Lanostane Triterpenoids with Glucose-Uptake-Stimulatory Activity from

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Cite This: J. Agric. Food Chem. 2019, 67, 7348−7364

Lanostane Triterpenoids with Glucose-Uptake-Stimulatory Activity from Peels of the Cultivated Edible Mushroom Wolf iporia cocos Baosong Chen,†,§,¶ Jinjin Zhang,†,§,¶ Junjie Han,† Ruilin Zhao,† Li Bao,*,† Ying Huang,‡ and Hongwei Liu*,†,§

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†

State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichenxi Road, Chaoyang District, Beijing 100101, P. R. China ‡ State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichenxi Road, Chaoyang District, Beijing 100101, P. R. China § Savaid Medicine School, University of Chinese Academy of Sciences, Beijing 100049, P. R. China S Supporting Information *

ABSTRACT: A chemical study on the peels of the cultivated edible mushroom Wolf iporia cocos led to the isolation and identification of 47 lanostane triterpenoids including 16 new compounds (1−16). The structures of the new compounds were determined by analysis of the NMR, MS, and electronic circular dichroism (ECD) data. Compounds 1 and 2 represent new members of the family of 4,5-secolanostane triterpenes. Compound 3 is a new aromatic lanostane triterpene with an unusual methyl rearrangement from C-10 to C-6. The absolute configurations of 1 and 8 were assigned by ECD spectra calculation. All compounds were evaluated for cytotoxicity (K562, SW480, and HepG2) and glucose-uptake-stimulating effects. Compounds 23, 25, 29, and 31 showed weak inhibition on the K562 cells with IC50 in the range of 25.7 to 68.2 μM, respectively. Compounds 21, 28, and 30 increased the glucose uptake in 3T3-L1 cells by 25%, 14%, and 50% at 5 μM, respectively. In addition, compounds 14, 23, 29, 35, and 43 showed insulin-sensitizing activity by increasing the insulin-stimulated glucose uptake at 2.5 μM in 3T3-L1 adipocytes. A preliminary structure−activity relationship analysis indicates that the 6/6/6/5 ring skeleton and the double bond between C-8 and C-9 are beneficial for the glucose-uptake-stimulating and insulin-sensitizing activities. Furthermore, the alkaline-insoluble fraction mainly containing compounds 22, 24, 28, and 31 were confirmed to have hypoglycemic and hypolipidemic activity on high-fat-diet-induced obese mice. This work confirms the potential of the peels’ extracts of W. cocos as a functional food or dietary supplements. KEYWORDS: Wolf iporia cocos, lanostane triterpene, cytotoxicity, glucose-uptake stimulation, insulin-sensitizing activity, structure−activity relationship

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INTRODUCTION Edible and medicinal mushrooms have attracted much attention from chemists and biologists because of their production of diverse bioactive components and their potential as nutritional supplements or medicinal agents.1−5 Wolf iporia cocos is a subterranean edible and medical fungus that grows on the roots of pine trees.6 The decorticated sclerotia of W. cocos, commercially called Fuling, is widely used as a functional food and traditional Chinese medicine in Asian countries because of its strong diuretic, sedative, and tonic effects.7−9 Polysaccharides and triterpenoids have been demonstrated to be the major bioactive constituents of W. cocos.10 Triterpenoids with various biological activities, such as poricoic acid C with antiinflammatory property,11 trametenolic acid with protective effect on PC12 cells against oxygen-glucose deprivation (OGD),12 dehydroeburicoic acid with antiproliferative activity against HL60,10 and pachymic acid promoting adipocytes differentiation of ST-13,13 were isolated from this species. Over 30 000 tons of Fuling are being produced each year by the large-scale cultivation of W. cocos in China. In each year, a large quantity of the peels of W. cocos is discarded in China, only with a small quantity being used as traditional Chinese medicine (named as Fuling-Pi). It has been reported that the © 2019 American Chemical Society

content of triterpenoids in the Fuling-Pi is about 10 times higher than that in Fu-Ling.14 Diabetes is a chronic, metabolic disease characterized by elevated levels of blood glucose, which is associated with serious complication such as cardiovascular and kidney diseases. Insulin resistance that leads to the decrease of glucose uptake into muscle or adipose cell is an important inducer of diabetes. In one recent report, pachymic acid from W. cocos was demonstrated to stimulate glucose uptake in 3T3L1 adipocytes cells through enhancing GLUT4 expression and translocation.15 To promote the applications of Fuling-Pi and obtain new natural products with glucose-uptake-stimulating activity from W. cocos, we conducted a detailed investigation on the peels’ extracts of W. cocos. As a result, 47 lanostane triterpenoids including 16 new compounds were identified. All triterpenes were evaluated for their cytotoxicity and stimulatory effect on glucose uptake. Received: Revised: Accepted: Published: 7348

April 25, 2019 June 5, 2019 June 10, 2019 June 10, 2019 DOI: 10.1021/acs.jafc.9b02606 J. Agric. Food Chem. 2019, 67, 7348−7364

Article

Journal of Agricultural and Food Chemistry Table 1. 1H and

13

C NMR Data for Compounds 1−3 1

2

3

position

δCa

δHb, mult (J in Hz)

δC a


δC c

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

23.3 40.5 214.2 41.1 133.8 129.7 121.9 143.7 130.7 136.1 123.5 136.2 48.5 49.7 41.6

2.91, dd (9.3, 7.0) 2.54, o

23.2 40.3 214.0 41.0 133.6 129.5 121.8 143.5 130.6 136.0 123.4 136.1 48.4 49.5 41.4

2.91, dd (9.3, 6.9) 2.55, m

25.9 36.6 213.9 47.6 143.4 131.3 119.6 145.1 129.8 131.2 123.6 137.4 48.4 50.3 42.2

16 17 18 19 20 21 22

78.2 52.3 17.7 20.0 46.4 180.1 30.4

23

32.3

24 25 26 27 28 29 30 31

155.0 33.8 22.0 21.9 18.3 18.3 25.3 107.3

2.48, o 6.98, d (7.5) 6.74, d (7.5)

6.47, d (10.0) 6.14, d (10.0)

1.79, 2.47, 4.33, 2.51, 0.81, 2.26, 2.58,

d (13.2) o dd (8.4, 5.7) o s s o

1.96, m 2.05, m 2.07, m 2. 18, m 2.26, 1.02, 1.04, 1.05, 1.07, 1.35, 4.75, 4.79,

m d (6.7) d (6.7) d (6.3) d (6.3) s s s

2.47, m 6.99, d (7.5) 6.74, d (7.5)

6.47, d (10.0) 6.14, d (10.0)

1.80, 2.47, 4.31, 2.48, 0.81, 2.26, 2.57,

78.1 52.3 17.5 19.8 46.0 179.8 31.9

d (13.3) m dd (8.3, 5.8) m s s m

1.88, m

26.1

2.04, m 2.13, m 5.14, d (7.0)

123.2 132.7 25.7 17.7 18.2 18.2 25.2

1.69, 1.62, 1.05, 1.07, 1.35,

s s d (6.3) d (6.3) s

77.3 52.5 17.8 14.9 48.2 178.4 31.1 32.9 155.7 33.8 21.8 21.6 27.3 27.1 25.5 106.9

δHd, mult (J in Hz) 2.88, q (6.5) 2.67, td (6.9, 2.1)

7.14, s

6.68, d (9.9) 6.89, d (9.9)

2.32, 2.81, 4.78, 3.21, 1.31, 2.14, 3.05,

m dd (12.8, 8.4) t (6.9) dd (11.3, 5.8) s s td (11.3, 3.2)

2.56, 2.75, 2.42, 2.58,

m m m m

2.31, 1.01, 1.02, 1.53, 1.49, 1.81, 4.88, 5.03,

m d (6.7) d (6.7) s s s s s̀

a Recorded at 125 MHz in CDCl3; bRecorded at 500 MHz in CDCl3; cRecorded at 125 MHz in pyridine−d5; dRecorded at 500 MHz in pyridine− d5.

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detector using an ODS column (C18, 250 × 9.4 mm, YMC Pak, 5 μm) at a flow rate of 2.0 mL/min. Fungal Material. The peels of W. cocos were collected from Baoshan City, Yunnan Province, China, and identified by Prof. Ruilin Zhao from State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences. A voucher specimen (HMAS 255464) had been deposited in the Herbarium of the Institute of Microbiology, Chinese Academy of Sciences, Beijing (HMAS). Extraction and Isolation. The dried and powdered peels of W. cocos (4.2 kg) were extracted under refluxing in ethanol for 2 h (30 L× 3). After filtration, the solvent was combined and concentrated under reduced pressure to yield about 350 g of residue. The ethanol extracts were subjected to silica gel CC eluted with a gradient of n-hexane/ ethyl acetate (100:0 to 2:1, v/v), followed by CH2Cl2/MeOH (100:0 to 0:100, v/v). The collected elution solvents were combined by thinlayer chromatography (TLC) analysis into 10 fractions (Fr. 1−Fr. 10). Fr. 5 (4.8 g) eluted with n-hexane/EtOAc (10:1) was further subjected to ODS CC eluted with MeOH in water (from 20 to 100%, v/v) to give 15 subfractions (Fr. 5-1−Fr. 5-16). Compounds 4 (6.2 mg, tR = 27.3 min), 5 (3.8 mg, tR = 32.5 min), 17 (7.2 mg, tR = 37.8 min), 21 (5.5 mg, tR = 43.2 min), and 34 (2.2 mg, tR = 48.2 min)

MATERIALS AND METHODS

General Experimental Procedures. Optical rotations were recorded on an Anton Paar MCP 200 Automatic Polarimeter. CD spectra were acquired using an Applied Photophysics Chirascan spectropolarimeter. IR and UV spectra were obtained on a Nicolet IS5 FT-IR spectrophotometer and on a Thermo Genesys-10S UV−vis spectrophotometer, respectively. NMR spectral data were recorded with a Bruker Avance-500 spectrometer in C5D5N (δH 8.74, 7.58 and 7.22/δC 150.4, 135.9 and 123.9) or CDCl3, (δH 7.26/δC 77.16). HSQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. HRESIMS data were obtained on an Agilent AccurateMass-Q-TOF LC/MS 6520 instrument. OD absorbance data were tested on a Spectra Max 190 microplate reader. Solvents including methanol, dichloromethane, and ethyl acetate used for extraction and chromatographic separation were of analytical grade. TLC was carried out on Silica gel HSGF254 plates and the spots were visualized by UV at 254 nm or spraying with 10% H2SO4 followed by heating. Silica gel (150−250 μm, Qingdao Haiyang Chemical Co., Ltd.), octadecylsilyl (ODS, 50 μm, YMC CO., LTD), and Sephadex LH-20 (Amersham Biosciences) were used for column chromatography (CC). HPLC separation was performed on an Agilent 1200 HPLC system with a DAD (Diode Array Detector) 7349

DOI: 10.1021/acs.jafc.9b02606 J. Agric. Food Chem. 2019, 67, 7348−7364

Article


13


5

6

position

δCa


δC a


δC a

1

36.7

36.8

29.1

1.54, dq (11.1, 8.0) 2.04, dt (13.1, 3.5) 1.95, m

37.1

2

1.51, m 2.03, dt (12.7, 2.9) 1.96, m

3 4 5 6

78.4 39.7 50.1 23.9

3.48, dd (9.3, 6.4)

78.4 39.7 50.1 23.9

3.49, dd (9.1, 6.7)

215.4 47.9 51.3 24.2

7 8 9 10 11 12

121.6 143.1 146.9 38.2 117.1 36.1

5.60, d (6.2)

121.6 143.2 147.0 38.3 117.2 36.7

5.60, d (6.2)

13 14 15

44.7 51.0 32.0

16

27.6

17 18 19 20 21 22

46.1 17.3 23.4 42.5 179.2 35.4

23

75.6

2.12, m 2.29, m 5.49, q (7.3)

24

124.8

25 26 27 28 29 30

139.3 26.0 18.6 29.2 17.0 26.1

1.29, dd (11.2, 4.4) 2.20, m

5.44, d (6.4) 2.25, m 2.65, dd (17.7, 6.4)

1.52, 1.78, 1.44, 2.08, 2.48, 0.92, 1.10, 2.87,

m td (11.7, 6.9) fm m q (9.0) s s td (9.2, 5.2)

29.1

1.31, dd (11.4, 4.4) 2.18, m

5.50, d (6.5) 2.36, m 2.62, dd (17.9, 6.5)

44.8 50.6 32.2

1.52, 1.77, 1.52, 1.96, 2.53, 0.80, 1.12, 2.86,

26.3 46.2 18.1 23.5 42.8 179.0 35.9

m m m m q (8.9) s s dt (12.2, 7.7)

35.3

121.4 142.7 145.0 37.6 125.0 73.8 50.8 50.7 44.9 75.5

o m dt (14.6, 3.8) td (14.6, 5.7)

1.61, 2.01, 2.16, 5.61,

dd (11.9, 3.7) m m o

5.62, br s 4.91, br s

1.94, d (13.1) 2.54, dd (13.1, 8.6) 4.64, dd (8.4, 3.4) 3.25, 1.11, 1.18, 3.21,

24.1

1.97, 2.18, 1.76, 1.88,

m m o m

1.37, 1.37, 1.15, 1.06, 1.53,

s s s s s

75.7

5.36, d (8.9)

124.6

5.31, d (8.8)

45.5

1.67, 1.68, 1.24, 1.15, 1.00,

139.6 25.5 18.6 29.2 17.1 26.0

1.68, 1.70, 1.24, 1.16, 1.05,

70.0 30.5 30.3 26.0 22.7 26.7

s s s s s

1.71, 2.21, 2.33, 2.74,

58.0 12.6 22.3 48.3 181.0 34.0

1.83, m 2.34, m 5.15, td (9.5, 5.6)

s s s s s


m s s m

2.45, m

a

Recorded at 125 MHz in pyridine−d5; bRecorded at 500 MHz in pyridine−d5. Fr. 7 (23.1 g) eluted with n-hexane/EtOAc (5:1) was further separated on ODS CC with MeOH in water (from 70 to 100%, v/v) to obtain 5 subfractions (Fr. 7-1−Fr. 7-5). Compounds 16 (8.4 mg, tR = 27.5 min), 37 (5.5 mg, tR = 33.1 min), 38 (12.2 mg, tR = 36.2 min) and 42 (17.4 mg, tR = 38.5 min) were obtained by RP-HPLC using 75% MeOH in water from Fr. 7-2 (300 mg, 80% MeOH elution). Compounds 46 (8.8 mg, tR = 16.5 min), 24 (8.6 mg, tR = 23.1 min), 40 (8.9 mg, tR = 29.2 min) and 43 (11.4 mg, tR = 40.5 min) were separated by RP-HPLC using 83% MeOH in water from Fr. 7-3 (440 mg, MeOH elution). Compounds 20 (8.9 mg, tR = 37.6 min), 22 (15.2 mg, tR = 43.1 min), 28 (11.3 mg, tR = 51.8 min), 31 (6.7 mg, tR = 57.4 min), 33 (11.5 mg, tR = 63.0 min) , and 36 (6.8 mg, tR = 72.2 min) were obtained by RP-HPLC using 75% MeOH in water from Fr. 7-4 (200 mg, 90% MeOH elution). Fr. 8 (4.4 g) eluted with n-hexane/EtOAc (5:1) was further subjected to ODS CC with MeOH in water (from 20 to 70%, v/v) to give 5 subfractions (Fr. 8-1−Fr. 8-5). Fr. 8-3 (150 mg, 50% MeOH elution) was separated by RP-HPLC using acetonitrile in water to give 7 (3.4 mg, tR = 28.1 min), 13 (3.2 mg, tR = 37.3 min), and 45 (7.5 mg, tR = 44.5 min).

were purified from Fr. 5-6 (159 mg, 80% MeOH elution) by RPHPLC using 75% MeOH in water. Fr. 5-13 (550 mg) eluted by 90% MeOH-H2O was separated by RP-HPLC using 80% MeOH in water to give 19 (4.3 mg, tR = 20.1 min), 23 (8.2 mg, tR = 28.3 min), 26 (4.1 mg, tR = 33.4 min), 27 (5.2 mg, tR = 37.2 min), 30 (3.2 mg, tR = 44.3 min), and 35 (8.5 mg, tR = 50.5 min). Fr. 5-15 (350 mg, 95% MeOH elution) was separated by RP-HPLC using 88% MeOH in water to give 18 (15.6 mg, tR = 37.1 min), 25 (37.5 mg, tR = 44.6 min), 29 (8.3 mg, tR = 53.4 min), 32 (22.2 mg, tR = 68.4 min), and 39 (6.7 mg, tR = 74.9 min). Fr. 6 (11.2 g) eluted with n-hexane/EtOAc (8:1) was further separated on ODS CC with MeOH in water (from 40 to 80%, v/v) to give 8 subfractions (Fr. 6-1−Fr. 6-8). Compounds 2 (3.4 mg, tR = 38.5 min) and 3 (7.8 mg, tR = 44.2 min) were obtained by RP-HPLC using 56% acetonitrile in water from Fr. 6-5 (320 mg, 65% MeOH elution). Fr. 6-6 (140 mg, 70% MeOH elution) was further separated by RP-HPLC using 60% acetonitrile in water to give compounds 1 (8.7 mg, tR = 35.1 min), 41 (4.8 mg, tR = 43.2 min), and 47 (11.3 mg, tR = 50.0 min). 7350


Article


13


position

δCa

1

39.2

2

30.6

3 4 5 6

177.2 149.6 51.1 29.1

7 8 9 10 11 12

118.3 142.3 137.9 36.8 120.7 37.4

13 14 15

46.0 49.7 44.2

16 17 18 19 20 21 22

76.7 58.1 18.7 22.7 49.3 179.3 32.3

23

30.1

24 25 26 27 28 29

134.5 128.1 20.9 20.5 22.6 112.5

30 31

25.2 61.9

9

δHb, mult (J in Hz) 1.91, 2.14, 2.42, 2.56,

2.31, 2.05, 2.56, 5.28,

m m m m

1.82, 2.41, 4.51, 2.86, 1.08, 1.03, 2.96,

d (13.2) dd (13.2, 8.5) t (7.2) m s s td (11.0, 3.3)

2.46, 2.71, 2.75, 2.86,

m m m m

s s s d d s d d

1.88, 2.13, 2.46, 2.54,

30.6

d (7.2) dd (19.5,4.5) m br, s

177.0 149.6 51.1 29.0 118.3 142.3 137.9 39.2 120.7 37.4 46.0 49.7 44.2 76.7 58.0 18.7 22.7 49.3 179.1 32.0 30.6

(2.6) (2.6) (11.8) (11.8)

10


36.8

5.33, br, s 2.48, m 2.51, m

1.76, 1.67, 1.73, 4.84, 4.77, 1.49, 4.43, 4.47,

δC a

137.2 134.0 63.1 17.2 22.6 112.5 25.2 61.4

2.32, 2.03, 2.54, 5.25,

m m dd (13.3, 8.5) o

d (7.3) m m d (3.9)

5.32, d (5.1) 2.48, m 2.68, m

1.78, 2.38, 4.51, 2.86, 1.07, 1.01, 2.96,

d (13.1) dd (13.1, 8.5) t (7.2) m s s dt (11.3, 3.3)

2.51, 2.75, 2.88, 2.76,

m o m m

4.48, 2.08, 1.72, 4.81, 4.77, 1.48, 4.55, 4.59,

3-OCH3

s s s d d s d d

δC a 36.2 29.9 174.9 149.5 51.0 28.8 118.3 142.1 137.5 39.1 120.8 37.4 46.0 49.3 44.1 76.7 58.0 18.7 22.5 49.6 179.1 32.1 30.6

(2.6) (2.6) (11.9) (11.9)

137.2 134.0 63.1 17.2 22.5 112.6 25.2 61.4 51.8

δHb, mult (J in Hz) 2.00, m 1.73, m 2.36, m

2.28, 2.06, 2.53, 5.26,

d (7.4) m m br s

5.30, br s 2.50, m 2.69, d (18.1)

1.81, 2.41, 4.53, 2.89, 1.08, 0.98, 2.99,

d (12.9) m t (7.4) m s s m

2.53, 2.78, 2.80, 2.90,

m m m m

4.50, 2.11, 1.73, 4.75, 4.80, 1.44, 4.57, 4.63, 3.61,

s s s br s br s s d (11.9) d (11.9) s

a

Recorded at 125 MHz in pyridine−d5. bRecorded at 500 MHz in pyridine−d5. Daedaleanic Acid E, 2. White gum, [α]25D = +8.4 (c 0.05, MeOH); UV (MeOH) λmax (log ε) = 228 (3.34), 272 (1.21) nm; CD (c 1.1 × 10−3 M, CH2Cl2) λmax (Δε) = 208 (−4.61), 223 (4.37), 243 (−0.76), 275 (0.05), 302 (−1.17) nm; IR (neat) νmax = 3232, 3049, 2967, 2928, 2874, 1705, 1456, 1383, 1012, 815, 801 cm−1; 1H and 13 C NMR data, see Table 1. Positive HRESIMS m/z 489.2985 [M + Na]+ (calcd for C30H42O4Na, 489.2981). Daedaleanic Acid F, 3. White gum, [α]25D = 54.2 (c 0.05, CH2Cl2); UV (MeOH) λmax (log ε) = 228 (3.48), 272 (1.23) nm; CD (c 1.1 × 10−3 M, CH2Cl2) λmax (Δε) = 221 (2.34), 237 (−2.73), 283 (1.99), 310 (−0.67) nm; IR (neat) νmax = 3311, 2971, 2926, 2872, 1704, 1462, 1275, 1203, 1137, 1017, 727 cm−1; 1H and 13C NMR data, see Table 1. Positive HRESIMS m/z 479.3158 [M + H]+ (calcd for C31H43O4, 479.3161). Porilactone A, 4. White gum, [α]25D = +5.9 (c 0.05, CH2Cl2); UV (CH2Cl2) λmax (log ε) = 247 (3.36) nm; CD (c 1.1 × 10−3 M, CH2Cl2) λmax (Δε) = 246 (4.17) nm; IR (neat) νmax = 3371, 2966, 2931, 2877, 1762, 1445, 1375, 1182, 1037, 992, 914, 814, 736 cm−1;

Fr. 9 (3.2 g) eluted with n-hexane/EtOAc (3:1) was further subjected to ODS CC with MeOH in water (from 30 to 100%, v/v) to give 8 subfractions (Fr. 8-1−Fr. 8-8). Fr. 8-6 (240 mg, 50% MeOH elution) was separated by RP-HPLC using 45% acetonitrile in water to give 6 (2.8 mg, tR = 18.1 min), 8 (2.9 mg, tR = 25.2 min), 11 (2.4 mg, tR = 28.3 min), 12 (2.6 mg, tR = 33.3 min), 14 (2.2 mg, tR = 37.3 min), and 15 (3.5 mg, tR = 44.5 min). Fr. 8-8 (150 mg, 80% MeOH elution) was separated by RP-HPLC using 42% acetonitrile in water to give 9 (3.4 mg, tR = 31.1 min), 10 (2.4 mg, tR = 34.1 min), and 44 (3.9 mg, tR = 47.3 min). Daedaleanic Acid D, 1. White gum. [α]25D = +16.6 (c 0.05, MeOH), UV (MeOH) λmax (log ε) = 226 (3.43), 272 (1.23) nm; CD (c 1.2 × 10−3 M, MeOH) λmax (Δε) = 208 (−0.30), 221 (5.15), 240 (−2.28), 272 (0.63), 302 (−2.09) nm; IR (neat) νmax = 3443, 2960, 2874, 1701, 1466, 1381, 1071, 892 cm−1; 1H and 13C NMR data, see Table 1. Positive HRESIMS m/z 481.3322 [M + H]+ (calcd for C31H45O4, 481.3318). 7351


Article


13


13

position

δC a

1

36.8

2

30.6

3 4 5 6

177.0 149.6 51.1 28.9

7 8 9 10 11 12

118.3 142.3 137.9 39.2 120.7 37.5

13 14 15

46.0 49.4 44.2

16 17 18 19 20 21 22

76.6 57.9 18.8 22.6 49.7 179.2 27.2

23

33.5

2.49, m 2.83, m 2.33, m

24 25 26

76.3 33.8 17.9

2.28, m 1.17, d (6.5)

27 28 29

17.8 22.7 112.5

30 31

25.3 66.3

δHb, mult (J in Hz) 1.90, 2.14, 1.29, 2.53,

2.34, 2.06, 2.55, 5.28,

ddd (13.4, 10.9, 5.8) m m m

m m m d (3.8)

5.34, d (5.1) 2.48, m 2.71, d (18.0)

1.81, 2.34, 4.49, 2.92, 1.06, 1.03, 2.93,

1.19, 1.73, 4.76, 4.83, 1.51, 4.02, 4.06,

d (13.0) m dd (8.5, 5.0) m s s m

d s d d s d d


δCa

36.8

1.93, m 2.16, td (12.4, 4.8) 2.54, m

36.7

30.6 177.0 149.7 51.1 28.9

2.36, 2.10, 2.58, 5.30,

1 18.3 142.2 137.9 39.2 120.7 37.4

(2.6) (2.6) (10.9) (10.9)

d (7.5) dd (19.2, 4.5) m s

5.36, s 2.50, m 2.71, o

46.0 49.7 44.2 76.8 57.9 18.7 22.7 48.9 179.1 32.2 30.7

(6.5)

14

δC a

155.5 76.4 70.2

1.82, 2.42, 4.53, 2.90, 1.09, 1.05, 3.03,

d (13.1) dd (13.1, 8.5) t (7.3) dd (11.2, 5.7) s s td (11.2, 3.3)

2.61, 2.84, 2.69, 2.90,

td (12.5, 3.4) m m dd (11.6, 5.7)

3.96, 4.05, 1.66, 1.75, 4.77, 4.84, 1.50, 5.31, 5.60,

25.5 22.6 112.5 25.3 109.5

d (10.7) d (10.7) s s s s s br s br s


30.6 177.0 149.6 51.1 30.0 118.3 142.3 137.9 39.2 120.7 37.4 46.0 49.5 44.2 76.6 58.0 18.8 22.6 49.7 179.4 31.2 31.4 79.7 73.0 26.3

d (11.7) m o o

2.34, 2.07, 2.56, 5.28,

o d (18.9) o br s

5.34, br s 2.49, o 2.69, d (18.3)

1.80, 2.38, 4.53, 2.91, 1.08, 1.02, 2.98,

d (13.5) o br s m s s m

2.42, 2.50, 1.98, 3.18, 3.84,

o o m m d (10.9)

1.42, s

26.3 22.6 112.5 25.3

1.90, 2.14, 2.30, 2.54,

1.49, 1.71, 4.76, 4.82, 1.50,

s s br s br s s

a

Recorded at 125 MHz in pyridine−d5; bRecorded at 500 MHz in pyridine−d5.

1

H and 13C NMR data, see Table 2. Positive HRESIMS m/z 453.3364 [M + H]+ (calcd for C30H45O3 453.3369). Porilactone B, 5. White gum, [α]25D=+16.9 (c 0.05, CH2Cl2); UV (CH2Cl2) λmax (log ε) = 246 (3.42) nm; CD (c 1.2 × 10−3 M, CH2Cl2) λmax (Δε) = 246 (1.18) nm; IR (neat) νmax = 3371, 2966, 2931, 2877, 1762, 1445, 1375, 1182, 1037, 992, 922, 844, 808, 740 cm−1; 1H and 13C NMR data, see Table 2. Positive HRESIMS m/z 453.3365 [M + H]+ (calcd for C30H45O3 453.3369). Pinicolic Acid F, 6. White gum, [α]25D = +4.9 (c0.05, MeOH); UV (MeOH) λmax (log ε) = 248 (3.59) nm; CD (c 1.1 × 10−3 M, MeOH) λmax (Δε) = 240 (−2.35) nm; IR (neat) νmax = 3384, 2965, 1745, 1385, 1024, 855 cm−1; 1H and 13C NMR data, see Table 2. Positive HRESIMS m/z 503.3368 [M + H]+ (calcd for C30H47O6 503.3373). 3,15-O-Diacetyl-dehydrotrametenolic Acid, 7. White gum, [α]25D = +44.0 (c 0.05, CH2Cl2); UV (CH2Cl2) λmax (log ε) = 243 (3.51) nm; CD (c 1.1 × 10−3 M, CH2Cl2) λmax (Δε) = 238 (3.49) nm; IR (neat) νmax = 3451, 2962, 2925, 2863, 1733, 1377, 1249, 1032 cm−1; 1 HNMR (500 MHz, pyridine-d5): δ 1.37 (td, J = 13.2, 3.5, H-1α),

1.82 (dt, J = 13.4, 3.4, H-1β), 1.66 (m, H-2α), 1.73 (m, H-2β), 4.67 (dd, J = 11.7, 4.2, H-3), 1.22 (dd, J = 11.7, 4.2, H-5), 1.92 (m, H-6α), 2.02 (m, H-6β), 5.79 (d, J = 6.3, H-7), 5.26 (overlap, H-11), 2.34 (m, H-12α), 2.62m12β5.44 (dd, J = 9.7, 5.5, H-15), 1.94 (m, H-16α), 2.41 (dd, J 14.7, 9.2, H-16β), 2.64 (m, H-17), 1.01 (s, H-18), 0.95 (s, H-19), 2.58 (m, H-20), 1.66 (m, H-22α), 1.9 (m, H-22β), 2.24 (m, H-23α), 2.32 (dd, J = 17.3, 6.1, H-23β), 5.28 (overlap, H-24), 1.65 (s, H-26), 1.59 (s, H-27), 0.83 (s, H-28), 0.95 (s, H-29), 1.26 (s, H-30), 2.02 (s, H-3−OCOCH3),2.17 (s, H-15−OCOCH3); 13CNMR (125 MHz, pyridine-d5): δ 35.2 (C-1), 24.1 (C-2), 80.2 (C-3), 37.4 (C-4), 49.0 (C-5), 22.8 (C-6), 121.3 (C-7), 140.5 (C-8), 145.6 (C-9), 35.8 (C-10), 116.5 (C-11), 37.3 (C-12), 44.3 (C-13), 51.2 (C-14), 77.0 (C-15), 36.2 (C-16), 45.9 (C-17), 16.2 (C-18), 22.5 (C-19), 48.2 (C20), 178.0 (C-21), 32.9 (C-22), 26.3 (C-23), 124.3 (C-24), 131.5 (C25), 25.4 (C-26), 17.4 (C-27), 27.8 (C-28), 16.8 (C-29), 18.5 (C30), 170.2 (C-3-OCOCH3), 20.8 (C-3−OCOCH3), 170.7 (C-15OCOCH3), 20.9 (C-15−OCOCH3); Positive HRESIMS m/z 555.3690 [M + H]+ (calcd for C34H51O6, 555.3686). 7352


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13


position

δC a

1

36.1

2

30.0

3 4 5 6

176.3 149.0 50.4 28.3

7 8 9 10 11 12

120.1 141.6 137.2 38.6 117.7 36.8

13 14 15 16 17 18 19 20 21 22

49.0 45.4 43.5 75.9 57.2 18.1 22.0 48.8 178.6 26.6

23

31.4

24 25 26 27 28 29

76.1 76.5 25.8 25.7 22.0 111.9

30 31

24.6 65.2

16 δHb, mult (J in Hz)

1.87, 2.11, 2.44, 2.56,

2.31, 2.03, 2.53, 5.31,

δCa

ddd (13.3, 11.0, 5.9) ddd (13.3, 11.0, 5.0) m m

120.2 141.7 137.3 38.6 117.7 37.0

5.24, br s 2.47, m 2.68, d (18.1)

m m m s s m

2.54, 2.95, 2.30, 2.42,

m m m m

1.56, 1.61, 1.70, 4.73, 4.79, 1.46, 4.28,

30.0 176.4 149.1 50.6 28.4

m m dd (19.1, 4.4) br s

2.33, 4.47, 2.88, 1.03, 1.00, 2.91,

36.2

49.1 45.5 43.5 76.1 56.9 18.3 22.1 48.5 178.6 26.6 31.1 76.4 76.2 25.9 25.9 22.1 112.0

s s s d (2.6) d (2.6) s s

24.7 65.6

δHb, mult (J in Hz) 1.87, 2.13, 2.45, 2.54,

dq (17.0, 5.9) td (12.4, 4.9) m m

2.31, 2.03, 2.53, 5.31,

m m dd (19.3, 4.4) d (5.1)

5.25, d (3.9) 2.44, m 2.67, d (18.8)

2.36, 4.47, 2.92, 1.03, 1.00, 2.93,

m dd (8.4, 5.0) m s s m

2.72, 2.82, 2.36, 2.48,

m m m m

1.59, 1.63, 1.70, 4.73, 4.79, 1.46, 4.28,

s s s d (2.6) d (2.6) s s

a

Recorded at 125 MHz in pyridine−d5; bRecorded at 500 MHz in pyridine−d5. IR (neat) νmax = 3410, 2953, 2928, 2880, 1732, 1659, 1437, 1378, 1269, 1198, 898, 736 cm−1; 1HNMR (500 MHz, pyridine-d5): δ 1.66 (m, H-1α), 1.96 (m, H-1β), 2.26 (m, H-2α), 2.32 (m, H-2β), 2.23 (m, H-5), 2.02 (m, H-6α), 2.48 (m, H-6β), 5.23 (overlap, H-7), 5.24 (overlap, H-11), 2.44 (m, H-12), 1.36 (m, H-15α), 1.71 (m, H-15β), 1.42 (m, H-16α), 2.06 (m, H-16β), 2.45 (m, H-17), 0.97 (s, H-18), 0.92 (s, H-19), 2.61 (td, J = 11.1, 3.4 Hz, H-20), 1.74 (m, H-22α), 1.91 (m, H-22β), 2.25 (m, H-23α), 2.34 (m, H-23β), 5.29 (t, J = 7.3 Hz, H-24), 1.64 (s, H-26), 1.59 (s, H-27), 1.68 (s, H-28), 4.72 (s, H29α), 4.77 (s, H-29β), 0.93 (s, H-30), 3.62 (s, H-3-OCH3); 13CNMR (125 MHz, pyridine-d5): δ 35.6 (C-1), 29.3 (C-2), 174.3 (C-3), 148.9 (C-4), 50.5 (C-5), 28.2 (C-6), 117.8 (C-7), 141.6 (C-8), 137 (C-9), 38.4 (C-10), 120.4 (C-11), 36.5 (C-12), 44.4 (C-13), 50 (C-14), 30.7 (C-15), 27 (C-16), 48 (C-17), 16.7 (C-18), 21.9 (C-19), 48.6 (C20), 178.2 (C-21), 33 (C-22), 26.5 (C-23), 124.6 (C-24), 131.5 (C25), 25.5 (C-26), 17.5 (C-27), 21.8 (C-28), 111.9 (C-29), 23.9 (C30), 51.1 (C-3-OCH3); Positive HRESIMS m/z 483.3478 [M + H]+ (calcd for C31H47O4, 483.3475). Poricoic Acid K, 12. White gum, [α]25D = +19.8 (c 0.05, CH2Cl2); UV (CH2Cl2) λmax (log ε) = 241 (3.54) nm; CD (c 1.1 × 10−3 M, CH2Cl2) λmax (Δε) = 241 (3.67) nm; IR (neat) νmax = 3411, 2964,

Poricoic Acid I, 8. White gum, [α]25D = −10.9 (c0.05, CH2Cl2); UV (CH2Cl2) λmax (log ε) = 240 (3.48) nm; CD (c 1.1 × 10−3 M, CH2Cl2) λmax (Δε) = 240 (3.98) nm; IR (neat) νmax = 3350, 2959, 2923, 1709, 1679, 1448, 1205, 1143, 1021, 886 cm−1; 1H and 13C NMR data, see Table 3. Positive HRESIMS m/z 515.3377 [M + H]+ (calcd for C31H47O6, 515.3373). Poricoic Acid J, 9. White gum, [α]25D = +17.9 (c 0.05, MeOH); UV (MeOH) λmax (log ε) = 243 (3.43) nm; CD (c 1.2 × 10−3 M, MeOH) λmax (Δε) = 240 (4.48) nm; IR (neat) νmax = 3353, 2973, 1709, 1434, 1203, 1033, 887 cm−1; 1H and 13C NMR data, see Table 3. Positive HRESIMS m/z 531.3328 [M + H]+ (calcd for C31H47O7, 531.3322). Poricoic Acid JM, 10. White gum, [α]25D = +15.9 (c 0.05, MeOH); UV (MeOH) λmax (log ε) = 243 (3.29) nm; CD (c 1.2 × 10−3 M, CH2Cl2) λmax (Δε) = 242 (3.35) nm; IR (neat) νmax = 3420, 2951, 2849, 1717, 1436, 1379, 1201, 1018, 892 cm−1; 1H and 13C NMR data, see Table 3. Positive HRESIMS m/z 545.3482 [M + H]+ (calcd for C32H49O7, 545.3478). 16-Deoxyporicoic Acid BM, 11. White gum, [α]25D = +16 (c 0.05, CH2Cl2); UV (CH2Cl2) λmax (log ε) = 243 (3.38) nm; CD (c 1.1 × 10−3 M, CH2Cl2) λmax (Δε) = 224 (1.47), 248 (0.75), 293 (2.31) nm; 7353


Article

Journal of Agricultural and Food Chemistry 1706, 1455, 1381, 1199, 1023, 897 cm−1; 1H and 13C NMR data, see Table 4. Positive HRESIMS m/z 533.3483 [M + H]+ (calcd for C31H47O7, 533.3478). Poricoic Acid L, 13. White gum, [α]25D = +11.9 (c 0.05, CH2Cl2); UV (CH2Cl2) λmax (log ε) = 243 (3.52) nm; CD (c 1.1 × 10−3 M, CH2Cl2) λmax (Δε) = 240 (3.65) nm; IR (neat) νmax = 3370, 2959, 2920, 2855, 1715, 1679, 1263, 1204, 1032 cm−1; 1H and 13C NMR data, see Table 4. Positive HRESIMS m/z 553.3148 [M + Na]+ (calcd for C31H46O7Na, 553.3141). Poricoic Acid M, 14. White gum, [α]25D = +4.9 (c 0.05, CH2Cl2); UV (CH2Cl2) λmax (log ε) = 243 (3.44) nm; CD (c 1.1 × 10−3 M, CH2Cl2) λmax (Δε) = 240 (3.07) nm; IR (neat) νmax = 3316, 2962, 2919, 2849, 1709, 1407, 1203, 1025, 739 cm−1; 1H and 13C NMR data, see Table 4. Positive HRESIMS m/z 541.3144 [M + Na]+ (calcd for C30H46O7Na, 541.3141). Poricoic Acid N, 15. White gum, [α]25D = +78.9 (c 0.05, MeOH); UV (MeOH) λmax (log ε) = 243 (3.38) nm; CD (c 1.1 × 10−3 M, MeOH) λmax (Δε) = 238 (3.46), 293 (1.46) nm; IR (neat) νmax = 3398, 2970, 2855, 1705, 1381, 1201, 1025, 954, 897 cm−1; 1H and 13 C NMR data, see Table 5. Positive HRESIMS m/z 571.3251 [M + Na]+ (calcd for C31H48O8Na, 571.3247). Poricoic Acid O, 16. White gum, [α]25D = −15.9 (c 0.05, MeOH); UV (MeOH) λmax (log ε) = 243 (3.33) nm; CD (c 1.1 × 10−3 M, MeOH) λmax (Δε) = 238 (2.38), 287 (0.53) nm; IR (neat) νmax = 3354, 2973, 2923, 2866, 2648, 1713, 1434, 1386, 1208, 1032, 886 cm−1; 1H and 13C NMR data, see Table 5. Positive HRESIMS m/z 571.3254 [M + Na]+ (calcd for C31H48O8Na, 571.3247). Preparation of the Alkaline-Insoluble Fraction (AIF) Mainly Containing Compounds 22, 24, 28, and 31 from the Peels’ Extracts of W. cocos. The ethanol extract (100 g) of the peels of W. cocos was suspended in the 2 L NaOH-H2O (1M) solution followed by ultrasound at room temperature for 1 h. The precipitates were collected by centrifugation and further washed with distilled water (1 L) for three times to give the alkaline-insoluble fraction (AIF) (18.2 g). The composition of the AIF was analyzed by HPLC analysis (chromatographic column: YMC-pack ODS-A column; mobile phase: 85% acetonitrile and 15% water with 0.01% trifluoroacetic acid; flow: 1 mL/min; temperature: 35 °C, Figure S5). Computation Section.16 Systematic conformational searches of structure 1a and 8a were performed by employing MMFF94 force field using CONFLEX. The MMFF minima were optimized with DFT calculations at the B3LYP/6-31G(d) level in methanol using the Gaussian 09 program. The 40 lowest electronic transitions were calculated using the time-dependent density-functional theory (TDDFT) methodology at the B3LYP/6-31G(d) level. The overall ECD spectra were then generated according to Boltzmann weighting of each conformer. Preparation of (S)- and (R)-MTPA Esters of 4 and 5. To a solution of 4 (1 mg) in pyridine (0.5 mL) was added (R)-MTPA chloride (25 μL), and the mixture was allowed to stand overnight at room temperature. The reaction was stopped by the addition of 1.0 mL of H2O, and the mixture was subsequently extracted with EtOAc (3 × 1.0 mL). The combined EtOAc layers were dehydrated over anhydrous NaSO4, and further evaporated to dryness. The residue was subjected to HPLC using 90% methanol in water to yield the (S)MTPA ester of 4(0.6 mg). The same procedure was used to prepare the (R)-MTPA ester of 4 (0.6 mg) with (S)-MTPA chloride.17 (S)MTPA ester of 5 (0.7 mg from 1 mg) and (R)-MTPA ester of 5 (0.7 mg from 1 mg) were prepared by the method as described for 4. (S)-MTPA Ester of 4. 1HNMR (500 MHz, pyridine-d5): δ 1.400 (m, H-1), 2.007 (m, H-1), 1.877 (m, H-2), 4.894 ((dd, J = 11.9, 4.0 Hz, H-3), 1.252 (m, H-5), 2.141 (m, H-6), 5.522 (m, H-7), 5.476 (m, H11), 2.448 (m, H-12), 2.201 (m, H-12), 2.448 (m, H-17), 0.869 (s, H18), 0.962 (s, H-19), 2.844 (m, H-20), 2.326 (m, H-22), 2.106 (m, H22), 5.451 (m, H-23), 5.374 (m, H-24), 1.644 (s, H-26), 1.644 (s, H27), 0.943 (s, H-28), 0.923 (s, H-29), 0.981 (s, H-30), 3.662 (s, HMPTA-OMe), 7.791 (m), 7.482 (m), 7.440 (m). (R)-MTPA Ester of 4. 1HNMR (500 MHz, pyridine-d5): δ 1.411 (m, H-1), 2.042 (m, H-1), 1.913 (m, H-2), 4.896 ((dd, J = 12.0, 4.5 Hz, H-3), 1.238 (m, H-5), 2.126 (m, H-6), 5.52 (m, H-7), 5.479 (m, H-

11), 2.461 (m, H-12), 2.207 (m, H-12), 2.451 (m, H-17), 0.86 (s, H18), 0.965 (s, H-19), 2.845 (m, H-20), 2.32 (m, H-22), 2.11 (m, H22), 5.458 (m, H-23), 5.375 (m, H-24), 1.644 (s, H-26), 1.644 (s, H27), 0.89 (s, H-28), 0.873 (s, H-29), a 0.973 (s, H-30), 3.618 (s, HMPTA-OMe), 7.81 (m), 7.485 (m), 7.438 (m). (S)-MTPA Ester of 5. 1HNMR (500 MHz, pyridine-d5): δ 1.485 (m, H-1), 1.999 (m, H-1), 1.941 (m, H-2), 4.95 (dd, J = 11.9, 4.0 Hz, H3), 1.307 (m, H-5), 2.076 (m, H-6), 5.543 (d, J = 6.3 Hz, H-7), 5.429 (d, J = 6.3 Hz, H-11), 2.631 (m, H-12), 2.378 (m, H-12), 1.767 (m, H-15), 1.544 (m, H-15), 1.972 (m, H-16), 2.534 (q, J = 8.3 Hz, H17), 0.771 (s, H-18), 1.05 (s, H-19), 2.871 (dt, J = 12.5, 7.7 Hz, H20), 2.343 (m, H-22), 1.838 (m, H-22), 5.16 (m, H-23), 5.327 (d, J = 8.8 Hz, H-24), 1.697 (s, H-26), 1.833 (s, H-27), 0.993 (s, H-28), 0.963 (s, H-29), 1.022 (s, H-30), 3.653 (s, H-MTPA-OMe), 7.828 (m), 7.472 (m), 7.514 (m). (R)-MTPA Ester of 5. 1.498 (m, H-1), 2.037 (m, H-1), 1.974 (m, H-2), 4.946 (dd, J = 12.2, 4.5 Hz, H-3), 1.293 (m, H-5), 2.049 (m, H6), 5.527 (d, J = 4.3 Hz, H-7), 5.435 (d, J = 6.3 Hz, H-11), 2.639 (m, H-12), 2.381 (m, H-12), 1.766 (m, H-15), 1.519 (m, H-15), 1.971 (m, H-16), 2.544 (q, J = 8.5 Hz, H-17), 0.776 (s, H-18), 1.052 (s, H19), 2.872 (dt, J = 12.2, 7.8 Hz, H-20), 2.35 (m, H-22), 1.847 (m, H22), 5.157 (m, H-23), 5.327 (d, J = 8.0 Hz, H-24), 1.698 (s, H-26), 1.834 (s, H-27), 0.929 (s, H-28), 0.891 (s, H-29), 1.022 (s, H-30), 3.695 (s, H-MTPA-OMe), 7.845 (m), 7.516 (m), 7.472 (m). Determination of the Absolute Configurations in the Diol Moiety of 12−14 Secondary Alcohol. According to the published literature,18−20 compounds 12, 13, and 14 (each 0.5 mg) were dissolved in a stock solution of Mo2(OAc)4 complex (1.70 mg) in DMSO (600 μL). The CD spectra were monitored within 20−30 min until stabilization. The inherent CD of the compounds was subtracted. The absolute configurations of the secondary alcohol moieties were determined by the sign at approximately 310 nm in the observed CD spectra. Cytotoxicity. Cell lines K562 (human bone marrow chronic myelogenous leukemia), SW480 (human colon cancer cells), and HepG2 (liver hepatocellular cells) were purchased from the National Infrastructure of Cell Line Resource. Fetal bovine serum (FBS) was purchased from Gibco. Roswell Park Memorial Institute (RPMI 1640), Dulbecco’s modified eagle medium (DMEM), and PBS were purchased from hyclone. Cytotoxicity assay was performed according to the method previously reported.21 After treating cells with tested compounds at different dose (the maximum final concentration of DMSO is 0.5%) for 48 h, 5 μL of CCK8 was added to each well, and incubated for another 4 h. Cisplatin (J&K Scientific Ltd., purity ≥98%) were used as positive controls. The assay plate was read at 450 nm using a microplate reader. The inhibition rate was calculated with the following formula: inhibitory rate (%) = [1 − (OD treated/ OD control)] × 100% and IC50 was calculated by plotting inhibitory rate vs test concentrations. Glucose Uptake Assay. The 3T3-L1 preadipocytes cell line were obtained from the National Infrastructure of Cell Line Resource and cultured in DMEM medium supplemented with 100 IU/ml penicillin, 100 μg/mL streptomycinm and 10% calf serum (CS, Gibco) in 5% CO2 at 37 °C. A differentiation experiment was performed as previously reported.15,22 Briefly, 3T3-L1 preadipocytes were induced by treating the cells with differentiation medium which contains DMEM, 0.5 mM 3-isobutyl-1-methylxanthane (IBMX, J&K Scientific Ltd.), 0.25 μM dexamethasone (J&K Scientific Ltd.), 10 μg/mL insulin (J&K Scientific Ltd.), and 10% FBS for 48 h. The cells were refed with DMEM supplemented with 10 μg/mL insulin and 10% FBS for the following 48 h and changed every 2 days. More than 90% of the cells expressed the adipocytes phenotype between 8 to 10 days after the initiation of differentiation and were used for the experiments. Glucose uptake by adipocytes cells was analyzed by measurement of 2-NBDG uptake.22 Briefly, differentiated cells were washed and starved in Krebs-Ringer phosphate HEPES (KRPH) buffer for 1 h. The cells were treated with or without 2.5−20 μM compounds for 2 h in the absence or presence of 100 nM insulin KRPH buffer. Then 2NBDG (100 μM in KRPH buffer) was added to each well followed by 7354


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Journal of Agricultural and Food Chemistry

Figure 1. Structures of compounds 1−47. incubation for 30 min at 37 °C. After the cells were washed, the fluorescence intensity of the cells containing 2-NBDG was analyzed immediately using a fluorescence microplate reader with excitation at 485 nm and emission at 535 nm. Protein concentration of each sample was quantified using BCA (bicinchoninic acid) protein assay kit. Animal Care and Experiments. The male C57BL/6J mice at the age of 9 weeks were purchased from the Experimental Animal Center,

Chinese Academy of Medical Sciences and fed with a high-fat diet (60 kcal% fat, 20 kcal% proteins, and 20 kcal% carbo-hydrates, Cat. D12492i, Research Diet, New Brunswick, NJ, U.S.A.). Mice were cared and treated according to recommendations in the Guide for the Care and Use of Laboratory Animals of the Institute of Microbiology, Chinese Academy of Sciences (IMCAS) Ethics Committee. Male C57BL/6J mice were divided into five groups (n = 10 each) based on their blood glucose levels and body weight. The AIF were suspended 7355


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Figure 2. Key 1H−1H COSY and HMBC correlations of compounds 1−16. curves (AUCs) generated from the data collected during the OGTT was calculated by Graphpad 7.0. Statistical Analysis. All results were representative of three independent experiments. Data were expressed as mean ± SD. For multiple comparisons, the statistical analysis was done with on-way or two-way ANOVA followed by the Turkey’s multiple comparison tests with Graph Pad 6.0. P < 0.05 was regarded as statistically significance.

in distilled water and daily administrated to DIO mice at the dose of 50 mg/kg and 100 mg/kg. Metformin was dissolved in distilled water and orally administrated at the dose of 100 mg/kg. Vehicle groups (normal diet and high-fat diet) were treated with an oral gavage of an equivalent volume of distilled water. Treatment was continued for 9 weeks. At the end of the study, mice were anesthetized by diethyl ether (Beijing Chemical Works, Beijing, China), and blood was sampled from the portal and cava veins. Levels of plasmatic glucose, the glycated hemoglobin A1C (HbA1C), the plasmatic insulin, plasmatic total cholesterol (TC), triglycerides (TG), and nonesterified fatty acid (NEFA) were measured at the indicated time by a commercial kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) as previously described.22,23 The insulin sensitivity index (ISI) was obtained by the measurement of fasting blood glucose (FBG, in mg/dL) and fasting blood insulin (FBI, in mU/L). ISI = 1/1000 (FBG × FBI). The insulin tolerance test (ITT) and the oral glucose tolerance test (OGTT) were conducted at the 56th and 59th day of treatment, as described in our earlier report.23,24The area under the

■

RESULTS AND DISCUSSION Structural determination of new triterpenes 1−16. Peels of the sclerotia of W. cocos (4.2 kg) were powdered and exhaustively extracted with ethanol. The 350 g ethanol extracts were separated by a combination of silica gel chromatography, LH20, ODS, and high-performance liquid chromatography (HPLC) to afford 16 new triterpenes (1−16) together with 31 known lanostane triterpenoids (Figure 1). By comparing NMR and MS data with the corresponding compounds in the literatures, known compounds were identified as dehydroebur7356


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Figure 3. Key NOE correlations of compounds 1, 3−6, and 9.

icoic acid (17),25 dehydroeburicoic acid monoacetate (18),25 dehydrosulphurenic acid (19),25 coriacoic acid B (20),11 dehydropachymic acid (21),26 dehydroeburiconic acid (22),27 polyporenic acid C (23),26 dehydrotrametenolic acid (24),28 3β-(acetyloxy)lanosta-7,9(11),24-trien-21-oic acid (25),29 dehydrotrametenonic acid (26),30 16α-hydroxy-3-oxolanosta7,9(11),24-trien-21-oic acid (27),31 eburicoic acid (28),32 eburicoic acid acetate (29), 33 pachymic acid (30),26 trematenolic acid (31),34 3-O-acetyl-16α-hydroxytrametenolic acid phthalimidomethyl ester (32),32 versisponic acid E (33),35 pinicolic acid A (34),36 pinicolic acid E (35),37 poricoic acid C (36),38 poricoic acid CM (37),38 poricoic acid A (38),39 poricoic acid AM (39),28 poricoic acid AE (40),27 16-deoxyporicoic acid B (41),38 poricoic acid BM (42),40 poricoic acid D (43),28 poricoic acid E (44),40 poricoic acid ZG (45),41 poricoic acid GM (46),42 and daedaleanic acid A (47). 43 The structures of the new compounds were determined by extensive spectroscopic experiments. Various bioactivities including cytotoxicity (17, 18, 21−24, 26, 28−31, 34, 37, 38, 41, 46, 47),26,38,43−48 anti-inflammatory activity (17, 19, 21, 23, 30),38,49,50 antibacterial activity (23, 34),51 antifibrotic effect (45),41 and inducing cell differentiation (24)13 have been reported for these known compounds. Daedaleanic acid D (1) gave a protonated molecule peak at m/z 481.3322 [M + H]+ (calcd for C31H45O4, 481.3318) in

the HREIMS spectrum, suggesting a molecular formula of C31H44O4 with a degree of unsaturation of 10. The 1H and 13C NMR (Table 1) spectra of 1 exhibited 3 singlet methyls at δH 0.81, 1.35, and 2.26; 4 doublet methyl signals at δH1.02 (d, J = 6.7 Hz), 1.04 (d, J = 6.7 Hz), 1.05 (d, J = 6.3 Hz), and 1.07 (d, J = 6.3 Hz); 1 oxygenated methine at δH 4.33 (dd, J = 8.4, 5.7 Hz); 2 broad singlet olefinic protons at δH 4.75 and 4.79; 2 pairs of mutually coupled olefinic protons at δH 6.14 (d, J = 10.0 Hz), 6.47 (d, J = 10.0 Hz), 6.74 (d, J = 7.5 Hz), and 6.98 (d, J = 7.5 Hz); and 31 carbon signals including 7 methyls, 5 methylenes, 5 methines, 2 sp3 quaternary carbons, a carboxylic group and a ketone (δC178.7 and 213.3), and 10 olefinic carbons. The above evidence together with 1H−1H COSY correlations (Figure 2) of H-6/H-7, H-11/H-12, H2-15/H-16/ H-17/H-20/H 2 -22/H2 -23, and H3 -26/H-25/H 3 -27 and HMBC correlations (Figure 2) of H-7 with C-5, C-6, C-8, C-9, and C-14; H-11 with C-8, C-9, and C-10; H3-18 with C12, C-13, C-14, and C-17; H3-19 with C-5 (δC 133.8), C-6 (δC 129.7), and C-10 (δC136.1); H-25 with C-24 and C-23; H-17, H-20, and H2-22 with C-21; H3-30 with C-13, C-14, and C-15; and H-11 and H-12 with C-13 indicated a substructure of 3a,7,9b-trimethyl-2,3,3a,9b-tetrahydro-1H-cyclopenta[a]naphthalen-2-ol linked to a 6-methyl-5-methyleneheptanoic acid side chain. A side chain of 2-methylpentan-3-one attached at C-10 was further deduced on the basis of 1H−1H COSY 7357


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Figure 4. Experimental ECD and calculated ECD spectra of 1 and 8. (a) Experimental ECD spectra of 1 and its calculated ECD spectra of related simplified possible stereoisomers 1a and 1b. (b) Experimental ECD spectra of 8 and its calculated ECD spectra of related simplified possible stereoisomers 8a and 8b.

new members of the family of 4,5-secolanostanes with the methyl rearrangement from C-19 to C-5. Daedaleanic acid F (3) was obtained as a white powder. On the basis of HRESIMS data at m/z 479.3158 [M + H]+ (calcd for C31H43O4, 479.3161), the molecular formula of 3 was determined to be C31H42O4. The 1H NMR spectrum (Table 1) of 3 showed signals for five singlet methyls at δH 1.31, 1.49, 1.53, 1.81, and 2.14; two doublet methys at δH1.01 (d, J = 6.7 Hz) and 1.02 (d, J = 6.7 Hz); two mutually coupled olefinic protons at δH 6.68 (d, J = 9.9 Hz) and 6.89 (d, J = 9.9 Hz); one singlet olefinic proton at δH7.14, two broad singlet olefinic protons at δH 4.88 and 5.03; and one oxygenated methane δH 4.78 (t, J = 6.9 Hz). In total, 31 carbon signals including 1 ketone carbonyl group (δC213.9), 1 carboxyl acid moiety (δC178.4), 1 oxygenated carbon (δC77.3), and 10 olefinic carbons were observed in the 13C NMR spectrum of 3, which in combination with characteristic proton resonances suggested an aromatic lanostane triterpenoid skeleton. Analysis of HMBC and 1H−1H COSY correlations of 3 (Figure 2) indicated the same 6/6/5 ring system and the identical side chain at C-17 as those of 1. The HMBC correlations from H328/29 to C-4, C-3, and C-5; from H3-19 to C-5, C-6, and C-7; and from H-1 to C-5, C-9, and C-10, as well as 1H−1H COSY of H2-1/H2-2 supported the formation of 2,2-dimethylcyclohex-3-en-1-one moiety and the substitution of a methyl group at C-6. Compound 3 was assigned to have the same relative and absolute configurations as those of 1 by the ROESY experiment (Figure 3) and the comparison of the CD spectrum. On the basis of the above evidence, the structure of 3 was confirmed as 16R-hydroxy-19(10 → 6)abeo-3oxolanosta-5,7,9,11,24(31)-pentaene-21-oic acid. The molecular formula of porilactone A (4) was determined to be C30H44O3 on the basis of the HRESIMS data at m/z 453.3364 [M + H]+ (calcd for C30H45O3 453.3369). The 1H and 13C NMR (Table 2) combined with the HSQC correlations revealed the presence of seven singlet methyls (δC/H 17.3/0.92, 23.4/1.10, 26.0/1.67, 18.6/1.68, 29.2/1.24, 17.0/1.15, and 26.1/1.00), three trisubstituted double bonds (δC/H 121.6/5.60, 117.1/5.44, and 124.8/5.36; and δC143.1, 146.9, and 139.3), two oxygenated methines (δC/H78.4/3.48

correlations of H2-1/H2-2, H3-28/H-4/H3-29, as well as HMBC correlations from the H3-28/29 and H2-2 to C-3 (δC 213.3), from H2-1 to C-9, and from H2-2 to C-10. The relative configurations of 1 were established by a ROESY experiment. Significant NOE correlations (Figure 3) of H3-18 with H-16 and H-20, H3-30 with H-17 indicated α-orientation of HO-16, H-17, and H3-30 and the β orientation for H3-18 and H-20. Thus, compound 1 was confirmed to possess a similar structure to that of daedaleanic acid A (47),37 belonging to the class of 4,5-seco-lanostane triterpenoids. To determine the absolute configurations using ECD calculation method, the structure of 1 was simplified to two stereoisomers 1a (13R/ 14R/16R/17R/20R) and 1b (13S/14S/16S/17S/20S). As seen from the Figure 4a, the calculated ECD curve of 1a was in good agreement with the experimental CD spectrum of 1. Thus, the absolute configuration of 1 was established as 13R, 14R, 16R, 17R, and 20R. From the above analysis, the structure of daedaleanic acid D was assigned as 16R-hydroxy-3-oxo19(10 → 5)abeo-4,5-secolanosta-5,7,9,11,24(31)-pentaene-21oic acid. Daedaleanic acid E (2) had the molecular formula of C30H42O4, as indicated by a sodium adduct ion peak at m/z 489.2985 [M + Na]+ (calcd for C30H42O4Na, 489.2981) in its HRESIMS spectrum. The 1H and 13C NMR (Table 1) of 2 resembled those of 1 except for the appearance of a trisubstituted double bond (δC/H 123.2/5.14, δC 132.7) and the loss of a terminal alkenyl group. The structure change in the side chain was further confirmed by the HMBC correlations of H-24 with C-23 and C-25, H3-26 and H3-27 with C-25 and C-24, and the 1H−1H COSY correlations of H215/H-16/H-17/H-20/H2-22/H2-23/H-24 (Figure 2). The ROESY spectrum (Figure S1) of 2 showed the same relative configurations with those of 1. The similar Cotton effects observed between 1 and 2 (Figure S2) confirmed the absolute configurations at C-13 and C-14 to be 13R and 14R, respectively. Considering the relative configuration determined, the absolute configuration of 2 was assigned as 13R, 14R, 16R, 17R, and 20R. Thus, the structure of 2 was assigned as 16R-hydroxy-3-oxo-19(10 → 5)abeo-4,5-secolanosta5,7,9,11,24-pentaene-21-oic acid. Compounds 1 and 2 are 7358


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Figure 5. Δδ (δ[(S)‑MPTA ester] − δ

[(R)‑MPTA ester])

values of the (S)- and (R)-MTPA esters of 4 and 5.

orientation for H-12, H-17, H3-28, and H3-30 in 6. Thus, the structure of 6 was determined as 12β,16α,25-trihydroxy-3oxolanosta-7,9(11)-dien-21-oic acid. The molecular formula of 3,15-O-diacetyl-dehydrotrametenolic acid (7) was determined to be C34H50O6 from its HRESIMS data at m/z 577.3500 [M + Na]+. The signals due to two additional acetyl groups and one oxygenated methine were observed in its 13C and 1H NMR spectra (see Materials and Methods). A comparison of NMR data between 7 and 24, as well as the detailed analysis of 2D NMR spectra of 7 (Figure 2) supported the attachment of two acetoxyl groups at C-3 and C-15 and assigned its structure as described. Poricoic acid I (8) was assigned the molecular formula of C31H46O6 by its HRESIMS ion peak at m/z 515.3377 [M + H]+ (calcd for C31H47O6, 515.3373). In the 13C and 1H NMR spectra (Table 3) of 8, the presence of two carboxyl groups (δC 177.2 and 179.3), a tetra-substituted double bond (δC 128.1 and 134.5), a conjugated diene moiety (δC 118.3, 120.7, 137.9, and 142.3; δH5.28 and 5.33), a terminal alkenyl group (δC 112.5, 149.6; δH4.77, 4.84), one oxygenated methylene (δC 61.9; δH4.43 and 4.47), one oxygenated methine (δC 76.7; δH 4.51), and six methyl groups (δC/H 18.7/1.08, 20.5/1.67, 20.9/ 1.76, 22.6/1.73, and 22.7/1.03), of which three were attached to the olefinic carbons, were observed. These aforementioned characteristic signals resembled with the NMR data of poricoic acid A (38),39 suggesting a 3,4-secolanostane-type triterpenoid skeleton. A detailed comparison of NMR spectrum between 8 and 38 indicated their structural difference in the side chain at C-17. The HMBC correlation from the oxygenated methylene to C-23, C-24, and C-25 and the requirement of molecular formula confirmed the presence of the hydroxyl methyl group at C-24 (Figure 2). The NOE correlations (Figure S1) of H318 with H-16, H3-19, H-15β (δH 2.38), and H-20; and H3-30 with H-5, H-15α (δH 1.78), and H-17, assigned the β orientation of H-16, H3-18, H3-19, and H-20 and the α orientation of H3-30, H-5, and H-17. An ECD calculation method was applied to determine the absolute configuration in 8. Two simplified stereo isomers, 8a and 8b, were generated. By comparing the calculated ECD curves and experimental CD spectrum of 8 (Figure 4b), the absolute configuration of 8 was assigned to be the same as that of 8a (5S/10S/13R/14R/16R/ 17R/20R). Finally, the structure of 8 was confirmed as 16Rhydroxy-24-(hydroxylmethyl)-3,4-secolanosta-4(29),7,9(11),24-tetraene-3,21-dioic acid. In the HRESIMS spectrum of poricoic acid J (9), the ion peak at m/z 531.3328 [M + H]+ (calcd for C31H47O7, 531.3322) confirmed a molecular formula of C31H46O7. The 13 C and 1H NMR spectra (Table 3) of 9 were similar with those of 8 except for the presence of an additional oxygenated

and 75.6/5.49), and one ester carbonyl (δC179.2). A further 1 H and 13C NMR data comparison between 4 and 24 revealed an identical substructure of lanostane-type triterpenoid with a 7,9(11) conjugated diene28 and a structural difference on the side chain at C-17. The HMBC correlations (Figure 2) of H23 with C-20 and C-21, the 1H−1H COSY correlations (Figure 2) of H-17/H-20/H2-22/H-23/H-24, and the requirement of unsaturation degree suggested the formation of γ-lactone ring between C-23 and C-21. The NOE correlations (Figure 3) of H-5 with H3-28 and H-3, H3-19 with H3-29 and H3-18, H3-18 with H-15β and H-20, H3-30 with H-15α and H-17, H-20 with H-24, and H-17 with H-23 demonstrated α orientation for H3, H-5, H-17, H-23, H3-28, H3-30 and β orientation for H3-18, H3-19, and H3-29. In order to unambiguously establish the absolute configurations of 4, a modified Mosher’s method using α-methoxy-α-(trifluoromethyl)phenylacetyl (MTPA) was applied. The (3S)- and (3R)-MTPA esters of 4 were successfully prepared according to Mosher’s MTPA ester method.17,52 The Δδ values of the (S)- and (R)-MTPA esters of 4 confirmed the 3S configuration (Figure 5). Taking the relative configuration determined, the absolute configuration of 4 was concluded to be 3S, 5R, 10S, 13R, 14R, 17R, 20R, 23R. Therefore, the structure of 4 was determined to be (20R,23R)20,23-epoxy-3S-hydroxy-20-oxo-lanosta-7,9(11),24-triene, as described in Figure 1. Porilactone B (5) was confirmed to have the same planar structure as that of 4 by analyzing the data of HRESIMS, 1D and 2D NMR (Table 2). The only difference between 4 and 5 lies in the stereochemistry of C-23, which was indicated by the NOE correlation (Figure 3) of H-17 with H-24, and H-20 with H-23 in 5. A 3S configuration was also determined in 5 by application of the same modified Mosher’s method as described in 4. Thus, the absolute configuration of 5 was assigned as 3S, 5R, 10S, 13R, 14R, 17R, 20R, 23S. A molecular formula of C30H46O6 was determined for pinicolic acid F (6) on the basis of HRESIMS data at m/z 503.3368 [M + H]+ (calcd for C30H47O6, 503.3373). Analysis of its 13C and 1H NMR spectra (Table 2) indicated the same chemical skeleton as that of the 16α-hydroxy-3-oxolanostane. The resonances due to one oxygenated quaternary carbon at δC 70.0 and two oxygenated methines at δC/H73.8/4.91 and 75.5/4.64, the 1H−1H COSY correlation (Figure 2) of H-12/ H-11 (δH 5.61) and H-16/H-15 and H-17, as well as the HMBC correlations from H-12 to C-10, C-11, C-13, C-14, and C-18; from H-16 to C-13, C-14, C-15, C-17, and C-20; and from H3-28/29 to C-24 and C-25 supported the substitution of a hydroxyl group at C-12, C-16, and C-25, respectively. The NOE correlations described in Figure 3 were used to assign the β orientation for H-16, H3-18, H3-19, H-20, and H3-29 and α 7359


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Journal of Agricultural and Food Chemistry methylene group at δC/H (63.1/4.48) and the absence of one singlet methyl group. The attachment of a hydroxyl methyl moiety at C-25 was proved by the HMBC correlations (Figure 2) from δH 4.48 to the methyl group at C-27 (δC 17.2) and the olefinic carbons C-24 (δC137.2) and C-25 (δC137.2). The NOE correlation (Figure 3) of H3-27 with H2-23 indicated the Z configuration for the double bond between C-24 and C-25. The similar CD spectrum between 8 and 9 indicated the same absolute configuration of 5S, 10S, 13R, 14R, 16R, 17R, and 20R. Finally, 9 was assigned to be 16R, 26-dihydroxy-24(hydroxylmethyl)-3,4-secolanosta-4(29),7,9(11),24Z-tetraene3,21-dioic acid. Compunds 10 and 11 were determined to be the methyl ester product of 9 and deoxyporicoic acid B (41) by MS and NMR data analysis, respectively (Table 3). They could be the artifacts formed during extraction and isolation. Poricoic acid K (12) gave a [M + H]+ ion peak in the HRESIMS at m/z 533.3483, indicating a molecular formula of C31H48O7. The 13C and 1H NMR spectra of 12 resembled those of 8(Table 4) except for the presence of one oxygenated quaternary carbon at δC 76.3 (C-24) and an extra methine and the loss of the tetra-substituted double bond in the side chain. Analysis of the 2D-NMR spectra (Figure 2), especially the 1 H−1H COSY correlations between the two doublet methyl groups (δH 1.78 and 1.79) and H-25 (δH 2.28) and the HMBC correlations from δH H-23 (δH 2.33) to C-24), C-23 (δC 33.8), and C-31 (δC 66.3); from H2-31 (δH 4.02/4.06) to C-23, C-24, and C-25; and from H3-26/27 to C-24 and C-25, indicated the substructure of 5-hydroxy-5-(hydroxymethyl)-6-methylheptanoic acid moiety in 12. The structure of 12 was finally assigned by detailed interpretation of 2D NMR spectra. The relative configurations of 12 were assigned by NOE correlations (Figure S1), the same as that of 8. The absolute configuration in the ring moiety of 12 was confirmed as 5S, 10S, 13R, 14R, 16R, 17R, and 20R, which is due to the same Cotton effects as that of 8 in the CD curve. For the existence of a vic-diols unit in the side chain, the absolute configuration of C-24 was established by the implement of a dimolybdenum tetraacetate [Mo2(OAc)4]-induced ECD experiment.19,53 As shown in Figure 6, the positive Cotton effect at 310 nm observed in the Mo2(OAc)4-induced ECD spectrum of 12 indicated the R configuration of C-24 on the basis of clockwise rotation in the

Newman form of the Mo-complexes.54 Thus, the structure of 12 was confirmed as 16R,24R-dihydroxy-24-(hydroxylmethyl)3,4-secolanosta-4(29),7,9(11)-triene-3,21-dioic acid. Poricoic acid L (13) had a molecular formula of C31H46O7, as determined by HRESIMS data at m/z 553.3148 [M + Na]+ (calcd for C31H46O7Na, 553.3141). The 13C and 1H NMR spectra of 13 (Table 4) showed much similarity with those of poricoic acid D (43) except for the loss of a singlet methyl group and the presence of one hydroxymethyl group (δC70.2 and δH 3.96/4.05). The structural changes in the side chain of 13 were confirmed by the HMBC correlations from δH 3.96/ 4.05 to C-24 (δC 155.5), C-25 (δC 76.4), and C-27; from H231 to C-23, C-24, and C-25; and from H-27 to C-24, C-25, and C-26. The relative configuration of 13 was determined by ROESY experiment (Figure S1). The absolute configurations of 5S, 10S, 13R, 14R, 16R, 17R, and 20R in 13 were determined by the CD spectrum comparison between 8 and 13 (Figure S2). In the Mo2(OAc)4-induced ECD spectrum of 13, a negative Cotton effect at 310 nm was noted (Figure 6), suggesting the R configurations of C-25 based on anticlockwise rotation in the Newman form of the Mo-complexes. Accordingly, compound 13 was assigned to be 16R,25R,26trihydroxy-3,4-secolanosta-4(29),7,9(11),24(31)-pentaene3,21-dioic acid. The HRESIMS of poricoic acid M (14) displayed a ion peak at m/z 541.3144 [M + Na]+ (calcd for C30H46O7Na, 541.3141), indicating a molecular formula of C30H46O7. The 13 C and 1H NMR spectra (Table 4) of 14 showed much similarity with those of poricoic acid ZG (45) except for an additional hydroxyl moiety. The HMBC correlations (Figure 2) from H-24 (δH3.84) to C-23 (δC 31.4) and C-25 (δC 73.0) indicated the substitution of a hydroxyl group at C-24. Detailed analysis of 2D-NMR dataconfirmed the planar structure of 14. The NOE correlations (Figure S1) in the ROESY spectrum together with the similar Cotton effects between 8 and 14 determined the absolute configurations of 5S, 10S, 13R, 14R, 16R, 17R, and 20R in the ring moiety of 14. A negative Cotton effect at 310 nm was observed in the Mo2(OAc)4-induced ECD spectrum of 14 (Figure 6), which indicated the R configuration of C-24. Finally, the structure of 14 was assigned as 16R,24R,25-trihydroxy-3,4-secolanosta4(29),7,9(11)-triene-3,21-dioic acid. Poricoic acid N (15) showed a molecular-related ion peak at m/z 571.3251 [M + Na]+ in its HRESIMS, indicating a molecular formula of C31H48O8. The 13C and 1H NMR spectral data (Table 5) of 15 were similar to those of 12 except for the absence of two doublet methyls (C-26 and C-27 in 12) and the presence of two additional singlet methyls and one oxygenated carbon in 15. A hydroxyl group attached at C-25 was deduced on the basis of the above analysis. HMBC correlations of H3-26/27 (δH 1.56/1.61), H2-31 (δH 4.28), H223 (δH 2.36/2.48) with C-25 (δC 76.5) and C-24 (δC 76.1) (Figure 3) indicated the substitution of three hydroxyl group at C-24, C-25, and C-31 in the side chain. Poricoic acid O (16) was determined to have the same planar structure as that of 15 on the basis of NMR and MS data interpretation. Compounds 15 and 16 differ from each other in thestereochemistry of C24. With little quantity in hand, the absolute stereochemistry at C-24 in 15 and 16 were left unsolved in this study. The relative configurations in 15 and 16 were solved by analysis of NOE correlations as depicted in Figure S1. Finally, the structures of 15 and 16 were assigned as 16α,24,25-trihydroxy-24-

Figure 6. Most stable conformer of compounds 12−14 complexed with Mo2(OAc)4 and their ECD spectra in DMSO. 7360


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Figure 7. Triterpenoids isolated from the peels of W. cocos enhanced glucose uptake in 3T3-L1 adipocytes. Differentiated adipocytes were treated with various concentrations of triterpenoids (2.5−20 μM) in the absence (a) or presence of 100 nM insulin (b) for 2 h before 2-NDBG uptake measurements. Data represent mean ± SD (n = 3). *p < 0.05, **p < 0.01 versus the untreated control group (Con) or the insulin-treated group (Ins).

compounds 3, 8, 10, 12, 13, 14, 21, 23, 28, 29, 30, 31, 33, 35, 37, and 43 at the concentration of 20 μM exhibited an insulin-sensitizing effect (Figure 7b) with the increase rate in the range of 16% to 39%, as compared with the level of glucose uptake in the cells treated by insulin at 100 nM. When the compound concentrations were decreased to 2.5 μM, only compounds 14, 23, 29, 35, and 43 showed insulin-sensitizing activities. The preliminary structure−activity relationship was analyzed for compounds 1−47 according to their glucose-uptakestimulating and insulin-sensitizing activities. It was found that the 6/6/6/5 ring skeleton and the double bond between C-8 and C-9 in the lanostane triterpenes greatly influenced the glucose-uptake-stimulating and insulin-sensitizing activities. Triterpenes of W. cocos with the 6/6/6/5 skeleton showed much stronger activities in both glucose-uptake-stimulating and insulin-sensitizing assay than triterpenes with the secolanostane skeleton. Compounds 29 and 30 possessing a double bond between C-8 and C-9 displayed stronger activities in stimulating the glucose uptake than compounds 18 and 21, which have a 7, 9(11)-diene moiety, respectively. In the insulin-sensitizing assay, compounds 29, 31, and 35 exhibited stronger activity than compounds 18, 24, and 27, respectively. In Vivo Hypoglycemic and Hypolipidemic Activity of the Alkaline-Insoluble Fraction (AIF) Derived from the EtOAc Extract. Compounds 22, 24, 28, and 31 were detected as the main components in the EtOAc extract from the peels of W. cocos. In this study, we found that they were poorly soluble in

(hydroxlmethy)-3,4-secolanosta-4(29),7,9(11)-triene-3,21dioic acid. Evaluation of Biological Activities. Cytotoxicity of Compounds 1−47. All compounds were tested for cytotoxicity toward cell lines K562 (human bone marrow chronic myelogenous leukemia), SW480 (human colon cancer cells), and HepG2 (liver hepatocellular cells). Compounds 23, 25, 29, and 31 showed the weak cytotoxicity on K562 cells with the IC50 values 68.2 ± 2.2, 45.3 ± 1.2, 33.1 ± 0.5, and 25.7 ± 1.7 μM, respectively, while positive drug cisplatin with the IC50 value 3.8 ± 0.2 μM. The other compounds displayed no cytotoxicity against K562 cells at the concentration of 100 μM. In assays with SW480 and HepG2 cells, none of tested compounds showed cytotoxicity at the dose of 100 μM. Glucose Uptake-Stimulating and Insulin-Sensitizing Activities of Compounds 1−47. To evaluate the hypoglycemic activity of triterpenes isolated from the peels of W. cocos, compounds 1−47 were tested for their stimulatory effects on glucose uptake using mammalian 3T3-L1 adipocytes fed with a fluorescent glucose analogue, 2-[N-(7-nitrobenz-2-oxa-1,3diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG).22 At the dose of 20 μM, compounds 3, 8, 10, 12, 14, 21, 23, 28, 29, 30, 33, 35, and 44 showed bioactivity in stimulating the glucose uptake in the 3T3-L1 adipocytes when compared with the control group (Figure 7a). Among them, compounds 21, 28, and 30 showed the most promising activity with the increase of glucose uptake by 25%, 14%, and 50% at 5 μM, respectively. In the assay of insulin-sensitizing activity, 7361


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Figure 8. Effects of the alkaline-insoluble fraction on blood glucose level, insulin resistance, and lipid metabolism. (a) Free diet blood glucose. (b) Ratio of HbA1c to Hb, (c) OGTT, and (d) area under curve (AUC) on the 59th day of treatment. (e) ITT and (f) AUG of ITT on the 56th day of treatment. (g) Plasmic insulin. (h) Insulin sensitivity index (ISI). (i) Plasmatic total triglyceride (TG). (j) Plasmatic total cholesterol (TC). (k) Plasmatic nonesterified fatty acid (NEFA). Con, C57BL/6J mice control; Mod, DIO model; Met, metformin treated mice, 100 mg/kg; AIF-L, the alkaline-insoluble fraction at the dose of 50 mg/kg; AIF-H, the alkaline-insoluble fraction at the dose of 100 mg/kg; N = 10 mice per group. Statistical analysis was done using one-way ANOVA followed by the Tukey post hoc test. * P < 0.05; ** P < 0.01, *** P < 0.001.

alkaline solution and can be easily separated from other triterpenoid acids by extracting the EtOAc extract with 1.0 M NaOH solvent. In above assay, compounds 28 and 31 have shown a good insulin-sensitizing effect on mammalian 3T3-L1 adipocytes. Therefore, the AIF mainly containing compounds 22, 24, 28, and 31 (Figure S5) was further evaluated for its hypoglycemic effects on high-fat-diet-induced obese (DIO) mice. DIO mice were treated with AIF at the concentration of 50 and 100 mg/kg by oral gavage, respectively. Metformin that exhibited strong effects on reducing hyperglycemia and improving glucose tolerance and insulin resistance was used as the positive control. After 8 weeks of treatment, AIF (at 100 mg/kg) significantly reduced the level of free diet plasmatic glucose and glycosylated hemoglobin A1c (HbA1c) (Figure 8a,b), which is the gold standard for glucose control in clinics.55 Treatment with AIF also improved glucose tolerance and insulin resistance. The mean area under curve (AUC) values during the oral glucose tolerance test (OGTT) and the insulin tolerance test (ITT) in the AIF-treated DIO mice were much lower than that of the vehicle-treated DIO mice (Figure 8c−f), which was comparable to that of metformin-treated group. In addition, treatment with AIF caused a reduction of the plasma insulin level (Figure 8g). An elevation of 53% and 37% in the insulin sensitivity index was observed in the metformin-treated group and the AIF-treated group, respectively, as compared with that of the model group (Figure 8h). Treating the DIO mice with AIF also decreased levels of plasma total cholesterol (TC), nonesterified fatty acid (NEFAs), and triglycerides (TG) in a dose-dependent manner

(Figure 8i−k). Particularly, the plasma TC level in the AIFtreated mice (100 mg/kg) was decreased to 78% of the model mice (Figure 8j). Treatment with AIF has no effect on the body weight gain and food intake in DOI mice. Conclusions. In summary, 47 triterpenoids including 16 new compounds were obtained from the peels of W. cocos. Compounds 1 and 2 belong to 4, 5-secolanostanes triterpenes that are rarely reported in nature.43,56 Compound 3 was a novel aromatization lanostane triterpenoid with an unusual methyl rearrangement from C-10 to C-6. Compounds 3, 8, 10, 12, 14, 21, 23, 28, 29, 30, 33, and 35 exhibited the glucoseuptake-stimulating activity and insulin-sensitizing activity. The alkaline-insobule fraction prepared from the peels of W. cocos showed in vivo hypoglycemic and hypolipidemic activity. Most triterpenes from the peels of W. cocos were noncytotoxic toward K562, SW480, and HepG2 cells. Only compounds 23, 25, 29, and 31 showed weak cytotoxicity against K562 cells. The bioactive lanostane-type triterpenes contained in the peels of W. cocos deserve further investigation for their potentials in regulating hyperglycemia. The low cytotoxicity and good hypoglycemic effect of triterpenes from the peels extract of W. cocos support its potential as food supplement for diabetes patients or people with insulin resistance.

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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.jafc.9b02606. 7362


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Key NOE correlations of new compounds, NMR spectra of 1−16, CD spectrum of new compounds, structures of conformers 1 and 8 at B3LYP/6-31G(d)level in MeOH for calculated ECD, and HPLC chromatograph of AIF (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: +86 10 64806074. Fax: +86 10 64807515. *E-mail: [email protected]. ORCID

Hongwei Liu: 0000-0001-6471-131X Author Contributions ¶

B.C., J.Z.: These authors contributed equally to this work.

Funding

This work was financially supported by the National Key R&D program of China (No. 2018YFD0400203), the Strategic Biological Resources Service Network program of CAS (Grant ZSTH-016), and the National Natural Science Foundation of China (Grant 81773614, 81673334). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Dr. Jinwei Ren and Wenzhao Wang (State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences) are appreciated for their help in measuring the NMR data and Mass data.

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Lanostane Triterpenoids with Glucose-Uptake-Stimulatory Activity from

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