Cucurbitane Glucosides from the Crude Extract of Siraitia grosvenorii

Apr 27, 2017 - Cucurbitane Glucosides from the Crude Extract of Siraitia grosvenorii with Moderate Effects on PGC-1α ... Prod. , 2017, 80 (5), pp 142...
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Cucurbitane Glucosides from the Crude Extract of Siraitia grosvenorii with Moderate Effects on PGC-1α Promoter Activity Biao Niu,†,‡,§,◆ Chang-Qiang Ke,†,◆ Bo-Han Li,§,∥ Yuanyuan Li,⊥ Yongji Yi,⊥ Yongwei Luo,⊥ Lin Shuai,§,∥ Sheng Yao,† Li-Gen Lin,¶ Jia Li,§,∥ and Yang Ye*,†,‡,§ †

State Key Laboratory of Drug Research and Natural Products Chemistry Department, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu-Chong-Zhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, People’s Republic of China ‡ School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, People’s Republic of China § University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, People’s Republic of China ∥ Chinese National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 189 Guoshoujing Road, Zhangjiang Hi-Tech Park, Shanghai 201203, People’s Republic of China ⊥ Guilin Layn Natural Ingredients Corp, Guilin 541199, People’s Republic of China ¶ State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Avenida da Universidade, Taipa, Macao 999078, People’s Republic of China S Supporting Information *

ABSTRACT: Seven new cucurbitane glucosides, 11-oxomogrosides III E and IV (1 and 2), 11-oxoisomogroside V (3), 7-oxomogrosides III E and IV (4 and 5), and mogrosides VI A and VI B (6 and 7), were separated from the crude extract of Siraitia grosvenorii. The new structures were defined by analysis of their 1H and 13C NMR, 2D NMR, and HRESIMS data. Especially, the band-selective constant time HSQC and band-selective constant time HMBC techniques were recuited to elucidate the structures of the complex glucoside moieties. Using the PGC-1α promoter driven luciferase reporter assay, the isolated compounds were examined for PGC-1α promoter activity.

P

transcriptional activators, seven new cucurbitane glucosides were identified from the crude extract of S. grosvenorii. Their structures were elucidated by comprehensive analysis of 1D and 2D NMR and HRESIMS data, especially band-selective constant time HMBC (CT-HMBC) and band-selective constant time HSQC (CT-HSQC) data. Herein, the isolation and structure elucidation of seven new cucurbitane glucosides, as well as their effects on activating PGC-1α transcription, are described.

GC-1α (peroxisome proliferator-activated receptor-γ coactivator-1α) plays an important role in energy homeostasis in high energy demand tissues, which is highly expressed in skeletal muscle and in the heart and brain.1−3 PGC-1α regulates a variety of physiological processes, such as mitochondrial biosynthesis, fatty acid metabolism, and fibertype switching in skeletal muscle.4 It was proven that increased PGC-1α expression protected the development of insulin resistance and improved metabolic responses in animals.5,6 Thus, identification of small-molecule PGC-1α activators is a promising way to find new therapeutic agents for treatment of metabolic diseases. Siraitia grosvenorii Swingle, belonging to the family Cucurbitaceae, is mainly distributed in the south of China and the north of Thailand, and its fruits are used as traditional herbal medicine for the treatment of dry cough, extreme thirst, and sore throat, as well as a natural sweetener.7 A number of cucurbitane glycosides have been identified from the fruits of this species as a table sugar substitute for obese and diabetic patients.8−13 In addition, the cucurbitane glycosides were reported with broad bioactivities, including antihyperglycemic effect,14 anticarcinogenic activity,13,15 and inhibitory effects on radical and lipid peroxidation.16 In searching for PGC-1α © 2017 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION A phytochemical isolation was carried out on the crude extract of the fruits of S. grosvenorii. Seven new cucurbitane glucosides, 11-oxomogrosides III E and IV (1 and 2), 11-oxoisomogroside V (3), 7-oxomogrosides III E and IV (4 and 5), and mogrosides VI A and VI B (6 and 7) (Chart 1), were isolated. The absolute configuration of the sugar components was determined as described previously.17 The sugars were converted into the thiazolidine derivatives and then into the arylthiocarbamates Received: November 29, 2016 Published: April 27, 2017 1428

DOI: 10.1021/acs.jnatprod.6b01086 J. Nat. Prod. 2017, 80, 1428−1435

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supported by IR, ROESY, 1H−1H COSY, and TOCSY data. On the basis of the outcome of the X-ray diffraction data generated for the aglycone of compound 6 (vide infra), the absolute configuration of 1 was defined as shown. The molecular formula of compound 2, C54H90O24, was deduced from the deprotonated molecular ion at m/z 1121.5770 [M − H]− in its HRESIMS. The 1H and 13C NMR data of 2 (Tables 2 and 1) were superimposable on those of 1 except that four glucosyl moieties [anomeric protons: δH 4.83 (d, J = 7.9 Hz), 5.08 (d, J = 7.7 Hz), 5.17 (d, J = 9.0 Hz), and 5.38 (d, J = 7.8 Hz)] rather than three were present in 2. In the HMBC spectrum, the cross-peaks between H-3 (δH 3.69, br s) and C-1 (δC 107.3) of glucosyl-GI, between H-1 (δH 4.83) of glucosyl-GI and C-3 (δC 86.9) of the aglycone, between H-1 (δH 5.17) of glucosyl-GII and C-6 (δC 70.8) of glucosyl-GI, between H-24 (δH 3.93, d, J = 8.9 Hz) of the aglycone and C-1 (δC 102.2) of glucosyl-GIII, and between H-1 (δH 5.38) of glucosyl-GIV and C-2 (δC 84.2) of glucosyl-GIII were observed. The above evidence, together with the analysis of the 1H−1H COSY, ROESY, and TOCSY spectra, defined the structure of 2, named 11-oxomogroside IV. Compound 3, a white powder, had the deprotonated molecular ion at m/z 1283.6239 [M − H]− in the HRESIMS, consistent with a molecular formula of C60H100O29. The 1H and 13 C NMR data of 3 (Tables 1 and 2) closely resembled those of 11-oxomogroside V.18,19 The 1H NMR spectrum of 3 displayed signals for five anomeric protons at δH 4.80 (d, J = 7.0 Hz), 4.87 (d, J = 8.0 Hz), 4.91 (d, J = 8.0 Hz), 5.20 (d, J = 7.7 Hz), and 5.51 (d, J = 7.3 Hz). The HMBC spectrum of 3 displayed crosspeaks between H-3 (δH 3.57, br s) of the aglycone and C-1 (δC 107.2) of glucosyl-GI, between H-1 (δH 5.20) of glucosyl-GII and C-4 (δC 81.6) of glucosyl-GI, between H-24 (δH 3.75, d, J = 9.6 Hz) of the aglycone and C-1 (δC 104.0) of glucosyl-GIII, between H-1 (δH 5.51) of glucosyl-GV and C-2 (δC 82.4) of glucosyl-GIII, and between H-1 (δH 4.87) of glucosyl-GIV and C-6 (δC 70.5) of glucosyl-GIII. The above evidence suggested the GII-(1→6)-GI moiety in 11-oxomogroside V was replaced by a GII-(1→4)-GI moiety in 3. In combination with the HSQC, 1H−1H COSY, ROESY, and TOCSY data, the structure of 3, 11-oxoisomogroside V, was defined. Compound 4 was obtained as a white, amorphous powder, and the protonated molecular ion at m/z 977.5293 [M + H]+ in the HRESIMS indicated the molecular formula as C48H80O20. The 13C and 1H NMR data of compound 4 (Tables 1 and 2) were quite similar to those of 7-oxomogroside II E,13 except for one additional glucose moiety in 4. This was supported by the presence of three anomeric protons at δH 4.89 (d, J = 7.8 Hz), 5.10 (d, J = 7.8 Hz), and 5.37 (d, J = 7.8 Hz). The HMBC cross-peaks between H-3 (δH 3.78, br s) of the aglycone and C1 (δC 107.5) of glucosyl-GI, between H-24 (δH 3.93, d, J = 7.4 Hz) of the aglycone and C-1 (δC 102.3) of glucosyl-GII, and between H-1 (δH 5.37) of glucosyl-GIII and C-2 (δC 84.3) of glucosyl-GII defined the structure of 4, 7-oxomogroside III E. Analysis of the 1H−1H COSY, ROESY, and TOCSY spectra further confirmed the structure of 4. The molecular formula of compound 5, C54H90O25, was deduced by the protonated molecular ion at m/z 1139.5808 [M + H]+ in the HRESIMS. The 1H and 13C NMR data of 5 (Tables 1 and 2) were highly similar to those of compound 4, except for one additional glucosyl moiety in 5. It was supported by the presence of four anomeric protons at δH 4.83 (d, J = 7.9 Hz), 5.08 (d, J = 7.7 Hz), 5.17 (d, J = 9.0 Hz), and 5.38 (d, J = 7.8 Hz) in the 1H NMR spectrum. The linkage of sugar

Chart 1. Structures of Compounds 1−7

using L-cysteine methyl ester and O-tolylisothiocyanate. By comparing the retention times with the standard sugars using HPLC-ELSD chromatography, all the sugar moieties in the new compounds were identified as D-glucose. Compound 1 was isolated as a white powder and exhibited the deprotonated molecular ion at m/z 959.5193 [M − H]− in its HRESIMS, compatible with a molecular formula of C48H80O19. The 13C and 1H NMR data (Tables 1 and 2) of 1 suggested the presence of eight methyl groups at δH 0.72 (s)/ δC 17.3, δH 0.98 (s)/δC 18.6, δH 1.11 (s)/δC 28.7, δH 1.16 (s)/ δC 20.5, δH 1.47 (s)/δC 27.4, δH 1.50 (s)/δC 26.2, δH 1.56 (s)/ δC 26.2, and δH 0.99 (d, J = 6.4 Hz)/δC 18.9, two oxymethines at δH 3.63 (br s)/δC 87.4 and δH 3.93 (d, J = 8.4 Hz)/δC 88.4, an olefinic methine at δH 5.50 (d, J = 5.4 Hz)/δC 118.8, and a carbonyl group (δC 214.2). The 1H NMR data of 1 (Table 2) exhibited signals for three anomeric protons of β-D-glucopyranosyl moieties at δH 4.88 (d, J = 7.7 Hz), 5.08 (d, J = 7.7 Hz), and 5.40 (d, J = 7.8 Hz). The 1H and 13C NMR spectra of the aglycone of 1 resembled those of 11-oxomogrol18 besides the glucosylation shifts of the C-3 (δC 87.4) and C-24 (δC 88.4), suggesting the sugar moieties might be attached to C-3 and C24. In the HMBC spectrum, the cross-peaks between H-3 (δH 3.64) and C-1 (δC 107.7) of the glucosyl (GI) moiety and between H-1 (δH 5.08) of the glucosyl (GII) unit and C-24 (δC 88.4) indicated that two glucose moieties were attached to C-3 and C-24. Furthermore, the HMBC cross-peaks between H-2 (δH 4.17) of glucosyl-GII and C-1 (δC 106.6) of glucosyl-GIII as well as H-1 (δH 5.40) of glucosyl-GIII and C-2 (δC 84.2) of glucosyl-GII constructed the GIII-(1→2)-GII moiety. Thus, the structure of 1, 11-oxomogroside III E, was defined and further 1429

DOI: 10.1021/acs.jnatprod.6b01086 J. Nat. Prod. 2017, 80, 1428−1435

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Table 1. 13C NMR Data for Compounds 1−7 (125 MHz, δ in ppm, in Pyridine-d5) position aglycone 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 22.5 28.8 87.4 42.3 141.5 118.8 24.3 44.3 49.4 36.2 214.2 49.1 49.3 50.0 34.9 28.6 50.0 17.3 20.5 36.8 18.9 33.9 28.6 88.4 72.7 26.2 27.4 28.7 26.2 18.6

2 22.6 28.9 86.9 42.3 141.6 118.8 24.3 44.3 49.3 36.2 214.2 49.1 49.4 50.0 34.9 28.6 50.1 17.3 20.6 36.9 19.1 33.9 28.6 88.5 72.7 26.1 27.4 28.7 26.2 18.6

3 22.4 28.7 87.6 42.3 141.4 118.8 24.4 44.3 49.3 36.2 214.3 49.1 49.3 50.0 34.9 28.7 50.3 17.4 20.6 36.5 19.0 33.4 29.5 92.5 73.1 24.9 27.4 28.6 26.2 18.7

4 27.4 29.3 88.2 44.2 170.9 125.5 201.6 60.4 41.0 39.6 77.1 41.0 47.4 49.0 34.9 28.9 50.9 17.2 26.2 37.3 19.3 34.2 29.0 88.5 72.7 26.2 27.4 27.8 26.0 19.9

5 27.4 29.3 87.6 44.2 171.1 125.5 201.6 60.4 41.0 39.5 77.1 41.1 47.4 49.0 34.9 28.9 50.9 17.2 26.9 37.4 19.3 34.2 29.1 88.6 72.7 26.2 27.4 27.8 25.9 19.9

6 27.2 29.9 87.8 42.6 144.7 118.7 24.9 43.9 40.5 37.1 78.3 41.4 47.8 50.0 34.9 28.9 51.4 17.5 26.6 36.7 19.4 33.5 29.8 92.4 73.1 24.9 27.4 28.0 26.6 19.7

position

7 27.2 30.0 87.1 42.7 144.7 118.6 24.9 43.9 40.5 37.1 78.3 41.4 47.8 50.0 34.9 28.9 51.4 17.5 26.6 36.7 19.5 33.5 29.9 92.4 73.1 24.9 27.4 28.0 26.6 19.7

sugar GI1 GI2 GI3 GI4 GI5 GI6 GII1 GII2 GII3 GII4 GII5 GII6 GIII1 GIII2 GIII3 GIII4 GIII5 GIII6 GIV1 GIV2 GIV3 GIV4 GIV5 GIV6 GV1 GV2 GV3 GV4 GV5 GV6 GVI1 GVI2 GVI3 GVI4 GVI5 GVI6

1

2

3

4

5

6

7

107.7 75.9 78.8 72.1 78.7 63.6 102.2 84.2 79.1 71.7 78.6 63.3 106.6 76.6 78.8 72.5 78.6 62.9

107.3 75.7 78.9 72.1 77.7 70.8 105.8 75.6 78.7 72.1 78.9 63.6 102.2 84.2 79.0 71.8 78.8 63.1 106.6 76.6 78.8 72.5 78.7 62.9

107.2 75.8 78.8 81.6 77.2 63.8 105.2 75.3 78.4 71.8 78.4 62.9 104.0 82.4 79.1 71.8 76.7 70.5 105.8 76.3 78.7 72.8 78.8 62.7 105.2 75.1 78.4 71.9 78.5 62.6

107.5 75.6 78.8 72.0 78.7 63.6 102.3 84.3 79.1 71.2 78.6 62.8 106.7 76.6 78.8 72.5 78.8 63.3

107.1 75.6 78.8 72.0 78.8 70.7 105.9 75.6 78.7 72.0 78.6 63.0 102.3 84.2 79.0 71.7 77.7 62.8 106.7 76.6 78.9 72.5 78.8 63.6

107.4 75.7 78.9 71.8 77.3 70.4 105.9 75.5 78.7 71.9 78.9 70.5 105.8 75.4 78.7 71.9 76.8 63.0 104.0 82.8 79.0 71.9 78.4 70.5 105.2 75.8 78.8 71.8 77.6 62.9 105.9 76.3 78.6 72.9 78.4 63.9

107.0 75.8 78.9 71.9 77.1 70.8 103.4 76.3 78.4 72.1 78.8 62.8 104.0 82.9 79.2 72.9 76.9 70.5 106.0 85.2 79.0 71.7 78.4 63.9 105.2 75.9 78.9 71.8 78.6 62.9 107.0 76.8 78.7 71.5 78.5 62.8

and C-24 (δC 92.4), suggesting the sugar moieties might be attached to C-3 and C-24. Additionally, the 1H NMR data of 6 (Table 3) suggested the presence of six β-D-glucopyranosyl anomeric protons at δH (4.82, d, J = 9.2 Hz), 4.88 (d, J = 7.6 Hz), 4.93 (d, J = 7.3 Hz), 5.08 (d, J = 7.7 Hz), 5.11 (d, J = 7.8 Hz), and 5.48 (d, J = 7.6 Hz), correlating with carbons at δC 107.4, 105.2, 104.0, 105.9, 105.8, and 105.9, respectively, in the HSQC spectrum. The resonances of the six β-D-glucopyranosyl moieties were heavily overlapped using conventional NMR techniques. Thus, the band-selective CT-HSQC (constant time HSQC) and band-selective CT-HMBC (constant time HMBC) experiments were carried out to elucidate the structure of 6. The pulse programs were used with a 2-fold low-pass Jfilter to suppress one-bond correlations. The high-resolution HMBC spectrum (Figure 1) displayed correlations between H3 (δH 3.70) of the aglycone and C-1 (δC 107.4) of glucosyl-GI, between H-1 (δH 5.08) of glucosyl-GII and C-6 (δC 70.4) of glucosyl-GI, between H-1 (δH 5.11) of glucosyl-GIII and C-6 (δC 70.5) of glucosyl-GII, between H-24 (δH 3.76) of the aglycone and C-1 (δC 104.0) of glucosyl-GIV, between H-1 (δH 5.48) of glucosyl-GVI and C-2 (δC 82.8) of glucosyl-GIV, and between H-1 (δH 4.88) of glucosyl-GV and C-6 (δC 70.5) of

moieties was determined by the HMBC experiment, exhibiting cross-peaks between H-3 (δH 3.83, br s) of the aglycone and C1 (δC 107.1) of glucosyl-GI, between H-1 (δH 4.83) of glucosylGI and C-3 (δC 87.6) of the aglycone, between H-1 (δH 5.17) of glucosyl-GII and C-6 (δC 70.7) of glucosyl-GI, between H-24 (δH 3.92, m) of the aglycone and C-1 (δC 102.3) of glucosylGIII, and between H-1 (δH 5.39) of glucose-GIV and C-2 (δC 84.2) of glucosyl-GIII. In combination with the 1H−1H COSY, ROESY, and TOCSY data, the structure of 5, 7-oxomogroside IV, was defined. Compound 6 was isolated as a white, amorphous powder. Its molecular formula, C66H112O34, was inferred by the deprotonated molecular ion at m/z 1447.6986 [M − H]− in the HRESIMS. The 1H and 13C NMR data (Tables 3 and 1) indicated the presences of eight methyl groups at δH 0.91 (s)/ δC 17.5, 0.92 (s)/δC 19.7, 1.09 (s)/δC 28.0, 1.34 (s)/δC 26.6, 1.34 (s)/δC 27.4, 1.46 (s)/δC 24.9, 1.52 (s)/δC 26.6, and 1.09 (d, J = 6.3 Hz)/δC 19.4, three oxymethines at 3.70 (br s)/δC 87.8, 4.17 (m)/δC 78.3 and 3.76 (d, J = 7.9 Hz)/δC 92.4, and an olefinic methine at 5.48 (m)/δC 118.7. The 1H and 13C NMR data of the aglycone moiety of 6 were similar to those of mogrol18 except for the glucosylation shifts of C-3 (δC 87.8) 1430

DOI: 10.1021/acs.jnatprod.6b01086 J. Nat. Prod. 2017, 80, 1428−1435

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Table 2. 1H NMR Data for Compounds 1−5 (500 MHz, δ in ppm, in Pyridine-d5) no. aglycone 1 2 3 6 7 8 10 11 12 15 16 17 18 19 20 21 22 23 24 26 27 28 29 30 sugar GI1 GI2 GI3 GI4 GI5 GI6 GII1 GII2 GII3 GII4 GII5 GII6 GIII1 GIII2 GIII3 GIII4 GIII5 GIII6 GIV1 GIV2 GIV3 GIV4 GIV5 GIV6

1 1.54, 1.94, 1.83, 2.41, 3.64, 5.50, 1.69, 2.16, 1.78, 2.47,

m m m m br s d (5.4) m m m m

2

3

1.63, m 1.94, m 1.83, m 2.51, m 3.69 (br s) 5.50, d (5.6) 1.71, m 2.15, m 1.77, m 2.44, m

1.53, m 1.92, m 1.77, m 2.27, m 3.57 (br s) 5.51, m 1.71, m 2.16, m 1.76, m 2.42, m

2.50, d (14.3) 2.93, d (14.3) 1.14, m 1.28, m 1.81, m 1.94, m 1.81, m 0.72, s 1.16, s 1.45, m 0.99, d (6.4) 1.83, m 1.82, m 2.05, m 3.93, d (8.4) 1.50, s 1.47 (s) 1.11 (s) 1.56 (s) 0.98 (s)

2.51, d (14.2) 2.93, d (14.2) 1.16, m 1.28, m 1.83, m 2.06, m 1.82, m 0.72(s) 1.17(s) 1.43, m 1.01, d (6.4) 1.81, m 1.77, m 2.06, m 3.93, d (8.9) 1.52 (s) 1.47 (s) 1.07 (s) 1.52 (s) 0.96 (s)

2.53, d (14.2) 3.00, d (14.2) 1.14, m 1.27, m 1.72, m 2.08, m 1.86, m 0.74(s) 1.16(s) 1.44, m 1.00, d (6.8) 1.81, m 1.56, m 1.83, m 3.75, d (9.6) 1.46 (s) 1.34 (s) 1.08 (s) 1.52 (s) 1.00 (s)

4.88, 4.00, 4.24, 4.21, 3.97, 4.41, 4.56, 5.08, 4.17, 4.36, 4.20, 3.96, 4.34, 4.56, 5.40, 4.14, 4.33, 4.21, 3.97, 4.39, 4.56,

4.83, 3.90, 4.20, 4.19, 3.92, 4.34, 4.84, 5.17, 4.03, 4.23, 4.18, 3.95, 4.34, 4.52, 5.08, 4.16, 4.27, 4.16, 3.92, 4.34, 4.52, 5.38, 4.17, 4.26, 4.20, 3.94, 4.34, 4.52,

4.80, 3.84, 4.22, 4.26, 3.94, 4.38, 4.51, 5.20, 4.04, 4.21, 4.24, 3.91, 4.34, 4.53, 4.91, 4.15, 4.25, 4.13, 3.94, 4.34, 4.50, 5.51, 4.14, 4.23, 4.19, 3.97, 4.32, 4.54,

d (7.7) m m m m m m d (7.7) m m m m m m d (7.8) m m m m m m

d (7.9) m m m m m m d (9.0) m m m m m m d (7.7) m m m m m m d (7.8) m m m m m m 1431

d (7.0) m m m m m m d (7.7) m m m m m m d (8.0) m m m m m m d (7.3) m m m m m m

4

5

2.09. m 3.05, m 2.08, m 2.43, m 3.78 (br s) 6.30 (s)

2.12, m 3.12, m 2.18, m 2.50, m 3.83 (br s) 6.29 (s)

2.54 (s) 3.24, m 4.14, m 2.14, m

2.54(s) 3.24, m 4.12, m 2.17, m

1.70, m 1.83, m 1.64, m 2.08, m 1.71, m 0.90 (s) 1.29 (s) 1.50, m 1.06, d (6.4) 1.81, m 1.83, m 2.07, m 3.93, d (7.4) 1.50 (s) 1.47 (s) 1.20 (s) 1.59 (s) 0.94 (s)

1.66, m 1.81, m 1.60, m 2.08, m 1.73, m 0.90(s) 1.29(s) 1.49, m 1.08, d (6.3) 1.81, m 1.80, m 2.05, m 3.92, m 1.51 (s) 1.46 (s) 1.16 (s) 1.54 (s) 0.94 (s)

4.89, 4.00, 4.20, 4.21, 3.96, 4.40, 4.56, 5.10, 4.15, 4.31, 4.18, 3.97, 4.32, 4.55, 5.37, 4.12, 4.30, 4.23, 3.97, 4.39, 4.55,

4.83, 3.95, 4.11, 4.20, 3.97, 4.32, 4.84, 5.17, 4.06, 4.28, 4.21, 3.97, 4.40, 4.55, 5.08, 4.15, 4.30, 4.04, 3.96, 4.33, 4.54, 5.38, 4.08, 4.20, 4.15, 3.96, 4.35, 4.53,

d (7.8) m m m m m m d (7.8) m m m m m m d (7.8) m m m m m m

d (7.9) m m m m m m d (9.0) m m m m m m d (7.7) m m m m m m d (7.8) m m m m m m

DOI: 10.1021/acs.jnatprod.6b01086 J. Nat. Prod. 2017, 80, 1428−1435

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Table 2. continued no.

1

2

3

GV1 GV2 GV3 GV4 GV5 GV6

4.87, 4.16, 4.27, 4.22, 3.99, 4.34, 4.55,

4

5

d (8.0) m m m m m m

Table 3. 1H NMR Data for Compounds 6 and 7 (500 MHz, δ in ppm, in Pyridine-d5) no. aglycone 1 2 3 6 7 8 10 11 12 15 16 17 18 19 20 21 22 23 24 26 27 28 29 30

6

7

2.00, m 2.99, m 2.08, m 2.47, m 3.70 (br s) 5.48, m 1.65, m 2.28, m 1.62, m 2.80, m 4.17, m 2.15, m

1.97, m 2.93, m 2.11, m 2.39, m 3.89 (br s) 5.47, m 1.61, m 2.26, m 1.60, m 2.77, m 4.17, m 2.14, m

1.05, m 1.13, m 1.48, m 2.13, m 1.79, m 0.91 (s) 1.34 (s) 1.54, m 1.09, m 1.81, m 1.61, m 1.90, m 3.76 (d) 7.9) 1.46 (s) 1.34 (s) 1.09 (s) 1.52 (s) 0.92 (s)

1.04, m 1.12, m 1.47, m 2.14, m 1.81, m 0.90 (s) 1.30 (s) 1.54, m 1.09, d (6.3) 1.81, m 1.61, m 1.90, m 3.77, m 1.45(s) 1.34(s) 1.12(s) 1.55(s) 0.90(s)

6 sugar GI1 GI2 GI3 GI4 GI5 GI6 GII1 GII2 GII3 GII4 GII5 GII6 GIII1 GIII2 GIII3 GIII4 GIII5 GIII6 GIV1 GIV2 GIV3 GIV4 GIV5 GIV6 GV1 GV2 GV3 GV4 GV5 GV6 GVI1 GVI2 GVI3 GVI4 GVI5 GVI6

4.82, 3.91, 4.18, 4.24, 4.06, 4.35, 4.83, 5.08, 4.07, 4.26, 4.26, 3.93, 4.36, 4.82, 5.11, 3.99, 4.23, 4.26, 4.06, 4.39, 4.43, 4.93, 4.21, 4.25, 3.96, 4.10, 3.99, 4.93, 4.88, 4.09, 4.27, 4.28, 3.94, 4.39, 4.52, 5.48, 4.13, 4.21, 4.14, 3.98, 4.34, 4.55,

d (9.2) m m m m m m d (7.7) m m m m m m d (7.8) m m m m m m d (7.3) m m m m m m d (7.6) m m m m m m d (7.6) m m m m m m

7 4.95, 4.22, 4.31, 4.09, 3.98, 4.31, 4.69, 5.26, 4.11, 4.23, 4.12, 3.91, 4.36, 4.49, 4.92, 4.18, 4.09, 4.23, 3.98, 3.97, 4.91, 5.48, 4.11, 4.21, 4.11, 3.96, 4.33, 4.53, 4.87, 4.24, 4.26, 4.07, 3.92, 4.36, 4.50, 5.27, 4.19, 4.21, 4.12, 3.90, 4.34, 4.51,

d (7.8) m m m m m m d (7.4) m m m m m m d (6.7) m m m m m m d (8.0) m m m m m m d (7.7) m m m m m m d (7.3) m m m m m m

mogroside VI A (6) as shown. This assignment also defines the absolute configurations of compounds 1−5 and 7. Compound 7 was obtained as a white, amorphous powder. It has the same molecular formula as 6, based on the deprotonated molecular ion at m/z 1447.6962 [M − H]− in the HRESIMS. The 1H and 13C NMR data of 7 (Tables 3 and

glucosyl-GIV, which determined the glucosyl linkage patterns of 6. Compound 6 was hydrolyzed, and the absolute configuration of the aglycone, mogrol, was determined by single-crystal X-ray diffraction (Figure 2). These data, together with the 1H−1H COSY, ROESY, and TOCSY spectra, defined the structure of 1432

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Figure 1. Partial CT-HSQC and CT-HMBC spectra of compound 6.

Table 4. Activating Effects of Compounds 1−7 on PGC-1α Transcriptional Activity normalized luciferase activity (%)a compound

10 μM

20 μM

1 2 3 4 5 6 7 forskolin (5 μM) dexamethasone (100 nM)

107.18 ± 1.84 128.84 ± 6.30 133.79 ± 2.28b 97.77 ± 2.97 105.03 ± 1.36 105.78 ± 7.01 104.17 ± 4.60 145.34 ± 1.25b 172.36 ± 2.82b

122.87 ± 3.94b 132.32 ± 1.84b 143.81 ± 0.91b 121.57 ± 2.51b 125.81 ± 9.62 118.42 ± 9.17 124.70 ± 2.76b

a

DMSO was used as a blank control (100%). bP < 0.05 was regarded as statistically significant compared with DMSO.

Figure 2. X-ray crystallographic structure (ORTEP drawing) of mogrol.

1) were similar to those of 6. In the band-selective CT-HSQC and band-selective CT-HMBC experiments, the HMBC crosspeaks between H-3 (δH 3.89, br s) of the aglycone and C-1 (δC 107.0) of glucosyl-GI, between H-1 (δH 5.26, J = 7.4 Hz) of glucosyl-GII and C-6 (δC 70.8) of glucosyl-GI, between H-24 (δH 3.77, m) of the aglycone and C-1 (δC 104.0) of glucosylGIII, between H-1 (δH 5.48, d, J = 8.0 Hz) of glucosyl-GIV and C-2 (δC 82.9) of glucosyl-GIII, between H-1 (δH 4.87, d, J = 7.7 Hz) of glucosyl-GV and C-6 (δC 70.5) of glucosyl-GIII, and between H-1 (δH 5.27, d, J = 7.3 Hz) of glucosyl-GVI and C-2 (δC 85.2) of glucosyl-GIV determined the linkage between the sugar moieties and the aglycone. In combination with the 1 H−1H COSY, ROESY, and TOCSY spectra, the structure of 7, mogroside VI B, was defined as shown. PGC-1α plays a key role in regulating mitochondria biogenesis and metabolism. Compounds effecting PGC-1α may serve as new agents to regulate glucose and fatty acid metabolism, fiber-type switching in skeletal muscle and heart, and have the potential to treat metabolic disorders and type 2 diabetes mellitus.1−6 The HEK293 cell line containing luciferase expression driven by the PGC-1α promoter was created previously.20 Using the luciferase reporter assay, the activating effect of the isolated compounds on PGC-1α transcriptional activity was examined. Compound 3 showed PGC-1α promoter activity of 1.3-fold at the concentration of 10 μM. At the concentration of 20 μM, compounds 1−4 and 7 demonstrated PGC-1α promoter activity ranging from 1.2- to 1.4-fold. Forskolin (5 μM) and dexamethasone (100 nM) were used as positive controls, with the normalized luciferase activities of 1.4- and 1.7-fold, respectively (Table 4). In a

previous study, compound ZLN027 was found to increase the PGC-1α promoter by 1.7-fold compared to forskolin (5 μM) and dexamethasone (100 nM), having stimulatory effects at 1.4and 2.0-fold, respectively.20 These data suggested that compounds 1−7 showed moderate effects when compared with ZLN027. Examination of the structures of these compounds revealed that the isolated compounds contain three different aglycone moieties of cucurbitane glucosides with a different number of sugar moieties. The relationship between their structures and effects on the PGC-1α promoter activity needs to be unveiled by further investigations.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Rudulph Autopol VI automatic polarimeter. IR spectra were recorded on a Nicolet Magna FTIR-750 spectrometer. NMR spectra were recorded on a Bruker Avance III 500 NMR spectrometer (Bruker, Ettlingen, Germany) using tetramethylsilane as internal standard. HRESIMS were measured on a Waters Xevo QTof mass detector. TLC was performed on precoated silica gel GF254 plates (Merck Chemical Co. Ltd., Shanghai, China). MCI (polystyrene) gel (CHP20P, 75−150 μm, Mitsubishi Chemical Industries, Tokyo, Japan), silica gel (Qingdao Marine Chemical Industrials, Qingdao, Shandong, People’s Republic of China), polyamide (Shanghai Chemical Reagents Co. Ltd.), and macroporous resin AB-8 (Shandong Lu Kang Chemical Industrials, Jinan, Shandong, People’s Republic of China) were used for column chromatography (CC). Analytical HPLC was applied on a Waters 2695 instrument (Milford, MA, USA) coupled with a 2998 PDA, a Waters 2424 ELSD, and a Waters 3100 MS detector. Preparative HPLC was performed on a Varian PrepStar pump with an Alltech 3300 ELSD (Columbia, MD, USA) using a 1433

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Waters SunFire RP C18, 5 μm, 30 × 150 mm column. All solvents used for CC and HPLC were of analytical grade (Shanghai Chemical Reagents Co. Ltd., Shanghai, People’s Republic of China) and gradient grade (Merck KGaA, Darmstadt, Germany), respectively. Plant Extract. The crude extract of S. grosvenorii was obtained from Guilin Rhine Biological Polytron Technologies Inc., Guangxi Province, People’s Republic of China. The HPLC-ELSD analysis result showed the extract contained mogroside V as the main constituent (2%) (Figures S75 and S76, Supporting Information). It was fully authenticated as the extract of S. grosvenorii according to the Chinese Pharmacopoeia. Isolation. The crude extract (990 g) was completely dissolved in 2 L of water through stirring, applied to CC over AB-8 macroporous resin, and eluted with aqueous EtOH in a step manner (0, 30, 60, 95% EtOH in H2O). The 60% EtOH fraction (200 g) was chromatographed over silica gel (300−400 mesh) eluted with CHCl3−MeOH− H2O (65:35:10 to 60:40:10, v/v) to afford seven fractions (Fr1 to Fr7). Fr3 was subject to CC over polyamide (100−200 mesh), eluted with H2O and 20% MeOH, successively, to yield Fr3.1 and Fr3.2, respectively. Fr3.1 was further separated with MCI gel, eluted with MeOH−H2O (0:1 to 1:0, v/v), to afford subfraction Fr3.1.1 to Fr3.1.20. Fr3.1.20 was purified by preparative HPLC (CH3CN−H2O, 5:95 to 40:60, v/v) to obtain compounds 1 (40 mg) and 2 (41 mg). Fr4 was subjected to CC over polyamide (100−200 mesh) eluted with H2O to afford subfraction Fr4.1. Compound 3 (18 mg) was isolated from Fr4.1 by preparative HPLC (CH3CN−H2O, 10:90 to 40:60, v/ v). Fr7 was subjected to CC over polyamide (100−200 mesh) eluted with H2O to give subfraction Fr7.1. Fr7.1 was purified by CC over MCI gel eluted with H2O−MeOH (1:0 to 0:1, v/v), and further preparative HPLC (CH3CN−H2O, 5:95 to 30:70, v/v), to give compounds 4 (12 mg), 6 (15 mg), and 7 (16 mg), as well as an impure sample of compound 5 (9 mg). Subsequent semipreparative HPLC (CH3CN−H2O, 18:72 to 18:72, v/v) afforded compound 5 (4 mg). 11-Oxomogroside III E (1): white, amorphous powder; [α]25D +32 (c 0.5, MeOH); IR (KBr) νmax 3430, 2924, 1687, 1467, 1385, 1173, 1077, 1034 cm−1; 1H and 13C NMR, see Tables 1 and 2; ESIMS m/z 959.7 [M − H]−; HRESIMS m/z 959.5193 [M − H]− (calcd for C48H79O19, 959.5216). 11-Oxomogroside IV (2): white, amorphous powder; [α]25D +17 (c 0.5, MeOH); IR (KBr) νmax 3426, 2930, 1687, 1467, 1385, 1171, 1077, 1036 cm−1; 1H and 13C NMR, see Tables 1 and 2; ESIMS m/z 1121.9 [M − H]−; HRESIMS m/z 1121.5770 [M − H]− (calcd for C54H89O24, 1121.5744). 11-Oxoisomogroside V (3): white, amorphous powder; [α]25D +17 (c 0.3, MeOH); IR (KBr) νmax 3426, 2924, 1687, 1465, 1385, 1169, 1075, 1036 cm−1; 1H and 13C NMR, see Tables 1 and 2; ESIMS m/z 1284.0 [M − H]−; HRESIMS m/z 1283.6239 [M − H]− (calcd for C60H99O29, 1283.6272). 7-Oxomogroside III E (4): white, amorphous powder; [α]25D +16 (c 0.2, MeOH); IR (KBr) νmax 3422, 2926, 1638, 1385, 1171, 1075, 1034 cm−1; 1H and 13C NMR, see Tables 1 and 2; ESIMS m/z 977.6 [M + H]+; HRESIMS m/z 977.5293 [M + H]+ (calcd for C48H81O20, 977.5316). 7-Oxomogroside IV (5): white, amorphous powder; [α]25D +1 (c 0.2, MeOH); IR (KBr) νmax 3428, 2924, 1640, 1385, 1171, 1075, 1034 cm−1; 1H and 13C NMR, see Tables 1 and 2; ESIMS m/z 1139.58 [M + H]+; HRESIMS m/z 1139.5808 [M + H]+ (calcd for C54H91O25, 1139.5844). Mogroside VI A (6): white, amorphous powder; [α]25D −6 (c 0.3, MeOH); IR (KBr) νmax 3422, 2924, 1640, 1465, 1383, 1173, 1077, 1036 cm−1; 1H and 13C NMR, see Tables 1 and 3; ESIMS m/z 1147.7 [M − H]−; HRESIMS m/z 1447.6986 [M − H]− (calcd for C66H111O34, 1447.6962). Mogroside VI B (7): white, amorphous powder; [α]25D −2 (c 0.3, MeOH); IR (KBr) νmax 3424, 2926, 1638, 1465, 1383, 1171, 1075, 1032 cm−1; 1H and 13C NMR, see Tables 1 and 3; ESIMS m/z 1148.9 [M − H]−; HRESIMS m/z 1447.6962 [M − H]− (calcd for C66H111O34, 1447.6962).

CT-HSQC and CT-HMBC. The 1H−13C CT-HMBC and CTHSQC spectra were recorded on a Bruker AVANCE III 500 instrument. The CT-HMBC experiments were carried out at 298 K with the following parameters: number of data points, 2048 for 1H and 220 for 13C; spectral width, 8012.8 Hz for 1H; acquisition time, 0.128 s; delay time, 1.0 s; dummy scan, 16; number of scan, 160; dwell time, 62.400 μs; prescan delay, 10.00 μs; 1J(XH)min, 120 Hz; 1J(XH)max, 170 Hz; nJ(XH), 8 Hz. The CT-HSQC experiments were carried out at 298 K with the following parameters: number of data points, 1024 for 1 H and 128 for 13C; spectral width, 8012.8 Hz for 1H; acquisition time, 0.064 s; delay time, 1.0 s; dummy scan, 16; number of scan, 4; dwell time, 62.400 μs; prescan delay, 10.00 μs. General Procedure for Acid Hydrolysis and Determination of Sugar Configuration in Compounds 1−7. Each compound (1 mg) was hydrolyzed by heating at 60 °C in 2 M HCl (0.5 mL) for 1.5 h. After lyophilization, the residue was dissolved in pyridine (500 μL) and reacted with L-cysteine methyl ester HCl (5 mg) at 60 °C for 1.5 h. Then O-tolylisothiocyanate (25 μL) was added to the mixture and reacted at 60 °C for an additional 1.5 h. The reaction mixture was analyzed by HPLC (SunFire C18 5 μm, 4.6 × 150 mm; 20% MeCN− 0.1% formic acid in water to 35% MeCN−0.1% formic acid in water, 0−25 min, 1 mL/min). The sugar was identified as D-glucose in compounds 1−7 (tR, 13.08 to 13.27 min) [authentic samples, Dglucose (tR, 13.17 min) and L-glucose (tR, 12.35 min)]. Single-Crystal X-ray Diffraction. Colorless crystals of mogrol were obtained by recrystallization in MeOH−H2O at room temperature. The X-ray crystal data were collected on a Bruker APEX-II CCD diffractometer with graphite-monochromated Cu Kα radiation (λ = 1.541 78 Å) (operated in the φ−ω scan mode). The structure of mogrol was refined with full-matrix least-squares calculations on F2 using the SHELXL program (Sheldrick, 2015). Crystallographic data for mogrol have been deposited at the Cambridge Crystallographic Data Centre (deposition no.: CCDC 1517510). Copies of these data can be obtained free of charge via the Internet at www.ccdc.cam.ac.uk/ conts/retrieving.html or on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [tel: (+44) 1223-336-408; fax: (+44) 1223336-033; e-mail: [email protected]]. PGC-1α Transcriptional Activity Luciferase Reporter Assay. Luciferase reporter gene driven by the PGC-1a promoter was stably expressed in HEK293 cells.20 The cells were maintained in highglucose Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. About 2800 cells were seeded into each well of a 384-well plate. After 24 h of incubation, compounds with a final concentration of 10 or 20 μM were added to a designated well. The same value of DMSO was added as a blank control. Forskolin (5 μM) and dexamethasone (100 nM) were used as positive controls. After incubating overnight, luciferase substrate was added to each well. The luminescence intensity was recorded by an EnVision spectrometer (PerkinElmer). The luciferase activity of each compound was normalized to that of DMSO. The results are presented as the mean ± SEM. Differences between the groups were analyzed with the Student’s t test.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b01086. 1D and 2D NMR, IR, and HRESIMS spectra of compounds 1−7 (PDF) Crystallographic data (CIF)

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +86 21 50806726. E-mail: [email protected] (Y. Ye). 1434

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ORCID

Li-Gen Lin: 0000-0002-6799-5327 Yang Ye: 0000-0003-1316-5915 Author Contributions ◆

B. Niu and C.-Q. Ke contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the National Science and Technology Major Project “Key New Drug Creation and Manufacturing Program” (2015ZX09103002), the National Natural Science Funds of China (81673327), Science and Technology Commission of Shanghai Municipality (15DZ2291600), Guangxi Science and Technology Department (14125008-229), the State Key Laboratory of Drug Research (SIMM1501ZZ-03), and the Research Fund of University of Macau (MYRG2014-00020-ICMS-QRCM and MYRG201500153-ICMS-QRCM) is gratefully acknowledged.

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