Structural characterization and in vitro antitumor activity of A polysaccharide from Artemisia annua L. (Huang Huahao)

Article abstract of DOI:10.1016/j.carbpol.2019.02.081

One water-soluble polysaccharide (AAP), with a molecular weight of 6.3 × 104 Da, was isolated from Artemisia annua L. Structrual analysis indicated that AAP was found to be a 1, 3-α-linked and 1, 3, 6-α-linked Glcp backbone, with a branch of 1, 6-α-linked Glcp and terminal 1-linked-L-Rhap along the main chain in a ratio of 1: 1: 1: 1. MTT assay showed that AAP reduced the cell viability of HepG2 cells in a concentration-dependent manner. DAPI staining and Flow cytometric analysis revealed that AAP suppressed cells proliferation, not most at least via inducing p65-dependent mitochondrial signaling pathway, as evidenced by more activation of caspase-3 and -9, down-regulation of Bcl-2 protein, up-regulation of Bax protein and release of cytochrome c from mitochondria into cytosol, as well as suppression of the nuclear factor-κB (NF-κB) p65. These data confirmed AAP inhibits HepG2 cell growth via inducing caspase-dependent mitochondrial apoptosis and inhibition of NF-κB p65.

Full text of DOI:10.1016/j.carbpol.2019.02.081

Accepted Manuscript
Title: Structural characterization and in vitro antitumor activity
of A polysaccharide from Artemisia annua L. (Huang Huahao)
Authors: Liang Yan, Chuan Xiong, Pan Xu, Jing Zhu, Zhirong
Yang, Hong Ren, Qiang Luo
PII:
S0144-8617(19)30223-1
DOI:
Reference:
https://doi.org/10.1016/j.carbpol.2019.02.081
CARP 14654
To appear in:
Received date:
Revised date:
Accepted date:
18 October 2018
29 January 2019
24 February 2019
Please cite this article as: Yan L, Xiong C, Xu P, Zhu J, Yang Z, Ren
H, Luo Q, Structural characterization and in vitro antitumor activity of A
polysaccharide from Artemisia annua L. (Huang Huahao), Carbohydrate Polymers
(2019), https://doi.org/10.1016/j.carbpol.2019.02.081
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Structural characterization and in vitro antitumor activity of A
polysaccharide from Artemisia annua L. (Huang Huahao)
c
a
b
d
LIANG YAN a b, CHUAN XIONG , PAN XU , JING ZHU , ZHIRONG YANG ,
HONG REN a, QIANG LUO a,*
a
Institute for Viral Hepatitis, Key Laboratory of Molecular Biology for Infectious
Diseases, Chinese Ministry of Education, Department of Infectious Diseases, Second
Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China
b
Pediatric Research Institute, Children's Hospital of Chongqing Medical University,
Ministry of Education Key Laboratory of Child Development and Disorders,
Chongqing 400014, China; China International Science and Technology Cooperation
Base of Child Development and Critical Disorders, Chongqing 400014, China.
c
Biotechnology and Nuclear Technology Research Institute, Sichuan Academy of
Agricultural Sciences, Chengdu 610061, China
d Sichuan Province Key Laboratory of Nature Resources Microbiology and Technique,
College of Life Sciences, Sichuan University, Chengdu, 610064, China
*Corresponding Author: QIANG LUO
Email: qiangluo@email.com
Highlights
9


A polysaccharide was isolated and characterized from Artemisia annua L. (Huang
Huahao)


This polysaccharide induced apoptosis of HepG2 cell
This polysaccharide-induced apoptosis is done via mitochondrial signaling pathway
Abstract
One water-soluble polysaccharide (AAP), with a molecular weight of 6.3 × 104Da,
was isolated from Artemisia annua L. Structrual analysis indicated that AAP was
found to be a 1, 3-α-linked and 1, 3, 6-α-linked Glcp backbone, with a branch of 1,
6-α-linked Glcp and terminal 1-linked-L-Rhap along the main chain in a ratio of 1: 1:
1: 1. MTT assay showed that AAP reduced the cell viability of HepG2 cells in a
concentration-dependent manner. DAPI staining and Flow cytometric analysis
revealed that AAP suppressed cells proliferation, not most at least via inducing
p65-dependent mitochondrial signaling pathway, as evidenced by more activation of
caspase-3 and -9, down-regulation of Bcl-2 protein, up-regulation of Bax protein and
release of cytochrome c from mitochondria into cytosol, as well as suppression of the
nuclear factor-κB (NF-κB) p65. These data confirmed AAP inhibits HepG2 cell
growth via inducing caspase-dependent mitochondrial apoptosis and inhibition of
NF-κB p65.
Keywords: Artemisia annua L.; Huang Huahao; Polysaccharide; Antitumor activity;
10

Apoptosis; Nuclear factor-κB (NF-κB) p65
1. Introduction
Cancer is a leading dominant cause of human death globally. Chemotherapy is
one of the most commonly applied and effective protocols for cancer treatment (Yang,
Xiao, & Sun, 2013). However, the search for selective anticancer chemotherapeutic
agents has not been fruitful to date, mainly due to their non-specific cytotoxicity and
adverse effects, such as multidrug resistance of tumor cells, and especially, toxicity to
normal cells (Bagchi et al., 2000; Krishna & Mayer, 2000). Recently, research interest
has focused on polysaccharides from various plants and herbs with anitumor
properties, which have been used in the prevention or treatment of cancer in many
countries (Li et al., 2015; Liu, Sheng, Zhang, & Sun, 2016). Most polysaccharides
derived from various natural sources are believed that have superior anti-tumor
activities and do not causes significant side effects compared with synthetic
compounds (Chen et al., 2013; Sun et al., 2014; Wang et al., 2013). Therefore,
discovery and evaluation of polysaccharides with antitumor properties and low
11

toxicity have emerged as one of the most attractive strategies against cancer.
Hua Huahao (Artemisia annua L.) is an annual plant native of the Asian
continent and has been traditionally used throughout the ages for the treatment of
malaria (Räth et al., 2004). In addition, A. annua are also reported to have significant
pharmacological activities such as anti-imflammatory, anti-cancer, and anti-obesity
activities that contribute to the therapeutic effects of the herb (Ho, Peh, Chan, & Wong,
2014; Kim, Seo, Liu, & Kim, 2014; Ko et al., 2016). Many bioactive chemical
constituents from A. annua including artemisinin, flavonoids, terpenoids and their
volatile oils have been well investigated (van der Kooy & Sullivan, 2013), but the
high-molecular components such as polysaccharides have rarely been reported. Initial
investigations indicated that A. annua polysaccharides can effectively inhibit
hepatoma cell growth by improving the human body’s cellular immune function and
facilitating cell apoptosis (Chen, Wang, & Liu, 2014). However, the underlying
molecular mechanism is still not elucidated. Therefore, in the present study, we first
carried out experiments to isolate and characterize a purified polysaccharide from this
plant, and then the underlying apoptotic mechanism of this polysaccharide was
investigated in human hepatoblastoma G2 (HepG2) cell line. The results will be
helpful for developing novel anti-tumor drugs.
2. Materials and methods
2.1. Materials and chemicals
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The dried ground part of Artemisia annua L. (Hua Huahao) was purchased from
a local grocery store in Chongqing (China). The Annexin V‑ fluorescein
isothiocyanate (FITC)‑ propidium iodide (PI) Apoptosis kit was purchased from
NanJing KeyGen Biotech Co., Ltd. (Nanjing, China). MTT (3-(4, 5-dimethyl-2-yl)-2,
5-diphenyl tetrazolium bromide), DAPI (2-(4-amidinophenyl)-6-indolecarbamidine
dihydrochloride), T-series dextran (T-2000, T-70, T-40, T-20, and T-10) and
monosaccharide standards (arabinose, rhamnose, galactose, glucose, mannose, fucose,
xylose, glucoturonic acid and galacturonic acid) were obtained from Sigma Chemical
Co. (St. Louis, MO). DEAE Sepharose fast flow and Sepharose CL-6B were
purchased from Pharmacia Co. (Sweden). Antibodies for β-actin, Bcl-2, Bax,
cytochrome c and NF-kappa B (kB)-p65 were purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA, U.S.A.). Horseradish peroxidase conjugated
secondary antibodies and the enhanced chemiluminescence (ECL) detection system
was purchased from Tiangen Biotech Co. (Beijing, China). Caspase-3, -8 and -9
activity assay kits were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).
Tissue culture media (RPMI 1640 and Eagle’s Minimum Essential Medium) and fetal
bovine serum (FBS) were from obtained GIBCO (Life Technologies, NY). Other
reagents (NaBH4, acetic anhydride, NaCl, phenol, sulphuric acid, chloroform, butanol,
ethanol, NaNO3, and 0.2% NaN3 ) were obtained in the highest purity grade available
commercially.
2.2. Isolation and purification of polysaccharide from A. annua
The dried A. annua L. (500 g) were powdered and defatted with 95% ethanol
13

(6000 mL) for three times and 2h each time. The residue (452g, 90.4% of dried raw
material) was then air-dried at room temperature and extracted with boiling water
(8000 mL) for 2 h and repeated for a total of three times. The extracts (20,230 mL)
were filtered, combined, concentrated by evaporation and centrifuged at 1700 × g for
10 min at 4 °C. The resulting supernatant (5680 mL, 28.08% of the original total
extract) was then concentrated and precipitated with 95% ethanol (4500 mL) for 24h
at 4 °C. Following centrifugation (1700 × g, 10 min) at 4 °C, the obtained
precipitation (20.5g, 4.1% of dried raw material) was re-dissolved in water in 400 mL
and added with 100 mL of Sevag reagent (chloroform:butanol 4:1, v/v) to remove free
protein in the samples for five times (Sevag, Lackman, & Smolens, 1938). The
combined supernatant (255 mL) was further concentrated and dialyzed at –40 °C
under 25 Psi pressure against running water for three days. The retentate was
concentrated, centrifuged, and lyophilized under the same conditions to give crude A.
annua polysaccharides (CAAP, 7.7g).
CAAP (7.7 g) was dissolved in 20 mL deionized water, centrifuged, and the
supernatant was loaded on a DEAE Sepharose fast flow column (2.0 cm × 40cm, Cl−
form), eluted with deionized water and a gradient of 0–1.0 M NaCl solution at a flow
rate of 2 mL/min to give three fractions (CAAP1, CAAP2 and CAAP3). All tubes (5
mL/each) were quantified by phenol-sulphuric acid method. The deionized
water-eluted fraction CAAP1 (4.12g) was collected and dissolved in 20 mL deionized
water, and centrifuged again to prepare the supernatant. Then each aliquot of this
supernatant (5ml) was further purified by gel chromatography on a Sepharose CL-6B
14

column (2.5 × 80 cm) eluted with deionized water at a flow rate of 0.3 mL/min to
yield a white power fraction, designated as AAP. This operation was repeated for four
times to yield of total 3.25g of AAP.
2.3. Physicochemical and structural analysis of polysaccharide
2.3.1. Chemical component analysis
Total carbohydrate content of the polysaccharide was measured according to the
phenol–sulfuric acid method (Dubois et al., 1956). Protein content of the
polysaccharide was quantified by the method of Bradford (1976). Uronic acid content
of the polysaccharide was detected by m-hydroxydiphenyl colorimetric method
(Blumenkrantz & Asboe-Hansen, 1973).
2.3.2. Monosaccharide composition analysis
A unit of 10 mg samples was hydrolyzed with 2 M TFA (2 ml) at 120° C for 2 h
in a sealed test tube. After removing TFA with methanol, the mixture of neutral
monosaccharides was reduced with NaBH4 and acetylated with acetic anhydride
(York, Darvill, McNeil, Stevenson, & Albersheim, 1985). After those procedures, the
acetylated products were re-dissolved in chloroform and performed on Shimadzu
2014 GC instrument (Shimadzu Co., Kyoto, Japan) equipped with a flame ionization
detector (FID) and a HP-1 capillary column (30 m × 0.25 mm × 0.25 µm). The GC
temperature was programmed at 120 °C for 3 min, followed by a 3◦C/min gradient up
to 220 °C and held at 220 °C for 3 min. N2 was used as the carrier gas and
myo-inositol was used as an internal standard at a constant flow rate of 1 mL/min.
15

2.3.3. Homogeneity and molecular weight determination
The homogeneity and averaged molecular weight was determined by HPGPC
instrument with an SHIMADZU HPLC system equipped with a TSK-G3000PWXL
column (7.8 mm ×300 mm) and a SHIMADZU RID-10A refractive index detector.
The mobile phase was composed of 0.1 mol/L NaNO3 and 0.2% NaN3, and the flow
rate through the HPLC column was 0.7 ml/min at 40 °C with 1.6 mpa. The samples
were dissolved in mobile phase as 0.3% (w/v) solution, centrifuged and 20 µl of the
supernatant was injected in each run. The averaged molecular weight was estimated
from the calibration curve of the elution volume of Dextran standards (T 130, 80, 50,
20, 10).
2.3.4. Ultraviolet (UV) spectroscopy analysis
The contents of protein and nucleic acid were determined using a Shimadzu
UV-2401 spectrometer (Shimadzu, Japan) and scanned in the range of 190-500 nm
(Makarova, Patova, Shakhmatov, Kuznetsov, & Ovodov, 2013).
2.3.5. IR spectroscopy analysis
Fourier-transform infrared (FTIR) spectrum of the polysaccharide was recorded
with a Nicolet Nexus 470 spectrometer (Thermo Nicolet Co., Madison, WI, USA) by
KBr method. Briefly, the polysaccharide dried at 35–44 °C in vacuum over P2O5 for
48 h was mixed with KBr powder, ground and then pressed into a 1-mm pellet for
FTIR spectral measurements in the frequency range of 400–4000 cm−1 (Zhao, Dong,
Chen, & Hua, 2010).
16

2.3.6. Methylation analysis
The vacuum-dried polysaccharide were methylated three times as described by
Needs and Selvendran (1993) with a minor modification. Complete methylation was
confirmed by the disappearance of the hydroxyl peak in the IR spectrum. The
resulting permethylated product was depolymerized with 90% HCOOH at 100 °C for
3 h followed by hydrolysis with 2 M TFA at 100 °C for another 4 h. completely
methylated polysaccharide was hydrolyzed with 2 ml of 2 M TFA, then, then reduced
and acetylated as described by Sweet, Shapiro, and Albersheim (1975). A gas
chromatograph–mass spectrometer (GC-MS) was used to analyse the glycosidic
linkage on the basis of relative retention time and fragmentation pattern, and the
molar ratios were calibrated using the peak areas and response factors of the FID in
GC.
2.3.7. Nuclear magnetic resonance (NMR) analysis
The polysaccharide samples were kept with P2O5 in vacuum for one week before
exhaustively being exchanged with 99.96% D2O using intermediate freeze drying
method. The deuterium-exchanged polysaccharides (60 mg) were dissolved in 0.6 ml
of 99.96% D2O in which 30μl acetone-d6 was added and then placed in 5 mm NMR
tubes. Then, the 1H, 13C and 2D NMR (HMBC) spectra of AAP were performed by
Bruker spectrometer (Bruker Corp., Zurich, Switzerland) at 400 MHz at 30 °C.
2.4. Cell lines and culture conditions
Human hepatoblastoma HepG2, human ovarian cancer cells SKOV3, breast
17

cancer cells MCF-7 and lung adenocarcinoma cells A549 were obtained from the
Committee on Type Culture Collection of Chinese Academy of Sciences (Shanghai,
China). SKOV3 and A549 cells were maintained in RPMI 1640 supplemented with
10% heat-inactivated FBS, 100 IU penicillin/ml and 100 µg streptomycin/ml. MCF-7
and HepG2 cancer cells were maintained in Eagle’s minimum essential medium with
10% FBS, 100 IU penicillin/ml and 100 µg streptomycin/ml. All cultured cells were
incubated at 37 °C in a humidified atmosphere containing 5% CO2 in the air.
2.5. Cell proliferation assay
Cell viability was measured quantitatively by using MTT, showed the activity of
living cells (Plumb, Milroy, & Kaye, 1989). Briefly, cancer cells were cultured to 80%
confluence in 96-well culture plates (1×105 cells/well) and then treated with indicated
concentrations of AAP (0, 5, 10, 20, 40, 80, and 160 µg/mL) for different indicated
period. 50 µl of 5 mM MTT solution was then added to each well and the cell was
cultured in dark for another 4 h. Thereafter, culture fluid was removed and 100 µL
dimethyl sulfoxide (DMSO) was added to each well to dissolve purple formazan
crystals, and then the absorbance was read at 540 nm on a 96-well plate reader. The
absorbance of untreated cells was considered as 100%. The results were determined
by three independent experiments.
2.6. DAPI staining assay
Morphological observation of nuclear change was assayed with a DNA-binding
fluorescent dye DAPI to determine whether AAP induces apoptosis or not. After
18

treatment with or without AAP (40 µg/mL) for 24 h, the cells were washed three times
with ice-cold PBS, fixed in a 4% formaldehyde solution for 20 min at 4 °C, then
stained with 5 µg/ml DAPI for 1 h in the dark prior to visualization of blue
fluorescence under a fluorescence microscope (OLYMPUS, Essex, U.K.).
2.7. Cell apoptosis assay by flow cytometry
The extent of apoptosis was determined using Annexin V‑ FITC‑ PI Apoptosis
kit as described by the manufacture’s instruction. Briefly, after exposure to AAP (10,
20, and 40 µg/mL) for 24 hours, cells were harvested, washed twice with PBS and
followed by gentle centrifugation. The resulting cell pellet was resuspended in
binding buffer (2.5 μl Annexin V-FITC, 5 μl PI). Non- AAP treated cells were
collected as the control. After being incubated in the dark for 15 min, samples were
assessed by a FACScan flow cytometer (Coulter EPICS XL, Beckman Coulter Inc.,
CA) and 10,000 cells were evaluated in each sample. Annexin V-positive and
PI-positive cells were recognized as necrotic cells, while Annexin V-positive but
PI-negative cells were regarded as apoptotic cells. The percentage of cells undergoing
apoptosis was determined by three independent experiments
2.8. Analysis of caspase activities
Activity of caspase--3, -8 or -9 was quantified using a colorimetric assay kits
according to the manufacturer’s protocol. Briefly, cancer cells treated with AAP (10,
20, and 40 µg/mL) for 24 h or untreated control cells were lysed in 50 ml of cold lysis
buffer and then cell lysates were collected and centrifuged at 12, 000 × g for 10 min at
19

4 °C. The resulting supernatants were collected to evaluate caspase activity. For each
reaction, 10 μg total proteins were added to 50μl reaction buffer, followed by the
additional 5µL of substrates specific for caspase-3, -8, or -9, respectively, and
transferred into a 96-well plate for 4 h incubation at 37 °C in a CO2 incubator. Finally,
absorbance was measured at 405 nm. Caspase activities were expressed relative to
theoretical density value (OD).
2.9. Preparation of cytosolic and mitochondrial fractionations and Western blotting
analysis
For isolation of total protein fractions, treated or untreated cells were collected,
washed twice with ice-cold PBS, and resuspended in cell lysis buffer and then
incubated on ice for 15 min. The cell lysates were harvested by centrifugation at
12,000 g for 20 min at 4 °C to yield the supernatants for Western blot analysis. To
examine the cytochrome c release from mitochondria, the cytosolic and mitochondrial
extracts were prepared using mitochondrial fraction kit as per the manufacturer’s
instruction. Protein concentrations of the samples were quantified by the method of
Bradford (1976).
Western blotting analyses were performed as described by Xin et al. 9 In brief,
each equivalent amount of total proteins (30 µg) were electrophoresed on 10–12%
SDS-PAGE gel and transferred onto nitrocellulose membranes, and then blocked with
5% (w/v) non-fat dry milk and incubated with specific primary antibodies
(anti-β-actin, anti-Bcl-2, anti-Bax, anti-cytochrome c and anti-NF-kB) at a 1:1000
20

dilution in blocking solution. After thrice washings in TBST for each 5min,
membranes were incubated with horseradish peroxidase conjugated secondary
antibodies at a dilution of 1:500 in blocking solution for 1 h at room temperature.
Finally protein bands were visualized with the aid of an enhanced chemiluminescence
system (ECL) system.
2.10. RT-PCR
Total RNA was extracted from AAP-treated cells with a Trizol RNA isolation kit
(Invitrogen Inc, CA, USA) and stored at −80 °C for subsequent analysis.
First-stranded cDNA was synthesized with 5 μg total RNA by RT-PCR kit according
to the manufacturer’s instruction. The sequences of primers used in RT-PCR were as
follows: Bax, 5’-TCCACCAAGAAGCTGAGCGA-3’ (forward) and
5’-GTCCAGCCCATGATGGTTCT-3’ (Reverse); Bcl-2,
5’-CTACGAGTGGGATGCGGGAGATG-3’ (forward) and
5’-GGTTCAGGTACTCAGTCATCCACAG3’ (Reverse); NF-kB,
5’-ATCCCAGGTTTCTTGCCTCT-3’ (forward) and
5’-GGAGCACAGATACCACCAAGA-3’ (Reverse); β-actin,
5’-TCACCCTGAAGTACCCCATC-3’ (forward) and
5’-CCATCTCTTGCTGCAAGTCC-3’ (Reverse). All of the primers used in RT-PCR
were used to produce the correlated products, respectively. Amplification condition:
one denaturing cycle at 94 °C (5min), 30 cycles at 94 °C (45 s), at appropriate Tm (1
min), at 72 °C (45 s), and a final extension at 72 °C (7 min). Finally, PCR products
were run on 1.5 percent agarose gel electrophoresis and DNA bands were visualized
21

by ethidium bromide staining. All experiments were performed in triplicate.
2.11. Statistical analysis
The results were expressed as mean ± SD (standard deviation). Statistical
differences were evaluated using the two-tailed Student’s t-test and values of p < 0.05
were considered significantly different.
3. Results and discussion
3.1. Isolation, purification and general chemical properties of the polysaccharide
Crude polysaccharides, CAAP, were obtained with a yield of 1.54% (w/w) from
A. annua through ethanol degreasing, hot-water extraction followed by ethanol
precipitation, deproteinization by Sevag method, dialysis with water and
lyophilization. CAAP was separated and fractionated on a DEAE Sepharose fast flow
column stepwise with distilled water and a NaCl gradient (0–1.0 M) to give three
elution peaks (titled as CAAP1, CAAP2 and CAAP3) (Fig. 1A). CAAP1 was further
purified by a Sepharose CL-6B column eluted with deionized water to yield on
purified fraction (Fig. 1B), named as AAP, with a yield of 0.65% (w/w). AAP
contained 93.8% of carbohydrates as determined by phenol–sulfuric acid assay.
Determination of protein and uronic acid suggested that AAP was free of proteins and
was a neutral polysaccharide. The HPGPC profile of AAP exhibited a single and
symmetrically sharp peak, which revealing it is a homogeneous polysaccharide (Fig.
1C). Based on the calibration with standard dextrans, its average molecular weight
22

was estimated to be about 6.3 × 104 Da. Moreover, GC analysis revealed that AAP
consisted of glucose (Glc) and rhamnose (Rha) in the molar ratios of 4: 1 (Fig. 2),
which indicated that the AAP was heterogeneous.
Fig. 1. (A) Isolation and purification of CAAP on a DEAE Sepharose fast flow
column; (B) Isolation and purification of CAAP1 on a Sepharose CL-6B gel
23

filtration column. (C) HPGPC chromatogram of AAP
Fig. 2. (A) Gas chromatograms of the mixed standard monosaccharides. (B) Gas
chromatograms of AAP
3.2. UV and IR spectrum analysis
A negative response to the Bradford test and no significant absorption peak in the
UV spectrum between wavelengths from 190 to 500 nm illustrated the absence of
protein and nucleic acid (Fig. 3A). FT-IR spectrum of AAP shown in Fig. 3B
exhibited the typical absorption of polysaccharides. The characteristic strong broad
24

band at 3408.52 cm−1 and the weak bands 2936.67 cm−1 were the stretch vibrations of
O-H and C-H, respectively (Zha et al., 2014). The absorption band at 1370.61 cm−1
was assigned to C-H bending vibration and the band at 1636.82 cm−1 was due to the
presence of bound water. The peaks in the range of 1036.21–1146.85 cm−1 were
attributed to the stretching vibration of C-O-C group. The freedom of band around
1720 and 1250 cm−1 indicated the absence of uronic acids and ester sulfate in the
sample, which supported the results of uronic acids assay and UV analysis. Moreover,
a characteristic absorption at 616.70 cm−1 was also observed, indicating the
α-configuration of the sugar units.
Fig. 3. (A) UV spectrum of AAP. (AB) FT-IR spectrum of AAP
25

3.3. Glycosyl linkages analysis
To determine the linkages of the monosaccharides in AAP, the fully methylated
product of AAP was depolymerized, hydrolyzed, acetylated, and analyzed by GC–MS
analysis. The individual peaks of methylated sugars from the GC-MS analysis were
identified according to their retention times and by comparison with the mass
spectrum. The GC–MS results indicated that the backbone chains were mainly 1,
4-linked Glcp (Residue-A, 2,3,6-Me3-Glc) and 1, 3, 6-linked Glcp (Residue-B,
2,4-Me2-Glc). The side chain attached to the O-6 position of Residue-B contained 1,
6-linked-Glcp (Residue-C, 2,3,4-Me3-Glc), which was ended with single
non-reducing terminal α-L-Rhap residue (Residue-D, 2,3,4-Me3-Rha). According to
the peak areas, four types of residues were seen in the ratio of 1: 1: 1: 1. The molar
ratio of each monosaccharide was also in accordance with the results of GC.
The structural characteristics of AAP were further confirmed with 1H and 13C
NMR spectral analysis. The proton signals between δ 3.0–5.5 and the carbon signals
locating in the range of δ 60–110 are the characteristic signals of polysaccharide in
NMR spectroscopy (Hu, Liang, & Wu, 2015). All 1H NMR and 13C NMR signals
were summarized in Table 1 based on the data available in literature (Liu, Sun, Yu, &
Liu, L. 2012; Liu, Sun, Liu, & Yu, 2012; Maity et al., 2017; Molaei & Jahanbin, 2018;
Ow, Green, Hao, & Mak, 2008; Shakhmatov, Belyy, & Makarova, 2018; Shakhmatov,
Toukach, Kuznetsov, & Makarova, 2014) and depicted in Fig. 4A and B, respectively.
It was clear from this figure that signals related to anomeric carbon were located in
the range δ 97–101 ppm for 13C NMR and the peaks of anomeric protons fell in the
26

range of δ 5.0-5.2 ppm for 1H NMR spectra, indicated that the presence of
α-glycosidic configuration in AAP (Huang, Li, Li, & Wang, 2011). This result was in
agreement with the glycosidic linkage types determined by IR. The signals at δ 99.56,
97.45, 99.96 and 101.35 ppm in 13C NMR spectrum were attributed to the anomeric
carbon atoms of Residue-A, Residue-B, Residue-C and Residue-D. The peaks at δ
78.45, 75.9, 69.8, and 68.75 ppm were coming from C4 of Residue-A, C3 of
Residue-B, C6 of Residue-B and C6 of Residue-C, respectively. Similarly, the
anomeric proton signals at δ5.10, 5.06, 5.03, and 5.15 in the 1H NMR revealed the
existence of Residue-A, Residue-B, Residue-C, and Residue-D. The strong signal at δ
4.70 ppm presented in the spectrum was attributed to HDO. In addition, a signal
detected at δ 1.25 ppm in 1H NMR and approximately δ 19.01 ppm in 13C NMR
which can be ascribed to H-6/C-6 methyl group of Rhap (Leone, Molinaro, Dubery,
Lanzetta, & Parrilli, 2007).
The sequences of different sugar residues and the substitution sites were
analyzed by observing the cross-peaks of both anomeric protons and carbons of
glycosyl residues in HMBC spectrum after the assignments of 1H and 13C chemical
shifts of all sugar residues. From the HMBC spectrum (Fig. 4C), we can observed
correlations between the following anomeric protons and carbons at the linkage
positions: C-4 (78.45) of residue A and H-1 (δ 5.03) of residue C (A C-4/C H-1), C-1
(99.96) of residue C and H-4 (δ 3.62) of residue A (C C-1/A H-4), C-3 (75.90) of
residue B and H-1 (δ 5.10) of residue A (B C-3/A H-1), C-1 (99.56) of residue A and
H-3 (δ 3.78) of residue B (A C-1/B H-3), C-6 (68.75) of residue C and H-1 (δ 5.06) of
27

residue B (C C-6/B H-1), C-1 (97.45) of residue B and H-6 (δ 3.87) of residue C (B
C-1/C H-6). All these cross peaks suggested that there was a repeating structure
fragment [→6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→3,6)-α-D-Glcp-(1→) probably
connected alternately in AAP as the linear backbone. Meanwhile the cross peaks at (δ
69.8/ 5.15) and e (δ 101.35/3.64) showed the correlation between C-6 of residue B
and H-1 of residue D, and the correlation between C-1 of residue D and H-6 of
residue B, respectively, indicating that the residue D was attached to C-6 of the
residue B.
28

Fig. 4. (A) 1H NMR spectrum of AAP. (B) 13C NMR spectrum of AAP. (C) HMBC
spectrum of AAP.
29

Table 1 Results of methylation analysis and 1H and 13C NMR chemical shifts of AAP
Chemical shifts, δ (ppm)
Methylated sugar
2,3,6-Me3-Glcp
Linkage type
Molar ratios
1
MS fraction
C1/(H1)
C2/(H2)
C3/(H3)
C4/(H4)
C5/(H5)
C6/(H6)
Residue A
43,87,101,113,
99.56/5.10
72.12/3.50 72.78/3.74 78.45/3.62 71.06/3.87 62.54/3.62
117,129,131,233
→4)-α-D-Glcp-(1→
2,4-Me2-Glcp
Residue B
1
1
57,87,99,117,129,1
59,173,233
97.45/5.06
99.96/5.03
72.7/3.45
75.9/3.78
72.7/3.61
72.0/3.76
69.8/3.64
→3,6)-α-D-Glcp-(1→
2,3, 4-Me3-Glcp
43,71,87,101,117,1
29,161,173,
Residue C
72.31/3.89 74.62/3.76 71.04/3.59 69.99/3.85 68.75/3.87
→6)-α-D-Glcp-(1→
189,233
2,3,4-Me3-Rhap
Residue D
1
43,87,101,129,143,
189,203
101.35/5.15 72.08/4.06 72.92/3.60 73.32/3.67 69.87/4.03 19.01/1.25
α-L-Rhap-(1→
30

On the basis of monosaccharide composition, methylation analysis and 1D and
2D NMR spectroscopy, the main structure of AAP was tentatively proposed in Fig. 5
as follows:
Fig. 5. Structure of AAP
3.4. AAP reduced viability of various cancer cells in vitro
To investigate the potential cytotoxicity of AAP, the comparative cytotoxic
effects of AAP on cell viability were evaluated in four different cell lines: HepG2,
SKOV3, MCF-7 and A549 cells by MTT assay (Fig. 6A). Exposure to AAP for 24
hours resulted in a decrease in cell viability in a dose-dependent manner in all cell
lines, with an IC50 range of 20.87-69.05 μg/ml. The average IC50 for HepG2,
SKOV3, MCF-7 and A549 were approximately 20.87, 43.25, 69.05, 58.54 μg/ml,
respectively. Clearly, human ovarian cancer HepG2 cell line was the most susceptible
cell line to AAP treatment. Thus further investigations were performed using HepG2
cells to evaluate antitumor activity of AAP (10, 20 and 40μg/ml) and to identify its
underlying molecular mechanism.
3.5. AAP induced the apoptosis of HepG2 cells
31

To assess whether the growth inhibitory effects of AAP on HepG2 cells was
associated with apoptosis, both control and AAP-treated cells were stained with the
fluorescent DAPI nuclear dye and visualized by a fluorescent microscope. As shown
in Fig. 6B, typical characteristics of apoptotic morphologic alterations, such as
nuclear chromatin condensation, nuclear fragmentation, and formation of apoptotic
bodies, were apparent in cells after exposure to AAP (40 µg/ml) for 24 h; in contrast,
no evidence of abnormal nuclear morphology was present in the control group, which
displayed intact and healthy nuclei. It suggested that AAP might induce ovarian
cancer cell apoptosis.
To further quantify the apoptosis caused by AAP on HepG2 cells, the
percentages of apoptotic cells were analyzed using flow cytometry (Fig. 7A). After 24
hours treatment with different concentrations of AAP, the numbers of early and late
apoptotic cells were significantly dose-dependently increased as compared to control
group (P<0.05 or P<<0.01). The percentage of total apoptotic cells in control cells
was 0.64%, 36.85% in cells treated with 10 μg/ml of AAP, 48.24% in cells treated
with 10 μg/ml of AAP and 64.78% in cells treated with 40 μg/ml of AAP (Fig. 7B).
All these results demonstrated that HepG2 cells undergo apoptosis in response to AAP
treatment.
32

Fig. 6. (A) Effect of AAP on the proliferation of various human cancer cells. (B)
Nuclear morphological changes induced by AAP in HepG2 cells after DAPI
staining (×100). (a) Control (untreated) cells; (b) cells treated with 10μg/ml AAP;
(c) cells treated with 20μg/ml AAP; (d) cells treated with 40μg/ml AAP. The
experiments were repeated three times.
33

Fig. 7. (A) Flow cytometric analysis of AAP induced apoptosis in HepG2 cells
using annexinV-FITC and PI double staining. (a) Control (untreated) cells; (b)
cells treated with 10μg/ml AAP; (c) cells treated with 20μg/ml AAP; (d) cells
treated with 40μg/ml AAP. (B) Quantitative presentation of apoptosis induced by
AAP in HepG2 cells. The experiments were repeated three times. * P < 0.05, ** P <
0.01 compared to the control group
3.6. AAP activated caspase-3, -8 and -9
A molecular hallmark of apoptosis is the activation of caspases, which are a
family of intracellular aspartate-specific cysteine proteases that execute cell death
through proteolytic cleavage in the induction of apoptosis (Stennicke & Salvesen,
1998). Once initiator caspases are activated, such as caspase 8 or caspase 9, which
would activate other downstream caspase-3 in response to pro-apoptotic signals, thus
lead to the execution stage of apoptosis (Philchenkov, 2004; Salvesen & Dixit, 1999).
Different initiator caspases are usually associated with specific pathways. For
34

example, caspase 9 is involved in the intrinsic (mitochondrial-dependent) apoptotic
pathway and caspase 8 is typically linked with the initiation of the extrinsic (death
receptor-dependent) pathway (Cho & Choi, 2002; Gogvadze, Orrenius, &
Zhivotovsky, 2006; Scaffidi et al., 1998). However, there is increasing evidence for
interaction and crosstalk between these two pathways (Chen & Wang, 2002; Michael,
2000). To examine whether caspase activation contributes to AAP-induced apoptosis,
the activities of initiator caspase (caspase-8 and-9) and effector caspase (caspase- 3)
were assessed by colorimetric assay kits after treatment of HepG2 cells with AAP (10,
20 and 40 µg/ml) for 24 h. The results showed a concentration-dependent increase in
the activities of caspase-8, -9, and -3 in AAP-treated cells (Fig. 8A). Treatment of
HepG2 cells with AAP at 10, 20 and 40µg/ml for 24 h increased 1.4-fold, 1.6-fold and
1.7-fold of the caspase-8 activity, and 1.9-fold, 2.2-fold and 2.4 fold of the caspase-9
activity, respectively. A similar change of caspase 3 activity was also observed and
increased 2.3-fold, 2.7-fold and 2.9-fold when the cells were exposed to 10, 20 and
40µg/ml of AAP after 24 h treatment. These data suggest the simultaneous
involvement of both the extrinsic and intrinsic apoptotic pathways in AAP-induced
apoptosis in HepG2 cells; however this apoptotic mechanism is more dependent on
intrinsic pathway as illustrated by a much more pronounced increase of caspase-9
activity and less activation of caspase 8.
3.7. AAP attenuated Bcl-2/Bax ratio and NF-kB expression, as well as mitochondrial
release of cytochrome c
The event of the release of cytochrome c from mitochondria into the cytosol
35

plays an important role in mitochondria-dependent apoptotic pathway (Gogvadze,
Orrenius, & Zhivotovsky, 2006). Once cytosol cytochrome c binds to apoptotic
protease activating factor 1 (Apaf-1), the formation of Apaf-1/cytochrome c
complexes allows the recruitment of caspase-9, then the latter trigger caspase-3
activation, thus resulting in execution of cell death (Green & Reed, 1998). What’s
more, Bid, a proapoptotic member of the Bcl-2 family, was induced to the cleaved
form by caspase-8 also trigger cytochrome c release (Green, 1998). To confirm
whether AAP induced apoptosis through a mitochondria-dependent pathway, proteins
in both mitochondrial and cytosolic fractions were prepared and analyzed with
western blot. Treatment of HeLa cells with 10, 20 and 40 µg/ml of AAP caused
concentration dependent release of cytochrome c from mitochondria to cytosol, as
evidenced by a gradual decrease in mitochondrial cytochrome c and a concomitant
increase in the cytosolic fraction (Fig. 8B). These results indicate that AAP can
promote cytochrome c release from mitochondria into cytosol.
36

Fig. 8. (A) Effect of AAP on caspase-3, -8 and -9 activities in HepG2 cells. (B)
Effect of AAP on the protein expression of cytochrome c in HepG2 cells. The
experiments were repeated three times. * P < 0.05, ** P < 0.01 compared to the
control group.
It is well known that the release of cytochrome c from mitochondria to cytosol is
37