Two pentasaccharide resin glycosides from Argyreia acuta

Article abstract of DOI:10.1080/14786419.2015.1030739

Two new compounds of acutacosides 1 and 2, pentasaccharide resin glycosides were isolated from the aerial parts of Argyreia acuta. The core of the two compounds was operculinic acid A, and they were esterfied at the same position, just one substituent group was linked at C-2 of Rha. The absolute configuration of the aglycone in the two compounds was established by Mosher's method, which was (11S)-hydroxyhexadecanoic acid (jalapinolic acid). Their structures were established by a combination of spectroscopic and chemical methods.

Full text of DOI:10.1080/14786419.2015.1030739

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Two pentasaccharide resin glycosides
from Argyreiaacuta
Yong-Qin Yina, Jie-Tao Pana, Bang-Wei Yua, Hong-Hua Cuia, You-
Shao Yana & Yan-Fen Chena
a Department of Traditional Chinese Medicinal Chemistry,
Guangdong Pharmaceutical University, Guangzhou510006, People's
Republic of China
Published online: 30 Apr 2015.
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To cite this article: Yong-Qin Yin, Jie-Tao Pan, Bang-Wei Yu, Hong-Hua Cui, You-Shao Yan & Yan-Fen
Chen (2015): Two pentasaccharide resin glycosides from Argyreiaacuta, Natural Product Research:
Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1030739
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Natural Product Research, 2015
http://dx.doi.org/10.1080/14786419.2015.1030739
Two pentasaccharide resin glycosides from Argyreia acuta
Yong-Qin Yin*, Jie-Tao Pan, Bang-Wei Yu, Hong-Hua Cui, You-Shao Yan and Yan-Fen Chen
Department of Traditional Chinese Medicinal Chemistry, Guangdong Pharmaceutical University,
Guangzhou 510006, People’s Republic of China
(Received 28 November 2014; final version received 13 March 2015)
Two new compounds of acutacosides 1 and 2, pentasaccharide resin glycosides were
isolated from the aerial parts of Argyreia acuta. The core of the two compounds was
operculinic acid A, and they were esterfied at the same position, just one substituent
group was linked at C-2 of Rha. The absolute configuration of the aglycone in the two
compounds was established by Mosher’s method, which was (11S)-hydroxyhexade-
canoic acid (jalapinolic acid). Their structures were established by a combination of
spectroscopic and chemical methods.
Keywords: resin glycoside; operculinic acid A; Argyreia acuta
1. Introduction
Argyreia acuta L. is a member of the genus Argyreia (Convolvulaceae; Editorial Committee of
China flora of Chinese Academy of Sciences 1997). A. acuta is a common folk herbal
medicine, with the efficacies of dispelling wind and eliminating dampness, relieving cough and
reducing sputum, stopping the bleeding and promoting tissue regeneration, relaxing and
activating the tendons, removing toxicity for eliminating carbuncles. Now a few compounds
were found, and recent research found it has hemostasis properties (Cai & Xu 2013). The resin
glycoside showed characteristics of Convolvulaceae; however, so far no resin glycoside has
been reported from A. acuta. In this study, two new pentasaccharide resin glycosides,
designated as acutacosides A (1) and acutacosides B (2), were isolated from the aerial parts of
A. acuta. The new compounds are maclactones of operculinic acid A, partially esterified with
Mba, Cna, Dodeca and Deca of different fatty acids. The lactone esterification site of the
aglycone, jalpinolic acid, was attached to the second saccharide at C-2 in 1 and 2 (Figure 1).
Their structures were elucidated on the basis of extensive spectroscopic data interpretation and
chemical degradation.
*Corresponding author. Email: yongqinyin@126.com
q 2015 Taylor & Francis

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Y.-Q. Yin et al.
Figure 1. The structures of compounds 1–3.
2. Results and discussion
A 95% EtOH extract of the dried aerial parts of A. acuta was partitioned between CHCl3 and
H2O to afford a resinous fraction which was chromatographed over Si gel, Rp-18, Sephadex LH-
20 and purified by preparative HPLC to afford compounds 1 and 2, which were hydrolysed with
alkaline and acid to afford organic acid and operculinic acid A (3). Peaks in the chromatograms
were detected from the alkaline hydrolysis mixtures and identified by comparison with authentic
samples as 2-methylbutyric acid methyl ester (tR 4.39 min) m/z [M þ H]þ 117 (5), 101 (23), 88
(96), 57 (100), 41 (55), 29 (45), 27 (19), and transcinnamic acid methyl ester (tR 13.29 min) m/z
[M]þ 162 (40), 131 (100), 103 (66), 77 (32), from 1 to 2 was identified. n-Decanoic acid (tR
12.37 min): m/z 172 [M]þ (4), 155 (5), 143 (30), 129 (5), 87 (59), 74 (100), 55 (18) from 1 was
identified. n-Dodecanoyl acid methyl ester (tR 15.17 min) m/z [M]þ 200 (1), 172 (1), 168 (10),
157 (15), 143(18), 129 (7), 87 (64), 74 (100), 55 (25), 43 (20), 41 (18) from 2 was identified. The
2-methylbutanoic acid was proved to be S-configuration by comparing the specific rotation with
that of authentic 2S-methylbutanoic acid (Yin et al. 2009). Acidic hydrolysis of operculinic acid
A liberated the aglycone, 11-hydroxyhexadecanoic acid, which was identified S-configuration
(Yin et al. 2008) and the monosaccharides mixture was derivatised and detected with GC–MS
(gas chromatography-mass spectrometer) by comparison with those of authentic samples to
improve as D-fucose, L-rhamnose and D-glucose (Luo et al. 2008).
Acutacosides A (1) and B (2), obtained as a white, amorphous powder. In HR-TOF-MS, a
pseudomolecular ion peak at m/z 1391.7322 (100%) [M þ Na]þ (calculated for 1391.7339) was
corresponding to the molecular formula C70H112O26 (1) and a pseudomolecular ion peak at m/z
1419.7686 (100%) [M þ Na]þ (calculated for 1419.7652) was determined as the molecular
formula C72H116O26 (2). Its IR spectrum gave peaks of hydroxyl (3443 cm21), carbonyl
(1723 cm21) and aromatic (1637 cm21) groups from 1 and peaks of hydroxyl (3442 cm21),
carbonyl (1722 cm21) and aromatic (1637 cm21) groups from 2. Alkaline hydrolysis of 1 and 2
afforded operculinic acid A (3) and dodecanoic, decanoic, 2-methylbutyric and trans-cinnamic
acids. 2-Methylbutyric acid was found to have the S-configuration by comparison of its optical
rotation value with that of an authentic sample. Acid hydrolysis afforded the monosaccharides
mixture, which was derivatised and detected with GC–MS by comparing with those of authentic
samples to improve as D-fucose, L-rhamnose and D-glucose. The 13C NMR data of 1 and 2 were
all the same from 177 to 41 ppm. All these evidences suggested that 1 and 2 were the substituents
attaching the same sites, the only difference was in the decanoic group in 1 and dodecanoic
group in 2. The 1H NMR data of 1 (Table S1) exhibited two trans-coupled olefinic protons at dH
6.60 (d, J ¼ 16.0 Hz, H-2 of Cna) and 7.87 (d, J ¼ 16.0 Hz, H-3 of Cna), a multiplet due to five

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protons at dH 7.27–7.45 (m, C6H5 of Cna) and at dH 0.81 (t, J ¼ 7.0 Hz, H-4 of Mba), 1.15 (d,
J ¼ 7.4 Hz, CH3-2 of Mba) and 2.48 (m, H-2 of Mba). Also a methyl triplet signal at 0.83 ppm
and a triplet-like signal in a methylene group at CH2-2 (2.32 ppm) of a decanoyl group, and two
signals at 2.28 (1H, m) and 2.44 (1H, m) ppm of the non-equivalent protons of the methylene
group at C-2 in the aglycone moiety were observed, suggesting a macrocyclic lactone-type
structure.
The 13C NMR spectrum of 1 exhibited five signals at dC 105.8, 104.6, 103.6, 100.6 and 98.9
assigned to anomeric carbons of five sugar units and dC 176.3, 173.8, 173.4, and 166.8 for four
ester carbonyl carbons. Then the anomeric protons at 5.09 (d, J ¼ 7.5 Hz), 4.74 (d, J ¼ 7.4 Hz),
6.27 (br s), 5.84 (br s) and 5.52 (br s) by HSQC data, respectively. All protons in each saccharide
system were assigned by 2D NMR TOCSY, HMBC and HSQC experiments, leading to the
identification of one glucopyranosyl unit, one fucopyranosyl unit and three rhamnopyranosyl
units as the monosaccharides present in 1. The interglycosidic connectivities were determined
from the following HMBC (Figure S1): C-2 (80.2 ppm) of fucose with H-1 (5.52 ppm) of
rhamnose; C-4 (82.5 ppm) of rhamose with H-1 (5.84 ppm) of rhamnose0; C-4 (80.1 ppm) of
rhamnose0 with H-1 (6.27 ppm) of rhamnose00 and C-3 (79.3 ppm) of rhamnose0 with H-1
(5.09 ppm) of glucose. Esterification positions was determined by HMBC data, between H-4 of
rhamnose00 (dH 6.09) and dC 176.0 (C-1 of Mba); H-3 of rhamnose00 (dH 6.01) and dC 166.4 (C-1
of Cna); H-2 of rhamnose0 (dH 6.33) and dC 173.8 (C-1 of Deca) and H-2 of rhamnose (dH 5.93)
and dC 173.4 (C-1 of aglycone), respectively. The position of the jalapinolic acid unit was finally
determined by HMBC between jalapinolic acid H-11 (3.83 ppm) and fucose C-1 (104.6 ppm),
with which correlations established the structure of 1 (Figure S1). From these observations, the
structure of acutacoside A (1) was elucidated as (S)-jalapinolic acid 11-O-a-L-rhamnopyranosyl-
(1 ! 3)-O-[3-O-trans-cinnamoyl-4-O-(S)-2-methylbutyryl-a-L-rhamnopyranosyl-(1 ! 4)]-O-
[2-O-n-decanoy]-a-L-rhamnopyranosyl-(1 ! 4)-O-a-L-rhamnopyranosyl-(1 ! 2)-O-b-D-fuco-
pyranoside, intramolecular 1,200-ester (Figure 1).
The NMR spectra (Table S1) of 2 were similar to those of 1, the interglycosidic
connectivities and esterification sites were all the same though by 2D NMR TOCSY, HMBC and
HSQC, just one group of decanoyl was different by GC–MS experiments. Accordingly, the
structure of 2 was elucidated as (S)-jalapinolic acid 11-O-a-L-rhamnopyranosyl-(1 ! 3)-O-[3-
O-trans-cinnamoyl-4-O-(S)-2-methylbutyryl-a-L-rhamnopyranosyl-(1 ! 4)]-O-[2-O-n-dodeca-
noy]-a-L-rhamnopyranosyl(1 ! 4)-O-a-L-rhamnopyranosyl-(1 ! 2)-O-b-D-fucopyranoside,
intramolecular 1,200-ester (Figure 1).
3. Experimental
3.1. General
NMR spectra were recorded on INOVA 500 spectrometers (1H NMR, HSQC and HMBC at
500 MHz; 13C NMR at 125 MHz) using C5D5N as solvent with tetramethylsilane as internal
reference. The chemical shifts were given in d (ppm) and coupling constants in Hz. HR-TOF-MS
experiments were performed on AB SCIEX Triple TOF 5600 plus MS spectrometer. UV on a
Shimadzu UV-2550 spectrophotometer and IR spectra were measured on a Shimadzu FTIR
Bruker-TENSOR 37 spectrophotometer. GC–MS experiment was performed on a TRACE GC
ULTRA DSQII intrument. Optical rotations were measured with an Anton Paar-MCP600
polarimeter in MeOH solution. The centrifugation was applied with D05 (Hunan Hexi Instrument
Co., Ltd, Changsha, China). Adsorbents for column chromatography were silica gel (200–
300 mm, Qingdao Marine Chemical Co., Ltd, Qingdao, China), Sephadex LH-20 (75–150 mm,
Pharmacia, Uppsala, Sweden), ODS (octa decylsilyl silicion) (40–63 mm, FuJi, Tokyo, Japan).
Preparative HPLC was performed using a Shimadzu LC-6AD series instrument equipped with a
UV detector at 280 nm and Shim-Park RP-C18 column (20 £ 200 mm i.d.). Thin-layer

4
Y.-Q. Yin et al.
chromatography was performed on pre-coated silica gel GF254 plates (Qingdao Marine Chemical
Co., Ltd) and detected by spraying with 10% H2SO4–EtOH.
3.2. Plant material
The aerial parts of A. acuta L. were collected at Guilin City, Guangxi Province, China, in August
2013, and identified by Prof. Jizhu Liu. A voucher specimen (no. 2013-2) is deposited at
Department of Traditional Chinese Medicinal Chemistry, Guangdong Pharmaceutical
University.
3.3. Extraction and isolation
The aerial parts (15 kg) of A. acuta were cut to pieces and were extracted two times with 95%
EtOH under reflux for 2 h. The extract (1.2 kg) was partitioned into CHCl3 and H2O-soluble
fractions. The CHCl3 extract (0.3 kg) was subjected to column chromatography on silica gel and
eluted with CH2Cl2–MeOH (20:1, 5:1) to give two subfractions. The first subfraction (20.1 g)
was purified by Sephadex LH-20 column with MeOH as eluent, factions of 6–27 (4.8 g) was
purified by RPHPLC and were eluted with 97% MeOH/H2O, two peaks were collected from
fraction 1, to afford acutacoside A (1, 35 mg, tR 48.25 min) and acutacosid B (2, 13.8 mg, tR
38.41 min).
3.4. Spectral data
25
Acutacoside A (1): A white powder; m.p. 140.1–141.18C; ½aꢀD 50 (c 0.01, MeOH). UV lmax
(MeOH) nm (log e): 279.5 (0.97), 217 (0.67) nm; IR (KBr) nmax cm21: 3443 (hydroxyl), 1724
(carbonyl group), 1637 (aromatic moiety). For 1H (C5D5N, 500 MHz) and 13C (C5D5N,
125 MHz) NMR spectral data, see Table S1. HR-TOF-MS: m/z 1391.7322 [M þ Na]þ
(calculated for 1391.7339, C70H112O26Na).
25
Acutacoside B (2): A white powder; m.p. 137.5–140.08C; ½aꢀD 17.3 (c 0.02, MeOH). UV
l
max (MeOH) nm (log e): 279.5 (1.21), 217.5 (0.82) nm; IR (KBr) nmax cm21: 3443 (hydroxyl),
1
1722 (carbonyl group), 1637 (aromatic moiety). For H (C5D5N, 500 MHz) and 13C (C5D5N,
125 MHz) NMR spectral data, see Table S1. HR-TOF-MS: m/z 1419.7686 [M þ Na]þ
(calculated for 1419.7652, C72H116O26Na).
3.5. Hydrolysis
3.5.1. Alkaline hydrolysis of 1 and 2
Compounds 1 and 2 (10 mg each) in 5% KOH (3 mL) were refluxed at 908C for 2 h, respectively.
The reaction mixture was acidified to pH 4.0 with 2 N HCl and extracted with hexane (3 mL £ 2)
and n-BuOH (3 mL £ 2). The organic layer was washed with H2O, dried over anhydrous
Na2SO4, then methylated by following method (She 2004): the hexane extracted was mixed
with 0.1 mL 0.5 mol CH3ONa solution, rocked for 5 min at room temperature, then 5 mL
CH3COOH and 1 g anhydrous CaCl2 powder were added, stewed for 1 h, centrifuged for 2–3 min
at 2000–3000 rpm min21, then the supernatant was analysed by GC–MS. GC–MS on a TRACE
GC ULTRA DSQII instrument under the following conditions: 30 m £ 0.25 mm £ 0.25 mm, TG-
5MS (Thermo) column; He, 0.8 mL min21; 408C, 3 min; 50–3108C, D108C min21, 70 eV.
3.5.2. Acid hydrolysis and sugar analysis
The glycosidic acid (3, 3 mg, from alkaline hydrolysis) was methylated with MeOH and catalysed
with 0.5 N H2SO4 to give operculinic acid A methyl ester (4). Compound 4 was hydrolysed with

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1 N H2SO4 and extracted with ether to obtain 11-hydroxyhexadecanoic acid methyl ester (5). The
aqueous layer of acidic hydrolysis was concentrated under reduced pressure to give a residue of
the sugars. The residue was dissolved in pyridine (0.1 mL), to which 0.08 M L-cysteine methyl
ester hydrochloride in pyridine (0.15 mL) was added. The mixture was kept at 608C for 1.5 h.
After the reaction mixture was dried in vacuo, the residue was trimethylsilylated with
1-trimethylsilylimidazole (0.1 mL) for 2 h. The mixture was partitioned between n-hexane and
H2O (0.3 mL each) and then the hexane extract was analysed by GC–MS on a TRACE GC
ULTRA DSQII instrument under the following conditions: 30 m £ 0.25 mm £ 0.25 mm,
TG-5MS (Thermo) column; He, 0.8 mL min21; 608C, 3 min; 60–1808C, D108C min21 keep
3 min, 180–2058C, D38C min21 keep 5 min, 205–3008C, D208C min21 keep 5 min, 70 eV. In the
acid hydrolysate of operculinic acid A methyl ester, D-fucose, L-rhamnose and D-glucose were
confirmed by comparing the retention times of their derivatives with those of authentic D-fucose
(tR 30.35 min), L-rhamnose (tR 30.14 min) and D-glucose (tR 31.65 min) derivatives prepared in
the same way, respectively.
3.5.3. Preparation of Mosher’s esters
The procedures for the preparation of Mosher’s esters, which was determined the absolute
configuration of 11S of the aglycone, were same as described previously for resin glycosides
from Ipomoea batatas.
4. Conclusion
In conclusion, investigation of the aerial parts of A. acuta afforded two new compounds. Their
structures were established by different spectroscopic analyses.
Supplementary material
Supplementary material relating to this article is available online, alongside Figures S1–S17.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the National Natural Science Foundation of China [grant number 81202885];
Natural Science Foundation of Guangdong province of China [grant number S2013040013819].
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