Anti-Influenza a virus of a new oligosaccharide citric acid derivative isolated from Vigna angularis (ohwi et ohashi. var. Dainagon) Seeds

Article abstract of DOI:10.1080/07328303.2019.1615499

A new oligosaccharide citric acid derivative was isolated by hot aqueous extraction at 85 °C from Vigna angularis seeds, and it was purified using anion exchange chromatography DE-52. The structure was inferred by using UV, 1 D and 2 D NMR spectroscopy and HR-MALDI/MS, and identified as α-D-galactopyranosyl-(1→6)-α-D-galactopyranosyl-(1→6)-α-D-glucopyranosyle-(1→2)-β-D-fructofuranosyle citric acid. The isolated oligosaccharide citric acid derivative revealed promising anti-influenza A virus with 50% inhibition concentration (IC₅₀) of 0.162 nM/mL and 50% cytotoxic dose (CC₅₀) of >2.38 nM/mL.

Full text of DOI:10.1080/07328303.2019.1615499

Journal of Carbohydrate Chemistry
ISSN: 0732-8303 (Print) 1532-2327 (Online) Journal homepage: https://www.tandfonline.com/loi/lcar20
Anti-Influenza a virus of a new oligosaccharide
citric acid derivative isolated from Vigna angularis
(ohwi et ohashi. var. Dainagon) Seeds
Magdy M. D. Mohammed & Nobuyuki Kobayashi
To cite this article: Magdy M. D. Mohammed & Nobuyuki Kobayashi (2019) Anti-Influenza
a virus of a new oligosaccharide citric acid derivative isolated from Vignaꢀangularis (ohwi
et ohashi. var. Dainagon) Seeds, Journal of Carbohydrate Chemistry, 38:4, 234-245, DOI:
10.1080/07328303.2019.1615499
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JOURNAL OF CARBOHYDRATE CHEMISTRY
2019, VOL. 38, NO. 4, 234–245
https://doi.org/10.1080/07328303.2019.1615499
Anti-Influenza a virus of a new oligosaccharide citric
acid derivative isolated from Vigna angularis (ohwi et
ohashi. var. Dainagon) Seeds
Magdy M. D. Mohammeda,b
and Nobuyuki Kobayashib
aPharmacognosy Department, National Research Centre, Cairo, Dokki, Egypt; bLaboratory of
Molecular Biology of Infectious Agents, Graduate School of Biomedical Sciences, School of
Pharmaceutical Sciences, Nagasaki University, Nagasaki, Japan
ABSTRACT
ARTICLE HISTORY
Received 6 March 2019
Accepted 2 May 2019
A new oligosaccharide citric acid derivative was isolated by
hot aqueous extraction at 85 ꢀC from Vigna angularis seeds,
and it was purified using anion exchange chromatography
DE-52. The structure was inferred by using UV, 1 D and 2 D
NMR spectroscopy and HR-MALDI/MS, and identified as a-D-
galactopyranosyl-(1!6)-a-D-galactopyranosyl-(1!6)-a-D-gluco-
pyranosyle-(1!2)-b-D-fructofuranosyle citric acid. The isolated
oligosaccharide citric acid derivative revealed promising anti-
influenza A virus with 50% inhibition concentration (IC50) of
0.162nM/mL and 50% cytotoxic dose (CC50) of >2.38 nM/mL.
KEYWORDS
Anti-influenza A virus; citric
acid; fabaceae/leguminosae;
stachyose; Vigna angularis
Introduction
Vigna angularis (Ohwi et Ohashi. var. Dainagon) belongs to the family of
Fabaceae (Leguminosae). V. angularis is associated with different common
names i.e., Azuki (Adzuki) Beans or Red Mung Beans. Its origin back to
East Asia (Korea, Japan, etc.),[1] but it can be cultivated elsewhere in tem-
perate zones.[2] Seeds of V. angularis have been used by native inhabitants
for the treatment of kidney diseases, abscesses, tumors and constipation.[2]
Recently, water extract of V. angularis seeds proved promising antibacter-
ial,[3] hepatoprotective,[4] and antifungal activities.[5]
Reviewing the available literature about phytoconstituents isolated from
V. angularis seeds reflects little attention in the area. Briefly, Iida and cow-
orkers reported the isolation of three triterpenoidal saponins from its hypo-
cotyls,[6] whereas Kojima and coworkers studied the glyceroglycolipid
content[7] and oligoglycosterols and saponins[8] isolated from Azuki bean.
In
addition,
a
digalactosyl
ononitol,[9]
anthocyanins
and
CONTACT Magdy M. D. Mohammed
melhenawy111@gmail.com
Department of Pharmacognosy, National
Research Centre, Dokki-12622, Cairo, Egypt
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lcar.
Supplemental data for this article can be accessed on the publisher’s website.
ß 2019 Taylor & Francis Group, LLC

JOURNAL OF CARBOHYDRATE CHEMISTRY
235
Figure 1. HPLC chromatograms: A-Oligosaccharide hydrolysate; B-Standard sugars.
proanthocyanidins,[10–13] and sesquiterpene glucoside ‘vignoside’,[14] were
also isolated from Adzuki seeds.
Based on the fact that the soaking solution of V. angularis seeds can pro-
tect against influenza, as reported by native inhabitants who use this soak-
ing solution for flu treatment, our interest was to clarify the antiviral
activity of the hot water extract of V. angularis seeds, which revealed potent
activity with 50% inhibition concentration (IC50) of 0.036 lg/mL and 50%
cytotoxic dose (CC50) of 1.911 lg/mL. Further chromatographic purification
resulted in the isolation of a new oligosaccharide citric acid derivative iden-
tified as a-D-galactopyranosyl-(1!6)-a-D-galactopyranosyl-(1!6)-a-D-glu-
copyranosyle-(1!2)-b-D-fructofuranosyle citric acid by using extensive
UV, 1 D-, 2 D-NMR and HR-MALDI/MS. Biological evaluation of the iso-
lated oligosaccharide citric acid revealed promising anti-influenza A virus
activity with IC50 of 0.162 nM/mL and CC50 of >2.38 nM/mL.
Results and discussion
Isolation and purification of the oligosaccharide
V. angularis seeds were extracted with hot water at 85 ꢀC, and the obtained
seed water extract (SWE) was concentrated till complete dryness. SWE was
tested for its anti-influenza A virus activity. The results revealed promising

236
M. M. D. MOHAMMED AND N. KOBAYASHI
OH
O
HO
O
HO
O
O
OH
HO
HO
O
Fru
Gal-T
HO
O
O
HO
O
HO
O
HO
O
OH
Glc
HO
O
Gal-I
HO
OH
HO
OH
HMBC (H
NOESY (H
C)
H)
Figure 2. Structure of SWE-1 and selected HMBC and NOESY correlations.
activity with IC50% of 0.036 lg/mL and CC50% of 1.911 lg/mL (S1), which
encouraged us to further unveil the active constituent.
Firstly, SWE (65 mg) was prepared for rough NMR and UV analysis.
1
The preliminary results of both H and 13C NMR suggested the possible
presence of a carbohydrate moiety (S2). Anion exchange chromatography
using DE-52 was selected for further purification. Thus, SWE was uploaded
over DE-52, and the column was eluted using NaOH (1.25 M).[15] The
resulted elute SWE-1 was the one with promising anti-influenza A virus
activity, nearly the same as that of SWE. Moreover, rough NMR and UV
analysis of purified SWE-1 suggested an oligosaccharide moiety, assumed
to be a raffinose family-oligosaccharides (RFOs) based on the report of
Tsuchida et al.[16]
Acid hydrolysis of the purified oligosaccharide residue (SWE-1) revealed
the presence of galactose, glucose, and fructose, based on paper chromato-
graphic analysis and comparison with authentic samples (S13). Moreover,
HPLC analysis of the hydrolysate (SWE-1) confirmed the presence of gal-
actose, glucose, and fructose residues (Figure 1 and Figure S14) (see experi-
mental section for more details).
NMR characterization of the oligosaccharide citric acid derivative
Extensive NMR study of purified SWE-1 led to its identification as an
oligosaccharide citric acid derivative, namely, a-D-galactopyranosyl-(1!6)-
a-D-galactopyranosyl-(1!6)-a-D-glucopyranosyle-(1!2)-b-D-fructofura-
nosyle citric acid (Figure 2).

JOURNAL OF CARBOHYDRATE CHEMISTRY
237
Table 1. NMR assignments of the oligosaccharide citric acid.
1Ha
13C
DEPT & HMQC
Fru
1
2
3
4
5
6
3.43 (2H, m)
62.88
105.18
77.80
75.37
82.82
62.41
CH2
C
CH
CH
CH
CH2
3.95 (1H, d, J ¼ 5 Hz)
3.80 (1H, m)
3.62 (1H, m)
3.50, 3.54 (2H, m, overlapped)
Glc
1
2
3
4
5
6
5.24 (1H, d, J ¼ 2.4 Hz)
3.51 (1H, m)
3.82 (1H, m)
3.28 (1H, m)
3.80 (1H, m)
93.45
74.06
72.61
70.93
72.49
67.79
CH
CH
CH
CH
CH
CH2
3.43, 3.61 (2H, m, overlapped)
Galactose Internal(Gal-I)
1
2
3
4
5
6
4.79 (2H, brs)
3.71 (1H, m)
3.30 (1H, m)
3.57 (1H, m)
3.57 (1H, m)
3.47, 3.82 (2H, m, overlapped)
99.69
72.37
72.32
69.75
69.63
67.22
CH
CH
CH
CH
CH
CH2
Galactose Terminal (Gal-T)
1
2
3
4
5
6
4.79 (2H, brs)
3.90 (1H, m)
3.58 (1H, m)
3.58 (1H, m)
3.72 (1H, m)
99.39
70.08
70.88
70.66
70.55
63.79
CH
CH
CH
CH
CH
CH2
3.63 (2H, m, overlapped)
Citric acid moiety
1, 10 (CHb)
1, 10 (CHa)
1, 10 (CH2COOH)
2 (COOH)
2 OCCOOH
2.33 (2H, d, J ¼ 12.8 Hz)
2.47 (2H, d, J ¼ 12.8 Hz)
47.26
47.26
180.15
182.99
76.51
CH2
CH2
C
C
C
aAssignment based on 2 D COSY and TOCSY.
1H, 13C NMR, and HMQC spectra (S5-7) and data (Table 1) revealed the
presence of an anomeric proton appeared at dH 5.24 (1 H, d, J ¼ 2.4 Hz, H-1)
characteristic for a hexose moiety in a-configuration according to J value meas-
urement,[17] which correlated through HMQC with a carbon signal appeared
at dC 93.45, suggesting to be a glucosyl moiety (Glc) as reported.[18,19] An
anomeric H signal appeared at dH 4.79 (2H, brs) and correlated in HMQC
with two carbons displayed at dC 99.69 and 99.39 were assigned to be the
anomeric carbons of galactose moieties ꢀGal-I and Gal-Tꢁ, respectively, as
reported.[18,19] Moreover, the 1JCH coupling constants forꢀGlc, Gal-I and Gal-Tꢁ
anomeric positions were all around 170Hz, which confirmed their
a-configuration.[18,20]
Careful inspection of the 13C NMR, DEPT and HMQC spectra (Figures
S5,7,8) revealed the presence of a quaternary carbon signal appeared at dC
105.18, which was assigned as the fructose (Fru) C-2.[18,19] Two methylene
carbons were recognized at dC 62.88 and 62.41 and assigned for CH2-1 and
-6 of fructose, respectively. Furthermore, CH2-1 at dC 62.88 gave HMQC

238
M. M. D. MOHAMMED AND N. KOBAYASHI
correlation with a broad signal at dH 3.43 (2 H, m), which was assigned for
H-1 of fructose based on its HMBC correlation with C-2 at dC 105.18.
Finally, the three distinct methylene groups were appeared at dC 63.79, 67.
22, and 67.79 were assigned for CH2-6 of Gal-T, Gal-I, and Glc,
respectively.
COSY and TOCSY (Figures S9, 10), the spin resonances for the remain-
ing protons of Fru, Glc, Gal-I, and Gal-T were established (Table 1). The
identified anomeric protons represent the keys for tracing connectivity of
all ring spin-systems. The interlinkage assignments of sugar residues were
established through three-bond correlation via HMBC experiment (Figure
S11). Briefly, H-1 of Glc at dH 5.24 was correlated with C-2 of Fru as con-
firmed with its correlation with CH2-1 of Fru, confirming theꢀGlc(1!2)Fruꢁ
interlinkage. The anomeric protons of Gal-I and Gal-T at dH 4.79 revealed
two long range correlations with CH2-6 at dC 67.79 and 67.22 of Glc and
Gal-I, respectively, confirming the ꢀGal-T(1!6)Gal-I(1!6)Glcꢁ interlinkages.
Hence, we can conclude that the interlinkages in this oligosaccharie are as
followingꢀGal-T(1!6)Gal-I(1!6)Glc(1!2)Fruꢁ.
Moreover, the 13C NMR spectrum revealed the presence of a sharp signal
appeared at dC 47.26, which was classified as a methylene carbon based on
the DEPT and HMQC spectra. A strong cross-peak appeared in the
HMQC spectrum revealed the correlation of this signal at dC 47.26 with
two sets of doublets appeared at dH 2.47 and 2.33 (2 H, d, J ¼ 12.8 Hz,
1
each) in the H NMR spectrum, corresponding to a-CH1,1ꢀ and b-CH1,1ꢀ,
respectively, suggesting the possible presence of two geminal methylene
groups.[21] The DEPT and HMQC spectra showed further three more qua-
ternary carbons, categorized as: one hydroxyl carbon appeared at dC 76.51,
along with two carbonyl carbons appeared at dC 180.15 and 182.99 with as
intensity ratio 2:1, which suggested a symmetrical configuration as reported
by Mohammed et al.[22] HMBC experiment disclosed a detailed long rang
correlations of the geminal methylene protons (a-CH1,1ꢀ and b-CH1,1ꢀ)
appeared at dH 2.47 and 2.33. Through cross-peak correlation with the
hydroxyl carbon at dC 76.51, and the two carbonyl carbons at dC 180.15
and 182.99, we confirmed the presence of a citric acid moiety with a sym-
metric configuration.[23] In addition, a cross-peak appeared in the HMBC
spectrum revealed the correlation of Gal-T methylene (CH2-6) appeared at
dH 3.63 with the hydroxyl carbon of citric acid moiety at dC 76.51, which
confirmed the attachment position of the citric acid moiety on the oligosac-
charide backbone to be at the downfield shift CH2-6 of Gal-T (Figure 2).
The exact configuration of the oligosaccharide citric acid derivative was
achieved based on the J value measurements and chemical shifts of anome-
ric protons and their corresponding carbons, which suggested a structure
of a-Gal-T(1!6)a-Gal-I(1!6)a-Glc(1!2)b-Fru. This was further

JOURNAL OF CARBOHYDRATE CHEMISTRY
239
confirmed using NOESY experiment (Figure S12), which gave raise to
strong cross-peaks between proton pairs close in space. Briefly, strong
cross-peaks were observed between H1/H2, H2/H4, and H3/H5 of Glc; a
strong cross-peak was also observed between H1/H2 of both Gal moieties,
which clarifying the a-configuration of Glc, Gal-I and Gal-T.[18,19]
Furthermore, the NOESY spectrum showed that H3 of Fru correlated with
both H1 and H5 of Fru and that H1 of Fru correlated with H1 of a-Glc,
which confirmed the b-configuration of the fructofuranosyle residue.[19,20]
Finally, based on detailed comparison with reported data, the obtained
oligosaccharide derivative was identified as a-D-galactopyranosyl-(1!6)-
a-D-galactopyranosyl-(1!6)-a-D-glucopyranosyle-(1!2)-b-D-fructofura-
nosyle citric acid (Figure 2).
Anti-influenza a virus activity
The relationship between natural saccharides (poly- and oligosaccharides)
and their antiviral activity gained attention during the last decades, and it
continues till today aiming to find new therapeutics to overcome the drug-
resistant viruses.[24,25] Oligosaccharides are mainly distributed within
legumes and vegetables, which represent promising plant oligosaccharides-
based natural antiviral agents. These oligosaccharides belong to the raffin-
ose family (RFOs).[26] RFOs considered as prebiotics which impart health
effects by activating probiotics selectively (i.e., bifidobacteria).[27] Moreover,
they reported to reduce colon cancer,[28] help in immune system modula-
tion,[29] and contribute to bone health improvement.[30]
The antiviral activity of different varieties of azuki beans against rabies
virus, HSV type-1, and IFV (PR8) has been reported by Kawai and
Fujita,[31] but it was not chemically substantiated. Moreover, Yamada and
coworkers[32,33] deduced that the increase in anti-HIV activity was influ-
enced by MW differences of O-acylated derivatives of carrageenan oligosac-
charides. The increasing-solubility of oligosaccharides enhances their
potential use as antiviral agents. In addition, Lombardi and coworkers[34]
reported that citric acid could effectively inactivate low pathogenic avian
influenza virus (LPAIV) on porous and nonporous surfaces.
Accordingly, we tested the anti-influenza (IFV) activity of theꢀhot water
extractꢁ of V. angularis, which resulted in promising outcomes (IC50%
¼
0.036 lg/mL and CC50% ¼ 1.911 lg/mL). Further chromatographic investi-
gation to find out the active principle led to the isolation of stachyose citric
acid derivative SWE-1, which showed potential anti-influenza A virus activ-
ity with IC50% ¼ 0.162 nM/mL and CC50% ¼ > 2.38 nM/mL. The mode of
action by which the isolated ꢀstachyose citric acidꢁ exerts anti-IFV activity
was assumed to be affecting the viral replication cycle at the early step,[31]

240
M. M. D. MOHAMMED AND N. KOBAYASHI
based on the fact that oligosaccharides can interact with proteins on the
cell surface to prevent virus attachment, and in the case, citric acid moiety
may also be able to disinfect the lipid envelope of IFV.[34,35]
Experimental
General experimental procedures
UV was measured with a Shimadzu UV-3100PC spectrophotometer.
Specific optical rotations were measured with a “KRUSS, OPTRONIC,
1
P8000 GmbH” polarimeter. NMR: 1 D spectra for H, 13C NMR and
1
DEPT-135 and 2 D spectrum for H-1H COSY were obtained using a pulse
sequence supplied from JEOL JNM-AL-400MHz (JEOL Ltd., Tokyo, Japan)
in DMSO-d6. 2 D spectra (HMQC, HMBC, TOCSY, and NOESY) were
obtained using a pulse sequence supplied from Varian Gemini VNMR-
500 MHz NMR spectrometer (Varian, Palo Alto, CA, USA). Chemical shifts
are given in ppm relative to trimethylsilane as an internal reference. High-
resolution MALDI-MS spectra were obtained with a JOEL JMS-700N
(JEOL Ltd., Tokyo, Japan) machine for electronic ionization or on JEOL
JMS-T100 TD (JEOL Ltd., Tokyo, Japan) for electrospray ionization, using
cyano-4-hydroxycinnamic acid as a matrix (m/z 189.17). All used solvents
were of analytical grade.
Plant materials
Vigna angularis (Ohwi et Ohashi. var. Dainagon) seeds were commercially
purchased from a local market in Nagasaki.
Extraction of plant materials
V. angularis seeds (500 g) were soaked in distilled H2O (1.5 L) and 15 g of
NaHCO3 overnight, and then allowed to simmer till 85 ꢀC for 10 min. The
supernatant was collected after cooling, filtered and subjected for concen-
tration till dryness to afford 25 g of a solid material, called ꢀcrude
ꢁ
oligosaccharide. Further purification of the oligosaccharide was performed
using anion exchange resin diethylaminoethyl (DEAE) cellulose-52
(Whatman Co., Maidstone, UK). Briefly, 2.5 g of the crude oligosaccharide
was dissolved in 25 mL of distilled H2O and then uploaded onto a DEAE-
52 cellulose column (1.8 cm ꢁ 30 cm) equilibrated with distilled water. The
oligosaccharide was eluted from the resin using 5% NaOH (w/v, 1.25 M)
(50 mg NaOH þ 1 mL H2O). Elutes were monitored by the phenol-sulfuric
acid method.[36] Carbohydrate-containing fractions were dialyzed against

JOURNAL OF CARBOHYDRATE CHEMISTRY
241
deionized water, freeze-dried, and kept for further analysis. In all experi-
ments we used sample concentration of 100 mg/mL.
Acid hydrolysis of the oligosaccharide derivative
The oligosaccharide derivative (10 mg) was hydrolyzed with 5 mL of 1.5 N
H2SO4 for 10 h at 100 ꢀC in a sealed glass tube. The hydrolysate was neu-
tralized with barium carbonate, and then filtered to remove the precipitate.
The resulted filtrate was chromatographed over paper chromatography
(Whatman No. 1 mm) with glucose, galactose, and fructose as the mono-
saccharide standards (50 lg/ml, Sigma-Aldrich). The solvent systems were:
(a) n-butanol – acetic acid – water (4:1:5 v/v, upper layer) and (b) water –
saturated phenol þ 1% ammonia, sugars were detected on paper by using
silver nitrate reagent.[37]
HPLC analysis
It was performed using a Waters 2690 separation module system (Waters
Associates, Milford, MA) equipped with Waters model 996 photodiode
array detector. Chromatographic separation was carried out using YMC
Packed Steel Column. D-ODS-5, 120 Å ODS (YAMAMURA Chemical
Laboratory Co., LTD.), with a 5 lm, 3.9 ꢁ 20 mm, C18 guard column
(Waters), operated at room temperature (23 ꢀC). Mobile phase was con-
sisted of 0.5% trifluoroacetic acid in water (A) and 100% methanol (B),
with an isocratic to linear elution at a flow rate of 1 ml/min. The elution
program was as follows: isocratic elution with 30% B (0–1 min); linear gra-
dient from 30 to 50% B (1–30 min); then isocratic elution with 50% B
(30–35 min); again linear gradient 50–100% B (35–40 min); isocratic with
100% B (40–50 min); finally, linear elution 100–20% B (50–55 min). The
injection volume for oligosaccharide hydrolysate sample was prepared
(5 mg/ml) and filtered through 0.45 lm membrane. The UVvis absorption
spectra of the column eluent were recorded from 211 to 900 nm.
Cells and viruses
Mardin-Darby canine kidney cells (MDCK) were grown in minimum
essential medium (MEM) supplemented with 5% fetal bovine serum (FBS)
and 1% penicillin/streptomycin (unless otherwise stated) at 37 ꢀC in a 5%
CO2 incubator. Original virus solution was influenza virus A/WSN/33
(3.72 ꢁ 107 TCID50/mL) with 100 TCID50/well infection.

242
M. M. D. MOHAMMED AND N. KOBAYASHI
Cytopathic effect inhibition assay
MDCK cells were seeded (100 cells/well ¼ 3.0 ꢁ 104 cells/well) in 96-well
plates and cultured in MEM/10% FBS for 2 d at 37 ꢀC to >90% confluence.
Then, 40 lL of the test sample (4% solution, 2 fold dilutions) were added
to 960 lL of MEM (-), and from the mixture 120 lL/well was added to the
each well of 96-wells. Cells were then washed with FBS and infected with
approximately 50 plaque forming units (PFU) of influenza virus (1000
TCID50/mL). Aliquot of the cell suspension (100 lL) was added to each
well of a 96-well flat-bottomed microtitre tray containing 100 lL of various
concentrations of the test sample.
After 3 d of incubation at 37 ꢀC in 5% CO2, the number of viable cells
was determined by the MTT method.[38] The cytotoxicity was evaluated in
parallel with the antiviral activity, which was based on the viability of
mock-infected cells, as monitored by the MTT method. The antiviral IC50
and CC50 of the sample were determined. The absorbances were deter-
R
V
mined with Tecan Infinite 200 PRO Modular Microplate Readers at a test
wavelength of 560 nm.
Compound characterization
a-D-Galactopyranosyl-(1!6)-a-D-galactopyranosyl-(1!6)-a-D-glucopyra-
nosyle-(1!2)-b-D-fructofuranosyle citric acid was a whitish amorphous
solid: [a]2D0 ¼ þ40ꢀ (c 0.025, H2O); UV absorption band at 195 nm (S4)
characteristic for a carbohydrate moiety;[39] HR-MALDI/MS (S3), at m/z
866.25125 [M þ Na þ 3H]þ (calcd 866.25099 for C30H51O27Na).
Acknowledgements
The authors are grateful to the National Research Center, Cairo, Egypt, for funding the experi-
mental work, including specific optical rotation, TOCSY and HMBC analysis [Project (No.
AR110210)], and to the Nagasaki University for providing facilities for instrumental analysis.
Conflict of Interest: The authors declare that they have no conflict of interest.
ORCID
Magdy M. D. Mohammed
http://orcid.org/0000-0001-5606-3498
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