International Journal of Biological Macromolecules 85 (2016) 505–513

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Immunostimulating activity of polyhydric alcohol isolated from Taxus

cuspidata
Choon Guen Lee a,1 , Jisun Lee b,1 , Da Gyung Lee a , Joo Won Kim a , Mawadda Alnaeeli c ,
Yong Il Park b , Jae Kweon Park a,∗
a
Department of Life Sciences, Gachon University, Seongnamdaero 1342, Seongnam-si, Gyeonggi-do 461-701, South Korea
b
Department of Biotechnology, The Catholic University of Korea, Bucheon 420-743, South Korea
c
Division of Infectious Diseases, School of Medicine, University of Louisville, Louisville, KY 40202, USA

a r t i c l e i n f o a b s t r a c t

Article history: A polyhydric alcohol (PAL) was isolated from Taxus cuspidata and its immunostimulatory activities were
Received 9 December 2015 assessed. The primary monosaccharide composition of the PAL was determined to be glucose, where
Received in revised form 4 January 2016 HPAEC analysis showed no significant amount of any other sugars. However, glycerol and xylitol were
Accepted 6 January 2016
identified as the main sugar alcohols. Fourier-transform infrared (FT-IR) analysis indicated that the
Available online 11 January 2016
purified PAL is a complex glycitol, which structurally contains significant amount of hydroxyl groups.
MALDI-TOF mass spectroscopy also demonstrated that PAL is a complex glycitol built in hexose poly-
Keywords:
merization. Enzyme linked immunosorbent assay showed that the PAL stimulates the release of the
Taxus cuspidata
Polyhydric alcohol
proinflammatory cytokines TNF-␣ and IL-6 in a dose-dependent manner. Furthermore, treatment of
Immune-stimulation RAW 264.7 cells with PAL for 24 h remarkably increased the phosphorylation levels of ERK, p38 and JNK
in a dose-dependent manner, whereas the total protein levels of ERK (t-ERK), p38 (t-p38) and JNK (t-
JNK) remained unchanged. These results clearly demonstrate that PAL stimulates the immune response
in RAW 264.7 cells through the activation of MAPKs (ERK, p38 and JNK) signaling pathway. To the best
of our knowledge, this is the first study to demonstrate the primary structure and immune-stimulating
activities of PAL from the fruit of T. cuspidata.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction
In commercial foodstuffs, sugar alcohols (SALs) are commonly
implicated as monosaccharide based alcohols such as sorbitol,
xylitol and maltitol, and used in place of table sugar (sucrose)
worldwide to counter the low sweetness [1]. Unlike common sug-
ars, however, it is believed that SALs do not contribute to the
formation of tooth cavities [2], due to their poor metabolism by
oral bacteria. Since SALs are not as sweet and have less food energy
than sucrose, SALs can be used to mask the unpleasant aftertastes
of some sweeteners [3,4]. The simplest SALs such as ethylene gly-
col is known as a notoriously toxic chemical, which is used as a
reagent of the antifreeze. Among SAL, xylitol is perhaps one of the
most popular that is obtained from natural sources due to its simi-
larity in visual appearance and sweetness to sucrose. SALs are often
obtained by hydrogenation of sugars under specific chemical pro-
∗ Corresponding author. Fax: +82 317508573.
E-mail addresses: jkpark@gachon.ac.kr, jamyeong-2@nate.com (J.K. Park).
1
These authors contributed equally to this work.
cess using immobilized cultures [5,6]. Recently, more than a million
tons of sorbitol has been produced through this process [7]. More-
over, xylitol has also been obtained using similar processes [5,8].
In contrast, polyhydric alcohols called PALs, also called [polyol,
polyalcohol, or glycitol], which are generally water-soluble solids
of multi-component of SALs that occur naturally. It is known that
the more complex PALs are for the most part nontoxic [9]. There-
fore PALs must be distinguished from the common SALs depending
on the size and complexity. Apart from industrial uses, PALs have
emerged in recent years as a rich and important source of bioactive
natural compounds. However less is known about the relationship
between the structural complexity and biological activities of PALs
[9]. For these reasons, isolation and characterization of these PALs
or unique PALs have been increasingly important topics for research
focus.
Seeking a unique sugary source from several candidates, Taxus
cuspidata came into focus. It is considered a potent plant produc-
ing several kinds of biological substances that may have potential
anticancer, antioxidant, and immunostimulating activities. T. cusp-
idata has been extensively investigated as a result of the discovery
of taxol, a chemical compound isolated from its stem barks with its

http://dx.doi.org/10.1016/j.ijbiomac.2016.01.027
0141-8130/© 2016 Elsevier B.V. All rights reserved.
506 C.G. Lee et al. / International Journal of Biological Macromolecules 85 (2016) 505–513
effective anticancer activity for breast and ovarian cancers world-
wide [10–12]. Although T. cuspidata has been used as a traditional
medicine, and the extensive studies on the chemistry of T. cuspidata
have been carried out [12], less is known about the immunostim-
ulating activity. Therefore, the aim of this research is to isolate the
glycans from T. cuspidata and evaluate its biological activities.
Immune-stimulation is regarded as one of the important strate-
gies to enhance the physical defense systems in patients having
an impaired immune response, including the elderly and cancer
patients [13]. The decline in immune function with aging results
in heightened susceptibility to infection and reduced vaccine effi-
cacy [14,15]. In cancer patients, it is also important that immune
cells such as natural killer cells and macrophages are activated
for host defense against tumor cell growth [16]. Thus, search-
ing for effective immunostimulators that can activate the immune
response of these cells has been an increasingly important topic of
research. Many studies have previously reported on the immunos-
timulating activities of natural compounds or extracts, including
flavonoids and polysaccharides [17–19]. Macrophages are strate-
gically located throughout the body tissues and are key players in
the innate and adaptive immune responses, which are essential
for the support of homeostasis and host defense against intra-
cellular parasitic bacteria, pathogenic protozoa and fungi as well
as against tumors, especially metastatic tumors [20,21]. In these
immune responses, activated macrophages produce large quanti-
ties of the proinflammatory molecules, including nitric oxide (NO),
tumor necrosis factor-␣ (TNF-␣), and interleukin-6 (IL-6), which
play a major role in the regulation of the immune system, inflam-
mation and oncogenesis [22]. Despite the vast number of extensive
studies performed focusing on this topic, there has not been enough
attention paid to the SALs or PALs in T. cuspidata berry fruit. This
is probably because of the fact that the quantity of one of the SALs
or PALs constitutes a small percentage of the total carbohydrates
contained in T. cuspidata berry fruit, most of which would be in
glucose-like glycans. In addition, it is difficult to purify and define
the complete structure of molecules, showing biologically different
and significant activities. Since many of these sugars and SALs are
available commercially as simple supplements to one’s diet as well
as for improving the immune response, the importance of under-
standing the nutritional intakes of the free sugars, SALs and/or PALs
has increased in during several decades. Therefore, the goal of the
present study was to isolate and characterize the primary structure
of PALs and their biological activities. Herein, we demonstrate that
the T. cuspidata-derived glycan designed to PALs is able to enhance
immunostimulating activity in murine RAW264.7 macrophages.
2. Materials and methods
2.1. Materials
Monosaccharides, sugar alcohols and trifluoroacetic acid (TFA)
were purchased from Sigma Chemical Co., (St. Louis, MO, USA). Sil-
ica gel 60/Kieselguhr F254 thin-layer chromatography (TLC) plates
were purchased from Merck KGaA (Darmstadt, Germany) and other
materials were also purchased from Samchun Chemical Co., Ltd.,
(Gyeonggi-do, Korea), or Sigma Chemical Co., (St. Louis, MO, USA)
in analytical grade. For the spectorphotometric analysis, ELISA
(Quant, BIO-TEK instruments, Inc., Winooski, VT, USA) was used
for the measurement of absorbance to calculate the relative quan-
tity of sugars. Fetal bovine serum (FBS), penicillin–streptomycin,
and trypsin–EDTA were purchased from Lonza (Walkersville,
MD, USA). 3-(4,5-Dimethylthiazol-yl)-diphenyl tetrazoliumbro-
mide (MTT) was provided by DUCHEPA Biochemie (Haarlem,
Netherlands). Dulbecco’s modified eagle’s media (DMEM) were
purchased from WelGENE (Seoul, Korea). The antibodies against
inducible nitric oxide synthase (iNOS) total p38 (t-p38), phospho-
p38 (p-p38), total ERK (t-ERK), phospho-ERK (p-ERK), total-JNK
(tJNK), phospho-JNK (p-JNK), ␤-actin, goat anti-mouse IgG-HRP
and goat anti- rabbit IgG-HRP were obtained from Cell Signaling
Technology (Danvers, MA, USA).
2.2. Collection of T. cuspidata berry fruit
T. cuspidata berry fruits were collected in InCheon-city
(126:38E/37:28N), South Korea, and used for the isolation of water-
soluble sugars. T. cuspidata berry fruits (50 g) were washed twice
with distilled water and rinsed quickly with pre-cooled ethanol
at −20 ◦ C. Air-dried fruits were then separated into five groups
and allowed to stand in 50 mL of n-hexan, iso-propanol, acetone,
methanol and distilled water for over-night at room temperature,
respectively. On the other hand, same amount of T. cuspidata berry
fruits were transferred to the solvents in step-wise method to
separate water-soluble glycans depending on the solubility. After
centrifugation for 30 min at 8,000 × g (Hanil, Korea), the resulting
precipitate was removed and the supernatant was collected for
further study.
2.3. Purification of water-soluble glycan
The crude glycans in the supernatant was further lyophilized
(Shin Il, Co., Ltd., Korea). Fifty milligrams of crude glycans were
dissolved in 5.0 mL of distilled water and was applied to a size-
exclusion column (1 × 25 cm) pre-equilibrated extensively with
distilled water. Each fraction (3 mL) of chromatography was then
assayed by phenol–sulfuric acid method to collect he carbohydrate-
positive fractions, which were pooled together and lyophilized. In
order to observe the purity of the water-soluble glycan, a thin layer
chromatography (TLC) using Silica gel 60/Kieselguhr F254 thin-layer
plate was performed using the solvent consisting of 1-propanol,
25% NH4 OH, H2 O (7:1:2, v/v). Sugar or sugar compounds on TLC
were detected under UV 254/365 nm or confirmed by baking at
200 ◦ C using a detecting reagent consisting of 3% cupric acetate and
8% H2 PO4 in distilled water.
2.4. Monosaccharide composition of water-soluble glycan
The monosaccharide composition of the purified water-soluble
glycan was analyzed by HPAEC after acidic hydrolysis with 2 M
trifluoroacetic acid (TFA) at 100 ◦ C for 4 h. In order to remove
the residual acid, dissolving of hydrolysates in distilled water
and repeating vacuum drying using a Speed-Vac (Biotron, Korea)
were performed. Hydrolysates from glycan were analyzed using
a CarboPac PA-100 column by HPAEC-PED (high performance
anion-exchange chromatography—pulsed electrochemical detec-
tion), using a Dionex DX600 chromatography system (Dionex Corp.,
Sunnyvale, California) with a pulsed electrochemical detector
(PED50), following the instructor’s instruction. Also sugar alcohols
(PALs) of glycan from T. cuspidata berry fruit were analyzed under
the same conditions, performed at 0.8 mL/min with the concentra-
tion gradient of 1 M NaNO3 in a constant concentration of NaOH
(0.2 M). Commercialized monosaccharide and sugar alcohols were
used as standard substances.
2.5. MALDI-TOF mass analysis and Fourier-transform infrared
(FT-IR) spectroscopy assessment
The average molecular weight of the PALs purified from T. cus-
pidata berry fruit was determined based on matrix-assisted laser
desorption/ionization-time of flight mass spectrometry (ABSCIEX)
(4800 MALDI TOF/TOF instrument, InCheon Technopark Bio Cen-
ter, Korea) analysis with 4000 series explorer v3.5 software. The
 C.G. Lee et al. / International Journal of Biological Macromolecules 85 (2016) 505–513
␣-Cyano-4-hydroxycinnamic acid (CHCA) matrix solution was pre-
pared at 10 mg/mL in 50% acetonitrile (CAN) containing 0.1%
trifluoroacetic acid (TFA). Subsequently, sample was mixed with
CHCA matrix solution (1:1) for MS analysis. Data were acquired in
positive reflector mode over a mass range of 800–8000 m/z, for a
total of 1050 laser shots by an Nd:YAG laser operating at 355 nm
and 200 Hz using external calibration spots with TOF/TOF cal mix.
Data explorer (ABSCIEX) was used to process MS data.
FT-IR spectrum (spectral region 4000–400 cm−1 , and resolution
4 cm−1 ) of the PALs dissolved in distilled water using ZnSe windows
with 0.1 mL of the PALs solution was recorded by accumulation of
at least 32/64-scans on FTIR-7600 spectrometer (Lambda scientific
Pty., Ltd., Austria)
2.6. Cell culture
The murine macrophage cell line RAW 264.7 was purchased
from the Korea Cell Line Bank (Seoul, Korea) and grown in DMEM
(WelGENE, Seoul, Korea) supplemented with 10% (v/v) FBS, 2 mM
l-glutamine and 100 U/mL penicillin/streptomycin (PEST). The cells
were incubated with varying concentrations (25–200 ␮M) of T. cus-
pidata for 24 h. Lipopolysaccharide (LPS) was used as a positive
control for activating the macrophages.
2.7. Cytotoxicity assay
The cytotoxicity of T. cuspidata was assessed by measuring
the cell viability using the 3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide (MTT) assay. The cells (5 × 104
cells/well) precultured in DMEM were loaded on 96-well
microplates. Different concentrations (25–200 ␮M) of T. cuspidata
were prepared by serial dilutions. After 24 h of incubation, 20 ␮l of
MTT solution (5 mg/ml) was added to each well, and the plates were
further incubated for 4 h. After the removal of the media, 200 ␮l of
DMSO was added into each well to dissolve the formazan crys-
tals. The absorbance was read at 570 nm using a microplate reader
(Molecular Devices Co., CA, USA).
2.8. Assay for TNF-˛ and IL-6 secretion
The concentration of TNF-␣ and IL-6 released from the cells
treated with T. cuspidata was determined using Mouse TNF-␣ or
IL-6 Enzyme-Linked Immunosorbent Assay (ELISA) kits (ENZO Life
Sciences, PA) according to the manufacturer’s protocol. Briefly, the
cells subcultured in DMEM media on 60-mm culture dishes were
incubated for 24 h with various concentrations of T. cuspidata. The
culture supernatants were harvested, and the levels of TNF-␣ and
IL-6 were determined by measuring optical density.
2.9. Measurement of NO production
The RAW 264.7 cells were cultured for 24 h in DMEM supple-
mented with 10% FBS and 1% PEST. The cells were then treated
with different concentrations of the T. cuspidata (25–200 ␮M) for
24 h. NO levels in the culture supernatants were determined using
Griess reagent (Sigma, St. Louis, MO). Briefly, nitrite in the culture
supernatants was mixed with an equal volume of Griess reagent
[0.1% naphthyl ethylenediamine (w/v) and 1% sulfanilamide (w/v)
in 5% phosphoric acid (v/v)], and the absorbance at 540 nm was
then measured. Sodium nitrite (NaNO2 , Sigma, St. Louis, MO) was
used as a reference.
2.10. Western blot analysis
Cell lysates were collected by scraping the cells in RIPA lysis
buffer (50 mM Tris, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100,
507
0.1% SDS, pH 7.8) containing protease and phosphatase inhibitor
cocktails (Roche, Germany) and then centrifuged at 14,000 rpm for
15 min at 4 ◦ C. The supernatants were used for Western blot analy-
sis. Upon measuring the protein concentration using the Bradford
assay, rapid colorimetric assay for cellular growth and survival was
performed [23]. The protein (30 ␮g) was loaded onto 12% SDS-
PAGE gels and then transferred to nitrocellulose membranes. The
membranes were incubated with the indicated primary antibod-
ies overnight at 4 ◦ C. The membranes were washed three times,
incubated with horseradish peroxidase-conjugated antibodies for
1.5 h and then visualized using the enhanced chemiluminescence
method (AbClon, Seoul, Korea).
For the statistical analysis, data from the experiment were
expressed as the means values ± S.D., unless representative data
were indicated as the averages from the experiments. The signifi-
cant importance of P-values was considered less than 0.05 through
all experiments.
3. Results and discussion
3.1. Isolation and purification of glycan
Since most of Taxus species are known as potent plant sources of
new bioactive natural compounds with various important biolog-
ical activities [24,25,12], isolation and characterization of unique
biomaterials from such plants have become an important part of
research in pre-pharmaceutical science field. Upon embarking on
the purification of biologically potent substances, we isolated and
characterized a water-soluble glycan from berry fruit of T. cuspidata
following treatment with organic solvents. As a result, the water-
soluble glycan isolated and purified by the procedure described in
the method section accounts for 25.4% of yield from the dry mass
of T. cuspidata berry fruit. Although the active form of molecules
measured by UV absorbance was obtained by fractionation using
organic solvents (data not shown), analysis of its preliminary struc-
ture is necessary to extend for better understanding for biological
activity. Instead of using step-wise purification with organic sol-
vent, the direct isolation of the water-soluble glycan using distilled
water was performed. We found that the water-soluble glycan
appeared in a single spot on the TLC plate (Fig. 1 inset). Water-
soluble glycan in distilled water was further purified using several
organic solvents. However, no significant difference was observed
before or after the purification. We further purified water-soluble
glycan using size-exclusion gel-filtration column chromatography,
no remarkable separation was observed (data not shown), evi-
dent in the absence of UV absorption reading. This suggests that
water-soluble glycan may be in its linear form, which does not
contain cyclic or phenolic compounds (Fig. 1B or C). Therefore
water-soluble glycan in distilled water was used as a purified glycan
for further study.
3.2. Determination of molecular weight of the water-soluble
glycan
Several experiments were performed in different scales of iso-
lation and purification to characterize the preliminary structure of
the water-soluble glycan isolated from the dry mass of T. cuspi-
data berry fruit. We first obtained the precise molecular weight
of the purified water-soluble glycan by using MALDI-TOF mass
spectroscopy. As shown in Fig. 1, we found that the purified water-
soluble glycan consisted of bigger than penta glycosylglycan, not a
single molecule observed unexpectedly. A couple of major peaks
were observed ranging from 5 to over 15 glycosylglycans with
differences of 162 m/z for each molecule respectively, thus indi-
cating that the molecule consisted of same unit of glycosylglycans.
508 C.G. Lee et al. / International Journal of Biological Macromolecules 85 (2016) 505–513

Fig. 1. MALDI-TOF mass analysis of PALs. PALs was analyzed and identified in full scanned scale ranging from 799 to 8023 m/z by MALDI-TOF mass spectrometry. TLC analysis
of PALs extracted from Taxus berry using ethanol was illustrated as an inset. Samples obtained from each preparation were baked at 200 ◦ C to detect saccharide in the samples
(A), irradiated under UV 254 nm (B) and irradiated under UV 365 nm (C). Glu, glucose; Fru, Fructose; GlcN, Glucosamine.

In general, glycosylglycans can be isolated depending on the size 3.4. HPLC analysis for sugar alcohols (SALs)
of molecules even using simple TLC. However, as shown in Fig. 1,
the purified water-soluble glycan appeared in almost a single spot The primary purpose of this analysis was to quantify the amount
on the TLC, and its size was estimated to be slightly bigger than of SALs in the water-soluble glycan. As shown in Fig. 3. it appears
monosaccharides which were tested under the same condition. that there are several patterns of SALs observed on the HPAEC
Probably mobile phase solution prepared for the separation of chromatograms. The SALs are shown with retention times for each
common sugars was not appropriate to the purified water-soluble compound identical with the standard mix consisted of glycerol,
glycan to complete separation of each molecule. Several mobile erythritol, xylitol, arabitol, sorbitol and mannitol, respectively. The
phase solutions were then applied to separate water-soluble gly- data for the water-soluble glycan are shown in the upper chro-
can by size without success, likely due to failure to create optimal matogram of Fig. 3. The glycerol was identified as the major SAL,
separation conditions. However we focused on the hypersolubil- following xylitol which was identified as the second most plentiful
ity of water-soluble glycan in distilled water, which can migrate SAL (Fig. 3A). We observed other minor SALs including unidenti-
upward on the TLC under the mobile phase solution. This did not fied compounds separated with retention times close to those of
allow us to easily separate the molecule based on size. erythritol, arabitol, sorbitol, mannitol and glucose (Table 1). Collec-
tively, Fig. 3 demonstrates that the water-soluble glycan is a type of
polyol, termed polyhydric alcohols (PALs) hereafter, based on the
3.3. HPLC analysis for monosaccharide composition size determination using MALDI-TOF mass and HPAEC analyses for
SALs.
As described above, the water-soluble glycan exhibited hyper-
solubility in distilled water, comparable with glucose used as a 3.5. Vibration spectra of PALs
standard material. Based on the results of MALDI-TOF mass anal-
ysis and hypersolubility of the purified water-soluble glycan, we FT-IR spectra of PALs are demonstrated in Fig. 4, according
first determined the monosaccharide composition by using HPAEC to the literature for the band assignment. Broad IR band ranging
analysis. As shown in Fig. 2, the major monosaccharide of the from 3050 to 3650 cm−1 arises from O H stretching vibrations of
water-soluble glycan was determined to be glucose (99.4% in mole
percentage). Even using high concentration of sample, no sig-
nificant amount of other sugars was observed. Accordingly, as Table 1
shown in Fig. 1, its relative molecular masses were determined Quantification analysis of monosaccharide and alcohol sugar in Taxus berry extracts
by using HPLC.
to be approximately ranging from 830 to 4,150 kDa, which are big
enough for high resolution separation from other monosaccharides Samples Retention time (min) pmol/1 ␮g sample
on TLC analysis. However no significant difference of migration of Monosaccharide Glucose 18.52 1506.2
the water-soluble glycan compared with other monosaccharides Alcohol Glycerol 10.60 33.16
tested by TLC analysis was observed. These differences in migra- sugar m-Erythritol 14.35 23.23
l-(−)-Arabitol 22.42 10.40
tion on TLC are likely due to the differences in isolates or different
d-Sorbitol 25.40 21.46
composition of the water-soluble glycan. Collectively, we propose d-Mannitol 29.03 32.04
that the water-soluble glycan- unlike common oligosaccharides or Unknown 1 11.43 –
polysacchairdes is a heteropolysaccharide consisting of hypersolu- Unknown 2 17.50 –
ble compounds likely sugar alcohols (SALs). Unknown 3 21.83 –
 C.G. Lee et al. / International Journal of Biological Macromolecules 85 (2016) 505–513
Fig. 2. HPAEC analysis for monosaccharide composition of the PALs.
Fuc, fucose; Rha, rhamnose; Ara, arabinose; Gal, galactose; Glc, glucose; Man, mannose; Xyl, xylose (Sigma, USA) as standard (A), A single peak derived from the Taxus berry
extracted by ethanol.
hydroxyls of glucose and fructose used as common sugars, and
water used as the solvent for PALs. A moderate IR band at 3400 cm−1
arises from stretching vibrations of primary amine for glucosamine
and PALs, which has shown also much intense IR bands at 3150
and 3650 cm−1 arises from asymmetric and symmetric stretching
vibrations of hydroxyls of PALs, respectively. Intense IR bands of
PALs appeared through 500–3700 cm−1 were assigned to asymmet-
ric and symmetric stretching vibrations of differences compared
to common sugars tested in this analysis. Upon investigating the
vibration spectra of PALs, we found that PALs have more inten-
sive and several O H stretching vibrations of hydroxyls compared
with glucose and fructose used as common sugars. These results
demonstrate the complexity of PALs, which consists of several sugar
alcohols with high quantity of glucose as the major monosaccha-
ride.
3.6. Effects of PALs on TNF-˛ and IL-6 productions
To determine whether PALs influence RAW 264.7 cells survival,
the effects of PALs on the viability of RAW 264.7 cells was examined
by MTT assay after treatment with PALs (25–200 ␮M) for 24 h. As
shown in Fig. 5, PALs treatment did not exhibit any cytotoxicity
in RAW 264.7 cells at concentrations up to 200 ␮M. Therefore, we
determined the dosage dependence of PALs as 200 ␮M for further
experimental investigations.
The pro-inflammatory cytokines such as TNF-␣ and IL-6 are
known as the potent immunomodulators derived from activated
monocytes, macrophages and T lymphocytes [26,27]. It has been
reported that the release of proinflammatory cytokines from
immune cells stimulates the innate immune response [28]. Thus,
509
the effects of PALs on TNF-␣ and IL-6 secretion from RAW264.7
cells were examined using ELISA. Our results show that PALs sig-
nificantly induced TNF-␣ and IL-6 secretions in a dose-dependent
manner (Fig. 6). TNF-␣ and IL-6 productions in cells treated with
200 ␮M of PALs increased approximately to be 2.3 and 27.2-fold,
respectively, compared with the untreated control (Fig. 7A and B).
Based on these data, we concluded that PALs were a strong stim-
ulator of TNF-␣ and IL-6 secretions and identifying PALs as new
immunostimulatory agent which can lead to the stimulation of the
innate immune response. Further work is needed to characterize
PALs as an immunostimulatory agent.
3.7. Effects of PALs on the iNOS expression and NO production
Nitric oxide (NO), a short-lived radical generated by the
inducible NO synthase (iNOS), is an essential molecule in the reg-
ulation of the immune response [21,29]. Herein, we examined
the effects of PALs on iNOS expression and NO production. After
incubation with PALs (25–200 ␮M) for 24 h, the expression lev-
els of iNOS were determined by Western blot analysis. As shown
in Fig. 8A, PALs significantly induced iNOS expression in a dose-
dependent manner. This result was further confirmed by measuring
the level of NO production. Consistent with results from the iNOS
expression level, when the cells were treated with varying con-
centrations of PALs (25–200 ␮M) for 24 h, the levels of NO were
significantly enhanced in a dose-dependent manner, with 76, 237,
302 and 445 nM increases, respectively, compared with untreated
cells (Fig. 7B). These results suggested that NO can be a mediator
in the immune signaling modulation by PALs. It is well known that
NO plays a dual role as a beneficial and/or detrimental molecule
510 C.G. Lee et al. / International Journal of Biological Macromolecules 85 (2016) 505–513

Fig. 3. HPAEC analysis for alcohol sugar composition of the PALs.

Glycerol, erythritol, xylitol, arabitol, sorbitol and mannitol as standard (B), A single peak derived from the Taxus berry extracted by ethanol (A).

in the inflammatory processes [30]. A high level of NO might be
associated with anticancer, bactericidal and antiparasitic effects
presumably through the formation of reactive radicals including
peroxynitrite [31], whereas a physiologically active low level of
NO production would be effective in the host-defense mechanism
against microbial pathogens in innate immune cells [32,33]. There-
fore, our results suggest that PALs could be a crucial switch, which
could serve as immunostimulator by producing physiological levels
of NO as a signal messenger molecule.
3.8. Molecular mechanism of immunostimulating activity
induced by PALs
The MAPK signaling pathway, which includes proteins such
as ERK1/2, JNK, and p38 is a major pathway stimulating the

Fig. 4. FT-IR spectra of the PALs. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
FT-IR spectrum (spectral region 4000–400 cm−1 ) of the PALs dissolved in distilled water using ZnSe windows was recorded by accumulation of at least 32/64-scans on
FTIR-7600 spectrometer (Lambda scientific Pty., Ltd., Austria). Red line: Taxus berry extracts, Purple line: Glucose. O H stretch, free hydroxyl: 3625 cm−1 , O H stretch,
H-bond: 3397 cm−1 , O H stretch: 3159 cm−1 , C C stretch: 2206 cm−1 , N H bend (1◦ amine): 1650 cm−1 , C H wag ( CH2 X): 1284 cm−1 , C N stretch (aliphatic amine):
1041 cm−1 , Aromatics: 809 cm−1 , C Cl stretch: 586 cm−1 .
 C.G. Lee et al. / International Journal of Biological Macromolecules 85 (2016) 505–513 511

Fig. 5. Effects of PALs side on the viability of RAW 264.7 cells.

Cell viability was assessed by MTT assay after treatment with PALs ranging from 0 to 200 ␮M for 24 h. The data were expressed as the percent normalized to the un-treated
control (Con, 0 ␮M). Data = mean ± SD, n = 3.

production of various cytokines, including TNF-␣, IL-6 and NO,
in macrophages [34]. Therefore, we investigated the association
of PALs (25–200 ␮M) with MAPKs (ERK, p38 and JNK) signaling
pathway by Western blot analysis. Treatment of PALs for 24 h
remarkably increased the phosphorylation levels of ERK, p38 and
JNK in a dose-dependent manner, whereas the total protein levels
of ERK (t-ERK), p38 (t-p38) and JNK (t-JNK) remained unchanged
(Fig. 8). These results demonstrate that PALs stimulates the immune
response in RAW 264.7 cells through the activation of MAPKs
(ERK, p38 and JNK) signaling pathway. Therefore, collectively our
results indicate that PALs can be a potential immunostimula-
tory agent, and may be a potential candidate for combinatorial
remedies against impaired immune system. Since earlier studies
demonstrated that T. cuspidata is considered as a potent plant pro-
ducing several kinds of biological substances showing anticancer,
antioxidant and immunostimulating activities, there have been
large numbers of studies performed extensively. Further studies
of their metabolism and absorption are needed to better define
their potential importance. However no report on the structural
analysis of PALs and its immunostimulating activities were demon-
strated up to date. Herein we demonstrated that the PALs activate
macrophage-mediated immune responses via mitogen activated
protein kinases (MAPKs) signaling pathway [34,35]. To the best of
our knowledge, we are the first to report on the isolation and char-

Fig. 6. Effects of PALs on the secretion of TNF-␣ and IL-6.

Cells were incubated with the indicated doses of PALs ranging from 0 to 200 ␮M for 24 h. The levels of (A) TNF-␣ and (B) IL-6 in the culture supernatants were determined
by ELISA. Data = mean ± SD, n = 3. *p < 0.05; **p < 0.01; ***p < 0.001, student’s t-test compared to the control.
512 C.G. Lee et al. / International Journal of Biological Macromolecules 85 (2016) 505–513
Fig. 7. Effects of PALs on the expression of iNOS and NO production in murine macrophages. RAW264.7 cells were incubated with the indicated doses of the PALs ranging
from 0 to 200 ␮M or LPS (0.1 ␮g/mL) for 24 h. (A) The expression levels were determined by Western blot analysis. (B) Nitrite levels in the culture media of PALs-stimulated
cells were measured using the Griess reaction. Data = mean ± SD, n = 3. **p < 0.01, student’s t-test compared to the control.
Fig. 8. Effects of PALs on the activation of MAPKs pathway in RAW 264.7 cells.
(A) Whole cell lysates were collected after treatment with increasing concentrations
of PALs ranging from 0 to 200 ␮M for 24 h. Total ERK (t-ERK), p38 (t-p38) and JNK (t-
JNK) were used as a loading control for the Western blot analysis of phosphorylated
ERK (p-ERK), p38 (p-p38) and JNK (p-JNK). LPS (0.1 ␮g/mL) was used as a positive
control.
acterization of PALs from T. cuspidata, and uncover PALs potential to
modulate the immune function of RAW264.7 murine macrophages
as immune stimulators.
4. Conclusions
A water-soluble glycan designated as PALs from T. cuspidata was
isolated and characterized by monosaccharide and sugar alcohol
compositions using HPAEC analysis, and MALDI-TOF mass spec-
tra and FT-IR analyses. The main monosaccharide component of
PALs was identified as the glucose, and glycerol and xylitol were
detected as the primary sugar alcohols. Concerning the isolation
and purification of PALs from T. cuspidata expending to other biore-
sources such as macroalga and microalga, our results demonstrated
good potential for use regarding process the scale-up for various
applications including food industry. To the best of our knowledge,
this is first study to demonstrate the primary structure of PAL, and
immune-stimulating activities from the fruit of T. cuspidata.
Conflict of interest
The authors declare that there is no conflict of interest.
Acknowledgement
This research was financially supported by a grant from the grant
(GCU-2015-0057) of Gachon University, Korea.
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