New steroidal saponins from the roots of Solanum melongena L.

Article abstract of DOI:10.1016/j.fitote.2018.04.021

Phytochemical investigation of the roots of Solanum melongena L. resulted in the isolation of six new steroidal saponins, including five new cholestane saponins (1-5) and one new steroidal alkaloid (6), along with one new natural product (7) and three know steroids (8-10). The structures of all isolated compounds were determined by 1D and 2D NMR experiments and by comparison of their spectroscopic and physical data with literature values. The inhibitory activities on nitric oxide (NO) production stimulated by lipopolysaccharide (LPS) in a RAW 264.7 cell line were assayed for all the isolated compounds. Compounds 1, 2 and 4–9 exhibited moderate inhibition of NO production with IC₅₀ values ranging from 12.6 to 59.5 μM.

Full text of DOI:10.1016/j.fitote.2018.04.021

Fitoterapia128(2018)12–19
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
New steroidal saponins from the roots of Solanum melongena L.
Bing-You Yang, Xin Yin, Yan Liu, Dong-Ying Zhao, Hai-Xue Kuang⁎
Key Laboratory of Chinese Materia Medica, Ministry of Education, Heilongjiang University of Chinese Medicine, Harbin 150040, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Solanaceae
Natural product
Steroids
Cholestane saponins
Steroidal alkaloid
Phytochemical investigation of the roots of Solanum melongena L. resulted in the isolation of six new steroidal
saponins, including five new cholestane saponins (1-5) and one new steroidal alkaloid (6), along with one new
natural product (7) and three know steroids (8-10). The structures of all isolated compounds were determined by
1D and 2D NMR experiments and by comparison of their spectroscopic and physical data with literature values.
The inhibitory activities on nitric oxide (NO) production stimulated by lipopolysaccharide (LPS) in a RAW 264.7
cell line were assayed for all the isolated compounds. Compounds 1, 2 and 4–9 exhibited moderate inhibition of
NO production with IC50 values ranging from 12.6 to 59.5 μM.
1. Introduction
for 1H NMR and 100 MHz for 13C NMR). Semi-preparative HPLC was
performed with a Waters SunFireTM C18 (250 × 10 mm, 5 μm) column
Solanum melongena L. (Solanaceae) is widely distributed in Southern
Asia, the Middle East and Northern Africa [1], and its unripe fruit is
primarily used as a vegetable. As a traditional Chinese medicine, the
roots of Solanum melongena L. are used to treat chilblains, beriberi,
pruritus, toothache [2], asthma, syphilis [3] and so on. A series of
biological activities, such as anti-inflammatory, sedative, hypnotic,
analgesic, neuroprotective and blood circulation-promoting effects
[4,5] have been reported for pure compounds and crude extracts from
the roots of Solanum melongena L. Previous phytochemical investiga-
tions have afforded the isolation of alkaloids [6], steroids [7], flavo-
noids [8], phenylpropanoid amides [9], coumarins [9] and lignans
[10]. In this paper, the phytochemical composition of the roots of So-
lanum melongena L. were studied, resulting in six new steroidal saponins
(1-6), one new natural product (7) and three know steroids (8-10)
(Fig. 1). The isolation and structural determination of the new com-
pounds are described here. The inhibitory activities of these compounds
on nitric oxide (NO) production were stimulated with lipopoly-
saccharide (LPS) in the RAW 264.7 cell line.
(Waters Corporation), and the HPLC system was equipped with a
Shimadzu CBM-20A, RID (reflective index detector) detector and LC-
6AD pump (Shimadzu Corporation, Japan). Column chromatography
was performed using Sephadex LH-20 (Pharmacia) and silica gel
(100–120 mesh and 200–300 mesh, Qingdao Marine Chemical Co.,
Qingdao, China). The thin-layer chromatography used GF254, and spots
were detected by spraying the plates with 10% H2SO4-EtOH reagent
followed by heating at 120 °C for 5 min.
2.2. Plant material
The roots of Solanum melongena L. were collected from Anguo of
Hebei Province (China), and identified by Prof. Rui-Feng Fan of
Heilongjiang University of Chinese Medicine. The voucher specimen
(No. 20160918) was deposited at Heilongjiang University of Chinese
Medicine.
2.3. Extraction and isolation
2. Experimental
The air-dried roots of Solanum melongena L (12 kg) were cut into
approximately 2 cm pieces and extracted under reflux with EtOH-H2O
(70:30 v/v) (3 × 120 L, 3 h each). The resulting extracts (940 g) were
concentrated under vacuum (40 °C), suspended in H2O (4.0 L), and
partitioned successively with petroleum ether (PE) (3 × 4.0 L, 24 h
each), EtOAc (3 × 4.0 L, 24 h each), and n-BuOH (3 × 4.0 L, 24 h each)
to give EtOAc (88 g) and n-BuOH (135 g) fractions. The n-BuOH-soluble
portion (135 g) was separated by silica gel column chromatography
2.1. General experimental procedures
High-resolution electrospray ionization mass spectrometry
(HRESIMS) was conducted using a Waters Xevo-TOF-MS™ instrument.
Optical rotations were measured on a JASCO P-2000 instrument. The
NMR spectra were recorded on a Bruker DPX 400 instrument (400 MHz
⁎
Corresponding author at: College of Pharmacy, Heilongjiang University of Chinese Medicine, 24 Heping Road, Xiangfang District, Harbin 150040, China.
E-mail address: hxkuang@hotmail.com (H.-X. Kuang).
https://doi.org/10.1016/j.fitote.2018.04.021
Received 5 March 2018; Received in revised form 24 April 2018; Accepted 27 April 2018
Availableonline30April2018
0367-326X/©2018PublishedbyElsevierB.V.

B.-Y. Yang et al.
Fitoterapia128(2018)12–19
Fig. 1. Chemical structures of compounds 1-10.
Table 1
1H and 13C NMR data for aglycons of compounds 1-5 (δ in ppm).
1a
2b
3a
4a
5a
Position
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
1
2
37.7
30.6
1.66 (m)
0.91 (m)
2.03 (m)
1.68 (m)
3.85 (m)
2.67 (dd, 13.2, 2.3)
2.42 (dd, 13.2, 2.5)
-
5.28 (br.d, 4.9)
1.84 (m)
1.50 (m)
1.34 (m)
0.90 (m)
-
38.5
30.7
1.88 (m)
1.08 (m)
1.92 (m)
1.61 (m)
3.61 (m)
2.45 (dd, 13.2, 2.4)
2.29 (dd, 13.2, 2.8)
-
5.37 (br.d, 4.9)
1.95 (m)
1.55 (m)
1.45 (m)
0.99 (m)
-
1.55 (m)
1.48 (m)
1.98 (m)
1.37 (m)
-
1.40 (m)
1.64 (m)
1.51 (m)
3.91 (m)
1.51 (m)
0.76 (s)
37.8
30.6
1.69 (m)
0.90 (m)
2.03 (m)
1.82 (m)
3.84 (m)
2.86 (overlap)
2.70 (overlap)
-
5.28 (br.d, 4.8)
1.83 (m)
1.48 (m)
1.33 (m)
0.87 (m)
-
37.3
30.2
1.63 (m)
0.89 (m)
2.02 (m)
1.66 (m)
3.82 (m)
2.68 (dd, 11.8, 2.2)
2.42 (dd, 11.8, 2.4)
-
5.29 (br.d, 4.7)
1.88 (m)
1.50 (m)
1.36 (m)
0.87 (m)
-
1.31 (m)
1.23 (m)
1.58 (m)
1.22 (m)
-
1.51 (m)
1.86 (m)
1.74 (m)
4.30 (m)
2.15 (m)
0.72 (s)
37.4
30.1
1.69 (m)
0.90 (m)
2.03 (m)
1.80 (m)
3.82 (m)
2.85 (overlap)
2.70 (overlap)
-
5.28 (br.d, 4.4)
1.88 (m)
1.53 (m)
1.37 (m)
0.87 (m)
-
1.32 (m)
1.22 (m)
1.58 (m)
1.20 (m)
-
1.51 (m)
1.88 (m)
1.75 (m)
4.31 (m)
2.14 (m)
0.72 (s)
3
4
78.6
39.7
79.2
39.5
78.5
39.4
78.2
39.3
78.0
38.9
5
6
7
141.3
122.2
32.6
141.9
122.6
32.9
141.2
122.3
32.6
140.9
121.8
32.2
140.8
121.9
32.2
8
9
10
11
32.0
50.7
37.3
21.3
32.6
51.6
37.9
21.7
32.1
50.8
37.4
21.3
31.6
50.3
36.9
20.8
31.6
50.3
37.0
20.8
1.37 (m)
1.36 (m)
12
40.3
1.85 (m)
1.22 (m)
-
41.0
40.3
1.84 (m)
1.22 (m)
-
38.9
39.1
13
14
15
44.5
54.4
37.5
45.1
54.9
37.1
44.5
54.4
37.5
43.6
53.6
37.6
43.5
53.6
37.6
1.42 (m)
1.78 (m)
1.65 (m)
4.26 (m)
1.85 (m)
0.67 (s)
0.89 (s)
2.79 (m)
1.17 (d, 6.8)
-
1.44 (m)
1.78 (m)
1.65 (m)
4.27 (m)
1.84 (m)
0.67 (s)
1.03 (s)
2.76 (m)
1.18 (d, 6.8)
-
16
17
18
19
20
21
22
23
76.4
63.7
13.8
19.8
49.9
17.2
215.4
39.6
77.1
63.8
13.7
19.8
50.5
16.9
218.1
39.7
76.4
63.7
13.8
19.8
49.8
17.2
215.2
39.4
75.0
62.1
14.5
19.4
47.3
17.5
214.7
39.1
75.0
62.1
14.5
19.4
47.3
17.4
214.8
39.1
1.02 (s)
0.87 (s)
1.01 (s)
2.68 (m)
1.14 (d, 6.8)
-
2.81 (m)
1.47 (d, 6.9)
-
2.83 (m)
2.73 (m)
2.14 (m)
1.68 (m)
1.88 (m)
3.76 (overlap)
2.80 (m)
1.46 (d, 6.9)
-
2.82 (m)
2.74 (m)
2.12 (m)
1.69 (m)
1.88 (m)
3.75 (overlap)
2.85 (m)
2.59 (m)
2.85 (m)
24
28.3
2.15 (m)
1.76 (m)
1.90 (m)
3.75 (dd, 10.4, 5.8)
3.68 (dd, 10.4, 6.1)
1.06 (d, 6.6)
28.0
1.62 (m)
1.36 (m)
1.58 (m)
3.39 (overlap)
3.33 (overlap)
0.90 (d, 6.6)
28.5
1.98 (m)
1.71 (m)
1.97 (m)
3.92 (dd, 9.4, 5.1)
3.58 (dd, 9.4, 5.8)
0.96 (d, 6.4)
27.9
27.9
25
26
36.6
67.9
36.4
68.1
33.9
75.6
36.1
67.4
36.1
67.4
27
a
17.8
17.0
18.0
17.2
1.08 (d, 6.7)
17.2
1.08 (d, 6.7)
Measured in C5D5N(1H: 400 MHz;13C: 100 MHz).
Measured in CD3OD(1H: 400 MHz;13C: 100 MHz).
b
with CH2Cl2-MeOH mixtures of increasing polarity to give ten fractions
(A1-A10). Fraction A2 (9 g) was subjected to ODS column chromato-
graphy with MeOH-H2O (1:9 to 1:0) to afford sub-fractions A2A-A2I.
Sub-fraction A2G (890 mg) was subjected to a Sephadex LH-20 column
eluted with MeOH (flow rate: 0.8 mL/min) to yield subfraction A2G4
and then purified by semi-preparative HPLC using Waters SunFireTM
13

B.-Y. Yang et al.
Fitoterapia128(2018)12–19
Table 2
1H and 13C NMR data for sugar moieties of compounds 1-5 (δ in ppm).
1a
2b
3a
4a
5a
Position
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
Glc I
1
2
3
4
5
6'
102.9
76.0
77.1
78.7
77.6
61.9
4.94 (d, 7.7)
3.98 (dd, 9.3, 7.7)
4.23 (m)
4.47 (m)
3.73 (m)
100.4
79.3
78.0
79.9
76.6
61.9
4.49 (d, 7.8)
3.38 (m)
3.59 (m)
3.51 (m)
3.32 (m)
100.7
79.0
77.4
78.4
78.2
61.7
4.93 (d, 6.2)
4.40 (m)
3.64 (m)
4.21 (m)
4.21 (m)
102.4
75.5
76.7
78.2
77.1
61.5
4.93 (d, 7.7)
3.98 (dd, 8.8, 7.7)
4.22 (m)
4.48 (m)
3.72 (m)
100.3
78.5
77.0
78.0
77.7
61.2
4.90 (d, 7.1)
4.39 (m)
3.62 (m)
4.20 (m)
4.20 (m)
4.26 (dd, 12.0, 5.1)
4.13 (dd, 12.0, 3.4)
3.79 (dd, 12.2, 3.2)
3.65 (dd, 12.2, 5.1)
4.18 (overlap)
4.02 (overlap)
4.26 (dd, 12.3, 4.9)
4.13 (dd, 12.3, 3.5)
4.19 (overlap)
4.07 (overlap)
Rha I
1
2
3
4
5
6
103.1
73.1
73.2
74.4
70.8
19.0
5.91 (br.s)
4.71 (m)
4.58 (dd, 9.2, 3.3)
4.35 (dd, 9.3, 9.2)
5.05 (m)
103.0
72.3
72.1
73.9
70.6
17.8
4.83 (d, 1.5)
3.91 (m)
3.62 (m)
3.38 (m)
3.92 (m)
103.4
73.0
73.3
74.6
70.9
19.0
5.86 (br.s)
4.83 (m)
4.56 (m)
4.33 (m)
4.92 (m)
102.7
72.6
72.8
74.0
70.3
18.5
5.91 (br.s)
4.71 (m)
4.59 (dd, 9.2, 3.3)
4.35 (dd, 9.3, 9.2)
5.04 (m)
102.9
72.5
72.8
74.1
70.4
18.5
5.85 (br.s)
4.82 (m)
4.53 (m)
4.32 (m)
4.93 (m)
1.72 (d, 6.2)
1.25 (d, 6.2)
1.62 (d, 6.2)
1.72 (d, 6.2)
1.62 (d, 6.2)
Rha II
1
2
3
4
5
6
102.3
72.1
72.4
73.7
69.7
17.9
5.20 (d, 1.4)
3.92 (m)
3.65 (m)
3.41 (m)
4.12 (m)
102.5
73.0
73.2
74.4
70.0
19.1
6.40 (br.s)
4.83 (m)
4.62 (m)
4.31 (m)
4.94 (m)
102.0
72.6
72.8
74.0
69.5
18.7
6.39 (br.s)
4.82 (m)
4.61 (m)
4.32 (m)
4.94 (m)
1.23 (d, 6.3)
1.76 (d, 6.2)
1.74 (d, 6.2)
Glc II
1
2
3
4
5
6
105.4
75.7
79.0
72.1
79.1
63.3
4.81 (d, 7.8)
4.02 (m)
4.38 (m)
4.22 (m)
4.24 (m)
4.53 (overlap)
4.38 (overlap)
a
Measured in C5D5N(1H: 400 MHz;13C: 100 MHz).
Measured in CD3OD(1H: 400 MHz;13C: 100 MHz).
b
C18 column (MeOH-H2O, 73:27; flow rate: 3 mL min−1) to yield com-
pound 10 (24 mg, tR 20.5 min). Sub-fraction A2H (782 mg) was also
separated by semi-preparative HPLC (MeOH-H2O, 75:25; flow rate:
3 mL min−1) to give compound 7 (18 mg, tR 22.5 min). Fraction A7
(8.7 g) was subjected to ODS column chromatography with MeOH-H2O
(1:9 to 1:0) to afford sub-fractions A7A-A7J. Sub-fraction A7E was
isolated by a Sephadex LH-20 column eluted with MeOH (flow rate:
0.8 mL/min) to afford subfraction A7E4, which was further purified by
semi-preparative HPLC (MeOH-H2O, 63:37; flow rate: 3 mL min−1) to
yield compound 6 (9.0 mg, tR 21.4 min). Subfraction A7F (2.8 g) was
chromatographed by semi-preparative HPLC (MeOH-H2O, 59:41; flow
rate: 3 mL min−1) to give compounds 4 (12.3 mg, tR 18.5 min), 5
(6.8 mg, tR 23.0 min) and 8 (18.3 mg tR 32.5 min). Subfraction A7G
(1.3 g) was also isolated by semi-preparative HPLC (MeOH-H2O, 66:34;
flow rate: 3 mL min−1) to afford compounds 2 (10.1 mg, tR 23.4 min)
and 1 (15.6 mg, tR 28.5 min). Fraction A8 (7.8 g) was subjected to ODS
column chromatography with MeOH-H2O (1:9 to 1:0) to give sub-
fractions A8A-A8H. Sub-fraction A8F (560 mg) was separated by semi-
preparative HPLC (MeOH-H2O, 61:39; flow rate: 3 mL min−1) to yield
compounds 3 (7.8 mg, tR 27.8 min) and 9 (9.8 mg, tR 37.5 min).
data for the sugar moiety, see Table 2; HRESIMS: m/z 909.4825 [M
+Na]+, (calcd. for C45H74NaO17, m/z 909.4824)
2.3.3. Abutiloside R (3)
White amorphous powder; [α]D = −31.0, (c = 0.1, MeOH); 1H
22
and 13C NMR data for the aglycon moiety, see Table 1; 1H and 13C NMR
data for the sugar moiety, see Table 2; HRESIMS: m/z 1071.5344 [M
+Na]+, (calcd. for C51H84NaO22, m/z 1071.5352)
2.3.4. Abutiloside S (4)
White amorphous powder; [α]D24 = -23.0, (c = 0.1, MeOH); 1H and
13C NMR data for the aglycon moiety, see Table 1; 1H and 13C NMR data
for the sugar moiety, see Table 2; HRESIMS: m/z 763.4255 [M+Na]+
(calcd. for C39H64NaO13, m/z 763.4245)
,
2.3.5. Abutiloside T (5)
White amorphous powder; [α]D21 = -55.0, (c = 0.2, MeOH); 1H and
13C NMR data for the aglycon moiety, see Table 1; 1H and 13C NMR data
for the sugar moiety, see Table 2; HRESIMS: m/z 909.4846 [M+Na]+
(calcd. for C45H74NaO17, m/z 909.4824)
,
2.3.1. Abutiloside P (1)
2.3.6. Abutiloside U (6)
22
White amorphous powder; [α]D = −33.0, (c = 0.1, MeOH); 1H
White amorphous powder; [α]D = −12.0, (c = 0.1, MeOH); 1H
and 13C NMR spectroscopic data, see Table 3; HRESIMS: m/z 804.4138
[M+Na]+, (calcd. for C40H63NaNO14, m/z 804.4146)
22
and 13C NMR data for the aglycon moiety, see Table 1; 1H and 13C NMR
data for the sugar moiety, see Table 2; HRESIMS: m/z 763.4243 [M
+Na]+, (calcd. for C39H64NaO13, m/z 763.4245)
2.4. Acid hydrolysis of compounds 1-6
2.3.2. Abutiloside Q (2)
White amorphous powder; [α]D = −22.0, (c = 0.1, MeOH); 1H
The compounds (1-6) (1 mg each) were hydrolyzed by refluxing
with 1 M HCl (1 mL) for 4 h. After cooling, the dried residues obtained
22
and 13C NMR data for the aglycon moiety, see Table 1; 1H and 13C NMR
14

B.-Y. Yang et al.
Fitoterapia128(2018)12–19
Table 3
2.5. Cell culture and cell viability assay
1H and 13C NMR data for compounds 6 and 7 (δ in ppm).
RAW 264.7 cells (mouse macrophages) (Shanghai Institutes for
Biological Sciences) were maintained in DMEM supplemented with
10% FBS and antibiotics (100 units/mL penicillin and 100 μg/mL
streptomycin) in a humidified atmosphere containing 5% CO2 at 37 °C.
An MTT assay was used to determine cell viability [13]. In brief, RAW
264.7 macrophages were seeded in 96-well plastic plates and treated
with 1–80 μM steroids from the roots of Solanum melongena L. for 24 h,
respectively. MTT (5 mg/mL) reagent was then added to the wells and
incubated for 4 h. The formazan formed in the cell pellets was dissolved
by adding 100 μL of DMSO, and the absorbance at 570 nm was mea-
sured with a microplate reader.
6a
7b
Position
δC
δH (J in Hz)
δC
δH (J in Hz)
1
2
37.4
30.2
1.67 (m)
0.94 (m)
2.06 (m)
1.24 (m)
3.86 (m)
2.68 (dd, 10.5, 2.7)
2.40 (dd, 10.5, 7.1)
-
5.30 (br.d, 5.1)
1.86 (m)
1.50 (m)
1.52 (m)
0.83 (m)
-
1.41 (m)
2.03 (m)
1.00 (m)
-
0.69 (overlap)
2.39 (m)
0.99 (m)
5.67 (ddd, 4.8, 8.2, 8.2)
2.43 (d, 8.2)
1.38 (s)
38.4
32.3
1.87 (m)
1.10 (m)
1.79 (m)
1.49 (m)
3.40 (m)
2.22 (m)
3
4
78.1
39.3
72.4
43.0
5
6
7
140.9
121.8
32.1
142.3
122.2
32.9
-
5.34 (br.d, 5.1)
1.97 (m)
1.62 (m)
1.51 (m)
1.02 (m)
-
1.58 (m)
1.96 (m)
1.40 (m)
-
1.44 (m)
1.66 (m)
1.51 (m)
3.91 (m)
1.62 (m)
0.77 (s)
2.6. Inhibitory assay of NO production
8
9
10
11
12
31.0
50.5
37.0
20.7
38.3
32.7
51.6
37.7
21.8
41.0
An inhibitory assay of NO production was examined in accordance
with a method described previously [14] with minor modifications.
Briefly, RAW 264.7 cells (1 × 105 cells in 100 μL) were seeded into a
96-well microplate, grown for 24 h, then stimulated with 1 μg/mL LPS
in a serum-free medium containing various concentrations of the
compounds (1, 5, 10, 20, 40 and 80 μM) for 24 h. Then the supernatant
of culture medium was transferred to a new 96-well microplate and
50 μL of 0.15% N-(1-naphtyl)ethylenediamine in H2O and 1.5% sulfa-
nilamide in 7.5% phosphoric acid were added. The absorbance at
570 nm was measured with a microplate reader.
13
14
15
42.4
54.3
37.6
45.1
54.9
37.2
16
17
18
19
20
21
22
23
24
74.5
67.8
14.8
19.4
206.5
31.3
167.4
97.2
40.7
77.1
63.8
13.7
19.8
50.0
17.0
218.1
39.7
28.0
0.89 (s)
-
2.27 (s)
-
1.02 (s)
2.68 (m)
1.15 (d, 6.9)
-
2.59 (m)
1.65 (m)
1.36 (m)
1.59 (m)
3.40 (dd, 10.8, 5.0)
3.35 (dd, 10.8, 4.5)
0.91 (d, 6.7)
3. Results and discussion
-
Compound 1 was obtained as a white amorphous solid. High re-
solution-election impact (HRESIMS) showed a molecular ion at m/z
763.4243 ([M+Na]+, calcd. for 763.4245), which agreed with the
2.08 (m)
1.59 (m)
2.37 (m)
3.22 (overlap)
2.76 (overlap)
0.73 (d, 6.6)
25
26
26.0
49.0
36.4
68.1
molecular formula C39H64O13
.
The 1H-NMR spectrum of 1 (Tables 1 and 2) showed four methyl
proton signals of a typical steroidal skeleton at δ 1.06 (3H, d,
J = 6.6 Hz, H3 -27), δ 1.17 (3H, d, J = 6.8 Hz, H3-21), δ 0.67, δ 0.89
(3H each, both s, H3-18, 19), an olefinic hydrogen δ 5.28 (1H br.d,
27
18.6
17.1
Glc I
1
2
3
4
5
6
102.4
75.6
76.7
78.2
77.2
61.5
4.95 (d, 7.8)
3.98 (dd, 8.7, 7.8)
4.22 (dd, 9.3, 8.7)
4.47 (dd, 9.4, 9.3)
3.73 (m)
4.26 (dd, 12.2, 5.1)
4.13 (dd, 12.2, 3.4)
J = 4.9 Hz, H-6), together with glucopyranosyl
J = 7.7 Hz, HGlc I-1) and one rhamnopyranosyl δ 5.91 (1H, br. s, HRha I
δ 4.94 (1H, d,
-
1). The above 1H NMR data, together with olefinic carbons signals at (δ
141.3, C-5) and (122.2, C-6) and a carbonyl carbon signal at (δ 215.4,
C-22) in the 13C-NMR spectrum, suggested 1 to be a Δ5,6-cholestane
skeleton in the aglycone with two sugar units. The 13C-NMR data of 1
were similar to abutiloside G obtained previously from the Solanum
abutiloides [7], and the major difference between their 13C-NMR spectra
was the absence of a group of glucosyl carbon signals in compound 1. In
addition, further comparative study of 13C-NMR spectrum between 1
and abutilosides G found that the chemical shifts of C-25 (δ 36.6)
moved to the low field and the chemical shifts of C-26 (δ 67.9) moved
to the high field. The chemical shifts of C-26 (δ 67.9) and C-25 (δ 36.6)
suggested that C-26 was linked with a hydroxyl group. Starting from the
two anomeric protons, the exact identity of the monosaccharides and
the sequence of the disaccharide chain were also determined by ana-
lysis of a combination of DEPT, 1H-1H COSY, HSQC, and HMBC spectra.
The connectivity of the two sugars was mainly based on the HMBC
correlations: HGlc I-1 (δ 4.94, 1H, d, J = 7.7 Hz) with C-3 (δ 78.6) of the
aglycone, HRha I-1 (δ 5.91, 1H, br. s) with CGlc I-4 (δ 78.7) (Fig. 2). The
anomeric configuration of glucose was determined to be β on the basis
of the J value of the anomeric proton in glucose (J = 7.7 Hz), the α-
configuration of rhamnopyranosyls was determined by the two sets of
chemical shifts: CRha I-3 (δ 73.2), CRha I-5 (δ 70.8) based on the litera-
ture [15]. The two sugars, D-glucose and L-rhamnopyranosyls were
identified by GC analysis after derivatization.
Rha I
1
2
3
4
5
6
MeO
102.7
72.7
72.8
74.0
70.4
18.5
49.9
5.90 (br.s)
4.70 (m)
4.58 (dd, 9.2, 3.3)
4.34 (dd, 9.4, 9.2)
5.03 (m)
1.72 (d, 6.2)
3.39 (s)
Measured in C5D5N(1H: 400 MHz;13C: 100 MHz).
Measured in CD3OD(1H: 400 MHz;13C: 100 MHz).
a
b
were partitioned between ethyl acetate and water. The residue from the
water part was dissolved in 1-(trimethylsilyl)imidazole and pyridine
(1 mL), and was maintained at 60 °C for 5 min. After drying the solu-
tion, the residue was partitioned between H2O and CHCl3. The CHCl3
layer was analyzed by GC [detector, flame ionization detector (FID);
detector temperature, 280 °C; injection temperature, 250 °C; DB-5 ca-
pillary column, 30 m × 0.25 mm × 0.25 μm; column temperature,
100 °C for 2 min and then increased to 280 °C at a rate of 10 °C/min;
final temperature, 280 °C for 5 min; and carrier gas, N2]. By comparison
with the retention time of authentic sugars, the absolute configurations
of sugar components were determined (D-glucose, 19.50 min; L-rham-
nopyranose, 18.32 min) [11,12].
The NOESY correlations (Fig. 3) of Me-19/H-1β, H-1α/H-3, Me-18/
H-16, H-20 indicated the α-orientation of H-3 and OH-16. The absolute
configuration of 25R in 1 was established by the chemical shift of H2-26
15

B.-Y. Yang et al.
Fitoterapia128(2018)12–19
Fig. 2. Key HMBC and 1H-1H COSY correlations of compounds 1–7.
Fig. 3. Key NOESY correlations of compound 1.
(δ 3.75 and 3.68 ppm, Δδ = 0.07) (Δδ ≤ 0.57 ppm) [16]. Thus, 1 was
inferred as (25R) 3β, 16α, 26-trihydroxy-5-en-cholestan-22-one-3-O-α-L-
rhamnopyranosyl- (1 → 4)-β-D-glucopyranoside, and named Abutiloside
P.
Compound 2 was obtained as a white amorphous powder with a
molecular formula of C45H74O17 as determined by the HRESIMS (m/z
909.4825 [M+Na]+ calcd. for 909.4824). The NMR spectra of 2
(Tables 1 and 2) were nearly identical to those of 1, with one major
difference due to the presence of one more sugar in 2 as indicated by
the addition of anomeric proton and the carbon signal at δ 4.83 (1H, d,
J = 1.5 Hz, HRha II-1) and δ 103.0 (CRha II-1), respectively. This sug-
gested that 2 contained a diglycosidic moiety. The attachment of the
rhamnopyranosyl units to C-2 of the glucose was confirmed by the
molecular formula of 3 was established as C51H84O22 from its HRESIMS
at m/z 1071.5344 [M+Na]+ (calcd. for 1071.5352). In the 1H and 13C-
NMR spectrum of 3 (Tables 1 and 2), signals due to an aglycone moiety
were similar with those of 2, although the signals due to the sugar
moiety were not identical. A comparative study of the 13C-NMR of 2
with that of 3 indicated the presence of an additional glucosyl unit in 3,
which was linked to C-26 hydroxy group of aglycone moiety, because
the chemical shifts of C-25 (δ 33.9) moved to the high field and the
chemical shifts of C-26 (δ 75.6) moved to the low field. The long-range
correlations were observed between the anomeric proton at δ 4.81 (1H,
d, J = 7.8, HGlc II-1) with the carbon at C-26 (δ 75.6) from the HMBC
experiment (Fig. 2). The relative configurations of 3 were determined
by a NOESY experiment, which were consistent with those of 2. Con-
sequently, the structure of 3 was determined to be (25R) 26-O-β-D-
glucopyranoside 3β, 16α, 26-trihydroxy-5-en-cholestan-22-one-3-O-α-L-
rhamnopyranosyl-(1 → 2) -[α-L-rhamnopyranosyl-(1 → 4)]-β-D-gluco-
pyranoside, and named Abutiloside R.
HMBC correlations observed between δ 4.83 (1H, d, J = 1.5 Hz, HRha II
-
1) and δ 79.3 (CGlc I-2) (Fig. 2). The relative configurations of 2 were
determined to be the same as those of 1 by a NOESY experiment. Fi-
nally, the structure of 2 was established as (25R) 3β, 16α, 26-trihy-
droxy-5-en-cholestan-22-one-3-O-α-L-rhamnopyranosyl-(1 → 2)-[α-L-
rhamnopyranosyl-(1 → 4)]-β-D- glucopyranoside, and named Abutilo-
side Q.
Compound 4 possessed a molecular formula of C39H64O13 on the
basis of the HRESIMS with [M+Na]+ at m/z 763.4255 (calcd. for
763.4245). The NMR spectroscopic data of 4 (Tables 1 and 2) were
closely related to those of 1. A comparison of the 13C-NMR data of these
Compound 3 was separated as a white amorphous powder. The
16

B.-Y. Yang et al.
Fitoterapia128(2018)12–19
Fig. 4. Key NOESY correlations of compound 4.
Fig. 5. Key NOESY correlations of compound 6.
two compounds indicated that they have same planar structures. The
key differences were the signals from carbons around C-16, which
suggested that these two compounds should be different orientations of
C-16. The orientation of H-16 was deduced by the NOESY observed for
Me-18/Me-21, H-15β and H-15α/H-16 (Fig. 4), indicating a β-orienta-
tion of OH-16. The rest of the configurations of 4 were determined to be
the same as those of 1. Thus, compound 4 was determined to be (25R)
3β,16β,26-trihydroxy-5-en-cholestan-22-one-3-O-α-L-rhamnopyranosyl-
(1→4)-β-D-glucopyranoside, and named Abutiloside S.
Compound 5 was obtained as a white amorphous powder. Its mo-
lecular formula was deduced as C45H74O17 by HRESIMS at (m/z
909.4846 [M+Na]+ calcd. for 909.4824). Comparison of the NMR data
(Tables 1 and 2) of 5 with those of 4 showed the close similarity be-
tween the structures of both compounds, except for the presence of one
additional sugar in 5 as indicated by the addition of an anomeric proton
to be (25R) 3β, 16β, 26-trihydroxy-5-en-cholestan-22-one-3-O-α-L-
rhamnopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1 → 4)]-β-D-glucopyr-
anoside, and named Abutiloside T.
Compound 6 was obtained as an amorphous powder and its mole-
cular formula was C40H63NO14 according to the HRESIMS at m/z
804.4138 [M+Na]+ (calcd. for 804.4146). The 1H-NMR spectrum
(Table 3) of 6 showed signals due to two tertiary methyl groups (δ 1.38,
3H, s; δ 0.89, 3H, s), two secondary methyl groups (δ 1.72, 3H, d,
J = 6.2 Hz; δ 0.73, 3H, d, J = 6.6 Hz), one acetyl group (δ 2.27, 3H, s),
one methoxyl group (δ 3.39, 3H, s), and one olefinic proton (δ 5.30, 1H,
br.d, J = 5.1 Hz). The 13C-NMR spectrum (Table 3) of 6 showed 40
carbon signals including two carbonyl carbons (δ 206.5, 167.4), two
olefinic carbons (δ 140.9, 121.8), one nitrogen- and oxygen-bearing
carbon (δ 97.2), and two anomeric carbons (δ 102.7, 102.4), suggesting
6 to be a disaccharide of a steroidal alkaloid. The 1H- and 13C-NMR
signals of aglycone moiety of 6 were superimposable on those of abu-
tiloside O, which was obtained previously from the underground parts
of Solanum sodomaeum [17]. The 1H- and 13C-NMR signals of the sugar
moiety were very similar to those of 1. In the HMBC spectrum, key
correlations of 6 were observed as illustrated in (Fig. 2). The stereo-
chemistry of C-16 and C-17 was determined by analysis of the coupling
and the carbon signal at δ 6.39 (1H, br. s, HRha II-1) and δ 102.0 (CRha II
-
1), respectively. The attachment of the rhamnopyranosyl units to the C-
2 of the glucose was determined by the HMBC correlations observed
between δ 6.39 (1H, br. s, HRha II-1) and δ 78.5 (CGlc I-2) (Fig. 2). The
relative configurations of 5 were established to be the same as those of
4 by a NOESY experiment. Therefore, the structure of 5 was determined
17

B.-Y. Yang et al.
Fitoterapia128(2018)12–19
Fig. 6. Hypothetical biogenetic pathway of compound 6.
previously synthesized by Robert et al. [18]. This was the first report of
its natural occurrence.
Table 4
NO inhibitory activities of compounds 1-10 in RAW 264.7 cell line.
The three known steroids, abutiloside G (8) [7], solaviaside B (9)
[19] and tumacone B (10) [20], were also identified by comparison of
their spectroscopic data with literature values.
Compounds
IC50 (μM)
Compounds
IC50 (μM)
1
2
3
4
5
6
12.6
26.8
> 80
26.2
38.2
52.8
1.3
2.3
7
8
9
10
59.5
58.8
37.4
> 80
39.6
4.7
3.7
3.1
Natural products containing 2,5-epimino-2-methoxy-4-pentanoi-
cacid ester steroidal alkaloids were previously isolated from some
Solanum plants, exemplified by abutiloside O [17,21] and solasodoside
E [22]. Here, the hypothetical biogenetic pathways of 6 have been
briefly discussed. Cycloartenol is a key precursor metabolized by de-
methylation, desaturation, isomerization and reduction reactions to
generate cholesterol [23–25]. It is transformed into furostanol steroidal
alkaloid through repeated steps of hydroxylation, oxidation and trans-
amination via cholesterol [25], followed by biogenetic route B [26] to
generate a steroidal alkaloid with an ester side chain at the 16-position.
Finally, the 2,5-epimino-2-methoxy-4-pentanoicacid ester steroidal al-
kaloid was produced by intramolecular cyclization and oxidization re-
action (Fig. 6).
Nitric oxide (NO) is a relevant target of inflammation, and plays a
key role in the pathogenesis of inflammation [27]. The inhibition of NO
release may be considered a therapeutic agent in the inflammatory
diseases [28]. The purified compounds 1-10 were tested for inhibition
of NO production induced by LPS in the macrophage cell line RAW
264.7. All isolates exhibited no cytotoxicity against RAW 264.7 cells
line tested by the MTT method at the concentration of 80.0 μM. The
inhibitory activities against the production of nitric oxide (NO) were
summarized in (Table 4). Compounds 1, 2 and 4-9 exhibited moderate
inhibition of NO production with IC50 values ranging from 12.6 to
59.5 μM. Compounds 3 and 10 were very weak or inactive (IC50 va-
lues > 80 μM). Due to the limited numbers of active compounds, only
the superficial and primary structure-activity relationships were dis-
cussed. Compared with compound 4, 1 showed better activity, while
the activity of 2 was better than that of 5, which suggested that the α-
orientation of OH-16 resulted in higher inhibitory activity. The IC50 of 2
2.6
2.9
4.2
Indomethacina
2.2
a
Positive control.
constant values of the signals due to H-16 (δ 5.69, 1H, ddd, J = 4.8, 8.2,
8.2 Hz) and H-17 (δ 2.43, 1H, d, J = 8.2 Hz), which were similar to
those of 16β-[(4S)-5-(β-D-glucopyranosyloxy)-4-methyl-1-oxopentyl]
oxy]-3β-[(O-α-L-rhamnopyranosyl-(1→2)-O-[α-L-rhamnopyranosyl-(1→
3)]-β-D-glucopyranosyl)oxy]pregn-5-en-20-one [17]. The correlations
of Me-19/H-1β, H-1α/H-3, Me-18/H-15β, H-15α/H-16α observed in the
NOESY spectrum (Fig. 5) further confirmed the β-orientation of C-3 and
C-16. However, the configurations of C-23 and C-25 have not been
confirmed. Consequently, 6 was determined to be 3β,16β-dihydroxy-
pregn-5-en-20-one-16-O-(2,5-epimino-2-methoxy-4-pentanoicacid)-
ester-3-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside, and named
Abutiloside U.
Compound 7 was obtained as a white powder and found to have the
molecular formula
C27H44O4 as inferred from its HRESIMS m/z
455.3144 [M+Na]+ (calcd. for 455.3137). In the 1H- and 13C-NMR
spectrum of 7 (Table 3), signals due to an aglycone moiety were similar
to those of 2. The 1H- and 13C-NMR shifts of C-2, C-3, and C-4 in 2
slightly changed in 7. A comparative study of 1H- and 13C-NMR found
that the disaccharide signals in 2 were absent in 7. The relative con-
figurations of 7 were determined by a NOESY experiment, which were
consistent with those of 2. Therefore, the structure of 7 was determined
to be (25R) 3β, 16α, 26-trihydroxy-5-en-cholestan-22-one, which was
18

B.-Y. Yang et al.
Fitoterapia128(2018)12–19
and 5 was higher than that of 1 and 4, respectively, which demon-
strated that the number of glycosyl groups may affect the activity.
In summary, our chemical investigation of the roots of Solanum
melongena L. resulted in the isolation and identification of ten steroids,
including six new steroidal saponins, named Abutilosides P-U (1-6) and
one new natural product (7). These compounds were investigated for
inhibitory activities on nitric oxide (NO) production stimulated by li-
popolysaccharide (LPS) in the RAW 264.7 cell line. Most of the isolates
displayed moderate inhibitory activity, except for inactive compounds
3 and 10. The present investigation suggested that roots of Solanum
melongena L. could be a potential source of natural anti-inflammatory
agents and their steroidal saponins might be responsible for inhibition
of NO production.
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Conflict of interest
The authors declare no competing financial interests.
Acknowledgements
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We thank Mr. Weiguo Zhu from the Analytical and Testing Center of
Zhengzhou University (10459) for collecting spectroscopic data.
Appendix B. Supplementary data
The spectroscopic data for compounds 1-6 are available as
Supporting information. Supplementary data to this article can be
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19