Antiproliferative and anti-inflammatory polyhydroxylated spirostanol saponins from Tupistra chinensis

Abstract

Tupistra chinensis is widely distributed in southwestern China and its rhizome is a famous folk medicine for the treatment of carbuncles and pharyngitis. Its chemical identity of potent antiproliferative and anti-inflammatory constituents has been carried out in this study. Twenty-three polyhydroxylated spirostanol saponins, including nine novels, were isolated and identified. The new spirostanol saponins were elucidated as spirost-25(27)-en-1β,2β,3β,4β,5β-pentol-2-O-β-D-xylopyranoside (1), spirost-25(27)- en-1β,2β,3β,4β,5β-pentol-2-O-α-L-arabinopyranoside (2), spirost-25(27)-en- 1β,3α,5β-triol (12), spirost-25(27)-en-1β,3α,4β,5β,6β-pentol (13), spirost-25(27)-en- 1β,2β,3β,5β-tetraol-5-O-β-D-glucopyranoside (16), 5β-spirost-25(27)-en-1β,3β-diol- 3-O-β-D-glucopyranosyl-(1 → 4)-β-D-glucopyranoside (17), (25R)-5β-spirostan- 1β,3β-diol-3-O-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranoside (18), (25R)-5β- spirostan-1β,3β-diol-3-O-β-D-fructofuranosyl-(2 → 6)-β-D-glucopyranoside (19), 5β-spirost-25(27)-en-3β-ol-3-O-β-D-glucopyranosyl-(1 → 4)-β-D-glucopyranoside (20). The antiproliferative effects against seven human cancer cell lines and inhibitory activities on nitric oxide (NO) production induced by lipopolysaccharide (LPS) in a macrophage cell line RAW 264.7 were assayed for all the isolated compounds. Compounds 17, 19 and 21 exhibited potential antiproliferative activities against all of human cancer cell lines tested. Compounds 21 showed significant inhibition on NO production with IC₅₀ values of 11.5 μM. These results showed that the spirostanol saponins isolated from the dried rhizomes of T. chinensis have potent antiproliferative and anti-inflammatory activities and T. chinensis might be used as anticancer and.anti-inflammatory supplement.

Medicinal plants have been used for treating human diseases and they produced a large variety of secondary metabolites such as alkaloids, flavonoids, triterpenoids and steroidal glycosides¹. Steroidal glycosides as the important secondary metabolites of medicinal plants have been reported to possess a wide range of biological activities including anticancer, anti-inflammatory, platelet aggregation inhibition, antihypertensive, cholesterol lowering, antifungal and antiviral². Additionally, steroidal glycosides have a wide variety of commercial uses such as surfactants, foaming agents and precursors for the industrial production of pharmaceutical drugs³,⁴.

A series of widespread chronic diseases such as cancer are now the leading causes of morbidity and mortality worldwide. Many cancers arise from sites of infection, chronic irritation and inflammation. Recent data have expanded the concept that inflammation is a critical component of tumor progression. Anti-inflammatory therapy is efficacious towards early neoplastic progression and malignant conversion⁵,⁶. Many studies have showed that biological activities of phytochemicals are comparable to previous synthetic compounds, and they have low side effects. Therefore, recent interests in medicinal plants have been focused on identifying active compounds and elucidating underlying molecular mechanisms of action⁷,⁸.

The genus Tupistra (Liliaceae) has 12 species in southern China. These species possess similar morphologic characteristics and some can be substituted for each other as a folk medicine to treat pharyngolaryngitis, rheumatic diseases and snake-bite⁹. Tupistra chinensis is widely distributed in southwestern China and its dried rhizome is a famous folk medicine for the treatment of carbuncles and pharyngitis. Previous phytochemical investigations on T. chinensis have led to the isolation of a variety of biologically active compounds, including steroidal sapogenins and their glycosides¹⁰,¹¹,¹²,¹³,¹⁴,¹⁵, cardenolides¹⁶, a pregnane genin and its glycoside¹⁷,¹⁸, flavonoids¹⁷,¹⁸,¹⁹, which have anti-inflammatory²⁰, cytotoxicity²¹,²²,²³ and antifungal activities²⁴,²⁵. Steroidal saponins that consist of spirostanol saponins and furostanol saponins were the most abundant active constituents in T. chinensis.

In the course of our continuing search for bioactive leading compounds from the medicinal herbs, we focused on steroidal saponins from the dried rhizomes of T. chinensis and obtained twenty-three polyhydroxylated spirostanol saponins including nine new compounds. The structures of all the isolated compounds were elucidated on the basis of spectroscopic data and chemical methods, including IR, NMR, MS, and GC analysis. Moreover, all of the isolated compounds were evaluated for their antiproliferative activity against seven human cancer cell lines and the inhibitory activities on NO production induced by LPS in a macrophage cell line RAW 264.7.

Results and Discussion

Structure identification

Nine new spirostanol saponins (1, 2, 12, 13, 16–20) and fourteen congeners (3–11, 14, 15, 21–23) were obtained from the 60% ethanol extract of the rhizomes of T. chinensis. Their chemical structures are shown in Fig. 1. The structures of all the isolated compounds were elucidated on the basis of spectroscopic data and chemical methods, including IR, NMR, MS, and GC analysis. The fourteen known spirostanols were identified by comparison of their reported spectroscopic data as spirost-25(27)-en-1β,3β,4β,5β-tetrol-5-O-β-D-glucopyranoside (3)²³, neopentrogenin (4)²⁶, Δ²⁵⁽²⁷⁾-pentrogenin (5)¹², neopentrogenin-5-O-β-D- glucopyranoside (6)²⁶, spirost-25(27)-en-1β,2β,3β,4β,5β-pentol-5-O-β-D- glucopyranoside (7)²⁶, spirost-25(27)-en-1β,2β,3β,4β,5β,7α-hexaol-6-one (8)¹⁵, wattigenin A (9)²⁷, spirost-25(27)-en-1β,2β,3β,4β,5β,6β,7α-heptol (10)²⁸, spirost-25(27)-en-1β,2β,3β,4β,5β,7α-hexaol-6-one-4-O-β-D-xylopyranoside (11)²⁹, tupichigenin D (14)¹⁸, spirost-25(27)-en-1β,3β,5β-triol-5-O-β-D-glucopyranoside (15)²³, (25S)-5β-spirostan-3β-ol-3-O-β-D-glucopyranosyl-(1 → 4)-β-D- glucopyranoside (21)³⁰, (25R)-5β-spirostan-3β-ol-3-O-β-D-glucopyranosyl-(1 → 4)- β-D-glucopyranoside (22)³⁰, wattoside I (23)⁹. Compounds 4, 6, 7, 21 and 22 are reported for the first time from T. chinensis, to our knowledge. The NMR data of the known compounds could be found in the supplementary material.

Figure 1. Structures of isolated compounds 1–23.

Compound 1 was isolated as white amorphous powder, −43.3 (c 0.25, MeOH). The molecular formula was inferred as C₃₂H₅₀O₁₁ according to the positive-ion HRESI-MS peak at m/z [M + H]⁺ 611.3437 (calcd for C₃₂H₅₁O₁₁ [M + H]⁺, 611.3431). The IR spectrum showed a strong absorption band at 3364 cm⁻¹, ascribable to hydroxyl functionalities. Acid hydrolysis and GC analysis of 1 gave D-xylose. The ¹H NMR spectrum showed three typical steroidal methyl signals at δ_H 1.59 (3H, s, Me-19), 1.09 (3H, d, J = 6.9 Hz, Me-21) and 0.84 (3H, s, Me-18) (Table 1). Four methane protons indicative of secondary alcoholic functions at δ_H 4.57 (1H, brs, H-1), 4.41 (1H, m, H-2), 5.03 (1H, brs, H-3) and 4.31 (1H, brs, H-4) were observed. The germinal protons at C-27 were observed at δ_H 4.57 and 4.82 as two singlets, which are characteristic of an exocyclic methylene. The ¹³C NMR spectrum exhibited characteristic spirostanol carbon signal at δ_C 109.8 (C-22) and three methyl (δ_C 16.8, 13.9, and 15.3), and two olefinic carbons δ_C 144.7 and 109.1 were assigned to the C-25 and C-27 positions, respectively, diagnostic of C-25 (27)-unsaturated sapogenin. The NMR spectrum of 1 showed an anomeric proton signal at δ_H 5.15 (1H, d, 7.5 Hz), corresponding to an anomeric carbon at δ_C 103.5 in the HSQC spectrum, indicated the presence of one sugar moiety. Unambiguous complete assignments for the ¹H and ¹³C NMR signals were made by combination of DEPT, ¹H − ¹H COSY, HSQC, HMBC, and NOESY spectra. The ¹H − ¹H COSY spectrum showed that the oxymethine proton δ_H 4.41 (H-2) was coupled to δ_H 4.57 (1H, brs, H-1) and 5.03 (1H, brs, H-3), and the oxymethine proton δ_H 5.03 (H-3) was coupled to the oxymethine proton at δ_H 4.31 (H-4). These findings supported the location of the hydroxyl groups at C-1, C-2, C-3, C-4, and C-5, together with the long-range correlations observed in the HMBC spectrum (Fig. 2). The HMBC correlations of H-1 (δ_H 5.15) of xylose with C-2 (δ_C 74.4) of the aglycon revealed the sugar residue attached to C-2 of the aglycon. The stereochemistry of the ring junctions and substituents of the aglycon of 1 were determined via cross-peaks observed in a NOESY spectrum according to the refs ^12,15 and ²³ (Fig. 2). The key correlations between H-4_α and H-7_α/H-9_α, and between H-2_α/H-9_α, supported the A/B cis ring junction pattern. Thus, the hydroxyl groups at C-5 and C-2 have a β-orientation. Additional NOE correlations between H-1_α and Me-19/H-11, and between H-3_α and H-2_α/H-4_α, were indicative of β-orientations for OH-1, OH-3 and OH-4. Thus, compound 1 was elucidated as spirost-25(27)-en-1β,2β,3β,4β,5β-pentol-2-O-β-D-xylopyranoside.

Table 1. NMR spectroscopic data for compounds 1 and 2 (pyridine-d₅).

Position	1		2
	δC	δH (J in Hz)	δC	δH (J in Hz)
1	77.8	4.57, brs	77.8	4.58, m
2	74.4	4.41, m	74.0	4.41, m
3	72.8	5.03, brs	72.8	5.03, brs
4	68.7	4.31, brs	68.7	4.31, brs
5	78.2		78.2
6	30.5	2.48, d (13.2)	30.5	2.48, d (13.2)
		1.67, m		1.67, m
7	28.8	1.54, m	28.8	1.50, m
		1.14, m		1.10, m
8	35.2	1.71, m	35.2	1.71, m
9	45.7	1.22, m	45.7	1.20, m
10	45.5		45.5
11	21.9	1.58, m	21.9	1.57, m
		1.45, m		1.41, m
12	40.2	1.59, m	40.2	1.58, m
		1.03, m		1.01, m
13	40.9		40.9
14	56.4	1.03, m	56.4	1.01, m
15	32.5	2.03, m	32.5	2.02, m
		1.44, m		1.43, m
16	81.7	4.59, m	81.8	4.58, m
17	63.3	1.81, m	63.4	1.80, m
18	16.8	0.84, s	16.8	0.83, s
19	13.9	1.59, s	13.9	1.58, s
20	42.2	1.96, m	42.2	1.95, m
21	15.3	1.09, d (7.0)	15.3	1.09, d (7.0)
22	109.8		109.8
23	33.5	1.81, m	33.5	1.76–1.79, m
24	29.3	2.70, m	29.3	2.70, m
		2.25, d (11.5)		2.24, d (11.5)
25	144.7		144.7
26	65.4	4.47, d (12.1)	65.4	4.47, d (12.1)
		4.04, d (12.1)		4.04, d (12.1)
27	109.1	4.82, s	109.1	4.81, s
		4.78, s		4.78, s
	2-O-Xyl		2-O-Ara
1	103.5	5.15, d (7.5)	103.4	5.13, d (7.0)
2	75.4	4.08, m	72.8	4.56, m
3	78.7	4.18, m	74.8	4.22, m
4	71.4	4.24, m	69.7	4.36, m
5	67.7	4.40, m	67.3	4.38, m
		3.76, m		3.85, m

Figure 2. Selected ¹H − ¹H COSY, HMBC and NOESY correlations of compounds 1, 12 and 13.

Compound 2 was isolated as white amorphous powder, with the same molecular formula of C₃₂H₅₀O₁₁ as 1 on the basis of HRESIMS (m/z 611.3412 [M + H]⁺). Acid hydrolysis of 2 gave L-arabinose. From a comparison of ¹H and ¹³C NMR data of 2 with those of 1 (Table 1), it was apparent that 2 contained the same aglycon as 1, except for a little different in the monosaccharide chain. Instead of the signals for a xylopyranosyl moiety, signals assignable to an α-L-arabinopyranosyl residue were observed at δ_H 5.13 (1H, d, J = 7.0 Hz, H-1), 4.56 (1H, m, H-2), 4.22 (1H, m, H-3), 4.36 (1H, m, H-4), 4.38 (1H, m, H-5a) and 3.85 (1H, m, H-5b), and δ_C 103.4 (C-1), 72.8 (C-2), 74.8 (C-3),69.7 (C-4), 67.3 (C-5). The linkage position of the arabinosyl moiety to C-2 of the aglycone was confirmed by an HMBC correlation between the anomeric proton at δ_H 5.13 and the C-2 carbon of the aglycone at δ_C 74.0. Therefore, the structure of 2 was elucidated as spirost-25(27)-en-1β,2β,3β,4β,5β-pentol- 2-O-α-L-arabinopyranoside.

Compound 12 was isolated as white amorphous powder, −33.1 (c 0.25, MeOH). The HRESIMS gave an ion at m/z 447.3112 [M + H]⁺ (calcd for C₂₇H₄₃O₅, 447.3110), consistent with the molecular formula C₂₇H₄₂O₅. The ¹H NMR spectrum showed three typical steroidal methyl signals at δ_H 1.50 (3H, s, Me-19), 1.12 (3H, d, J = 7.0 Hz, Me-21) and 0.87 (3H, s, Me-18) (Table 2). The ¹³C NMR spectrum exhibited characteristic spirostanol carbon signal at δ_C 109.8 (C-22) and three methyl (δ_C 16.9, 13.7, and 15.4), and two olefinic carbons δ_C 144.8 and 109.1 were observed, diagnostic of C-25 (27)-unsaturated sapogenin. The ¹H − ¹H COSY spectrum showed that two methylene protons at δ 2.12 (H-2_α) and 2.61 (H-2_β) were coupled to δ 4.31 (H-1) and 5.13 (H-3), and the oxymethine proton δ 5.13 (H-3) was coupled to two methylene protons at δ 2.26 (H-4_β) and 2.61 (H-4_α). These findings indicated the location of the hydroxyl groups at C-1, C-3, and C-5, together with the long-range correlations observed in the HMBC spectrum (Fig. 2). The key NOESY correlations (Fig. 2) between H-4_α and H-7_α/H-9_α, and between H-2_α and H-9_α, supported the A/B cis ring junction pattern. Thus, the hydroxyl group at C-5 has a β-orientation. Additional NOE correlations between H-1_α and Me-19/H-11, and between H-3_β and H-2_β/H-4_β, were indicative of β-orientation for OH-1 and α-orientation for OH-3. On the basis of the above analysis, compound 12 was finally assigned to be spirost-25(27)-en-1β,3α,5β-triol.

Table 2. NMR spectroscopic data for compounds 12 and 13 (pyridine-d₅).

Position	12		13
	δC	δH (J in Hz)	δC	δH (J in Hz)
1	75.9	4.31, brs	76.2	4.19, brs
2	38.9	2.61, m	36.6	2.62, m
		2.12, t (11.1)		2.23, m
3	63.7	5.13, m	69.2	4.85, m
4	44.0	2.61, m	75.9	4.34, d (9.1)
		2.26, m
5	77.4		80.7
6	36.7	1.98, m	69.5	4.94, s
		1.58, d (13.2)
7	29.2	1.49, m	35.4	2.07, m
		1.07, m		1.55, m
8	35.4	1.66, m	30.7	2.38, d (8.4)
9	45.8	1.45, m	45.9	1.50, m
10	43.3		44.9
11	21.7	1.38, m	21.4	1.50, m
				1.40, m
12	40.3	1.66, m	40.1	1.66, m
		0.99, m		1.00, m
13	41.1		41.1
14	56.6	0.97, m	56.3	1.02, m
15	32.5	2.05, dd (12.1, 5.5)	32.5	2.07, m
		1.46, m		1.43, m
16	81.8	4.65, dd (14.6, 7.7)	81.8	4.62, dd (14.5, 7.6)
17	63.4	1.85, m	63.4	1.85, m
18	16.9	0.87, s	16.9	0.87, s
19	13.7	1.50, s	16.9	1.86, s
20	42.3	1.98, m	42.2	1.96, m
21	15.4	1.12, d (7.0)	15.4	1.10, d (7.0)
22	109.8		109.8
23	33.6	1.80, m	33.6	1.77, m
24	29.3	2.73, td (13.2, 5.8)	29.3	2.69, td (13.6, 5.5)
		2.26, m		2.23, m
25	144.8		144.8
26	65.4	4.51, d (12.0)	65.4	4.47, d (12.1)
		4.06, d (12.0)		4.03, d (11.9)
27	109.1	4.83, s	109.1	4.81, s
		4.80, s		4.78, s

Compound 13 was isolated as white amorphous powder, −34.9 (c 0.25, MeOH). The molecular formula, C₂₇H₄₂O₇, was deduced from the HRESI-MS peak (m/z [M + H]⁺ 479.3018, calcd for C₂₇H₄₃O₇, 479.3009). The ¹H NMR spectrum showed three typical steroidal methyl signals at δ_H 1.86 (3H, s, Me-19), 1.10 (3H, d, J = 7.0 Hz, Me-21) and 0.87 (3H, s, Me-18) (Table 2). The down-field shifted hydrogen chemical shift of Me-19 in combination with an oxymethine proton (δ_H 4.94) at B ring confirmed the presence of the OH group attached at C-6 in compound 13, which was consistent with previous reports¹². The ¹³C NMR spectrum exhibited characteristic spirostanol carbon signal at δ_C 109.8 (C-22) and three methyl (δ_C 16.9, 16.9, and 15.4), and two olefinic carbons δ_C 144.8 and 109.1 were observed, diagnostic of C-25 (27)-unsaturated sapogenin. These ¹H-NMR data and ¹³C-NMR signals suggested that 13 was a C-25(27) unsaturated spirostane type steroidal sapogenin. The ¹H-¹H COSY spectrum showed that two methylene protons at δ 2.23 (H-2_α) and 2.62 (H-2_β) were coupled to δ 4.19 (H-1) and 4.85 (H-3), and the oxymethine proton δ 4.34 (H-4) was in turn coupled with the oxygenated methane proton at δ 4.85 (H-3). The oxygenated methane proton at δ 4.94 (H-6) was coupled with two methylene protons at δ 1.55 (H-7_α) and 2.07 (H-7_β). These findings supported the location of the hydroxyl groups at C-1, C-3, C-4, and C-6. The key NOESY correlations (Fig. 2) between H-4_α and H-7_α/H-9_α, and between H-2_α and H-9_α, supported the A/B cis ring junction pattern. Thus, the hydroxyl groups at C-4 and C-5 have β-orientation. Additional NOE correlations between H-1_α and Me-19/H-11, and between H-3_β and H-2_β, between H-6_α and H-7_α, were indicative of β-orientation for OH-1, OH-6 and α-orientation for OH-3. On the basis of the above analysis, compound 13 was formulated as spirost-25(27)-en-1β,3α,4β,5β,6β-pentol.

Compound 16 was isolated as white amorphous powder. The molecular formula of C₃₃H₅₂O₁₁ was determined by the positive-ion HRESIMS peak at m/z [M + H]⁺ 625.3552 (calcd for C₃₃H₅₃O₁₁, 625.3588). The ¹H NMR spectrum showed three typical steroidal methyl signals at δ_H 1.62 (3H, s, Me-19), 1.08 (3H, d, J = 7.0 Hz, Me-21) and 0.83 (3H, s, Me-18) (Table 3). The ¹H NMR spectrum of 16 was similar to that of 1, except for the absence of one oxymethine proton. This suggests that both compounds possessed the same C-25(27) unsaturated spirostane type skeleton. The ¹³C NMR spectrum exhibited characteristic spirostanol carbon signal at δ_C 109.8 (C-22) and three methyl (δ_C 16.8, 14.1, and 15.3), and two olefinic carbons δ_C 144.8 and 109.1 were assigned to the C-25 and C-27 positions, respectively. Compared the NMR data with those of spirost-25(27)-en-1β,2β,3β,5β-tetraol-5-O-β-D-galactopyranoside³¹, the NMR features of those two compounds were almost same except for the signals of the sugar moiety. The NMR spectrum of 16 showed an anomeric proton signal at δ_H 5.29 (1H, d, J = 7.7 Hz, H-1-Glc), corresponding to an anomeric carbon at δ_C 97.7 in the HSQC spectrum, indicated the presence of one sugar moiety. The sugar was identified as D-glucose by GC analysis of its chiral derivatives after acid hydrolysis. Thus, compound 16 was inferred as spirost-25(27)-en-1β,2β,3β,5β-tetraol-5-O-β-D- glucopyranoside.

Table 3. NMR spectroscopic data for compound 16 (pyridine-d₅).

Position	δC	δH (J in Hz)	Position	δC	δH (J in Hz)
1	78.0	4.20, d (7.8)	18	16.8	0.83, s
2	68.4	3.95, m	19	14.1	1.62, s
3	71.4	4.51, brs	20	42.3	1.96, m
4	37.0	2.50, m	21	15.3	1.08, d (7.0)
5	83.4		22	109.8
6	31.0	2.11, td (13.5)	23	33.6	1.82, m
		2.03, m			1.78, m
7	29.3	1.53, d (13.7)	24	29.3	2.72, td (13.4)
		0.88, dd (12.7, 3.9)			2.25, d (11.5)
8	34.8	1.59, m	25	144.8
9	45.9	1.17, m	26	65.4	4.48, d (12.2)
10	47.0				4.05, d (12.2)
11	21.8	1.42, m	27	109.1	4.83, s
12	40.2	1.59, m			4.79, s
		1.02, m	5-O-Glc
13	40.8		1	97.7	5.29, d (7.7)
14	56.3	1.02, m	2	75.9	3.95, m
15	32.5	2.03, m	3	79.1	4.26, t (8.6)
		1.45, m	4	72.0	4.08, m
16	81.8	4.61, m	5	79.1	4.08, m
17	63.3	1.84, m	6	63.1	4.60, m
					4.31, dd (11.5)

Compound 17 was isolated as white amorphous powder. The HRESIMS of 17 showed the ion at m/z 755.4275 [M + H]⁺, and its molecular formula was inferred as C₃₉H₆₂O₁₄ on the basis of the analysis of ¹H and ¹³C NMR and DEPT spectra. Acid hydrolysis and GC analysis of 17 gave D-glucose. Compared the ¹H and ¹³C NMR data with those of isorhodeasapogenin 3-O-β-D-glucopyranosyl-(1 → 4)-β-D- glucopyranoside³², the NMR features of those two compounds were almost same except for the signals of the F-ring, which was suggested the same A-E ring substitution in the two compounds. The signals due to the methyl of C-27 in isorhodeasapogenin 3-O-β-D-glucopyranosyl-(1 → 4)-β-D-glucopyranoside were replaced by the signals assigned to the C-25(27) exo-methylene group at δ_H 4.79 (1H, s) and 4.82(1H, s), and δ_C 144.8 (C-25) and 109.1 (C-27) in 17. Hence, the structure of 17 was elucidated as 5β-Spirost-25(27)-en-1β,3β-diol-3-O-β-D-glucopyranosyl- (1 → 4)-β-D-glucopyranoside.

Compound 18 was obtained as white amorphous powder. The molecular formula of C₃₉H₆₄O₁₄ was assigned from the positive-ion HRESIMS peak (m/z 757.4403 [M + H]⁺, calcd for C₃₉H₆₅O₁₄ [M + H]⁺, 757.4374). The IR spectrum showed the characteristic absorption of hydroxyl group at 3411 cm⁻¹ and the four steroidal sapogenins absorption bands 986, 919, 899, 873 cm⁻¹, and the 899 band was stronger than the 919 band. It was suggested that 18 was a sapogenin with “iso” configuration of the F ring³³. Acid hydrolysis and GC analysis of 18 gave D-glucose. From a comparison of ¹H and ¹³C NMR data of 18 (Table 4) with those of isorhodeasapogenin 3-O-glucopyranosyl-(1 → 4)-β-D-glucopyranoside³², it was apparent that 18 contained the same aglycon as isorhodeasapogenin 3-O-glucopyranosyl-(1 → 4)-β-D- glucopyranoside, except for a little different in the saccharide chains. The linkage of the sugar units was established from the following HMBC correlations: H-1 (δ_H 5.02) of the additional glucose′ with C-6 (δ_C 70.7) of the glucose, H-1 (δ_H 4.93) of the glucose with C-3 (δ_C 76.0) of the aglycon. Thus, compound 18 was elucidated as (25R)-5β-spirostan-1β,3β-diol-3-O-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranoside.

Table 4. NMR spectroscopic data for compounds 17–20 (pyridine-d₅).

Position	17		18		19		20
	δC	δH (J in Hz)	δC	δH (J in Hz)	δC	δH (J in Hz)	δC	δH (J in Hz)
1	72.7	3.89, d (9.1)	72.3	3.95, m	72.3	3.94, brs	31.3	1.70, m
								1.46, m
2	32.3	2.30, m	31.7	2.35, d (11.3)	31.8	2.39, d (14.7)	27.3	1.91, m
		1.84, m		1.82, m		1.80, m		1.52, m
3	75.2	4.53, m	76.0	4.55, m	75.6	4.52, brs	74.9	4.32, m
4	29.5	1.90, m	30.4	1.92, m	30.1	1.89, m	30.9	1.79, m
		1.82, m		1.74, m		1.73, m		1.71, m
5	31.3	2.38, d (11.5)	31.4	2.40, d (14.9)	31.4	2.34, d (12.4)	37.3	2.04, m
6	26.8	1.73, m	26.8	1.72, m	26.8	1.69, m	27.3	1.73, m
		1.15, m		1.10, m		1.10, m		1.06, m
7	26.8	1.28, m	26.6	1.23, m	26.7	1.23, m	27.1	1.24, m
		1.01, m		0.94, m		0.97, m		0.96, m
8	36.1	1.59, m	36.1	1.56, m	36.1	1.56, m	35.9	1.49, m
9	42.5	1.20, m	42.2	1.15, m	42.3	1.15, m	40.6	1.29, m
10	40.8		40.6		40.6		35.6
11	21.4	1.28, m	21.4	1.23, m	21.4	1.23, m	21.5	1.32, m
								1.19, m
12	40.5	1.66, m	40.5	1.61, m	40.6	1.62, m	40.6	1.68, m
		1.06, m		1.00, m		1.03, m		1.08, m
13	41.0		41.0		41.0		41.3
14	56.7	1.08, m	56.6	1.05, m	56.6	1.06, m	56.8	1.08, m
15	32.5	2.03, m	32.5	2.02, m	32.5	2.03, m	32.5	2.04, m
		1.43, m		1.42, m		1.14, m		1.42, m
16	81.8	4.60, m	81.5	4.60, m	81.5	4.62, d (7.3)	81.9	4.62, dd (14.7, 7.5)
17	63.5	1.84, m	63.5	1.83, m	63.5	1.86, m	63.5	1.85, m
18	17.0	0.84, s	17.0	0.81, s	17.0	0.81, s	16.9	0.82, s
19	19.5	1.26, s	19.5	1.24, s	19.4	1.23, s	24.2	0.84, s
20	42.2	1.97, m	42.3	1.95, m	42.4	1.96, m	42.2	1.97, m
21	15.4	1.10, d (7.0)	15.4	1.14, d (6.9)	15.4	1.14, d (7.0)	15.4	1.10, d (6.9)
22	109.8		109.6		109.6		109.8
23	33.6	1.78, m	32.2	1.68, m	32.2	1.68, m	33.6	1.78, m
24	29.3	2.72, m	29.6	1.56, m	29.6	1.56, m	29.3	2.72, td (13.5, 5.4)
		2.26, m
25	144.8		30.9	1.56, m	31.0	1.56, m	144.8
26	65.4	4.48, d (12.1)	67.2	3.58, m	67.2	3.60, m	65.4	4.49, m
		4.05, d (12.1)	3.51, t (10.5)	3.51, m		3.51, m		4.05, m
27	109.1	4.82, s	17.7	0.68, d (5.6)	17.7	0.69, d (5.6)	109.1	4.82, s
		4.79, s						4.78, s
	3-O Glc		3-O Glc		3-O Glc		3-O Glc
1	101.3	4.97, d (7.8)	102.4	4.93, d (7.8)	102.1	4.89, d (7.8)	103.2	4.91, d (7.8)
2	74.9	3.96, m	75.2	3.93, m	75.3	3.91, m	75.1	4.08, m
3	77.2	4.31, m	78.8	4.20, m	78.8	4.20, m	77.3	4.32, m
4	81.5	4.33, m	72.2	4.05, m	72.4	4.07, m	81.7	4.39, t (9.1)
5	77.0	3.96, m	77.3	4.15, m	77.1	4.07, m	76.8	3.92, dt (9.5, 3.2)
6	62.4	4.58, m	70.7	4.89, d (9.1)	63.1	4.85, d (9.1)	62.6	4.60, m
		4.31, m		4.18, m		4.55, m		4.51, m
	Glc(1 → 4)		Glc(1 → 6)		Fru(2 → 6)		Glc(1 → 4)
1′	105.3	5.21, d (7.9)	105.6	5.02, d (7.7)	63.2	4.37, m	105.3	5.23, d (7.9)
						4.26, d (8.0)
2′	75.1	4.11, m	75.8	4.06, m	106.2		75.2	4.13, t (8.3)
3′	78.8	4.02, m	78.7	3.93, m	79.8	5.21, m	78.6	4.24, m
4′	71.9	4.22, m	71.8	4.25, m	77.3	5.01, t (7.9)	71.8	4.23, m
5′	78.6	4.22, m	78.8	4.24, m	84.4	4.57, m	78.8	4.01, m
6′	62.8	4.53, m	63.0	4.53, m	64.4	4.37, m	62.7	4.51, m
		4.31, m		4.39, m				4.32, m

Compound 19 was isolated as white amorphous powder. The HRESIMS of 19 showed the ion at m/z 757.4401 [M + H]⁺, and its molecular formula was inferred as C₃₉H₆₄O₁₄ on the basis of the analysis of ¹H and ¹³C NMR and DEPT spectra. On acid hydrolysis, 19 liberated D-fructose and D-glucose, identified by GC analysis of their chiral derivatives. From comparison of the NMR data of 19 with those of 18, it was indicated that 19 contained the same aglycone as 18. The HMBC correlations between the H-6 (δ_H 4.85, 4.55) of the glucose and C-2 (δ_C 106.2) of the fructose indicated the saccharide attached to C-3 of the aglycone was β-D-fructofuranosyl-(2 → 6)-β-D- glucopyranosyl. Thus, compound 19 was elucidated as (25R)-5β-spirostan-1β,3β-diol- 3-O-β-D-fructofuranosyl-(2 → 6)-β-D-glucopyranoside.

Compound 20 was isolated as white amorphous powder. The HRESIMS of 20 showed the ion at m/z 739.4254 [M + H]⁺, and its molecular formula was inferred as C₃₉H₆₂O₁₃ on the basis of the analysis of ¹H and ¹³C NMR and DEPT spectra. Compared the ¹H and ¹³C NMR data with those of (25R)-5β-spirostan-3β-ol-3-O-β-D- glucopyranosyl-(1 → 4)-β-D-glucopyranoside³⁴, the NMR features of those two compounds were almost same except for the signals of the F-ring, which was suggested the same A-E ring substitution in the two compounds. The signals due to the methyl of C-27 in (25R)-5β-spirostan-3β-ol-3-O-β-D-glucopyranosyl-(1 → 4)-β-D- glucopyranoside were replaced by the signals assigned to the C-25(27) exo-methylene group at δ_H 4.78 (1H, s) and 4.82(1H, s), and δ_C 144.8 (C-25) and 109.1 (C-27) in 20. Hence, the structure of 20 was elucidated as 5β-Spirost-25(27)-en-3β-ol-3-O-β-D- glucopyranosyl-(1 → 4)-β-D-glucopyranoside.

Antiproliferative activities

TB, TC, TD and all the isolated compounds were evaluated for antiproliferative activities against seven human tumor cell lines including FaDu (human hypopharyngeal carcinoma), Detroit 562 (human metastatic pharyngeal squamous cell carcinoma), CNE-1 (high differentiation human nasopharyngeal carcinoma), CNE-2 (low differentiation human nasopharyngeal carcinoma), HepG2 (human hepatocellular carcinoma), K562 (human chronic leukemia), SPC-A-1 (human lung adenocarcinoma) with a modified MTT method according to reported protocols, and cis-dichlorodiammineplatinum (II) was used as a positive control. The results were shown in Table 5 and Table S2 for the isolated compound and the crude extract of T. chinensis, respectively. TD showed moderate activity against five out of seven cancer cell lines (Fadu, Detroit 562, HepG2, K562 and SPC-A-1) with IC₅₀ values of 20.9–29.3 μg/mL. TC exhibited selective antiproliferative activity against Fadu, Detroit 562, and K562 with IC₅₀ values of 6.7 ± 0.5, 45.9 ± 1.7, and 6.5 ± 2.3 μg/mL, respectively. Compounds 17, 19 and 21 exhibited moderate antiproliferative effects against all of human cancer cell lines tested. Compound 22 showed moderate inhibitory activity against six out of seven human cancer cell lines tested excepted CNE-2. Compound 18 displayed weak inhibitory activities against Detroit 562, CNE-2 and HepG2 with IC₅₀ values of 39.4 ± 0.7, 40.7 ± 3.1 and 46.5 ± 1.0 μM, respectively. Compound 20 showed moderate antiproliferative activity against FaDu, Detroit 562 and HepG2. Compound 4 exhibited moderate inhibitory activity against FaDu and HepG2 with IC₅₀ values of 27.6 ± 3.6 and 29.7 ± 3.1 μM, respectively. Compounds 3, 5 and 12 exhibited potential antiproliferative activities against FaDu with IC₅₀ values of 28.6 ± 1.6, 41.2 ± 1.8 and 12.1 ± 1.2 μM, respectively. The other compounds exhibited IC₅₀ values larger than 50.0 μM against the seven tested cell lines and were considered to be inactive. Compounds 17–22 showed higher antiproliferative activities against human tumor cell lines than other compounds, suggesting that glycosylation at C-3 of the aglycon play an important role in the growth inhibition on cancer cells of spirostanol saponins. The structure-activity relationship of the isolated compounds (1–23) indicated that the glycosylation at C-3 of the aglycon was the most important structural feature for the high antiproliferative activity and other structural features (e.g., OH position and number) played a modified role in enhancing or reducing the activity. However comparison of antiproliferative activity is only an approximation as there are notable differences in IC₅₀ values between control compounds. A choice of reference and cell lines may lead to various conclusions with respect to the potency of antiproliferative compounds³⁵.

Table 5. Antiproliferative activities of some compounds from T. chinensis against human cancer cell lines.

Compound	IC50 (μM)a
	FaDu	Detroit 562	CNE-1	CNE-2	HepG2	K562	SPC-A-1
3	28.6 ± 1.6*	>50.0	>50.0	>50.0	>50.0	>50.0	>50.0
4	27.6 ± 3.6***	>50.0	>50.0	>50.0	29.7 ± 3.1***	>50.0	>50.0
5	41.2 ± 1.8***	>50.0	>50.0	>50.0	>50.0	>50.0	>50.0
12	12.1 ± 1.2	>50.0	>50.0	>50.0	>50.0	>50.0	>50.0
17	20.3 ± 2.1	20.2 ± 2.0	33.6 ± 0.3***	23.6 ± 1.9***	28.6 ± 1.0***	27.4 ± 0.2***	24.8 ± 0.7***
18	>50.0	39.4 ± 0.7***	>50.0	40.7 ± 3.1***	46.5 ± 1.0***	>50.0	>50.0
19	61.4 ± 2.5***	21.5 ± 1.1***	50.9 ± 1.1***	29.5 ± 0.9***	34.0 ± 0.5***	40.2 ± 2.1***	39.4 ± 1.0***
20	20.1 ± 0.4	20.1 ± 0.6***	>50.0	>50.0	28.0 ± 0.7***	>50.0	>50.0
21	12.7 ± 1.7*	22.7 ± 0.5**	17.4 ± 0.5***	14.5 ± 1.1**	14.3 ± 1.5***	22.2 ± 0.2***	22.2 ± 0.2***
22	20.5 ± 3.4	29.1 ± 0.6*	33.7 ± 0.6***	>50.0	17.7 ± 0.3***	33.8 ± 0.1***	26.3 ± 1.0***
CISb	18.1 ± 0.7	26.2 ± 1.5	10.8 ± 0.4	7.9 ± 1.1	6.5 ± 0.3	13.5 ± 0.8	3.8 ± 0.3

^aValues are presented as means ± SD (n = 3).

^bPositive control.

^*p < 0.05, **p < 0.01, ***p < 0.001 versus positive control.

Anti-inflammatory activities

Nitric oxide (NO) is a signaling molecule that plays a key role in the pathogenesis of inflammation³⁶. NO production by immune cells has been used as an indicator of the presence and extent of inflammation as well as the effectiveness of anti-inflammatory agents³⁷.

Compounds 1–23 were tested for their inhibitory effects on NO production induced by LPS in a macrophage cell line RAW 264.7. Cell viability was first determined by the MTT method to find whether inhibition of NO production was due to the cytotoxicity of the tested samples. The anti-inflammatory activities were summarized in Table 6. All the tested samples exhibited no cytotoxicity against RAW 264.7 macrophage cells at their effective concentrations. The crude extracts of T. chinensis TB, TC and TD showed moderate inhibition on NO production with IC₅₀ values of 32.6 ± 3.7, 37.5 ± 5.4 and 36.6 ± 1.8 μg/mL (see Table S3 in the supporting information). Compound 21 showed significant inhibition on NO production with IC₅₀ values of 11.5 μM, comparable to that of the positive control indomethacin.at 47.4 μM³⁸. Compounds 1, 4, 9, 10, 13, 14, 17 and 20 exhibited moderate inhibition with IC₅₀ values between 21.7 and 33.6 μM. Compounds 5, 7, 15, 18 and 19 displayed inhibition activity, but quite limited with IC₅₀ values of 45.9 ± 3.1, 45.2 ± 5.7, 46.1 ± 13.1, 41.6 ± 13.5 and 45.4 ± 11.2 μM, respectively. Other compounds showed weak activity with IC₅₀ value above than 50 μM. The structure-activity relationship of the isolated spirostanol saponins shows that small changes such as different positions or the number of the hydroxyl groups, R/S configuration on the aglycone led to changes in activity. The most active compound is 21, and comparison with 22 shows that change in the orientation or of the methyl group at C-25 from axial in 21 to equatorial in 22 diminishes the activity³⁹. Comparison of the activity of 4 and 5 shows that 4 is more active than 5 due to the exocyclic double in 5, which can be illustrated by the relative higher IC₅₀ value of compound 20 and the relative lower IC₅₀ value of compound 21. The kinds, the number and the linkage of sugar also play important roles in their anti-inflammatory activity⁴⁰.

Table 6. Inhibitory effects of some compounds from T. chinensis on NO production induced by LPS in macrophagesa^c.

Compound	IC50 (μM)a	Compound	IC50 (μM)a
1	28.7 ± 2.1	15	46.1 ± 13.1
4	21.7 ± 1.3	17	21.0 ± 0.8
5	45.9 ± 3.1	18	41.6 ± 13.5
7	45.2 ± 5.7	19	45.4 ± 11.2
9	33.6 ± 1.7	20	22.6 ± 5.0
10	28.7 ± 4.5	21	11.5 ± 1.3
13	26.1 ± 0.5	Indomethacinb	47.4 ± 4.5
14	29.1 ± 0.6

^aValues are presented as means ± SD (n = 3).

^bPositive control.

^cCompounds 2, 3, 6, 8, 11, 12, 16, 19, 22, 23 with IC₅₀ > 50.0 μM.

Conclusion

Twenty-three polyhydroxylated spirostanol saponins, including nine new compounds (1, 2, 12, 13, 16–20) were isolated from the rhizomes of T. chinensis and their structures were determined by spectroscopic methods. Among the isolated compounds, Compounds 17, 19 and 21 exhibited potential antiproliferative activities against all of human cancer cell lines tested. Compound 21 showed significant inhibition on NO production with IC₅₀ values of 11.5 μM. The present investigation suggested that T. chinensis can be a potential source of natural antiproliferative and anti-inflammatory agents and its spirostanol saponins may be responsible for the biological activity. However, further studies are required, not only to assess the bioactivities of the isolates in vivo, but also to investigate the mechanisms underlying the observed biological activities of isolated compounds.

Materials and Methods

Chemicals

HPLC-grade methanol was purchased from Oceanpak Chemical Co. (Gothenburg, Sweden). D101 macroporus resin (Xi′an Lanxiao Resin Corporation Ltd., Xi′an, China), silica gel (200–300 mesh, Anhui Liangchen Silicon Material Co. Ltd., Lu’an, China) and ODS (40–60 μm, Merck KGaA, Darmstadt, Germany) were used for column chromatography. All solvents used for column chromatography were of analytical grade and purchased from Sinopharm Chemical Reagent Co. (Shanghai, China). Foetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from HyClone Laboratories (Logan, UT, USA). Sugar reagents used for GC analysis, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cis-dichlorodiammineplatinum (II) and indomethacin were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

General experimental procedures

Optical rotations were measured with a JASCO P-1020 digital polarimeter. IR spectra were recorded on a PerkinElmer 100 IR spectrometer with KBr. NMR spectra were obtained on a Bruker Ultrashield 500 Plus instrument, using tetramethylsilane as internal standard. Waters AQUITY UPLC/Q-TOF mass spectrometer was used to record HRESIMS spectra. Semi-preparative HPLC was performed using a RAININ pump equipped with a Gilson 133 refractive index detector. A semi-preparative column (COSMOSIL-Pack 5C₁₈-MS-II, 10ID × 250 mm, 5 μm) was used for HPLC. Microplate reader (Kehua Technologies, Inc., Shanghai, China) was used for bioassay.

Plant material

The rhizomes of T. chinensis Baker was purchased from Shennongjia Forest District (Shennongjia, China), and identified by Prof. Xiangjiu He, the School of Pharmacy, Guangdong Pharmaceutical University. A voucher specimen (No. GDPU-NPR-2013002) was deposited in the Department of Medicinal Chemistry, Guangdong Pharmaceutical University, Guangzhou, China.

Extraction and isolation

The air-dried rhizomes of T. chinensis (17.0 kg) were extracted four times with 60% EtOH (each 70 L × 4 h) under reflux. The combined EtOH extracts were evaporated under reduced pressure (for complete removal of organic solvent) to yield an aqueous suspension (20 L), and then partitioned between H₂O (20 L) and EtOAc (20 L ×  4) to give an EtOAc fraction (218.0 g). The aqueous fraction was applied to a D101 macroporous resin column, eluted with H₂O (50 L), 20% EtOH (50 L), 60% EtOH (50 L) and 80% EtOH (50 L), repectively. The solvents were evaporated under vacuum to yield 20% EtOH eluted fraction (TB, 194.5 g) and 60% EtOH eluted fraction (TC, 458 g) and 80% EtOH eluted fraction (TD, 83.8 g).

A half part of fraction TC (200.0 g) was subjected to silica gel column chromatography (200 ~ 300 mesh, 3 kg, 95 × 1140 mm) eluted with a gradient of CHCl₃/MeOH (20:1 to 2:1, followed by MeOH) to yield 18 fractions (C1-C18). Fraction C3 (108.2 mg) was further purified by ODS and semi-preparative HPLC (3.0 mL/min, 85% MeOH in H₂O isocratic elution) to afford compound 12 (2.7 mg). Fraction C4 (157.5 mg) was separated by an ODS column eluted with a gradient of MeOH-H₂O (3:7 to 5:5, v/v) to afford compound 14 (23.0 mg). Fraction C5 (625.8 mg) was separated by an ODS column eluted with a gradient of MeOH-H₂O (1:9 to 2:8, v/v) to afford compound 13 (20.9 mg). Fraction C12 (5.1 g) was subjected to ODS eluted with a gradient of MeOH-H₂O (3:7 to 8:2, v/v) to afford eight fractions (C12-1 to C12-8). Subfraction C-12-3 (335.5 mg) was further purified by semi-preparative HPLC (3.0 mL/min, 60% MeOH in H₂O isocratic elution) to afford compound 7 (194.7 mg).

Fraction TD (83.3 g) was separated by silica gel column chromatography (200 ~ 300 mesh, 2 kg, 95 × 745 mm) eluted with a gradient of CHCl₃/MeOH (100:1 to 1:1, v/v) to obtain twenty-one fractions (D1-D21). Fraction D13 (15.8 g) was separated by an ODS column eluted with a gradient of MeOH-H₂O (1:9 to 7:3, v/v) to afford nine fractions (D13-1 to D13-9). Compound 5 (15.3 mg) was crystallized from subfraction D13-4 (90.2 mg) in MeOH. Fraction D14 (21.5 g) was subjected to ODS eluted with a gradient of MeOH-H₂O (1:9 to 8:2, v/v) to afford nine fractions (D14-1 to D14-9). Compound 8 (18.6 mg) was crystallized from subfraction D14-5 in MeOH. Subfraction D-14-7 (335.5 mg) was further purified by semi-preparative HPLC (3.0 mL/min, 70% MeOH in H₂O isocratic elution) to afford compound 4 (14.3 mg) and compound 15 (32.4 mg). Fraction D15 (1.8 g) was purified by ODS to obtain eleven fractions (D15-1 to D15-11). Subfraction D15-6 (87.4 mg) was purified by semi-preparative HPLC (3.0 mL/min, 80% MeOH in H₂O isocratic elution) to yield compound 3 (51.8 mg). Fraction D16 (1.5 g) was purified by ODS to obtain three fractions (D16-1 to D16-3). Subfraction D16-3 (1.18 g) was purified by semi-preparative HPLC (3.0 mL/min, 75% MeOH in H₂O isocratic elution) to yield compounds 20 (17.2 mg), 21 (14.2 mg) and 22 (23.7 mg). Fraction D18 (11.4 g) was subjected to ODS to obtain seventeen fractions (D18-1 to D18-17). Subfraction D18-9 (1.3 g) was further purified by semi-preparative HPLC (3.0 mL/min, 60% MeOH in H₂O isocratic elution) to yield compounds 9 (87.3 mg). Subfraction D18-10 (649.5 mg) was further purified by semi-preparative HPLC (3.0 mL/min, 60% MeOH in H₂O isocratic elution) to yield compounds 6 (55.3 mg) and 16 (25.5 mg). Subfraction D18-14 (243.8 mg) was further purified by semi-preparative HPLC (3.0 mL/min, 75% MeOH in H₂O isocratic elution) to yield compounds 17 (27.7 mg), 18 (22.0 mg) and 19 (58.1 mg). Fraction D19 (3.6 g) was subjected to silica gel column chromatography (300 ~ 400 mesh, 150 g, 95 × 745 mm) eluted with a gradient of CHCl₃/MeOH (10:1 to 3:1, v/v) to obtain eight fractions (D19-1 to D19-8). Subfraction D19-7 was further purified by semi-preparative HPLC (3.0 mL/min, 70% MeOH in H₂O isocratic elution) to yield compounds 1 (45.4 mg) and 23 (10.1 mg). Fraction D20 (4.9 g) was subjected to ODS eluted with a gradient of MeOH-H₂O (1:9 to 8:2, v/v) to obtain ten fractions (D20-1 to D20-10). Subfraction D20-6 was further purified by semi-preparative HPLC (3.0 mL/min, 70% MeOH in H₂O isocratic elution) to yield compound 2 (28.1 mg). Fraction D21 (4.6 g) was subjected to ODS eluted with a gradient of MeOH-H₂O (1:9 to 7:3, v/v) to obtain eleven fractions (D21-1 to D21-11). Subfraction D21-6 (1.2 g) was further purified by semi-preparative HPLC (3.0 mL/min, 60% MeOH in H₂O isocratic elution) to yield compounds 10 (62.0 mg) and 11 (29.2 mg).

Spirost-25(27)-en-1β,2β,3β,4β,5β-pentol-2-O-β-D-xylopyranoside (1) White amorphous powder; −43.3 (c 0.25, MeOH); IR (KBr) ν_max 3364, 2940, 1645, 1454, 1380, 1045, 920cm⁻¹; ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z [M + H]⁺ 611.3437 (calcd for C₃₂H₅₁O₁₁, 611.3431).

Spirost-25(27)-en-1β,2β,3β,4β,5β-pentol-2-O-α-L-arabinopyranoside (2) White amorphous powder; −49.2 (c 0.25, MeOH); IR (KBr) ν_max 3422, 2949, 1650, 1452, 1380, 1047, 923 cm⁻¹; ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z [M + H]⁺ 611.3412 (calcd for C₃₂H₅₁O₁₁, 611.3431).

Spirost-25(27)-en-1β,3α,5β-triol (12) White amorphous powder; −33.1 (c 0.25, MeOH); IR (KBr) ν_max 3300, 2950, 1650, 1592, 1455, 1381, 1046, 926, 899, 878 cm⁻¹; ¹H and ¹³C NMR data, see Table 2; HRESIMS m/z [M + H]⁺ 447.3112 (calcd for C₂₇H₄₃O₅, 447.3110).

Spirost-25(27)-en-1β,3α,4β,5β,6β-pentol (13) White amorphous powder; −34.9 (c 0.25, MeOH); IR (KBr) ν_max 3398, 2954, 1717, 1655, 1450, 1381, 1043, 924 cm⁻¹; ¹H and ¹³C NMR data, see Table 2; HRESIMS m/z [M + H]⁺ 479.3018 (calcd for C₂₇H₄₃O₇, 479.3009).

Spirost-25(27)-en-1β,2β,3β,5β-tetraol-5-O-β-D-glucopyranoside (16) White amorphous powder; −44.9 (c 0.50, MeOH); IR (KBr) ν_max 3424, 2941, 1651, 1452, 1372, 1046, 922 cm⁻¹; ¹H and ¹³C NMR data, see Table 3; HRESIMS m/z [M + H]⁺ 625.3552 (calcd for C₃₃H₅₃O₁₁, 625.3588).

5β-Spirost-25(27)-en-1β,3β-diol-3-O-β-D-glucopyranosyl-(1 → 4)-β-D-glucopyranoside (17) White amorphous powder; −52.9 (c 0.25, MeOH); IR (KBr) ν_max 3419, 2921, 1645, 1452, 1047, 920 cm⁻¹; ¹H and ¹³C NMR data, see Table 4; HRESIMS m/z [M + H]⁺ 755.4275 (calcd for C₃₉H₆₃O₁₄, 755.4218).

(25R)-5β-Spirostan-1β,3β-diol-3-O-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranoside (18) White amorphous powder; −27.0 (c 0.12, MeOH); IR (KBr) ν_max 3411, 2935, 1641, 1451, 1050, 986, 919, 899, 873 cm⁻¹; ¹H and ¹³C NMR data, see Table 4; HRESIMS m/z [M + H]⁺ 757.4403 (calcd for C₃₉H₆₅O₁₄, 757.4374).

(25R)-5β-Spirostan-1β,3β-diol-3-O-β-D-fructofuranosyl-(2 → 6)-β-D-glucopyranoside (19) White amorphous powder; −66.8 (c 0.50, MeOH); IR (KBr) ν_max 3391, 2929, 1632, 1451, 1053, 984, 918, 899, 865 cm⁻¹; ¹H and ¹³C NMR data, see Table 4; HRESIMS m/z [M + H]⁺ 757.4401 (calcd for C₃₉H₆₅O₁₄, 757.4374).

5β-Spirost-25(27)-en-3β-ol-3-O-β-D-glucopyranosyl-(1 → 4)-β-D- glucopyranoside (20) White amorphous powder; −30.6 (c 0.50, MeOH); IR (KBr) ν_max 3423, 2925, 1634, 1452, 1043, 921, 896 cm⁻¹; ¹H and ¹³C NMR data, see Table 4; HRESIMS m/z [M + H]⁺ 739.4254 (calcd for C₃₉H₆₃O₁₃, 739.4269).

Acid hydrolysis and GC analysis

Compounds 1, 2, 12, 13 and 16–20 (1–2 mg) were dissolved in 5 mL 2 M HCl and heated at 90 °C for 8 h. The reaction mixture was extracted with EtOAc (5 mL × 3) and the aqueous residue was evaporated under vacuum at 60 °C. Then 5 mg NH₂OH.HCl was added to the residue and the mixture was dissolved in 600 μL pyridine. After heating at 90 °C for 30 min, 300 μL Ac₂O was added. After homogenized, the mixture was heated at 90 °C for another 1 h. The reaction mixture was analyzed by GC using standard aldononitrile peracetates as reference samples⁴¹.

Measurement of inhibition activity on tumor cell proliferation

Antiproliferative activities against human nasopharyngeal cancer cells (CNE-1, CNE-2, FaDu, Detroit 562), human liver cancer cells (HepG2), human chronic leukemia cells (K562) and human lung adenocarcinoma cells (SPC-A-1) of the pure spirostanol saponins isolated from T. chinensis were measured by the modified MTT assay as described previously⁴².

Measurement of anti-inflammatory activity

Determination of NO production was performed by measuring the accumulation of nitrite in the culture supernatant using the Griess reagent, as previously described⁴³.

Statistical analysis

All the experiments were conducted for three independent replicates, and data were expressed as mean ± SD. Statistical analyses were performed by one-way ANOVA. Dunnett’s Multiple Comparison Test was used to determine the significance of differences between the groups. Differences at P < 0.05 were considered statistically significant.

Additional Information

How to cite this article: Xiang, L. et al. Antiproliferative and anti-inflammatory polyhydroxylated spirostanol saponins from Tupistra chinensis. Sci. Rep. 6, 31633; doi: 10.1038/srep31633 (2016).