Steroids from the Root Bark of - ACS Publications - American

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Bioactive Octahydroxylated C21 Steroids from the Root Bark of Lycium chinense Ya-Wen An,† Zhi-Lai Zhan,†,‡ Jing Xie,† Ya-Nan Yang,† Jian-Shuang Jiang,† Zi-Ming Feng,† Fei Ye,† and Pei-Cheng Zhang*,† †

State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China ‡ State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, People’s Republic of China S Supporting Information *

ABSTRACT: Lyciumsterols A−K (1−11), 11 new octahydroxylated C21 steroids, were isolated from the root bark of Lycium chinense, along with 15 known compounds. Characterization of these C21 steroids showed the presence of eight hydroxy groups on the C21 steroid skeleton with a (2E,4E)-5-phenyl-2,4-pentadienoate group at C-12 or C-20 and various 2,6-deoxy sugar residues at C-3. The structures of these compounds were elucidated using spectroscopic data interpretation. Compounds 2, 3, and 7 exhibited dose-dependent protective effects on pancreatic islet cells and may help to improve cell viability. In addition, it was found that compounds 7, 8, 9, and 11 exhibited autophagy activation.

n Chinese medicine, “Lycii Cortex” is a traditional medicinal herb derived from the root bark of Lycium chinense Mill. or Lycium barbarum L. (Solanaceae)1 and has been used for the treatment of diabetes. Modern pharmacological studies of these species have revealed a broad range of biological effects, including antihypertensive, antioxidative, heptaprotective, antifungal, α-galactosidase inhibitory, and hypochlolesterolemic activities. 2−10 Previous phytochemical investigations of L. chinense and L. barbarum have led to the isolation of more

I

than 110 compounds, including alkaloids, lignanamides, cyclopeptides, lignans, sterols, and other compounds.11−19 The present investigation focused specifically on the constituents in the ethyl acetate-soluble portion of L. chinense root bark, which was obtained from an 80% EtOH extract. This study resulted in the isolation of 11 new octahydroxylated C21 steroids (lyciumsterols A−K, 1−11) and 15 known compounds. Octahydroxylated C21 steroids have been previously reported, with all of these having been isolated from Orthenthera viminea20 and Cynanchum saccatum.21−23

Received: December 8, 2015 © XXXX American Chemical Society and American Society of Pharmacognosy

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DOI: 10.1021/acs.jnatprod.5b01087 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Table 1. 1H NMR Spectroscopic Data (500 MHz, δ in ppm, J in Hz) for 1−4 in DMSO-d6 position

1

2

3

4

1a 1b 2a 2b 3 4a 4b 6 7a 7b 9 11a 11b 12 15a 15b 16a 16b 18 19 20 21 2′, 6′ 3′, 5′ 4′ 7′ 8′ 9′ 10′ 1″ 2a″ 2b″ 3″ 4″ 5″ 6″

1.75, m 1.59, m 1.58, m 1.37, m 3.85, m 1.89, t (12.0) 1.41, m 3.45, m 1.95, dd (3.5, 14.5) 1.63, m 1.67, m 1.63, m 1.24, m 3.21, m 1.74, m 1.57, m 1.79, m 1.12, m 1.18, s 1.13, s 5.02, q (6.0) 1.13, d (6.0) 7.56, d (7.5) 7.38, dd (7.5, 7.5) 7.31, t (7.5) 6.99, d (16.5) 7.13, dd (16.5, 16.5) 7.32, dd (16.5,16.5) 5.98, d (16.5)

1.71, m 1.58, m 1.59, m 1.36, m 3.86, m 1.91, t (12.5) 1.42, m 3.46, m 1.99, dd (3.5, 14.5) 1.71, m 1.80, m 1.81, m 1.47, m 4.52, m 1.70, m 1.59, m 1.80, m 1.12, m 1.40, s 1.16, s 3.28, q (6.0) 0.97, d (6.0) 7.56, d (7.5) 7.38, dd (7.5, 7.5) 7.32, t (7.5) 7.03, d (16.0) 7.11, dd (16.0, 16.0) 7.42, dd (16.0, 16.0) 6.09, d (16.0)

1.72, m 1.57, m 1.68, m 1.42, m 4.01, m 1.84, t (12.0) 1.55, m 3.48, m 1.98, dd (3.0, 14.0) 1.71, m 1.79, m 1.80, m 1.45, m 4.53, m 1.77, m 1.67, m, 1.58, m 1.14, m 1.40, s 1.15, s 3.28, overlap 1.00, d (6.0) 7.56, d (7.5) 7.38, dd (7.5, 7.5) 7.32, t (7.5) 7.03, d (16.0) 7.08, dd (16.0, 16.0) 7.43, dd (16.0, 16.0) 6.08, d (15.5) 4.81, dd (1.5, 9.5) 1.47, m 1.71, m 3.82, d (2.5) 2.97, br d (8.0) 3.59, m 1.11, d (6.5)

1.60, m 1.52, m 1.59, m 1.60, m 4.00, m 1.87, m 1.54, m 3.78, m 1.80, m 1.55, m 1.67, m 1.75, m 1.44, m 4.58, m 1.98, dd (3.5, 14.5) 1.67, m 1.68, m 1.13, m 1.41, s 0.93, s 3.32, overlap 0.97, d (6.0) 7.56, d (7.5) 7.39, dd (7.5, 7.5) 7.34, t (7.5) 7.04, d (16.5) 7.11, dd (16.5, 16.5) 7.43, dd (16.5, 16.5) 6.08, d (16.5) 4.81, dd (1.5, 9.5) 1.80, m 1.54, m 3.83, m 3.01, m 3.61, m 1.12, d (6.5)

7.38 (2H, dd, J = 7.5, 7.5 Hz), and 7.31 (1H, dd, J = 7.5, 7.5 Hz); two sets of trans conjugated olefinic protons at δH 6.99 (1H, d, J = 16.5 Hz), 7.13 (1H, dd, J = 16.5, 16.5 Hz), 7.32 (1H, dd, J = 16.5, 16.5 Hz), and 5.98 (1H, d, J = 16.5 Hz); and three methyl signals at δH 1.18 (3H, s), 1.13 (3H, s), and 1.13 (3H, d, J = 6.0 Hz). In the 13C NMR spectrum (Table 2), 32 carbon resonances were observed, with 11 of these resonances attributed to a (2E,4E)-5-phenyl-2,4-pentadienoate moiety, which was verified by HMBC correlations from H-8′ to C-1′, C-7′, and C-9′ and from H-9′ to C-8′, C-7′, and C-11′. In combination with the observed HSQC correlations, the remaining 21 carbon signals were designated as three methyls, seven methylenes, five methines, and six quaternary carbons. After we considered the remaining four degrees of unsaturation and the molecular formula, the skeleton of 1 was elucidated as a C21 sterol with eight hydroxy groups. In the HMBC spectrum, a key correlation from H-20 (δH 5.02) to C-11′ (δC 165.1) suggested that the (2E,4E)5-phenyl-2,4-pentadienoate moiety is located at C-20. To determine the relative configuration of 1, the ROESY and 13C NMR data were analyzed. The characteristic ROESY correlations from OH-5 to H-3 and H-9, from OH-6 to CH3-19 and OH-8, from H-9 to H-12 and H-14, from OH-8 to CH3-18, and from OH-14 to OH-17, combined with the chemical shifts of C-5, C-6,

Considering the traditional medical uses for L. chinense root bark, the compounds isolated were tested for their protective effects on injured pancreatic islet cells. Furthermore, a literature survey revealed that autophagy plays a vital role in maintaining normal cell function and protecting cells from various forms of cellular stress including endoplasmic reticulum (ER) stress, the unfolded protein responses (UPR), islet amyloid polypeptides (IAPP), and inflammation during insulin resistant states,24−30 prompting the testing of the isolates for the ability to activate autophagy. In this paper, reported are the isolation and structural elucidation of new octahydroxylated C21 steroids as well as the biological evaluation of these compounds.

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RESULTS AND DISCUSSION Compound 1 was obtained as a white amorphous powder. Its molecular formula was determined to be C32H44O9 based on the protonated molecular ion peak at m/z 573.3057 [M + H]+ (calcd for C32H45O9, 573.3064) observed in the HRESIMS, which indicated 11 degrees of unsaturation. The IR spectrum showed the presence of hydroxy (3418 cm−1) and carbonyl (1692 cm−1) groups, as well as an aromatic ring (1624 and 1449 cm−1). The 1H NMR spectrum of 1 (Table 1) exhibited the presence of monosubstituted aromatic protons at δH 7.56 (2H, d, J = 7.5 Hz), B



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Table 2. 13C NMR Spectroscopic Data (125 MHz, δ in ppm) for 1−11

a13

position

1a

2a

3a

4a

5a

6a

7b

8b

9b

10b

11b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 1′ 2′, 6′ 3′,5′ 4′ 7′ 8′ 9′ 10′ 11′ 1″ 2″ 3″ 4″ 5″ 6″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ OCH3-3‴ 1‴′ 2‴′ 3‴′ 4‴′ 5‴′ 6‴′ OCH3-3‴′ 1‴″ 2‴″ 3‴″ 4‴″ 5‴″ 6‴″ OCH3-3‴″

33.3 30.2 65.6 40.3 74.1 75.7 33.0 77.3 38.5 37.5 27.5 69.7 57.7 87.4 31.8 33.4 86.4 9.5 17.5 74.0 14.9 136.0 126.8 128.8 128.9 139.3 127.1 143.4 122.9 165.1

32.9 30.2 65.5 40.2 74.0 75.5 33.2 76.8 38.2 37.6 23.6 73.7 56.0 87.4 32.8 31.9 87.1 11.2 17.3 69.7 18.9 135.9 127.2 128.8 129.2 140.1 126.6 144.6 121.9 165.7

32.7 27.8 73.3 36.9 73.8 75.5 33.2 76.7 38.2 37.6 23.5 73.6 56.0 87.4 32.0 32.8 87.1 11.1 17.2 69.6 18.9 135.9 127.2 128.8 129.0 140.1 126.6 144.6 121.9 165.7 95.4 38.8 67.1 72.8 68.8 18.4

27.2 25.5 73.5 27.3 76.3 67.3 37.9 75.1 35.0 40.5 23.9 73.6 55.9 87.5 32.8 32.2 87.4 11.3 19.3 69.5 18.7 135.9 127.2 128.8 129.1 140.1 126.6 144.6 121.9 165.7 96.2 38.5 67.0 72.6 69.1 18.3

32.8 27.8 73.5 36.9 73.7 75.7 33.2 77.2 38.4 37.5 27.4 69.6 57.7 87.4 31.7 33.4 86.4 9.4 17.3 74.1 14.8 136.0 127.1 128.8 129.0 139.3 126.8 143.3 122.9 165.0 95.4 38.5 67.4 81.7 66.1 18.0 98.9 34.7 77.5 72.8 69.6 18.4 57.6

27.2 25.5 73.5 27.4 76.4 67.7 38.0 75.5 35.3 40.4 27.6 69.7 57.7 87.4 32.7 32.6 86.9 9.7 19.5 74.0 14.8 136.0 127.1 128.8 128.8 139.4 126.8 143.6 122.7 164.9 96.0 38.0 67.4 81.4 66.1 17.9 98.8 34.7 77.5 72.8 69.6 18.4 57.6

33.9 29.1 74.1 38.3 75.3 77.9 34.5 78.6 39.6 39.1 24.9 75.1 57.4 88.6 33.0 33.6 88.4 12.1 18.2 71.0 19.6 136.7 127.7 129.2 129.3 140.6 127.0 145.3 122.8 167.0 96.0 39.3 67.5 83.5 68.4 18.6 99.8 35.6 78.8 74.1 71.0 18.9 58.1

33.8 29.1 74.0 38.3 75.2 77.9 34.5 78.6 39.6 39.1 24.9 75.1 57.4 88.6 33.0 33.6 88.4 12.0 18.2 71.0 18.6 136.7 127.7 129.2 129.3 140.6 127.0 145.3 122.0 167.0 96.0 39.3 67.5 83.4 68.4 18.7 99.8 36.7 77.7 83.2 69.1 19.6 58.8 102.2 37.2 81.4 76.1 72.9 18.5 57.0

28.2 26.7 75.8 29.0 78.0 68.9 39.0 76.6 36.7 41.9 25.2 75.2 57.3 88.9 32.9 34.1 88.5 12.3 20.3 70.8 18.6 136.6 127.7 129.2 129.4 140.7 126.9 145.4 122.7 167.0 97.9 38.9 67.5 83.1 68.7 18.7 99.8 36.7 77.7 83.0 69.1 19.4 58.8 102.2 37.2 81.4 76.1 72.9 18.5 57.0

33.9 29.2 74.2 38.4 75.3 78.0 34.6 78.9 40.1 39.0 28.6 71.1 59.3 88.6 33.0 34.5 87.8 10.6 18.3 75.6 15.7 136.8 128.6 129.2 129.6 139.6 127.2 143.9 122.1 166.4 96.1 39.1 67.7 81.1 68.7 18.6 99.0 29.2 75.9 77.0 63.2 16.8 56.5 100.5 36.6 77.8 83.3 69.1 18.6 58.8 102.2 37.2 81.4 76.2 72.9 18.7 57.1

33.8 29.1 74.1 38.4 75.1 77.8 34.5 78.6 39.6 39.0 24.9 75.3 57.4 88.4 33.0 33.6 88.6 12.0 18.2 71.0 19.6 136.7 127.7 129.2 129.3 140.6 127.1 145.3 122.9 167.0 96.0 39.1 67.7 81.2 68.7 18.6 98.9 28.6 75.9 77.0 63.2 16.8 56.5 100.5 36.6 77.9 83.3 69.1 18.6 58.8 102.2 37.2 81.4 76.2 72.9 18.6 57.0

C NMR spectra were measured in DMSO-d6.

b13

C NMR spectra were measured in pyridine-d5.

and C-20, indicated that CH3-18, CH3-19, OH-3, OH-6, OH-8, OH-14, and OH-17 are β-oriented, whereas OH-5, H-9, and H-12 are α-oriented.

The absolute configuration of C-20 in 1 was determined by comparing its experimental and calculated ECD data. Conformational analysis of 1a and its diastereoisomer 1b (Figure 1) were C



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Figure 1. Experimental ECD spectrum of compound 1 and calculated ECD spectra of 1a and 1b in MeOH.

performed using the MMFF94 molecular mechanics force field. The preferred conformers were optimized further at the B3LYP/ 6-31G(d) level. The ECD spectra of different conformers were simulated using a Gaussian function with a half-bandwidth of 0.4 eV. The calculated ECD spectra were obtained according to the Boltzmann weighting of each of the conformers with different populations. The overall theoretical ECD data of 1b matched the experimental ECD data of 1 (Figure 1) very well. Accordingly, the structure of compound 1 was assigned as pregnane-3β,5α,6β,8β,12β,14β,17β,20(S)-octol-20-O-[(2E,4E)5-phenyl-2,4-pentadienoate], and this compound was given the trivial name lyciumsterol A. Compound 2 was isolated as a white amorphous powder. A molecular formula of C32H44O9 was deduced by HRESIMS [M + Na]+ at m/z 595.2876, which indicated 11 degrees of unsaturation. A comparison of the spectroscopic data between 2 and 1 demonstrated that the difference between these two compounds is in the linkage position of the (2E,4E)-5-phenyl2,4-pentadienoate group. In the HMBC spectrum of 2, the key correlation of H-12 (δH 4.52) to C-11′ (δC 165.7) confirmed that the (2E,4E)-5-phenyl-2,4-pentadienoate moiety is located at C-12. On the basis of the analysis described above, 2 (lyciumsterol B) was characterized as pregnane-3β,5α,6β,8β,12β,14β,17β,20(S)octol-12-O-[(2E,4E)-5-phenyl-2,4-pentadienoate]. Compound 3 was obtained as a white amorphous powder. Its molecular formula was determined to be C38H54O12 by negative HRESIMS (m/z 701.3518 [M − H]−). The spectroscopic data of 3 were similar to those of 2, except for the resonances of six additional carbon signals (δC 95.4, 38.8, 67.1, 72.8, 68.8, and 18.4), which indicated the occurrence of a 2,6-deoxygenated sugar moiety. Based on the HSQC and HMBC spectra of 3, the 1 H- and 13C NMR signals in this sugar moiety were assigned as shown in Tables 1 and 2. In combination with data from the literature,31 the sugar was identified as digitoxopyranose. According to the 1H NMR spectrum, an anomeric proton at δH 4.81 (1H, dd, J = 1.5, 9.5 Hz) indicated a β-linkage for this sugar unit. Acid hydrolysis of 3 yielded D-digitoxopyranose, 31 which was identified by its specific rotation value ([α]20 D + 35). 13 When compared to the C NMR data of 2, the downfield shift of C-3 (δC 73.3) in 3 suggested that the linkage point of digitoxopyranose is at the C-3 position. This was confirmed by the correlation from H-1″ (δH 4.81) to C-3 (δC 73.3) in the HMBC spectrum. The absolute configuration of 3 was determined to be the same as those of 2 based on NOESY correlations (Figure 2) and the ECD spectrum (Figure S14, Supporting Information). Thus, the structure of compound 3 (lyciumsterol C) was established as pregnane-3β,5α,6β,8β, 12β,14β,17β,20(S)-octol-12-O-[(2E,4E)-5-phenyl-2,4-pentadienoate]-3-O-β-D-digitoxopyranoside.

Figure 2. Key ROESY correlations of compound 3.

The molecular formula of compound 4 was established as being the same as 3 (C38H54O12), based on the positive HRESIMS ion observed at m/z 725.3505 [M + Na]+. Analysis of the UV, IR, and NMR data with those of 3 suggested that these two compounds possess the same planar structure. In the 13C NMR spectrum (Table 2), the resonances for C-5 (δC 76.3) and C-7 (δC 37.9) in 4 exhibited a significant downfield shift, whereas the resonance for C-6 (δC 67.3) displayed an upfield shift compared to those of 3. These results, when combined with the NOESY correlation (Figure 3) between H-6 (δH 3.78) and H-19

Figure 3. Key HMBC correlations of compounds 3 and 4.

(δH 0.93), demonstrated that the OH-6 group is α-oriented.32 Therefore, compound 4 (lyciumsterol D) was proposed as pregnane-3β,5α,6α,8β,12β,14β,17β,20(S)-octol-12-O-[(2E,4E)5-phenyl-2,4-pentadienoate]-3-O-β-D-digitoxopyranoside. The HRESIMS analysis of 5 showed a deprotonated ion at m/z 845.4306 [M − H]− (calcd for C45H65O15, 845.4323). The spectroscopic data for 5 revealed that the aglycone is also an octahydroxylated C21 sterol almost identical to that of 2. The observation of two anomeric proton signals [δH 4.78 (1H, dd, J = 1.5, 9.5 Hz, H-1″), 4.68 (1H, dd, J = 1.5, 9.5 Hz, H-1‴)] and a methoxy group [δH 3.48 (3H, s)], as well as two methyl proton signals [δH 1.10 (1H, d, J = 6.5 Hz, H-6″), 1.09 (1H, d, J = 6.5 Hz, H-6‴)], revealed the occurrence of two β-linked D



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Table 3. 1H NMR Spectroscopic Data (500 MHz, δ in ppm, J in Hz) for 5−7 in DMSO-d6 position

5

6

7

1a 1b 2a 2b 3 4a 4b 6 7a 7b 9 11a 11b 12 15a 15b 16a 16b 18 19 20 21 2′, 6′ 3′, 5′ 4′ 7′ 8′ 9′ 10′ 1″ 2a″ 2b″ 3″ 4″ 5″ 6″ 1‴ 2a‴ 2b‴ 3‴ 4‴ 5‴ 6‴ OCH3-3‴

1.80, m 1.55, m 1.64, m 1.40, m 3.99, m 1.83, m 1.55, m 3.46, m 1.93, m 1.70, m 1.71, m 1.40, m 1.22, m 3.21, m 1.75, m 1.63, m 1.63, m 1.14, m 1.18, s 1.11, s 5.08, q (6.0) 1.13, d (6.0) 7.55, d (7.5) 7.37, dd (7.5, 7.5) 7.32, dd (7.5, 7.5) 6.99, d (16.0) 7.12, dd (16.0, 16.0) 7.30, dd (16.0, 16.0) 5.98, d (16.0) 4.78, dd (1.5, 9.5) 1.67, m 1.50, m 3.67, m 3.10, m 4.01, m 1.10, d (6.5) 4.68, dd (1.5, 9.5) 2.04, m, 1.48, m 3.48, m 3.06, m 3.59, m 1.09, d (6.5) 3.48, s

1.68, m 1.47, m 1.56, m 1.55, m 4.06, m 1.86, m 1.62, m 3.69, m 1.79, m 1.55, m 1.54, m 1.24, m 1.24, m 3.24, m 1.80, m 1.68, m 1.80, m 1.18, m 1.19, s 0.91, s 5.05, q (6.0) 1.14, d (6.0) 7.56, d (7.5) 7.37, dd (7.5, 7.5) 7.32, dd (7.5, 7.5) 6.99, d (16.0,) 7.12, dd (16.0, 16.0) 7.39, dd (16.0, 16.0) 5.98, d (16.0) 4.81, dd (1.5, 9.5) 1.78, m 1.56, m 3.69, m 3.15, m 4.04, m 1.09, d (6.5) 4.69, dd (1.5, 9.5) 2.06, m 1.49, m 3.49, m 3.05, m 3.60, m 1.10, d (6.5) 3.33, s

2.28, m 1.89, m 2.26, m 2.02, m 4.89, m 2.70, m, 2.29, m 4.19, br s 2.72, m, 2.47, m 2.55, m 2.46, m 2.15, m 5.24, dd (4.0, 11.0) 2.16, m 2.03, m 1.89, m 1.46, m 2.12, s 1.68, s 4.03, m 1.34, d (6.0) 7.47, d (7.5) 7.33, dd (7.5, 7.5) 7.28, dd (7.5, 7.5) 6.94, d (16.5) 6.94, dd (16.0, 16.0) 7.93, dd (16.0, 16.0) 6.42, d (16.5) 5.41, d (9.5) 2.04, m 2.34, m 4.60, m 3.46, m 4.07, m 1.33, d (6.5) 5.10, d (9.5) 1.67, m, 2.33, m 3.70, m 3.47, m 4.03, m 1.43, d (6.5) 3.43, s

2,6-deoxygenated sugar units. The 1H and 13C NMR chemical shifts of each sugar unit (Tables 2 and 3) were unambiguously assigned through analysis of the HSQC, HMBC, TOCSY, and ROESY spectra. As a result, the sugar moiety was established as a β-cymaropyranosyl-(1 → 4)-β-digitoxopyranosyl unit.31,33 The D-configurations of these two sugar moieties were confirmed by the same method with that of 3. In addition, the key HMBC correlation of H-1″ to C-3 (δC 73.5) suggested that the sugar moiety occurred at the C-3 position. On the basis of the above analysis, the structure of compound 5 (lyciumsterol E) was characterized as pregnane-3β,5α,6β,8β,12β,14β,17β,20(S)octol-20-O-[(2E,4E)-5-phenyl-2,4-pentadienoate]-3-O-β-Dcymaropyranosyl-(1 → 4)-O-β-D-digitoxopyranoside. Compound 6 was isolated as a white amorphous powder with a molecular formula of C45H66O15 (m/z 845.4298 [M − H]− for

C45H65O15) as determined by the HRESIMS. A comparison of the NMR spectra of 6 and 5 revealed that these two compounds have the same planar structure, with the difference between them being the configuration of C-6. In the 13C NMR spectrum, the characteristic chemical shifts of C-5 (δC 76.4), C-6 (δC 67.7) and C-7 (δC 38.0) indicated the presence of a OH-6α group in 6.32 This was supported by ROESY correlations observed from H-6 (δH 3.69) to CH3-19 (δH 0.91) and OH-8 (δH 3.34). Hence, compound 6 (lyciumsterol F) was deduced as pregnane3β,5α,6α,8β,12β,14β,17β,20(S)-octol-20-O-[(2E,4E)-5-phenyl2,4-pentadienoate]-3-O-β-D-cymaropyranosyl-(1 → 4)-O-β-Ddigitoxopyranoside. The spectroscopic data obtained for compound 7 (lyciumsterol G) were in agreement with those of 5, except for the position of the (2E,4E)-5-phenyl-pentadienoate group. In the E



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analysis, these four sugar units were elucidated as D-digitoxopyranose, L-cymaropyranose, D-cymaropyranose, and D-oleandropyranose. The sequence of this tetrasaccharide moiety was determined to be β-D-oleandropyranosyl-(1 → 4)-O-β-Dcymaropyranosyl-(1 → 4)-O-α-L-cymaropyranosyl-(1 → 4)-Oβ-D-digitoxopyranosyl based on correlations from H-1‴″ to C-4‴′, H-1‴′ to C-4‴, and H-1‴ to C-4″ in the HMBC spectrum. In addition, the HMBC correlation from H-1″ to C-3 (74.2) suggested that the tetrasaccharide moiety is attached at the C-3 position. On the basis of the above information, the structure of 10 was assigned as shown, and this compound was given the trivial name lyciumsterol J. Compound 11 gave the same molecular formula (C59H90O21) as that of 10. Similar to 1 and 2, the difference between 10 and 11 was found to be the location of the (2E,4E)-5-phenylpentadienoate group. In the HMBC spectrum of 11, the correlation from H-12 (δH 5.24) to C-11′ (δC 167.0) confirmed the location of the (2E,4E)-5-phenyl-2,4-pentadienoate moiety as C-12. From the above analysis, 11 (lyciumsterol K) was fully characterized as shown. The structures of 15 known compounds isolated from the root bark of L. chinense were identified by comparing their spectroscopic data to those found in the literature. They were determined to be 1,2-dihydro-6,8-dimethoxy-7-hydroxy-l-(3,4dihydroxyphenyl)-N1,N2-bis-[2-(4-hydroxyphenyl)ethyl]-2,3naphthalene dicarboxamide,35 1,2-dihydro-6,8-dimethoxy-7-hydroxy-l-(3,5-dimethoxy-4-hydroxylphenyl)-N 1,N 2-bis-[2-(4hydroxyphenyl)ethyl]-2,3-naphthalene dicarboxamide,35 melongenamide,36 (E)-2-(4,5-dihydroxy-2-{3-[(4-hydroxyphenethyl)amino]-3-oxopropyl}phenyl)-3-(4-hydroxy-3,5-dimethoxyphenyl)-N-(4-hydroxylphenethyl)acrylamide,3 N-cis-ferulolytyramine dimer,37 N-trans-ferulolytyramine dimer,37 trans-Ncaffeoyltyramine,5 trans-N-isoferuloyltyramine,14 trans-N-p-hydroxy cinnamoyltyramine,14 dihydro-N-caffeoyltyramine,5 N-cisferuloyloctopamine,37 wogonin,38 apigenin,39 scopoletin,40 and xanthyletin.41 The protective activities of 1−26 were tested using an injured pancreatic islet cell model. As shown in Figure 4, compounds 2, 3, and 7 ameliorated cell proliferation in a dose-dependent manner. The relative OD values of compound 2 at 1, 5, and 10 μM were enhanced by −3.1%, 4.2%, and 8.8%, respectively, when compared to that of the DMSO control. In turn, the relative OD values for compound 3 at 1, 5, and 10 μM increased by 1.5%, 4.0%, and 11.2%, respectively. Notably, for compound 7 at 1, 5, and 10 μM, the relative OD values increased by 0.2%, 81.7%, and 87.7%, respectively, which was nearly 2-fold more than the DMSO control level at a final concentration of 5 or 10 μM. For the positive control liraglutide, the relative OD values increased by 19.1% at 1 μM. Related studies have shown that the activation of autophagy can increase the viability of pancreatic islet cells, improve insulin resistance, and maintain pancreatic islet cell function.24−30 Thus, all of the isolates were screened for autophagy modulators using an autophagy modulator screening system. As shown in Figure 5, after treatment with compounds 7, 8, 9, and 11 at a concentration of 25 μM, the cells exhibited significant increases in GFP-LC3-II fluorescence, besides, the addition of bafilomycin A1 further increased GFP-LC3-II accumulation, which indicated the activation of autophagic flux. Compounds 7, 8, 9, 11, and the positive control curcumin increased autophagic activity by approximately 2.1-, 2.5-, 3.1-, 1.3-, and 1.7-fold, when compared to the blank control, respectively.

C NMR spectrum, the resonance for C-12 (δC 75.1) of 7 was shifted significantly downfield when compared to that of 5 (C-12 at δC 69.6). This implied that the (2E,4E)-5-phenyl-pentadienoate group is located at C-12 in 7 instead of C-20 in 5, which was supported by an observed correlation from H-12 (δH 5.24) to C-11′ (δC 167.0) in the HMBC spectrum of 7. Based on these data, compound 7 was designated as pregnane-3β,5α,6β,8β, 12β,14β,17β,20(S)-octol-12-O-[(2E,4E)-5-phenyl-2,4-pentadienoate]-3-O-β-D-cymaropyranosyl-(1 → 4)-O-β-D-digitoxopyranoside. Compound 8 (lyciumsterol H) was obtained as a white amorphous powder, and the molecular formula C52H78O18 was defined by HRESIMS. Comparison of the NMR spectra with those of 7 revealed these compounds to differ by the presence of an additional oleandropyranose unit in 8. The 1H NMR spectrum revealed three anomeric proton signals [δH 5.41 (1H, d, J = 9.5 Hz), δH 5.15 (1H, d, J = 9.5 Hz), and δH 4.73 (1H, d, J = 9.5 Hz)], which confirmed the presence of three β-linked 2,6-deoxygenated sugar units. Acid hydrolysis of 8 resulted in three sugar moieties. On the basis of their characteristic rotation values ([α]20 D + 41, + 33, − 9, respectively), the β-digitoxopyranose, β-cymaropyranose, and β-oleandropyranose moieties were deduced to be in D-configurations.31 The sequence of these sugars was determined by the key HMBC correlations from H-1‴ (δH 5.15) to C-4″ (δC 83.4) and from H-1‴′ (δH 4.73) to C-4‴ (δC 83.2). Moreover, the HMBC correlation from H-3 (δH 4.89) to C-1″ (δC 96.0) confirmed that the sugar moiety was at the C-3 position. Consequently, the structure of 8 was determined as pregnane-3β,5α,6β,8β,12β,14β,17β,20(S)-octol20-O-[(2E,4E)-5-phenyl-2,4-pentadienoate]-3-O-β-D-oleandropyranosyl-(1 → 4)-O-β-D-cymaropyranosyl-(1 → 4)-O-β-Ddigitoxopyranoside. Differences in the spectroscopic data between the aglycones of 9 and 8 (Tables 2 and 4) were found to be similar to those between the aglycones of 4 and 3. This suggested that the OH-6 group is in an α-configuration in compound 9 and in a β-configuration in compound 8. These conclusions were supported by the ROESY correlations between H-6 (δH 4.76) and H-19 (δH 1.58) in 9. Consequently, compound 9 (lyciumsterol I) was characterized as pregnane-3β,5α,6α,8β,12β,14β,17β,20(S)octol-20-O-[(2E,4E)-5-phenyl-2,4-pentadienoate]-3-O-β-D-oleandropyranosyl-(1 → 4)-O-β-D-cymaropyranosyl-(1 → 4)-O-βD-digitoxopyranoside. The molecular formula of 10 was established as C59H90O21 from the sodiated molecular ion peak observed at m/z 1157.5853 [M + Na]+ in the HRESIMS. Analysis of the 1D NMR data for 10 revealed the same aglycone as present in compound 1. Additionally, four anomeric protons at δH 5.32 (d, J = 9.5 Hz), 5.09 (br s), 5.13 (d, J = 10.5 Hz), and 4.76 (d, J = 9.5 Hz), three methoxy groups at δH 3.51 (s), 3.44 (s), and 3.38 (s), and four methyl groups at δH 1.55 (d, J = 6.0 Hz), 1.41 (d, J = 6.5 Hz), 1.28 (d, J = 6.0 Hz), and 1.24 (d, J = 6.5 Hz) were observed in the 1 H NMR spectrum of 10. These data indicated that there are four 2,6-deoxygenated sugar units with one α-linkage and three β-linkages in this compound. Further analysis of the HSQC, HMBC, and TOCSY data allowed for the assignment of the chemical shifts for the tetrasaccharide moiety (Tables 2 and 4). Acid hydrolysis of 10 yielded digitoxopyranose, cymaropyranose, and oleandropyranose. The D-configurations of the digitoxopyranose and the oleandropyranose were determind by their characteristic rotation values ([α]20 D + 42 and −10, respectively).31,34 The cymaropyranose was obtained as a 1:1 racemate on the basis of the rotation values ([α]20 D 0). From the above F



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Table 4. 1H NMR Spectroscopic Data (500 MHz, δ in ppm, J in Hz) for 8−11 in Pyridine-d5 position

8

9

10

11

1a 1b 2a 2b 3 4a 4b 6 7a 7b 9 11a 11b 12 15a 15b 16a 16b 18 19 20 21 2′, 6′ 3′, 5′ 4′ 7′ 8′ 9′ 10′ 1″ 2a″ 2b″ 3″ 4″ 5″ 6″ 1‴ 2a‴ 2b‴ 3‴ 4‴ 5‴ 6‴ 1‴′ 2a‴′ 2b‴′ 3‴′ 4‴′ 5‴′ 6‴′ 1‴″ 2a‴″ 2b‴″ 3‴″ 4‴″ 5‴″ 6‴″ OCH3-3‴ OCH3-3‴′ OCH3-3‴″

1.81, m 1.49, m 2.25, m 2.02, m 4.89, m 2.69, m 2.28, m 4.18, m 2.72, m 2.48, m 2.56, m 2.46, m 2.15, m 5.24, dd (4.0, 11.0) 2.15, m 2.03, m 1.88, m 2.28, m 2.13, s 1.68, s 4.18, m 1.33, d (6.0) 7.48, d (7.5) 7.32, dd (7.5, 7.5) 7.29, dd (7.5, 7.5) 6.94, d (16.5) 6.93, dd (16.5, 16.5) 7.92, dd (16.5, 16.5) 6.42, d (16.5) 5.41, d (9.5) 2.36, m 2.05, m 4.59, m 3.47, m 4.06, m 1.32, d (6.5) 5.15, d (9.5) 2.29, m 1.75, m 4.03, m 3.45, m 4.19, m 1.32, d (6.5) 4.73, d (9.5) 2.52, m 1.69, m 3.47, m 3.47, m 3.56, m 1.55, d (6.5)

2.17, m 1.61, m 2.09, m 1.89, m 4.34, m 2.68, m 2.09, m 4.76, m 2.73, m 1.93, m 2.28, m 2.50, m 2.16, m 5.29, dd (4.0, 11.0) 1.97, m 1.97, m 2.37, m 2.20, m 2.15, s 1.58, s 4.09, q (6.0) 1.33, d (6.0) 7.46, d (7.5) 7.33, dd (7.5, 7.5) 7.28, dd (7.5, 7.5) 6.90, d (16.5) 6.91, dd (16.5, 16.5) 7.86, dd (16.5, 16.5) 6.36, d (16.5) 5.38, d (9.5) 2.33, m 1.95, m 4.59, m 3.47, m 4.22, m 1.40, d (6.5) 5.15, d (9.5) 2.30, m 1.71, m 4.03, m 3.43, m 4.18, m 1.33, d (6.5) 4.73, d (9.5) 2.53, m 1.70, m 3.58, m 3.47, m 3.57, m 1.55, d (6.5)

3.56, s 3.44, s

3.55, s 3.44, s

1.64, m 2.28, m 2.00, m 2.54, m 4.87, m 2.72, m 2.27, m 4.07, m 2.74, m 2.49, m 2.44, m 2.40, m 1.90, m 3.81, dd (3.5, 10.5) 2.09, m 2.17, m 1.94, m 2.02, m 2.06, s 1.71, s 5.83, q (6.0) 1.63, d (6.0) 7.42, d (7.5) 7.35, dd (7.5, 7.5) 7.25, dd (7.5, 7.5) 6.71, d (16.0) 6.90, dd (16.0, 16.0) 7.62, dd (16.0, 16.0) 6.24, d (16.0) 5.32, d (9.5) 2.37, m 2.03, m 4.43, m 3.44, m 3.94, m 1.28, d (6.0) 5.09, br s 2.23, m 2.03, m 4.01, m 3.69, m 4.51, m 1.24, d (6.5) 5.13, d (10.5) 2.36, m 1.87, m 4.08, m 3.51, m 4.17, m 1.41, d (6.5) 4.76, d (9.5) 1.76, m 2.56, m 3.45, m 3.46, m 3.57, m 1.55, d (6.0) 3.38, s 3.51, s 3.44, s

1.50, m 2.26, m 1.25, m 2.02, m 4.84, m 2.27, m 2.69, m 4.18, m 2.72, m 2.48, m 2.55, m 2.45, m 2.14, m 5.24, m 2.04, m 2.16, m 1.89, m 1.82, m 2.13, s 1.68, s 4.02, m 1.32, d (6.0) 7.48, d (7.5) 7.33, dd (7.5, 7.5) 7.27, dd (7.5, 7.5) 6.94, d (16.0) 6.94, dd (16.0, 16.0) 7.92, dd (16.0, 16.0) 6.42, d (16.0) 5.33, d (9.5) 2.37, m 2.02, m 4.44, m 3.43, m 3.92, m 1.26, d (6.0) 5.10, br s 2.24, m 2.09, m 4.02, m 3.69, m 4.52, m 1.24, d (6.5) 5.12, d (10.5) 2.38, m 1.88, m 4.07, m 3.52, m 4.16, m 1.40, d (6.5) 4.75, d (9.5) 1.72, m 2.54, m 3.46, m 3.45, m 3.57, m 1.54, d (6.0) 3.38, s 3.55, s 3.44, s

G



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Figure 4. Protective activities of compounds 2, 3, and 7 on injured pancreatic islet cells.

Figure 5. Autophagy activation of compounds 7, 8, 9, and 11.

Flash chromatography was conducted using a Combiflash RF200 apparatus (Teledyne Isco Corp., Lincoln, NE, U.S.A.). Preparative HPLC was performed using a Shimadzu LC-6AD instrument with an SPD-10A detector (Shimadzu Corp., Tokyo, Japan), using a YMC-Pack ODS-A column (250 mm × 20 mm, 5 μm; YMC Corp., Kyoto, Japan). Preparative HPLC-ELSD was performed using an Agilent 1260 series system with a TSKgel G3000PWXL column (300 mm × 7.8 mm, 5 μm; TOSOH Corp., Tokyo, Japan). HPLC-DAD analysis was performed using an Agilent 1260 series system with an Apollo C18 column (250 mm × 4.6 mm, 5 μm). Column chromatography was performed on silica gel (100−200, 200−300 mesh, Qingdao Marine Chemical Inc., Qingdao, People’s Republic of China), RP-C18 (50 μm, YMC Corp.), and Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala, Sweden). Plant Material. The root bark of Lycium chinense (three years old) was collected from Ningan Town, Zhongning County, Ningxia Hui autonomous region, People’s Republic of China, in March 2012. The plant material was identified by L. Ma (Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing). A voucher specimen (ID number: ID-S-2592) was deposited in the Herbarium of the Institute of Materia Medica, Chinese Academy of Medical Science, Beijing.

Analysis of preliminary structure−activity relationships has revealed that there are two features of the C21 steroids obtained that appear to contribute to their biological activities. First, the (2E,4E)-5-phenyl-pentadienoate group needs to occur at C-12. Second, two or more sugar moieties on the C21 steroids seem to contribute to the activation of autophagy, such as in compounds 7, 8, 9, and 11. Two or fewer sugar moieties on the C21 steroids contribute to the protective activity of injured pancreatic islet cells, as observed for compounds 2, 3, and 7.

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EXPERIMENTAL SECTION General Experimental Procedures. The optical rotations were measured on a JASCO P-2000 polarimeter (JASCO, Easton, MD, U.S.A.). UV spectra were detected on a JASCO V650 spectrometer. ECD spectra were recorded with a JASCO J-815 spectrometer. Infrared (IR) spectra were recorded on a Nicolet 5700 spectrometer (Thermo Scientific, Waltham, MA, U.S.A.) using an FT-IR microscope transmission method. 1 H NMR (500 MHz), 13C NMR (125 MHz), and 2D NMR spectra were recorded with Bruker 500 MHz NMR spectrometer (Bruker-Biospin, Billerica, MA, U.S.A.) using TMS as internal standard and the values are given in ppm. High-resolution electrospray ionization mass spectrometry (HRESIMS) was performed on an Agilent 1100 series LC/MSD ion trap mass spectrometer (Agilent Technologies, Waldbronn, Germany). H



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Extraction and Isolation. The root bark of L. chinense (100 kg) was extracted three times with 80% EtOH (600 L) under reflux. After the solvent was evaporated, the residue (8.0 kg) was suspended in water (45 L) and then extracted with EtOAc (45 L) three times. The dried EtOAc fraction (1.5 kg) was subjected to column chromatography on silica gel and continuously eluted with petroleum ether (fraction A, 600 g), EtOAc (fraction B, 220 g), acetone (fraction C, 40 g), and methanol (fraction D, 80 g), successively. Fraction B was further separated on silica gel chromatography column and eluted with petroleum ether-EtOAc (from 100:0 to 0:100) in a gradient to afford 10 fractions (fractions B1−B10). Fraction B3 (20 g) was subjected to passage over an ODS flash chromatography column that was eluted with MeOH-H2O (from 5:95 to 100:0) to yield 26 fractions (fractions B3-1−B3-26). Fraction B3-5 was purified with preparative RP-HPLC using MeOH-H2O (45:55) as mobile phase to yield compounds 13 (21 mg) and 14 (17 mg). Fraction B3-8 was purified by preparative RP-HPLC using MeOH-H2O (48:52) as mobile phase to yield compounds 16 (12 mg) and 17 (16 mg). Fraction B4 (5 g) was subjected to Sephadex LH-20 column and eluted with MeOH-H2O (from 60:40 to 100:0) to afford five fractions (fractions B4-1−B4-5). Fraction B4-3 was further purified using preparative RP-HPLC with MeOH-H2O (60:40) as the mobile phase to yield 26 (6 mg). Fraction B4-4 was further purified with preparative RP-HPLC with MeOHH2O (80:20) as mobile phase to obtain compound 23 (11 mg). Fraction B5 (9 g) was separated using Sephadex LH-20 eluted with CHCl3-MeOH (from 50:50 to 0:100) to yield six fractions (fractions B5-1−B5-6). Fraction B5-2 were further purified by preparative RP HPLC using MeOH-H2O (45:55) to yield compounds 12 (33 mg), 18 (40 mg) and 19 (20 mg). Fraction B6 (3.0 g) was subjected to passage over Sephadex LH-20 by elution with 45% MeOH to afford compounds 20 (16 mg) and 21 (54 mg). Fraction B7 (25 g) was subjected to flash chromatography with a RP-C18 column (55 cm × 8 cm, 20 μm; YMC Corp.) and eluted with MeOH-H2O (from 5:95 to 100:0) to afford 10 fractions (fractions B7-1−B7-10). Compound 22 (5 mg) was obtained from fraction B7-3 by recrystallization. Fraction B7-8 was further purified by preparative RP-HPLC using MeOH-H 2 O (60:40) as mobile phase to obtain compounds 24 (10 mg), 25 (50 mg), and 26 (13 mg). Fraction B8 (4.4 g) was subjected to flash chromatography with a RP-C18 column (50 cm × 4.6 cm, 20 μm; YMC Corp.) that was eluted with MeOH-H2O (from 5:95 to 100:0) to yield 15 fractions (fractions B8-1−B8-15). Fraction B8-4 was purified using preparative RP HPLC with MeOH-H2O (45:55) as the mobile phase to yield 15 (41 mg). Fraction B8-11 was purified using preparative RP HPLC with MeOH-H2O (70:30) as the mobile phase to yield 2 (15 mg). Fraction B9 (28.2 g) was chromatographed over a RP-C18 column (55 cm × 8 cm, 20 μm; YMC Corp.) and eluted with MeOH-H2O (from 5:95 to 100:0) to yield 26 fractions (fractions B9-1−B9-26). Fraction B9-14 was purified by preparative RP HPLC with MeOH-H2O (60:40) as mobile phase to obtained compound 1 (8 mg). Fraction B10 (14.4 g) was further separated using a RP-C18 column eluted with MeOH-H2O (from 10:100 to 100:0) in a gradient manner, to yield fractions B10-1−B10-25. Fraction B10-8 was purified by preparative RP-HPLC with MeOH-H2O (65:35) to obtain compound 3 (15 mg). Fraction B10-10 was purified by preparative RP-HPLC with MeOH-H2O (70:30) to furnish compound 4 (19 mg). Fraction B10-12 was purified by preparative RP-HPLC with MeOH-H2O (70:30) to yield 5 (7 mg) and 6 (8 mg). Fraction B10-14 was purified by

preparative RP-HPLC with MeOH-H2O (75:25) to obtain compound 7 (13 mg). Fraction B10-15 was purified by RP-C18 preparative HPLC with MeOH-H2O (80:20) to produce 8 (33 mg) and 9 (22 mg). Fraction B10-18 was purified by RP-C18 preparative HPLC with MeOH-H 2O (85:15) to afford compounds 10 (12 mg) and 11 (19 mg). Lyciumsterol A (1). White amorphous powder; [α]20 D + 58 (c 0.05 MeOH); UV (MeOH) λmax (log ε) 308 (4.06) nm; ECD (MeOH) λ max(Δε) 300 (+2.76), 256 (−0.26), 218 (+1.09) nm; IR (KBr) νmax 3418, 2943, 1692, 1624, 1449, 1382, 1246, 1055, 1002, 978, 694 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 573.3057 [M + H]+ (calcd for C32H45O9, 573.3064). Lyciumsterol B (2). White amorphous powder; [α]20 D + 9 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 312 (4.21) nm; ECD (MeOH) λmax (Δε) 308 (+1.62), 216 (−0.76) nm; IR (KBr) νmax 3429, 2937, 1694, 1627, 1241, 1131, 1051, 1000, 877, 691 cm−1; 1 H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 595.2876 [M + Na]+ (calcd for C32H44O9Na, 595.2883). Lyciumsterol C (3). White amorphous powder; [α]20 D + 24 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 313 (4.40) nm; ECD (MeOH) λmax (Δε) 311 (+3.29), 218 (−0.91) nm; IR (KBr) νmax 3441, 2938, 1700, 1623, 1594, 1453, 1421, 1238, 1077, 702 cm−1; 1 H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 701.3518 [M − H]− (calcd for C38H53O12, 701.3543). Lyciumsterol D (4). White amorphous powder; [α]20 D + 19 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 313 (4.30) nm; ECD (MeOH) λmax (Δε) 304 (+2.04), 220 (−0.64) nm; IR (KBr) νmax 3426, 2933, 1701, 1623, 1369, 1241, 1133, 1073, 997, 875 cm−1; 1 H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 725.3505 [M + Na]+ (C38H54O12Na, calcd for 725.3513). Lyciumsterol E (5). White amorphous powder; [α]20 D + 41 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 308 (4.36) nm; ECD (MeOH) λmax (Δε) 307 (+4.08), 240 (−0.47), 219 (+ 0.99) nm; IR (KBr) νmax 3432, 2969, 2935, 1697, 1626, 1450, 1362, 1087, 1002, 869 cm−1, 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 845.4306 [M − H]− (calcd for C45H65O15, 845.4323). Lyciumsterol F (6). White amorphous powder; [α]20 D + 54 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 308 (4.30) nm; ECD (MeOH) λmax (Δε) 309 (+4.74), 238 (−0.90) nm; IR (KBr) νmax 3433, 2970, 2937, 1699, 1626, 1451, 1313, 1084, 1002, 871 cm−1; 1 H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 845.4298 [M − H]− (calcd for C45H65O15, 845.4323). Lyciumsterol G (7). White amorphous powder; [α]20 D + 44 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 313 (4.43) nm; ECD (MeOH) λmax (Δε) 309 (+3.30), 209 (−0.79) nm; IR (KBr) νmax 3441, 2935, 1702, 1624, 1450, 1371, 1166, 1086, 1001, 977, 871 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 869.4295 [M + Na]+ (calcd for C45H66O15Na, 869.4299). Lyciumsterol H (8). White amorphous powder; [α]20 D + 26 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 313 (4.45) nm; ECD (MeOH) λmax (Δε) 309 (+3.12), 210 (−1.14) nm; IR (KBr) νmax 3441, 2934, 1702, 1624, 1450, 1370, 1165, 1086, 999, 874 cm−1; 1 H and 13C NMR data, see Tables 2 and 4; HRESIMS m/z 989.5091 [M − H]− (calcd for C52H77O18, 989.5110). Lyciumsterol I (9). White amorphous powder; [α]20 D + 8 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 310 (4.17) nm; ECD (MeOH) λmax (Δε) 306 (+1.88) nm; IR (KBr) νmax 3441, 2934, 1702, 1624, 1450, 1370, 1165, 1086, 999, 874 cm−1; 1H and 13 C NMR data, see Tables 2 and 4; HRESIMS m/z 1013.5100 [M + Na]+ (calcd for C52H78O18Na, 1013.5086). I



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Lyciumsterol J (10). White amorphous powder; [α]20 D − 7 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 307 (4.33) nm; ECD (MeOH) λmax (Δε) 302 (+3.17), 250 (−0.30), 222 (+1.65) nm; IR (KBr) νmax 3456, 2935, 1702, 1625, 1450, 1366, 1087, 1002, 869, 696 cm−1; 1H and 13C NMR data, see Tables 2 and 4; HRESIMS m/z 1157.5853 [M + Na]+ (calcd for C59H90O21Na, 1157.5872). Lyciumsterol K (11). White amorphous powder; [α]20 D − 9 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 313 (4.41) nm; ECD (MeOH) λmax (Δε) 308 (+3.57), 221 (−0.96) nm; IR (KBr) νmax 3451, 2934, 1703, 1624, 1450, 1367, 1165, 1087, 1002, 907, 874 cm−1; 1H and 13C NMR data, see Tables 2 and 4; HRESIMS m/z 1157.5850 [M + Na]+ (calcd for C59H90O21Na, 1157.5872). Acidic Hydrolysis of Compounds 3−11. Compound 10 (6 mg) was dissolved in dioxane (2 mL). Then, 2 N HCl (0.05 mL) was added to the solution and refluxed for 2 h. The mixture was concentrated under vacuum, and the residue was subjected to preparative HPLC-ELSD with a TSKgel G3000PWXL column (300 mm × 7.8 mm, 5 μm) and eluted with H2O at a flow of 0.2 mL/min to yield digitoxopyranose, oleandropyranose, and cymaropyranose, which showed the retention time of 52.2, 54.1, and 55.5 min, respectively. Hydrolysis of 3−9, 11 was performed according to the procedure described for 10. These monosaccharides were determined by comparing the retention time of authentic samples, and their absolute configurations were established by comparing the experimental and reported rotation values.31 Protective Effects of the Compounds on Injured Pancreatic Islet Cells. The MIN6 cell line, a pancreatic β cell line derived from the mouse, was cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 25 mM glucose, 100 IU/mL penicillin, 100 μg/mL streptomycin, 50 μM β-mercaptoethanol, and 10% fetal bovine serum (FBS) at 37 °C with a 95% air-5% CO2 mixture. Cell monolayers were plated in a poly-L-lysine-coated 96-well plate at a density of 1 × 104 cells/ well for 24 h until 80−90% confluency was reached. The cells were then stimulated with 0.4 mM palmitic acid (final concentration) without or with a test sample (compound or positive control, liraglutide) dissolved in 0.1% DMSO (final concentration) in serum-free DMEM for 24 h. Cells that were cultured in serum-free DMEM only were used as the normal control (Con). Cells that were stimulated with palmitic acid and 0.1% DMSO were used as the model control (Model). Cell proliferation was determined using CCK-8 agents, by measuring the value of absorbance (OD) at a wavelength of 450 nm.42,43 The OD values of compounds with the same concentrations in serum-free DMEM were used as blank readings. The OD value of Con was set to 1, and the relative OD value of each well was calculated using the following formula:

without any stimulation were used as blank controls. Cells that were treated with 20 μM curcumin were used as positive controls. After an 8 h incubation, the cells were harvested with trypsin, washed with PBS, followed by PBS containing 0.1% saponin and were then suspended in 1 mL PBS.44 Fluorescence intensity at 488 nm was measured using flow cytometry. More than 15 000 events were captured for each sample, and data analysis was carried out using a FACS Express 5. The compounds that led to an increase in the fluorescence intensity of LC3-II were subjected to a second round of screening. The lysosomotropic inhibitor bafilomycin A1 (0.1 μM) was added, and fluorescence intensities with or without the addition of bafilomycin A1 were compared.45

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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.5b01087. UV, IR, NMR, MS spectra for compounds 1−11 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86-10-63165231. Fax: +86-10-63017757. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The research described in this publication was supported by the National Natural Science Foundation of China (No. 81303207) and the Beijing Natural Science Foundation (No. 7144227). The authors thank Professor L. Ma (Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College) for the plant identification and Ms. Y.-H. Wang (Analytical and Testing Center, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences) for conducting the NMR experiments.

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REFERENCES

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relative proliferation (%) = (OD(sample) − ODmodel )/ODmodel × 100

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