydroxytamoxifen as the positive control (Table 3). Gypenosides 2, 3, 5, and 6 showed a cytotoxic effect against all cell lines, with IC50 values ranging from 10.5 ± 1.4 to 28.1 ± 2.6 μM. On the basis of the obtained data, gypenoside 5 exhibited the strongest PTP1B inhibitory activity, with an IC50 value of 8.2 ± 0.9 μM, and moderate cytotoxicity, with IC50 values from 10.5 ± 1.4 to 14.2 ± 2.6 μM, which was probably due to the presence of an hydroperoxy group in compound 5. Similar structure−activity relationships were found regarding the PTP1B inhibitory activity and cytotoxicity. The gypenosides 5 and 6 that possessed four sugar units revealed stronger PTP1B inhibitory activity, with IC50 values of 8.2 ± 0.9 and 12.5 ± 1.8 μM, respectively, and stronger cytotoxicity, with IC50 values ranging from 10.5 ± 1.4 to 25.6 ± 1.0 μM. The gypenosides 2−4 with two sugar units in their structures showed less PTP1B inhibitory activity, with IC50 values ranging from 18.2 ± 1.3 to 28.6 ± 2.8 μM, and less cytotoxicity, with IC50 values ranging from 15.3 ± 1.0 to 28.1 ± 2.6 μM. The gypenoside 1 possessing only one sugar unit showed negligible activity.

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Notes

The authors declare no competing financial interest.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b01477. HRESIMS, IR spectra, 1H, 13C, heteronuclear singlequantum correlation (HSQC), HMBC, and rotatingframe Overhauser effect spectroscopy (ROESY) NMR spectra of the new gypenosides 1−6 (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Telephone/Fax: +86-931-4968385. E-mail: [email protected]. ORCID

Jun-Li Yang: 0000-0001-7199-0214 Pan-Pan Zhou: 0000-0001-8111-8155 Yan-Ping Shi: 0000-0001-6517-9556 Funding

This work was financially supported by the National Natural Science Foundation of China (81673325), the CAS Pioneer 5933


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Further New Gypenosides from Jiaogulan (Gynostemma pentaphyllum

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