Steroidal constituents isolated from the seeds of Withania somnifera

Article abstract of DOI:10.1080/14786419.2019.1667351

A new withanolide glycoside (1), two new ergostanol glycosides (2 and 3), and a new furostanol glycoside (4), along with nine known steroidal derivatives (5–12) were isolated from the seeds of Withania somnifera. The structures of the new compounds were determined using spectroscopic analysis and hydrolysis. The cytotoxic activities of the isolated compounds were evaluated against Ca9-22 human gingival carcinoma cells, HSC-2 human mouth carcinoma cells, and HL-60 human promyelocytic leukemia cells. Only 12 exhibited cytotoxic activity against these cell lines with IC₅₀ values of 0.38, 0.54, and 1.5 μM, respectively.

Full text of DOI:10.1080/14786419.2019.1667351

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Steroidal constituents isolated from the seeds of
Withania somnifera
Tomoki Iguchi, Minpei Kuroda, Mai Ishihara, Hiroshi Sakagami & Yoshihiro
Mimaki
To cite this article: Tomoki Iguchi, Minpei Kuroda, Mai Ishihara, Hiroshi Sakagami & Yoshihiro
Mimaki (2019): Steroidal constituents isolated from the seeds of Withaniaꢀsomnifera, Natural
Product Research, DOI: 10.1080/14786419.2019.1667351
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1667351
Steroidal constituents isolated from the seeds
of Withania somnifera
Tomoki Iguchia, Minpei Kurodaa, Mai Ishiharaa, Hiroshi Sakagamib and
Yoshihiro Mimakia
aDepartment of Medicinal Pharmacognosy, School of Pharmacy, Tokyo University of Pharmacy and
b
Life Sciences, Tokyo, Japan; Research Institute of Odontology (M-RIO), Meikai University,
Saitama, Japan
ABSTRACT
ARTICLE HISTORY
Received 21 July 2019
Accepted 31 August 2019
A new withanolide glycoside (1), two new ergostanol glycosides
(2 and 3), and a new furostanol glycoside (4), along with nine
known steroidal derivatives (5–12) were isolated from the seeds
of Withania somnifera. The structures of the new compounds
were determined using spectroscopic analysis and hydrolysis. The
cytotoxic activities of the isolated compounds were evaluated
against Ca9-22 human gingival carcinoma cells, HSC-2 human
mouth carcinoma cells, and HL-60 human promyelocytic leukemia
cells. Only 12 exhibited cytotoxic activity against these cell lines
with IC50 values of 0.38, 0.54, and 1.5 lM, respectively.
KEYWORDS
Withania somnifera;
Solanaceae; seed;
withanolide glycoside;
ergostanol glycoside;
furostanol glycoside;
cytotoxic activity
1. Introduction
Withania somnifera is an evergreen shrub belonging to the Solanaceae family, which is
found in diverse areas of Africa, the countries of the Mediterranean littoral, and India.
W. somnifera has been frequently used in Ayurvedic medicine for over 3,000 years and
is often referred to as “Ashwagandha” or “Indian ginseng” (Jana and Charan 2018).
The extracts of W. somnifera exhibit antibacterial, antifungal, and anticancer properties
(John 2014; Dar et al. 2015). The chemical constituents in the leaves and fruits of
W. somnifera have been well studied, and a variety of withanolide derivatives, such as
withaferin A, and alkaloids have been isolated and identified (John 2014). Currently,
the withaferin A content of Sardinian plant of W. somnifera has been shown to be
CONTACT Tomoki Iguchi
iguchit@toyaku.ac.jp
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1667351.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
T. IGUCHI ET AL.
higher than that of the Indian and Sicilian ones (Scartezzini et al. 2007). However, the
chemical composition of the seeds of W. somnifera have not been fully surveyed,
which prompted our group to carry out a phytochemical study on the seeds of this
plant. As a result, a new withanolide glycoside (1), two new ergostanol glycosides (2
and 3), a new furostanol glycoside (4), and nine known steroidal derivatives (5–12)
were isolated from W. somnifera seeds. The new compounds (1–4) were identified
based on the analysis of their spectroscopic data and hydrolysis. The cytotoxic activ-
ities of the isolated compounds against Ca9-22 human gingival carcinoma cells, HSC-2
human mouth carcinoma cells, and HL-60 human promyelocytic leukemia cells
were evaluated.
2. Results and discussion
The seeds of W. somnifera (1.0 kg) were extracted with hot MeOH. The concentrated
MeOH extract (90 g) was passed through a Diaion HP-20 porous polymer polystyrene
resin column and successively eluted with MeOH/H2O (1:4), EtOH, and EtOAc. The
EtOH eluted fraction (45 g) was repeatedly separated using silica gel and octadecyl-
silanized (ODS) silica gel column chromatography (CC), and preparative ODS HPLC to
obtain 12 compounds (1–12). The structure of the known compounds were elucidated
as (20S,22R)-1a,3b,27-trihydroxywitha-5,24(25)-dienolide (5) (Sahai 1985), (20S,22R)-3b-
[(b-D-glucopyranosyl)oxy]-1a-hydroxywitha-5,24(25)-dienolide (6) (Nakano et al. 2013),
(20S,22R)-3b-[(O-b-D-glucopyranosyl-(1!6)-b-D-glucopyranosyl)oxy]-1a-hydroxywitha-5,
24(25)-dienolide (7) (Matsuda et al. 2001), (20S,22R)-3b-[(O-b-D-glucopyranosyl-(1!6)-
b-D-glucopyranosyl)oxy]-1a,20-dihydroxywitha-5,24(25)-dienolide (8) (Matsuda et al.
2001),
(20S,22R)-3b-[(O-b-D-glucopyranosyl-(1!6)-b-D-glucopyranosyl)oxy]-1a,27-
dihydroxywitha-5,24(25)-dienolide (9) (Matsuda et al. 2001), (20S,22R)-3b-[(O-b-D-gluco-
pyranosyl-(1!6)-b-D-glucopyranosyl)oxy]-1a,27-dihydroxywitha-5,24(25)-dienolide (10)
(Zhao et al. 2002), (20S,22R)-3b-[(O-b-D-glucopyranosyl-(1!6)-b-D-glucopyranosyl)oxy]-
27-[(O-b-D-glucopyranosyl-(1!6)-b-D-glucopyranosyl)oxy]-1a-hydroxy-5,24(25)-dienolide
(11) (Zhao et al. 2002), and (20S,22R)-5b,6b-epoxy-4b,27-dihydroxy-1-oxowitha-2,24(25)-
dienolide (12) (Fuska et al. 1982) (Figure 1).
Compound 1 was obtained as an amorphous powder and its molecular formula
was identified as C52H82O25 based on high-resolution electrospray ionization time-of-
flight mass spectroscopy (HRESITOFMS; 1129.5044 [M þ Na]þ, calculated for
1
C52H82O25Na: 1129.5043) and 13C-NMR spectroscopy. The H- and 13C-NMR spectra of
1
1 were similar to those of 11, showing the following H- and 13C-NMR features: Four
methyl groups [dH 2.26 (3H, s, Me-28), 0.96 (3H, s, Me-19), 0.92 (3H, d, J ¼ 6.4 Hz, Me-
21), and 0.55 (3H, s, Me-18); dC 20.9 (C-28), 19.4 (C-19), 13.4 (C-21), and 11.6 (C-18)], an
a,b-unsaturated d-lactone group [dH 4.46 (1H, m, H-22), 2.43 (1H, dd, J ¼ 17.1, 14.0 Hz,
H-23a), and 2.07 (1H, dd, J ¼ 17.1, 8.1 Hz, H-23b); dC 166.1 (C-26), 158.1 (C-24), 123.2
(C-25), 78.3 (C-22), and 30.0 (C-23)], an olefinic group [dH 5.55 (br s, H-6); dC 139.2 (C-5)
and 123.9 (C-6)], two oxygenated methine groups [dH 4.75 (1H, m, W1/2 ¼ 21.8 Hz, H-3)
and 4.05 (1H, br s, H-1); dC 74.3 (C-3) and 72.3 (C-1)], an oxygenated methylene group
[dH 5.02 (d, J ¼ 11.4 Hz, H-27a) and 4.82 (d, J ¼ 11.4 Hz, H-27b); dC 63.3 (C-27)], two qua-
ternary carbons [dC 42.7 (C-13) and 41.9 (C-10)], and four anomeric protons and

NATURAL PRODUCT RESEARCH
3
Figure 1. Structures of 1–12.
carbons [dH 5.34 (d, J ¼ 7.7 Hz), 5.13 (d, J ¼ 7.7 Hz), 5.03 (d, J ¼ 7.9 Hz), and 4.92 (d,
J ¼ 7.7 Hz); dC 105.6, 105.3, 103.1, and 102.4]. Enzymatic hydrolysis of 1 with naringi-
nase gave 5 and D-glucose. A comparison of the 1H- and 13C-NMR spectra of the
sugar moieties in 1 with those of 11 suggested that 1 was a bisdesmoside of 5, which
differed from 11 in the sugar chain sequence of the diglycoside attached to C-26 in
the aglycone. The 1H-1H correlation spectroscopy (COSY), total correlation spectros-
copy (TOCSY), heteronuclear single quantum coherence (HSQC), and HSQC-TOCSY
1
data obtained for 1 allowed the sequential assignment of the H- and 13C-NMR signals
observed for each sugar unit, indicating that the sugar moieties in 1 consist of two
terminal b-D-glucopyranosyl units [Glc00: dH 5.13 (d, J ¼ 7.7 Hz, H-100); dC 105.3, 75.2,
78.3, 71.4, 78.2, and 62.4 (C-100–600); Glc0000: dH 5.34 (d, J ¼ 7.7 Hz, H-10000); dC 105.6, 76.3,
78.1, 71.3, 78.2, and 62.5 (C-10000–60000)], a 2-monosubstituted inner b-D-glucopyranosyl
unit [Glc000: dH 5.03 (d, J ¼ 7.9 Hz, H-1000); dC 102.4, 83.3, 78.4, 71.0, 78.1, and 62.3
(C-1000–C-6000)], and a 6-monosubstituted inner b-D-glucopyranosyl unit [Glc0: dH 4.92 (d,
J ¼ 7.7 Hz, H-10); dC 103.1, 75.0, 78.4, 71.3, 76.9, and 69.6 (C-10–C-60)]. The heteronuclear
multiple bond correlation (HMBC) spectrum of 1 exhibited long-range correlations
between H-100 of Glc00 (dH 5.13) and C-60 of Glc0 (dC 69.6), H-10 of Glc0 (dH 4.92) and C-3
of the aglycone (dC 74.3), H-10000 of Glc0000 (dH 5.34) and C-2000 of Glc000 (dC 83.3), and
between H-1000 of Glc000 (dH 5.03) and C-27 of the aglycone (dC 63.3). Thus, 1 was

4
T. IGUCHI ET AL.
established to be (20S,22R)-3b-[(O-b-D-glucopyranosyl-(1!6)-b-D-glucopyranosyl)oxy]-27-
[(O-b-D-glucopyranosyl-(1!2)-b-D-glucopyranosyl)oxy]-1a-hydroxywitha-5,24(25)-dienolide.
1
Compound 2 (C40H66O14) was obtained as an amorphous powder. The H-NMR spec-
trum of 2 exhibited signals for five typical steroidal methyl protons [dH 2.09 (3H, s, Me-27),
2.00 (3H, s, Me-28), 1.20 (3H, d, J ¼ 6.8Hz, Me-21), 1.14 (3H, s, Me-19), and 0.71 (3H, s, Me-
18)], an olefinic proton [dH 5.65 (1H, br d, J ¼ 4.4 Hz, H-6)], and two anomeric protons [dH
5.13 (1H, d, J ¼ 7.8 Hz) and 4.86 (1H, d, J ¼ 7.7 Hz)]. The 13C-NMR spectrum showed signals
for five steroid methyl carbons [dC 19.9 (C-19), 19.0 (C-28), 17.5 (C-27), 13.0 (C-21), and 12.0
(C-18)], two pairs of olefinic carbons [dC 140.2 (C-5), and 123.8 (C-6); 133.2 (C-25) and 127.3
(C-24)], and two anomeric carbons [dC 105.5 and 103.1]. The spectral features of 2 are
closely related to those observed for (22R,24Z)-1a,3b,22,26-tetrahydroxyergosta-5,24(25)-
diene 26-O-b-glucopyranoside (cilistol v), which was isolated from Solanum cilistum (Zhu
et al. 2001). However, the molecular formula of 2 was larger than that of cilistol v by
C6H10O5, which corresponds to a hexosyl unit. Enzymatic hydrolysis of 2 with naringinase
yielded an aglycone (2a) whose 1H- and 13C-NMR spectra were in agreement with those of
the aglycone of cilistol v and D-glucose. The above-mentioned data implied 2 was a
1
derivative of cilistol v bearing one more D-glucopyranosyl moiety. Analysis of the H-1H
COSY and HSQC spectra of the sugar moieties in 2 revealed that it was composed of a 6-
monosubstituted inner b-D-glucopyranosyl unit (Glc0) and a terminal b-D-glucopyranosyl
unit (Glc00). In the HMBC spectrum of 2, long-range correlations were observed between
H-100 of Glc00 (dH 4.86) and C-60 of Glc0 (dC 70.1), and between H-10 of Glc0 (dH 5.13) and C-26
of the aglycone (dC 70.2). Therefore, 2 was deduced to be (22R,24Z)-1a,3b,22-trihydroxyer-
gosta-5,24(25)-dien-26-yl O-b-D-glucopyranosyl-(1!6)-b-D-glucopyranoside.
Compound 3 was obtained as an amorphous powder. The HRESITOFMS and 13C-NMR
data allowed the molecular formula of 3 to be assigned as C52H86O24, which was larger
than that of 2 by C12H20O10, indicating the presence of two additional hexosyl groups.
1
The H- and 13C-NMR spectra of 3 exhibited four anomeric protons and carbons at dH
5.11 (d, J ¼ 7.8 Hz, H-100 of Glc00)/dC 105.1, 5.10 (d, J ¼ 7.8 Hz, H-10000 of Glc0000)/dC 105.2,
4.91 (d, J ¼ 7.7 Hz, H-10 of Glc0)/dC 103.0, and 4.85 (d, J ¼ 7.8Hz, H-1000 of Glc000)/dC 103.7.
1
Enzymatic hydrolysis of 3 was carried out to give 2a and D-glucose. When the H- and
13C-NMR spectra of 3 were compared with those of 2, the 13C-NMR signal assigned
to C-3 was shifted downfield by 8.1 ppm, whereas those corresponding to C-2 and
C-4 moved upfield by 1.4 and 5.7 ppm, respectively, which implied that both the C-3
and C-26 hydroxy groups were glycosylated. The 1H-1H COSY, TOCSY, HSQC, and
HSQC-TOCSY spectra obtained for 3 indicate the presence of two 6-monosubstituted
b-D-glucopyranosyl units (Glc0 and Glc000) and two terminal b-D-glucopyranosyl units
(Glc00 and Glc0000). HMBC correlations were observed from H-100 of Glc00 [dH 5.11
(d, J ¼ 7.8 Hz)] to C-60 of Glc0 (dC 69.5), H-10 of Glc0 [dH 4.91 (d, J ¼ 7.7Hz)] to C-3 of the
aglycone (dC 74.2), H-10000 of Glc0000 [dH 5.10 (d, J ¼ 7.8 Hz)] to C-6000 of Glc000 (dC 69.8), and
H-1000 of Glc000 [dH 4.85 (d, J ¼ 7.8Hz)] to C-26 of the aglycone (dC 70.0) in 3. Thus, 3 was
assigned as (22R,24Z)-26-[(b-D-glucopyranosyl-(1!6)-b-D-glucopyranosyl)oxy]-1a,22-dihy-
droxyergosta-5,24(25)-dien-3b-yl b-D-glucopyranosyl-(1!6)-b-D-glucopyranoside.
Compound 4 was obtained as an amorphous powder. The HRESITOFMS and 13C-NMR
1
data showed 4 had a molecular formula of C45H76O19. In the H- and 13C-NMR spectra
recorded for 4, signals for four characteristic steroidal methyl groups [dH 1.27 (3H, d,

NATURAL PRODUCT RESEARCH
5
J ¼ 7.0Hz, Me-21), 0.99 (3H, d, J ¼ 6.6 Hz, Me-27), 0.82 (3H, s, Me-18), and 0.62 (3H, s,
Me-19); dC 17.2 (C-27), 16.5 (C-18), 16.2 (C-21), and 12.1 (C-19)] and three anomeric pro-
tons and carbons [dH 5.23 (1H, d, J ¼ 7.9 Hz), 4.89 (1H, d, J ¼ 7.4 Hz), and 4.78 (1H, d,
J ¼ 7.8 Hz); dC 106.4, 104.7, and 102.3] were observed. In addition, the hemiacetal carbon
signal observed at dC 110.5 and a positive color reaction in Ehrlich’s test implied that 4
was a furostanol glycoside. Compound 4 was treated with b-D-glucosidase, which gave
the corresponding spirostanol glycoside (4a) (Mahato et al. 1981) and D-glucose. On the
other hand, acid hydrolysis of 4 with 1 M HCl gave (25S)-5a-spirostan-3b-ol (4 b)
1
(Sashida et al. 1992), D-galactose, and D-glucose. The NMR data, including H-1H COSY,
TOCSY, HSQC, and HSQC-TOCSY spectra, indicated that the sugar moieties in 4 consist
of two terminal b-D-glucopyranosyl units [Glc0: dH 5.23 (d, J ¼ 7.9 Hz, H-10); dC 106.4,
75.7, 78.2, 71.8, 78.2, and 62.6 (C-10–60) and Glc00: dH 4.78 (d, J ¼ 7.8Hz, H-100); dC 104.7,
74.9, 78.1, 71.3, 78.2, and 62.3 (C-100–600)] and a 4-monosubstituted b-D-galactopyranosyl
unit [Gal: dH 4.89 (d, J ¼ 7.4 Hz, H-1000); dC 102.3, 73.1, 74.9, 79.6, 75.2, and 60.9 (C-
1000–6000)]. In the HMBC spectrum of 4, long range correlations were observed from H-10
of Glc0 (dH 5.23) to C-4000 of Gal (dC 79.6), H-1000 of Gal (dH 4.89) to C-3 of the aglycone
(dC 77.2), and H-100 of Glc00 (dH 4.78) to C-26 of the aglycone (dC 75.2). The configuration
of the C-22 hydroxy group in 4 was determined to be 22a based on the NOE correl-
ation observed between the signals corresponding to H-20 (dH 2.18) and H2-23 (dH
2.06). Accordingly, 4 was determined to be (25S)-26-[(b-D-glucopyranosyl)oxy]-22a-
hydroxy-5a-furostan-3b-yl O-b-D-glucopyranosyl-(1!4)-b-D-galactopyranoside.
The cytotoxic activities of the isolated compounds were evaluated against Ca9-22,
HSC-2, and HL-60 cells. As expected from the previously reported data, 12 (withaferin
A) showed considerable cytotoxic activity against Ca9-22, HSC-2, and HL-60 cells when
compared with the positive controls, etoposide and doxorubicin, with IC50 values of
0.38, 0.54, and 1.5 lM, respectively. Ca9-22 and HSC-2 oral cancer cells were found to
be more sensitive to 12 than HL-60 leukemia cells. The other compounds did not
show any cytotoxic activity (IC50 >20 lM).
3. Experimental
See supplementary material.k
4. Conclusions
A phytochemical investigation of the seeds of W. somnifera gave 12 steroidal deriva-
tives (1–12), including four new steroidal glycosides (1–4). The structures of the new
compounds were determined using extensive spectroscopic analysis, including two-
dimensional NMR spectroscopy and hydrolysis. The cytotoxic activities of the isolated
compounds against Ca9-22, HSC-2, and HL-60 cells were evaluated. Only 12 showed
cytotoxic activity against Ca9-22, HSC-2, and HL-60 cells with IC50 values of 0.38, 0.54,
and 1.5 lM, respectively.

6
T. IGUCHI ET AL.
Acknowledgements
We thank Ms. Chiseko Sakuma, Tokyo University of Pharmacy and Life Sciences, for acquiring
NMR spectra.
We thank Mr. Haruhiko Fukaya, Tokyo University of Pharmacy and Life Sciences, for acquiring
mass spectra.
Disclosure statement
The authors declare no conflict of interest.
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