Colocynthenins A–D, Ring-A seco-Cucurbitane Triterpenoids from the

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Colocynthenins A−D, Ring‑A seco-Cucurbitane Triterpenoids from the Fruits of Citrullus colocynthis Yushuang Liu,†,‡ Guangying Chen,§ Xiaoyu Chen,†,‡ Shi-Xin Chen,∥ Li-She Gan,∥ and Tao Yuan*,†

J. Nat. Prod. Downloaded from pubs.acs.org by UNIV OF SOUTH DAKOTA on 09/05/18. For personal use only.

†

Key Laboratory of Plant Resources and Chemistry of Arid Zone and State Key Laboratory of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi 830011, People’s Republic of China ‡ University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China § Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou 571158, People’s Republic of China ∥ College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, People’s Republic of China S Supporting Information *

ABSTRACT: Four ring-A seco-cucurbitane triterpenoids, colocynthenins A−D (1−4), together with seven known cucurbitane triterpenoids (5−11), were isolated from the fruits of Citrullus colocynthis. Their structures and absolute configurations were elucidated based on spectroscopic analysis and quantum chemical ECD calculations. Compound 1 possesses an unprecedented 2,11-lactone moiety, while compound 2 is the first reported cucurbitane triterpenoid with an unusual cyano group. Compounds 1 and 3 showed acetylcholinesterase inhibitory activities in a standard in vitro assay, with IC50 values of 2.6 and 3.1 μM, respectively.

C

colocynthis fruits.10 As part of an ongoing project on bioactive compounds from traditional Uygur medicines,11 in the present study four new seco-cucurbitane triterpenoids, named colocynthenins A−D (1−4), as well as seven known cucurbitane triterpenoids (5−11) have been isolated from C. colocynthis fruits. The anti-acetylcholinesterase (AChE) activities of these compounds have been evaluated.

ucurbitane triterpenoids are a group of tetracyclic triterpenoids characterized by a 19-(10→9β)-abeo-10αlanost-5-ene skeleton that were originally obtained from the plant family Cucurbitaceae.1 Cucurbitane triterpenoids from Nature have been isolated with a variety of oxygenation functionalities at different positions. This class of triterpenoids has attracted the attention of the medicinal chemistry community attributed to their significant cytotoxic potencies.2 In addition to cytotoxicity, cucurbitane triterpenoids also show other bioactivities such as anti-inflammatory, antityrosinase, and antidiabetic effects.3 Citrullus colocynthis L. (Cucurbitaceae), also known as colocynth, desert gourd, and bitter apple, is a vine that is distributed widely in deserts across the world. In the People’s Republic of China, it is mainly planted in the Xinjiang Uygur Autonomous Region. C. colocynthis has been used traditionally to treat jaundice, diabetes, asthma, cancer, and pain.4−6 Pharmacological research has indicated that C. colocynthis shows broad biological effects, such as cytotoxicity and antioxidant, antidiabetic, and antimicrobial activities.7−9 Previous phytochemical investigations of this plant indicated that cucurbitane triterpenoids are the main components of C. © XXXX American Chemical Society and American Society of Pharmacognosy

Compound 1 was yielded as a light yellow, amorphous solid with [α]25D +13 (c 0.1, MeOH). The molecular formula of 1 Received: June 6, 2018

A

DOI: 10.1021/acs.jnatprod.8b00461 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products Table 1. 1H and

13

Note

C NMR Data of 1−4 (in CD3OD) 1a

no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Ac

δH (multi, J in Hz) 5.78 (s)

6.41 2.72 2.27 2.31

(dd, 2.9, 5.2) (m) (m) (dd, 3.4, 6.9)

2.35 (d, 13.8) 2.25 (d, 13.8)

1.83 1.28 4.56 2.37 1.19 1.14

(dd, 8.6, 12.7) (d, 12.7) (t, 7.6) (d, 6.4) (3H, s) (3H, s)

1.42 (3H, s) 6.83 (d, 15.8) 6.97 (d, 15.8) 1.56 1.57 1.43 1.48 1.26 2.03

(3H, (3H, (3H, (3H, (3H, (3H,

s) s) s) s) s) s)

2b δC 110.6 167.3 181.1 47.1 138.6 135.7 26.7 40.9 44.0 159.0 107.9 45.1 48.1 50.2 45.7 72.3 61.1 20.6 22.7 80.7 25.5 205.5 122.9 151.7 81.2 26.8 26.7 26.9 27.0 19.7 22.0 172.0

δH (multi, J in Hz) 5.00 (s)

6.41 (t, 3.9) 2.68 (m) 2.36 (dd, 4.7, 21.1)

3.21 (d, 14.3) 2.64 (d, 14.3)

1.89 1.42 4.55 2.59 0.89 1.22

(dd, 8.9, 13.2) (d, 13.2) (t, 7.7) (d, 7.2) (3H, s) (3H, s)

1.41 (3H, s) 6.85 (d, 15.9) 7.01 (d, 15.9) 1.58 1.59 1.49 1.55 1.30 2.02

(3H, (3H, (3H, (3H, (3H, (3H,

s) s) s) s) s) s)

3a δC 92.9 118.7 180.9 47.9 138.2 137.1 26.6 44.1 56.7 159.8 215.8 52.4 50.3 52.0 45.4 72.0 60.5 20.7 25.7 80.3 25.3 205.1 122.7 152.0 81.2 27.1 26.8 27.1 29.9 19.5 22.0 172.0

OMe

δH (multi, J in Hz) 5.37 (s)

6.24 2.43 2.27 2.09

(dd, 2.7, 6.1) (m) (dt, 3.1, 19.3) (dd, 3.4, 8.5)

3.09 (d, 16.7) 2.70 (d, 16.7)

1.88 1.31 4.51 2.51 0.96 1.17

(dd, 9.2, 13.2) (d, 13.2) (t, 7.7) (d, 7.2) (s) (3H, s)

1.38 (3H, s) 6.82 (d, 16.0) 6.97 (d, 16.0) 1.52 1.55 1.28 1.41 1.20 1.99

(3H, (3H, (3H, (3H, (3H, (3H,

s) s) s) s) s) s)

4b δC 117.9 169.8 180.9 47.5 141.8 134.4 26.4 44.1 57.1 156.0 217.9 53.8 50.3 49.7 46.2 72.0 61.4 21.5 27.4 80.3 25.4 205.3 122.7 151.9 81.2 27.0 26.6 26.8 30.2 19.3 22.0 172.0

δH (multi, J in Hz) 5.36 (s)

6.27 2.47 2.30 2.12

(dd, 2.6, 6.1) (m) (dt, 2.9, 19.5) (dd, 2.7, 8.5)

3.12 (d, 16.6) 2.71 (d, 16.6)

1.91 1.31 4.54 2.52 0.97 1.19

(dd, 9.1, 13.5) (d, 13.5) (t, 7.6) (d, 7.1) (3H, s) (3H, s)

1.40 (3H, s) 6.84 (d, 16.0) 6.97 (d, 16.0) 1.55 1.57 1.27 1.45 1.22 2.00

(3H, (3H, (3H, (3H, (3H, (3H,

s) s) s) s) s) s)

3.65 (3H, s)

δC 117.8 169.5 179.3 47.4 141.5 134.8 26.4 44.0 57.1 155.6 217.8 53.7 50.4 49.7 46.2 72.0 61.3 21.4 27.0 80.3 25.4 205.3 122.8 151.9 81.2 26.8 26.6 26.8 30.6 19.3 22.0 172.0 52.7

a

Recorded at 600 or 150 MHz for 1H and 13C, respectively. bRecorded at 400 or 100 MHz for 1H and 13C, respectively.

assigned to their corresponding carbons. The 1H−1H COSY (Figure 1a) correlations of H-6/H-7/H-8 disclosed the connection of C-6 to C-8 (bold bonds in Figure 1a). The HMBC spectrum (Figure 1a) was then used to establish the structure of the A′, B, and C rings. The HMBC correlations from H3-19 to C-8 (δC 40.9), C-9 (δC 44.0), C-10 (δC 159.0), and C-11 (δC 107.9); from H3-18 to C-12 (δC 45.1), C-13 (δC 48.1), and C-14 (δC 50.2); and from H3-30 to C-8, C-13, and C-14 supported both the structure proposed for the C ring and the connection of the C-6−C-8 fragment with C-9 and C-14 via C-8. The HMBC correlations from H-7 to C-5 (δC 138.6), from H-6 to C-10, and from H3-28 and H3-29 to C-3 (δC 181.1), C-4 (δC 47.1), and C-5 indicated that the B ring possesses a 2-carboxyl-2-propyl fragment at C-5. The C-2−C1−C-10 fragment was constructed by the HMBC correlations from H-1 to C-2 (δC 167.3), C-5, and C-9. The functionalities elucidated by the above-mentioned 1D and 2D NMR data accounted for 10 DBEs, and the remaining DBE required the presence of an additional ring in 1. Analysis of the 13C NMR data of C-2 (δC 167.3) and C-11 (δC 107.9) suggested that C-2 and C-11 are connected via an ester to form a lactone ring (A′)

was determined as C32H44O10 by its HRESIMS peak at m/z 611.2829 [M + Na]+ (calcd for C32H44O10Na, 611.2832), corresponding to 11 double-bond equivalents (DBEs). The IR spectrum of 1 displayed absorption bands for hydroxy (3380 cm−1) and carbonyl groups (1683 cm−1). The 1H NMR spectrum (Table 1) of 1 exhibited signals for eight tertiary methyl groups at δH 1.57, 1.56, 1.48, 1.43, 1.42, 1.26, 1.19, and 1.14 (each 3H, s), a trans double bond at δH 6.97 (d, J = 13.8 Hz) and 6.83 (d, J = 13.8 Hz), two olefinic protons at δH 6.41 (dd, J = 5.2, 2.9 Hz) and 5.78 (s), and an acetyl group at δH 2.03 (3H, s). Analysis of the 13C NMR and HSQC spectra revealed the presence of 32 carbons comprising nine methyls, three methylenes, seven methines (including four sp2 carbons), and 13 quaternary carbons (four of which are carbonyls). The above-mentioned spectroscopic data implied that compound 1 is a cucurbitane triterpenoid.12 Extensive analysis of its 1D and 2D NMR data disclosed that 1 has the same D ring and C-17 side chain as cucurbitacin E (5).12 Therefore, the key points of structure elucidation of 1 constructed its unusual tricyclic moiety (the A′, B, and C rings). The HSQC spectrum allowed all of the protons to be B

DOI: 10.1021/acs.jnatprod.8b00461 J. Nat. Prod. XXXX, XXX, XXX−XXX

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correlations. The HMBC correlations from H-6 to C-10, from H-7 to C-5, and from H3-19 to C-8, C-9, and C-10 supported the construction proposed for ring B. A 2-carboxyl-2-propyl moiety was assigned at C-5 based on the HMBC correlations from H3-28 and H3-29 to C-3, C-4, and C-5. A double bond, Δ1(10), was determined based on the HMBC correlations from H-1 to C-5, C-9, and C-10 and the 13C NMR data. The HMBC correlation from H-1 to a quaternary carbon (δC 118.7, C-2) suggested the linkage between C-1 and C-2. The fragments elucidated from these spectroscopic data accounted for C32H43O8 of the molecular formula, and the remaining nitrogen atom and quaternary carbon (C-2) were thus connected via a triple bond to form an unusual cyano group. The IR absorption band at 2216 cm−1 also supported the presence of a cyano group. Therefore, the planar structure of 2 was elucidated as depicted. The Δ1(10) double bond was determined to be in the E configuration by the NOESY correlation between H-1 and H-12. The relative configurations of other chiral centers were established as being the same as those of cucurbitacin E based on the NOESY correlations observed. Compound 3 was assigned a molecular formula of C32H44O10 on the basis of its HRESIMS ion at m/z 611.2821 [M + Na]+ (calcd for C32H44O10Na, 611.2832). Detailed analysis of its 1H and 13C NMR data (Table 1) indicated that the structure of 3 is similar to that of 2, with the only difference being the functional group at C-2. A carboxylic acid group at C-2 in 3 instead of the cyano group in 2 was determined by the HMBC correlation from H-1 (δH 5.37, s) to a carbon at δC 169.8 (C-2), which was also supported by the notable changes in the chemical shifts of the adjacent positions (C-1, C-5, and C-10). The structure of 3 was thus elucidated as depicted. The molecular formula of compound 4 was determined to be C33H46O10 based on the HRESIMS signal at m/z 625.2984 [M + Na]+ (calcd for C33H46O10Na, 625.2989), which was 14 mass units more than that of 3. The 1H and 13C NMR spectra of 4 were almost the same as those observed for 3, and the only obvious differences were the presence of signals for an additional methoxy group (δH 3.65, s; δC 52.7) in 4. This functionality is connected to C-3 based on the HMBC correlation from the methoxy proton signal to C-3. Thus, compound 4 was determined to be the methyl ester of 3. The absolute configurations of compounds 2−4 were assigned tentatively as depicted based on the biogenetic relationship of cucurbitane triterpenoids1 and the relative configurations discussed above. To confirm the assignments, quantum chemical time-dependent density functional theory (TDDFT) calculations of the theoretical electronic circular dichroism (ECD) spectra of compounds 1−3 were carried out.13 As shown in Figure 3, both the calculated and experimental ECD spectra of compounds 1−3 showed the same major Cotton effects at approximately the same wavelengths. Although the calculated ECD spectrum of 1 was somewhat shifted, the main Cotton effects were similar to those in the experimental spectrum. Therefore, qualitative analysis of the calculated and experimental ECD spectra allowed the assignments of the absolute configurations of compounds 1−3 in accordance with the above assignment. Accordingly, compound 4 has the same absolute configuration as 3. Therefore, the structures and absolute configurations of compounds 1−4 were determined as depicted, and they were given the trivial names colocynthenins A−D (1−4).

Figure 1. (a) Key 1H−1H COSY (bold lines) and selected HMBC correlations (H → C) of 1. (b) Key ROESY correlations of 1.

with a hemiketal at C-11. Thus, the planar structure of 1 was elucidated to be a ring-A seco-cucurbitane triterpenoid, as depicted in Figure 1. The relative configuration of 1 was established from its ROESY spectrum (recorded in DMSO-d6). The ROESY correlations (Figure 1b) from OH-11 to H3-18 and H3-19; H3-19 to H-8; H-8 to H3-18; and H3-18 to H-16 indicated that these groups are cofacial, and so they were assigned as βoriented. Consequently, the ROESY correlations from H3-18 to H3-21 and from H-17 to H3-30 indicated that H-17 and CH3-30 are α-oriented. As compound 1 has the same C-17 side chain as that of cucurbitacin E (5), the C-20 was assigned tentatively as R* based on the ROESY correlation from OH-20 to H-16. Compound 2 showed a sodiated molecular ion [M + Na]+ at m/z 592.2877 (calcd for C32H43NO8Na, 592.2886) in its HRESIMS, corresponding to the molecular formula C32H43NO8. Comparison of the NMR (Table 1) data of 2 with those of cucurbitacin E indicated that they have the same C and D rings as well as the same C-17 side chain. Extensive analysis of the 2D NMR data of 2 permitted the determination of its structure. Similar to compound 1, the connection of C-6 to C-8 was determined from the 1H−1H COSY (Figure 2)

Figure 2. Key 1H−1H COSY (bold lines) and selected HMBC correlations (H → C) of 2. C

DOI: 10.1021/acs.jnatprod.8b00461 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 3. Experimental (black line) and B3LYP/6-311++G(2d,2p)//B3LYP/6-31G(d) calculated (red line) ECD spectra of 1−3. with tetramethylsilane as an internal standard. HRESIMS data were acquired using a micrOTOF-Q II mass spectrometer (Bruker). Semipreparative HPLC separations were performed on a Hitachi Chromaster system consisting of a 5110 pump, 5210 autosampler, 5310 column oven, 5430 diode array detector, and YMC C18 column (250 × 10 mm, S-5 μm, YMC Co., Kyoto, Japan) all operated by EZChrom Elite software. All solvents were of ACS or HPLC grade and were obtained from Tianjin Zhiyuan Chemical (Tianjin, People’s Republic of China) and Sigma-Aldrich (St. Louis, MO, USA), respectively. Silica gel (300−400 mesh), C18 reversed-phase silica gel (150−200 mesh, Merck), and MCI gel (CHP20P, 75−150 μM, Mitsubishi Chemical Industries Ltd.) were used for column chromatography, and precoated silica gel GF254 plates (Qingdao Marine Chemical Plant, Qingdao, People’s Republic of China) were used for TLC. 5,5′-Dithiobis-2-nitrobenzoic acid, acetylcholinesterase, and sodium dodecyl sulfate were purchased from Sigma, acetylthiocholine iodide was purchased from Adamas (Shanghai, People’s Republic of China), and huperzine A was purchased from Naturestandard (Shanghai, People’s Republic of China). Plant Material. The fruits of Citrullus colocynthis were purchased from Xinjiang Uyghur Medicine Hospital (Xinjiang, People’s Republic of China) and were identified by Prof. Yan Wei, College of Grassland and Environment Sciences, Xinjiang Agricultural University. A voucher specimen (CC-201710) was deposited in the Key Laboratory of Plant Resources and Chemistry of Arid Zone, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences (Xinjiang, People’s Republic of China). Extraction and Isolation. An air-dried powder of C. colocynthis fruit (2.0 kg) was extracted with methanol (8 L × 3) by maceration at room temperature (7 days each time) to afford 335.7 g of a crude methanol extract. The extract was suspended in distilled water and then extracted successively with petroleum ether, ethyl acetate, and nbutanol. The ethyl acetate fraction (37.4 g) was chromatographed over MCI gel (MeOH−H2O, 30:70 to 100:0, v/v) to yield 10 fractions (A−J). Fraction F (4.2 g) was chromatographed over an

In addition, seven known compounds were identified as cucurbitacin E (5),14 6′-acetyl-2-O-β-D-glucocucurbitacin E (6),14 arvenin I (7),15 arvenin II (8),15 cucurbitacin B (9),15 23,24-dihydrocucurbitacin B (10),15 and coloynthoside A (11),10a by comparison of their NMR data with the reported data in the literature. All of the isolates and a positive control, huperzine A, were tested for their AChE inhibitory activities. As is well known, AChE is a target for Alzheimer’s disease. The results indicated that compounds 1 and 3 showed significant anti-AChE activities with IC50 values of 2.6 and 3.1 μM, respectively, which were somewhat less potent than that of huperzine A (IC50 0.4 μM). The other tested compounds exhibited no discernible inhibitory activities at 10 μM. In summary, this work presents the discovery of four new ring-A-modified seco-cucurbitane triterpenoids, and, among these compounds, 1 possesses an unprecedented 2,11-lactone moiety, while 2 was isolated as the first cucurbitane triterpenoid with an unusual cyano group. Biosynthetic analysis suggested that an enzymatic Baeyer−Villiger reaction is a key step in the generation of these new compounds. Biological evaluation showed that compounds 1 and 3 exhibited antiAChE activities, when evaluated in vitro.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on an Autopol VI automatic polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA) at room temperature. The IR spectra were recorded on a Nicolet 6700 (Thermo Fisher Scientific) spectrometer. The UV spectra were measured on a Shimadzu UV-2550 UV−visible spectrophotometer. 1D and 2D NMR data were recorded on Varian 400 and 600 MHz instruments D

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open ODS column eluted with MeOH−H2O (40%, 60%, 80%, 95%, v/v) to give fractions F1−F6 and compound 7 (333.2 mg). Fraction F3 (0.9 g) was subjected to separation over a silica gel chromatographic column eluted with CH2Cl2−MeOH (30:1 to 5:1 v/v) and then purified by semipreparative HPLC (MeOH−H2O, 0−25 min: 49:51; 25−26 min: 62:38 to 100:0; 26−27 min: 100:0; 27−28 min: 100:0 to 49:51; 28−35 min: 49:51; v/v, 3 mL/min) to yield compounds 9 (79.7 mg) and 11 (18.4 mg). Fraction F5 (1.1 g) was separated on a silica gel column eluted with CH2Cl2−MeOH (20:1 to 15:1 v/v) to yield compound 8 (796.0 mg). Fraction I (2.3 g) was further separated on a column of RP-18 silica gel (MeOH−H2O, 60:40 to 80:20, v/v) to give nine fractions (I1−I9). Fraction I6 was subjected to silica gel column chromatography (CC) and eluted with a gradient of CH2Cl2−MeOH (50:1 to 5:1 v/v) to obtain seven fractions (I6a−I6g). Purification of fraction I6g (124.0 mg) by semipreparative HPLC, eluting with MeOH−H2O (0−21 min: 60:40; 21−22 min: 60:40 to 100:0; 22−23 min: 100:0; 23−24 min: 100:0 to 60:40; 24−31 min: 60:40; v/v, 3 mL/min), yielded compounds 3 (11.8 mg) and 5 (30.8 mg). Fraction I6d (0.8 g) was separated by silica gel CC eluted with a gradient of CH2Cl2−MeOH (40:1 to 5:1 v/v) afforded six fractions (I6d1−I6d6). Fraction I6d5 (99.3 mg) was purified by semipreparative HPLC, eluting with MeOH−H2O (0−21 min: 60:40; 21−22 min: 60:40 to 100:0; 22−23 min: 100:0; 23−24 min: 100:0 to 60:40; 24−31 min: 60:40; v/v, 3 mL/min), to yield compound 1 (4.1 mg). Fraction J (1.7 g) was chromatographed over a column of RP-18 silica gel (MeOH−H2O, 60:40 to 100:0, v/v) to afford six subfractions (J1−J6). Fraction J5 (760.9 mg) was purified by silica gel CC and eluted with a gradient of CH2Cl2−MeOH (70:1 to 3:1 v/v) to give five fractions (J5a−J5e). Fraction J5e (299.8 mg) was purified on a column of Sephadex LH-20 eluted with MeOH to give four fractions (J5e1−J5e4). Purification of fraction J5e2 (80.3 mg) by semipreparative HPLC, eluting with MeOH−H2O (0−25 min: 66:34; 25−26 min: 66:34 to 100:0; 26−27 min: 100:0; 27−28 min: 100:0 to 66:34; 28−35 min: 66:34; v/v, 3 mL/min), yielded compounds 4 (8.7 mg) and 2 (5.5 mg). Compound 10 (18.0 mg) was obtained from fraction J5e4 (287.0 mg) through semipreparative HPLC by elution with MeOH−H2O (0−23 min: 64:36; 23−24 min: 64:36 to 100:0; 24−25 min: 100:0; 26−27 min: 100:0 to 64:36; 27− 34 min: 64:36; v/v, 3 mL/min). Fraction J6 (120.6 mg) was purified on a Sephadex LH-20 column eluted with MeOH to yield compound 6 (69.6 mg). Colocynthenin A (1): yellow, amorphous solid; [α]20D +13 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 235 (4.19), 284 (4.05) nm; IR νmax 3380, 2973, 1683, 1654, 1456, 1261, 1050, 800 cm−1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 611.2829 [M + Na]+ (calcd for C32H43O10Na, 611.2832). Colocynthenin B (2): yellow, amorphous solid; [α]20D +84 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 205 (4.26), 225 (4.31), 242 (4.25), 274 (4.10) nm; IR νmax 3421, 2975, 2879, 2216, 1696, 1684, 1456, 1368, 1261, 1203, 1022, 800 cm−1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 592.2877 [M + Na]+ (calcd for C32H42NO8Na, 592.2886). Colocynthenin C (3): colorless, amorphous solid; [α]20D +106 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 205 (4.40), 225 (4.33), 274 (4.10) nm; IR νmax 3446, 2975, 1684, 1418, 1368, 1261, 1019, 799, 722 cm−1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 611.2821 [M + Na]+ (calcd for C32H43O10Na, 611.2832). Colocynthenin D (4): yellow, amorphous solid; [α]20D +91 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 205 (4.23), 225 (4.31), 242 (4.29), 274 (3.88) nm; IR νmax 3482, 2937, 1688, 1629, 1435, 1368, 1262, 1202, 1137, 1022, 840, 722 cm−1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 625.2984 [M + Na]+ (calcd for C33H45O10Na, 625.2989). In Vitro AChE Inhibitory Activity Assay. The AChE inhibitory activities of compounds 1−11 were measured by the Ellman method.16 Detailed procedures are shown in the Supporting Information.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00461. Copies of spectra for compounds 1−4 and ECD calculations of compounds 1−3 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax (T. Yuan): 86-991-3690335. E-mail: yuantao@ms. xjb.ac.cn. ORCID

Tao Yuan: 0000-0001-7746-3714 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (81773998, U1703331, 21502222), Xinjiang Key Research and Development Program (2016B03038-3), and the Recruitment Program of Global Experts (to T.Y.), China.

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REFERENCES

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DOI: 10.1021/acs.jnatprod.8b00461 J. Nat. Prod. XXXX, XXX, XXX−XXX