Polyhydroxy cucurbitane triterpenes from Hemsleya penxianensis tubers

Ten new cucurbitane triterpenoids, hemsleyacins A–J (1–10), together with three known cucurbitane triterpenoids, dihydrocucurbitacin F (11), scandenogenin D (12), and jinfushanencin F (13), were separated from ethanolic tuber extracts of Hemsleya penxianensis. The absolute configurations of the new compounds were established based on NMR, HRESIMS, and CD spectra. Compounds 7 and 10–12 were evaluated in terms of their antifeedant activity against Plutella xylostella larvae. The result showed that compound 10 exhibited potent antifeedant activity against P. xylostella larvae after 48 h of treatment. Furthermore, the MTT test showed that compound 11 exhibited potent inhibition toward the UMUC-3 and T24 cell lines with IC50 values of 29.12 and 35.62 μM, respectively, compared to the positive control cisplatin IC50 values of 8.27 and 13.72 μM. Western blot analysis revealed that compound 11 treatments substantially inhibited the phosphorylation of IκBα.

The proposed structure was further confirmed by an evaluation of the 2D NMR data, including 1 H-1 H COSY, HSQC, and HMBC experiments. In the HSQC spectra, the olefinic proton signal at H-6 (δ H 5.71) was correlated with the olefinic carbon signal at C-6 (δ C 119.0). Together with the HMBC correlations (Fig. 2) from H-7 (δ H 1.87) and H-8 (δ H 1.93) to the olefinic carbon (C-6, δ C 119.0), this suggested that the double bond was linked to C-5 and C-6. One of the carbonyl carbon signals at C-11 (δ C 213.7) was verified by the HMBC correlations from both H 3 −19 and H-10, while the other carbonyl carbon signal at C-22 was correlated with H 3 -21. The positions of the methyl groups were established based on the HMBC correlations from δ H 1.16 (CH 3 -29) and δ H 1.73 (CH 3 -28) to C-3 (δ C 81.9) and C-4 (δ C 43.2), δ H 1.28 (CH 3 -19) to C-11(δ C 213.7), δ H 1.44 (CH 3 -30) to C-14 (δ C 54.3), δ H 2.36 (CH 3 -21) to C-17 (δ C 121.8), C-20 (δ C 146.6), and C-22 (δ C 200.4), and δ H 1.10 (CH 3 -26) and δ H 1.09 (CH 3 -27) to C-25 (δ C 79.0). An examination of the HMBC spectrum revealed that the cross-peaks from CH 3 -21 to C-17 (δ C 121.8), C-20 (δ C 146.6), and C-22 (δ C 200.4) were indicative of an α,β-unsaturated carbonyl moiety. Furthermore, the correlations from H-16 (δ H 3.93) to C-17 (δ C 121.8), and C-20 (δ C 146.6) and C-23 (δ C 71.7) suggested that the six-membered E-ring was closed via C-16/O/C-23, which is consistent with the degree of unsaturation. Finally, three hydroxyl groups were present at C-2, C-3, and C-25, respectively, based on the downfield chemical shifts of δ C 71.2, 81.9, and 79.0 together with the molecular formula C 30 H 44 O 6 above. The NOESY experiment and coupling constants determined the relative configuration of compound 1 in which NOESY correlations of H-19 and H-3, and H-10 and H-2 suggested an α-configuration for OH-3 and β-configuration for OH-2 (Fig. 3). The   configuration has been fully determined, indicated the S, R, R, R, and S configurations of C-8, C-9, C-10, C-13, and C-14, respectively. Compound 1 has a new cucurbitacin triterpene skeleton with an α, β-unsaturated carbonyl six-membered ring through an oxygen bridge between C-16 and C-23. Biogenetically, compound 1 could be converted from the hypothetical precursor 1a (Fig. 4). Oxidation of the C-23/C-24 double bond will produce the epoxy derivative 1b. Dehydration in C-16 forms a six-membered ring (E-ring) in 1c. Then, 1c immediately undergoes dehydration and finally generates compound 1. Thus, the structure of 1 was identified and it was called hemsleyacin A. . The 13 C NMR spectrum contained 32 signals, including nine angular methyls, five methylenes, seven methines (one sp 2 methines and four oxygenated methines), and 11 quaternary carbons (four sp 3 carbons, one sp 2 carbon, four carbonyl carbons, and two oxygenated carbons). 1 Based on a comparison of their NMR data, compound 2 has the same tetracyclic triterpenic nucleus as, but they differ in their side chains. The NMR spectral data indicated that the ring of the side chain in compound 1 was cracked in compound 2, and an acetyl group was present at C-16 as was confirmed by the HMBC correlations from H-16 (δ H 5.84) to an oxygenated carbon δ C 171.0. Furthermore, the data suggested that the double bond at C-17/C-20 had disappeared, and hydroxyl groups were located at C-20 and C-24, respectively. Further analysis of the HMBC data, the correlations from H-26 and H-27 to δ C 75.0 (C-24), H-21 to δ C 55.2 (C-17) confirmed the above deduction. The cross peaks of H-17/Me-21 and H-16/ Me-30/H-20 in the NOESY spectrum suggested that the side chain H-16, and H-20 were β-oriented, respectively. Compared with the carbon data of leucopaxillones A at C-24 (δ C 75.0), the relative configuration of H-24 was determined as that of α-orientation 12 . Thus, the structure of compound 2 was determined and it was called hemsleyacin B.
Compound 3 was separated as a white amorphous powder and had a molecular formula of C 33 H 52 O 9 based on HR-ESIMS (m/z 615.3527 [M + Na] + , calculated for C 33 H 52 O 9 Na, 615.3509). The 1 H NMR and 13 C APT data were www.nature.com/scientificreports www.nature.com/scientificreports/ similar to those of 2 (Tables 1 and 2). One difference was that the carbon signal of 2 at C-24 (δ C 75.0) was moved downfield by δ C 85.0 (C-24) in 3, and the molecular weight of 3 in HR-ESIMS was larger by 14 units than that of 2, both of which suggested that the hydroxyl group at C-24 in 2 was replaced by an oxymethyl group in 3. The NMR spectra revealed an extra proton signal at δ H 3.62 (3 H, s) and a carbon signal at δ C 60.5, and the HMBC spectrum (Fig. 2) revealed a correlation between δ H 3.62 (3 H, s) and δ C 85.0 (C-24), supporting the deduction above. The relative configuration of 3 was consistent with 2 based on a comparison of their NOESY spectral data. Therefore, the structure of compound 3 was determined, and it was called hemsleyacin C.
Compound 4 was isolated as a white powder. Its molecular formula was C 32 H 52 O 8, as inferred from the HR-ESIMS (m/z 587.3577 [M + Na] + , calculated for C 33 H 52 O 9 Na, 587.3560). The 1 H NMR and 13 C APT data of 4 were comparable with those of 2 (Tables 1 and 2). A comparison of 4 and 2 demonstrated that the carbon signals at C-16 (δ C 70.6), C-11 (δ C 77.0), and C-24 (δ C 35.3) were shifted upfield, while the signal at C-25 (δ C 81.4) was shifted downfield. These differences suggested that in 4, the carbonyl group at C-11 in 2 was substituted by a hydroxyl group, the acetyl group at C-16 and hydroxyl group at C-25 found in 2 exchanged their positions and the methoxyl group at C-24 was absent. The determination of the relative configurations of the methyl groups and other protons in the tetracyclic ring based on the significant NOE correlation between H-3 (δ H 3.47) and H 3 -29 (δ H 1.33), H-11 and CH 3 -19 revealed their β-orientation. Considering the identical biogenetic of cucurbitacin triterpenes, the absolute configuration of C-11 was R. Thus, compound 4 was identified and was called hemsleyacin D.
Compound 5 was isolated as a white powder and corresponded to the molecular formula C 32 H 48 O 9 based on the pseudomolecular ion at m/z 599.3196 (calculated for C 32 H 48 O 9 Na [M + Na] + , 599.3212), indicating nine degrees of unsaturation. The IR spectrum showed absorption bands corresponding to hydroxy (3411 cm -1 ) and unsaturated carbonyl (1719 cm −1 ) groups. The 1 H and 13 C APT spectra (Tables 1 and 2) of 5 were similar to those of 4 but differed in that the protons at C-7 and hydroxyl group at C-11 in 4 were all oxidized to carbonyl groups in 5. The characteristic carbon signals in the 13 C APT spectrum at C-7 (δ C 200.2) and C-11 (δ C 211.5) in combination with the HMBC correlations from H-6 (δ H 6.63, s) to and H 3 −19 (δ H 1.27, s) to C-8 (δ C 59.3) supported the above result. Based on this, the structure of compound 5 was determined and it was called hemsleyacin E.
Compound 6 possessed a molecular formula of C 34 H 50 O 10 , as indicated by the HR-ESIMS and NMR examinations. The 1 H and 13 C NMR (Tables 1 and 2) data of 6 were similar to those of compound 5, even though the  www.nature.com/scientificreports www.nature.com/scientificreports/ hydroxyl group at C-16 found in 5 was replaced by an acetyl group in 6. The extra NMR signals at δ H 2.13, and δ C 21.6 and 170.9 confirmed the existence of an additional acetyl group, while the HMBC correlations (Fig. 2) from H-16 to δ C (170.9) supported the position of the acetyl group at C-16. Therefore, compound 6 was identified and was called hemsleyacin F.  H 2 -24), δ C 151.9 (C-24), and δ C 121.6 (C-23) and the upfield chemical shift of carbonyl carbon at C-22 (δ C 204.5). These differences suggested the existence of a double bond between C-23 and C-24 that was conjugated with the carbonyl carbon at C-22. The HMBC correlations (Fig. 2) from C-16 to C-23 validated the above result. Compound 7 was thus identified and was named hemsleyacin G.
Compound 8 had a pseudomolecular ion peak at m/z 599.3143 (calculated for C 32 H 48 O 9 Na, 599.3196) [M + Na] + in the positive ion HR-ESIMS with a molecular formula of C 32 H 48 O 9 . The 1 H and 13 C APT spectroscopic data (Tables 1 and 2) of 8 matched those of 7, except for the rearrangement of an α, β-unsaturated ketone at C-5/C-6/C-7. The olefinic data of H-6 (δ H 6.37, d, J = 9.0 Hz) and H-7 (δ H 5.65, m), C-6 (δ C 131.7) and C-7 (δ C 130.4) suggested that the carbonyl group at C-7 in 7 was reduced and dehydrated and formed this double bond in 8. The downfield chemical shift of C-5 (δ C 73.9) in combination with the molecular formula C 32 H 48 O 9 revealed that the double bond at C-5/C-6 in 7 was also reduced, and one hydroxyl group was placed at C-5 in 8. The HMBC correlations (Fig. 2)  www.nature.com/scientificreports www.nature.com/scientificreports/ fewer degrees of unsaturation than 8. A comparison of the NMR spectra of 9 with that of 8 indicated that their structures were similar, except that the carbon signals of C-16 (δ C 71.1), C-23 (δ C 33.1), and C-24 (δ C 38.9) in 9 were shifted upfield. These differences suggested that the double bond at C-23/C-24 in 8 was absent in 9 and that at C-16, a hydroxyl group was present in 9 instead of the acetyl group in 8. In the HMBC spectrum, the correlation between the proton signals of H 2 -24 (δ H 2.18) and C-22 (δ C 216.2), and H 2 -23 (δ H 3.28) and C-25 (δ C 72.5) confirmed the lack of a double bond at C-23/C-24 in 9. The relative configuration of 9 was deduced by the NOESY spectrum. Based on these results, compound 9 was named hemsleyacin I.
Compound 10 was purified as a white powder with a molecular formula of C 32 H 50 O 9 based on the HR-ESIMS ion peak at m/z 601.3328 [M + Na] + (calculated for C 32 H 50 O 9 Na, 601.3353). The NMR spectroscopic data of 10 closely matched those of 9, except that the appearance of an additional acetyl signal (δ C 82.0, C-25). C-25 (δ C 82.0) was downfield when compared with C-25 (δ C 69.5) of 9, suggesting that the hydroxyl group at C-25 in 9 was substituted by a single acetyl group in 10 in the 13 C APT NMR spectrum. The HMBC correlations (Fig. 2) from H 3 -26 (δ H 1.53, s) to δ C 170.6 together with the molecular formula confirmed the above deduction. Thus, compound 10 was identified and named hemsleyacin J.
The antifeedant activities of compounds 7 and 10-12 were evaluated against Plutella xylostella larvae. The result showed that compound 10 exhibited potent antifeedant activity against P. xylostella larvae after 48 h of treatment with the highest antifeedant rate of 43.57% at the concentration of 0.5% (Table 3). Furthermore, the cytotoxicity of all of the isolated compounds was evaluated against the UMUC-3 and T24 bladder cancer cell lines according to the MTT procedure. The results (  13.72 μM). To assess whether the inhibitory activity of dihydrocucurbitacin F on bladder cancer cells was associated with the cessation of the NFκB pathway, the phosphorylation of IκBα in T24 and UMUC-3 cells was detected by western blot analysis with p-IκBα-specific antibodies after the cells were exposed to dihydrocucurbitacin F for 48 h. The results revealed that dihydrocucurbitacin F treatment substantially inhibited the phosphorylation of IκBα (Fig. 5).
Methods experimental procedures. IR spectra and UV absorption spectra were measured using FTIR-8400S and Shimadzu UV2550 spectrometers, respectively. Optical rotations were determined using a Jasco P-1010 polarimeter. TNMR spectra were recorded with a Bruker AV III 600 NMR spectrometer using trimethylsilyl (TMS) as the internal standard. HR-ESIMS spectra were obtained using a LTQ-Obitrap XL spectrometer. Preparative HPLC was performed using a Shimadzu analytic LC furnished with two LC-6AD pumps, a SPD-20A UV detector,    where T is the consumption of the leaf in the treatment and C is the consumption of the leaf in the control. cytotoxicity assays. The cytotoxicities of the tested compounds (1-13) were determined using the MTT assay. The UMUC-3 and T24 cancer cells were cultivated in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum and cultured at a density of 1.2 × 104 cells/mL in a 96-well microtiter plate. Subsequently, five different concentrations of each compound, resuspended in dimethyl sulfoxide (DMSO), were placed into the wells. Three replicates of each concentration were tested. Following incubation at 5% CO 2 at 37 °C for 48 h, 10 μL of MTT (4 mg/mL) was inserted into each well, following which the cells were incubated for a further 4 h. The liquid in each well was then extracted and replaced with DMSO (200 μL). Using a microplate reader, absorbance was measured at a wavelength of 570 nm.
Western blot analysis. The total proteins of the UMUC-3 and T24 cells were extracted with ice-cold RIPA buffer combined with protease and phosphatase inhibitor cocktail (Cowin Biotech Co., Ltd., Beijing, China). Equal amounts of total cell lysates were boiled in Laemmli SDS sample buffer, separated by 10% SDS-PAGE and then transferred to polyvinylidene difluoride membranes (PVDF). Following blocking with 5% nonfat milk for 2 h, the membrane was probed with primary antibody at 4 °C overnight and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody. Using a commercial ECL kit (Cowin Biotech Co., Ltd), the target bands were visualized by enhanced chemiluminescence.