Accepted Manuscript

Anti-inflammatory steroids from the rhizomes of Dioscorea septemloba Thunb

Yi Zhang, Hai-Yang Yu, Li-Ping Chao, Lu Qu, Jing-Ya Ruan, Yan-Xia Liu,
Yong-Zhe Dong, Li-Feng Han, Tao Wang

PII: S0039-128X(16)30039-3
DOI: http://dx.doi.org/10.1016/j.steroids.2016.05.007
Reference: STE 7978

To appear in: Steroids

Received Date: 6 January 2016

Revised Date: 29 April 2016
Accepted Date: 20 May 2016

Please cite this article as: Zhang, Y., Yu, H-Y., Chao, L-P., Qu, L., Ruan, J-Y., Liu, Y-X., Dong, Y-Z., Han, L-F.,
Wang, T., Anti-inflammatory steroids from the rhizomes of Dioscorea septemloba Thunb, Steroids (2016), doi:
http://dx.doi.org/10.1016/j.steroids.2016.05.007

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Anti-inflammatory steroids from the
rhizomes of Dioscorea septemloba Thunb

Yi Zhang1,a, Hai-Yang Yu1,a, Li-Ping Chao2, Lu Qu1, Jing-Ya Ruan2, Yan-Xia Liu1,

Yong-Zhe Dong2, Li-Feng Han2, and Tao Wang 1*

1. Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of

Traditional Chinese Medicine, 312 Anshanxi Road, Nankai District, Tianjin, 300193,

China

2. Tianjin Key Laboratory of TCM Chemistry and Analysis, Institute of Traditional

Chinese Medicine, Tianjin University of Traditional Chinese Medicine, 312 Anshan

Road, Nankai District, Tianjin, 300193, China

a
Y. Zhang and H.-Y. Yu contributed equally to this work.

* Corresponding authors. Tel./fax: +86 22 5959 6163. E-mail addresses:

wangtao@tjutcm.edu.cn (T. Wang)

1
ABSTRACT

Seven new steroid glycosides, dioscorosides A1 (1), A2 (2), B1 (3), B2 (4), C1 (5), C2

(6), and D (7), together with 22 known ones (829) were isolated from the rhizomes

of Dioscorea septemloba, their structures were elucidated by chemical and

spectroscopic methods. All isolates were evaluated for in vitro anti-inflammatory

potential using LPS-stimulated RAW 264.7 murine macrophages. Among them,

spirostane glycosides 18 and 2124 exhibited significant inhibition of nitrite

production. Moreover, the structure-activity relationship was summarized.

Key words: Dioscorea septemloba Thunb; steroids; anti-inflammatory activity

2
1. Introduction

The genus Dioscorea belongs to the Dioscoreaceae family with more than 600

species of flowering plants, and is found in tropical and sub-tropical regions in the

world. Dioscorea septemloba Thunb, as a species of this genus, is widely distributed

in Zhejiang, Jiangxi, Fujian, Hubei, Hunan, Guangdong, and Guanxi provinces in

China. The dried rhizomes of it have been used as a common Chinese traditional

medicine (TCM) for treatment of rheumatism, urethra, renal infection, and so on for a

long time.

Steroid glycosides, have been found to be the characteristic secondary metabolites

in Dioscorea genus plants, which have broad biological activities such as anti-tumor,

anti-fungal, and anti-inflammatory [1-4]. In our on-going program of screening the

anti-inflammatory agents from TCM, the 70% ethanol-water extract of D. septemloba

showed in vitro anti-inflammatory activity. Then, the isolation and biological activity

of D. septemloba rhizomes was studied, which led to the isolation of seven new

steroid glycosides, dioscorosides A1 (1), A2 (2), B1 (3), B2 (4), C1 (5), C2 (6), and D

(7), along with 22 known ones, spongioside B (8) [4], dioscoreavilloside A (9) [5],

protodioscin (10) [6], methyl protodioscin (11) [7], protogracillin (12) [8], methyl

protogracillin (13) [8], pseudoprotodioscin (14) [9], pseudoprotogracillin (15) [10],

26-O--D-glucopyranosyl-3,23,26-triol-25(R)-furosta-5,20(22)-dien-3-O--L-rhamn

opyranosyl(14)-[-L-rhamnopyranosyl(12)]--D-glucopyranoside (16), diosgenin

(17) [11], prosapogenin A of dioscin (18) [12], diosgenin

3-O--D-glucopyranosyl(13)--D-glucopyranoside (19) [8], prosapogenin B of

3
dioscin (20) [13], dioscin (21) [6], gracillin (22) [14], 7-oxodioscin (23) [15],

(25R)-spirost-5-en-3,7-diol-3-O--L-arabinofuranosyl(14)-[-L-rhamnopyranosyl

-(12)]--D-glucopyranoside (24) [16], orbiculatoside B (25) [17], borassoside B (26)

[18], stigmasterol (27) [19], -sitosterol (28) [19], and hypoglaucin G (29) [20]. In

this paper, their structures were determined by analysis of physical data, spectroscopic

analysis, and chemical methods. And all isolates were evaluated for in vitro

anti-inflammatory potential using LPS-stimulated RAW 264.7 murine macrophages.

Chart 1.

2. Experimental procedures

2.1. General

Optical rotations were recorded on Rudolph Autopol IV automatic polarimeter. IR

spectra were run on Varian 640-IR FT-IR spectrophotometer, respectively. NMR

spectra were obtained on Bruker 500 MHz NMR spectrometer at 500 MHz for 1H and

125 MHz for 13C NMR (internal standard: TMS). Negative-ion HRESI-TOF-MS were

made on Agilent Technologies 6520 Accurate-Mass Q-Tof LC/MS spectrometer.

Column chromatographies (CC) were performed on macroporous resin D101

(Haiguang Chemical Co., Ltd., Tianjin, China), silica gel (74149 m, Qingdao

Haiyang Chemical Co., Ltd., Qingdao, China), ODS (50 m, YMC Co., Ltd., Tokyo,

4
Japan), and Sephadex LH-20 (Ge Healthcare Bio-Sciences, Uppsala, Sweden).

Preparative high performance liquid chromatography (PHPLC) column, Cosmosil

5C18-MS-II (20 mm i.d. 250 mm, Nakalai Tesque, Inc., Tokyo, Japan) were used to

separate the constituents.

2.2. Plant material

The rhizomes of D. spongiosae were purchased at a market in Anguo country,

Heibei province, China, and identified by Dr. Li Tianxiang. The voucher specimen was

deposited at the Academy of Traditional Chinese Medicine of Tianjin University of

TCM.

2.3. Extraction and isolation

The dried roots of D. spongiosae (7.8 kg) were refluxed with 70% ethanol-water

for two times. Evaporation of the solvent under pressure provided a 70%

ethanol-water (1150.1 g). The residue was dissolved in H2O, and subjected to D101

CC (H2O 95% EtOH Acetone) to afford H2O (725.2 g), 95% EtOH (245.5 g),

and acetone (1.9 g) eluent, respectively.

The EtOH eluent (150.0 g) was subjected to silica gel CC [CHCl3-MeOH (100:1

100:3 100:7, v/v) CHCl3-MeOH-H2O (10:3:1 7:3:1 6:4:1, v/v/v, lower

layer) ] to yield 12 fractions (Fr. 112). Fraction 2 (11.0 g) was separated by silica gel

5
CC [Petroleum ether-EtOAc (50:1 30:1 20:1 10:1 5:1, v/v)

CHCl3-MeOH (100:1 100:5, v/v)], and 16 fractions (Fr. 2-12-16) were obtained.

Fraction 2-8 (383.6 mg) was purified by PHPLC [MeOH-H2O (98:2, v/v)] to yield 27

(7.0 mg) and 28 (9.0 mg). Fraction 2-10 (544.0 mg) was recystallized from MeOH to

give 17 (218.8 mg). Fraction 6 (14.0 g) was subjected to ODS CC [MeOH-H2O

(20:80 30:70 40:60 50:50 60:40 70:30 80:20 100:0, v/v)], and

yieled 18 fractions (Fr. 6-16-18). Fraction 6-15 (500.0 mg) was purified by PHPLC

[MeOH-1% CH3COOH (82:18, v/v) ] to provide 18 (158.9 mg). Fraction 6-16 (378.8

mg) was separated by PHPLC [MeOH-1% CH3COOH (84:16, v/v)] to give 19 (25.1

mg) and 20 (118.5 mg). Fraction 7 (8.0 g) was isolated by ODS CC [MeOH-H2O

(20:80 30:70 40:60 50:50 60:40 70:30 80:20 100:0, v/v)] to give

14 fractions (Fr. 7-17-14). Fraction 7-9-4 (34.2 mg) was purified by PHPLC

[CH3CN-1% CH3COOH (26:74, v/v)], and 9 (4.2 mg) was obtained. Fraction 7-13

(178.9 mg) was separated by PHPLC [MeOH-1% CH3COOH (75:25, v/v)] to yield 23

(26.7 mg). Fraction 8 (4.5 g) was isolated by ODS CC [MeOH-H2O (20:80 30:70

40:60 50:50 60:40 70:30 80:20 100:0, v/v)], 16 fractions (Fr.

8-1Fr. 8-16) were obtained. Fraction 8-11 (193.1 mg) was further separated by

PHPLC [CH3CN-1% CH3COOH (36:64, v/v)] to give 25 (20.0 mg). Fraction 9 (8.3 g)

was isolated by ODS CC [MeOH-H2O (30:70 40:60 50:50 60:40 70:30

80:20 100:0, v/v)] to provide 15 fractions (Fr. 9-19-15). Fraction 9-9 (427.9

mg) was subjected to PHPLC [MeOH-H2O (55:45, v/v)] to yield 3 (23.3 mg).

Fraction 9-10440.0 mgwas separated by PHPLC [MeOH-H2O (55:45, v/v)] to give

6
8 (70.5 mg) and 26 (17.1 mg). Fraction 9-16 (186.7 mg) was further isolated by

PHPLC [CH3CN-1% CH3COOH38:62, v/v] to obtain 5 (30.4 mg), 6 (14.7 mg), and

24 (12.4 mg). Fraction 9-17 (439.3 mg) was further purified by PHPLC [MeOH-H2O

(82:18, v/v)], as a result, 21 (22.2 mg) and 22 (19.8 mg) were obtained. Fraction 10

(15.0 g) was separated by PHPLC [MeOH-H2O (30:70 40:60 50:50 60:40

70:30 100:0, v/v)] to yield 29 fractions (Fr. 10-110-29). Fraction 10-20 (119.9

mg) was purified by PHPLC [MeOH-H2O (50:50, v/v)] to yield 4 (41.7 mg). Fraction

10-21157.3 mgwas separated by PHPLC [MeOH-H2O (50:50, v/v)] to give 29

(39.5 mg). Fraction 10-23 (206.3 mg) was further subjected to PHPLC [CH3CN-1%

CH3COOH (26:74, v/v)] to obtain 2 (77.6 mg). Fraction 10-23-7 (34.6 mg) was

further isolated by PHPLC [CH3CN-1% CH3COOH (26:74, v/v)], and 16 (11.8 mg)

was yielded. Fraction 10-28 (4.0 g) was subjected to PHPLC [MeOH-H2O (50:50,

v/v)] to give 22 fractions (Fr. 10-28-110-28-22). Fraction 10-28-12 (200.0 mg) was

isolated by PHPLC [MeOH-H2O (68:32, v/v)] to produce 7 (33.1 mg), 11 (13.7 mg),

and 13 (11.4 mg). Fraction 10-28-17 (200.0 mg) was further separated by PHPLC

[MeOH-H2O (68:32, v/v)], and 10 (5.7 mg) was obtained. Fraction 11 (11.0 g) was

subjected to ODS CC [MeOH-H2O (10:90 20:80 30:70 40:60 50:50

60:40 70:30 80:20 90:10 100:0, v/v)] to give 14 fractions (Fr.

11-111-14). Fraction 11-12 (3.2 g) was purified by PHPLC [CH3CN-1% CH3COOH

(25:75, v/v)] to produce 12 (46.4 mg). Fraction 11-14 (335.1 mg) was further isolated

by PHPLC [MeOH-1% CH3COOH (65:35, v/v)] to yield 14 (19.0 mg) and 15 (23.0

mg).

7
2.3.1. Doscoroside A1 (1)

White powder; []25 D 34.2 (c 0.76, MeOH); IR (KBr) max 3407, 2936, 1700,

1647, 1457, 1381, 1129, 1074, 1040, 815, 691 cm1; 1H and 13C NMR data, see Table

1; HRESI-TOF-MS Negative-ion mode m/z 947.4885 [M + COOH] (calcd for

C46H75O20, 947.4847).

2.3.2. Doscoroside A2 (2)

White powder; []25D 45.2 (c 0.89, MeOH); IR (KBr) max 3411, 2935, 1700,

1647, 1455, 1383, 1039, 811, 697 cm1; 1H and 13

C NMR data, see Table 2;

HRESI-TOF-MS Negative-ion mode m/z 1093.5472 [M + COOH] (calcd for

C52H85O24, 1093.5436).

2.3.3. Doscoroside B1 (3)

White powder; []25 D 31.1 (c 0.97, MeOH); IR (KBr) max 3398, 2936, 1457,

1375, 1128, 1074, 1041, 690 cm1; 1

H and 13
C NMR data, see Table 3;

HRESI-TOF-MS Negative-ion mode m/z 949.5012 [M + COOH] (calcd for

C46H77O20, 949.5014).

2.3.4. Doscoroside B2 (4)

White powder; []25 D 48.9 (c 0.94, MeOH); IR (KBr) max 3398, 2935, 1647,

1456, 1365, 1131, 1073, 1041, 812 cm1; 1H and 13

C NMR data, see Table 4;

8
HRESI-TOF-MS Negative-ion mode m/z 1095.5612 [M + COOH] (calcd for

C52H87O24, 1095.5593).

2.3.5. Doscoroside C1 (5)

White powder; []25 D 52.8 (c 0.77, MeOH); IR (KBr) max 3401, 2930, 1451,

1385, 1242, 1130, 1044, 981, 813 cm1; 1H and 13

C NMR data, see Table 5;

HRESI-TOF-MS Negative-ion mode m/z 937.4579 [M + Cl] (calcd for C45H74O18Cl,

937.4569).

2.3.6. Doscoroside C2 (6)

White powder; []25 D 59.3 (c 0.61, MeOH); IR (KBr) max 3399, 2931, 1647,

1378, 1242, 1127, 981, 814 cm1; 1

H and 13
C NMR data, see Table 6;

HRESI-TOF-MS Negative-ion mode m/z 963.4790 [M + COOH] (calcd for

C46H75O21, 963.4806).

2.3.7. Doscoroside D (7)

White powder; []25 D 82.2 (c 0.81, MeOH); IR (KBr) max 3400, 2936, 1645,

1454, 1379, 1132, 1043, 911, 813 cm1; 1H and 13

C NMR data, see Table 7;

HRESI-TOF-MS Negative-ion mode m/z 1081.4974 [M + Cl] (calcd for

C51H82O22Cl, 1081.4992).

2.3.8. Acid hydrolysis for 17

9
 Compounds 17 (each 1.5 mg) were refluxed with in 1 M HCl (1.0 mL) on a

water bath for 3 h, respectively. The reaction mixture was neutralized with Amberlite

IRA-400 (OH form) and removed by filtration. The aqueous layer was subjected to

HPLC analysis using the similar method as reference [21]: HPLC column, Kaseisorb

LC NH2-60-5, 4.6 mm i.d.250 mm (Tokyo Kasei Co., Ltd., Tokyo, Japan); detection,

optical rotation [Shodex OR-2 (Showa Denko Co., Ltd., Tokyo, Japan); mobile phase,

CH3CN-H2O [(75:25, v/v; flow rate 1.0 mL/min)]. L-rhamnose (8.8 min, negative

optical rotation) and D-glucose (17.0 min, positive optical rotation) were identified for

17 by comparison of their retention times and optical rotations with those of

authentic samples.

2.4. In vitro anti-inflammatory assay

2.4.1. Materials

LPS, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and

L-N6-(1-Iminoethyl) lysine (L-NIL) were purchased from Sigma Chemical (St. Louise,

MO).

2.4.2. Cell culture and cell viability assay

RAW 264.7 macrophages (IBMS, CAMS/PUMC, Beijing China) were maintained

in DMEM supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, and

100 g/mL streptomycin in a humidified atmosphere containing 5% CO2 at 37C. An

10
MTT assay was used to determine cell viability. In brief, RAW 264.7 macrophages were

seeded in 24-well plastic plates and treated with 030 M steroids from D. septemloba

rhizomes for 24 h, respectively. The medium was removed and the cells were incubated with

0.5 mg/mL of MTT solution. After 3 h incubation, the supernatant was removed and

formation of formazan, and the absorbance at 540 nm was measured with a microplate reader

[25].

2.4.3. Measurement of NO levels

As nitrite is a major stable product of NO, the concentration of NO in culture

supernatants was determined by measuring the nitrite levels by using Griess reagent

(St. Louise, MO). Cells were pretreated with steroids from D. septemloba rhizomes, for 1

h, and then stimulated with LPS (500 ng/mL) for 24 h. After incubation, aliquots of

100 L of each culture medium was mixed with an equal volume of Griess reagent.

Nitrite levels were determined using an ELISA plate reader at 540 nm, and

concentrations were calculated by reference to a NaNO2 standard calibration curve

[24].

3. Results and discussion

Doscoroside A1 (1) was a white powder with molecular formula C45H74O18,

established by negative-ion HRESI-TOF-MS at m/z 947.4885 [M + COOH] (calcd

for C46H75O20, 947.4847). The IR spectrum showed absorption bands ascribable to

11
hydroxyl (3407 cm1), carbonyl (1700 cm1), olefinic bond (1647 cm1), and

O-glycosidic linkage (1074 cm1). Acid hydrolysis of 1 with 1 M HCl afforded

D-glucose and L-rhamnose, whose absolute configurations were determined by HPLC

analysis [21]. The 1H and 13

C NMR assignment (Table 1) indicated 1 was a

cholestane-type steroid with five methyls [ 0.92 (6H, d, J = 5.5 Hz, H3-26 and 27),

1.10, 1.32 (3H each, both s, H3-19, 18), 1.25 (3H, d, J = 7.0 Hz, H3-21)], an olefinic

hydrogen [ 5.20 (br. d, ca. J = 5 Hz, H-6)], together with two -D-glucopyranosyl [

4.76 (1H, d, J = 7.5 Hz, H-1'''), 4.99 (1H, d, J = 7.0 Hz, H-1')], and one

-L-rhamnopyranosyl [ 6.35 (1H, br. s, H-1'')]. The 1H1H COSY spectrum of 1

suggested the presence of six partial structures written in bold lines as shown in Fig.

1. According to the long-range correlations from H-4 to C-5, C-6, C-10; H-6 to C-8,

C-10; H3-18 to C-1214, C-17; H3-19 to C-1, C-5, C-9, C-10; H3-21 to C-17, C-20,

C-22; H3-26 to C-24, C-25, C-27; H3-27 to C-2426; H-1' to C-3; H-1'' to C-2'; H-1'''

to C-16, the planar structure of 1 was determined, which was very close to that of

dioscoreavilloside B,

(22R)-3,16-di-(O--D-glucopyranosyl)-22-hydroxycholest-5-en-12-one [5,22],

except for the -L-rhamnopyranosyl substitution at 2'-position of -D-glucopyranosyl

linked with 3-position in 1, Consequently, the structure of doscoroside A1 (1) was

elucidated and named as

(22R)-3-O-[-L-rhamnopyranosyl(12)--D-glucopyranosyl]-16-O--D-glucopyra

nosyl-22-hydroxycholest-5-en-12-one.

12
 Fig. 1.

Table 1.

Doscoroside A2 (2) was obtained as white powder, and its molecular formula was

deduced as C51H84O22 from [M + COOH] quasi-molecular ion at m/z 1093.5472

(calcd for C52H85O24, 1093.5436) in the negative-ion HRESI-TOF-MS spectrum. The

1 13
H, C NMR (Table 2) and 2D NMR (1H1H COSY, HSQC, HMBC) spectra

indicated that 2 possessed the same functional groups as 1:

(22R)-3,16-di-(O--D-glucopyranosyl)-22-hydroxycholest-5-en-12-one. Moreover,

there was another -L-rhamnopyranosyl [ 5.82 (1H, br. s, H-1''')] in 2. Meanwhile,

the linkages of sugar parts in 2 were clarified by the HMBC correlations observed

from H 4.92 (H-1') to C 77.7 (C-3); H 6.36 (H-1'') to C 77.8 (C-2'); H 5.82 (H-1''')

to C 78.4 (C-4'); and H 4.77 (H-1''') to C 81.8 (C-16). On the other hand, to assign

the badly overlapped protons in sugar chemical shift range, HSQC-TOCSY

experiment was developed. In the HSQC-TOCSY spectra, the correlations between

the following proton and carbon pairs were observed: C 100.2 (C-1') and H 3.64

(H-5'), 4.19 (H-2' and 3'), 4.35 (H-4'), 4.92 (H-1'); H 4.35 (H-4') and C 61.2 (C-6');

C 101.9 (C-1'') and H 4.61 (H-3''), 4.82 (H-2''), 6.36 (H-1''); H 1.75 (H-6'') and C

18.6 (C-6''), 69.4 (C-5''), 72.7 (C-3''), 74.0 (C-4'''); C 102.8 (C-1''') and H 4.54

(H-3'''), 4.68 (H-2'''), 5.82 (H-1'''); H 1.61 (H-6''') and C 18.4 (C-6''), 70.3 (C-5''),

72.6 (C-3''), 73.8 (C-4'''); C 106.9 (C-1'''') and H 3.85 (H-5''''), 4.02 (H-2''''), 4.17

(H-3''''), 4.25 (H-4''''), 4.77 (H-1''''); H 4.25 (H-4'''') and C 62.8 (C-6''''). Finally, the

13
structure of 2 was determined.

Table 2.

Doscoroside B1 (3) was a white powder with negative optical rotation []D25

31.1 (c = 0.97, MeOH)]. The molecular formula C45H76O18 of it was determined by

negative-ion HRESI-TOF-MS (m/z 949.5012 [M + COOH] (calcd for C46H77O20,

949.5014). The combined analysis of the 1D and 2D NMR spectra suggested that 3

was a cholestane glycoside with four oxygen substitutions at C-3 (C 78.2), C-12 (C

78.2), C-16 (C 84.7), and C-22 (C 75.9), and three sugar residues (two

-D-glucopyranosyl and one -L-rhamnopyranosyl units). The NMR data of 3 were

found to be close to those of 1 except that the resonances for C-12 oxo group (C

214.1) and C-22(R) [C 33.8 (C-23), 35.3 (C-20), 73.2 (C-22)] were replaced by those

of oxygenated methine (C 78.2) and C-22(S) [C 35.2 (C-23), 38.1 (C-20), 75.9

(C-22)], respectively in 3 [5,22]. The NMR data for aglycon of 3 were close to those

of dioscoreavilloside A,

(22S)-3,16-di-(O--D-glucopyranosyl)-12,22-dihydroxycholest-5-ene [22,23].

Then, the structure of doscoroside B1 (3) was determined as

(22S)-3-O-[-L-rhamnopyranosyl(12)--D-glucopyranosyl]-16-O--D-glucopyra

nosyl-12,22-dihydroxycholest-5-ene.

Table 3.

14
 The molecular formula of doscoroside B2 (4) was established as C51H86O22 based

on the [M + COOH] quasimolecular ion at m/z 1095.5612 (calcd for C52H87O24,

1095.5593) in the HRESI-TOF-MS spectrum. The 1H and 13

C NMR data assignment

(Table 4) based on 1H 1H COSY, HSQC, HMBC, and HSQC-TOCSY spectroscopic

data analyses indicated that 4 was comprised of the same aglycon and sugar parts as 3

and 2, respectively. Finally, on acid hydrolysis and identification with HPLC analysis,

the presences of D-glucose and L-rhamnose were clarified [21]. The structure of it was

elucidated as shown in Chart 1.

Table 4.

Doscoroside C1 (5) was isolated as white powder. In the negative-ion

HRESI-TOF-MS of it, quasimolecular ion peak was observed at 937.4579 [M + Cl]

(calcd for C45H74O18Cl, 937.4569), and the molecular formula was revealed to be

C45H74O18. The 1H NMR spectrum of 5 showed signals of two tertiary methyl groups

at 0.89, 1.61 (3H each, both s, H3-18, 19), four secondary methyl groups at 0.68

(3H, d, J = 5.5 Hz, H3-27), 1.12 (3H, d, J = 7.0 Hz, H3-21), 1.59 (3H, d, J = 6.5 Hz,

H3-6'''), and 1.74 (3H, d, J = 6.0 Hz, H3-6''), as well as three anomeric proton

resonances at 4.81 (1H, d, J = 8.0 Hz, H-1'), 5.79 (1H, br. s, H-1'''), and 6.34 (1H, br.

s, H-1''). Acid hydrolysis of 5 with 1 M HCl afforded D-glucose and L-rhamnose,

whose absolute configurations identified with HPLC analysis [21], and the molar ratio

15
of them was 1:2. Therefore, the resonances at 1.59 and 1.74 were due to the methyl

13
groups of two rhamnose. The C NMR and DEPT spectra exhibited 45 carbon

resonances, 27 of which were attributed to the aglycon. A characteristic quaternary

carbon signal at C 109.2 indicated that 5 was a spirostanol glycoside. The signals

appearing from rings D, E, and F of its aglycon were essentially agree with those of

dioscin (21) [6] in the 13C NMR spectrum (Table 5), which suggested that 5 possessed

a 25R-spirostane skeleton. On the other hand, two oxygenated methine resonances [C

75.9 (C-3), 76.1 (C-6)] and a hydroxy-bearing quaternary carbon signal [C 75.5 (C-5)

were observed. And the locations of the hydroxy groups on the aglycon were

determined by means of 1H1H COSY, HSQC, HMBC, and HSQC-TOCSY spectra.

Especially in the HSQC-TOCSY spectrum of it, the correlations between C 75.9 (C-3)

and H 1.45, 2.04 (H2-1), 2.04, 2.18 (H2-2), 2.45, 2.89 (H2-4), 4.81 (H-3); C 76.1 (C-6)

and H 1.88, 2.14 (H2-7), 2.30 (H-8), 4.13 (H-6) were clearly found. And in the

HMBC spectrum, the long-range correlations from H 1.61 (H3-19), 2.45, 2.89 (H2-4)

to C 75.5 (C-5) were observed, which made the locations of hydroxy groups be

assigned at C-3, 5, and 6 positions. Moreover, the NOE correlations (Fig. 2) between

H 0.89 (H3-18) and H 2.30 (1H, m, H-8); H 1.61 (H3-19) and H 2.30 (H-8), 2.04

(1H, m, H-2), 2.89 (1H, dd, J = 12.0, 13.0 Hz, H-4) displayed in the NOESY

spectrum indicated an A/B trans ring junction. On the other hand, the NOE

correlations between H 2.45 (1H, dd, J = 4.5, 13.0, H-4) and H 4.13 (1H, m, H-6),

4.81 (1H, m, H-3) suggested both of the two hydroxyls at position 3 and 6 were in

-configuration. Therefore, the aglycon of 5 was identified as

16
(3,5,6,25R)-spirostane-3,5,6-triol. The linkage positions and sequence of the sugar

moieties could be assigned by analysis of the HMBC spectrum, in which the

correlations from H 4.81 (H-1') to C 75.9 (C-3); H 6.34 (H-1'') to C 77.9 (C-2'); H

5.79 (H-1''') to C 78.5 (C-4') were observed. Thus, the structure of doscoroside C1 (5)

was elucidated.

Fig. 2.

Table 5.

The HRESI-TOF-MS of doscoroside C2 (6) exhibited a quasimolecular ion at m/z

963.4790 [M + COOH] (calcd for C46H75O21, 963.4806), corresponding to a

molecular formula C45H74O19. The 1H, 13

C (Table 6) and NOESY (Fig. 2) spectra for

aglycon of 6 were very close to those of 5, which indicated 6 had the same aglycon

((3,5,6,25R)-spirostane-3,5,6-triol) as 5. Acid hydrolysis on 6 afforded D-glucose

and L-rhamnose with the molar ratio 2:1, being agreement with three anomeric proton

resonances at 4.83 (1H, d, J = 7.5 Hz, H-1'), 5.05 (1H, d, J = 7.5 Hz, H-1'''), and

6.35 (1H, br. s, H-1''). According to the HMBC correlations from anomeric protons to

their linked carbons (Fig. 1), the linkage positions and sequence of the sugar moieties

were assigned.

Table 6.

17
 Doscoroside D (7) was obtained as white powder. Its molecular formula was

revealed to be C51H82O22 by HRESI-TOF-MS at m/z 1081.4974 [M + Cl] (calcd for

13
C51H82O22Cl, 1081.4992). The C NMR and DEPT spectra exhibited 51 carbon

resonances, 27 of which were attributed to the aglycon. A characteristic quaternary

carbon signal at C 109.5 suggested that 7 was a spirostanol glycoside, too.

Comparison of the 1H and 13

C NMR spectroscopic data (Table 7) of 7 with those of

pratioside C [23], identified the aglycon of 7 as

(3,25S)-spirost-5-en-27-hydroxymethyl-3-ol. Acid hydrolysis on 7 afforded

D-glucose and L-rhamnose. Moreover, compared with compounds 2, 4 and 5, it

showed the presence of 2,4-di--L-rhamnopyranosyl--D-glucopyranosyl group.

Meanwhile, there was another -D-glucopyranosyl in 7. Furthermore, the linkage

positions of the two sugar moieties were assigned by long-range correlations observed

in HMBC spectrum (Fig. 1).

Table 7.

To investigate the effects of steroids from D. septemloba rhizomes on cell viability,

RAW 264.7 macrophages were treated with LPS in the presence or absence of

compounds, and cell numbers were assessed by MTT assays. As all of them did not

significantly affect cell viability, even at a concentration of 30 M (S52). 30 M was

used in subsequent experiments.

The potential anti-inflammatory effects of compounds on LPS-stimulated NO

18
production were examined in RAW 264.7 macrophages by pretreating cells with

various compounds for 1 h before stimulation with 500 ng/mL LPS for 24 h. NO

concentrations in the culture medium were measured by using Griess reagent. As

shown in Table 8, NO production was markedly induced in LPS-stimulated RAW

264.7 macrophages when compared to unstimulated normal (negative control). The

70% ethanol-water extract of D. septemloba showed in vitro anti-inflammatory

activity at 100 g/mL. Compounds 18, 21 and 22 displayed significant inhibitory

activities on LPS-induced NO production at 30 M, but 23 and 24 showed slight

activities on it at same concentration Positive control cells treated with NIL (30 M),

a selective inhibitor of inducible nitric oxide synthase (Table 8) [24]. These results

suggested that compounds 18 and 2124 may have potent anti-inflammatory activity.

In summary, four kinds of steroids such as cholestane (14, 8, 9, 27, 28),

spirostane (57, 1726), furostane (1016), and pregnane (29) steroids were obtained

from the rhizomes of D. septemloba, and all isolates were evaluated for in vitro

anti-inflammatory potential using LPS-stimulated RAW 264.7 murine macrophages.

Among them, cholestane, furostane, and pregnane glycosides showed no

anti-inflammatory activity on the cells. On contrast, spirostane glycosides (18, 2124)

showed strong activity on NO production, but the aglycon (17) of them showed no

effect on it, which indicated spirostane type glycosides are the major

anti-inflammation compounds in the rhizomes of D. septemloba. Moreover, compared

the activities of 1726, we can conclude that

2--L-rhamnopyranosyl--D-glucopyranosyl moiety substitution at 3 position will

19
enhance the activity. On the other hand, the evidence of comparison between

compounds 5 and 21, 6 and 22 indicated double bond at position 5 is essential moiety

for NO product inhibition.

Table 8.

Acknowledgments

This work was financed by Program for New Century Excellent Talents in

University (NCET-12-1069), Program for Tianjin Innovative Research Team in

University (TD12-5033).

20
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22
[17] Shen P, Wang SL, Liu XK, Yang CR, Cai B, Yao XS. A new steroidal saponin

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[18] Yoshikawa M, Xu F, Morikawa T, Pongpiriyadacha Y, Nakamura S, Asao Y,

Kumahara A, Matsuda H. Medicinal flowers. XII.(1) New spirostane-type steroid

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[20] Hu K, Yao XS, Dong AJ, Kobayashi H, Iwasaki S, Jing YK. A new pregnane

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23
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24
Legends:

Chart 1.

Fig. 1. The main 1H 1H COSY and HMBC correlations of 17.

Fig. 2. The main NOE correlations for AD rings of 5 and 6.

1
Table 1 H (500 MHz) and 13C NMR (125 MHz) data for 1 in C5D5N

1
Table 2 H (500 MHz) and 13C NMR (125 MHz) data for 2 in C5D5N

1
Table 3 H (500 MHz) and 13C NMR (125 MHz) data for 3 in C5D5N

1
Table 4 H (500 MHz) and 13C NMR (125 MHz) data for 4 in C5D5N

1
Table 5 H (500 MHz) and 13C NMR (125 MHz) data for 5 in C5D5N

1
Table 6 H (500 MHz) and 13C NMR (125 MHz) data for 6 in C5D5N

1
Table 7 H (500 MHz) and 13C NMR (125 MHz) data for 7 in C5D5N

Table 8 Inhibitory effects of 70% ethanol-water extract of D. septemloba (0) and compounds

129 on NO production in RAW 264.7 macrophages.

25
Chart 1.

26
 OH OH OH OH
21 22 26
23
O 18 20
24
25 O OH 22 OH
12
12 16 O 27
O O O
19 11 17
13
1 9
2 10 14 15
8
3 HO HO
7
O 5 O O O
4 6
HO O HO HO O HO
HO 4'
OH HO 4'
OH
O 1' O O 1' O
O OH O 2' OH O OH O 2' OH
1'''' 1''''
OH 1'''
HO O O OH 1'''
HO O O
1' HO HO 1' HO HO
HO 2' OH 1''' OH HO 2' OH 1''' OH
O O
HO OH HO OH
HO O HO O HO O HO O
1'' 1'' 1'' 1''

HO OH 1 HO OH 2 HO OH 3 HO OH 4

21 O 26 O O
27
18 20 22 23 25 27
12
O 24
O O O
19 11 13
17
16
1 9
15
2 10 14
8 HO
3 5
7
HO O HO O HO O O
4 OH 6 OH OH
O OH O OH O 1''''

4'
OH O 4' OH HO
1' 2' 1' 1'
O HO O OH
2' HO 2'
3'
HO O O O HO O O
O
1''' OH 1'''
1'''
HO
HO OH OH HO OH
HO O HO O HO O
1
H 1H COSY :
1'' 1'' 1''
HMBC :
HO OH 5 HO OH 6 HO OH 7

Fig. 1. The main 1 H

1H COSY and HMBC correlations of 1
7.

27
 D rings of 5 and 6.
Fig. 2. The main NOE correlations for A

28
Table 1
1
H and 13C NMR data for 1 in C5D5N (500 MHz for 1H and 125 MHz for 13C).
No. C H (J in Hz) No. C H (J in Hz)
1 37.1 0.99 (m), 1.51 (m) 24 36.8 1.65 (m), 1.95 (m)
2 29.9 1.82 (m), 2.01 (m) 25 28.9 1.64 (m)
3 78.2 3.85 (m) 26 23.0 0.92 (d, 5.5)
4 38.7 2.69 (dd, 12.5, 12.5) 27 23.1 0.92 (d, 5.5)
2.80 (dd, 5.5, 12.5) 1' 100.3 4.99 (d, 7.0)
5 141.0 2' 77.8 4.23 (m, overlapped)
6 121.7 5.20 (br. d, ca. 5) 3' 79.6 4.28 (dd, 8.0, 9.0)
7 31.6 1.32 (m), 1.71 (m) 4' 71.8 4.14 (dd, 9.0, 9.0)
8 32.0 1.62 (m) 5' 77.7 3.88 (m)
9 54.6 1.32 (m) 6' 62.6 4.32 (m, overlapped)
10 38.0 4.50 (m, overlapped)
11 38.1 2.22 (dd, 5.5, 12.5) 1'' 102.0 6.35 (br. s)
2.71 (dd, 12.5, 12.5) 2'' 72.5 4.79 (br. d, ca. 3)
12 214.1 3'' 72.8 4.61 (dd, 3.0, 9.0)
13 57.3 4'' 74.1 4.32 (m, overlapped)
14 57.2 1.17 (m) 5'' 69.4 4.94 (m)
15 37.0 1.54 (m), 2.41 (m) 6'' 18.6 1.76 (d, 6.0)
16 81.8 4.55 (m) 1''' 106.9 4.76 (d, 7.5)
17 49.3 3.03 (dd, 8.0, 11.5) 2''' 75.6 4.01 (dd, 7.5, 8.5)
18 13.3 1.32 (s) 3''' 78.7 4.17 (dd, 8.5, 9.5)
19 19.0 1.10 (s) 4''' 71.6 4.23 (m, overlapped)
20 35.3 2.54 (m) 5''' 78.2 3.85 (m)
21 13.2 1.25 (d, 7.0) 6''' 62.8 4.40 (dd, 4.5, 11.5)
22 73.2 4.32 (m, overlapped) 4.50 (m, overlapped)
23 33.8 1.86 (m), 1.92 (m)

29
Table 2
1
H and 13C NMR data for 2 in C5D5N (500 MHz for 1H and 125 MHz for 13C).
No. C H (J in Hz) No. C H (J in Hz)
1 37.1 0.87 (m), 1.54 (m) 27 23.0 0.92 (d, 5.0)
2 29.9 1.79 (m), 2.00 (m) 1' 100.2 4.92 (d, 7.5)
3 77.7 3.80 (m) 2' 77.8 4.19 (m, overlapped)
4 38.6 2.67 (dd, 12.5, 12.5) 3' 78.2 4.19 (m, overlapped)
2.80 (dd, 5.5, 12.5) 4' 78.4 4.35 (dd, 9.0, 9.0)
5 140.4 5' 76.8 3.64 (m)
6 121.8 5.23 (br. d, ca. 5) 6' 61.2 4.08 (dd, 5.0, 12.0)
7 31.6 1.32 (m), 1.74 (m) 4.21 (br. d, ca. 12)
8 32.0 1.61 (m) 1'' 101.9 6.36 (br. s)
9 54.6 1.33 (m) 2'' 72.4 4.82 (br. d, ca. 3)
10 37.9 3'' 72.7 4.61 (dd, 3.0, 9.5)
11 38.1 2.23 (dd, 5.5, 12.0) 4'' 74.0 4.36 (dd, 9.5, 9.5)
2.72 (dd, 12.0, 12.0) 5'' 69.4 4.92 (m)
12 214.0 6'' 18.6 1.75 (d, 6.5)
13 57.2 1''' 102.8 5.82 (br. s)
14 57.1 1.17 (m) 2''' 72.4 4.68 (br. d, ca. 3)
15 37.0 2.03 (m), 2.42 (m) 3''' 72.6 4.54 (dd, 3.0, 8.5)
16 81.8 4.56 (m) 4''' 73.8 4.33 (dd, 8.5, 9.5)
17 49.3 3.03 (dd, 8.0, 10.5) 5''' 70.3 4.89 (m)
18 13.3 1.33 (s) 6''' 18.4 1.61 (d, 6.0)
19 18.9 1.10 (s) 1'''' 106.9 4.77 (d, 7.5)
20 35.3 2.54 (m) 2'''' 75.5 4.02 (dd, 7.5, 8.5)
21 13.2 1.26 (d, 7.0) 3'''' 78.6 4.17 (dd, 8.5, 9.0)
22 73.2 4.32 (m) 4'''' 71.5 4.25 (dd, 9.0, 9.0)
23 33.7 1.87 (m) 5'''' 77.9 3.85 (m)
24 36.8 1.65 (m), 1.95 (m) 6'''' 62.8 4.40 (dd, 5.0, 12.0)
25 28.8 1.65 (m) 4.51 (dd, 2.0, 12.0)
26 23.0 0.92 (d, 5.0)

30
Table 3
1
H and 13C NMR data for 3 in C5D5N (500 MHz for 1H and 125 MHz for 13C).
No. C H (J in Hz) No. C H (J in Hz)
1 37.5 0.93 (m), 1.71 (m) 24 36.5 1.49 (m), 1.71 (m)
2 30.1 1.84 (m), 2.06 (m) 25 28.6 1.58 (m)
3 78.2 3.88 (m) 26 22.9 0.88 (d, 6.5)
4 38.9 2.69 (dd, 12.0, 12.0) 27 23.0 0.86 (d, 6.5)
2.77 (dd, 5.0, 12.0) 1' 100.3 4.98 (d, 7.0)
5 140.8 2' 77.8 4.21 (dd, 7.0, 8.5)
6 121.9 5.26 (br. d, ca. 5) 3' 79.6 4.23 (dd, 8.5, 9.0)
7 31.8 1.41 (m), 1.77 (m) 4' 71.8 4.12 (dd, 9.0, 9.0)
8 30.6 1.34 (m) 5' 77.9 3.86 (m)
9 49.9 1.02 (m) 6' 62.7 4.32 (dd, 6.5, 11.5)
10 37.1 4.46 (br. d, ca. 12)
11 31.0 1.66 (m), 1.86 (m) 1'' 102.0 6.32 (br. s)
12 78.2 3.67 (dd, 4.5, 11.5) 2'' 72.5 4.75 (br. d, ca. 3)
13 48.9 3'' 72.8 4.57 (dd, 3.0, 9.0)
14 53.9 0.79 (m) 4'' 74.1 4.29 (dd, 9.0, 9.5)
15 36.0 1.99 (m), 2.38 (m) 5'' 69.4 4.93 (m)
16 84.7 4.47 (m) 6'' 18.6 1.72 (d, 6.0)
17 63.1 1.81 (m) 1''' 107.2 4.83 (d, 7.5)
18 10.9 1.34 (s) 2''' 75.5 4.01 (dd, 7.5, 8.5)
19 19.4 1.03 (s) 3''' 78.9 4.17 (dd, 8.0, 8.5)
20 38.1 2.83 (m) 4''' 71.7 4.18 (dd, 8.0, 9.5)
21 13.8 1.62 (d, 7.0) 5''' 78.4 3.89 (m)
22 75.9 4.37 (m) 6''' 63.0 4.37 (dd, 6.5, 12.0)
23 35.2 1.72 (m), 1.84 (m) 4.53 (dd, 2.5, 12.0)

31
Table 4
1
H and 13C NMR data for 4 in C5D5N (500 MHz for 1H and 125 MHz for 13C).
No. C H (J in Hz) No. C H (J in Hz)
1 37.5 0.96 (m), 1.71 (m) 27 23.0 0.87 (d, 6.5)
2 30.1 1.80 (m), 2.00 (m) 1' 100.2 4.90 (d, 7.0)
3 78.1 3.83 (m) 2' 77.7 4.19 (m, overlapped)
4 38.9 2.69 (dd, 11.5, 11.5) 3' 77.9 4.19 (m, overlapped)
2.78 (dd, 5.0, 11.5) 4' 78.6 4.34 (dd, 9.5, 9.5)
5 140.7 5' 76.9 3.62 (m)
6 122.0 5.28 (br. d, ca. 5) 6' 61.3 4.06 (dd, 5.0, 12.0)
7 31.8 1.41 (m), 1.76 (m) 4.19 (m, overlapped)
8 30.6 1.31 (m) 1'' 101.9 6.36 (br. s)
9 49.9 1.03 (m) 2'' 72.5 4.80 (br. d, ca. 3)
10 37.1 3'' 72.8 4.59 (dd, 3.0, 9.0)
11 31.0 1.67 (m), 1.87 (m) 4'' 74.1 4.32 (dd, 9.0, 9.0)
12 78.1 3.68 (dd, 3.5, 11.5) 5'' 69.5 4.91 (m)
13 48.9 6'' 18.6 1.73 (d, 7.0)
14 53.8 0.81 (m) 1''' 102.8 5.82 (br. s)
15 35.9 1.99 (m), 2.40 (m) 2''' 72.5 4.66 (br. d, ca. 3)
16 84.7 4.49 (m) 3''' 72.7 4.52 (dd, 3.0, 9.0)
17 63.0 1.80 (m) 4''' 73.8 4.32 (dd, 9.0, 9.0)
18 10.9 1.35 (s) 5''' 70.4 4.88 (m)
19 19.3 1.04 (s) 6''' 18.5 1.60 (d, 6.0)
20 38.1 2.85 (m) 1'''' 107.2 4.85 (d, 8.0)
21 13.8 1.64 (d, 7.5) 2'''' 75.5 4.02 (dd, 8.0, 8.5)
22 75.9 4.39 (m) 3'''' 78.9 4.21 (dd, 8.5, 8.5)
23 35.2 1.72 (m), 1.85 (m) 4'''' 71.7 4.23 (dd, 8.0, 8.0)
24 36.4 1.50 (m), 1.70 (m) 5'''' 78.4 3.92 (m)
25 28.6 1.56 (m) 6'''' 63.0 4.40 (dd, 5.5, 11.5)
26 22.9 0.88 (d, 6.5) 4.54 (dd, 3.0, 11.5)

32
Table 5
1
H and 13C NMR data for 5 in C5D5N (500 MHz for 1H and 125 MHz for 13C).
No. C H (J in Hz) No. C H (J in Hz)
1 33.0 1.45 (m), 2.04 (m) 23 31.8 1.63 (m), 1.68 (m)
2 29.7 2.04 (m), 2.18 (m) 24 29.2 1.54 (m)
3 75.9 4.81 (m) 25 30.6 1.55 (m)
4 38.6 2.45 (dd, 4.5, 13.0) 26 66.8 3.47 (dd, 10.5, 10.5)
2.89 (dd, 12.0, 13.0) 3.57 (dd, 3.0, 10.5)
5 75.5 27 17.3 0.68 (d, 5.5)
6 76.1 4.13 (1H, m) 1' 100.6 4.81 (d, 8.0)
7 35.6 1.88 (m, overlapped) 2' 77.9 4.17 (dd, 8.0, 9.0)
2.14 (m) 3' 78.0 4.06 (dd, 9.0, 9.0)
8 30.8 2.30 (m) 4' 78.5 4.33 (dd, 9.0, 9.0)
9 45.8 1.88 (m, overlapped) 5' 76.8 3.42 (m)
10 39.2 6' 61.2 4.01 (dd, 3.0, 12.0)
11 21.5 1.47 (m) 4.07 (br. d, ca. 12)
12 40.6 1.19 (dd, 3.5, 12.5) 1'' 101.7 6.34 (br. s)
1.75 (m) 2'' 72.3 4.75 (br. d, ca. 3)
13 41.0 3'' 72.6 4.63 (dd, 3.0, 9.0)
14 56.5 1.28 (m) 4'' 74.3 4.31 (m, overlapped)
15 32.3 1.44 (m), 2.09 (m) 5'' 69.2 5.01 (m)
16 81.2 4.54 (br. dd, ca. 7, 14) 6'' 18.6 1.74 (d, 6.0)
17 63.1 1.82 (dd, 6.5, 8.5) 1''' 102.8 5.79 (br. s)
18 16.7 0.89 (s) 2''' 72.5 4.66 (br. d, ca. 3)
19 17.1 1.61 (s) 3''' 72.7 4.51 (dd, 3.0, 9.0)
20 42.0 1.95 (m) 4''' 73.8 4.31 (m, overlapped)
21 15.0 1.12 (d, 7.0) 5''' 70.3 4.85 (m)
22 109.2 6''' 18.4 1.59 (d, 6.5)

33
Table 6
1
H and 13C NMR data for 6 in C5D5N (500 MHz for 1H and 125 MHz for 13C).
No. C H (J in Hz) No. C H (J in Hz)
1 33.0 1.47 (m), 2.05 (m) 24 29.3 1.54 (m)
2 29.6 2.11 (m), 2.24 (m) 25 30.6 1.55 (m)
3 75.2 4.91 (m) 26 66.9 3.48 (dd, 11.0, 11.0)
4 38.2 2.43 (dd, 4.5, 12.0) 3.56 (dd, 4.5, 11.0)
2.91 (dd, 12.0, 12.0) 27 17.3 0.68 (d, 5.5)
5 75.5 1' 100.0 4.83 (d, 7.5)
6 76.3 4.14 (m) 2' 77.1 4.18 (dd, 7.5, 8.5)
7 35.7 1.91 (m), 2.18 (m) 3' 89.5 4.05 (dd, 8.5, 8.5)
8 30.9 2.32 (m) 4' 69.4 4.04 (dd, 8.5, 8.5)
9 45.9 1.92 (m) 5' 77.7 3.61 (m)
10 39.3 6' 62.4 4.21 (dd, 6.5, 12.0)
11 21.5 1.49 (m) 4.33 (dd, 2.0, 12.0)
12 40.6 1.19 (dd, 4.0, 12.5) 1'' 102.0 6.35 (br. s)
1.76 (m) 2'' 72.4 4.80 (br. d, ca. 3)
13 41.1 3'' 72.6 4.57 (dd, 3.0, 9.0)
14 56.5 1.31 (m) 4'' 74.4 4.27 (dd, 9.0, 9.0)
15 32.3 1.45 (m), 2.12 (m) 5'' 69.5 5.01 (m)
16 81.2 4.56 (m) 6'' 18.7 1.72 (d, 6.5)
17 63.2 1.84 (dd, 6.0, 8.5) 1''' 104.4 5.05 (d, 7.5)
18 16.7 0.89 (s) 2''' 75.0 3.98 (dd, 7.5, 8.0)
19 17.1 1.64 (s) 3''' 78.5 4.17 (dd, 8.0, 9.0)
20 42.0 1.96 (dd, 7.0, 7.0) 4''' 71.5 4.08 (dd, 8.5, 9.0)
21 15.0 1.13 (d, 7.0) 5''' 78.7 4.01 (m)
22 109.2 6''' 62.4 4.24 (dd, 5.5, 12.0)
23 31.8 1.63 (m), 1.67 (m) 4.54 (br. d, ca. 12)

34
Table 7
1
H and 13C NMR data for 7 in C5D5N (500 MHz for 1H and 125 MHz for 13C).
No. C H (J in Hz) No. C H (J in Hz)
1 37.5 0.99 (m), 1.75 (m) 27 72.0 3.45 (dd, 8.0, 9.5)
2 30.1 1.87 (m), 2.06 (m) 3.94 (dd, 5.5, 9.5)
3 78.1 3.88 (m) 1' 100.3 4.94 (d, 7.0)
4 39.0 2.72 (dd, 11.5, 11.5) 2' 77.9 4.18 (dd, 7.0, 8.0)
2.80 (dd, 3.0, 11.5) 3' 77.8 4.22 (dd, 8.0, 9.0)
5 140.8 4' 78.5 4.38 (dd, 9.0, 9.0)
6 121.8 5.33 (br. d, ca. 5) 5' 76.9 3.64 (m)
7 32.3 1.44 (m), 1.89 (m) 6' 61.3 4.09 (br. d, ca. 12)
8 31.7 1.56 (m) 4.21 (dd, 5.0, 12.0)
9 50.3 0.90 (m) 1'' 102.0 6.38 (br. s)
10 37.1 2'' 72.5 4.83 (br. d, ca. 3)
11 21.1 1.44 (m) 3'' 72.8 4.62 (dd, 3.0, 9.0)
12 39.8 1.11 (m), 1.69 (m) 4'' 74.1 4.36 (dd, 9.0, 9.5)
13 40.4 5'' 69.5 4.95 (m)
14 56.6 1.05 (m) 6'' 18.6 1.76 (d, 6.0)
15 32.2 1.44 (m), 2.03 (m) 1''' 102.9 5.84 (br. s)
16 81.1 4.50 (m) 2''' 72.5 4.68 (br. d, ca. 3)
17 62.8 1.78 (m) 3''' 72.7 4.53 (dd, 3.0, 9.0)
18 16.3 0.82 (s) 4''' 73.9 4.33 (dd, 9.0, 9.5)
19 19.4 1.05 (s) 5''' 70.4 4.91 (m)
20 42.0 1.93 (dd, 7.0, 7.0) 6''' 18.5 1.62 (d, 6.0)
21 15.0 1.11 (d, 7.0) 1'''' 105.0 4.77 (d, 7.5)
22 109.5 2'''' 75.1 4.02 (dd, 7.5, 9.5)
23 31.3 1.67 (m) 3'''' 78.6 4.24 (m, overlapped)
24 23.9 1.67 (m) 4'''' 71.6 4.24 (m, overlapped)
25 36.7 2.04 (m) 5'''' 78.5 3.96 (m)
26 63.6 3.72 (dd, 11.0, 11.0) 6'''' 62.8 4.39 (br. d, ca. 12)
4.04 (dd, 4.0, 11.0) 4.57 (dd, 5.0, 12.0)

35
Table 8
Inhibitory effects of 70% ethanol-water extract of D. septemloba (0) and compounds 129 on NO
production in RAW 264.7 macrophages.

NRC (%) NRC (%)

Normal 11.00.7** 14 101.11.2
Control 100.03.1 15 101.40.6
Positive Control 12.60.8** 16 102.60.6
0 46.83.8* 17 97.00.7
1 90.23.3 18 26.81.4**
2 102.20.4 19 89.85.0
3 91.34.3 20 98.02.0
4 95.01.1 21 11.90.8**
5 95.20.6 22 11.30.6**
6 93.41.1 23 81.31.9*
7 101.60.6 24 84.24.7*
8 92.61.0 25 107.53.6
9 100.61.4 26 93.70.7
10 101.70.5 27 95.50.9
11 101.40.3 28 101.11.0
12 105.00.8 29 101.10.8
13 101.30.6

Nitrite relative concentration (NRC): percentage of control group, which set as 100%. Values
represent the mean SD of three determinations. *P < 0.05; **P < 0.01 (Differences between
compound-treated group and control group). N = 6. Final concentration of 0 was 100 g/mL, 129
was 30 M.

36
Graphical abstract

37
Highlights:

1. Seven new steriods were isolated from the rhizomes of Dioscorea septemloba, their

structures were elucidated by chemical and spectroscopic methods.

2. Several compounds displayed significant inhibitory activities on LPS-induced NO

production, which suggested that these compounds may have potent

anti-inflammatory activity.

3. The structure-activity relationship of steroids isolated from Dioscorea septemloba

was summarized

38