Five New Cucurbitane-Type Triterpenoid Glycosides from the Rhizomes of Hemsleya penxianensis with Cytotoxic Activities.

Five new cucurbitane-typetriterpenoid glycosides, named Xuedanoside F-J (1-5), were obtained from the rhizomes of Hemsleya penxianensis (Xue dan), which belongs to the family of Cucurbitaceae. These new compounds were elucidated byspectroscopic analysis, including 1D, 2D NMR, and HR-ESI-MS spectra. Additionally, all the isolates were evaluated for cytotoxicity against three human cancer cell lines (Hela, MCF-7, and A-549) with the IC50 ranging from 2.25 to 49.44 µM in vitro with treatment 48 h and showed low cytotoxicity in human normal liver L-02 cells (IC50 > 50 µM). Compound 5 showed the most significant cytotoxic activity with the IC50 value of 2.25, 4.72, and 5.33 µM in 48 h, respectively.

1. Introduction

Hemsleya pengxianensis W.J. Chang, a native plant andwidely distributed in the south-west provinces of China, belongs to the genus of Hemsleya in Cucurbitaceae family [1]. It is also called “Xue dan” dialectally and has been used as a traditional Chinese medicine for a long time [2]. The tubers of H. pengxianensis have been dispensed for a variety of ailments including bacillary dysentery, sore throats, stomachaches, toothaches, diarrhea, ulcers, jaundice, bronchitis, chronic cervicitis, and tuberculosis [2,3,4].

Previous phytochemical reports have indicated that Hemsleya spp.possess rich terpenoid compounds including diterpenes, triterpenes, and particularly cucurbitane triterpenoid glycosides, which are efficient in the cureof all kinds of inflammation and cancers [5,6,7,8,9,10]. In prior research, our studies led to the disclosure of nineteen new cucurbitane-type triterpenoids that have shown significant anti-tumor cytotoxicity [11,12,13]. Recently, further study of H. pengxianensis has found another five new cucurbitane triterpenoid saponins named Xuedanoside F-J (1–5) (Figure 1), which were isolated from the rhizomes of H. penxianensis. In this paper, we report the isolation and structure identification of Xuedanoside F-J and evaluate their cytotoxic activity against human cancer cell lines.

Figure 1

Structures of compounds 1–5.

2. Results

Compound 1 was isolated as an amorphous white powder with +60.5 (c 0.1, MeOH). The molecular formula was determined as C36H56O11 according to the molecular ion peak at m/z [M + Na]+ 687.3725in the HR-ESI-MS (calculated for 687.3720 C36H56NaO11). Its IR data displayed absorptions for hydroxyl (3565–3340 cm-1) and carbonyl (1651 and 1687 cm-1) groups. Acid hydrolysis of 1 with HCl gave D-glucose as the constituent unit, which was tested by GC analysis. D-Glucose (t = 25.5 min) was detected by comparison with authentic monosaccharide. The configuration of the glycosidic bond was β on the basis of the coupling constant of the anomeric proton at δH 4.80 (d, J = 6.0 Hz). The 1H-NMR data (Table 1) revealed the existence of seven angular methyl signals at δH 1.26 (s), 1.21 (s), 1.44 (s), 1.91 (s), 1.34 (s), 1.28 (s), and 1.45 (s); four oxygenated methines at δH4.08 (m), 4.20 (m), 3.40 (d, J = 12.0 Hz), 5.06 (m), and 5.19 (t, J = 6.0 Hz); and two olefinic proton signals at δH 5.68 (m) and 6.89 (d, J = 6.0 Hz). The 13C APT NMR data (Table 2) showed 36 carbon signals due to 7 methyls (δC21.4, 21.8, 23.2, 22.4, 23.6, 26.7, 31.4), 6 methylenes, 9 methines and 8 quaternary carbons (including 2 olefinic carbon at δC 143.7, 135.7 and one carbonyl carbons at δC 214.4), of which 30 were assigned to the aglycon part, and the remaining 6 were ascribed to the sugar moiety. All assignments of proton signals achieved by 1H and 13C correlations in the HSQC spectrum. The IR and 1H and 13C-NMR spectra data identified that compound 1 is an oxygenated cucurbitane triterpenoid glycoside derivative [11]. The connectivities of compound 1 were deduced mainly by 1H-1H COSY and HMBC spectra (Figure 2). Analysis of the HMBC spectrum (Supplementary Materials), the correlations from δH3.40 (H-3) to δC 72.2 (C-2) and δC 44.1 (C-4), and δH 2.95 (H-17) to δC 80.8 (C-20) suggested the presence of hydroxyl groups at C-2, C-3, and C-20, respectively. Besides, HMBC correlations of H-6 with C-5 (δC 143.7) and C-7 (δC 22.5), H-24 with C-23 (δC 71.8), C-25 (δC 135.7), and C-27 (δC 23.2), H-12 with C-11 (δC 214.4) implied that olefinic groups were at C-5 and C-25, and a carbonyl group was at C-11. Comprehensive comparison of the NMR data of 1 with those of the known compound hemslelis A [10] suggested that compound 1 was an analogue of hemslelis A, except that compound 1 contained one D-glucose and lost a carbonyl group at C-7. The location of the sugar unit was located at C-26 by an O atom due to the HMBC correlations (Figure 2) from the proton signal at δH 4.85 (H-26) to anomeric carbon at δC 103.9, and the signal for C-26 revealed a powerful downfield shift to δ 68.0 (+6.8 ppm). In the NOESY spectrum (Supplementary Materials), correlations from H-2 to H-10, H-3 to H-19 indicated that OH-2 was β-oriented, and OH-3 was α-oriented, respectively. Furthermore, the 3J coupling constant (J = 12.0 Hz) verified the antiperiplanar link between H-2 and H-3. NOESY correlations from H3-18 to H-16 corroborated that these protons were inthe β-orientation, and the coupling constant (J = 12.0 Hz) also supported the antiperiplanar relationship between H-16 and H-17. The six-member ring through O atom between C-16 and C-23 suggested the synperiplanar conformation of H-16 and H-23. Therefore, taken along with 1H-1H COSY, HSQC, HMBC, and NOESY spectra (Supplementary Materials), the structure of compound 1 was established as 2β, 3α, 20β-trihydroxycucurbita-16α-23α-epoxy-5, 24(E)-diene-11-one-26-O- D-glucopyranoside and it was named Xuedanoside F.

Table 1

1H-NMR Spectra Data (600 MHz, pyridine-d5) for Compounds 1–5 (δH in ppm, J in Hz).

Position	1	2	3	4	5
1	1.52 (1H, m)2.44 (1H, m)	2.07 (1H, m)1.72 (1H, m)	1.75 (1H, m)1.67 (1H, m)	2.92 (1H, m)2.01 (1H, m)	2.06 (1H, m)1.62 (1H, m)
2	4.08 (1H, m)	2.05 (1H, m) 1.89 (1H, m)	2.37 (1H, m)1.90 (1H, m)	2.41 (1H, m) 2.02 (1H, m)	1.85 (1H, m)2.38 (1H, m)
3	3.40 (1H, d, 12.0)	3.70 (1H, s)	3.62 (1H, s)	3.67 (1H, s)	3.70 (1H, s)
6	5.68 (1H, m)	5.66 (1H, d, 12.0)	5.52 (1H, d, 6.0)	5.49 (1H, d, 6.0)	6.31 (1H, s)
7	1.85 (1H, m)2.28 (1H, m)	2.24 (1H, m)1.78 (1H, m)	1.81 (1H, m)1.94 (1H, m)	2.29 (1H, m)1.70 (1H, m)
8	1.93 (1H, m)	1.84 (1H, m)	1.80 (1H, m)	1.62 (1H, m)	2.62 (1H, s)
10	2.66 (1H, m)	2.54 (1H, d, 14.4)	2.47 (1H, m)	2.79 (1H, d, 10.8)	2.98 (1H, m)
11				4.18 (1H, m)
12	2.64 (1H, m)3.17 (1H, d, 12.0)	3.21 (1H, d, 14.4)2.68 (1H, d, 14.4)	2.49 (1H, m)2.94 (1H, d, 12.0)	2.12 (1H, m)2.07 (1H, m)	2.94 (1H, d, 18.0)2.52 (1H, d, 12.0)
15	1.61 (1H, m)1.92 (1H, m)	1.90 (1H, m)1.62 (1H, m)	1.30 (1H, m)1.38 (1H, m)	1.24 (1H, m)1.07 (1H, m)	1.40 (1H, m)1.80 (1H, m)
16	5.06 (1H, m)	5.21 (1H, t, 6.0)	1.27 (1H, m)2.13 (1H, m)	1.87 (1H, m)1.18 (1H, m)	1.26 (1H, m)1.88 (1H, m)
17	2.14 (1H, d, 12.0)	2.16 (1H, d, 9.0)	1.68 (1H, m)	1.61 (1H, m)	1.64 (1H, m)
18	1.26 (3H, s)	1.27 (3H, s)	0.70 (3H, s)	0.89 (3H, s)	0.68 (3H, s)
19	1.21 (3H, s)	1.24 (3H, s)	1.14 (3H, s)	1.31 (3H, s)	1.12 (3H, s)
20			1.45 (1H, m)	1.58 (1H, m)	1.38 (1H, m)
21	1.44 (3H, s)	1.45 (3H, s)	0.89 (3H, s)	0.94 (3H, s)	0.80 (3H, s)
22	1.79 (1H, m)2.07 (1H, q, 6.0)	2.09 (1H, m)1.81 (1H, m)	1.52 (1H, m)1.18 (1H, m)	1.45 (1H, m)1.09 (1H, m)	1.17 (1H, m)1.58 (1H, m)
23	5.19 (1H, t, 6.0)	5.11 (1H, m)	2.18 (2H, m)	2.11 (1H, m)1.98 (1H, m)	2.16 (1H, m)2.30 (1H, m)
24	6.89 (1H, d, 6.0)	6.92 (1H, d, 9)	5.72 (1H, t, 6.0)	5.65 (1H, t, 7.2)	5.88 (1H, t, 6.0)
26	4.85 (1H, d, 6.0)4.44 (1H, d, 6.0)	4.86 (1H, d, 12.0)4.45 (1H, d, 12.0)	4.31 (2H, s)	4.32 (2H, s)	4.73 (2H, s)
27	1.91 (3H, s)	1.91 (3H, s)	1.83 (3H, s)	1.80 (3H, s)	4.70 (2H, s)
28	1.34 (3H, s)	1.37 (3H, s)	0.98 (3H, s)	0.91 (3H, s)	1.05 (3H, s)
29	1.28 (3H, s)	1.13 (3H, s)	1.10 (3H, s)	1.15 (3H, s)	1.18 (3H, s)
30	1.45 (3H, s)	1.41 (3H, s)	1.54 (3H, s)	1.56 (3H, s)	1.58 (3H, s)
Glc
1′	4.80 (1H, d, 6.0)	4.81 (1H, d, 7.8)	4.83 (1H, d, 6.0)	4.91 (1H, d, 7.8)	4.86 (1H, d, 6.0)
2′	4.02 (1H, m)	4.05 (1H, m)	3.95 (1H, m)	3.98 (1H, m)	3.97 (1H, m)
3′	4.17 (1H, m)	4.22 (1H, m)	4.18 (1H, m)	4.21 (1H, m)	4.21 (1H, m)
4′	4.18 (1H, m)	4.21 (1H, m)	4.16 (1H, m)	4.21 (1H, m)	4.20 (1H, m)
5′	3.89 (1H, m)	3.95 (1H, m)	3.92 (1H, m)	3.93 (1H, m)	3.95 (1H, m)
6′	4.55 (1H, d, 6.0)4.35 (1H, m)	4.58 (1H, d, 12.0)4.39 (1H, m)	4.50 (1H, d, 12.0)4.35 (1H, m)	4.57 (1H, d, 12.0)4.41 (1H, m)	4.55 (1H, d, 12.0)4.40 (1H, m)

Table 2

13C-NMR (150MHz, pyridine-d) spectral data of compounds 1–5.

Position	1	2	3	4	5
1	35.9	21.6	22.6	27.2	22.5
2	72.2	30.3	28.8	30.0	28.4
3	82.7	76.0	87.7	88.3	87.2
4	44.1	42.4	42.5	42.8	43.9
5	143.7	141.9	141.7	144.7	168.3
6	120.0	119.4	118.9	118.9	125.4
7	25.5	24.7	24.6	25.0	199.6
8	44.1	43.5	44.4	43.9	60.0
9	50.50	50.2	49.5	40.5	49.5
10	35.5	36.1	36.4	37.3	38.0
11	214.4	213.8	214.2	78.6	211.7
12	50.1	49.4	49.2	41.5	49.1
13	50.0	49.3	49.4	47.8	48.9
14	49.9	49.2	50.0	50.1	49.7
15	42.9	42.2	35.0	34.9	35.2
16	72.1	71.2	28.5	28.7	28.2
17	57.3	56.5	50.1	51.0	49.6
18	21.4	20.5	17.4	19.2	17.4
19	21.8	20.8	20.8	26.7	21.3
20	73.8	73.0	36.4	36.5	36.3
21	31.4	30.6	18.7	19.7	18.8
22	47.7	47.0	36.8	37.5	37.0
23	71.8	71.0	25.1	25.2	24.8
24	133.5	132.7	125.4	127.7	127.7
25	135.7	134.8	136.7	136.7	141.3
26	68.0	67.1	68.5	68.5	65.8
27	23.2	22.4	14.5	17.4	58.9
28	22.4	21.6	19.0	28.1	18.9
29	23.6	28.2	28.9	26.8	28.6
30	26.7	26.7	26.3	22.3	25.6
Glc
1′	103.9	103.0	107.8	107.8	107.6
2′	76.3	75.6	75.9	75.9	75.9
3′	79.9	79.1	79.1	79.1	79.1
4′	73.0	72.2	72.1	72.2	72.1
5′	79.8	79.0	78.6	78.2	78.8
6′	64.1	63.3	63.4	63.4	63.4

Figure 2

1H-1H COSY and HMBC correlations of compounds 1 ( 1H-1H COSY; HMBC).

Compound 2 was obtained as a shapeless white powder with + 83.8 (c 0.1, MeOH). Its molecular formula was established as C36H56O10 based on its HR-ESI-MS spectrum at m/z [M + Na]+ 671.3768 (calculated for C30H46NaO4, 671.3771). An analysis of the 1H and 13C-NMR data (Table 1 and Table 2) displayed that the structure of 2 was similar to that of 1. An unambiguous comparison the data of 2 with 1 shown that oxymethine at C-2 in 2 was absent. Furthermore, it was observed that the carbon signal at C-3, in comparison with 1, evidently shifted to δC 76.0 (–12.7 ppm) in 13C-NMR data of 2. Additionally, in the HMBC spectrum (Supplementary Materials), correlations from H-2 to C-4 proved the deficiency of the group. The significant NOESY (Supplementary Materials) correlations from H-10 (δH 2.54) to H-3 (δH 3.70), from H-3 (δH 3.70) to H3-29 (δH 1.13) confirmed the relative configurations of methyl groups and other protons in the tetracyclic rings. The coupling constant of J = 12.0 Hz further confirmed the antiperiplanar relationship between H-16 and H-17.Taken together with the analysis of NOE spectra between the two compounds, compound 2 was elucidated as 3β, 20β-dihydroxycucurbita-16α-23α-epoxy-5, 24(E)-diene-11-one-26-O-d- glucopyranoside and it was named Xuedanoside G.

Compound 3 was determined to be a molecular formula of C36H58O8, as established with its HR-ESI-MS data at m/z [M + Na]+ 641.4021 (calculated for C36H58NaO8, 641.4029). Comparing its 1H and 13C-NMR data (Table 1 and Table 2) with that of 2 showed that their structures were close, with the exception of the presence of the sugar group at C-3 (δC 87.7) in 3 instead of the sugar group at C-26 (δC 67.1) in 2. Similarly, itlackedthe a hydroxyl group at C-20 (δC 36.4) and the loss of an ether bond between H-16 and C-23 in 3. In the HMBC spectrum (Supplementary Materials), the sugar unit was linked at C-3 according to the correlation from the proton signal at δH 3.62 (H-3) to anomeric carbon at δC 107.8 (C-1′), and the signal for C-3 indicated a significant downfield shift to δ 87.7 (+11.7 ppm). Similarly, in comparison to 2, the signals for C-16, C-20, and C-23 revealed the powerful upfield shift to δ 28.5 (–42.7 ppm), δ 36.4 (–36.6 ppm), and δ 25.1 (–45.9 ppm), respectively, while a hydroxyl group and an ether bond were absent. Compound 3 was eventually determined to be 26-hydroxycucurbita-5, 24(E)-diene-11-one-3-O-β-d-glucopyranoside, and it was named Xuedanoside H.

Compound 4 had a molecular formula C36H60O8 (Calcd for C36H60NaO8, 643.4186) on the basis of ion peak at m/z [M + Na]+ 643.4178 in HR-ESI-MS. The 1D NMR signals (Table 1 and Table 2) were tightly connected to those of 3, with the difference of the carbonyl group of C-11 (δC 214.2) in 3, where it was substituted for a hydroxy group at δC 78.6 in compound 4. This difference was verified by 2D NMR spectra (Supplementary Materials). In the HMBC spectrum, the correlations from H-11 at δH 4.18 to C-8 (δC 43.9), C-10 (δC 37.3), and C-13 (δC 47.8) revealed that the hydroxyl group was located at C-11. Within the NOESY spectrum, from H3-19 to H-11 and from H-11 to H3-18 suggested that H-11 was β-oriented, and the structure of compound 4 was established as 11α, 26-dihydroxycucurbita-5, 24(E)-diene-3-O-β-d-glucopyranoside, and it was named Xuedanoside I.

Compound 5 possesses a molecular formula of C36H56O10on the basis of HR-ESI-MS at m/z [M + Na]+ 671.3779 (calculated for C36H56NaO10, 671.3771) and NMR spectra. Its 1H and 13C-NMR (Table 1 and Table 2) data are similar to those of compound 3, with the exception of the addition of a carbonyl group at C-7 (δC 199.6) and a hydroxyl group at C-27 (δC 58.9) in 5, respectively. In the HMBC spectrum (Supplementary Materials), the correlations of H-6 (δH 6.31) with the downfield carbon C-7 (δC 199.6) (compared with C-7 in 3) implied acarbonyl group at C-7. Furthermore, in comparison to 3, the signal for C-27 revealed a powerful downfield shift to δ 58.9 (+44.4 ppm), while a hydroxyl group was added at C-27. The form of 5 was confirmed by spectra of 1H-1H COSY, HSQC, HMBC, and NOESY (Supplementary Materials); it wasidentified as 26, 27-dihydroxycucurbita-5,24(E)- diene-7,11-dione-3-O-β-d-glucopyranoside and was named Xuedanoside J.

Furthermore, the cytotoxicity of all isolates was assessed with three human tumor cell lines (Hela, MCF-7, and A-549) according to the MTT procedure, and doxorubicin was used as the positive control. The results of cytotoxicity were displayed in Table 3. Compound 5 exhibited remarkable cytotoxicity against Hela, MCF-7, and A-549 cell lines with IC50 values from 2.25 to 5.33 μM in 48 h. Compounds 3–4 showed moderate cytotoxicity with the IC50 values between 7.55 and 18.72 μM in 48 h, whereas compounds 1 and 2 had weak effects with IC50> 30 μM. Meanwhile, the results revealed that tested compounds had low cytotoxic activity with the IC50 value more than 50.0 μM in normal human liver L-02 cells when compared to the control drug, doxorubicin (IC50 = 15.42 μM).

Table 3

Cytotoxicity (IC50, μM ± SD) of compounds 1–5 against three human cancer cell lines.

Compounds	Hela		MCF-7		A-549		L-02
	48 h	24 h	48 h	24 h	48 h	24 h	48 h	24 h
1	34.38±2.05	50.56 ± 4.28	45.09 ± 3.52	57.85 ± 5.16	49.44 ± 2.67	68.82 ± 4.33	>100	>100
2	31.75 ± 1.45	40.32 ± 2.56	45.88 ± 0.92	60.74 ± 4.73	47.58 ± 0.84	80.65 ± 5.16	>100	>100
3	7.55 ± 1.75	13.15 ± 1.88	10.88 ± 2.77	26.12 ± 1.22	8.55 ± 1.78	20.12 ± 1.08	68.25 ± 3.78	>100
4	14.77 ± 2.15	25.38 ± 3.72	12.54 ± 1.32	25.44 ± 3.15	18.72 ± 2.35	40.18 ± 3.02	89.55 ± 4.60	>100
5	2.25 ± 0.42	4.88 ± 1.05	4.72 ± 0.54	12.65 ± 2.36	5.33 ± 0.68	12.45 ± 1.28	50.52 ± 2.15	>100
doxorubicin	1.32 ± 0.03	2.15 ± 0.06	2.45 ± 0.05	3.02 ± 0.04	3.85 ± 0.05	6.10 ± 0.26	15.42 ± 0.28	26.56 ± 1.35

3. Discussion

Cucurbitane triterpene and its glycoside derivatives widely exist in the genus of Hemsleya, which are the effective constituents and show potent anti-tumor cytotoxicity. As a result, we evaluated all the isolates for their cytotoxic activity against three human cancer cell lines. Compared to the doxorubicin positive control group, all compounds showed moderate cytotoxicity due to their 24-ethylenic linkage substituent [8], with the value of IC50 ranging from 2.25 to 49.44 µM. Compound 5 displayed the most significant cytotoxic activity, which may be related to the carbonyl group at C-7 as a characteristic structural unit compared to its derivatives. Compounds 1 and 2 revealed the weak cytotoxic activity when compared with the other isolates, which may be caused by the formation of ether bond between C-16 and C-23. In brief, the A ring and branch chain had dramatic effects on potency against human tumor cell lines. All compounds showed low cytotoxic activity in human normal liver L-02 cells when compared to doxorubicin. Based on these promising results, compounds 3 and 5 could serve as potential anti-cancer agents for future cancer chemotherapy.

4. Materials and Methods

4.1. General Experimental Procedures

1D and 2D NMR spectra were obtained with a Bruker AV 600 NMR spectrometer(chemical shifts are presented asδ values with TMS as the internal standard) (Bruker, Billerica, Germany). HR-ESI-MS were performed on a Q-tof spectrometer (Waters, Milford, MA, USA). UV and IR data were done using a Shimadzu UV2550 and FTIR-8400Sspectrometer (Shimadzu, Kyoto, Japan), respectively. Thin-layer chromatography (TLC) was performed on pre-coated silica gel GF254 (Zhi Fu Huang Wu Pilot Plant of Silica Gel Development, Yantai, China). Semi-preparative HPLCwas conducted on an analytic LC equipped with a pump of P230, a DAD detector of 230+ (Ellte, Dalian, China) with a C18 ODS-A (5 µm, YMC, Kyoto, Japan). Column chromatography with silica gel was used (100-200 and 200-300 mesh, Qingdao Marine Chemical plant, Qingdao, China). All solvents used were of analytical grade (Beijing Chemical Plant, China).

4.2. Plant Material

The rhizomes of Hemsleya penxianensis (Cucurbitaceae) were collected in the Jinfuo mountain, Nanchuan district of Chongqing City, China, on September 2014, and were identified by Prof. Si-Rong Yi, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, where the voucher specimen (CS140921) was stored. The plant drug was dried in the shade, powdered, and contained in an airtight container.

4.3. Extraction and Isolation

The rhizomes of H. penxianensis (10.0 kg) were extracted with 95% EtOH under reflux (3 h × 60 L × 3). The EtOH extract was evaporated at 50 °C, and the crude extracts were dissolved in water. The aqueous extraction was re-extracted with EtOAc, and an EtOAc fraction was obtained. The fraction of EtOAc (200 g) was subjected to silica gel column chromatography and eluted with a gradient system of CH2Cl2-MeOH to obtain 12 fractions (Fr. A-Fr. L).

The fraction J (16.3 g) was subjected to column chromatography on silica gel and eluted with CH2Cl2-MeOH gradient (60:1, 40:1, 30:1, 20:1, 10:1, 5:1 v/v), to obtain 6 fractions (Fr. I-VI). The Fr. IV (3.2 g) was further separated by MCI-gel column chromatography with methanol-water (10:90, 20:80, 30:70, 40:60, 50:50, 70:70, 90:10, 100:0) gradient elution, giving 8 fractions (Fr. IV.1–IV.8). Fraction IV.3 was subjected to semi-preparative HPLC with CH3CN-H2O as the mobile phase (18:82, v/v) by the YMC-Pack ODS-A column to acquire compound 1 (8.7 mg, t = 12.4 min) and 2 (8.8 mg, t = 17.8 min). Fraction IV.4 was prepared by semi-preparative HPLC eluting with CH3CN-H2O (16:84, v/v) to give compound 3 (6.7 mg, t= 15.2 min), 4 (9.5 mg, t = 22.8 min), and 5 (8.7 mg, t = 26.4 min).

The structures of compounds 1-5 were determined by HR-ESI-MS, UV, IR, 1D, and 2D NMR spectra.

Xuedanoside F (1). C36H56O11,+ 60.5 (c 0.1, MeOH), white amorphous powder; IR (KBr) νmax cm-1: 1651, 1687, 3565-3340; UV λmax (MeOH) nm (log ε): 205.8 (5.80); HR-ESI-MS m/z [M + Na]+ 687.3725(calcd. 687.3720); 1H and 13C-NMR spectra data, see Table 1 and Table 2.

Xuedanoside G (2). C36H56O10, + 83.8 (c 0.1, MeOH), white amorphous powder; IR (KBr) νmax cm-1: 1675, 1689, 3569-3254; UV λmax (MeOH) nm (log ε): 210.5 (5.68); HR-ESI-MS m/z [M + Na]+ 671.3768(calcd. 671.3771); 1H and 13C-NMR spectra data, see Table 1 and Table 2.

Xuedanoside H (3). C36H58O8, + 69.7 (c 0.1, MeOH), white amorphous powder; IR (KBr) νmax cm-1: 1170, 1661, 1723, 3633-3354; UV λmax (MeOH) nm (log ε): 202.8 (5.10); HR-ESI-MS m/z [M + Na]+ 641.4021(calcd. 641.4029); 1H and 13C-NMR spectra data, see Table 1 and Table 2.

Xuedanoside I (4). C36H60O8, + 30.9 (c 0.1, MeOH), white amorphous powder; IR (KBr) νmax cm-1: 1145, 1640, 3658-3385; UV λmax (MeOH) nm (log ε): 202.4 (5.08); HR-ESI-MS m/z [M + Na]+ 643.4178 (calcd.643.4186); 1H and 13C-NMR spectra data, see Table 1 and Table 2.

Xuedanoside J (5). C36H56O10, + 59.7 (c 0.1, MeOH), white amorphous powder; IR (KBr) νmax cm-1: 1195, 1651, 1680, 3650-3460; UV λmax (MeOH) nm (log ε): 209.6 (5.60); HR-ESI-MS m/z 671.3779 [M + Na]+ (calcd.671.3771); 1H and 13C-NMR spectra data, see Table 1 and Table 2.

Acid hydrolysis of Compounds 1–5 were accomplished by the procedure described previously [14,15].

4.4. Cytotoxicity Assays

Compounds 1–5 isolated from H. penxianensis were screened for cytotoxicity against three human cancer cell lines, including Hela, breast cancer MCF-7, lung cancer A-549, and the normal liver L-02 cells. This used the MTT method as described in previously published literature [16,17]. Briefly, the cells, at a density of 1.1 × 105 cells/mL in 96-well microtiter plate, were cultured in DMEM medium with 10% fetal bovine serum at 37 °C in a 5% CO2 incubator overnight. Then, the cells were treated with the test compounds at five concentrations in triplicate. After 24 h and 48 h of treatment, the cells were incubated with 10 μL of MTT (4 mg/mL) for another 4 h. The residual liquid was removed, and 200 µL DMSO was added. The absorbance was tested using a microplate reader at a wavelength of 570 nm.