Two new iridoid glycosides from the root barks of Sambucus williamsii Hance

Article abstract of DOI:10.3390/molecules16053869

Chemical investigation of the ethanol extract of the root barks of Sambucus williamsii Hance collected in the Heilongjiang province of China resulted in the isolation of two new iridoid glycosides, williamsoside A (1) and williamsoside B (2). Their structures were elucidated on the basis of extensive spectroscopic analysis (1D, 2D-NMR and HRESIMS) and chemical studies. Iridoid glycosides have for a long time been considered as characteristic ingredients of S. williamsii. However, the presence of iridoid glycosides with apiofuranosyl moieties in S. williamsii is reported for the first time in this study.

Full text of DOI:10.3390/molecules16053869

Molecules 2011, 16, 3869-3874; doi:10.3390/molecules16053869
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Two New Iridoid Glycosides from the Root Barks of Sambucus
williamsii Hance
Zhen-Yue Wang †, Hua Han †, Bing-You Yang, Yong-Gang Xia and Hai-Xue Kuang *
Key Laboratory of Chinese Materia Medica, Heilongjiang University of Chinese Medicine, Ministry of
Education, Harbin 150040, China
†
These two authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail: hxkuang@hotmail.com;
Tel.: +86-45182193001; Fax: +86-45182110803.
Received: 15 April 2011; in revised form: 28 April 2011 / Accepted: 29 April 2011 /
Published: 9 May 2011
Abstract: Chemical investigation of the ethanol extract of the root barks of Sambucus
williamsii Hance collected in the Heilongjiang province of China resulted in the isolation
of two new iridoid glycosides, williamsoside A (1) and williamsoside B (2). Their structures
were elucidated on the basis of extensive spectroscopic analysis (1D, 2D-NMR and
HRESIMS) and chemical studies. Iridoid glycosides have for a long time been considered as
characteristic ingredients of S. williamsii. However, the presence of iridoid glycosides with
apiofuranosyl moieties in S. williamsii is reported for the first time in this study.
Keywords: Sambucus williamsii Hance; root barks; iridoid glycosides
1. Introduction
Sambucus williamsii Hance, a tree widely distributed in China, belongs to the family Adoxaceae [1].
S. williamsii is also called in Chinese “Jie-gu-mu”, “Da-jie-gu-dan”, “Gong-lao-dao”, etc. [2]. The root
bark of this plant is a folk medicine with a long history of use in China for the treatment of fractures
and osteoporosis [2]. Phytochemical studies showed that triterpenoids, flavonoids, lignans and the
iridoid morroniside are major constituents of S. williamsii [3]. In our present work, investigation of its
ethanol extract led to the isolation of two new iridoid glycosides. In this paper, we present the isolation

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and structural characterization of the two new iridoid glycosides on the basis of the interpretation of
spectral data, including 1D, 2D NMR and HRESIMS data.
2. Results and Discussion
Compound 1 was obtained as a white amorphous powder and showed positive results for the
Molisch reagent, which was considered to be indicative of an iridoid glycoside. Its molecular formula
was established as C22H34O13 by the positive HRESIMS data, indicating six degrees of unsaturation.
The 1H-NMR spectrum of 1 (Table 1) showed the two characteristic signals of the common iridoid
compounds, namely a proton at δ 5.11 (1H, d, J = 6.2 Hz) and a singlet at δ 7.41 (1H) corresponding to
the C-1 and C-3 protons, respectively. In addition, the signals at δ 3.68 (3H, s) and 1.09 (3H, d, J = 6.7 Hz)
were attributed to Me-12 and Me-10, respectively. The typical downfield signals at δ 4.64 (1H, d, J = 7.9 Hz),
4.99 (1H, d, J = 2.3 Hz) were assigned to the anomeric H-atom of the β-glucopyranosyl and
α-apiofuranosyl moieties according to their coupling constants and splitting patterns [4].
Table 1. 1H and 13C-NMR data of 1 and 2 in CD3OD at 400 MHz and 100 MHz, J in Hz.
No.
1
2
δ H
δ C
98.2
152.8
112.8
δ H
δ C
98.2
152.3
113.8
32.4
42.8
75.0
42.4
46.5
13.7
169.5
51.6
100.3
74.7
77.9
71.6
77.2
68.5
110.9
77.9
80.5
75.0
1
3
4
5
6
7
8
9
10
11
12
1’
2’
3’
4’
5’
6’
1”
2”
3”
4”
5.11 (1H, d, J = 6.2)
5.15 (1H, d, J = 5.5)
7.39 (1H, d, J = 1.2)
7.41 (1H, s)
2.87 (1H, dd, J = 7.8, 15.7) 35.5 3.11 (1H, dd, J = 7.9, 16.2)
2.20 (1H, m), 1.35 (1H, m) 33.6 1.60 (1H, m), 2.23(1H, m)
1.90 (1H, m), 1.89 (1H, m) 34.1
1.99 (1H, q, J = 6.9, 7.3)
1.73 (1H, m)
4.05 (1H,dd, J= 4.4, 4.0)
36.6
1.89 (1H, m)
48.4 1.99 (1H, dt, J = 3.8, 5.0)
1.09 (3H, d, J = 6.7)
21.0
169.7
51.7
100.4
74.7
77.9
71.5
77.2
68.4
110.9
77.9
80.5
75.0
1.10 (3H, d, J = 6.8)
3.68 (3H, s)
4.64 (1H, d, J = 7.9)
3.19 (1H, m)
3.68(3H, s)
4.62 (1H, d, J = 7.9)
3.19 (1H, m)
3.35 (1H, m)
3.29 (1H, m)
3.42 (1H, m)
3.34 (1H, m)
3.26 (1H, m)
3.42 (1H, m)
3.68 (1H, m), 3.97 (1H, m)
4.99 (1H, d, J = 2.3)
3.88 (1H, m)
3.62 (1H,m), 3.98 (1H,m)
5.00 (1H, d, J = 2.4)
3.88(1H, m)
3.95 (1H, dd, J = 9.6)
3.75(1H, dd, J = 9.6)
3.55(1H, s)
3.75(1H, dd, J = 9.6)
3.94(1H, dd, J = 9.6)
3.55 (2H, s)
5”
65.6
65.5

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The 13C-NMR spectrum of 1 (Table 1) showed resonances for 22 C-atoms, including two
quaternary carbons (δ 112.8, 169.7), five methines (δ 98.2, 152.8, 35.5, 36.6, 48.4), two methylenes
(δ 33.6, 34.1), and two methyls (δ 21.0, 51.7) belonging to the aglycone moiety, a glucopyranosyl
group (δ 100.4, 74.7, 77.9, 71.5, 77.2, 68.4), and an apiofuranosyl group (δ 110.9, 77.9, 80.5, 75.0,
65.6). C-1 that was connected with C-3 through an oxygen atom on the basis of the chemical shifts of
C-1(δ 98.2) and C-3 (δ 152.8), which was also confirmed by the HMBC correlations from H-3/C-1 and
H-1/C-3. The HMBC spectrum was used to elucidate the connection of different structural fragments, as
well as to confirm the above assignments. In this spectrum, other key long-range correlations were
observed between Me-10/C-7, C-8 and C-9, and between Me-12/C-11 (Figure 1). The connection positions
of the glucopyranosyl and apiofuranosyl groups in 1 were established unambiguously by a HMBC
experiment in which long-range correlations between H-1’/C-1, H-1/C-1’, H-1”/C-6’, and H-6’/C-1”.
Figure 1. Key HMBC and NOESY correlations of 1.
Since the stereochemistry of the three asymmetric centers (C-1, C-5, and C-9) was the same in
practically all iridoids identified hitherto [5,6], the C-1 oxygen atom, H-5 and H-9 were assigned the
β-orientation in the iridoid skeleton. The stereo configuration of Me-10 was determined as a
β-orientation on the basis of the key NOESY correlations between Me-10/H-9, H-9/H-5, and H-8/H-1
(Figure 2). Thus, the structure of 1 was identified to be 6’-apiosyldeoxyloganin, with the structure
shown in Figure 2, and it was named williamsoside A.
Figure 2. Structures of 1 and 2.

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Compound 2 was obtained as a white amorphous powder and showed positive results for the
Molisch reagent, which was also considered indicative of another iridoid glycoside. Its molecular
formula was established as C22H34O14 by the positive HRESIMS, indicating six degrees of
1
unsaturation. The H-NMR spectrum of 2 showed distinct resemblance to that of 1. The only notable
difference was the change in the H-7 signal, which appeared as a double doublet at δ 4.05 (1H, dd, J = 4.4,
4.0 Hz), indicating that C-7 was substituted by a hydroxyl group. The 13C-NMR (DEPT) spectrum
showed an additional downfield C-atom signal at δ C 75.0 in 2, which was affirmatively assigned to
the C-7 carbon. The stereo configuration of the hydroxyl group at C-7 and methyl group at C-8 was
determined to be the β-orientation on the basis of the obvious NOESY correlations between H-7/H-8,
H-8/H-1, Me-10/H-9, and H-9/H-5. On the basis of above data, the structure of 2 was identified to be
as shown in Figure 2, and it was named williamsoside B.
3. Experimental
3.1. General
IR spectra were recorded on a Shimadzu FTIR-8400S spectrometer. NMR spectra were recorded on
a Bruker DPX 400 NMR instrument (at 400 MHz for 1H-NMR and 100 MHz for 13C-NMR). Chemical
shifts are given as δ values with reference to tetramethylsilane (TMS) used as internal standard, and
coupling constants are given in Hz. HRESIMS were carried out on Waters Xevo QTOF mass
spectrometer. Preparative HPLC (Waters, Delta 600-2487) was performed on a Hypersil-ODS II
(10 m, 20 × 300 mm, Yilite, Dalian, China).
3.2. Plant Material
The root barks of S. williamsii were collected in August 2008 from the Fangzheng district,
Heilongjiang Province, China, and identified by the author Zhen-Yue Wang. A voucher specimen
(20080079) has been deposited at Heilongjiang University of Chinese Medicine, Harbin, China.
3.3. Extraction and Isolation
The dried root barks (5.0 kg) of S. williamsii were extracted with 95% EtOH under reflux (2 × 10 L)
for 2 h (each time), and the combined soln. was filtered and concentrated under vacuum to an oily
residue, which was suspended in H2O. The suspension was passed through AB-8 crosslinked
polystyrene, and sequentially eluted with H2O, 50% EtOH, and 95% EtOH, respectively. The 50%
EtOH elution fraction was concentrated under vacuum to yield a residue (52.0 g), which was subjected
to silica gel column and eluted successively with CHCl3/MeOH (15:1→1:1) to give 10 fractions
(Fraction 1–10). Fraction 7 (5 g) was further separated by ODS column to afford 9 sub-fractions A1–A9.
The sub-fraction A9 was subjected to preparative HPLC (Hypersil-ODS Π column) eluted with
MeOH/H2O (2:3) to afford compounds 1 (28 mg) and 2 (49 mg).
Williamsoside A (1): White amorphous powder, [α]25 = −27.0 (c = 0.1, MeOH). IR (KBr): ν = 3303,
D
2945, 2831, 1448, 1417, 1114, 1035, 659 cm−1. HRESIMS (positive): m/z = 507.2057 (calc. for
C22H35O13, 507.2028, [M + H]+), 524.2352 (calc. for C22H38NO13, 524.2343, [M + NH4]+), 529.1880

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(calc. for C22H34NaO13, 529.1897, [M + Na]+) and 545.1628 (calc. for C22H34KO13, 545.1636, [M + K]+).
1H and 13C-NMR: see Table 1.
Williamsoside B (2): White amorphous powder, [α]25 = −16.0 (c = 0.1, MeOH). IR (KBr): ν = 3312,
D
2945, 2833, 1447, 1419, 1113, 1035, 660 cm−1. HRESIMS (positive): m/z = 523.2002 (calc. for
C22H35O14, 523.2027, [M + H]+), 540.2271 (calc. for C22H38NO14, 540.2292, [M + NH4]+), and
545.1849 (calc. for C22H34NaO14, 545.1846, [M + Na]+). 1H and 13C-NMR: see Table 1.
Acid Hydrolysis of 1 and 2. To a solution of 1 and 2 (each, 250 μg) in MeOH (1 mL) was added 5%
H2SO4 (1 mL) and the mixture was refluxed for 8 h. The reaction mixture was then neutralized with
saturated sodium carbonate and extracted with ethyl acetate (EtOAc, 2 × 5 mL) to give an aqueous
fraction containing sugars and an EtOAc fraction containing the aglycone part. The aqueous phase was
concentrated and compared with standard sugars using the TLC eluent systems EtOAc/n-butanol/water
(2:7:1) and CH2Cl2/MeOH/water (10:6:1) [7-9]; the two sugars were thus identified as apiose and glucose.
4. Conclusions
Iridoid glycosides represent a large group of cyclopentano[c]pyran monoterpenoids which have
been reported to be associated with diverse biological activities including choleretic, purgative, liver
protective, vasoconstrictive, antimicrobial, analgesic, antitumor, sedative and anti-inflammatory
properties [10]. As a part of our chemical investigation on S. williamsii, we have isolated two new
iridoid glycosides containing a 6’-apiofuranosyl moiety. Their structures were established on the basis
of spectroscopic evidence. This is the first time iridoid glycosides with apiofuranosyl moieties have
been reported in this species.
Acknowledgements
We would like to acknowledge the financial support from the State Key Creative New Drug Project of
11th Five-year Plan of China (2009ZX09103-383).
References and Notes
1. Tropicos. Missouri Botanical Garden. Available online: http://www.tropicos.org/
NameSearch.aspx?name= Sambucus/ (accessed on 18 April 2011).
2. Han, H.; Yan, Y.Y.; Kuang, H.X.; Dong, P.L. Advances in research of Sambucus williamsii
hance. Inform. Trad. Chin. Med. 2008, 25, 14-16.
3. Ouyang, F.; Liu, Y.; Li, R.; Li, L.; Wang, N.L.; Yao, X.S. Five lignans and an iridoid from
Sambucus williamsii. Chin. J. Nat. Med. 2011, 9, 26-29.
4. Garcia, J.; Chulia, A.J. Loganin and New iridoid glucosides in Gentiana pedicellata [J]. Planta
Med. 1986, 52, 327-329.
5. Zapesochnaya, G.G.; Kurkin, V.A.; Pervykh, L.N.; Karasartov, B.S. Velpetin—A new iridoid
glycoside from Nepeta velutina. Khim. Prir. Soedin. 1991, 6, 777-781.
6. Feng, C.L.; Gong, M.F.; Zeng, Y.B.; Dai, H.F.; Mei, W.L. Scyphiphin C, a New Iridoid from
Scyphiphora hydrophyllacea. Molecules 2010, 15, 2473-2477.

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7. Bedir, E.; Toyang, N.J.; Khan, I.A.; Walker, L.A.; Clark, A.M. A new dammarane type triterpene
glycoside from Polyscias fulva. J. Nat. Prod. 2001, 64, 95-97.
8. Chaturvedula, V.S.P.; Schilling, J.K.; Miller, J.S.; Andriantsiferana, R.; Rasamison, V.E.;
Kingston, D.G.I. New cytotoxic oleanane saponis from the infructescences of Polyscias amplifolia
from the Madagascar rainforest. Planta Med. 2003, 69, 440-444.
9. Huan, V.D.; Yamamura, S.; Ohtani, K.; Kasai, R.; Yamasaki, K.; Nham, N.T. Oleanane saponins
from Polyscias fructicosa. Phytochemistry 1998, 47, 451-457.
10. Ling, S.K.; Tanaka, T.; Kouno, T. Effects of iridoids on lipoxygenase and hyaluronidase activities
and their activation by β-glucosidase in the presence of amino acids. Biol. Pharm. Bull. 2003, 26,
352-356.
Sample Availability: Samples of williamsosides A and B are available from the authors.
© 2011 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).