Two new 23S,26R-hydroxylated spirostanoid saponins from the fruits of Solanum indicum var. recurvatum

Article abstract of DOI:10.1016/j.steroids.2019.108506

Solanum indicum var. recurvatum, distributed mainly in Hekou, Simao and Menghai areas of Yunnan province, China, is characterized by stellate pubescent leaves with irregular lengths and oblong, short, and downwardly curved anthers. The original species, S. indicum, has been used as an anti-inflammatory, wound-healing agent, an analgesic, and for the treatment of rhinitis, cough, and breast cancer in Chinese folk medicine. The variety was chemically studied for the first time, leading to the isolation of two new rare 23S,26R-hydroxylated spirostanoid saponins, together with five known steroidal components, two of which the rare natural ketosteroids 6-hydroxyandrosta-1,4-diene-3,17-dione and rostadienedione have been isolated from the genus Solanum for the first time. Elucidation of the structures was accomplished by extensive 1D and 2D NMR, UV, and HRESIMS spectroscopic methods.

Full text of DOI:10.1016/j.steroids.2019.108506

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Two new 23S,26R-hydroxylated spirostanoid saponins from the fruits of Sola-
num indicum var. recurvatum
Joseph Sakah Kaunda, Ying-Jun Zhang
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STE 108506
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Steroids
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Two new 23S,26R-hydroxylated spirostanoid saponins from the fruits of Solanum indicum
var. recurvatum
Joseph Sakah Kaundaa,b and Ying-Jun Zhanga,c,*
aState Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute
of Botany, Chinese Academy of Sciences, Kunming 650204, P. R. China
bGraduate School of the Chinese Academy of Sciences, Beijing 100039, P. R. China
cYunnan Key Laboratory of Natural Medicinal Chemistry, Kunming Institute of Botany, Chinese
Academy of Sciences, Kunming 650201, People's Republic of China
* Corresponding author:
Ying-Jun Zhang
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute
of Botany, Chinese Academy of Sciences, Kunming 650204, P. R. China
*Tel/Fax: +86-871-6522-3235. E-mail: zhangyj@mail.kib.ac.cn
1

ABSTRACT
Solanum indicum var. recurvatum, distributed mainly in Hekou, Simao and Menghai areas of
Yunnan province, China, is characterized by stellate pubescent leaves with irregular lengths and
oblong, short, and downwardly curved anthers. The original species, S. indicum, has been used as
an anti-inflammatory, wound-healing agent, an analgesic, and for the treatment of rhinitis, cough,
and breast cancer in Chinese folk medicine. The variety was chemically studied for the first time,
leading to the isolation of two new rare 23S,26R-hydroxylated spirostanoid saponins, together
with five known steroidal components, two of which the rare natural ketosteroids 6-
hydroxyandrosta-1,4-diene-3,17-dione and rostadienedione have been isolated from the genus
Solanum for the first time. Elucidation of the structures was accomplished by extensive 1D and
2D NMR, UV, and HRESIMS spectroscopic methods.
Keywords: Solanum indicum var. recurvatum; Solanaceae; 23S,26R-hydroxylated spirostanoid
saponins; ketosteroids
2

1. Introduction
The genus Solanum is regarded to be one of the largest among the Angiosperms and the most
representative and largest genus of the family Solanaceae [1]. It is comprised of about 1500
species distributed across subtropical and tropical regions of Asia, tropical Africa, non-arid
Africa, Americas, and Australia. Many species belonging to this genus possess a rich repertoire
of medicinal, economic, and ornamental importance [1-2]. Within the past 30 years, Solanum has
attracted significant attention in chemical and biological studies [1]. Previous phytochemical
investigations on Solanum species led to the identification of steroidal saponins, steroidal
alkaloids, terpenes, flavonoids, lignans, sterols, phenolics, coumarins, and so on.
Solanum indicum var. recurvatum, much the same as its original species, S. indicum, is
characterized by stellate pubescent leaves with irregular lengths and oblong, short, and
downwardly curved anthers [3]. It is distributed mainly in Hekou, Simao and Menghai areas of
Yunnan province, China [3]. Whereas S. indicum has been used in Chinese folk medicine as an
anti-inflammatory, wound-healing, and analgesic agents, as well as for the treatment of rhinitis,
cough, and breast cancer [4], the available literature data did not reveal any local uses of the
variety. Driven by the aspiration to unearth its chemical constituents and investigate
theirbioactivities, our study on methanolic extract of the fruits of S. indicum var. recurvatum
uncovered four steroidal saponins (1-4), one steroidal sapogenin (5) and two ketosteroids (6-7).
Compounds 1-2 are new rare 23S,26R-hydroxylated spirostanoid saponins. All the isolates were
obtained from the titled plant for the first time. The new isolates were also evaluated for their
cytotoxic, antifungal, and anti-inflammatory activities. Herein, we report the study.
2. Experimental
3

2.1. General
Optical rotations were measured on Rudolph Autopol VI polarimeter (Rudolph Research
Analytical, Hacketstown, NJ, USA). IR spectra were measured on a Bio-Rad FTS-135 series
spectrometer with KBr pellets. UV spectra were recorded on a UV 210A Shimadzu spectrometer.
CD spectra were obtained on a JASCO 810 spectrometer. One- and two-dimensional (1D and 2D)
NMR spectra were recorded in C5D5N with Bruker DRX-600 and Avance 800 spectrometer
13
operating at 600 and 800 MHz for 1H and at 150 and 200 MHz for C, respectively. Coupling
constants are expressed in hertz, and chemical shifts are given on a δ (parts per million, ppm)
scale with tetramethylsilane (TMS) as an internal standard. ESI mass spectra were recorded on a
VG Auto Spec-300 spectrometer. High-resolution (HR) ESI mass spectra were recorded on an
API QSTAR Pular-1 spectrometer. Column chromatography (CC) was performed on MCI gel
CHP20P (75−100 m, Mitsubishi Chemical Co., Ltd.), silica gel (200-300 mesh, Qingdao
Marine Chemical Co., Qingdao, China) and RP-18 gel (40−60 m) (Merck, Darmstadt,
Germany). Preparative HPLC and semi-preparative reversed-phase were performed using a
Capcell C18 (Agilent, America) column (10 mm × 250 mm) and Cosmosil cholester (Kyoto,
Japan) column (4.6 mm I.D. × 150 mm); the flow rate used for Preparative HPLC was 3.0
mL/min, and the separation and detection were achieved using Newstyle NU3000 SERIALS
UV/VIS detector (Jiangsu Hanbon Science & Technology Co. Ltd, China). Thin-layer
chromatography (TLC) was performed on precoated silica gel H plates, 0.20−0.25 mm thick
(Qingdao Haiyang Chemical Co.), with chloroform/methanol/water (7:3:0.5 or 8:2:0.2 v/v/v),
and spots were visualized under UV light and by spraying with 10% H2SO4 in EtOH followed by
heating. Water was purified in a Milli-Q (Millipore, America). Acetonitrile (chromatographic
grade) was purchased from Merck (Darmstadt, FR, Germany).
4

2.2. Plant material
Fruits of S. indicum var. recurvatum were collected in December 2018 in the vicinity of
Xiding township, Menghai county, Xishuangbanna, Yunnan province, China. The plant material
was identified by Prof. C.-R. Yang and a voucher specimen (KIB-Z-2018012) was deposited in
the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming
Institute of Botany, Chinese Academy of Sciences, Kunming, China.
2.3. Extraction and isolation
Dried fruits (1.5 kg) of S. indicum var. recurvatum were crushed into powder and extracted in
a Soxhlet apparatus with methanol for 72 h. The methanol extract (4.5 L) was then concentrated
under reduced pressure in a rotary evaporator to give a residue (122 g) which was suspended into
water (1000 mL) and extracted with n-butanol (each 500 mL, 4 times) to yield n-butanol (63 g)
and water (45 g) fractions, after concentration under reduced pressure. The butanol fraction (63 g)
was subjected to MCI gel, eluting with MeOH: H2O (0:1→1:0), to give 7 fractions, M1-M7.
Fraction M5 (5.5 g) was applied to silica gel (120 g) column (4.0 x 35 cm), eluting with
chloroform/methanol/water (100:0:0, 98:2:0, 96:4:0, 94:6:0, 92:8:0, 90:10:0, 90:10:1, 85:15:1,
80:20:2 v/v) to generate compounds 1 (6 mg) and 2 (7 mg), and fractions M5c-M5f. Fr. M5g
(850 mg) was applied to silica gel (36 g) column (3.5 x 30 cm) chromatography (CC), eluting
with chloroform/methanol/ water (100:0:0, 98:2:0, 96:4:0, 94:6:0, 92:8:0, 90:10:0, 90:10:1,
85:15:1, 80:20:2 v/v) to furnish 3 (27 mg) and 4 (43 mg). Fraction M6 (6 g) was applied to silica
gel CC (120 g, 4.0 x 30 cm), eluting with chloroform/methanol/water (100:0:0, 98:2:0, 96:4:0,
94:6:0, 92:8:0, 90:10:0, 90:10:1, 85:15:1, 80:20:2 v/v), to afford fractions M6a-M6h. Fr. M6a
(900 mg), was subjected to silica gel (36 g) CC (3.5 x 30 cm), eluting as outlined above, to
afford two fractions, M6a-1 (300 mg) and M6a-2 (420 mg). Fr. M6a-1 was purified on RP-18 to
5

bring forth fractions Fr. M6a-1a (29 mg), Fr. M6a-1b (25 mg), and Fr. M6a-1c (46 mg), which
were submitted for further purification by preparative HPLC with an isocratic mobile phase
system of acetonitrile/water. Fr. M6a-1a furnished 6 (tR = 26.3 min, 2.0 mg) (35:65, v/v), Fr.
M6a-1b gave 7 (tR = 38.7 min, 2.0 mg) (40:60, v/v), and Fr. M6a-1c afforded 5 (tR = 18.9 min,
7.0 mg) (25:75, v/v).
2.3.1 (22R,23S,25R,26R)-spirost-5-ene-3β,23,26-triol 3-O-α-L-rhamnopyranosyl-(1→4)-β-D-
glucopyranoside (1):
25
D
Yellow amorphous powder; [α]
-73.2 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 196.6 (0.54)
nm ; IR (KBr) νmax 3424, 2934, 2906, 2850, 1714, 1636, 1509, 1454, 1436, 1383, 1305, 1274,
1259, 1197, 1065, 1038, 914, 894, 874, 856, 837 cm−1; ECD (MeOH) λmax (Δε) 199 (1.00), 237
(-0.17), 250 (-0.07); 1H and 13C NMR (600/150 MHz, pyridine-d5) data, see Table 1; HRESIMS
m/z: 777.4031 [M+Na]+ (calcd for C39H62O14Na, 777.4032).
2.3.2 22R,23S,25R,26R)-spirost-5-ene-3 β,23,26-triol 3-O-β-D-glucopyranoside (2):
25
D
White amorphous powder; [α] -19.3 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 192.8 (0.61),
196.8 (0.78), 269.2 (0.09), 283.2 (0.10) nm ; IR (KBr) νmax 3431, 2933, 2853, 1712, 1634, 1516,
1454, 1436, 1384, 1318, 1274, 1258, 1159, 1074, 1044, 917, 895, 856, 837 cm−1; ECD (MeOH)
λmax (Δε) 202 (0.35), 297 (-0.35); 1H (600 MHz, pyridine-d5) and 13C NMR (150 MHz, pyridine-
d5) data, see Table 1; HRESIMS m/z: 631.3458 [M + Na]+ (calcd for C33H52O10Na, 631.3453).
2.3.3 Acid hydrolysis of compounds 1–2 and GC analysis
Compounds 1-2 (2 mg, each), were separately dissolved in 5% HCl (1 mL) and heated (90 °C)
for 2 h. HCl was then removed by evaporation in vacuum. The reaction mixtures were diluted
6

with H2O and extracted with EtOAc. The aqueous layer was neutralized with 0.1 M NaOH and
essentially dried to give the monosaccharide mixtures. In to the solution of the sugar mixtures in
pyridine (2 mL), was added L-cysteine methyl ester hydrochloride (about 2 mg) and the reaction
mixture kept at 60 °C for 1 h before addition of trimethylsilylimidazole (2 mL) in to the mixtures.
Following this, the reaction mixtures were further kept at 60 °C for 1 h, before stopping the
reaction and immediately submitting them for GC analysis [5], run on Hewlett Packard (HP)
5890 series II gas chromatography equipped with flame ionization detector (FID) and thermal
conductivity detector (TCD). The column was HP-5: column temperature: 280 °C, increasing at
the rate of: 3 °C/min; carrier gas: N2 (1.5 mL/min); injector and detector temperature: 250 °C;
injection volume: 1.0 L; and split ratio: 1/50. The retention times of the samples were compared
with those of the derivatives of authentic sugars, under the same condition. The sugar moieties of
1 were determined to be D-glucose (tR: 28.232 min) and L-rhamnose (tR: 24.265 min) by
crosschecking with the standards, D-glucose (tR: 28.418 min) and L-rhamnose (tR: 24.348 min),
respectively. The sugar moiety of 2 was resolved to be D-glucose (tR: 28.358 min) by matching.
2.4. Cytotoxicity assay
The cytotoxic activities of compounds 1–2 were evaluated against five human cancer cell lines
(HL-60, SMMC-7721, A-549, MCF-7 and SW480) which were cultured in RPMI-1640 or
DMEM medium by means of 4-{5-[3-(carboxymethoxy)phenyl]-2- (4,5-dimethyl-1,3- thiazol- 2-
yl)-2H-tetrazol-3-ium-3-yl}benzene-1-sulfonate (MTS) assay [6]. Cells in RPMI-1640 or
DMEM medium containing 10% fetal bovine serum and inoculated with 3000-15000 cells per
well, seeded into a 96-well cell culture plate with a volume of 100 L per well, and kept at 37 °C
in the incubator for 48 h. The compounds were dissolved in DMSO. After 48 h, 100 L
supernatant culture solutions from each well was discarded and 20 L MTS solution was added.
7

The samples were incubated further for 4 h. Cisplatin (DDP) and taxol compounds were used as
positive control in each experiment. Cell growth curve was plotted with the concentration as
abscissa and cell viability as ordinate. The optical density (OD) was measured at 492 nm using
MULTISKAN FC to determine inhibition rates of the 5 strains.
2.5. Antifungal assay
The test was performed in Potato Dextrose medium (Scientific Research Special) according to
the procedure of Y. Liu et al. [7]. A volume of 100 L aliquot from the stock solutions of the
samples which had been initially prepared was added into the wells of the 96-well plates. 100 L
of the inoculum was added to achieve a final inoculum concentration of 1 × 105 CFU/mL in each
well. The volume in each well was finally adjusted to 200 L. Amphotericin B (Sigma-Aldrich)
was used as reference substance. After incubation at 37 °C for 24 hours, plates were read at 625
nm to calculate the inhibitory rate of C. albicans - ATCC 10231 (Microbiologics).
2.6. Anti-inflammatory assay
As previously described by N. Soonthornsit et al. [8], RAW264.7 cells (Cell Bank of Chinese
Academy of Sciences, Shanghai, China) were inoculated into 96-well plates, and induced
stimulation with 1 g/mL LPS (Sigma-Aldrich), and a test compound (final concentration 50 M)
was added to prepare a drug-free group and an L-NMMA-positive drug group (Sigma-Aldrich)
as control. After the cells were cultured overnight, the medium was taken to detect NO
production, and the absorbance was measured at 570 nm. MTS was added to the remaining
medium for cell viability assay to eliminate the toxic effects of the compounds on the cells. NO
production inhibition rate (%) = (non-drug treatment group OD570 nm - sample group OD570 nm)
/ non-drug treatment group OD570 nm × 100%.
8

3. Results and discussion
The MeOH extract of the fruits of S. indicum var. recurvatum was subjected to repeated
column chromatography over MCI-gel CHP20P, silica gel, and semi-preparative HPLC, to yield
seven steroidal compounds (1-7). Among them, compounds 1-2 are new 23S,26R-hydroxylated
spirostanoid saponins. The known constituents were identified, on the basis of comparison of
their NMR spectroscopic and MS data with those reported in the literature, as two steroidal
glycosides, indioside A (3) [2,4] and 26-O-β-D-glucopyranosyl-22-methoxy-3,26-dihydroxy-
(25R)-furost-5-ene
3-O-L-rhamnopyranosyl-(1→2)-[-α-L-arabinopyranosyl-(1→3)]-β-D-
glucopyranoside (4) [9], one steroidal sapogenin indioside L (5) [10], and two ketosteroids, 6β-
hydroxyandrosta-1,4-diene-3,17-dione (6) and rostadienedione (7) [11,12] (Fig. 1). All of them
were reported from the titled plant for the first time. The only reports on the isolation of
23S,26R-hydroxylated spirostanoid glycoside were featured by Yohara et al. [2] and Mona et al.
[4] who obtained compound 3 from the fruits of S. indicum. Compounds 6-7 were identified as
rare ketosteroids in plants, only isolated from Aglaia rubiginosa (Meliaceae) [11]. They were
previously obtained by chemical synthesis and microbiological biotransformations [12].
Compound 1 was obtained as a yellow, amorphous powder. Its molecular formula was
assigned as C39H62O14Na based on a quasi-molecular ion peak at m/z 777.4031 [M+Na]+ in the
1
HR-ESI-MS (calcd for C39H62O14Na, 777.4032). The H NMR spectrum of 1 indicated two
tertiary methyls at δH 1.01 and 0.87 (each 3H, s, CH3-18, CH3-19) and two secondary methyls at
δH 1.30 (3H, d, J = 7.0 Hz, CH3-21) and 1.18 (3H, d, J = 6.2 Hz, CH3-27), which showed
characteristics of a typical steroidal glycoside [4]. The two anomeric proton signals at δH 4.98
(1H, d, J = 7.7 Hz) and 5.94 (1H, s) suggested the existence of two sugar moieties in 1. The 13C
NMR spectrum showed 27 signals that emerged from the aglycone of the glycoside; these signals
9

pointed out the presence of a hemiacetal [δC 96.6 (C-26)], a ketal [δC 114.2 (C-22)], three
oxygenated methines [δC 78.7 (C-3), 82.2 (C-16), 67.9 (C-23)], a tri-substituted double bond [δC
141.3 (C-5), 122.2 (C-6)], eight methylenes [δC 37.9 (C-1), 30.7 (C-2), 39.7 (C-4), 32.7 (C-7),
21.6 (C-11), 40.7 (C-12), 32.6 (C-15), 38.2 (C-24)], six methines [δC 32.0 (C-8), 50.7 (C-9), 57.2
(C-14), 63.0 (C-17), 36.0 (C-20), 38.9 (C-25)], four methyls (δC 17.1 (C-18), 19.8 (C-19), 15.3
(C-21), 17.9 (C-27), and two quaternary carbons [δ 37.5 (C-10), 41.5 (C-13)]. Furthermore, the
spectrum unveiled the sapogenol moiety as a spirostanol derivative containing one hemiacetal, a
13
ketal, and three oxygen-bearing carbons. Also displayed on the C NMR spectrum were 12
carbon signals that designated the existence of a glucosyl and one terminal rhamnosyl moieties,
which were determined to be D-glucose (tR: 28.232 min) and L-rhamnose (tR: 24.265 min) on the
basis of acidic hydrolysis, followed by GC analysis of the corresponding trimethylsilylated L-
cysteine adduct. The HMBC experiment showed correlations from δH 1.18 (H3-27) to δC 67.9
(C-23), 38.9 (C-25) and 96.6 (C-26), from δH 1.30 (H3-21) to δC 63.0 (C-17), 36.7 (C-20) and
114.2 (C-22) and from δH 4.04 (1H, dd, J = 4.9, 7.8 Hz, H-23) to C-22 (Fig. 2), suggesting that
the aglycone of 1 was a 3,23,26-trihydroxyspirost-5-ene derivative [4], even as confirmed by the
1H–1H COSY, HMBC and ROESY correlations (Fig. 2). The H-26 signal appeared as a doublet
at δH 5.22 (J = 8.0 Hz), expressing trans-diaxial coupling between H-26 and H-25 [3]. Moreover,
in the ROESY spectrum, correlations were observed between H-20 (δ 3.04) and H-23 (δ 4.04) as
well as between H-23 and H-25 (δ 2.02) (Fig. 2), conveying that C-22 and C-25 were both in the
R configuration [2, 4]. In the sugar moiety, HMBC correlations were observed from the
rhamnosyl H-1 (δ 5.94) to glucosyl C-4 (δ 77.8) and from the glucosyl H-1 (δH 4.98) to the
aglycone C-3 (δ 78.7). Except for the sugar moieties, the above NMR features of 1 were quite
similar to those of compound 3, the first 23S,26R-hydroxylated spirostanoid saponin with three
10

sugar moieties at aglycone C-3 obtained previously from the fruits of S. indicum [2,4]. Instead of
one rhamnosyl and one arabinosyl moieties linked to the inner glucosyl C-2 and C-3, resp, in 3,
compound 1 has only one rhamnosyl group linked to the inner glucosyl C-2. Accordingly, the
structure of 1 was characterized as (22R,23S,25R,26R)-spirost-5-ene-3β,23,26-triol 3-O-α-L-
rhamnopyranosyl-(1→4)-β-D-glucopyranoside .
Compound 2 was obtained as a white amorphous powder. Its molecular formula was
established as C33H52O10, in reference to the quasi-molecular ion peak at m/z 631.3458 [M+Na]+
in the HR-ESI-MS (calcd for C33H52O10Na, 631.3453). There was absolute similarity of signals
1
13
13
in H and C NMR spectra of 1 and 2, however, C NMR spectrum of 2 exhibited 33 signals
implying a loss of one hexosyl unit from the sugar chain in relation to compound 1. The absent
sugar moiety was determined to be α-L-rhamnopyranosyl, which existed in compound 1 at C-4"
13
(δ 77.8), whereas in compound 2, the C NMR chemical shift signal assignable to C-4" of
glucosyl was upfield shifted to δ 72.2. The single anomeric proton displayed at δH 5.06 (1H, d, J
= 7.7 Hz) indicated only one glycosidic moiety in 2[4], which was resolved to be D-glucose (tR:
28.358 min) by acidic hydrolysis, followed by GC analysis of the corresponding
trimethylsilylated L-cysteine adduct. The HMBC correlation was observed from the glucosyl H-
1 (δH 5.06) to C-3 (δ 79.0) of the aglycone moiety. Compound 2 was thus determined to be
(22R,23S,25R,26R)-spirost-5-ene-3 β,23,26-triol 3-O-β-D-glucopyranoside.
Compounds 1-2 were evaluated for their cytotoxicities on five human cancer cell lines (HL-
60, SMMC- 7721, A-549, MCF-7 and SW480), antifungal activity on Candida albicans (ATCC
10231), and inhibition activity on nitric oxide (NO) production; none of them was active at
concentrations of 40 M, 200 M, and 50 M, respectively.
11

It is opined that introducing two hydroxyl groups at C-23 and C-27 of progenin II or dioscin
considerably decreased their antimicrobial activities against Propionibacterium acnes by
comparison of the MIC values of chonglouoside SL-2 and progenin II or chonglouoside SL-3
and dioscin [13]. Regarding biological activities, these compounds mentioned above (progenin II
or dioscin) might not be the perfect examples to compare with compounds 1 and 2 owing to
different sugar moieties and positions of the hydroxy group. Bizarrely, however, they tend to
underscore why such compounds, like 1 and 2, with hydroxy groups attached directly to the F
ring display poor biological activities e.g. antimicrobial, cytotoxic and anti-inflammatory. In
order to shed more light on this, it should therefore be of great interest to investigate and
compare the biological activities of compound 1 and yamogenin 3-O-α-L-rhamnopyranosyl-
(1→4)-β-D-glucopyranoside [14], as well as compound 2 and (22S,25S)-spirost-5-en-3β-yl β-D-
glucopyranoside [15]. This is because the compounds are absolutely similar except for the
introduction of hydroxy groups at C-23 and C-26 in 1 and 2.
4. Conclusion
Two new rare 23S,26R-hydroxylated spirostanoid saponins (1-2), together with five known
steroidal constituents (3-7) were isolated from the methanolic extract of the fruits of Solanum
indicum var. recurvatum. The rare natural ketosteroids, 6-7, were isolated from the genus
Solanum for the first time. Compounds 1 and 2 exhibited no cytotoxicity, antifungal, and anti-
inflammatory activities; this should invite further studies to explore other biological effects in
future.
Acknowledgements
12

We are delighted to express our sincere gratitude to members of the analytical center of State
Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of
Botany, for spectroscopic data measurements. This work was partially supported by Yunnan Key
Laboratory of Natural Medicinal Chemistry (S2017-ZZ14).
Declaration of Competing Interest
The authors declare no conflict of interest.
Supplementary data
Supplementary data to this article can be found online at
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14

Table 1
1D NMR data of compounds 1-2 in pyridine-d5 (600 MHz/150 MHz)
1
2
Position δH (J in Hz)
1
2
3
4
5
6
7
δC
δH (J in Hz)
δC
37.9
1.71 m, 0.98 m
2.09 m, 1.71 m
3.89 m
37.9 1.71 m, 0.98 m
30.7 2.14 m, 1.74 m
78.7 3.98 overlapped
39.7 2.73 m, 2.46 m
141.3
122.2 5.29 d (5.1)
32.7 1.99 m, 1.48 m
32.0 1.50 m
30.7
79.0
39.8
141.4
122.2
32.7
32.0
50.7
37.5
21.6
40.7
41.5
57.2
32.6
82.2
63.1
17.1
19.9
36.7
15.4
114.2
67.9
38.2
38.9
96.6
17.9
2.72 m, 2.46 m
5.31 d (5.0)
2.00 m, 1.50 m
1.49 m
8
9
0.89 m
50.7 0.89 m
37.5
21.6 1.43 m
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
1.43 m
1.76 m, 1.17 m
40.7 1.77 m, 1.14 m
41.5
1.06 m
57.2 1.07 m
2.01 m, 1.81 m
4.71 m
32.6 2.01 m, 1.81 m
82.2 4.71 m
1.95 m
1.01 s
0.87 s
3.04 m
63.0 1.96 m
17.1 1.03 s
19.8 0.86 s
36.7 3.03 m
15.3 1.32 d (6.8)
114.2
67.9 4.02 m
1.30 d (7.0)
4.04 m
2.19 m, 2.03 m
2.02 m
38.2 2.18 m, 2.02 m
38.9 1.98 m
5.22 d (8.0)
1.18 d (6.2)
3-O-Glc
4.98 d (7.7)
4.02 m
4.27 m
3.89 m
3.75 m
96.6 5.21 d (8.0)
17.9 1.17 d (6.1)
1'
2'
3'
4'
5'
6'
102.9 5.06 d (7.7)
76.0 4.07 m
77.2 4.32 m
103.1
75.8
79.1
72.2
78.6
63.3
78.7 4.30 m
77.6 3.98 overlapped
62.0 4.57 m, 4.43 m
4.29 m, 4.17 m
4'-O-Rha
5.94 s
1"
2"
3"
4"
5"
6"
103.2
73.1
73.3
74.5
70.9
19.0
4.74 m
4.62 m
4.39 m
5.07 m
1.76 d (6.2)
15

OH
OH
27
O 26
23
21
O
20
17
25
22
18
OH
OH
O
11
9
O
19
10
16
H
13H
8 H
1
H
H
Glc
H
3
H
HO
HO
5
HO
O
O
O
O
O
1'
HO
HO
rha
OH
2
1
OH
1''
O
HO
HO
OGlc
OH
OH
R =
HO
OCH3
O
O
H
O
OH
HO
O
O
HO
HO
O
H
H
H
H
O
OH
HO
O
H
H
H
H
RO
RO
HO
3
4
OH
O
O
O
O
H
OH
H
O
H
H
H
H
OH
H
O
H
O
7
6
O
5
Fig. 1. Compounds 1-7 isolated from the fruits of Solanum indicum var. recurvatum
16

H
1H-1H COSY
HMBC H
OH
OH
H
H
C
H
OH
O
H
ROESY H
H
HO
O
O
O
HO
O
OH
HO
HO
OH
1
1
Fig. 2
1
. Key H- H COSY, HMBC, and ROESY correlations of
Graphical Abstract
Two new 23S,26R-hydroxylated spirostanoid saponins from the fruits of Solanum indicum var.
recurvatum
Joseph Sakah Kaunda and Ying-Jun Zhang
OH
OH
xyl
O
O
OH
O
glc
OH
OH
O
O
HO
O
H
H
1
2
R = rha
R = H
O
H
H
O
HO
HO
O
H
H
H
H
HO
HO
O
HO
rha
OH
O
O
S-O
OGlc
HO
RO
3
S
HO
OH
O
OCH3
O
O
H
H
OH
O
H
H
H
H
H
Solanum indicum
var. recurvatum
H
R
H
H
H
O
6
7
R =OH
R = H
S-O
O
5
4
Highlights
1. The fruits of S. indicum var. recurvatum were studied chemically.
17

2. Two new rare 23S,26R-hydroxylated spirostanoid saponins were obtained.
3. Two rare ketosteroids were isolated from the genus Solanum for the first time.
The first author, Joseph Sakah Kaunda: Investigation; Writing - original draft and revision.
Corresponding author, Ying-Jun Zhang: Funding acquisition; Supervision; Writing - review &
editing.
18