Structural elucidation of three novel oligosaccharides from Kunlun Chrysanthemum flower tea and their bioactivities

Article abstract of DOI:10.1016/j.fct.2021.112032

Coreopsis tinctoria is commonly called Kunlun Chrysanthemum and a plateau plant with tremendous commercial value in functional tea and medicinal applications. In folk medicine, Kunlun Chrysanthemum flower is often used as an adjunctive therapy for diabetes and Alzheimer's disease. To further explore the chemicals responsible for the health benefits of Kunlun Chrysanthemum flowers, three homogeneous oligosaccharides, CT70-1A, CT70-1B and CT70-2 were isolated, and their detailed structures were determined from chemical and spectral analyses. The three oligosaccharides were composed of glucose, mannose, galactose, and arabinose in different ratios. They showed dose-dependent α-amylase and α-glucosidase inhibitory effects. In addition, they showed NO production inhibitory activities in BV2 cells, with IC₅₀ values of 0.23, 0.24 and 0.27 mM, respectively. Taken together, these results suggested that Kunlun Chrysanthemum oligosaccharides might ameliorate hyperglycemia and neuroinflammation, which could prevent the development of diseases such as type 2 diabetes and Alzheimer's disease. This study provides chemical and bioactive perspectives that support the consumption of Kunlun Chrysanthemum flower tea for health benefits.

Full text of DOI:10.1016/j.fct.2021.112032

Food and Chemical Toxicology 149 (2021) 112032
Contents lists available at ScienceDirect
Food and Chemical Toxicology
journal homepage: www.elsevier.com/locate/foodchemtox
Structural elucidation of three novel oligosaccharides from Kunlun
Chrysanthemum flower tea and their bioactivities
,
b
,
,
,b,*
Qian Yua 1, Wei Chena 1, Jing Zhonga, Degang Qingc, Chunyan Yana
a Clinical Pharmacy of the First Affiliated Hospital of Guangdong Pharmaceutical University, Guangzhou, 510060, China
b School of Clinical Pharmacy, Guangdong Pharmaceutical University, Guangzhou, 510006, China
c Xinjiang Institute of Traditional Chinese Medicine and Ethnodrug, Urumqi, 830002, China
A R T I C L E I N F O
A B S T R A C T
Handling editor. Dr. Jose Luis Domingo
Coreopsis tinctoria is commonly called Kunlun Chrysanthemum and a plateau plant with tremendous commercial
value in functional tea and medicinal applications. In folk medicine, Kunlun Chrysanthemum flower is often used
as an adjunctive therapy for diabetes and Alzheimer’s disease. To further explore the chemicals responsible for
the health benefits of Kunlun Chrysanthemum flowers, three homogeneous oligosaccharides, CT70-1A, CT70-1B
and CT70-2 were isolated, and their detailed structures were determined from chemical and spectral analyses.
The three oligosaccharides were composed of glucose, mannose, galactose, and arabinose in different ratios.
Keywords:
Coreopsis tinctoria
Kunlun chrysanthemum
Oligosaccharides
α
α
-Glucosidase inhibitory activity
-Amylase inhibitory activity
They showed dose-dependent
α-amylase and α-glucosidase inhibitory effects. In addition, they showed NO
production inhibitory activities in BV2 cells, with IC50 values of 0.23, 0.24 and 0.27 mM, respectively. Taken
together, these results suggested that Kunlun Chrysanthemum oligosaccharides might ameliorate hyperglycemia
and neuroinflammation, which could prevent the development of diseases such as type 2 diabetes and Alz-
heimer’s disease. This study provides chemical and bioactive perspectives that support the consumption of
Kunlun Chrysanthemum flower tea for health benefits.
Anti-neuroinflammation activity
1. Introduction
proliferation, thus enhancing neurogenesis and mitigating the cognitive
deficits in mice with AD (Huang et al., 2017).
Numerous reports indicate that neuroinflammation, characterized by
the hyperactivity of microglia, is involved in the pathogenesis of some
neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkin-
son’s disease, and Huntington’s disease (Joakim et al., 2013; Zhang and
Jiang, 2015). Excessive microglia activation leads to increased proin-
flammatory cytokines, including nitric oxide (NO), tumor necrosis factor
Diabetes mellitus (DM) is a chronic metabolic disease and a major
disorder of insulin regulation. DM, especially type 2 diabetes (T2D), has
become a major public health concern. T2D causes serious complica-
tions, such as diabetic ketoacidosis, nonketotic hyperosmolar coma,
cardiovascular diseases, and chronic kidney failure (Campbell, 2011).
Researchers have reported that diabetes increases the risk of AD (Sridhar
et al., 2015). The underlying mechanisms are complicated and varied,
including inflammation, cerebrovascular changes, the deregulation of
brain insulin uptake, and Aβ accumulation (Weinstein et al., 2015;
Pasquier et al., 2006). Takeda et al. (2010) used an animal model to
investigate the relationship between AD and DM. APP+-ob/ob (express
amyloid precursor protein with hyperglycemia) mice showed severe
cerebrovascular changes and amyloid deposition, while the insulin level
in the APP+-ob/ob mice brains was significantly lower than that in the
control. It could be concluded that cerebrovascular inflammation and
alteration in brain insulin signaling may be pivotal factors for AD and
DM. Velazquez et al. (2017) also drew the same conclusion. These
α
(TNF-α), interleukin 1β (IL-1β), and reactive oxygen species (ROS)
(Hensley, 2010). These factors may aggravate the progressive damage of
neurodegenerative diseases. Therefore, the inhibition of the hyperac-
tivity of microglia, which could suppress the production and secretion of
proinflammatory cytokines, may be a potential strategy for the treat-
ment of AD mediated by neuroinflammation (Fang et al., 2018; Zhang
et al., 2016). It has been reported that compounds extracted from
Cannabis sativa Linn. significantly inhibited the TNF-α released from BV2
microglia, suggesting its ability to treat neurodegenerative diseases
(Zhou et al., 2018). Moreover, Ganoderma lucidum (Leyss. exFr.) Karst
polysaccharides were reported to promote neural progenitor cell
* Corresponding author. School of Clinical Pharmacy, Guangdong Pharmaceutical University, Guangzhou, 510006, China.
E-mail address: ycybridge@gdpu.edu.cn (C. Yan).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.fct.2021.112032
Received 3 November 2020; Received in revised form 22 December 2020; Accepted 26 January 2021
Available online 30 January 2021
0278-6915/© 2021 Elsevier Ltd. All rights reserved.

Q. Yu et al.
Food and Chemical Toxicology 149 (2021) 112032
studies suggested that anti-diabetic drugs may have direct implications
on the treatment of AD and dementia prevention (Barnes and Yaffe,
2011). Thus, control of hyperglycemia may be an effective way to pre-
vent neurodegeneration (Heneka et al., 2015). As key enzymes,
2.2. Extraction of Kunlun Chrysanthemum oligosaccharides
Twenty kilograms flowers of Kunlun Chrysanthemum were divided
into two parts. Each 10 kg were extracted with distilled water 1:10 (w/v)
at 80 ◦C for 3 h, and the extraction was repeated three times. The su-
pernatant was concentrated to 1/7 of the initial volume under reduced
pressure and was further precipitated with 95% ethanol to a final con-
centration of 50% over 24 h. The precipitates (P50) were collected. The
supernatants were then concentrated and precipitated by 95% ethanol
to a final concentration of 70% to obtain P70.
α
-amylase and α-glucosidase play important roles in controlling post-
prandial hyperglycemia and maintaining glucose levels in the normal
range. The inhibition of these enzymes will slow down the hydrolysis of
carbohydrates, as well as prolong the speed of blood glucose absorption
from the small intestine (Joshi et al., 2015). Therefore, the inhibitors of
α
-amylase and α-glucosidase function as effective drugs to control the
blood glucose level and treat T2D. However, these commercial drugs
come with some side effects; hence, the search for natural products with
enzyme inhibitory effects and minimal side effects are needed (Kwon
et al., 2006).
2.3. Isolation and purification of Kunlun Chrysanthemum
oligosaccharides
Coreopsis tinctoria Nutt. originates in North America and was intro-
duced into Xinjiang, China. It is now widely distributed in the Kunlun
Mountains and is called “Kunlun Chrysanthemum” by the local people.
As an industrial crop, Kunlun Chrysanthemum has been cultured on a
large scale, with a yield of almost 5000 kg per year. Moreover, it is used
as an ingredient in a well-known Compositae functional tea (Wang et al.,
2014), and also used to treat cold and fever, apart from removing toxins
(Wang et al., 2015). Therefore, it has become a popular herbal product
in the health tea/beverage industry in China. Recent pharmacological
studies have shown that it has various activities, such as hypoglycemic,
hypotensive, and anticancer (Dias et al., 2010; Wang et al., 2014). A
previous study reported that ethyl acetate extracts of Kunlun Chrysan-
themum flowers significantly controlled the blood glucose levels of the
STZ-induced diabetic rats (Yao et al., 2015). Cai et al. (2016) also re-
ported that extracts of Kunlun Chrysanthemum flower prevented high
blood glucose levels, improved glucose tolerance in high-fat mice, and
P70 was selected for further purification based on its water-solubility
and higher yield. After deproteinization and dialysis (molecular weight
cut-off 1000 Da), P70 was concentrated and freeze-dried. Subsequently,
P70 was dissolved in distilled water (20 mg/mL) and centrifuged (8124
g) for 5 min. The supernatants (10 mL) were separated on a DEAE-52
column (Ø 2.5 × 45 cm). The column was eluted with a gradient of
NaCl solution (0–1 M) at a flow rate of 1.2 mL/min. The elution was
collected via automatic fraction collector and monitored by the phenol-
sulfuric acid method (Dubois et al., 1956) to obtain two fractions. The
fraction eluted with distilled water, labeled as P70-1, was collected,
concentrated and dialyzed with cellulose membrane (molecular weight
cut-off 1000 Da) against distilled water in a 2 L beaker. The solution
outside the dialysis tube was collected, concentrated, freeze-dried and
further purified via Sephacryl S-100 gel-filtration column (Ø 1.6 × 100
cm), eluting with distilled water at a flow rate of 20 mL/h. Two sub-
fractions containing carbohydrates were collected and labeled as
CT70-1A and CT70-1B, respectively. Another fraction eluted with 0.015
M NaCl, labeled as P70-2, was collected, concentrated and dialyzed
(molecular weight cut-off 100 Da). The solution inside the dialysis tube
was collected, concentrated and freeze-dried. P70-2 (20 mg/mL) was
further purified via Sephacryl S-100 gel-filtration column (Ø 1.6 × 100
cm), eluting with distilled water at a flow rate of 20 mL/h. The eluate
was collected, freeze-dried, and labeled as CT70-2. The procedure of
extraction and purification of three oligosaccharides from the flowers of
Kunlun Chrysanthemum was shown in Scheme S1. The structures and
bioactivities of CT70-1A, CT70-1B, and CT70-2 were studied in the
following experiments.
exhibited
α-glucosidase inhibitory activity. Certain extracted compo-
nents of Kunlun Chrysanthemum, including phenolic acids, chalcones,
and flavonoids, inhibited NO production in N9 cells, showing
anti-neuroinflammatory activity (Li et al., 2015). Moreover, Kunlun
Chrysanthemum contains carbohydrates, amino acids, and flavonoids
(Guo et al., 2015a). Of these, carbohydrates are considered the major
biologically active components in Kunlun Chrysanthemum with phar-
macological functions, including antioxidant and anti-proliferative ac-
tivity (Jing et al., 2016). However, there are no studies on the effects of
Kunlun
Chrysanthemum
oligosaccharides,
in
terms
of
anti-neuroinflammation or
α
-glucosidase and -amylase inhibitory ac-
α
tivities. Additionally, there are only a few reports on the detailed
structures of purified oligosaccharides from Kunlun Chrysanthemum. In
this study, we isolated, purified, and characterized three oligosaccha-
rides from Kunlun Chrysanthemum and measured their
2.4. Determination of homogeneity, molecular weight and optical ratation
The homogeneity of oligosaccharide was determined using specific
optical rotation method (Qian et al., 2009). Eighty milligrams of oligo-
saccharide samples were dissolved in distilled water, and anhydrous
ethanol was added to a final concentration of 70%. One precipitate was
obtained after centrifugation (8124 g) for 5 min. Subsequently, anhy-
drous ethanol was added to the supernatant to obtain a final concen-
tration of 80%, and another precipitate was obtained. The two
precipitates were dissolved in distilled water to a final concentration of
2 mg/mL, respectively. The optical rotation of the solution was deter-
mined using a P8000 Kruss polarimeter (Germany) (Li et al., 2013).
The homogeneity and molecular weight distribution of samples were
further estimated by high-performance gel permeation chromatography
(HPGPC), equipped with a Waters 2414 refractive index detector and
TSK-GEL G-5000PWXL and G-3000PWXL gel column in series (Tosoh
Biosep, Japan) (Wang et al., 2017b). Twenty microliters of sample so-
lution (3 mg/mL) were eluted with 0.02 M KH2PO4 solution at a flow
rate of 0.5 mL/min. The columns were calibrated with T-series Dextran
standards (Dextran T1000, T500, T70, T40, T10, and T5). The average
molecular weight was estimated via the calibration curve obtained.
Matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry (MALDI-TOF-MS, Bruker ultraFleXtreme, Germany) was
used to detect the molecular weight distribution. The sample solution
anti-neuroinflammation activities, as well as the
-amylase inhibitory activities.
α-glucosidase and
α
2. Materials and methods
2.1. Materials and chemicals
The flowers of Kunlun Chrysanthemum were obtained from Minfeng
county, Hetian city, Xinjiang Autonomous Region, China. Voucher
specimens (No. 2017061101) were deposited in the School of Pharmacy
at Guangdong Pharmaceutical University. Cellulose DEAE-52 was pur-
chased from Whatman Co. (Maidstone, Kent, U.K.). Sephacryl S-100 HR
gel filtration medium was purchased from GE Healthcare Bio-Sciences
AB (Uppsala, Sweden). Standard monosaccharides, T-series dextran, 1-
phenyl-3-methyl-5-pyr-azolone (PMP), trifluoroacetic acid (TFA),
dimethyl-sulfoxide (DMSO), and Congo red were purchased from Sigma
Chemical Co. (St. Louis, MO, USA). α-Glucosidase and α-amylase were
purchased from Sigma Aldrich (Steinheim, Germany). BV2 cells were
purchased from the School of Basic Medical Science at Peking Union
Medical College. All other chemicals and solvents were of analytical
grade.
2

Q. Yu et al.
Food and Chemical Toxicology 149 (2021) 112032
(0.5
μ
L) was mixed with 0.5
μ
L of the matrix solution, and 1
μ
L of the
2.8. Nuclear magnetic resonance (NMR) analysis
mixture was applied to a stainless steel sample slide and dried under
vacuum. Mass spectra were acquired from 700 m/z to 3500 m/z in
positive linear mode (Arrizon et al., 2010).
Fifty milligrams of oligosaccharide were dissolved in 750 μL D2O.
The proton nuclear magnetic resonance (1H NMR), carbon-13 nuclear
magnetic resonance (13C NMR) and Two-dimensional (2D) spectros-
copy, including heteronuclear single-quantum coherence (HSQC) and
heteronuclear multiple-bond correlation (HMBC) were obtained from a
Bruker AV-500 spectrometer (Germany) with tetra-methylsilane (TMS)
as the internal standard.
2.5. Monosaccharide component analysis of Kunlun Chrysanthemum
oligosaccharides
The monosaccharide composition of oligosaccharide was determined
by PMP precolumn derivatization and HPLC method (Dai et al., 2010;
Wu et al., 2014). Briefly, 5 mg of each oligosaccharide was hydrolyzed
by 2 M TFA (2 mL) at 90 ◦C in sealed tubes for 30 min, respectively.
Then, the hydrolysate was co-distilled with methanol to remove excess
TFA until the pH returned to neutral.
2.9.
α
-Glucosidase inhibitory activity
-glucosidase inhibitory activity was determined according to a
The
α
previously published method (Striegel et al., 2015). A mixture of 40
μ
L
sample (dissolved in 0.1 M phosphate buffer), 20 μL α-glucosidase (0.2
The residues of CT70-1A and CT70-1B or monosaccharides standard
mixture (mannose, rhamnose, glucuronic acid, galacturonic acid,
glucose, galactose, xylose, arabinose, and fucose) were further mixed
U/mL), and 0.1 M phosphate buffer (pH 6.8, 50
μ
L) were incubated in
96-well plates at 37 ◦C for 5 min. Then, 50
μ
L of 10 mM p-nitro-
phenyl-α-D-glucopyranoside solution dissolved in 0.1 M phosphate
with 100 μL PMP methanol solution (0.5 M). The mixture was incubated
for 30 min at 70 ◦C and neutralized with 100
μL of 0.3 M HCl solution.
buffer (pH 6.8) were added to each well. The reaction mixtures were
incubated at 37 ◦C for 30 min. The catalytic reaction was terminated by
the addition of 1 mL Na2CO3 solution (0.1 M). Acarbose served as the
positive control. The reaction system without saccharides was used as
the blank. The absorbance was measured at 405 nm with a UV–Vis
spectrophotometer, and the inhibition percentage was expressed as
follows:
The aqueous solution of derivatization was extracted with equivalent
volume of chloroform and repeated for three times to remove the PMP
residues. Finally, samples were filtered through a 0.45 μm filter and
analyzed by an Agilent 1260 HPLC system (Agilent, Santa Clara, CA,
USA). The mobile phase was 0.05 M NaH2PO4–Na2HPO4 elution buffer
(pH 6.7) and acetonitrile (83:17, v/v) at a flow rate 1 mL/min. The
system was coupled with a ZOR-BAX Eclipse XDB C-18 column and an
ultraviolet detector (Jiang et al., 2016).
Ablank ꢀ Asample
Inhibition percentage (%)
=
× 100%
(1)
Ablank
The monosaccharide composition of CT70-2 was determined by
high-performance anion exchange chromatography with amperometric
detection (HEPAEC-PAD) (Wang et al., 2017a). The hydrolysate residue
of CT70-2 was re-dissolved in 1 mL of distilled water. The solution or
monosaccharide standard mixture (mannose, rhamnose, glucose,
galactose, xylose, arabinose, fructose, and fucose) was analyzed on an
HPAEC-PAD coupled with a CarboPac PA10 column (250 × 2 nm),
followed by elution with H2O and 250 mM NaOH (96:4, v/v) at a flow
rate 0.2 mL/min.
2.10.
α
-Amylase inhibitory activity
-amylase inhibitory activity was determined according to the
The
α
literature (Eleazu et al., 2016). Twenty-five microliters of sodium
phosphate buffer (20 mM, pH 6.8), 25 L sample solution (dissolved in
20 mM sodium phosphate buffer), and 0.5 mg/mL -amylase (dissolved
in 20 mM sodium phosphate buffer) were mixed in a microcentrifuge
tube. The mixture was incubated for 10 min at 25 ◦C. Then, 25
L of
0.5% starch solution in sodium phosphate buffer (20 mM, pH 6.8) was
added. The reaction mixture was kept at 25 ◦C for 10 min. Next, 50
L of
μ
α
μ
2.6. Fourier transform-infrared (FT-IR) spectrum analysis
μ
96 mM 3,5-dinitrosalicylic acid were added and heated in boiling water
for 5 min. The mixture was then cooled to room temperature. Acarbose
was used as the positive control. The reaction system without saccha-
rides was used as the blank. The absorbance was measured at 540 nm
with a UV–Vis spectrophotometer, and the inhibition percentage was
calculated.
Samples (1 mg) were mixed with KBr (100 mg), pressed into a pellet,
and then analyzed with a PerkinElmer FT-IR spectrometer from 4000 to
400 cmꢀ 1(Yan et al., 2019) .
2.7. Methylation of Kunlun Chrysanthemum oligosaccharides
The oligosaccharides were methylated according to the method re-
ported by Huang et al. (2015). The oligosaccharide (3.5 mg) was dis-
solved in 1 mL anhydrous methanol and evaporated. The residue was
dissolved in 5 mL DMSO and then methylated with DMSO/NaOH slurry
(5 mL/40 mg) and 7.5 mL CH3I in an ultrasonic bath to accelerate the
reaction. The complete methylation was confirmed by the disappear-
ance of the hydroxyl absorption peak in the IR spectrum. Then, the
methylated sample was hydrolyzed with 2 mL TFA (3 M) for 6 h at
120 ◦C and dehydrated again by adding 1 mL anhydrous methanol.
Later, the product was dissolved with 2 mL distilled water, reduced with
2.11. Effects of Kunlun Chrysanthemum saccharides on the viability and
NO production of BV2 cells
The viability of BV2 cells was examined by MTT method. BV2 cells
were maintained in DMEM medium supplemented with 10% fetal
bovine serum (FBS), 100 units/mL penicillin and 100 μg/mL strepto-
mycin. All cells were cultured in a humidified atmosphere containing
5% CO2 at 37 ◦C. BV2 cells were seeded into a 96-well plate with a
density of 3 × 104 cells/mL and cultured for 24 h. After being treated
with tested samples (10
μL) for 24 h, the BV2 cells were incubated with
20 mg NaBH4 for 30 min at 40 ◦C, acetylated with 100
μL acetic anhy-
20 μL MTT (5 mg/mL) for 4 h. Then, 150 μL of DMSO were added to each
dride–pyridine (1:1 molar ratio) for 120 min at 95 ◦C. The final product
was subjected to GC-MS (GC-MS-QP 2010; Shimadzu, Kyoto, Japan) to
analyze the methylated alditol acetates. The temperature programming
method was used for GC/MS assay. The temperature of column was kept
at 150 ◦C for 1 min, and increased from 150 ◦C to 180 ◦C at a rate of
10 ◦C/min and maintained at 180 ◦C for 1 min, then up to 260 ◦C at a
rate of 15 ◦C/min and kept at 260 ◦C for 5 min. The injection temper-
ature was 220 ◦C. The mass spectra were acquired from 40 m/z to 400
m/z.
well, and the absorbance was measured on a microplate reader at 490
nm.
The NO production of BV2 cells was determined according to a
previously published method (Nhiem et al., 2017). BV2 cells were
seeded into a 96-well plate with a density of 3 × 105 cells/mL and
incubated at 37 ◦C and 5% CO2 for 24 h. BV2 cells were treated with
different concentrations of P70 (30, 60, 120, 240
CT70-1B, CT70-2 (12.5, 25, 50, 100, 200
25, 50, 100
M) for 48 h at 37 ◦C in the presence of LPS (1
μ
g/mL), CT70-1A,
M), or indomethacin (12.5,
g/mL). Fifty
μ
μ
μ
3

Q. Yu et al.
Food and Chemical Toxicology 149 (2021) 112032
microliters of the cell supernatant were mixed with the I and II solutions
from the NO kits. The absorbance was measured on a microplate reader
at 540 nm. The percentage of NO inhibition was calculated as follows:
Inhibition percentage (%) = (Al ꢀ As)/(Al ꢀ Ac) × 100%,
(2)
where Ac is the absorbance of the control group (the reaction system
without sample or LPS); As is the absorbance of the sample group; and Al
is the absorbance of the LPS group (the reaction system without sample).
2.12. Statistical analysis
Results are expressed as mean ± SD. Statistical analysis of experi-
mental data was performed using one-way analysis of variance (one-way
ANOVA) for at least three independent biological replicates. All calcu-
lations were performed using Graph Pad Prism6 (GraphPad Software,
San Diego, CA). P < 0.05 was considered statistically significant.
3. Results and discussion
3.1. Basic properties of Kunlun Chrysanthemum oligosaccharides
Crude saccharides were isolated from Kunlun Chrysanthemum
through hot water extraction, ethanol precipitation, and deproteiniza-
tion. P70 was separated on a DEAE-cellulose 52 column and eluted with
distilled water and 0.015 M NaCl to gain P70-1 and P70-2, respectively.
P70-1 was dialyzed with a cellulose membrane (molecular weight cut-
off, 100 Da) and subsequently purified by using a Sephacryl S-100 col-
umn. Distilled water was used as the eluent to obtain CT70-1A and
CT70-1B. After dialysis, P70-2 was further purified by a Sephacryl S-100
column and eluted with distilled water to obtain CT70-2.
CT70-1A, CT70-1B and CT70-2 all showed a single symmetrical peak
by HPGPC detection (Fig. 1 and Figs. S1–S2), indicating that they were
all homogeneous oligosaccharides, and their molecular weights were
1221, 1318, and 1247 Da, respectively. The differentials of specific
optical rotation were no more than 5◦ between the two fractional pre-
cipitates, further indicating that each sample was a homogeneous
oligosaccharide, which were consistent with the results of HPGPC. Their
specific optical rotations were = -9◦, ꢀ 37◦, and ꢀ 20◦, respectively.
Fig. 2. HPLC analysis of the mixture of standard monosaccharides (A) and
component monosaccharides of CT70-1A (B). 1, mannose; 2, rhamnose; 3,
glucuronic acid; 4, galacturonic acid; 5, glucose; 6, galactose; 7, xylose; 8,
arabinose; 9, fucose.
3.2. Monosaccharide composition analysis of Kunlun Chrysanthemum
oligosaccharides
As shown in Fig. 2 and Figs. S3–S4, the results of HPLC and HPAEC-
PAD revealed that CT70-1A, CT70-1B, and CT70-2 were all composed of
mannose, glucose, galactose, and arabinose.
3.3. FT-IR analysis of Kunlun Chrysanthemum oligosaccharides
The IR spectra of Kunlun Chrysanthemum oligosaccharides are
shown in Fig. 3 and Figs. S5–S6. A broad bands at 3349 cmꢀ 1 (CT70-1A),
Fig. 1. The HPGPC chromatogram of CT70-1A.
4

Q. Yu et al.
Food and Chemical Toxicology 149 (2021) 112032
3:1:1:1:1. That also indicated the three terminal Glc were coincident.
Six residues of CT70-1B were present, including 1,5-di-O-acetyl-
2,3,4,6-tetra-O-methyl-D-glucitol, 1,2,3,5,6-penta-O-acetyl-4-O-methyl-
D-glucitol, 1,2,4-tri-O-acetyl-3,5-di-O-methyl-L-arabinitol, 1,5,6-tri-O-
acetyl-2,3,4-tri-O-methyl-D-galactitol, 1,3,5,6-tetra-O-acetyl-2,4-di-O-
methyl-D-mannitol, and 1,4-di-O-acetyl-2,3,5-tri-O-methyl-L-arabinitol.
These results indicate that the main residue linkages of CT70-1B were
Glcp-(1→, →2,3,6)-Glcp-(1→, →2)-Araf-(1→, →6)-Galp-(1→, →3,6)-
Manp-(1→ and Araf-(1 → .
Five residues of CT70-2 were identified, including 1,5-di-O-acetyl-
2,3,4,6-tetra-O-methyl-D-glucitol,
1,4,5-tri-O-acethyl-2,3,6-tri-O-
methyl-D-glucitol,
1,4-di-O-acetyl-2,3,5-tri-O-methyl-L-arabinitol,
1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl-D-galactitol and 1,3,5,6-tetra-O-
acetyl-2,4-di-O-methyl-D-mannitol. These results indicate that the main
residue linkages of CT70-2 were Glcp-(1→, →4)-Glcp-(1→, Araf-(1→,
→6)-Galp-(1→ and →3,6)-Manp-(1 → .
Fig. 3. IR spectrum of CT70-1A.
3358 cmꢀ 1 (CT70-1B), and 3393 cmꢀ 1 (CT70-2) were matched the hy-
droxyl stretching vibration of oligosaccharides. The peaks around 2927
cmꢀ 1 (CT70-1A), 2933 cmꢀ 1 (CT70-1B), and 2927 cmꢀ 1 (CT70-2) were
corresponded to the C–H stretching vibration. A weak bands appeared at
1639 cmꢀ 1(CT70-1A), 1643 cmꢀ 1 (CT70-1B), and 1638 cmꢀ 1 (CT70-2)
were due to absorption of water, because of hygroscopic properties of
this oligosaccharide. The bands around 1036 cmꢀ 1(CT70-1A), 1026
cmꢀ 1 (CT70-1B), and 1035 cmꢀ 1 (CT70-2) were the characteristic peaks
of pyranose configuration of the oligosaccharides. In addition, the peak
at 938 cmꢀ 1 indicated the existence of the furan ring in CT70-2 (Wang
et al., 2017a; Albuquerque et al., 2014).
3.5. NMR spectra of Kunlun Chrysanthemum oligosaccharides
The 1H and 13C NMR spectra of CT70-1A are shown in Fig. 4. In the
1H NMR spectrum, all chemical signals were distributed from 1.00 to
6.00 ppm. Those in the range of 4.00–6.00 ppm were associated with
anomeric protons. The
α and β configurations of the residues were
determined via the chemical shift and coupling constants of the
anomeric proton (Habibi et al., 2004). The 1H NMR data showed signals
at δH 4.41, 4.83, 5.04, and 5.36 ppm, which were attributed to charac-
teristic anomeric proton signals. An examination of the 13C NMR data
disclosed five anomeric carbon signals at δC 92.4, 100.1, 102.2, 103.1,
and 108.3 ppm, which substantiated that there were five residues in
CT70-1A. The result was consistent with the result of GC-MS. In the
HSQC spectra (Fig. 4C), we observed the correlations of 5.36 with 92.4,
5.36 with 108.3, 5.04 with 102.2, 4.83 with 100.1, and 4.41 with 103.1.
Comprehensive analyses of 1H NMR, 13C NMR, HSQC spectra and
GC-MC suggested that the glycosidic linkages were followed by
3.4. Methylation and GC-MS analysis
GC-MS analysis was performed to identify linkages in the sugar
residue. The methylated oligosaccharides were acid hydrolyzed and
acetylated prior to GC-MS analysis. Mass spectrum of methylated sugar
residues are shown in Figs. S7–S9. The results of methylation analysis by
GC-MS are shown in Table 1 and Tables S1–S2. Five residues of CT70-1A
were identified, including 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-D-
glucitol, 1,2,4-tri-O-acetyl-3,5-di-O-methyl-L-arabinitol, 1,5,6-tri-O-
acetyl-2,3,4-tri-O-methyl-D-galactitol, 1,3,5,6-tetra-O-acetyl-2,4-di-O-
methyl-D-mannitol, and 1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl-D-gluci-
tol. These results indicate that CT70-1A may be composed of Glcp-(1→,
→2)-Araf-(1→, →6)-Galp-(1→, →3,6)-Manp-(1→ and →6)-Glcp-(1→ at a
ratio of 11.34:4.21:3.05:4.93:4.01, respectively, which was about
(1→)-linked
α
-D-glucose, (1 → 2)-linked
α-L-arabinose, (1 → 6)-linked
β-D-galactose, (1 → 6)-linked
α
-D-glucose and (1 → 3, 6)-linked
β-D-mannose (Dertli et al., 2017; Sahragard and Jahanbin, 2017; Zhao
et al., 2017; Li et al., 2017). Furthermore, based on the correlations of
HMBC spectrum, the H2/C2, H3/C3, H4/C4, H5/C5, and H6/C6
chemical shifts of each residue were defined. Thus, the combined 1H,
13C, HSQC, and methylation data, along with data from the literature
(Wang et al., 2009; Westphal et al., 2010; Pattanayak et al., 2015; Guo
et al., 2015b), led to complete descriptions of the residues of CT70-1A
(Table 2).
The HMBC spectra showed cross-peaks in different residues, con-
firming the linkage sites and sequences among residues. Fig. 4D shows
cross-peaks that could be assigned as follows: H1 (5.36 ppm) of residue
A and C2 (84.0 ppm) of residue B (AH1/BC2), suggesting that the C-2 of
residue B was linked to the O-1 of residue A. The cross-peaks of 4.03/
108.3 ppm (DH3/BC1), 3.45/102.2 ppm (DH6/EC1), 5.36/72.3 ppm
(AH1/FC6), 4.83/72.1 ppm (FH1/EC6), and 5.36/103.1 ppm (AH1/
DC1) suggest that the C-1 of residue B was linked to the O-3 of residue D,
the C-1 of residue E was linked to the O-6 of residue D, the C-6 of residue
F was linked to the O-1 of residue A, the C-6 of residue E was linked to
the O-1 of residue F, and the C-1 of residue D was linked to the O-1 of
residue A.
Table 1
GC-MS data of the methylated products of CT70-1A.
PMAA
Molar
ratio
Mass fragments (m/z)
Linkage
1,5-di-O-acetyl-
2,3,4,6-tetra-
O-methyl-D-
glucitol
11.34
4.21
3.05
4.93
4.01
43,57,71,87,101,118,129,145,162
Glcp-(1→
1,2,4-tri-O-
acetyl-3,5-di-
O-methyl-L-
arabinitol
43,57,87,101,111,129,146,161
→2)-
Araf-(1→
1,5,6-tri-O-
acetyl-2,3,4-
tri-O-methyl-
D-galactitol
1,3,5,6-tetra-O-
acetyl-2,4-di-
O-methyl-D-
mannitol
43,57,87,99,101,117,129,169,189
43,57,87,101,117,129,139,189,234
43,57,71,99,101,117,129,143,161,173
→6)-
The 1H and 13C NMR spectra of CT70-1B are shown in Fig. S10. The
1H NMR data show the characteristic anomeric proton signals at δH 4.41,
4.36, 4.84, 5.16, and 5.36 ppm. An examination of the 13C NMR data
Galp-(1→
→3,6)-
Manp-
(1→
disclosed six anomeric carbon signals at δ 92.4, 99.7, 103.0, 103.2,
C
108.6, and 109.4 ppm, which indicated that there were six residues in
CT70-1B. The result was consistent with the result of GC-MS. In the
HSQC spectra (Fig. S10C), we observed the correlations of 5.36 with
92.4, 5.36 with 108.6, 5.16 with 109.4, 4.84 with 99.7, 4.41 with 103.0
and 4.36 with 103.2. A comprehensive analysis of 1H NMR, 13C NMR,
HSQC spectra, and GC-MC suggested that the glycosidic linkages were
1,5,6-tri-O-
acetyl-2,3,4-
tri-O-methyl-
D-glucitol
→6)-
Glcp-(1→
5

Q. Yu et al.
Food and Chemical Toxicology 149 (2021) 112032
Table 2
Chemical shifts for the resonances of glycosyl residues in CT70-1A, obtained
from 1H and13C NMR spectra.
Sugar residue
C1/
H1
C2/
H2
C3/
H3
C4/
H4
C5/
H5
C6/H6
α
A
-D-Glcp-(1→
92.4
5.36
72.3
3.70
75.1
3.87
73.1
3.30
75.3
3.83
65.1
3.72/
3.93
–
→2)-
α-L-Araf-(1→
108.3
5.36
84.0
4.01
72.0
79.3
4.19
79.3
80.7
4.37
75.1
67.2
3.96
72.6
B
–
→3,6)-β-D-Manp-
103.1
67.2
(1→
D
4.41
3.98
74.2
4.03
76.8
4.03
4.03
74.3
4.18
76.6
3.85
3.87
73.6
3.85
75.1
3.75
3.60
77.1
4.17
75.6
3.38
3.45
72.1
4.15
72.3
3.70/
3.64
→6)-β-D-Galp-(1→
102.2
5.04
E
→6)-
α-D-Glcp-(1→
100.1
4.83
F
followed by (1→)-linked
α
-D-glucose, (1 → 2)-linked
α
-L-arabinose,
(1→)-linked -L-arabinose, (1 → 2,3,6)-linked
α
α-D-glucose, (1 → 6)-
linked β-D-galactose, and (1 → 3, 6)-linked β-D-mannose (Dertli et al.,
2017; Sahragard and Jahanbin, 2017; Zhao et al., 2017; Li et al., 2017).
Furthermore, based on the correlations of the HMBC spectrum, the
H2/C2, H3/C3, H4/C4, H5/C5, and H6/C6 chemical shifts of each res-
idue were defined. Thus, based on the 1H, 13C, HSQC, and methylation
data, and in combination with data published from related studies
(Wang et al., 2009; Westphal et al., 2010; Pattanayak et al., 2015; Guo
et al., 2015b), we assigned a complete description of the residues of
CT70-1B (Table S3).
The HMBC spectra showed cross-peaks in different residues, con-
firming the linkage sites and sequences among residues. Fig. S10D shows
cross-peaks that could be assigned as follows: H1 (5.36 ppm) of residue
A and C2 (84.1 ppm) of residue F (AH1/FC2), suggesting that the C-2 of
residue F was linked to the O-1 of residue A. The cross-peaks of 4.08/
108.6 ppm (BH3/FC1), 5.36/80.4 ppm (AH1/EC3), 3.94/109.4 ppm
(BH2/GC1), 3.68/109.4 ppm (EH6/GC1), 3.86/103.0 ppm (BH6/EC1),
5.36/103.2 ppm (AH1/DC1), and 4.84/72.6 ppm (BH1/DC6) suggest
that the C-1 of residue F was linked to the O-3 of residue B, the C-3 of
residue E was linked to the O-1 of residue A, the C-1 of residue G was
linked to the O-2 of residue B, the C-1 of residue G was linked to the O-6
of residue E, the C-1 of residue E was linked to the O-6 of residue B, the
C-1 of residue D was linked to the O-1 of residue A, and the C-6 of residue
D was linked to the O-1 of residue B.
The 1H and 13C NMR spectra of CT70-2 are shown in Fig. S11. The 1H
NMR data showed characteristic anomeric proton signals at δ 4.41,
H
5.02, 5.10, 5.18 and 5.36 ppm. Examination of the 13C NMR data dis-
closed five anomeric carbon signals at δC 92.4, 99.9, 103.3, 107.4 and
109.2 ppm, indicating the presence of five residues in CT70-2. The result
was consistent with the result of GC-MS. In the HSQC spectra
(Fig. S11C), we observed the correlations of 5.36 with 92.4, 5.18 with
109.2, 5.10 with 99.9, 5.02 with 107.4 and 4.41 with 103.3. Compre-
hensive analyses of 1H NMR,13C NMR, HSQC spectra and GC-MC sug-
gested that the glycosidic linkages were followed by (1→)-linked
α-D-
glucose, (1→)-linked -L-arabinose, (1 → 4)-linked -D-glucose, (1 → 6)-
α
α
linked β-D-galactose, and (1 → 3, 6)-linked β-D-mannose (Dertli et al.,
2017; Sahragard and Jahanbin, 2017; Zhao et al., 2017; Li et al., 2017).
Furthermore, based on the correlations of HMBC spectrum, the H2/C2,
H3/C3, H4/C4, H5/C5 and H6/C6 chemical shifts of each residue were
defined. Thus, combined with the 1H, 13C, HSQC, and methylation data
as well as the related references (Wang et al., 2009; Westphal et al.,
2010; Pattanayak et al., 2015; Guo et al., 2015b), we assigned a com-
plete description of residues of CT70-2 (Table S4).
The HMBC spectra showed cross-peaks in different residues, con-
firming the linkage sites and sequences among residues. Fig. S11D shows
cross-peaks that could be assigned as follows: H1 (5.36 ppm) of residue
A and C6 (72.4 ppm) of residue D (AH1/DC6), suggesting that the C-6 of
Fig. 4. 1H (A), 13C (B), HSQC (C), and HMBC (D) spectra of CT70-1A.
6

Q. Yu et al.
Food and Chemical Toxicology 149 (2021) 112032
residue D was linked to the O-1 of residue A. The cross-peaks of 3.83/
109.2 ppm (BH4/EC1), 5.10/81.1 ppm (BH1/FC3), 3.64/107.4 ppm
(FH6/DC1), and 5.36/103.3 ppm (AH1/FC1) indicate that the C-1 of
residue E was linked to the O-4 of residue B, the C-3 of residue F was
linked to the O-1 of residue B, the C-1 of residue D was linked to the O-6
of residue F, and the C-1 of residue F was linked to the O-1 of residue A.
In addition, the 3.83/99.9 ppm (BH4/BC1) cross-peaks suggested that C-
1 of residue B was linked to the O-4 of residue B, indicating that residue
B was repeated in CT70-2.
In summary, the predicted structures of CT70-1A, CT70-1B and
CT70-2 were shown in Scheme 1. The three oligosaccharides were
composed of glucose, mannose, galactose, and arabinose in different
ratios. Interestingly, they were all composed of (1 → 6)-linked β-D-Galp,
(1 → 3, 6)-linked β-D-Manp and α-D-Glcp-(1 → . However, there were
also some different residues in them. CT70-1A also consisted of (1 → 2)-
linked -L-Araf and (1 → 6)-linked -D-Glcp. CT70-1B also included (1
→ 2)-linked -L-Araf, (1 → 2,3,6)-linked -D-Glcp and -L-Araf-(1 → .
And CT70-2 also consisted of (1 → 4)-linked -D-Glcp and -L-Araf-(1 →
In short, CT70-1A, CT70-1B and CT70-2 were all novel
oligosaccharides.
α
α
α
α
α
α
α
.
3.6.
α-Glucosidase inhibitory activity of Kunlun Chrysanthemum
saccharides
The inhibition of
α-glucosidase, a key carbohydrate hydrolyzing
enzyme, could serve as a promising target for the treatment of diabetes
by controlling the postprandial glucose levels and suppressing post-
prandial hyperglycemia (Ademiluyi et al., 2014). The
α-glucosidase
inhibitory activities of Kunlun Chrysanthemum saccharides are pre-
sented in Fig. 5. P70 is a crude saccharide obtained from Kunlun
Chrysanthemum. P70 was deproteinized to get the saccharide dP70. P70
and dP70 had almost the same effects on α-glucosidase inhibitory ac-
tivity, revealing that the protein was not the bioactive component in
crude saccharide P70. CT70-1A mildly inhibited α-glucosidase, with an
IC50 value of 2.68 ± 0.10 mM (3.27 ± 0.01 mg), while CT70-1B and
CT70-2 had IC50 values of 2.62 ± 0.06 mM (3.45 ± 0.08 mg) and 4.89 ±
0.02 mM (6.05 ± 0.02 mg), respectively. CT70-1B, CT70-2, and
CT70-1A had better α-glucosidase inhibitory activities than some re-
ported polysaccharides, such as PRM3 isolated from the Rhynchosia
minima root (IC50 value of 8.85 mg/mL) (Jia et al., 2017). The result
suggested that Kunlun Chrysanthemum oligosaccharides may be the
active components responsible for the anti-diabetic activities of Kunlun
Chrysanthemum.
3.7.
α
-Amylase inhibitory activity of Kunlun Chrysanthemum saccharides
α
-Amylase is one of the major secretory products of the pancreas and
salivary glands, and plays a role in the digestion of starch and glycogen.
The inhibition of α-amylase can control the breakdown of dietary starch
into smaller oligomers to delay glucose absorption, which is an impor-
tant aspect in the management of type 2 diabetes (Lordan et al., 2013).
The α-amylase inhibitory activity of Kunlun Chrysanthemum saccha-
rides is presented in Fig. 6. P70 and dP70 had similar effects on the
-amylase inhibitory activity, revealing the protein was not the bioac-
tive component in crude saccharides P70. CT70-1A significantly
α
inhibited α-amylase, with an IC50 value of 2.77 ± 0.10 mM (3.38 ± 0.01
Scheme 1. The predicted structures of CT70-1A, CT70-1B and CT70-2.
mg), while CT70-1B and CT70-2 had IC50 values of 2.75 ± 0.06 mM
(3.62 ± 0.08 mg) and 3.35 ± 0.03 mM (4.18 ± 0.04 mg), respectively.
These values were better than that of corn silk polysaccharide N-CSPS
(IC50 value was 10.07 mg/mL) (Chen et al., 2013).
Chrysanthemum crude saccharides P70 and 200
μ
M of homogeneous
saccharides had no cytotoxic effects on BV2 cells. The result indicated
that 0–240 μg/mL of crude saccharides and 0–200 μM of homogeneous
saccharides could be selected as the tested concentration in the
following experiment.
3.8. Effects of Kunlun Chrysanthemum saccharides on the viability and
NO production of BV2 cells
Extensive pathology studies revealed that AD is closely related to
neuroinflammation. In the occurrence and progress of neuro-
inflammation, excessive amounts of NO in the central nervous system
We determined the cytotoxicity of CT70-1A, CT70-1B, CT70-2 via
NO production of BV2 cells. As shown in Fig. 7A, 240 μg/mL of Kunlun
7

Q. Yu et al.
Food and Chemical Toxicology 149 (2021) 112032
Fig. 5.
α-Glucosidase inhibitory activity of Kunlun Chrysanthemum saccharides. (A) P70: Kunlun Chrysanthemum crude saccharide; dP70: the crude saccharide after
deproteinization of P70; (B) CT70-1A, CT70-1B, CT70-2: the homogeneous saccharides of Kunlun Chrysanthemum.
Fig. 6.
α-Amylase inhibitory activity of Kunlun Chrysanthemum saccharide. (A) P70: Kunlun Chrysanthemum crude saccharide; dP70: the crude saccharide after
deproteinization of P70; (B) CT70-1A, CT70-1B, CT70-2: the homogeneous saccharides of Kunlun Chrysanthemum.
(CNS) were released to stimulate microglia activation and inflammatory
response, and related studies revealed that compounds with NO inhib-
itory effects are potentially useful for the treatment of inflammatory and
related neurodegenerative diseases (Liu et al., 2018). The
anti-neuroinflammatory activities of Kunlun Chrysanthemum saccha-
rides were tested by measuring NO production in BV2 cells induced by
LPS. As shown in Fig. 7B–C, the Kunlun Chrysanthemum crude sac-
charides P70 moderately inhibited the NO production in BV2 cells.
Moreover, CT70-1A, CT70-1B and CT70-2 showed significant NO pro-
duction inhibitory activities, with IC50 values of 0.23, 0.24 and 0.27
mM, respectively. The three purified oligosaccharides obtained from
P70 exhibited synergistic effects to inhibit NO production in BV2 cells.
The results suggest that CT70-1A, CT70-1B and CT70-2 are the active
components in Kunlun Chrysanthemum that can be used for the treat-
ment of AD.
α
-D-Glcp1-6
1-1 -D-Glcp,
L-Araf1-3)( -L-Araf1-2)
1-6β-D-Galp1-6( -L-Araf1-[4
pectively. Kunlun Chrysanthemum oligosaccharides CT70-1A, CT70-1B,
α
-D-Glcp1-6β-D-Galp1-6(
-D-Glcp1-3( -L-Araf1-6)β-D-Manp1-6(
-D-Glcp1-6β-D-Galp1-1 -D-Glcp, and
-D-Glcp1]3-3)β-D-Manp1-1 -D-Glcp, res
α
-D-Glcp1-2
α
-L-Araf1-3)β-D-Manp
-D-Glcp1-2
-D-Glcp
α
α
α
α
α-
α
α
α
α
α
α
α
and CT70-2 exhibited concentration-dependent α-glucosidase inhibitory
activities, with IC50 values of 2.68 ± 0.10 mM, 2.62 ± 0.06 mM, and
4.89 ± 0.02 mM, respectively. The oligosaccharides also exhibited
α
-amylase inhibitory activities, with IC50 values of 2.77 ± 0.10 mM,
2.75 ± 0.06 mM, and 3.35 ± 0.03 mM, respectively. Moreover,
CT70-1A, CT70-1B, and CT70-2 showed NO production inhibitory ac-
tivities, with IC50 values of 0.23 mM, 0.24 mM and 0.27 mM, respec-
tively. The above results suggest that Kunlun Chrysanthemum
oligosaccharides are the active components responsible for the
anti-diabetic effects through
α-glucosidase and α-amylase inhibitory
activities, and the oligosaccharides exhibit anti-neuroinflammatory ef-
fects via the inhibition of NO production. Therefore, Kunlun Chrysan-
themum oligosaccharides could prevent the development of diseases
such as type 2 diabetes and Alzheimer’s disease, and further studies are
worthy to investigate the relative pharmacological mechanism.
4. Conclusions
In this study, we presented the characterization of three new oligo-
saccharides, CT70-1A, CT70-1B, and CT70-2, which were isolated from
the flowers of Kunlun Chrysanthemum. Their molecular weights were
1221 Da, 1318 Da and 1247 Da, respectively. CT70-1A consisted of (1 →
CRediT authorship contribution statement
2)-linked
→ 3, 6)-linked β-D-Manp and was terminated with
1B consisted of (1 → 2)-linked -L-Araf, (1 → 2,3,6)-linked
→ 6)-linked β-D-Galp, (1 → 3, 6)-linked β-D-Manp and was terminated
with -D-Glcp-(1→ and -L-Araf-(1 → . CT70-2 consisted of (1 → 4)-
linked -D-Glcp, (1 → 6)-linked β-D-Galp, and (1 → 3, 6)-linked β-D-
α
-L-Araf, (1 → 6)-linked β-D-Galp, (1 → 6)-linked
-D-Glcp-(1 → . CT70-
-D-Glcp, (1
α-D-Glcp, (1
α
Qian Yu: Writing - original draft, preparation, Writing - review &
editing, Validation, Supervision. Wei Chen: Methodology, Development
or design of methodology, Data curation, Writing - original draft,
preparation. Jing Zhong: Investigation. Degang Qing: Validation.
Chunyan Yan: Ideas, Supervision, Writing - review & editing, Funding
acquisition.
α
α
α
α
α
Manp, and was terminated with α-D-Glcp-(1→ and α-L-Araf-(1 → . Ac-
cording to the rules of International Union of Pure and Applied Chem-
istry (1979), the systematic names of three oligosaccharides were
8

Q. Yu et al.
Food and Chemical Toxicology 149 (2021) 112032
Fig. 7. The effects of Kunlun Chrysanthemum saccharides on the viability of BV2 cells (A). The effects of Kunlun Chrysanthemum saccharides on NO production of
BV2 cells induced by LPS (B and C).
Chen, S., Chen, H., Tian, J., Wang, Y., Xing, L., Wang, J., 2013. Chemical modification,
Declaration of competing interest
antioxidant and α-amylase inhibitory activities of corn silk polysaccharides.
Carbohydr. Polym. 98, 428–437.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Dai, J., Wu, Y., Chen, S.W., Zhu, S., Yin, H.P., Wang, M., Tang, G., 2010. Sugar
compositional determination of polysaccharides from Dunaliella salina by modified
RP-HPLC method of precolumn derivatization with 1-phenyl-3-methyl-5-pyrazolone.
Carbohydr. Polym. 82, 629–635.
ˆ ´
Dertli, E., Colquhoun, I.J., Cote, G.L., Gall, G.L., Narbad, A., 2017. Structural analysis of
the α-D-glucan produced by the sourdough isolate lactobacillus brevis E25. Food
Acknowledgments
Chem. 242, 45–52.
Dias, T., Bronze, M.R., Houghton, P.J., Mota-Filipe, H., Paulo, A., 2010. The flavonoid-
rich fraction of Coreopsis tinctoria promotes glucose tolerance regain through
pancreatic function recovery in streptozotocin-induced glucose-intolerant rats.
J. Ethnopharmacol. 132, 483–490.
This study was funded by the National Natural Science Foundation of
China (Nos. U1703110, 81673557 and 81973454).
Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric
method for determination of sugars and related substances. Anal. Chem. 28,
350–356.
Appendix A. Supplementary data
Eleazu, C.O., Eleazu, K.C., Iroaganachi, M., 2016. In vitro starch digestibility, α-amylase
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.fct.2021.112032.
and α-glucosidase inhibitory capacities of raw and processed forms of three varieties
of Livingstone potato (Plectranthus esculentus). Innov. Food Sci. Emerg. 37, 37–43.
Fang, Y., Xia, W., Cheng, B., Hua, P., Zhou, H., Gu, Q., Xu, J., 2018. Design, synthesis,
and biological evaluation of compounds with a new scaffold as anti-
neuroinflammatory agents for the treatment of Alzheimer’s disease. Eur. J. Med.
Chem. 149, 129–138.
References
Ademiluyi, A.O., Oboh, G., Boligon, A.A., Athayde, M.L., 2014. Effect of fermented
Guo, L., Zhang, W., Li, S., Ho, C.T., 2015a. Chemical and nutraceutical properties of
Coreopsis tinctoria. J. Funct. Foods. 13, 11–20.
soybean condiment supplemented diet on α-amylase and α-glucosidase activities in
streptozotocin-induced diabetic rats. J. Funct. Foods. 9, 1–9.
Guo, Q., Cui, S.W., Ding, H., Wang, Q., Wang, C., 2015b. Non-starch polysaccharides
from American ginseng: physicochemical investigation and structural
characterization. Food Hydrocolloids 44, 320–327.
˜
Albuquerque, P.B.S., Jr, W.B., Santos, G.R.C., Correia, M.T.S., Mourao, P.A.S., Teixeira, J.
A., Carneiro-da-Cunha, M.G., 2014. Characterization and rheological study of the
galactomannan extracted from seeds of Cassia grandis. Carbohydr. Polym. 104,
127–134.
Habibi, Y., Heyraud, A., Mahrouz, M., Vignon, M.R., 2004. Structural features of pectic
polysaccharides from the skin of Opuntia ficus-indica prickly pear fruits. Carbohydr.
Res. 339, 1119–1127.
Arrizon, J., Morel, S., Gschaedler, A., Monsan, P., 2010. Comparison of the water-soluble
carbohydrate composition and fructan structures of Agave tequilana plants of
different ages. Food Chem. 122, 123–130.
Heneka, M.T., Fink, A., Doblhammer, G., 2015. Effect of pioglitazone medication on the
incidence of dementia. Ann. Neurol. 78, 284–294.
Barnes, D.E., Yaffe, K., 2011. The projected impact of risk factor reduction on
Alzheimer’s disease prevalence. Alzheimers Dement 7, 819–828.
Cai, W., Yu, L., Yu, Z., Li, F., Kong, S., Tan, H., Xu, H., Huang, C., 2016. Extracts of
Coreopsis tinctoria Nutt. flower exhibit antidiabetic effects via the inhibition of
Hensley, K., 2010. Neuroinflammation in alzheimer’s disease: mechanisms, pathologic
consequences, and potential for therapeutic manipulation. J. Alzheimers Dis. 21,
1–14.
Huang, Y., Li, N., Wan, J.B., Zhang, D., Yan, C., 2015. Structural characterization and
antioxidant activity of a novel heteropolysaccharide from the submerged
fermentation mycelia of Ganoderma capense. Carbohydr. Polym. 134, 752–760.
α
-glucosidase activity. J. Diabetes Res. 23, 1–9.
Campbell, R.K., 2011. Clarifying the role of incretin-based therapies in the treatment of
type 2 diabetes mellitus. Clin. Therapeut. 33, 511–527.
9

Q. Yu et al.
Food and Chemical Toxicology 149 (2021) 112032
Huang, S., Mao, J., Ding, K., Zhou, Y., Zeng, X., Yang, W., Wang, P., Zhao, C., Yao, J.,
Xia, P., Pei, G., 2017. Polysaccharides from ganoderma lucidum promote cognitive
function and neural progenitor proliferation in mouse model of Alzheimer’s disease.
Stem Cell Rep 8, 84–94.
Sridhar, G.R., Lakshmi, G., Nagamani, G., 2015. Emerging links between type 2 diabetes
and Alzheimer’s disease. World J. Diabetes 6, 744–751.
Striegel, L., Kang, B., Pilkenton, S.J., Rychlik, M., Apostolidis, E., 2015. Effect of black
tea and black tea pomace polyphenols on α-glucosidase and α-amylase inhibition,
International Union of Pure and Applied Chemistry, 1979. Nomenclature of Organic
Chemistry. Pergamon, Oxford.
relevant to type 2 diabetes prevention. Front. Nutr. 2, 1–6.
Takeda, S., Sato, N., Uchioyamada, K., Sawada, K., Kunieda, T., Takeuchi, D.,
Kurinami, H., Shinohara, M., Rakugi, H., Morishita, R., 2010. Diabetes-accelerated
memory dysfunction via cerebrovascular inflammation and Aβ deposition in an
Alzheimer mouse model with diabetes. P. Natl. Acad. Sci. USA. 107, 7036–7041.
Velazquez, R., Tran, A., Ishimwe, E., Denner, L., Dave, N., Oddo, S., Dineley, K.T., 2017.
Central insulin dysregulation and energy dyshomeostasis in two mouse models of
Alzheimer’s disease. Neurobiol. Aging 58, 1–13.
Jia, X., Hu, J., He, M., Zhang, Q., Li, P., Wan, J., He, C., 2017. α- Glucosidase inhibitory
activity and structural characterization of polysaccharide fraction from Rhynchosia
minima root. J. Funct. Foods 28, 76–82.
Jiang, J., Kong, F., Li, N., Zhang, D., Yan, C., Lv, H., 2016. Purification, structural
characterization and in vitro antioxidant activity of a novel polysaccharide from
Boshuzhi. Carbohydr. Polym. 147, 365–371.
Jing, S., Chai, W., Guo, G., Zhang, X., Dai, J., Yan, L.J., 2016. Comparison of antioxidant
and antiproliferative activity between Kunlun Chrysanthemum flowers
polysaccharides (KCCP) and fraction PII separated by column chromatography.
J. Chromatogr. B 1019, 169–177.
Wang, Y., Wei, X., Jin, Z., 2009. Structure analysis of an acidic polysaccharide isolated
from green tea. Nat. Prod. Res. 23, 678–687.
Wang, W., Chen, W., Yang, Y., Liu, T., Yang, H., Xin, Z., 2014. New phenolic compounds
from Coreopsis tinctoria nutt. and their antioxidant and angiotensin I-converting
enzyme inhibitory activities. J. Agric. Food Chem. 63, 200–208.
Wang, T., Xi, M., Guo, Q., Wang, L., Shen, Z.M., 2015. Chemical components and
antioxidant activity of volatile oil of a compositae tea (Coreopsis tinctoria, nutt.) from
Mt. Kunlun. Ind. Crop. Prod. 67, 318–323.
Joakim, H., Sebastian, P., Lennart, M., Oskar, H., 2013. Tau pathology and parietal white
matter lesions have independent but synergistic effects on early development of
Alzheimer’s disease. Dement. Geriatr. Cogn. 3, 113–122.
Joshi, S.R., Eberhard, S., Nanwei, T., Parag, S., Sanjay, K., Rahul, R., 2015. Therapeutic
potential of
α
-glucosidase inhibitors in type 2 diabetes mellitus: an evidence-based
Wang, C., Zhang, D., Zhang, M., Jiao, Y., Jiang, K., Yan, C., 2017a. Structural
characterization of a novel oligosaccharide from Achyranthes bidentata and its anti-
osteoporosis activities. Ind. Crop. Prod. 108, 458–469.
review. Expet Opin. Pharmacother. 16, 1959–1981.
Kwon, Y.I., Vattem, D.A., Shetty, K., 2006. Evaluation of clonal herbs of lamiaceae
species for management of diabetes and hypertension. Asia Pac. J. Clin. Nutr. 15,
107–118.
Wang, X., Zhang, M., Zhang, D., Wang, S., Yan, C., 2017b. An O-acetyl-glucomannan
from the rhizomes of Curculigo orchioides: structural characterization and anti-
osteoporosis activity in vitro. Carbohydr. Polym. 174, 48–56.
Li, N., Yan, C., Hua, D., Zhang, D., 2013. Isolation, purification, and structural
characterization of a novel polysaccharide from Ganoderma capense. Int. J. Biol.
Macromol. 57, 285–290.
Weinstein, G., Maillard, P., Himali, J.J., Beiser, A.S., Au, R., Wolf, P.A., Seshadri, S.,
DeCarli, C., 2015. Glucose indices are associated with cognitive and structural brain
measures in young adults. Neurology 84, 2329–2337.
Li, N., Meng, D., Pan, Y., Cui, Q., Li, G., Ni, H., Sun, Y., Qing, D., Jia, X., Pan, Y., Hou, Y.,
2015. Anti-neuroinflammatory and NQO1 inducing activity of natural
phytochemicals from Coreopsis tinctoria. J. Funct. Foods. 17, 837–846.
Li, Q.M., Wang, J.F., Zha, X.Q., Pan, L.H., Zhang, H.L., Luo, J.P., 2017. Structural
characterization and immunomodulatory activity of a new polysaccharide from
jellyfish. Carbohydr. Polym. 159, 188–194.
Westphal, Y., Kühnel, S., Waard, P., Hinz, S.W.A., Schols, H.A., Voragen, A.G.J.,
Gruppen, H., 2010. Branched arabino-oligosaccharides isolated from sugar beet
arabinan. Carbohydr. Res. 345, 1180–1189.
Wu, X., Jiang, W., Lu, J., Yu, Y., Wu, B., 2014. Analysis of the monosaccharide
composition of water-soluble polysaccharides from Sargassum fusiforme by high
performance liquid chromatography/electrospray ionisation mass spectrometry.
Food Chem. 145, 976–983.
Liu, F., Dong, B., Yang, X., Yang, Y., Zhang, J., Jin, D.Q., Ohizumi, Y., Lee, D., Xu, J.,
Guo, Y., 2018. No inhibitors function as potential anti-neuroinflammatory agents for
AD from the flowers of Inula japonica. Bioorg. Chem. 77, 168–175.
Yan, C., Huang, D., Shen, X., Qin, N., Jiang, K., Zhang, D., Zhang, Q., 2019. Identification
and characterization of a polysaccharide from the roots of Morinda officinalis, as an
inducer of bone formation by up-regulation of target gene expression. Int. J. Biol.
Macromol. 133, 446–456.
Lordan, S., Smyth, T.J., Soler-Vila, A., Stanton, C., Ross, R.P., 2013. The α-amylase and
α
-glucosidase inhibitory effects of Irish seaweed extracts. Food Chem. 141,
2170–2176.
Nhiem, N., Tuong, N., Ky, P., Subedi, L., Park, S., Ngoc, T., Yen, P., Tai, B., Quang, T.,
Kiem, P., Kim, S., Kim, S., 2017. Chemical components from Phaeanthus vietnamensis
and their inhibitory NO production in BV2 cells. Chem. Biodivers. 14, e1700013.
Pasquier, F., Boulogne, A., Leys, D., Fontaine, P., 2006. Diabetes mellitus and dementia.
Diabetes Metab. 32, 403–414.
Yao, L., Li, L., Li, X., Li, H., Zhang, Y., Zhang, R., Wang, J., Mao, X., 2015. The anti-
inflammatory and antifibrotic effects of Coreopsis tinctoria nutt on high-glucose-fat
diet and streptozotocin-induced diabetic renal damage in rats. BMC Compl.
Alternative Med. 15, 314–326.
Zhang, F., Jiang, L., 2015. Neuroinflammation in Alzheimer’s disease. Neuropsychiatric
Dis. Treat. 14, 243–256.
Pattanayak, M., Samanta, S., Maity, P., Sen, I.K., Nandi, A.K., Manna, D.K., Mitra, P.,
Acharya, K., Islam, S.S., 2015. Heteroglycan of an edible mushroom Termitomyces
clypeatus: structure elucidation and antioxidant properties. Carbohydr. Res. 413,
30–36.
Zhang, Y., Yang, X., Jin, G., Yang, X., Zhang, Y., 2016. Polysaccharides from Pleurotus
ostreatus alleviate cognitive impairment in a rat model of Alzheimer’s disease. Int. J.
Biol. Macromol. 92, 935–941.
Qian, J.Y., Chen, W., Zhang, W.M., Zhang, H., 2009. Adulteration identification of some
fungal polysaccharides with SEM, XRD, IR and optical rotation: a primary approach.
Carbohydr. Polym. 78, 620–625.
Zhao, C., Diao, Y., Wang, C., Qu, W., Zhao, X., Ma, H., Shan, J., Sun, G., 2017. Structural
characters and protecting β-cells of a polysaccharide from flowers of Inula japonica.
Int. J. Biol. Macromol. 101, 16–23.
Sahragard, N., Jahanbin, K., 2017. Structural elucidation of the main water-soluble
polysaccharide from Rubus anatolicus roots. Carbohydr. Polym. 175, 610–617.
Zhou, Y., Wang, S., Lou, H., Fan, P., 2018. Chemical constituents of hemp (Cannabis
sativa, L.) seed with potential anti-neuroinflammatory activity. Phytochem. Lett. 23,
57–61.
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