Isolation and characterization of triterpenoid saponins from leaves of Aralia nudicaulis L

Article abstract of DOI:10.1016/j.phytol.2021.04.004

Three new oleanolic glycosides (1–3), along with seven known saponins from various plants (4–10) were isolated for the first time from the leaves of Aralia nudicaulis. Their structures were elucidated on the basis of spectroscopic evidence, including 1D and 2D NMR, and HRESIMS. Nudicauloside A and B (1–2) have shown moderate anti-inflammatory activity, as demonstrated by inhibition of LPS-induced NO production in raw 264.7 murine macrophages (IC₅₀ = 74–101 μM).

Full text of DOI:10.1016/j.phytol.2021.04.004

Phytochemistry Letters 43 (2021) 184–189
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Isolation and characterization of triterpenoid saponins from leaves of Aralia
nudicaulis L
Serge Lavoie*, Julie Pierra, Jean Legault, Diamondra Raminoson, Quentin Lion,
´
Vakhtang Mshvildadze, Andre Pichette
´
´
´ ´
´
´
Centre de recherche sur la borealie (CREB), Laboratoire d’analyse et de separation des essences vegetales (LASEVE), Departement des Sciences Fondamentales, Universite
´
`
´
´
du Quebec a Chicoutimi, 555 boulevard de l’Universite, Chicoutimi, Quebec, G7H 2B1, Canada
A R T I C L E I N F O
A B S T R A C T
Keywords:
Three new oleanolic glycosides (1–3), along with seven known saponins from various plants (4–10) were isolated
for the first time from the leaves of Aralia nudicaulis. Their structures were elucidated on the basis of spectro-
scopic evidence, including 1D and 2D NMR, and HRESIMS. Nudicauloside A and B (1–2) have shown moderate
anti-inflammatory activity, as demonstrated by inhibition of LPS-induced NO production in raw 264.7 murine
Aralia nudicaulis
Saponins
NMR spectroscopy
Arabinose
macrophages (IC50 = 74–101 μM).
Pharmacological activities
Anti-inflammatory
Nudicauloside
1. Introduction
Furthermore, their anticancer, antimicrobial, antioxidant, and
anti-inflammatory activities were evaluated in vitro.
Saponins are ubiquitous in plants. Some families have been studied
for centuries for their rich saponin composition, like Caryophyllaceae,
Leguminosae and Araliaceae (Sparg et al., 2004). The latter comprise
species of the genus Panax (ginseng) which were shown to contain
several bioactive saponins (Jia and Zhao, 2009). Several species of the
genus Aralia, especially A. elata (Table 1) which is an adaptogen (Pan-
ossian et al., 2021), also contain bioactive saponins explaining their
broad usage as an herbal remedy in Russia, Korea, Japan and China
2. Results and discussion
2.1. Chemical investigation of the A. nudicaulis leaves extract
ThemethanolicextractfromleavesofAralianudicauliswassubjectedtoa
number of MS-guided separation steps using Diaion HP-20, silica gel and
reversed phase C18. The final purification of the saponin compounds was
realized using semi-preparative HPLC, yielding three new (1–3) and seven
known compounds (4–10) identified for the first time on this specie (Fig. 1).
By comparison of NMR spectroscopic data with literature values, the known
compounds were identified as hemsgiganoside B (4) (Chen et al., 2003),
guaiacin B (5) (Shaker et al., 1999), araliasaponin I (6) (Song et al., 2000),
3-O-[β-D-glucopyranosyl-(1→2)]-[β-D-glucopyranosyl-(1→3)]--
´
(Shikov et al., 2016). In the province of Quebec in Canada, three species
of Aralia are indigenous: A. racemosa, A. hispida and A. nudicaulis. The
´
latter was used by aboriginal peoples of Quebec as root infusion or
decoction to treat several health problems such as stomach pain, kidney
disorder, cold, and cough (Moerman, 1998). More recently, poly-
acetylenes with bioactivity against Mycobacterium tuberculosis were
isolated from the roots of A. nudicaulis (Li et al., 2012). However, the
saponin content of A. nudicaulis was never reported.
α
-L-arabinopyranosyl hederagenin (7) (also known as prosapogenin) (Chen
et al., 1997), araliasaponin XII (8) (Ahmad et al., 1990), araliasaponin IX (9)
(Song et al., 2001), and araliasaponin III (10) (Song et al., 2000).
In the last 15 years, we dedicated some of our research effort toward
the synthesis of glycosides with molecular scaffold obtained from plant
material (Gauthier et al., 2009; Piochon et al., 2009). For this reason, we
have a strong interest for plants containing glycosides. In this study, we
report the isolation and structural elucidation of three new saponins,
nudicauloside A–C (1–3), from an Aralia nudicaulis leaf extract.
2.2. Compound 1
Compound 1 was isolated as a white amorphous powder with a
molecular formula determined as C53H86O22 based on a HRESIMS
* Corresponding author.
E-mail address: serge_lavoie@uqac.ca (S. Lavoie).
https://doi.org/10.1016/j.phytol.2021.04.004
Received 6 November 2020; Received in revised form 2 April 2021; Accepted 7 April 2021
Available online 21 April 2021
1874-3900/Crown Copyright © 2021 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe. All rights reserved.

S. Lavoie et al.
Phytochemistry Letters 43 (2021) 184–189
sodiated adduct at m/z 1097.5503. The 1H NMR spectrum shows typical
resonances for saponins with four anomeric protons at δ 5.38 (d, J
=8.1 Hz, H-1IV), 4.62 (d, J =7.7 Hz, H-1II), 4.57 (d, J =7.H8 Hz, H-1III)
and 4.28 (d, J =7.3 Hz, H-1I), one olefinic proton at δ 5.25 (t, J
=3.7 Hz, H-12), and seven methyl groups at δH 1.16 (s, H3-H27), 1.05 (s,
H3-23), 0.95 (s, H3-25), 0.93 (s, H3-30), 0.91 (s, H3-29), 0.84 (s, H3-24),
0.80 (s, H3-26) (Table 2). The 13C NMR spectrum indicated one carbonyl
123.8 (C-12). After careful examination of COSY, HSCQC and HMBC 2D
NMR spectra, the aglycone of 1 could be identified as an oleanolic acid,
typical of most saponins isolated from Aralia (Ma et al., 2013; Zhang
et al., 2013). The remaining 23 carbon signals accounted for three
hexoses and one pentose. The 1H and 13C chemical shifts were assigned
for each sugar using HSQC, COSY, and a series of 1D TOCSY spectra
obtained by selectively exciting the anomeric protons and using an
increasing mixing time (Table 3). With the help of literature data
(Agrawal, 1992), and considering that all anomeric signals were dou-
blets with J values > 7.5 Hz, three β-glucopyranoside and one
Table 1
Species of Aralia known for their saponin content.
Species
Part
Reference
A. elata
leaf
Zhang et al. (2013)
Song et al. (2001), Lee et al. (2005)
Nhiem et al. (2011)
Kawai et al. (1989), Ik et al. (2006)
Yu et al. (1994)
root bark
bark
A. cordata
aerial
root
carbon at δ 178.0 (C-28) and a pair of sp2 carbon at δ 144.9 (C-13) and
A. spinifolia
A. armata
C
root bark
aerial
root bark
root
Hu et al. (1995)
A. continentalis
A. taibaiensis
A. subcapitata
A. dasyphylla
A. decaisneana
Kim and Kang (1998)
Bi et al. (2012)
Zou et al. (2001)
root bark
root
Xiao et al. (1999)
Miyase et al. (1996a)
Tian et al. (2006)
bark
A. echinocaulis
A. chinensis
root
Li et al. (2016)
root
Miyase et al. (1996b)
Table 2
1H and 13C NMR spectroscopic data for the aglycone part of 1–3.a
1b
13C
2c
13C
3c
13C
position
1H
1H
1H
1.62 m
0.98 m
1.83 m
1.70 m
3.15 dd
(11.7, 4.3)
–
38.8 t
1.47 m
0.88 m
2.04 m
1.80 m
39.2 t
1.52 m
1.01 m
2.13 m
1.94 m
1
2
39.9 t
27.1 t
26.6 t
26.4 t
3
4
5
90.5 d
40.2 s
57.1 d
89.0 d
39.7 s
55.9 d
18.5 t
3.23 m
82.7 d
43.9 s
48.2 d
18.5 t
4.16 m
–
0.74
0.78 m
1.60 m
d (11.4)
1.48 m
1.31 m
1.43 m
1.32 m
–
1.54 m
1.41 m
1.48 m
1.32 m
–
1.58 m
–
1.89 m
5.25 t
(3.7)
1.68 m
1.36 m
1.57 m
1.30 m
6
19.3 t
33.2 t
33.2 t
7
34.0 t
8
40.7 s
49.0 d
37.9 s
24.6 t
123.8
d
39.9 s
48.1 d
37.0 s
23.8 t
122.9
d
40.3 s
48.5 d
37.2 s
24.2 t
123.3
d
9
1.61 m
–
1.88 m
5.44 t
(3.5)
1.73 m
10
11
1.91 m
12
5.42 br s
144.9
s
144.1
s
144.4
s
13
14
15
–
–
42.9 s
–
42.1 s
28.3 t
–
42.5 s
28.6 t
1.80 m
1.08 m
2.05 m
1.72 m
–
2.35 m
1.18 m
2.08 m
1.97 m
–
2.33 m
1.10 m
2.04 m
1.93 m
28.9 t
23.4 t
23.7 t
16
17
18
24.0 t
48.0 s
42.6 d
47.0 s
41.8 d
46.2 t
47.3 s
42.1 d
46.5 t
2.85 dd
(13.6, 4.8)
1.70 m
1.15 m
–
1.40 m
1.22 m
1.74 m
1.61 m
3.19 dd
(13.9, 4.9)
1.73 m
3.21 m
1.77 m
1.26 m
–
1.36 m
1.10 m
1.83 m
1.76 m
19
20
21
47.2 t
31.5 s
34.9 t
1.24 m
30.8 s
34.0 t
31.1 s
34.3 t
1.33 m
1.08 m
1.82 m
1.74 m
4.29 m
3.70
32.6 t
32.9 t
22
23
33.1 t
28.5 q
1.05 s
28.1 q
1.25 s
65.0 t
d (10.5)
1.06 s
0.93 s
1.12 s
1.18 s
24
25
26
27
17.0 q
16.0 q
17.7 q
26.3 q
178.0
s
0.84 s
0.95 s
0.80 s
1.16 s
16.7 q
15.6 q
17.5 q
26.1 q
176.4
s
1.08 s
0.83 s
1.10 s
1.25 s
13.7 q
16.5 q
17.9 q
26.4 q
176.8
s
28
–
–
29
30
33.5 q
24.0 q
0.91 s
0.93 s
33.2 q
23.7 q
0.92 s
0.89 s
33.5 q
24.0 q
0.90 s
0.88 s
a
Fig. 1. Structure of compounds1–10 from Aralia nudicaulis.
Measured at 400 MHz for 1–2, or 800 MHz for 3.
Recorded in CD3OD.
b
c
Recorded in pyridine-d5.
185

S. Lavoie et al.
Phytochemistry Letters 43 (2021) 184–189
α
-arabinopyranoside could be identified. To further confirm this
=7.8 Hz,H-1III),alongwithanolefinicprotonatδH 5.44(t,J=3.5 Hz,H-1).
conclusion, an acid hydrolysis of 1 was performed and the sugars were
acetylated and analyzed by chiral GC-FID. The retention time of the
sugars matched those for D-glucose and L-arabinose. Finally, the linkage
was elucidated with HMBC correlations from H-1I to δC 90.5 (C-3), from
H-1II to δ 83.9 (C-3I), from H-1III to δ 87.4 (C-3II), and from H-1IV to δ
The HSQC spectrum showed a correlation atδ 4.77 (m, H-1I) andδ 105.8
H
C
which was assigned to the anomeric proton of an additional sugar moiety.
Analysis of the 2D NMR spectra revealed that the aglycone of 2 was also an
oleanolic acid. Literature data (Agrawal et al., 1995) and acid hydrolysis
allowed the identification of four D-glucopyranosyl and one L-arabinopyr-
anoside moieties with linkage established from HMBC correlations be-
tween H-1I and δ 89.3 (C-3), H-1II and δ 83.6 (C-3I), H-1III and δ 88.3
178.0 (C-28). From all the above eviCdence, compound 1, named nudi-
C
C
cauloside
A,
was
identified
as
β-D-glucopyranosyl
3β
C
C
-O-[β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→3)
-L-arabinopyranosyl]-olean-12-en-28-oate.
(C-3II), H-1IV andCδC 176.8 (C-28), and H-1V and δC 77.6 (C-2I). Therefore,
compound 2, named nudicauloside B, was identified as
3β-O-[β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→3)
-
α
-[β-D-glucopyranosyl-(1→2)]-
α-L-arabinopyranosyl]-olean-12-en-28-oate.
2.3. Compound 2
2.4. Compound 3
Compound 2, obtained as a white amorphous powder, exhibited a
pseudo-molecular sodium adduct in its HRESIMS spectrum at m/z
1275.5978, indicative of the molecular formula C59H96O27. The 1H NMR
spectrarevealedonlyfouranomericprotonsignalsatδH 6.37(d, J=8.0 Hz,
H-1IV), 5.50 (d, J =7.7 Hz, H-1V), 5.32 (d, J =7.8 Hz, H-1II) and 5.20 (d, J
Compound 3 was obtained as a white amorphous solid, and its mo-
lecular formula was established as C59H96O28 based on a pseudo-
molecular sodiated ion in its HRESIMS spectrum at m/z 1275.5980. A
close inspection of the 2D NMR spectra revealed the same sugar pattern
as for 2, with almost the same chemical shifts for 1H and 13C. This
assertion was further confirmed by acid hydrolysis of 3 yielding the
same saccharide species as for 2. The largest difference was observed for
the anomeric proton of arabinose, which was suspected of being linked
to a different aglycone. The latter was identified as hederagenin, based
on the following evidence: an additional primary alcohol at δH 4.29 (m,
H2-23) and 3.70 (d, J =10.5 Hz, H2-23), together with the methyl at 1.06
(s, H3-24), showed HMBC correlations to δ 82.5 (C-3), 43.3 (C-4) and
48.2 (C-5). Additionally, NOESY crosspeakCs between H2-23, H-6eq and
H-5, H-3 and H-5, H-5 and H-9, and H-9 and H3-27 allowed to assign all
Table 3
1H and 13C NMR spectroscopic data for the sugar moieties of 1–3.a
position
1b
2c
3c
13C
1H
13C
1H
13C
1H
3-O-
α
-L-Arap
1I
107.1
d
4.28
105.4
d
4.77 m
104.7
d
5.00
d (7.3)
3.71 m
3.66 m
4.03 m
3.86 m
3.55 m
d (6.8)
4.70 t (7.8)
4.15 m
4.41 m
4.11 m
3.52
2I
3I
4I
5I
72.1 d
83.9 d
69.6 d
66.7 t
77.3 d
83.3 d
68.8 d
66.0 t
4.74 m
4.32 m
4.48 m
4.16 m
3.65 m
77.7 d
83.8 d
69.0 d
66.2 t
these protons on the
α face of the aglycone, while the protons of the β
face were located from correlations between H3-24 and H3-25, H3-25
and H3-26, and H-18 and H3-30. From all the above evidence, nudi-
cauloside C (3) was identified as 3β-O-[β-D-glucopyranosyl-(1→3)-β-D-
d (11.2)
β-D-Glc at O-3 of
α
-L-Arap
1II
105.0
4.62
104.6
d
5.32
104.8
d
5.24
glucopyranosyl-(1→3)-[β-D-glucopyranosyl-(1→2)]-α-L-arabinopyr-
d
d (7.7)
3.51 m
3.59 m
3.47 m
3.32 m
3.85 m
3.69 m
d (7.8)
4.01 m
4.22 m
4.11 m
3.90 m
4.41 m
4.24 m
d (7.7)
4.01 m
4.25 m
4.11 m
3.90 m
4.41 m
4.25 m
2II
3II
4II
5II
6II
74.8 d
87.4 d
69.7 d
78.2 d
62.3 t
73.9 d
87.9 d
69.7 d
78.1 d
62.2 t
74.3 d
88.2 d
70.0 d
78.4 d
62.5 t
anosyl]-23-hydroxyolean-12-en-28-oate.
2.5. Evaluation of pharmacological activities
Nudicauloside A–C (1–3) were subjected to several biological assays
to evaluate their pharmaceutical and cosmeceutical potential (Table 4).
At first, we have evaluated the biological activities of isolated com-
pounds (1–10) including cytotoxicity against human cancer cell lines
A549, DLD-1 and healthy human skin fibroblasts (WS1); antimicrobial
activities against Escherichia coli, Staphylococcus aureus, and Candida
albicans. The results show that all compounds tested are inactive at the
β-D-Glc at O-3 of β-D-Glc
1III
105.3
d
4.57
105.5
d
5.20
105.9
d
5.21
d (7.8)
3.28 m
3.39 m
3.28 m
3.35 m
3.89 m
3.64 m
d (7.8)
4.05 m
4.19 m
4.19 m
3.96 m
4.53 m
4.31 m
d (7.9)
4.06 m
4.21 m
4.19 m
3.97 m
4.53
2III
3III
4III
5III
6III
75.6 d
77.8 d
71.6 d
78.7 d
62.7 t
75.4 d
78.1 d
71.5 d
78.6 d
62.4 t
75.7 d
78.4 d
71.8 d
78.9 d
62.8 t
highest concentration of 160
μM. Kuang et al. (2013) found cytotoxicity
d (10.3)
4.31 m
against A549 with analogs of 1–3 having a β-D-glucopyranosyl in place
of the
α-L-arabinopyranosyl at C-3, suggesting the arabinose moiety
28-O-β-D-Glc
1IV
95.7 d
5.38
95.8 d
6.37
96.1 d
6.34
would be detrimental for this activity. The anti-inflammatory activity of
Aralia extracts was already reported (Lee et al., 2009; Suh et al., 2007).
For this reason, the inhibition of LPS-induced NO production by 1–3 in
raw 264.7 murine macrophages was studied (Table 4). Moderate ac-
d (8.1)
3.32 m
3.41 m
3.37 m
3.33 m
3.82 m
3.68 m
d (8.0)
4.22 m
4.30 m
4.38 m
4.05 m
4.48 m
4.42 m
d (8.0)
4.21 m
4.30 m
4.38 m
4.04 m
4.48
2IV
3IV
4IV
5IV
6IV
73.9 d
78.3 d
71.1 d
77.6 d
62.4 t
74.2 d
78.9 d
71.1 d
79.4 d
62.2 t
74.5 d
79.3 d
71.4 d
79.7 d
62.5 t
tivities were obtained for 1 and 2 with IC50 values of 74 ± 28 μM and
101 ± 20 μM respectively.
d (10.2)
4.42 m
3. Experimental
β-D-Glc at O-2 of
α
-L-Arap
1V
104.3
d
5.50
104.6
d
5.48
d (7.7)
4.06 m
4.19 m
4.18 m
3.70 m
4.36 m
4.30 m
d (7.8)
4.08 m
4.18 m
4.18 m
3.67 m
4.36 m
4.29 m
3.1. General experimental procedures
2V
3V
4V
5V
6V
76.2 d
78.6 d
72.4 d
77.4 d
63.2 t
76.5 d
78.9 d
72.3 d
77.8 d
63.3 t
Styrene-divinylbenzene polymer (Diaion HP-20, Sigma-Aldrich) and
silica gel (Silicycle, 40–63 m) adsorbents were used for column chro-
μ
matography. Low-pressure liquid chromatography was achieved using a
Sepacore flash system (Buchi) equipped with two pumps, a UV detector
and a fraction collector. Since saponins rarely have chromophore, the
fraction collector was set in time mode. TLC analyses were performed on
precoated silica gel plates (Kieselgel 60F254, Merck) using a CHCl3/
a
Measured at 400 MHz for 1–2, or 800 MHz for 3.
Recorded in CD3OD.
b
c
Recorded in pyridine-d5.
186

S. Lavoie et al.
Phytochemistry Letters 43 (2021) 184–189
3.3. Extraction and isolation
Table 4
Pharmacological activities of 1–3.
Dried and powdered leaves from A. nudicaulis (1.1 kg) were refluxed
sequentially in DCM (3 × 5 L), and MeOH (3 × 5 L), each time for
90 min. The solutions were filtered and concentrated under vacuum
yielding two fractions (DCM, 41.4 g; MeOH, 168 g). An aliquot from the
MeOH extract (45.3 g) was suspended in 500 mL H2O and partitioned
using 3 L of EtOAc (A, 4.5 g), then 3 L of n-BuOH (B, 9.2 g). An aliquot of
the BuOH extract (8.5 g) was separated by CC using Diaion HP-20 and a
step gradient of H2O/MeOH (1:0 → 0:1) yielding five fractions (B1–B5).
Fraction B4 (3.0 g) was subjected to flash chromatography on silica gel
and eluting isocratically with CHCl3/MeOH/H2O 26:14:3 to afford five
fractions (B4.1–B4.5). Fraction B4.2 was separated on CC using silica gel
and CHCl3/MeOH/H2O 50:15:1 as eluent yielding nine fractions
(B4.2.1–B4.2.9). Fraction B4.2.4 was purified by HPLC with 40 % of
eluent B to afford pure 5 (21.7 mg). Compounds 6 (17.2 mg) and 7
(5.8 mg) were obtained after purification of fraction B4.2.5 by HPLC
using 40 % of eluent B. Fraction B4.2.6 was purified by HPLC using 40 %
of eluent B to afford compound 1 (39.3 mg). Fraction B4.2.7 was sub-
jected to HPLC purification with 40 % of eluent B yielding pure 10
(17.9 mg). Compound 8 (21.5 mg) was obtained from fraction B4.2.8
following HPLC purification with 40 % of eluent B. Fraction B4.4 was
separated by CC on silica gel using CHCl3/MeOH/H2O 48:20:3 as the
eluent yielding five fractions (B4.4.1–B4.4.5), along with pure 4
(158.2 mg) and 3 (69.9 mg). Fraction B4.4.4 was subjected to HPLC
separation with 36 % of eluent B to afford compounds 2 (25.7 mg) and 9
(21.7 mg). Fraction B4.5 was purified by HPLC using a gradient of 20 %
B → 37 %B in 30 min to afford more 4 (83.6 mg)
1
2
3
Positive
controla
Cytotoxicity IC50
A549
(μM)
> 160
> 160
> 160
> 160
> 160
> 160
> 160
> 160
> 160
13 ± 2
6.2 ± 0.8
6 ± 2
DLD-1
WS1
Antimicrobial IC50
(μM)
E. coli
> 160
> 160
> 160
N.D.
N.D.
N.D.
> 160
> 160
> 160
0.012 ± 0.001
0.023 ± 0.005
1.1 ± 0.1
S. aureus
C. albicans
Antioxidant
ORAC (
μ
mol TE/
N.D.
N.D.
0.35 ± 0.09
93 ± 22
> 130
24 ± 3
μ
mol)
Cell based
N.D.
N.D.
0.081 ± 0.005
antioxidant
Anti-inflammatory IC50
74 ± 28
101 ± 20
(μM)
a
Positive controls were etoposide for A549, DLD-1, and WS1; gentamycin for
E. coli and S. aureus, amphotericin B for C. albicans, L-NAME for anti-
inflammatory, and quercetin for antioxidant. N.D.: not determined.
MeOH/H2O (26:14:3) solvent systems. Spots were detected by spraying
the plates with 20 % H2SO4 in MeOH, followed by heating at 110 ◦C for
10 min. Saponins content was monitored by LC–MS using an 1100 LC
unit and a G1946 VL MS instrument (Agilent Technologies). A dual ESI
and APCI ionization source ran in negative mode was used. A Kinetex
XB-C18 HPLC column (Phenomenex, 250 × 4.6 mm, 5 μm particle size)
was used with 1 mL/min of H2O and MeCN, both acidified with 0.1 %
HCOOH. Semi-preparative HPLC purifications were performed on an
Agilent 1100 Series equipped with two pumps, an autosampler, a UV
detector, and a fraction collector. The column used for separations was a
3.3.1. Nudicauloside A (1)
White amorphous solid; [α D25
]
+25.7◦ (c 0.19, MeOH); IR (film) νmax
:
3307, 2942, 2831, 1449, 1416, 1113, 1021 cmꢀ 1; 1H and 13C NMR data,
see Tables 2 and 3; HRESIMS m/z 1097.5560 [M + Na]+ (calcd for
Kinetex XB-C18 (Phenomenex, 250 × 21.2 mm, 5 μm particle size).
53H86O22Na+ 1097.5503).
Solutions of HCOOH 0.1 % in H2O (solvent A) and HCOOH 0.1 % in
MeCN (solvent B) were used as eluents with a flow rate of 20 mL/min.
Optical rotations were measured in methanol at 589 nm using an
Autopol IV polarimeter (Rudolph Research Analytical). FTIR spectra
were recorded from a thin film of compounds deposited on a NaCl
windows, and using a Cary 630 instrument (Agilent Technologies).
Chiral separations of acetylated monosaccharides were performed on a
7890A GC-FID instrument (Agilent Technologies) using a Chiramix
C
3.3.2. Nudicauloside B (2)
White amorphous solid; [α D25
+16.4 (c 0.23, MeOH); IR (film) νmax
]
3354, 2941, 2878, 1745, 1160, 1073, 1028 cmꢀ 1; 1H and 13C NMR data,
see Tables 2 and 3; HRESIMS m/z 1259.6013 [M + Na]+ (calcd for
C
59H96O27Na+ 1259.6031).
column (GL Sciences, 30 m × 0.25 mm ×0.25 μm), and helium as the
3.3.3. Nudicauloside C (3)
carrier gas (flow 1 mL/min). The injector temperature was maintained
at 230 ◦C, while the GC oven was kept at 120 ◦C for 1 min and raised at a
rate of 4 ◦C/min up to 180 ◦C, which was held constant for 120 min.
NMR spectra (1H, 13C, DEPT-135, DQF-COSY, edited-HSQC, HMBC and
NOESY) were recorded on a 9.4 T instrument (400 MHz for 1H and
100 MHz for 13C), equipped with a QNP 5 mm probe (Bruker) or a
18.8 T instrument (800 MHz for 1H and 200 MHz for 13C), equipped
with a 5 mm triple resonance broadband cryoprobe (Bruker). Spectra
were acquired in pyridine-d5 or methanol-d4, and chemical shifts were
reported in ppm (δ) relative to the residual solvent peaks (Gottlieb et al.,
1997). All spectra were processed using MestReNova 12.0. High reso-
lution mass spectrometry was conducted on a 6210 Q-TOF spectrometer
(Agilent Technologies) equipped with an electrospray source operating
in the positive ion mode.
White amorphous solid; [α D25
]
+15.9◦ (c 0.41, MeOH); IR (film) νmax
3370, 2942, 2361, 1747, 1625, 1074, 1030 cmꢀ 1; 1H and 13C NMR data,
see Tables 2 and 3; HRESIMS m/z 1275.5978 [M + Na]+ (calcd for
C59H96O28Na+ 1275.5980).
3.4. Acid hydrolysis
Compounds 1–3 (3 mg of each) were refluxed in aqueous HCl (10 %
v/v, 10 mL) during 4 h. Aglycons were recovered with CHCl3 and the
aqueous layers were neutralized with N,N-dioctylmethylamine (10 % in
CHCl3). The sugar residues were acetylated with pyridine/acetic anhy-
dride (1:1, 2 mL) under agitation during 24 h. The monosaccharide
acetates were extracted with EtOAc (3 mL), and the organic fraction was
washed successively with sat. aqueous NaHCO3 (3 × 2 mL), H2SO4 20 %
(3 × 2 mL) and water (3 × 1 mL). The samples were subjected to chiral
GC-FID analysis and compared to retention times of monosaccharide
acetates synthesized from authentic samples.
3.2. Plant material
The leaves of Aralia nudicaulis L. were collected in June and July
◦
′
′′
◦
′
′′
`
´
2016, near Laterriere, Quebec, Canada (48 23 07 N, 71 25 07 W), and
dried in the shade. The plant was authenticated by Mr Patrick Nadeau
3.5. Pharmacological assays
´
´
´
`
(Departement des sciences fondamentales, Universite du Quebec a
Chicoutimi). A voucher specimen (No. QFA0297432) has been depos-
3.5.1. Cell culture
´
ited at the Louis-Marie herbarium of Laval University (Quebec).
Human skin fibroblasts (WS1, ATCC CRL-1502), human lung carci-
noma (A549, ATCC CCL-185), human colon adenocarcinoma (DLD-1,
187

S. Lavoie et al.
Phytochemistry Letters 43 (2021) 184–189
ATCC CCL-221), and RAW 264.7 murine macrophage (ATCC TIB-71)
were cultured in Dulbecco’s minimum essential medium (DMEM) with
Earle’s salts and L-glutamine (Mediatech Cellgro), to which were added
vitamins, sodium pyruvate and non-essential amino acids (all at a 1:100
v/v dilution of supplied solutions), 10 % fetal bovine serum (Hyclone),
penicillin (100 IU/mL) and streptomycin (100 mg/mL). Cells were
maintained at 37 ◦C in a humidified environment containing 5 % CO2.
4. Conclusion
This research has allowed the identification of ten saponins,
including three new ones. Their chemical structures have been estab-
lished from 1D and 2D NMR spectroscopy, HRESIMS, and chemical
degradation. The isolated compounds were not found active in cyto-
toxicity and antimicrobial assays, but two of them (1 and 2) showed
moderate anti-inflammatory activities, as demonstrated by inhibition of
LPS-induced NO production in raw 264.7 murine macrophages.
3.5.2. Cytotoxic assay
Exponentially growing cells (WS1, A549 and DLD-1) were trans-
ferred in 96-well microplates (Costar, Corning Inc.) at a density of
Authors’ contributions
5 × 103 cells/well in 100
μL of culture medium and were allowed to
adhere for 16 h before treatment. Increasing concentrations of each
compound in DMSO were diluted with culture medium and added to the
JP, DR and QL carried out this study. SL, VM, JL and AP designed the
experiments and supervised the work. SL, JP, JL and AP wrote the
manuscript. All authors read and approved the final manuscript.
wells (100 μL/well). The final concentration of DMSO in the culture
medium was maintained at 0.5 % to avoid solvent toxicity. Cytotoxicity
was assessed after incubation for 48 h and using resazurin (λex 530 nm,
λem 590 nm) (O’Brien et al., 2000) on an automated 96-well Fluoroskan
Ascent F1 plate reader (Labsystems). Fluorescence was proportional to
the cellular metabolic activity in each well. Survival percentages were
defined as the fluorescence in experimental wells compared to that in
control wells after subtraction of blank values. Cytotoxicity results were
expressed as means ± standard deviation and represent the concentra-
tion inhibiting 50 % of cell growth (IC50). The values were compared to
etoposide, used as a positive control.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgments
The authors thank Mr. Patrick Nadeau for plant identification. Also,
Julia Kubanek and Les Gelbaum (Georgia Institute of Technology) are
kindly acknowledged for the high-field NMR spectroscopy experiments.
3.5.3. Anti-inflammatory activity
´
Finally, we thank Karl Girard-Lalancette and Audrey Belanger for the
Exponentially growing RAW 264.7 murine macrophages were
pharmacological assays.
transferred in 96-well microplates (Falcon, BD) at a density of 7.5 × 104
cells/well in 100 μL of culture medium and were allowed to adhere
Appendix A. Supplementary data
overnight. Cells were then incubated for 24 h with either culture me-
dium (blank), L-NAME (positive control, 250 M) or increasing con-
μ
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.phytol.2021.04.004.
centrations of 1–3 dissolved in DMSO. The final concentration of solvent
in the culture medium was maintained at 0.25 %. Cells were then
stimulated with 100 ng/mL LPS and incubated for 24 h. Cell-free su-
pernatants were collected, and the NO concentration was immediately
determined using the Griess reaction (Green et al., 1990) with minor
modifications (Legault et al., 2010).
References
Agrawal, P.K., 1992. NMR Spectroscopy in the structural elucidation of oligosaccharides
and glycosides. Phytochemistry 31, 3307–3330.
Agrawal, P.K., Jain, D.C., Pathak, A.K., 1995. NMR Spectroscopy of steroidal sapogenins
and steroidal saponins: an update. Magn. Reson. Chem. 33, 923–953.
Ahmad, V.U., Perveen, S., Bano, S., 1990. Saponins from the leaves of Guaiacum
officinale. Phytochemistry 29, 3287–3290.
3.5.4. Antioxidant activity
The procedure was modified from the method described by Ou et al.
(2001). Briefly, the ORAC assay was carried out with a Fluoroskan
Ascent Fl™ plate reader (Labsystems). Trolox was used as a control
standard. The experiment was conducted at 37.5 ◦C and pH 7.4, with a
blank sample in parallel. The fluorimeter was programed to record the
fluorescence of fluorescein every 1 min after addition of 2,2′-azobis
(2-amidinopropane) dihydrochloride (AAPH). The final results were
calculated by comparing the net areas under the fluorescein decay
curves between the blank and the samples. ORAC values were expressed
Banfi, E., Scialino, G., Monti-Bragadin, C., 2003. Development of a microdilution method
to evaluate Mycobacterium tuberculosis drug susceptibility. J. Antimicrob. Chemother.
52, 796–800.
Bi, L., Tian, X., Dou, F., Hong, L., Tang, H., Wang, S., 2012. New antioxidant and
antiglycation active triterpenoid saponins from the root bark of Aralia taibaiensis.
Fitoterapia 83, 234–240.
Chen, M., Wu, W.W., Nanz, D., Sticher, O., 1997. Leonticins D-H, five triterpene saponins
from Leontice kiangnanensis. Phytochemistry 44, 497–504.
Chen, Y., Chiu, M.H., Gu, K., Li, Z.R., 2003. Cucurbitacin and triterpenoid glycosides
from Hemsleya giganthy. Chin. Chem. Lett. 14, 475–478.
in micromoles of Trolox equivalents (TE) per milligram (μmol TE/mg).
´
Cote, H., Boucher, M.A., Pichette, A., Legault, J., 2017. Anti-inflammatory, antioxidant,
antibiotic, and cytotoxic activities of Tanacetum vulgare L. essential oil and its
constituents. Medicines 4.
The cell-based assay was evaluated using the DCFH-DA, as described by
´
Girard-Lalancette (Cote et al., 2017; Girard-Lalancette et al., 2009).
Gauthier, C., Legault, J., Rondeau, S., Pichette, A., 2009. Synthesis of betulinic acid acyl
glucuronide for application in anticancer prodrug monotherapy. Tetrahedron Lett.
50, 988–991.
Human skin fibroblasts WS1 were incubated with compounds dissolved
in DMSO at different concentrations. Quercetin was used as a positive
control and expressed with the IC50 values.
Georges, P., Legault, J., Lavoie, S., Grenon, C., Pichette, A., 2012. Diterpenoids from the
buds of Pinus banksiana lamb. Molecules 17, 9716–9727.
Girard-Lalancette, K., Pichette, A., Legault, J., 2009. Sensitive cell-based assay using
DCFH oxidation for the determination of pro- and antioxidant properties of
compounds and mixtures: analysis of fruit and vegetable juices. Food Chem. 115,
720–726.
3.5.5. Antibacterial and antifungal activities
Antibacterial and antifungal tests were performed following the
same method described by Banfi with some modifications (Banfi et al.,
2003; Georges et al., 2012). The compounds were incubated with
different strains depending on the kind of test. Antibacterial activity was
measured comparing E. coli and S. aureus results with gentamycin as
reference. Instead of the antifungal activity, which one is evaluated on
C. albicans and compared to amphotericin B. Results are all expressed at
Gottlieb, H.E., Kotlyar, V., Nudelman, A., 1997. NMR chemical shifts of common
laboratory solvents as trace impurities. J. Org. Chem. 62, 7512–7515.
Green, S.J., Meltzer, M.S., Hibbs Jr, J.B., Nacy, C.A., 1990. Activated macrophages
destroy intracellular Leishmania major amastigotes by an L-arginine-dependent
killing mechanism. J. Immunol. 144, 278–283.
Hu, M., Ogawa, K., Sashida, Y., Xiao, P.-G., 1995. Triterpenoid glucuronide saponins
from root bark of Aralia armata. Phytochemistry 39, 179–184.
Ik, S.L., Jin, W., Zhang, X., Tran, M.H., Kyung, S.S., Yeon, H.S., Bae, K., 2006. Cytotoxic
and COX-2 inhibitory constituents from the aerial parts of Aralia cordata. Arch.
Pharmacal Res. 29, 548–555.
the IC50
.
188

S. Lavoie et al.
Phytochemistry Letters 43 (2021) 184–189
Jia, L., Zhao, Y., 2009. Current evaluation of the millennium phytomedicine- ginseng (I):
etymology, pharmacognosy, phytochemistry, market and regulations. Curr. Med.
Chem. 16, 2475–2484.
Ou, B., Hampsch-Woodill, M., Prior, R.L., 2001. Development and validation of an
improved oxygen radical absorbance capacity assay using fluorescein as the
fluorescent probe. J. Agric. Food Chem. 49, 4619–4626.
Kawai, H., Nishida, M., Tashiro, Y., Kuroyanagi, M., Ueno, A., Satake, M., 1989. Studies
on the structures of udosaponins A,B,C,D,E, and F from Aralia cordata Thunb. Chem.
Pharm. Bull. 37, 2318–2321.
Panossian, A.G., Efferth, T., Shikov, A.N., Pozharitskaya, O.N., Kuchta, K., Mukherjee, P.
K., Banerjee, S., Heinrich, M., Wu, W., Guo, D., Wagner, H., 2021. Evolution of the
adaptogenic concept from traditional use to medical systems: pharmacology of
stress- and aging-related diseases. Med. Res. Rev. 41, 630–703.
Kim, J.S., Kang, S.S., 1998. Saponins from the aerial parts of Aralia continentalis. News
Physiol. Sci. 4, 45–50.
Piochon, M., Legault, J., Gauthier, C., Pichette, A., 2009. Synthesis and cytotoxicity
Kuang, H.X., Wang, Z.B., Wang, Q.H., Yang, B.Y., Xiao, H.B., Okada, Y., Okuyama, T.,
2013. Triterpene glucosides from the leaves of aralia elata and their cytotoxic
activities. Chem. Biodivers. 10, 703–710.
evaluation of natural α-bisabolol β-d-fucopyranoside and analogues. Phytochemistry
70, 228–236.
Shaker, K.H., Bernhardt, M., Elgamal, M.H.A., Seifert, K., 1999. Triterpenoid saponins
from Fagonia indica. Phytochemistry 51, 1049–1053.
Lee, E.B., Kim, O.J., Kang, S.S., Jeong, C., 2005. Araloside A, an antiulcer constituent
from the root bark of Aralia elata. Biol. Pharm. Bull. 28, 523–526.
Lee, J.H., Ha, Y.W., Jeong, C.S., Kim, Y.S., Park, Y., 2009. Isolation and tandem mass
fragmentations of an anti-inflammatory compound from aralia elata. Arch.
Pharmacal Res. 32, 831–840.
Shikov, A.N., Pozharitskaya, O.N., Makarov, V.G., 2016. Aralia elata var. mandshurica
(Rupr. & Maxim.) J.Wen: an overview of pharmacological studies. Phytomedicine
23, 1409–1421.
Song, S.J., Nakamura, N., Ma, C.M., Hattori, M., Xu, S.X., 2000. Four new saponins from
the root bark of Aralia elata. Chem. Pharm. Bull. 48, 838–842.
Legault, J., Girard-Lalancette, K., Grenon, C., Dussault, C., Pichette, A., 2010.
Antioxidant activity, inhibition of nitric oxide overproduction, and in vitro
antiproliferative effect of maple sap and syrup from Acer saccharum. J. Med. Food
13, 460–468.
Song, S.J., Nakamura, N., Ma, C.M., Hattori, M., Xu, S.X., 2001. Five saponins from the
root bark of Aralia elata. Phytochemistry 56, 491–497.
Sparg, S.G., Light, M.E., Van Staden, J., 2004. Biological activities and distribution of
plant saponins. J. Ethnopharmacol. 94, 219–243.
Li, H., O’Neill, T., Webster, D., Johnson, J.A., Gray, C.A., 2012. Anti-mycobacterial
diynes from the Canadian medicinal plant Aralia nudicaulis. J. Ethnopharmacol.
140, 141–144.
Suh, S.-J., Jin, U.-H., Kim, K.-W., Son, J.-K., Lee, S.H., Son, K.-H., Chang, H.W., Lee, Y.-C.,
Kim, C.-H., 2007. Triterpenoid saponin, oleanolic acid 3-O-β-D-glucopyranosyl(1 →
Li, Y., Yue, R., Liu, R., Zhang, L., Wang, M., 2016. Secondary metabolites from the root of
Aralia echinocaulis Hand. -Mazz. Rec. Nat. Prod. 10, 639–644.
Ma, Z.Q., Zhang, Y., Cai, C.K., Li, Q., Ni, J., 2013. Two new triterpenoid saponins from
the leaves of Aralia elata. J. Asian Nat. Prod. Res. 15, 849–854.
Miyase, T., Shiokawa, K.-I., Zhang, D.M., Ueno, A., 1996a. Araliasaponins I-IX, triterpene
saponins from the roos of Aralia decaisneana. Phytochemistry 41, 1411–1418.
Miyase, T., Sutoh, N., Zhang, D.M., Ueno, A., 1996b. Araliasaponins XII-XVIII, triterpene
saponins from the roots of Aralia chinensis. Phytochemistry 42, 1123–1130.
Moerman, D.E., 1998. Native American Ethnobotany. Timber Press Inc.
Nhiem, N.X., Lim, H.Y., Kiem, P.V., Minh, C.V., Thu, V.K., Tai, B.H., Quang, T.H.,
Song, S.B., Kim, Y.H., 2011. Oleanane-type triterpene saponins from the bark of
Aralia elata and their NF-κB inhibition and PPAR activation signal pathway. Bioorg.
Med. Chem. Lett. 21, 6143–6147.
3)-α-L-rhamnopyranosyl(1 → 2)-α-L-arabinopyranoside (OA) from Aralia elata
inhibits LPS-induced nitric oxide production by down-regulated NF-κB in raw 264.7
cells. Arch. Biochem. Biophys. 467, 227–233.
Tian, Z., Lin, G., Zheng, R.X., Huang, F., Yang, M.S., Xiao, P.G., 2006. Anti-hepatoma
activity and mechanism of ursolic acid and its derivatives isolates from Aralia
decaisneana. World J. Gastroenterol. 12, 874–879.
Xiao, K., Yi, Y.H., Wang, Z.Z., Tang, H.F., Li, Y.Q., Lin, H.W., 1999. A cytotoxic triterpene
saponin from the root bark of Aralia dasyphylla. J. Nat. Prod. 62, 1030–1032.
Yu, S.-S., Yu, D.-Q., Liang, X.-T., 1994. Triterpenoid saponins from the roots of Aralia
spinifolia. J. Nat. Prod. 57, 978–982.
Zhang, Y., Peng, Y., Li, L., Zhao, L., Hu, Y., Hu, C., Song, S., 2013. Studies on cytotoxic
triterpene saponins from the leaves of Aralia elata. Food Chem. 138, 208–213.
Zou, M.-L., Mao, S.-L., Sang, S.-M., Xia, Z.-H., Lao, A.-N., 2001. Two new triterpenoid
saponins from Aralia subcapitata. Nat. Prod. Lett. 15, 157–161.
O’Brien, J., Wilson, I., Orton, T., Pognan, F., 2000. Investigation of the Alamar Blue
(resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur. J.
Biochem. 267, 5421–5426.
189