Water-soluble polysaccharides from finger citron fruits (Citrus medica L. var. sarcodactylis)

Article abstract of DOI:10.1016/j.carres.2013.12.020

Four water-soluble polysaccharides, FCp-1, FCp-2, FCp-3, and FCp-4 were obtained from finger citron fruits (Citrus medica L. var. sarcodactylis) by hot-water extraction and ethanol precipitation, followed by routine separation procedure. Based on the calibration curve, molecular weights of them were estimated to be 113.9, 32.6, 140.3, and 177.1 kDa respectively. The acid hydrolysis, methylation, IR, GC-MS, and NMR experiments were used for composition analysis. FCp-1 was a heteropolysaccharide composed of arabinose, galactose, glucose, rhamnose, and xylose, with a molar ratio of 3.0:7.0:4.1:1.0:1.5. FCp-2 and FCp-4 were →4)-α-d-GalpA(1→ linking galacturonan differ in molecular weights. FCp-3 was a →6)-α-d-Glcp(1→ linking glucan. According to the results of in vitro assays, FCp-3 showed significantly and moderately enhancing capacities toward the proliferation of splenocytes and thymocytes respectively. Thus, FCp-3 or analogs may have further use as immunomodulatory agents.

Full text of DOI:10.1016/j.carres.2013.12.020

Carbohydrate Research 388 (2014) 100–104
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Water-soluble polysaccharides from finger citron fruits
(Citrus medica L. var. sarcodactylis)
Zhengchun He a,b, Fengjie Liang a, Yuyang Zhang a, Yuanjiang Pan a,
⇑
a Department of Chemistry, Zhejiang University, Hangzhou 310027, People’s Republic of China
b Department of Pharmacy, Dali College, Dali 671000, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 October 2013
Received in revised form 19 December 2013
Accepted 27 December 2013
Available online 8 January 2014
Four water-solublepolysaccharides, FCp-1,FCp-2,FCp-3,andFCp-4 were obtained from finger citronfruits (Citrus
medica L. var. sarcodactylis) by hot-water extraction and ethanol precipitation, followed by routine separation pro-
cedure. Based on the calibration curve, molecular weights of them were estimated to be 113.9, 32.6, 140.3, and
177.1 kDa respectively. The acid hydrolysis, methylation, IR, GC–MS, and NMR experiments were used for
composition analysis. FCp-1 was a heteropolysaccharide composed of arabinose, galactose, glucose, rhamnose,
and xylose, with a molar ratio of 3.0:7.0:4.1:1.0:1.5. FCp-2 and FCp-4 were ?4)-
a-D-GalpA(1? linking galactur-
Keywords:
Citrus medica L. var. sarcodactylis Hort
Polysaccharide
onan differ in molecular weights. FCp-3 was a ?6)- -Glcp(1?linking glucan. According to the results of in vitro
a-D
assays, FCp-3 showed significantly and moderately enhancing capacities toward the proliferation of splenocytes
and thymocytes respectively. Thus, FCp-3 or analogs may have further use as immunomodulatory agents.
Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved.
Immunological activity
The finger citron (Citrus medica L. var. sarcodactylis Hort) (FC) be-
longstothefamilyRutaceae, andiswidelycultivatedinOrientalcoun-
tries. FC fruit is used in traditional Chinese medicine as adjuvants in
the treatment of a variety of chronic diseases, including hypertension
and respiratory tract infections,1,2 and are also consumed as func-
tional foods, being considered beneficial to liver, pancreatic, and
stomach function.3 Previous studies have shown FC fruit contains sig-
nificant quantities of essential oil, with this material possessing anti-
oxidant activity that could possibly be commercially exploited,3,4 and
which have also demonstrated insulin secretagogue and anti-inflam-
matory activities.5 However, a detailed analysis of the structural and
biological properties of polysaccharides derived from FC fruit has yet
to be reported, except for research on extraction methods of crude
polysaccharide.3 Given the presence and diverse biological activities
of polysaccharides throughout nature,6–11 and their use in many
healthcare products, we decided to investigate the polysaccharide
profile of FC fruit. In this paper, we report the fractionation (Fig. 1),
compositional analysis, structural characterization, and immunolog-
ical activity of the polysaccharides (Fig. 2) from the FC fruit.
from KBr pellets in the 4000–400 cmÀ1 range on a Bruker Alpha-T
FT-IR instrument. 1H and 13C NMR spectra of 50 mg compound
samples were recorded at 25 °C in D2O on a Bruker 500 MHz spec-
trometer. Chemical shifts (d) are reported in ppm relative to Me4Si
(dH 0.0). Gas chromatography–mass spectrometry (GC–MS)
analyses were performed on a Thermo Fisher Scientific TRACE
DSQ Single Quadrupole GC–MS instrument, using an Agilent
HP-5 column (30 m  0.25 mm i.d). The ionization potential was
70 eV, and the temperature of the ion source was 200 °C.
1.2. Plant material
Fresh FC fruits (Citrus medica L. var. sarcodactylis Hort) were col-
lected from Jinhua, Zhejiang Province, China, in March 2012, and
identified by Dr. Ende Liu (Kunming Institute of Botany, Chinese
Academy of Sciences, Kunming, Yunnan Province, China).
1.3. Extraction and isolation of polysaccharides FCp-1, FCp-2,
FCp-3, and FCp-4
Whole FC fruit (1.2 kg) was ground to a pulp, and the resulting
material was divided into three portions. Each portion was treated
with deionized water (2.0 L), and the resulting mixtures were
boiled at 100 °C for 3 h and then filtered. The combined filtrates
were concentrated to dryness under reduced pressure at 50 °C.
The residue (210 g) was suspended in deionized water (800 mL)
and washed successively with petroleum ether, EtOAc and finally
n-BuOH. The remaining aqueous layer was concentrated to a vol-
ume of 200 mL under reduced pressure at 50 °C, and the resulting
1. Experimental
1.1. General procedures
Ultraviolet–visible (UV–vis) spectra were recorded on a SHIMA-
DZU UV-1800 UV–vis spectrophotometer. IR spectra were recorded
⇑
Corresponding author. Tel.: +86 571 87951264.
E-mail address: cheyjpan@zju.edu.cn (Y. Pan).
http://dx.doi.org/10.1016/j.carres.2013.12.020
0008-6215/Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved.

Z. He et al. / Carbohydrate Research 388 (2014) 100–104
101
A sample of FCp (1 g) was dissolved in deionized water (5 mL),
and the mixture was centrifuged. The supernatant was separated,
and then loaded onto a DEAE-Sepharose Fast Flow (Pharmacia)
chromatography column (2.6 Â 40 cm), and eluted successively
with deionized water, 0.1 M NaCl, 0.2 M NaCl, and 0.3 M NaCl (each
300 mL). The proportion of polysaccharide in each of these eluents
was determined by the phenol–sulfuric acid colorimetric
method.14 This analysis revealed that the desired polysaccharide
resided in the three NaCl eluents, and dialyzed in 3500 Da MWCO
tubing in deionized water at 37 °C for 48 h, respectively. The
solution remaining within the dialysis membrane was then con-
centrated to dryness under reduced pressure, and the residue
freeze-dried to give three crude solid. These materials were further
purified on a Sephadex G-100 (Pharmacia) gel-filtration chroma-
tography column (2.6 Â 40 cm) using 0.01 M NaCl as an eluent.
Appropriate fractions were combined, dialyzed, and freeze-dried
as before, to yield four homogeneous polysaccharides, denoted
FCp-1 (56 mg), FCp-2 (84 mg), FCp-3 (64 mg), and FCp-4
(220 mg) (Fig. 2).
Finger citron fruits
Extraction (Hot water)
Precipitation (Ethanol)
Deproteinization (Sevag reagent)
Decoloration (Macroreticular resin)
FCp
DEAE-Sepharose F F column
Water
0.1 M NaCl
FCp-A
0.2 M NaCl
FCp-1
FCp-2
0.3 M NaCl
FCp-4
FCp-3
Figure 1. Fractionation procedure of the polysaccharides.
1.4. Determination of purity and molecular weight of FCp-1,
FCp-2, FCp-3, and FCp-4 by high-performance gel-permeation
chromatography (HPGPC)
solution was added to 95% aqueous EtOH (3.0 L). This mixture was
left to stand for 12 h, and the resulting crude polysaccharide pre-
cipitate was collected by filtration. This precipitate was freeze-
dried to yield a crude polysaccharide mixture (18.5 g), a portion
(10 g) of which was then re-dissolved in deionized water
(300 mL). This solution was deproteinated by treatment with Sevag
reagent (chloroform/n-butanol at a ratio of 4:1, v/v), according to
literature methods.12 The deproteinated solution was decolorized
by passing through a macroreticular anion-exchange resin column
(D315) and washed with double column volume deionized water,
and the resulting solution was concentrated to a volume of
100 mL.13 This solution was dialyzed in 3500 Da MWCO (molecular
weight cut-off) tubing at 37 °C for 48 h, and the solution remaining
within the dialysis membrane was freeze-dried to yield polysac-
charide (FCp; 5.5 g).
HPGPC was used to determine the purity of FCp-1, FCp-2,
FCp-3, and FCp-4. Analyses were conducted on a Waters 515
instrument (Milford, PA, USA) equipped with a Waters 2410
refractive index detector and a TSKgel G4000SWxl (Tosoh Biosep,
Japan) size-exclusion analytical column (7.8 Â 300 mm), using
deionized water as the mobile phase and with a sample concen-
tration of 3 mg/mL. T-series dextran standards (Sigma–Aldrich)
with defined molecular masses ranging from 10 to 300 kDa were
used to calibrate the HPGPC system. All data obtained
were collected and analyzed using the Waters MillenniumÒ32
software package.
Figure 2. HPGPC profiles for obtained polysaccharides FCp-1 (PD = 1.16), FCp-2 (PD = 1.06), FCp-3 (PD = 1.22), and FCp-4 (PD = 1.41). (PD = Mw/Mn; PD, polydispersity; Mw,
weight-average molecular mass; Mn, number-average molecular mass).

102
Z. He et al. / Carbohydrate Research 388 (2014) 100–104
1.5. Component analysis of polysaccharides FCp-1, FCp-2, FCp-3,
and FCp-4
well and the plates were incubated for a further 4 h. The superna-
tant was then removed by aspiration, and the resulting formazan
crystals were dissolved in DMSO (150 lL). The absorbance of these
Samples of polysaccharides FCp-1 to FCp-4 (each 5 mg) were
hydrolyzed in 2 M trifluoroacetic acid (TFA; 5 mL) at 120 °C for
6 h, and then evaporated to dryness under reduced pressure. The
residue was divided into two portions. One portion was used for
paper chromatography (PC), which was performed on Whatman
3 MM filter paper, eluting with either (a) EtOAc/HOAc/H2O
(3:3:1) or (b) n-BuOH/pyridine/H2O (6:4:3) solvent systems.
Papers were visualized by aniline–diphenylamine–phosphoric acid
solution (a) or p-anisidine solution (b),15 and authentic standards
DMSO solutions was measured at 570 nm with a plate reader.19,20
Proliferation of splenocytes (% Splenocyte Proliferation Index or %
SPI) and thymocytes (% Thymocyte Proliferation Index or % TPI)
was expressed as mean standard deviations. All experiments
were conducted in quadruplicate, and repeated three times.
2. Results and discussion
Four water-soluble polysaccharides, FCp-1, FCp-2, FCp-3, and
FCp-4 were obtained from FC by hot-water extraction and ethanol
precipitation, followed by routine separation procedure. Based on
the calibration curve, molecular weights of them were estimated
to be 113.9, 32.6, 140.3, and 177.1 kDa respectively (Fig. 2). Sam-
ples were all white powders, and did not absorb at 280 and
260 nm in the UV–visible spectrum, suggesting the absence of
protein and nucleic acid. Samples also did not cause darkening of
iodine solution, suggesting the absence of starch.
Further chemical analysis of them was conducted by treating
samples with 2 M TFA at 120 °C for 8 h. PC of the resulting solu-
tions showed that only galacturonic acid was present in FCp-2
and FCp-4, which was confirmed by examination of its NMR spec-
tral data. Glucose was found to be present in FCp-3, which was fur-
ther confirmed by GC–MS and NMR analysis (see Supplementary
information). FCp-1 was found to be composed of arabinose,
galactose, glucose, rhamnose, and xylose, in a molar ratio of
3.0:7.0:4.1:1.0:1.5, This corresponded to the results of GC–MS
(Table 1). The absolute configuration of the polysaccharides was
determined by GLC of their acetylated glycosides, using (+)-2-buta-
nol, as per the literature method.21 The results showed that both
galacturonic acid in FCp-2 and FCp-4 and glucose in FCp-3 have
(L-arabinose,
D
-glucose, -galactose,
D
-mannose,
D
D-ribose, D-xylose,
L-rhamnose,
D-glucuronic acid and D-galactonic acid) were used as
reference compounds. The other portion of crude residue was trea-
ted with NaBH4 (15 mg), and the resulting mixture stirred at room
temperature for 12 h. The reaction mixture was then treated with a
mixture of Ac2O and pyridine (1.0 mL of each) at 120 °C for 6 h,16
and then concentrated to dryness under reduced pressure. The
resulting alditol acetates were re-dissolved in CH2Cl2 (1 mL) for
GC–MS analysis, from which their structures were identified by
their retention times and electron-impact profiles.
1.6. Methylation analysis of polysaccharides FCp-1 and FCp-3
Per-O-methylation of polysaccharide samples FCp-1 and FCp-3
was performed in a dry solution of DMSO and CH3I, using NaOH as
a catalyst.17 To a solution of polysaccharide (20 mg) in dry DMSO
(5 mL) and under a positive pressure of dry N2 was added dry
NaOH powder (200 mg), and the resulting mixture was stirred
overnight at room temperature. Subsequently, CH3I (2 mL) was
slowly added to the reaction mixture, and the resulting mixture
stirred for a further 4 h at room temperature. At this time, deion-
ized water (1 mL) was added to stop the reaction, and the mixture
was subjected to dialysis and freeze-drying conditions (as previ-
ously described). This whole process was repeated three times, un-
til the resulting residue displayed no –OH absorption band in the IR
spectrum, at which point the mixture was hydrolyzed by treat-
ment with 2 M TFA (5 mL) at 120 °C for 8 h. The residue was then
reduced with NaBH4 (15 mg) at room temperature and followed by
adding Ac2O and pyridine (1.0 mL of each) to acetylate at 120 °C for
8 h, then concentrated to dryness under reduced pressure. The
resulting partially methylated alditol acetates were re-dissolved
in CH2Cl2 (1 mL) for further GC–MS analysis.
the
FCp-1 have the
have the -configuration.
D
-configuration. And the galactose, glucose, and xylose in
D
-configuration, while the arabinose and rhamnose
L
The IR spectrum of FCp-2 and FCp-4 had strong absorption
bands at 3425, 2938, 1612, 1416, 1331, 1100, 1088, 1016, 953,
835, and 595 cmÀ1. The strong band at 1612 cmÀ1 was due to the
carbonyl group, while the band at 2938 cmÀ1 was ascribed to a
C–H stretching vibration. The strong band at 3425 cmÀ1 was
Table 1
Profile of partially O-methylated alditol acetates obtained from methylation analysis
of FCp-1 and FCp-3
1.7. Assay of effect of polysaccharides FCp-1, FCp-2, FCp-3, and
FCp-4 on splenocyte and thymocyte proliferation
Compound
O-Me-alditol acetate
mol (%)
FCp-1
2,3,5-Me3-Ara
2,3,4-Me3-Xyl
2,3,4-Me3-Ara
2,3,4-Me3-Rha
2,4-Me2-Ara
2,3,4,6-Me4-Glc
2,3-Me2-Ara
2,3,4,6-Me4-Gal
2,3-Me2-Xyl
2,3-Me2-Rha
2-Me1-Rha
2,3,6-Me3-Gal
2,3,6-Me3-Glc
2,3,4-Me3-Gal
2,3,4-Me3-Glc
3,6-Me2-Gal
2,3-Me2-Glc
2,3-Me2-Gal
2,4-Me2-Gal
1
2
1
1
11
1
5
2
7
4
1
3
7
12
14
1
3
8
Cells of the spleen and thymus were prepared from immunized
mice under aseptic conditions by homogenization in Hank’s
balanced salt solution (HBSS),18 followed by addition of Tris–NH4Cl
solution to lyse the erythrocytes. The resulting solution was centri-
fuged, and the pellet was re-suspended in complete RPMI 1640
(Sigma–Aldrich) medium, and the cell concentration was adjusted
with medium to 1 Â 106 cells/mL. The viability of cells was esti-
mated as being >90% by the Trypan Blue dye exclusion method.
Next, a sample of cells (180
well microtiter plate, and incubated with 20
trations (12.5, 25, 50, 100 and 200 g/mL) of samples FCp-1, FCp-2,
lL) was seeded in each well of a 96-
l
L of various concen-
l
FCp-3, and FCp-4 at 37 °C in a 5% CO2 incubator. Lipopolysaccha-
ride (LPS, L6511 of Salmonella enterica serotype Typhimurium, Sig-
ma–Aldrich, 4
Canavalia ensiformis, Sigma–Aldrich, 10
l
g/mL) and Concanavalin A (Con A, L7647 of
g/mL) were used as posi-
l
16
tive controls for splenocyte and thymocyte stimulation assays,
respectively. After 48 h, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyl tetrazolium bromide] (20 lL, 5 mg/mL) was added to each
FCp-3
2,3,4,6-Me4-Glc
2,3,4-Me3-Glc
Trace
100

Z. He et al. / Carbohydrate Research 388 (2014) 100–104
103
attributed to a hydroxyl stretching vibration, and the characteristic
absorption signal at 835 cmÀ1 indicated the presence of
a-pyra-
noid-type glycosidic linkage.22–24 In the IR spectrum of FCp-3, a
hydroxyl stretching vibration band was seen at 3422 cmÀ1, and a
C–H stretching vibration band at 2935 cmÀ1. The presence of three
absorption bands at 1098, 1084, and 1021 cmÀ1 indicated the
presence of a pyranoid ring, while the characteristic absorption
band at 838 cmÀ1 suggested the presence of
a glycosidic linkage
of the polysaccharide.22–24
GC–MS of per-O-methylated samples of FCp-3 showed one
main peak, which corresponded to 2,3,4-tri-O-methyl-Glc, while
a series of peaks were present in the GC–MS profile of FCp-1
(Table 1).
In the 1H NMR spectra (see Supplementary information), FCp-2
and FCp-4 displayed five strong proton signals at dH 5.09, 4.82,
4.44, 4.02, and 3.79. Six strong carbon signals at dC 175.2 (C6),
98.9 (C1), 77.7 (C4), 71.1 (C5), 68.6 (C3) , and 67.9 (C2) could be
observed in the corresponding 13C NMR spectrum, with the most
downfield of these obviously representing the C6-carbonyl group
of the alduronic acid unit, which was corresponding to PC results.
And heteronuclear correlations observed in the HMQC spectrum
(see Supplementary information) further confirmed FCp-2 and
FCp-4 as being ?4)-a-D-GalpA(1? linked galacturonans.
FCp-3 had seven groups of signals at dH 5.00, 4.00, 3.92, 3.77,
3.73, 3.59, and 3.54 in its 1H NMR spectrum. In its 13C NMR spec-
trum, six strong carbon signals at dC 97.6 (C1), 71.3 (C2), 73.3
(C3), 69.4 (C4), 70.1 (C5), and 65.5 (C6) could be observed, with
the most downfield of these being the anomeric carbon, which cor-
relates with the anomeric proton at dH 5.00 in the HMQC spectrum.
The two methylene protons (dH 4.00 and 3.77) of C6 (dC 65.5) were
coupled to the anomeric carbon at dC 97.6, demonstrating the
location of the connecting sugar unit, which could also be clearly
observed in the HMBC spectrum. The correlations of other protons
in the sugar unit with their neighboring carbon atoms were also
observed in the HMBC spectrum, indicating that the glucose units
Figure 3. Effects of different concentrations of FCp-1 and FCp-3 on splenocytes (A)
and thymocytes (B) proliferation; LPS (lipopolysaccharide) and Con A (concanavalin
A) were as positive control (⁄Significantly different from PBS control, p < 0.05).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.carres.2013.
12.020.
were linked in 1 ? 6 position in a ?6)-a-D-Glcp(1? linking mode.
This corresponded to the results of GC–MS.
In FCp-1, the anomeric protons signals were present at dH 5.0–5.5
in the 1H NMR spectrum, and the other protons (except for methyl
protons of rhamnose units) were at dH 3.2–4.8. In the 13C NMR
spectrum, anomeric carbons were present at dC 98.0–110, and other
carbons (except for methyl carbon of rhamnose units at dC 16.37)
were distributed in dC 60.0–85.0 (see Supplementary information).
The immunological activity of FCp-1, FCp-2, FCp-3, and FC-p4
was evaluated by splenocyte and thymocyte proliferation experi-
ments. The results are represented as mean standard deviation,
and are compared against the activity of a phosphate-buffered sal-
ine (PBS) control, according to a literature method.19 FCp-2 and
FCp-4 exhibited no effect on splenocyte or thymocyte proliferation,
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