Malonylginsenosides with Potential Antidiabetic Activities from the

Article pubs.acs.org/jnp

Malonylginsenosides with Potential Antidiabetic Activities from the Flower Buds of Panax ginseng Shi Qiu,†,§ Wen-Zhi Yang,†,§ Chang-Liang Yao,† Xiao-Jian Shi,† Jing-Ya Li,‡ Yang Lou,‡ Ya-Nan Duan,‡ Wan-Ying Wu,*,† and De-An Guo*,† †

Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Haike Road 501, Shanghai 201203, People’s Republic of China ‡ National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zuchongzhi Road 555, Shanghai 201203, People’s Republic of China S Supporting Information *

ABSTRACT: LC-MS-guided phytochemical isolation of malonylginsenosides, featuring neutral elimination of CO2 and C3H2O3 by the negative mode collision-induced dissociation, from the flower buds of Panax ginseng led to the isolation of 19 malonylsubstituted triterpenoid saponins. They include 15 new malonylginsenosides, malonylfloralginsenosides-Re1−Re3 (1−3), -Rb1 and -Rb2 (4, 5), -Rd1−Rd6 (6−11), and -Rc1−Rc4 (12−15), and the known m-Rb1, m-Rc, m-Rb2, and m-Rd (16−19). Compound 11 represents the first dimalonyl saponin isolated from the Panax genus, while 2−4, 9, and 10 are five ginsenosides with single malonylation at the C-20 sugar chain. The antidiabetic activities of nine of these malonyl-substituted ginsenosides (1, 3, 4, 8, 13, and 16−19) and five of the corresponding non-malonyl ginsenosides (Re, Rb1, Rb2, Rc, and Rd) were evaluated by L6 myotubes’ glucose consumption and AMPKα2β1γ1 activation. Ginsenoside Rb2, 1, and 18 promoted glucose consumption of differentiated L6 myotubes, while ginsenosides Rb1, Rb2, and Rd and the malonylginsenosides 4, 8, 13, 16, 17, and 19 activated AMPKα2β1γ1 (EC50: 0.0168−2.8 μM, fold: 1.7−4.7).

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lipidemia, and insulin resistance.7,8 However, due to their thermal instability,9 malonylginsenosides are rather difficult to isolate as pure compounds, and hitherto only seven compounds of this type were isolated from the Panax genus.10−13 Therefore, it is of considerable significance to discover more malonylginsenosides in order to probe their structural diversity and to provide candidate compounds for antidiabetic assessment. Malonylginsenosides can be rapidly characterized by mass spectrometry, while NMR spectroscopy enables confirmation of their malonylation sites. In particular, by HRMS, the loss of CO2 (43.9898 Da) and C3H2O3 (86.0004 Da; a malonyl group) in the negative-mode collision-induced dissociation (CID) is characteristic for malonylginsenosides, and the ginsenoside structures can be elucidated based on the typical loss of sugar residues and the sapogenin product ions. The malonyl-substituted ginsenosides from different parts of P. ginseng, P. quinquefolius, and P. notoginseng have been identified or tentatively characterized,14−18 and these malonylginsenoside analogues show taxonomic significance for the differentiation among the three Panax species.18 Comparison of the chemical composition of the roots, stems/leaves, flower buds, berries, and seeds of P. ginseng using UHPLC/QTOF-MS

iabetes mellitus is a metabolic disorder seriously jeopardizing the health of humans, which is characterized clinically by chronic hyperglycemia due to insufficient insulin action.1,2 The pathogenesis of diabetes mellitus involves both genetic and environmental factors. In accordance with the global rise in obesity, diabetes mellitus has become increasingly prevalent in the USA, Middle East, and South Asian communities.3 Medicinal herbs have been used to treat diabetes mellitus long before the advent of Western medicine and show fewer side effects, of which Panax ginseng C.A. Meyer has been utilized.4 The use of P. ginseng roots in the treatment of “Xiao-Ke” symptoms is recorded in many ancient treatises of traditional Chinese medicine. The therapeutic efficacy has been demonstrated by in vitro models, animal studies, and clinical trials and is closely related to the triterpenoid saponins, also known as ginsenosides. Ginsenosides, a group of bioactive molecules found abundantly in the genus of Panax L., are oligoglycosides of the dammarane-type triterpenoids protopanaxadiol (PPD) and protopanaxatriol (PPT), together with their derivatives resulting from modifications to the C-17 side chains or acylation on the appended sugar moieties.5 More than 80 ginsenosides have been isolated from the raw and processed materials of P. ginseng.6 Notably, a malonylginsenoside fraction prepared from P. ginseng showed potential in the therapy of type II mellitus diabetes by alleviating hyperglycemia, hyper© 2017 American Chemical Society and American Society of Pharmacognosy

Received: August 29, 2016 Published: March 27, 2017 899

DOI: 10.1021/acs.jnatprod.6b00789 J. Nat. Prod. 2017, 80, 899−908

Journal of Natural Products

Article

revealed that the flower buds had the highest content of malonylginsenosides,19 which indicated a natural source for discovering novel malonylginsenoside structures. A systematic isolation process targeting malonylginsenosides from the flower buds of P. ginseng was performed. Fifteen new (1−15) and four known (16−19) malonylginsenosides were obtained. Spectroscopic analysis and acidic hydrolysis followed by GC were used for structural elucidation. Pairing antidiabetic assessment of malonylginsenosides and their corresponding neutral saponins was performed by in vitro L6 myotubes’ glucose consumption and AMPKα2β1γ1 activation. L6 myotubes’ glucose consumption embodies the effect of drug(s) on glucose utility of cells, while AMPKα2β1γ1 is a new therapeutic target for type II mellitus diabetes.20

the presence of a malonyl group, two hexosyl units, and a methylpentosyl moiety presumably attached to a possible PPT sapogenin. Three anomeric protons [δH 5.28 (1H, d, J = 6.8 Hz), δH 5.11 (1H, d, J = 7.8 Hz), and δH 6.52 (1H, brs)] in the 1 H NMR spectrum, three methine carbons (δC 98.3, 102.2, and 102.3) in the 13C DEPT-135 spectrum, and GC determination after acidic hydrolysis (Figure S11, Supporting Information) showed the presence of two β-D-glucosyl units and an α-Lrhamnosyl moiety in compound 1. Eight methyl singlets [δH 0.98 (6H), 1.20, 1.37, 1.60, 1.66, 1.68, and 2.13] and one olefinic proton [δH 0.98 (1H, t, J = 7.0 Hz)] were observed as well. Thirty carbons (including eight methyl groups, eight methylenes, nine methines, and five quaternary carbons) in the 13 C NMR spectrum were assignable to the 20(S)-PPT sapogenin.5 Compared with ginsenoside Re,22 additional signals corresponding to two carbonyl carbons [δC 168.3 and 169.9] and one methylene group [δC 43.1/δH 3.83 (2H, dd, J = 15.6, 21.2 Hz)] were ascribed to a malonyl substituent,11,12 confirmed by the HMBC cross-peaks of δH 3.83/δC 168.3 and δH 3.83/δC 169.9 (Figure 1). The malonylation site was at the primary 6′-OH of the diglycosyl moiety due to the HMBC cross-peak from H-6′ [δH 5.16 (1H, dd, J = 1.5, 11.5 Hz)] to the carbonyl carbon at δC 168.3. In contrast to ginsenoside Re, the signals of C-6′ and C-5′ were deshielded and shielded by 2.8 and 2.9 ppm, respectively. The HMBC cross-peaks of H-1″ [δH 6.52 (brs)] to C-2′ (δC 78.9), H-1′ [δH 5.28 (d, J = 6.8 Hz)] to C-6 (δC 75.0), and H-1‴ [δH 5.11 (d, J = 7.8 Hz)] to C-20 (δC 83.8) confirmed the glycosylation pattern as the same as that of Re. The ROESY correlations H-6 with 18-CH3, 19CH3, and 29-CH3 indicated the α-orientation of the 6-Oglycosyl chain, while the correlations of H-17/30-CH3, H-3/H5, and H-5/28-CH3 supported the β-oriented 3-OH and C-17 side chain (Figure S10, Supporting Information). Assignment of the 1H and 13C NMR data (Table 1) was achieved through HSQC, HMBC, 1H−1H COSY, and ROESY experiments. Thus, the structure of compound 1 was established as 6-O-[α-Lrhamnopyranosyl(1→2)-(6-O-malonyl)-β-D-glucopyranosyl](3β,6α,12β,20S)-3,6,12,20-tetrahydroxydammar-24-ene-20-Oβ-D-glucopyranoside and given the trivial name malonylfloralginsenoside Re1. Compounds 2 and 3 were identified as two isomers of 1, having the same molecular formula C51H84O21 determined by their HRESIMS data. Both exhibited similar MS2 spectra (Figures S12 and S20, Supporting Information) compared to 1. The 1H and 13C NMR spectra of 2 and 3 gave resonances ascribable to nine methyl groups, one olefinic proton, and three anomeric protons [δH 5.27 (d, J = 7.0 Hz; H-1′), δH 6.51 (brs; H-1″), and δH 5.35 (d, J = 8.0 Hz; H-1‴) for 2; δH 5.27 (d, J = 7.0 Hz; H-1′), δH 6.51 (brs; H-1″), and δH 5.18 (d, J = 7.8 Hz; H-1‴) for 3] similar to those of Re.22 The presence of a malonyl substituent in 2 and 3 was confirmed due to the presence of three additional carbons (δC 43.6, 167.5, and 170.3 for 2; δC 43.4, 167.9, and 170.3 for 3) and two protons corresponding to a methylene group [δH 3.92 (m) for 2 and δH 3.77 (dd, J = 15.5, 19.3 Hz) for 3]. The malonyl substituent was assigned to 2‴-OH of the Glc located at C-20 (δC 76.6) for 2 based on the HMBC cross-peak from H-2‴ [δH 5.60 (dd, J = 8.1, 9.3 Hz)] to the carbonyl carbon (δC 167.5) (Figure 1), the deshielded C-2‴ (1.4 ppm), and the shielded C-1‴ (2.3 ppm) and C-3‴ (2.5 ppm) in contrast to Re. For compound 3, the HMBC cross-peak from H-4‴ [δH 5.68 (t, J = 9.6 Hz)] to the carbonyl carbon (δC 167.9) (Figure 1), combined with the deshielded C-4‴ (2.2 ppm) and shielded C-3‴ (3.0 ppm) and

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RESULTS AND DISCUSSION The CID of deprotonated malonylginsenosides leads to the elimination of 44 Da (CO2) and 86 Da (C3H2O3) fragments.14,15 On this basis, malonylginsenosides in Panax species can be easily discriminated from neutral ginsenosides or other acyl derivatives by LC-MS analysis. Thus, an LC-MS-guided strategy was employed for the targeted isolation of malonylginsenosides from the flower buds of P. ginseng, applying (1) extraction of the drug materials with 70% aqueous EtOH under ultrasonic conditions; (2) liquid−liquid extraction by n-butanol to prepare a fraction with enriched malonylginsenosides from the water layer; (3) column chromatographic separation using AB-8 resin and MCI gel; (4) semipreparative HPLC purification by reversed-phase and hydrophilic interaction chromatography (HILIC); and (5) structural elucidation by MS, NMR, and acidic hydrolysis/GC analysis. Using this approach, 15 new dammarane-type malonylginsenosides (1− 15) and four known saponins were isolated, all of which showed purity of >95% (Figure S1, Supporting Information). Four known malonylginsenosides were identified as m-Rb1 (16),10 m-Rc (17),10,21 m-Rb2 (18),10,21 and m-Rd (19).10 Compound 1 was obtained as a white, amorphous powder. Its molecular formula was determined as C51H84O21, evidenced from the HRESIMS data (m/z 1031.5435 [M − H]−, calcd for C51H83O21, 1031.5427), indicating 10 indices of hydrogen deficiency. The MS2 spectrum (Figure S2, Supporting Information) displayed a series of product ions due to the cleavage of a malonyl group and of sugar residues at m/z 987 (−44 Da), 945 (−86 Da), 799, 637, and 475, which suggested 900



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Figure 1. Key HMBC correlations of compounds 1−5.

anomeric protons [δH 5.33 (d, J = 7.6 Hz, H-1′), δH 5.41 (d, J = 7.6 Hz, H-1″), δH 5.15 (d, J = 7.7 Hz, H-1‴), and δH 5.12 (d, J = 7.7 Hz, H-1⁗)], combined with GC (Figure S36, Supporting Information) following acidic hydrolysis, confirmed the presence of four β-D-glucosyl moieties. These data were consistent with those of ginsenoside Rb1,23 except for the deshielded C-4″″ (2.1 ppm) and the shielded C-3″″ (3.1 ppm) and C-5″″ (2.9 ppm). The malonyl substituent, characterized by three additional carbons (δC 168.0, 43.5, and 170.5) and two methylene protons [δH 3.79 (m)], was assigned at 4″″-OH based on the HMBC cross-peak from H-4⁗ [δH 5.86 (t, J = 9.7 Hz)] to the carbonyl carbon (δC 168.0) (Figure 1). In addition, the H-1′/C-3, H-1″/C-2′, H-1‴/C-20, and H-1″″/C-6‴ HMBC cross-peaks provided evidence for the glycosylation sites. The structure of 4 was thus established as 3-O-[β-Dglucopyranosyl(1→2)-β- D -glucopyranosyl]-(3β,12β,20S)(3,12,20)-trihydroxydammar-24-ene-20-O-β-D-glucopyranosyl-

C-5‴ (1.7 ppm) compared with Re, indicated the malonylation at 4‴-OH of the C-20 glucosyl moiety. Accordingly, the structures of 2 and 3 were established as 6-O-[α- Lrhamnopyranosyl(1→2)-β-D-glucopyranosyl]-(3β,6α,12β,20S)3,6,12,20-tetrahydroxydammar-24-ene-20-O-(2-O-malonyl)-βD-glucopyranoside and 6-O-[α-L-rhamnopyranosyl(1→2)-β-Dglucopyranosyl]-(3β,6α,12β,20S)-3,6,12,20-tetrahydroxydammar-24-ene-20-O-(4-O-malonyl)-β- D -glucopyranoside and named malonylfloralginsenosides Re2 and Re3, respectively. Compound 4 exhibited the molecular formula C57H94O26 according to the HRESIMS data (m/z 1193.5927 [M − H]−, calcd for C57H93O26, 1193.5955), indicating 10 indices of hydrogen deficiency. Its MS2 spectrum (Figure S28, Supporting Information) exhibited ions at m/z 1149 (−44 Da), 1107 (−86 Da), 945, 783, 621, and 459, indicating one malonyl substituent and four hexose units linked to a possible PPD sapogenin. The 1 H and 13C NMR spectra showed resonances assignable to a 3,20-disubstituted 20(S)-PPD sapogenin. Additionally, four 901



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Table 1. NMR Spectroscopic Data (500 MHz, Pyridine-d5) for Malonylfloralginsenosides Re1−Re3 (1−3) m-floral-Re1 (1) position

δC, type

1

39.7, CH2

2

28.1, CH2

3 4 5 6 7

78.7, 40.4, 61.2, 75.0, 46.3,

CH C CH CH CH2

8 9 10 11

41.5, 49.9, 40.0, 31.0,

C CH C CH2

12 13 14 15

70.4, 49.4, 51.8, 31.3,

CH CH C CH2

16

27.0, CH2

17 18 19 20 21 22

51.8, 17.8, 18.0, 83.8, 22.4, 36.4,

23

23.3, CH2

24 25 26 27 28 29 30

126.4, CH 131.4, C 26.1, CH3 18.2, CH3 32.6, CH3 17.6, CH3 17.7, CH3 6-Glc 102.2, CH 78.9, CH 79.8, CH 72.8, CH 75.3, CH 66.0, CH2

1′ 2′ 3′ 4′ 5′ 6′

1″ 2″ 3″ 4″ 5″ 6″ 1‴ 2‴ 3‴ 4‴ 5‴

CH CH3 CH3 C CH3 CH2

2′-Rha 102.3, CH 72.6, CH 72.9, CH 74.5, CH 69.8, CH 19.1, CH3 20-Glc 98.3, CH 75.3, CH 79.5, CH 72.0, CH 78.7, CH

δH, mult (J in Hz) 1.70, 0.96, 1.90, 1.85, 3.47,

m m m m dd (11.7, 4.8)

1.41, 4.69, 2.25, 1.99,

m m m m

1.53, m 2.06, 1.53, 4.18, 1.97,

m m m m

1.53, 0.83, 1.78, 1.27, 2.52, 1.20, 0.98,

m m m m m s s

1.60, 2.36, 1.78, 2.59, 2.35, 5.34,

s m m m m t (7.0)

1.66, 1.68, 2.13, 1.37, 0.98,

s s s s s

5.28, 4.39, 4.37, 4.21, 4.04, 5.16, 4.70,

d (6.8) m m m m dd (11.5, 1.5) m

6.52, 4.81, 4.70, 4.34, 4.95, 1.79,

brs d (3.0) m m dq (9.6, 6.2) d (6.5)

5.11, 3.99, 4.17, 4.23, 3.98,

d (7.8) m m t (9.3) m

m-floral-Re2 (2) δC, type 39.6, CH2 28.1, CH2 78.5, 40.3, 61.1, 74.7, 46.2,

CH C CH CH CH2

41.4, 49.7, 39.9, 31.0,

C CH C CH2

70.8, 49.7, 51.8, 31.2,

CH CH C CH2

27.0, CH2 51.8, 17.9, 18.0, 83.4, 22.4, 36.3,


23.7, CH2 126.2, CH 131.3, C 26.1, CH3 18.1, CH3 32.5, CH3 17.5, CH3 17.5, CH3 6-Glc 102.1, CH 78.9, CH 79.8, CH 72.8, CH 78.7, CH 62.7, CH2 2′-Rha 102.3, CH 72.6, CH 72.9, CH 74.5, CH 69.8, CH 19.1, CH3 20-Glc 96.0, CH 76.6, CH 76.8, CH 71.8, CH 78.8, CH

902

δH, mult (J in Hz) 1.66, 1.00, 1.90, 1.84, 3.46,

m m m m dd (11.5, 4.5)

1.40, 4.69, 2.26, 2.00,

m m m m

1.55, m 1.99, 1.55, 4.00, 1.90,

m m m m

1.47, 0.83, 1.78, 1.29, 2.47, 1.18, 0.97,

m m m m m s s

1.56, 2.34, 1.87, 2.44, 2.23, 5.21,

s m m m m t (7.0)

1.61, s 1.64 s 2.12, s 1.36, s 0.99, s 5.27, 4.39, 4.37, 4.24, 3.97, 4.46, 4.29,

d (7.0) m m m m dd (11.8, 2.3) m

6.51, 4.81, 4.69, 4.36, 4.98, 1.79,

brs dd (3.4, 1.2) m m dq (9.6, 6.2) d (6.5)

5.35, 5.60, 4.33, 4.21, 3.96,

d (8.0) dd (8.1, 9.3) m m m

m-floral-Re3 (3) δC, type 39.7, CH2 28.1, CH2 78.7, 40.3, 61.1, 74.9, 46.3,

CH C CH CH CH2

41.5, 49.9, 40.0, 31.0,

C CH C CH2

70.8, 49.7, 51.7, 31.2,

CH CH C CH2

27.0, CH2 51.7, 17.8, 18.0, 83.9, 22.6, 36.3,


23.4, CH2 126.2, CH 131.3, C 26.1, CH3 18.1, CH3 32.5, CH3 17.6, CH3 17.6, CH3 6-Glc 102.1, CH 78.9, CH 79.8, CH 72.8, CH 78.7, CH 62.5, CH2 2′-Rha 102.3, CH 72.6, CH 72.9, CH 74.5, CH 69.8, CH 19.1, CH3 20-Glc 98.4, CH 75.4, CH 76.3, CH 73.9, CH 76.8, CH

δH, mult (J in Hz) 1.71, 0.96, 1.88, 1.82, 3.47,

m m m m dd (11.5, 4.5)

1.41, 4.68, 2.27, 2.00,

m m dd (12.4, 3.2) m

1.52, m 1.99, 1.55, 4.00, 1.90,

m m m m

1.49, 0.86, 1.76, 1.24, 2.53, 1.18, 0.97,

m m m m dd (19.7, 9.7) s s

1.60, 2.37, 1.76, 2.48, 2.21, 5.26,

s m m m m m

1.60, 1.58, 2.12, 1.37, 0.98,

s s s s s

5.27, 4.40, 4.39, 4.22, 3.99, 4.38, 4.21,

d (7.0) m m m m m m

6.51, 4.82, 4.70, 4.35, 4.97, 1.79,

brs dd (3.4, 1.1) dd (9.4, 3.6) m dq (9.5, 6.1) d (6.5)

5.18, 4.05, 4.37, 5.68, 4.03,

d (7.8) m m t (9.6) m



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Table 1. continued m-floral-Re1 (1) position

δC, type

6‴

63.4, CH2

−O−CO CH2 COOH

6′-Mal. 168.3, C 43.1, CH2 169.9, C

δH, mult (J in Hz) 4.53, dd (11.7, 2.7) 4.40, m

3.83, dd (2H, 21.2, 15.6)

m-floral-Re2 (2)

m-floral-Re3 (3)

δC, type

δH, mult (J in Hz)

δC, type

63.4, CH2

4.52, dd (11.5, 2.7) 4.39, m

63.4, CH2

2‴-Mal. 167.5, C 43.6, CH2 170.3, C

3.92, m

4‴-Mal. 167.9, C 43.4, CH2 170.3, C

δH, mult (J in Hz) 4.53, dd (11.5, 2.6) 4.40, m

3.77, dd (2H, 19.3, 15.5)

Figure 2. Key HMBC correlations of 6−11.

NMR data of 4 and 5 are given in Table S1, Supporting Information. Compounds 6−10 were identified as isomers with the molecular formula C51H84O21 deduced from their HRESIMS data (Figures S45, S53, S61, S70, and S78, Supporting Information). The 1H and 13C NMR spectra of compound 8 displayed resonances consistent with a 3,20-disubstituted 20(S)-PPD. Three anomeric protons [δH 4.90 (d, J = 7.6 Hz, H-1′), δH 5.42 (d, J = 7.7 Hz, H-1″), and δH 5.21 (d, J = 7.7 Hz, H-1‴)] in conjunction with the GC (Figure S69, Supporting Information) indicated the presence of three β-D-glucosyl units. Comparison of the 13C NMR data with those of ginsenoside Rd23 showed a deshielded C-4″ and shielded C-3″ and C-5″ in 8. The malonyl substituent, characterized by three carbons (δC 167.9, 43.4, and 170.4) and two methylene protons [δH 3.79 (dd, J = 15.4, 22.1 Hz], was linked to 4″-OH via the HMBC cross-peak from H-4″ [δH 5.86 (t, J = 9.6 Hz)] to the carbonyl carbon (δC 167.9). Other HMBC cross-peaks that supported the malonyl substituent and the glycosylation sites are indicated

(1→6)-(4-O-malonyl)-β-D-glucopyranoside and named malonylfloralginsenoside Rb1. Compound 5 was an isomer of 4 with the same molecular formula C57H94O26, as deduced from the HRESIMS data. By comparison of the 13C NMR data with those of Rb1,23 significant variations were observed with respect to the deshielded C-3″ (δC 80.2) and shielded C-2″ (δC 75.2) and C-4″ (δC 69.5), together with three additional carbons (δC 168.6, 43.6, and 170.7) ascribed to a malonyl substituent. The HMBC cross-peak from H-3″ [δH 5.98 (t, J = 9.7 Hz)] to the carbonyl carbon of the malonyl substituent (δC 168.6) confirmed the malonylation at 3″-OH. The glycosylation sites were in accordance with Rb1 according to the HMBC crosspeaks (Figure 1). The structure of 5 was established as 3-O-[(3O-malonyl)-β-D-glucopyranosyl(1→2)-O-β-D-glucopyranosyl](3β,12β,20S)-3,12,20)-trihydroxydammar-24-ene-20-O-β-Dglucopyranosyl(1→6)-β-D-glucopyranoside and named malonylfloralginsenoside Rb2. The assignments of the 1H and 13C 903



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Figure 3. Key HMBC correlations of 12−15.

3,12,20-trihydroxydammar-24-ene-20-O-β-D-glucopyranoside and 3-O-[(3-O-malonyl)-β-D-glucopyranosyl(1→2)-β-D-glucopyranosyl]-(3β,12β,20S)-3,12,20-trihydroxydammar-24-ene-20O-β-D-glucopyranoside and named malonylfloralginsenosides Rd1 and Rd2, respectively. Comparison of the 13C NMR data of 9 and 10 with those of Rd23 indicated that changes occurred on the glucosyl moieties attached to C-20. For 9, a deshielded C-3‴ and shielded C-1‴ and C-3‴ were observed. In contrast, C-6‴ in the spectrum of 10 was deshielded, while a shielding effect was observed for C5‴. The HMBC spectra displayed key cross-peaks from H-3‴ [δH 5.95 (t, J = 9.2 Hz)] to the carbonyl carbon (δC 169.1) of the malonyl group for 9 and from the terminal H-6‴ [δH 5.16 (dd, J = 1.8, 11.6 Hz) and δH 4.71 (dd, J = 7.6, 11.7 Hz)] to the carbonyl carbon (δC 168.4) for 10, respectively (Figure 2). On the basis of this evidence, the structures of compounds 9 and 10 were established as 3-O-[β-D-glucopyranosyl(1→2)-β-Dglucopyranosyl]-(3β,12β,20S)-3,12,20-trihydroxydammar-24ene-20-O-(3-O-malonyl)-β-D-glucopyranoside and 3-O-[β-Dglucopyranosyl(1→2)-β- D -glucopyranosyl]-(3β, 12β,20S)-

in Figure 2. These data established the structure of 8, malonylfloralginsenoside Rd3, as 3-O-[(4-O-malonyl)-β-Dglucopyranosyl(1→2)-β- D -glucopyranosyl]-(3β,12β,20S)3,12,20-trihydroxydammar-24-ene-20-O-β-D-glucopyranoside. On comparison of the 13C NMR data of 6/7 with those of ginsenoside Rd, significant changes were observed in three additional carbons ascribed to a malonyl substituent and the signals ascribable to the terminal glucosyl unit of the C-3 saccharide chain. For 6, the C-1″ and C-3″ resonances were deshielded by 4.3 and 2.6 ppm, respectively, while the C-2″ resonance showed negligible variation. The HMBC cross-peak of H-2″ [δH 5.73 (t, J = 8.8 Hz)] to the carbonyl carbon (δC 168.5) provided evidence for malonylation at 2″-OH. For 7, by comparison with the 13C NMR data of ginsenoside Rd, it was apparent that C-2″ and C-4″ were shielded, while C-3″ was slightly deshielded. A key HMBC cross-peak of H-3″ [δH 5.98 (t, J = 9.5 Hz)] to the carbonyl carbon (δC 168.6) indicated the malonyl group was linked to 3″-OH. Thus, the structures of 6 and 7 were established as 3-O-[(2-O-malonyl)-β- D glucopyranosyl(1→2)-β- D -glucopyranosyl]-(3β,12β,20S)904



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a characteristic deshielding, accompanied by shielded C-3″ and C-5″ resonances, while a deshielded C-3″ and shielded C-2″ and C-4″ resonances were observed for 14. These data indicated the malonylation sites of 13 and 14 at 4″-OH and 3″-OH, respectively. Accordingly, the HMBC cross-peaks from δH 5.87 (t, J = 9.6 Hz, H-4″) to the carbonyl carbon of the malonyl group (δC 168.2) for 13 and the proton of δH 5.99 (t, J = 9.6 Hz, H-3″) to the carbonyl carbon (δC 168.5) for 14 confirmed the malonylation sites. Therefore, the structures of 13 and 14 were defined as 3-O-[(4-O-malonyl)-β- D glucopyranosyl(1→2)-β- D -glucopyranosyl]-(3β,12β,20S)3,12,20-trihydroxydammar-24-ene-20-O-α-L-arabinopyranosyl(1→6)-β-D-glucopyranoside and 3-O-[(3-O-malonyl)-β-Dglucopyranosyl(1→2)-β- D -glucopyranosyl]-(3β,12β,20S)3,12,20-trihydroxydammar-24-ene-20-O-α-L-arabinopyranosyl(1→6)-β-D-glucopyranoside, named malonylfloralginsenosides Rc2 and Rc3, respectively. Contrary to 13/14, the carbon framework of 15 was similar to that of ginsenoside Rc by analysis of the 1H and 13C NMR spectra.23 In contrast to Rc, the C-3″ resonance was deshielded, and those of C-2″ and C-4″ were shielded, which indicated malonylation at 3″-OH. The HMBC cross-peak from H-3″ [δH 5.99 (t, J = 9.5 Hz)] to the carbonyl carbon (δC 168.6) of the malonyl group confirmed the malonylation site (Figure 3). Thus, the structure of 15 was established as 3-O-[(3-O-malonyl)-β-D-glucopyranosyl(1→2)β-D-glucopyranosyl]-(3β,12β,20S)-3,12,20-trihydroxydammar24-ene-20-O-α-L-arabinofuranosyl(1→6)-β-D-glucopyranoside and named malonylfloralginsenoside Rc4. The antidiabetic activities of the malonylginsenosides (1, 3, 4, 8, 13, and 16−19) and the corresponding nonmalonylated ginsenosides (Rb1, Rc, Rb2, Rd, and Re) were evaluated on the promotion of L6 myotubes’ glucose consumption and the activation of AMPKα2β1γ1. AMPK is a universal energy sensor influencing the cellular physiological process.25 Synthesized 20(S)-PPD derivatives were reported as new AMPKα2β1γ1 activators.20 As a result, ginsenoside Rb2, at 10 and 20 μM, and 1 and 18 at 20 μM promoted the glucose consumption of differentiated L6 myotubes. Nine potential AMPKα2β1γ1 activators were found, including three non-malonylated ginsenosides (Rb1, Rb2, and Rd) and six malonylginsenosides (4, 8, 13, 16, 17, and 19), with EC50 values varying between 0.0168 and 2.8 μM. In particular, ginsenoside Rb2 showed the strongest activation effect (EC50: 16.8 nM; fold: 4.7) and is more effective than the reported 20(S)-PPD derivatives. However, their activities were generally weaker than the positive control berberine (10 μM). Ginsenoside Rb2 showed antidiabetic activities on both in vitro models; however, malonylation of Rb2 (13 and 18) otherwise decreased the activity. These results indicated the potential antidiabetic properties of PPD-type ginsenosides to be operating through different pathways.

3,12,20-trihydroxydammar-24-ene-20-O-(6-O-malonyl)-β-Dglucopyranoside, respectively, named malonylfloralginsenosides Rd4 and Rd5, respectively. Compound 11 possessed the molecular formula C54H86O24, as determined by the HRESIMS data (m/z 1117.5416 [M − H]−, calcd for C54H85O24, 1117.5431). The MS2 spectrum (Figure S86, Supporting Information) showed ions at m/z 1029 (−88 Da), 945 (−172 Da), 765, 621, and 459, suggesting the presence of two malonyl substituents and three hexosyl residues attached to a PPD sapogenin. The 1H and 13C NMR spectra displayed the typical signals ascribed to a 3,20disubstituted 20(S)-PPD, i.e., three anomeric protons [δH 4.94 (d, J = 7.6 Hz, H-1′), δH 5.35 (d, J = 7.6 Hz, H-1″), and δH 5.14 (d, J = 7.8 Hz, H-1‴)], and four carbonyl carbons (δC 168.4, 168.7, 170.1, and 170.1) and two groups of methylene protons [δH 3.83 (4H, m)] assignable to two malonyl substituents. By comparing the 13C NMR data with those of ginsenoside Rd,23 the terminal C-6″ and C-6‴ resonances were deshielded, while those of C-5″ and C-5‴ were shielded. The malonylation sites at 6″-OH and 6‴-OH were confirmed by the HMBC cross-peaks of H-6″ [δH 4.99 (1H, dd, J = 5.0, 11.8 Hz) and δH 5.10 (1H, dd, J = 1.6, 11.8 Hz)] to the carbonyl carbon at δC 168.4, and the proton of H-6‴ [δH 4.72 (1H, dd, J = 7.4, 11.6 Hz) and δH 5.17 (1H, dd, J = 1.9, 11.8 Hz)] to the carbon of δC 168.7 (Figure 2). The structure of compound 11 was thus defined as 3-O-[(6-O-malonyl)-β-Dglucopyranosyl(1→2)-β- D -glucopyranosyl]-(3β,12β,20S)3,12,20-trihydroxydammar-24-ene-20-O-(6-O-malonyl)-β-Dglucopyranoside and named malonylfloralginsenoside Rd6. The molecular formula of compound 12 was determined as C56H92O25 based on its HRESIMS data (m/z 1163.5835 [M − H]−, calcd for C56H91O25, 1163.5849). The MS2 spectrum (Figure S94, Supporting Information) showed ions at m/z 1119 (−44 Da), 1077 (−86 Da), 945, 783, 621, and 459, indicating the presence of a malonyl group, a pentosyl unit, and three hexosyl moieties attached to a PPD sapogenin. The 1H and 13C NMR spectra displayed signals ascribable to a ginsenoside Rb3 framework,24 involving eight methyl singlets, three oxymethines [δC 78.6 (C-3), 70.6 (C-12), and 83.9 (C-20)], and two olefinic [δC 126.5 (C-24) and 131.4 (C-25)] carbon signals, and four anomeric protons/carbons [δH 4.93 (d, J = 7.5 Hz, H-1′)/δC 105.4 (C-1′); 5.35 (d, J = 7.7 Hz, H-1″)/105.4 (C-1″); 5.15 (d, J = 7.6 Hz, H-1‴)/98.6 (C-1‴); and 5.01 (d, J = 7.4 Hz, H1″″)/106.4 (C-1″″)]. Comparison with the 13C NMR data of Rb3 revealed three additional carbons assignable to a malonyl substituent (δC 169.4, 43.5, and 174.2), together with the deshielded C-6″ and the shielded C-5″. These spectroscopic changes suggested the malonylation of 6″-OH, which was supported by the HMBC cross-peak from H-6″ [δH 5.07 (d, J = 11.2 Hz) and δH 4.99 (m)] to the carbonyl carbon (δC 169.4) (Figure 3). Accordingly, the structure of 12 was defined as 3-O[(6-O-malonyl)-β-D-glucopyranosyl(1→2)-β-D-glucopyranosyl]-(3β,12β,20S)-3,12,20-trihydroxydammar-24-ene-20-O-β-Dxylopyranosyl(1→6)-β-D-glucopyranoside and named malonylfloralginsenoside Rc1. Compounds 13−15 were identified as three isomers of 12, all possessing a molecular formula of C56H92O25. In addition to the signals characteristic for a malonyl substituent, the 1H and 13 C NMR spectra of 13 and 14 showed resonances that could be assigned to the framework of ginsenoside Rb2.23 The 13C NMR data of 13 and 14 were similar, except for the signals ascribed to the terminal glucosyl moiety of the disaccharide chain attached to C-3. Compared with Rb3, C-4″ of 13 showed

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotation data were acquired on an Autopol VI polarimeter from Rudolph Research Analytical (Hackettstown, NJ, USA). IR data were determined on a Thermo Scientific Nicolet iS5 FT-IR spectrometer and processed using the Thermo Scientific OMNIC v9.1 software (San Jose, CA, USA). A Bruker Avance III HD Ascend 500 MHz spectrometer was used to determine the 1D and 2D NMR data (1H, 13C, HSQC, HMBC, 1H−1H COSY, and ROESY) in pyridine-d5. HRESIMS data were recorded on a Xevo G2-S QTOF system (Waters Corporation, Milford, MA, USA). Semipreparative reversed-phase HPLC [an Atlantis T3 column (10 × 250 mm, 5 μm; Waters, Milford, MA, 905



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Supporting Information; HRESIMS (negative) m/z 1193.5927 [M − H]− (calcd for C57H93O26, 1193.5955). Malonylfloralginsenoside Rb2 (5): white, amorphous powder; [α]20 D +11 (c 0.1, MeOH); IR (KBr) νmax 3423, 2962, 2925, 1745, 1635, −1 1 1541, 1454, 1385, 1261, 1084, 1043, 802 cm ; H (500 MHz, pyridine-d5) and 13C NMR (125 MHz, pyridine-d5) data, see Table S1, Supporting Information; HRESIMS (negative) m/z 1193.5939 [M − H]− (calcd for C57H93O26, 1193.5955). Malonylfloralginsenoside Rd1 (6): white, amorphous powder; [α]20 D +16 (c 0.1, MeOH); IR (KBr) νmax 3423, 2962, 2925, 1745, 1604, −1 1 1456, 1385, 1261, 1078, 1039, 802, 621 cm ; H (500 MHz, pyridined5) and 13C NMR (125 MHz, pyridine-d5) data, see Table S2, Supporting Information; HRESIMS (negative) m/z 1031.5435 [M − H]− (calcd for C51H83O21, 1031.5427). Malonylfloralginsenoside Rd2 (7): white, amorphous powder; [α]20 D +17 (c 0.1, MeOH); IR (KBr) νmax 3425, 2970, 2925, 1747, 1631, 1454, 1385, 1261, 1086, 1049, 802, 577 cm−1; 1H (500 MHz, pyridined5) and 13C NMR (125 MHz, pyridine-d5) data, see Table S2, Supporting Information; HRESIMS (negative) m/z 1031.5431 (calcd for C51H83O21, 1031.5427). Malonylfloralginsenoside Rd3 (8): white, amorphous powder; [α]20 D +17 (c 0.1, MeOH); IR (KBr) νmax 3423, 2960, 2925, 1743, 1628, 1456, 1387, 1261, 1082, 1038, 802, 579 cm−1; 1H (500 MHz, pyridined5) and 13C NMR (125 MHz, pyridine-d5) data, see Table S2, Supporting Information; HRESIMS (negative) m/z 1031.5437 [M − H]− (calcd for C51H83O21, 1031.5427). Malonylfloralginsenoside Rd4 (9): white, amorphous powder; [α]20 D +10 (c 0.1, MeOH); IR (KBr) νmax 3423, 2962, 2925, 1747, 1637, 1454, 1385, 1261, 1080, 1026, 802, 580 cm−1; 1H (500 MHz, pyridined5) and 13C NMR (125 MHz, pyridine-d5) data, see Table S3, Supporting Information; HRESIMS (negative) m/z 1031.5432 [M − H]− (calcd for C51H83O21, 1031.5427). Malonylfloralginsenoside Rd5 (10): white, amorphous powder; [α]20 D +21 (c 0.1, MeOH); IR (KBr) νmax 3423, 2962, 2925, 1747, 1635, 1456, 1387, 1261, 1080, 1026, 802, 577 cm−1; 1H (500 MHz, pyridine-d5) and 13C NMR (125 MHz, pyridine-d5) data, see Table S3, Supporting Information; HRESIMS (negative) m/z 1031.5435 [M − H]− (calcd for C51H83O21, 1031.5427). Malonylfloralginsenoside Rd6 (11): white, amorphous powder; [α]20 D +17 (c 0.1, MeOH); IR (KBr) νmax 3421, 2962, 2925, 1747, 1628, 1454, 1415, 1387, 1261, 1080, 1022, 802, 577 cm−1; 1H (500 MHz, pyridine-d5) and 13C NMR (125 MHz, pyridine-d5) data, see Table S3, Supporting Information; HRESIMS (negative) m/z 1117.5416 [M − H]− (calcd for [C54H86O24−H]−, 1117.5431). Malonylfloralginsenoside Rc1 (12): white, amorphous powder; [α]20 D +6 (c 0.1, MeOH); IR (KBr) νmax 3423, 2960, 2925, 1745, 1628, 1456, 1385, 1261, 1084, 1024, 802, 567 cm−1; 1H (500 MHz, pyridined5) and 13C NMR (125 MHz, pyridine-d5) data, see Table S4, Supporting Information; HRESIMS (negative) m/z 1163.5835 [M − H]− (calcd for C56H91O25, 1163.5849). Malonylfloralginsenoside Rc2 (13): white, amorphous powder; [α]20 D −5 (c 0.1, MeOH); IR (KBr) νmax 3421, 2960, 2925, 1745, 1637, 1456, 1414, 1387, 1261, 1081, 1039, 802, 569 cm−1; 1H (500 MHz, pyridine-d5) and 13C NMR (125 MHz, pyridine-d5) data, see Table S4, Supporting Information; HRESIMS (negative) m/z 1163.5837 [M − H]− (calcd for C56H91O25, 1163.5849). Malonylfloralginsenoside Rc3 (14): white, amorphous powder; [α]20 D +11 (c 0.1, MeOH); IR (KBr) νmax 3423, 2962, 2925, 1745, 1635, 1454, 1385, 1261, 1088, 1028, 802, 580 cm−1; 1H (500 MHz, pyridine-d5) and 13C NMR (125 MHz, pyridine-d5) data, see Table S4, Supporting Information; HRESIMS (negative) m/z 1163.5835 [M − H]− (calcd for C56H91O25, 1163.5849). Malonylfloralginsenoside Rc4 (15): white, amorphous powder; [α]20 D −1 (c 0.1, MeOH); IR (KBr) νmax 3423, 2962, 2925, 1745, 1635, 1456, 1385, 1261, 1086, 1045, 802, 600 cm−1; 1H (500 MHz, pyridined5) and 13C NMR (125 MHz, pyridine-d5) data, see Table S4, Supporting Information; HRESIMS (negative) m/z 1163.5837 [M − H]− (calcd for C56H91O25, 1163.5849). Acid Hydrolysis of Compounds 1, 4, 8, and 14. Compounds 1, 4, 8, and 14 (each 2 mg) were hydrolyzed in 1 M HCl (4 mL) under

USA); an Eclipse XDB-C18 column (9.4 × 250 mm, 5 μm; Agilent Technologies, Inc., Palo Alto, CA, USA); and an SB-C18 column (9.4 × 250 mm, 5 μm; Agilent Technologies)] and HILIC [an XAmide column (10 × 250 mm, 5 μm; Acchrom Technologies Co., Ltd., Beijing, China)] were performed on an Agilent 1100 HPLC system (Agilent Technologies, Inc., Waldbronn, Germany) for compound purification. AB-8 resin (Anhui Sanxing Resin Technology Ltd., Bengbu, China) and MCI gel (75−100 μm, Mitsubishi Chemical Co., Ltd., Tokyo, Japan) were used for column chromatography (CC). The solvents used for extraction and CC were all of analytical grade, while HPLC-grade solvents were used for semipreparative HPLC and UHPLC/QTOF MS analysis. Plant Material. The flower buds of P. ginseng were collected from Funan State Forest Farm (Baishan, Jilin Province, China) in August 2015. Authentication of the sample was performed according to the botany traits recorded in the Flora of China. A voucher specimen (No. 20150810-FBPG) was deposited in the herbarium at Shanghai Institute of Materia Medica. Extraction and Isolation. The fine, dry powder of the flower buds (2.5 kg) was extracted with 70% EtOH (20 L) using ultrasound assistance and constant stirring for 4 h at room temperature, to give, after evaporation, a crude extract (800 g). The residue was dissolved in H2O (4 L) and subjected to liquid−liquid partitioning using n-BuOH and H2O (4 L × 3). The water fraction (200 g) was further fractionated on an AB-8 resin column (140 cm × 33 cm), eluting with EtOH−H2O (0, 20, 30, 40, 50, and 90%). UV detection directed the combination of eluates, yielding five fractions (Frs. A−F). Frs. C and D contained the target malonylginsenosides. Semipreparative HPLC of Fr. C (8 g) on an SB-C18 column eluting with CH3CN−H2O (23:77) generated 1−3 (65, 14, and 18 mg, respectively). Fraction D (45 g) was separated on an MCI gel column (500 mL) eluting with EtOH− H2O (35:65 and 45:55), producing three subfractions (Frs. D1−D3). Fr. D1 (6 g) was initially separated by semipreparative HPLC on an HILIC column (CH3CN−H2O, 75:25), giving two fractions (Fr. D1-1 and Fr. D1-2). Another Eclipse XDB-C18 column (MeOH−H2O, 7:3) was used to further purify Fr. D1-2 (1.5 g) to obtain 4 (15 mg), 5 (6 mg), and 11 (15 mg). Frs. D2 (11 g) and D3 (5.5 g) were also separated by combining HILIC and RP semipreparative HPLC. After the HILIC fractionation, four (Fr. D2-1 to Fr. D2-4) and two (Fr. D31 to Fr. D3-2) subfractions were obtained from Frs. D2 and D3, respectively. Fr. D2-2 (400 mg) was separated on an Atlantis T3 column (CH3CN−H2O, 39:61), giving 6 (4.3 mg), 8 (20 mg), and 9 (4 mg). Semipreparative HPLC of Fr. D2-4 (240 mg) on an SB-C18 column yielded 7 (13 mg) and 10 (14 mg). Further purification of Fr. D3-1 and Fr. D3-2 by semipreparative HPLC separately on an SB-C18 column and an Eclipse C18 column (CH3CN−H2O, 34:66) afforded 12 (5 mg) and 13−15 (9, 10, and 10 mg, respectively). Malonylfloralginsenoside Re1 (1): white, amorphous powder; [α]20 D +2 (c 0.1, MeOH); IR (KBr) νmax 3417, 2962, 2927, 1732, 1639, 1454, 1385, 1261, 1078, 1026, 806, 621 cm−1; 1H (500 MHz, pyridine-d5) and 13C NMR (125 MHz, pyridine-d5) data, see Table 1; HRESIMS (negative) m/z 1031.5435 [M − H]− (calcd for C51H83O21, 1031.5427). Malonylfloralginsenoside Re2 (2): white, amorphous powder; [α]20 D −4 (c 0.1, MeOH); IR (KBr) νmax 3421, 2964, 2931, 1757, 1639, 1454, 1385, 1319, 1261, 1076, 1047, 808, 617 cm−1; 1H (500 MHz, pyridined5) and 13C NMR (125 MHz, pyridine-d5) data, see Table 1; HRESIMS (negative) m/z 1031.5431 [M − H]− (calcd for C51H83O21, 1031.5427). Malonylfloralginsenoside Re3 (3): white, amorphous powder; [α]20 D −1 (c 0.1, MeOH); IR (KBr) νmax 3423, 2964, 2929, 1747, 1635, 1454, 1385, 1313, 1261, 1080, 1047, 808 cm−1; 1H (500 MHz, pyridine-d5) and 13C NMR (125 MHz, pyridine-d5) data, see Table 1; HRESIMS (negative) m/z 1031.5437 [M − H]− (calcd for C51H83O21, 1031.5427). Malonylfloralginsenoside Rb1 (4): white, amorphous powder; [α]20 D +12 (c 0.1, MeOH); IR (KBr) νmax 3423, 2962, 2925, 1747, 1635, −1 1 1454, 1387, 1261, 1081, 1045, 802, 575 cm ; H (500 MHz, pyridined5) and 13C NMR (125 MHz, pyridine-d5) data, see Table S1, 906



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reflux for 4 h. The dried residue was dissolved in H2O and further extracted by EtOAc (3 × 5 mL). The aqueous layer was dried to produce a mixture of monosaccharides. The solution of the sugar mixture in pyridine (0.2 mL) was added to L-cysteine methyl ester hydrochloride (2.0 mg) and kept at 60 °C for 2 h. Trimethylsilylimidazole (1.5 mL) was added to the reaction mixture in an ice−water bath and heated at 60 °C for 2 h. The reaction mixture (4 μL) was directly analyzed by GC under the following conditions: HP-5 quartz capillary column (30 × 0.32 mm) and a H2 flame ionization detector; column temperature, 180−280 °C; programmed increase, 3 °C/min; carrier gas, N2 (1 mL/min); injector and detector temperature, 250 °C; and split ratio, 1/50. The configuration of the sugars was confirmed by comparing the retention times with those of the derivatized authentic samples. The retention times of D- and L-glucose were 20.58 and 21.02 min, respectively. All of the glucose residues from 1, 4, 8, and 14 were D-configured. In addition, the rhamnosyl unit in 1 and the arabinosyl moiety in 14 were L-configured (tR 17.21 and 15.61 min, respectively), showing retention times consistent with those of the authentic samples. Promotion Activity of Glucose Consumption of L6 Myotubes. L6 rat myotubes, purchased from ATCC (ATCC CRL1458, Rockefeller, MD, USA), were maintained in high-glucose (4500 mg/L) Dulbecco’s minimal essential medium (DMEM) with 10% fetal bovine serum (FBS, Millipore ES009B). For myotube differentiation, cells were maintained in high-glucose (4500 mg/L) DMEM with 2% FBS for 4−6 days. L6 rat myotubes were differentiated in a 96-well cell culture cluster. After differentiation, L6 myotubes were incubated in 0.5% bovine serum albumin and low-glucose (1000 mg/L) DMEM containing the compounds at the indicated concentrations for 12 h. After treatment, the glucose concentration in the medium was measured with the glucose assay reagent (Shanghai Mind Bioengineering Co. Ltd., Shanghai, China) based on the glucose oxidase method. The glucose concentration of the wells with cells was subtracted from the glucose concentration of the blank wells without cells to calculate the glucose consumption.26 Activation Activity of AMPKα2β1γ1. Recombinant AMPKα2β1γ1 and CaMKKβ protein were constructed, expressed (in BL21 strain), and purified before the test. AMPKα2β1γ1 protein was completely phosphorylated through incubation with CaMKKβ at room temperature for 3.5 h. The AMPK activity was tested using the HTRF KinEASE-STK kit (Cisbio, Codolet, France). Briefly, the reaction was carried out in 384-well plates in a reaction volume of 10 μL containing 32 mmol/L Tris-HCl, pH 7.5, 4 mmol/L MgCl2, 0.8 mmol/L DTT, 160 nmol/L substrate-1 peptide, and 4 μmol/L ATP. The reaction was initiated by addition of 2 μL of compound, 4 μL of STK substrate 1-biotin, and 4 μL of 1.6 nM/L recombinant AMPKα2β1γ1, followed by incubation for 45 min at 30 °C. The reaction was terminated by addition of the detection reagent containing 57.5 nmol/L XL-665 and STK-antibody labeled with Eu3+-cryptate. The fluorescence was measured at 615 nm (cryptate) and 665 nm (XL665). In primary screening, the compounds were tested at a concentration of 20 μM. Secondary screenings were conducted at 40, 20, 10, 5, 2.5, 1.25, and 0.625 μM among the compounds whose activation of AMPKα2β1γ1 protein was no less than 1.5-fold to obtain a dose−response curve using Graphpad Prism 6.01.

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Article

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Tel: +86-21-20231000, ext. 2221. Fax: +86-21-50272789. ORCID

Wan-Ying Wu: 0000-0001-9549-6448 Author Contributions §

S. Qiu and W.-Z. Yang contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support from National Natural Science Foundation of China (81503240, 81473344, and 81470166). The authors also acknowledge the assistance of Professor Emeritus G. A. Cordell, Natural Products Inc., Evanston, IL, USA, in reviewing the manuscript prior to publication.

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REFERENCES

(1) Seino, Y.; Nanjo, K.; Tajima, N.; Kadowaki, T.; Kashiwagi, A.; Araki, E.; Ito, C.; Inagaki, N.; Iwamoto, Y.; Kasuga, M.; Hanafusa, T.; Haneda, M.; Ueki, K. J. Diabetes Investig. 2010, 1, 212−228. (2) Sas, K. M.; Karnovsky, A.; Michailidis, G.; Pennathur, S. Diabetes 2015, 64, 718−732. (3) Perry, R. J.; Samuel, V. T.; Petersen, K. F.; Shulman, G. I. Nature 2014, 510, 84−91. (4) Li, K. K.; Gong, X. J. RSC Adv. 2015, 5, 47353−47366. (5) Yang, W. Z.; Hu, Y.; Wu, W. Y.; Ye, M.; Guo, D. A. Phytochemistry 2014, 106, 7−24. (6) Shin, B. K.; Kwon, S. W.; Park, J. H. J. Ginseng Res. 2015, 39, 287−298. (7) Liu, Z.; Wang, L. J.; Li, X.; Hu, J. N.; Chen, Y.; Ruan, C. C.; Sun, G. Z. Phytother. Res. 2009, 23, 1426−1430. (8) Liu, Z.; Li, W.; Li, X.; Zhang, M.; Chen, L.; Zheng, Y. N.; Sun, G. Z.; Ruan, C. C. J. Ethnopharmacol. 2013, 145, 233−240. (9) Liu, Z.; Li, Y.; Li, X.; Ruan, C. C.; Wang, L. J.; Sun, G. Z. J. Pharm. Biomed. Anal. 2012, 64−65, 56−63. (10) Kitagawa, I.; Taniyama, T.; Hayashi, T.; Yoshikawa, M. Chem. Pharm. Bull. 1983, 9, 3353−3356. (11) Ruan, C. C.; Liu, Z.; Li, X.; Liu, X.; Wang, L. J.; Pan, H. Y.; Zheng, Y. N.; Sun, G. Z.; Zhang, Y. S.; Zhang, L. X. Molecules 2010, 15, 2319−2325. (12) Sun, G. Z.; Li, X. G.; Liu, Z.; Wang, J. Y.; Zheng, Y. N.; Yang, X. W. Chem. J. Chin. Univ. 2007, 28, 1316−1318. (13) Wang, Y. S.; Jin, Y. P.; Gao, W.; Xiao, S. Y.; Zhang, Y. W.; Zheng, P. H.; Wang, J.; Liu, J. X.; Sun, C. H.; Wang, Y. P. J. Ginseng Res. 2016, 40, 245−250. (14) Yang, W. Z.; Ye, M.; Qiao, X.; Liu, C. F.; Miao, W. J.; Bo, T.; Tao, H. Y.; Guo, D. A. Anal. Chim. Acta 2012, 739, 56−66. (15) Qiu, S.; Yang, W. Z.; Shi, X. J.; Yao, C. L.; Yang, M.; Liu, X.; Jiang, B. H.; Wu, W. Y.; Guo, D. A. Anal. Chim. Acta 2015, 893, 65− 76. (16) Yang, W. Z.; Bo, T.; Ji, S.; Qiao, X.; Guo, D. A.; Ye, M. Food Chem. 2013, 139, 762−769. (17) Lai, C. J. S.; Tan, T.; Zeng, S. L.; Qi, L. W.; Liu, X. G.; Dong, X.; Li, P.; Liu, E. H. J. Pharm. Biomed. Anal. 2015, 109, 184−191. (18) Shi, X. J.; Yang, W. Z.; Qiu, S.; Yao, C. L.; Shen, Y.; Pan, H. Q.; Bi, Q. R.; Yang, M.; Wu, W. Y.; Guo, D. A. Anal. Chim. Acta 2017, 952, 59−70. (19) Qiu, S.; Yang, W. Z.; Yao, C. L.; Qiu, Z. D.; Shi, X. J.; Zhang, J. X.; Hou, J. J.; Wang, Q. R.; Wu, W. Y.; Guo, D. A. J. Chromatogr. A 2016, 1453, 78−87. (20) Liu, J. H.; Chen, D. K.; Liu, P.; He, M. N.; Li, J.; Li, J. Y.; Hu, L. H. Eur. J. Med. Chem. 2014, 79, 340−349.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00789. 1 H and 13C NMR, HSQC, HMBC, 1H−1H COSY, ROESY, IR, and CID-MS/MS spectra, purity determination of compounds 1−15, GC chromatograms of 1, 4, 8, and 14, and 1H and 13C NMR data assignments of 4 and 15 (PDF) 907



Article

(21) Sun, G. Z.; Liu, Z.; Li, X. G.; Zheng, Y. N.; Wang, J. Y. Chin. J. Anal. Chem. 2005, 33, 1783−1786. (22) Teng, R. W.; Li, H. Z.; Chen, J. T.; Wang, D. Z.; He, Y. N.; Yang, C. R. Magn. Reson. Chem. 2002, 40, 483−488. (23) Cho, J. G.; Lee, M. K.; Lee, J. W.; Park, H. J.; Lee, D. Y.; Lee, Y. H.; Yang, D. C.; Baek, N. I. J. Ginseng Res. 2010, 34, 113−121. (24) Li, P. Y.; Hao, X. H.; Li, X. Zhong Cao Yao 1999, 30, 563−565. (25) Zaks, I.; Getter, T.; Gruzman, A. Future Med. Chem. 2014, 6, 1325−1353. (26) Yin, J.; Hu, R. M.; Chen, M. D.; Tang, J. F.; Li, F. Y.; Yang, Y.; Chen, J. L. Metab., Clin. Exp. 2002, 51, 1439−1443.

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Malonylginsenosides with Potential Antidiabetic Activities from the

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