Food Chemistry 286 (2019) 8–16

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Chemical compositions of chrysanthemum teas and their anti-inflammatory T

and antioxidant properties
⁎
Yanfang Lia,b, Puyu Yanga, Yinghua Luob, Boyan Gaoa,b, Jianghao Sunc, Weiying Lua, Jie Liud, ,
⁎
Pei Chenc, Yaqiong Zhanga, Liangli (Lucy) Yub,
a
Institute of Food and Nutraceutical Science, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China
b
Department of Nutrition and Food Science, University of Maryland, College Park, MD 20742, United States
c
Food Composition and Methods Development Laboratory, Beltsville Human Nutrition Research Center, Agricultural Research Service, United States Department of
Agriculture, Beltsville, MD 20705, United States
d
Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology & Business University (BTBU), Beijing 100048, China

A R T I C LE I N FO A B S T R A C T

Keywords: Seventeen commercial chrysanthemum teas (Chrysanthemum morifolium and Coreopsis tinctoria) were extracted
Chrysanthemum tea with hot-H2O, and examined and compared to the 75% methanol extracts for their chemical compositions using
Chemical composition UPLC/Q-TOF-MS analysis. For the first time, 6, 8-C,C-diglucosylapigenin and eriodicyol-7-O-glucoside were
Anti-inflammatory detected in the Snow chrysanthemum, and acetylmarein was detected in HangJu, GongJu and HuaiJu. The
Antioxidant
extracts were also examined for their radical scavenging and anti-inflammatory activities in vitro. The hot-H2O
extract of Kunlunmiju 1 had the greatest total phenolic content, and relative DPPH and oxygen radical absor-
bance capacity values of 12.72 mg gallic acid equivalents/g, 105.48 and 1222.50 μmol Trolox equivalents/g,
respectively. In addition, all the hot-H2O extracts suppressed the lipopolysaccharide-induced interleukin-6, IL-1β
and cyclooxygenase-2 mRNA expressions, and H2O2-induced intracellular reactive oxygen species production in
cultured cells. The results from this research may be used to promote the consumption of chrysanthemum as a
functional tea.

1. Introduction mice. Interestingly, chrysanthemum has been primarily consumed as a

Chrysanthemum (Chrysanthemum morifolium and Coreopsis tinctoria)
has been cultivated for tea and food utilizations for many years. The tea
made from chrysanthemum flowers or the cooked dishes of fresh
chrysanthemum leaves are considered to be beneficial to human health.
These beneficial effects may include but are not limited to anti-in-
flammation (Lee et al., 2009; Zhang, Shi, Zhao, Chai, & Tu, 2013),
antioxidant (Duh, Tu, & Yen, 1999; Wang, Xi et al., 2015; Yang et al.,
2011), and antiallergic properties (Xie, Qu, Wang, Wang, Yoshikawa, &
Yuan, 2012). For example, Yao et al. (2015) found that ethyl acetate
extracts of C. tinctoria reduced the expression of kidney proin-
flammatory cytokines in high-glucose-fat diet and streptozotocin (STZ)-
induced diabetic rats. Duh et al. (1999) revealed that C. morifolium
(Harng Jyur) hot-H2O extracts had served as DPPH radical scavengers,
metal chelators, and active oxygen scavengers. In addition, Ukiya et al.
(2001) reported that the triterpene diol and triol constituents from C.
morifolium methanol extracts inhibited TPA-induced inflammation in
tea product, which is the hot-water infusion of the flowers. To date, few
studies have investigated the health beneficial effects of the hot-H2O
extracts of chrysanthemum, and the research results from its organic
solvent extracts cannot truly reflect the health effects of drinking
chrysanthemum tea.
The beneficial effects of chrysanthemum have been mainly attrib-
uted to its flavonoids (Lin & Harnly, 2010; Yang et al., 2016) and
phenolic acids (Lai, Lim, Su, Shen, & Ong, 2007; Yang et al., 2016). For
instance, Lai et al. (2007) identified 17 flavonoids and caffeoylquinic
acids in the 100% methanol extracts of C. morifolium. Later, Lin and
Harnly (2010) found 46 flavonoids and 17 caffeic acid derivatives in the
60% methanol extracts of C. morifolium and Yang et al. (2016) identi-
fied 26 flavonoids and four phenolic acids in the 70% methanol extracts
of C. tinctoria. Recently, Xie et al. (2012) found that 75% methanol
extracts of five cultivars of C. morifolium significantly differed in their
profile and quantity of flavonoid aglycones and glycosides. To our best
knowledge, no systematic study was reported about the chemical

⁎
Corresponding authors.
E-mail addresses: zoe_li@sjtu.edu.cn (Y. Li), gaoboyan@sjtu.edu.cn (B. Gao), jianghao.sun@ars.usda.gov (J. Sun), weiying.lu@sjtu.edu.cn (W. Lu),
liu_jie@btbu.edu.cn (J. Liu), pei.chen@ars.usda.gov (P. Chen), yqzhang2006@sjtu.edu.cn (Y. Zhang), lyu5@umd.edu (L.L. Yu).

https://doi.org/10.1016/j.foodchem.2019.02.013
Received 17 October 2018; Received in revised form 6 December 2018; Accepted 4 February 2019
Available online 12 February 2019
0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
Y. Li, et al.
profile of hot-H2O extracts of chrysanthemum, though this information
might better reflect the health components through chrysanthemum tea
consumption. Again, additional research is needed to investigate the
bioactive components in the hot-H2O extracts of chrysanthemum.
In the present study, hot-H2O and 75% methanol extracts of 17
commercially available chrysanthemum tea products, including 11 C.
morifolium and 6 C. tinctoria were investigated for their chemical
compositions using UPLC/Q-TOF-MS analysis, and their anti-in-
flammatory and antioxidant activities. 75% methanol extracts were
included, so that the chemical composition results from the present
study may be related and compared with previous studies. The anti-
inflammatory properties were measured as their abilities to suppress
the relative mRNA expressions of IL-6, IL-1β, and COX-2 in LPS-induced
RAW 264.7 macrophages. Total phenolic contents (TPC), and scaven-
ging capabilities against DPPH and oxygen radicals were examined as
the antioxidant activities, along with their inhibitory effects of H2O2-
induced intracellular reactive oxygen species (ROS) formation in the
cultured ARPE-19 cells. The results from this study might be used to
improve the production and consumption of chrysanthemum tea, thus
improve human health.
2. Materials and methods
2.1. Chemicals and reagents
Chlorogenic acid (3-O-caffeoylquinic acid, CA), luteolin, apigenin
and quercetin were purchased from Shanghai Institute of Material
Medica, Chinese Academy of Science (Shanghai, China). 2,2′-Azobis(2-
methylpropionamidine) dihydrochloride (AAPH) was purchased from
the J&K Scientific (Beijing, China). Fluorescein (FL), gallic acid, Folin-
Ciocalteu’s phenol reagent (FC), dimethyl sulfoxide (DMSO), 2,2-di-
phenyl-1-picrylhydrazyl radical (DPPH%), 6-hydroxy-2,5,7,8-tetra-
methylchroman-2-carboxylic acid (Trolox) and 2′,7′-dichlorofluorescin
diacetate (DCFH-DA) were obtained from Sigma-Aldrich (St. Louis, MO,
USA). Analytical grade acetone, methanol, sodium carbonate, sodium
dihydrogen phosphate (NaH2PO4), disodium hydrogen phosphate
(Na2HPO4) and thirty percent hydrogen peroxide (30%-H2O2) were
obtained from Sinopharm (Sinopharm International Co., Ltd., Shanghai,
China). The HPLC grade acetonitrile, methanol and formic acid (FA)
were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ultrapure
water was prepared using a Milli-Q purification system (Millipore
Laboratory, Bedford, MA, USA). Other chemicals or solvents were of
analytical grade and used without further purification.
2.2. Botanical materials
Five cultivars of commercial chrysanthemum tea products, 6 of
‘HangJu’, 2 of ‘GongJu’, 1 of ‘HuaiJu’, 2 of ‘FubaiJu’, and 6 of ‘Snow
chrysanthemum’ growing in the regions of Hangzhou, Anhui, Henan,
Hubei province and Xinjiang Uygur Autonomous Region were collected
from local markets. Detailed information of the 17 chrysanthemum
samples is provided in Supplementary data (Table S1).
2.3. Sample preparation
Authentic standards solutions were prepared by dissolving accu-
rately weighed standard compounds in methanol at the concentration
of 0.05 mg/mL. All of the solutions were stored at 4 °C before analysis.
The chrysanthemum samples were ground to a particle size < 20-
mesh prior to extraction. The ground sample (2 g) was mixed with
20 mL of boiling water and the mixture was kept at ambient tempera-
ture overnight. The mixture was centrifuged at 2360 g for 20 min, and
5 mL supernatant was accurately taken and dried using a freeze-dryer,
and the residue was weighted accurately. After re-dissolving in 5 mL of
H2O, 0.1 mL of each sample was taken and ten times diluted prior to
centrifugation at 13,400 g for 20 min. The supernatants were subjected
9
Food Chemistry 286 (2019) 8–16
to ultra-performance liquid chromatography (UPLC) and UPLC com-
bined with a quadrupole time-of-flight mass spectrometer UPLC/Q-
TOF-MS analyses. In addition, the residues were quantitatively re-dis-
solved in 50% aqueous acetone to a concentration of 1 mg/mL, and kept
at 4 °C for total phenolic content and free radical scavenging activity
evaluation. For in vitro bioactivity evaluation, the residues were
quantitatively re-dissolved in DMSO to a stock solution at concentration
of 100 mg/mL and kept at −80 °C and diluted to 20 μg/mL with culture
medium before tests.
Following the same procedure, another set of supernatants extracted
with 75% methanol were prepared. The solvent was dried under ni-
trogen. The residues were kept at −80 °C until chemical and bioactivity
evaluations.
2.4. UPLC and UPLC/Q-TOF-MS conditions
UPLC/Q-TOF-MS analysis was performed using an ACQUITY ultra-
performance liquid chromatography combined with a Xevo G2 quad-
rupole time-of-flight mass spectrometer (UPLC-Q-TOF-MS) (Waters,
Milford, Massachusetts, USA). An Acquity UPLC BEH C18 column
(2.1 mm i.d. × 100 mm, 1.7 μm) attached with a VanGuard precolumn
(2.1 mm i.d. × 5 mm, 1.7 μm) (Waters, Milford, MA, USA) was used. A
binary solvent system of A) 0.1% (v/v) formic acid in deionized water
and B) 0.1% (v/v) formic acid in acetonitrile was used. The gradient
elution program was 0–17.5 min, 10–30% B; 17.5–19 min, 30–55% B;
19–19.1 min, 55–95% B; 19.1–21.1 min, 95% B; 21.1–21.2 min,
95–10% B; 21.2–22.2 min, 10% B; with a stable flow rate of 0.3 mL/
min. The column temperature was 45 °C, and the injection volume was
2 μL. The electrospray ionization (ESI) source was operated under the
negative ion mode. The capillary voltage and the cone voltage were
2.5 kV and 30 V, respectively. The ionization temperature was 120 °C,
and desolvation temperature was 500 °C. The sodium formate (0.34 g/L
in 90% isopropanol, v/v) and leucine enkephalin (2 mg/L in 50%
acetonitrile, v/v) were used for Q-TOF-MS startup and lockspray cali-
bration, respectively. Two channels of MS measurements were re-
corded. The first channel was for the data collection of MS signals
ranging from 100 to 1000 Da with a 6 eV collision energy. The second
channel offered additional fragmentation information for the com-
pound identification, which collected mass fragments of 100–1000 Da
with a 30 eV collision energy. Each sample was tested in triplicate and
the methanol blank solution was injected to reduce sample cross-con-
taminations.
2.5. Determination of total phenolic content (TPC) of chrysanthemum
samples
The TPC was estimated as previously described following a la-
boratory protocol (Yu, Haley, Perret, & Harris, 2002). Briefly, 50 μL of
the sample solution or standard was mixed with 250 μL of Folin-Cio-
calteu (FC) reagent and 3 mL of ultrapure water, followed by adding
750 μL of 20% sodium carbonate to start the reaction. The absorbance
was measured at 765 nm using a Multimode Reader (TECAN, Männe-
dorf, Switzerland) after 2 h reaction in dark at ambient temperature.
The TPC is reported as milligram gallic acid equivalents (GAE)/g
flower.
2.6. Inhibition on IL-6, IL-1β, and COX-2 mRNA expression in RAW264.7
macrophages cells
RAW264.7 macrophages (American Type Culture Collection,
Manassas, VA, USA) were cultured as previously reported (Huang et al.,
2011). Briefly, RAW264.7 macrophages were cultured in DMEM media
supplemented with 10% fetal bovine serum (FBS) and containing 100
U/mL penicillin and 100 μg/mL streptomycin. Cell incubation was
performed at 37 °C in a humidified atmosphere of 5% CO2 and 95% air.
RAW 264.7 mouse macrophages (6 × 104 cells/mL) were seeded in
Y. Li, et al.
six-well plates and incubated for 24 h to reach the 80% confluence.
After pretreatment with culture medium containing 20 μg/mL extracts
for another 24 h incubation, 10 ng/mL lipopolysaccharide (LPS) was
added to the culture medium for another 4 h incubation, after which,
the culture medium was removed and cells were collected for the total
RNA isolation and real-time PCR.
The total RNA isolation and real-time PCR were performed fol-
lowing to a laboratory protocol previously reported (Huang et al.,
2011). Briefly, TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was
added for total RNA isolation. IScript reverse transcriptase kits (Bio-Rad
Laboratories, Hercules, CA, USA) were used to synthesis the first-strand
cDNA following the manufacturer’s protocol. The quantitative real-time
PCR was performed on an ABI 7900HT RT-PCR system using AB Power
SYBR Green PCR Master Mix. The amplification cycling parameters
used for PCR were 50 °C for 2 min, 95 °C for 10 min, and 40 cycles of
amplification at 95 °C for 15 s and 60 °C for 1 min. The results were
expressed as relative mRNA levels of IL-6, IL-1β and COX-2 using β-
actin as an internal control. All data were analyzed using the 2−ΔΔCt
method. Primers used in this study were as follows: IL-6 (forward,
5′-CACGGCCTTCCCTACTTCAC-3′; reverse, 5′-TGCAAGTGCATCATCGT
TGT-3′); IL-1β (forward, 5′-GTTGACGGACCCCAAAAGAT-3′; reverse,
5′-CCTCATCCTGGAAGGTCCAC-3′); COX-2 (forward, 5′-GGGAGTCTG
GAACATTGTGAA-3′; reverse, 5′-GCACGTTGATTGTAGGTGGAC
TGT-3′); β-actin (forward: 5′-TGTCCACCTTCCAGCAGATGT-3′; reverse:
5′-AGCTCAGTAACAGTCCGCCTAGA-3′).
2.7. Relative DPPH radical (DPPH%) scavenging capacity (RDSC)
The RDSC values were determined following a laboratory protocol
previously described (Cheng, Moore, & Yu, 2006). Briefly, 100 μL of the
sample solution or control or standard were mixed with 100 μL of
freshly prepared 0.2 mM DPPH% to initiate the reaction. The absorbance
was measured at 515 nm every minute for 90 min of reaction. The RDSC
values were calculated by the calibration curve of Trolox standards with
a linearity range from 7 to 36 μM. Results were reported as μmoles
Trolox equivalents (TE)/g of flower.
2.8. Oxygen radical absorbance capacity (ORAC)
The ORAC assay was performed as previously reported following a
laboratory protocol (Moore et al., 2005). Briefly, 81.63 nM fluorescein
(FL) and 0.36 mM AAPH working solutions were freshly prepared in
75 mM sodium phosphate buffer (pH 7.4), and the Trolox standard
solutions were prepared in 50% aqueous acetone. After 30 μL sample
solution, blank solvent or standard were mixed with 225 μL of FL
working solution in the wells of 96-well plates, and pre-incubated
20 min at 37 °C in the plate tray, 25 μL of freshly made AAPH working
solution were added to each of the reaction wells to initiate the anti-
oxidant-radical reactions. The fluorescence intensities were recorded
every minute for 2 h at 37 °C, with excitation and emission wavelengths
of 485 and 535 nm, respectively. Results were reported as μmoles TE/g
flower.
2.9. Reduction on H2O2-induced oxidative stress in ARPE-19 cells
Human ARPE-19 cells from ATCC were cultured in DMEM/F12
medium as previously reported (Feng, Chen, Sun, Wang, & Sun, 2014).
The levels of ROS were estimated using the standard operating proce-
dure adapted from the previously described procedure (Yerushalmi,
Dahl, Devereaux, Gumpricht, & Sokol, 2001). This assay was carried out
with a non-fluorescent compound di-hydrodichlorofluorescein diace-
tate (DCFDA), which would be oxidized by ROS to a highly fluorescent
compound of 2′,7′-dichlorofluorescein (DCF). ARPE-19 cells
(1 × 105 cells/mL) were seeded in 96-well plates and incubated for 24 h
to reach confluence. Then cells were pretreated with culture medium in
the presence or absence of 20 μg/mL sample extract for another 24 h
10
Food Chemistry 286 (2019) 8–16
and then washed once with 200 μL of Hanks’ balanced salt solution
(HBSS) and incubated with 200 μM H2O2 for another 60 min. After
washing with 200 μL of HBSS, the cells were incubated with 5 μM
DCFDA for another 30 min. The contents of DCF were monitored using
a fluorescence spectrophotometer, with excitation and emission wave-
lengths of 485 and 520 nm, respectively. The intracellular ROS levels
were estimated comparing to a standard curve using DCF, and was
normalized as pmol DCF/μg DNA. The DNA contents were determined
using a fluorescence spectrophotometer assay described in a previous
study (Rago, Mitchen, & Wilding, 1990).
2.10. Statistical analysis
Data were reported in mean ± standard deviation (SD) for tripli-
cate tea samples. Statistical significance was declared at P < 0.05
following by one-way analysis of variance (ANOVA) with Tukey’s post-
hoc test. The t-test was employed to identify differences between dif-
ferent solvents in the TPC, anti-inflammatory and antioxidant activity
assays. All statistical analyses were performed using SPSS software
(version 21.0, SPSS, Inc., Chicago, IL, USA). All figures were prepared
using GraphPad Prism software (Version 6.00, Graphpad Software Inc.,
San Diego, CA, USA).
3. Results and discussion
3.1. Chemical compositions of chrysanthemum samples
The chemical compositions of the 17 chrysanthemum samples were
characterized by UPLC/Q-TOF-MS analysis. A total of 28 peaks were
detected in the hot-water extracts (Table 1, Fig. S1), and 34 peaks were
detected in the 75% methanol extracts (Table 1, Fig. S2). Positive
identification of the peaks was made by comparing the retention time,
high-resolution molecular ions ([M−H]−) and major MS2 fragment
ions of the UPLC peaks with those of the authentic standards. For ex-
ample, peak 2 had the same retention time, molecular ion ([M−H]−) at
m/z 353.0867 and major fragment at m/z 191.0542 ([M−H]− of quinic
acid) with chlorogenic acid standard, suggesting that peak 2 is
chlorogenic acid. Therefore, 2 peaks were positively identified as
chlorogenic acid (peak 2) and luteolin-7-O-glucoside (peak 11) in both
hot-water and 75% methanol extracts (Table 1), and a third peak was
positively identified as luteolin (peak 28) in the 75% methanol extracts
(Table 1).
The other 31 peaks were tentatively identified according to the re-
tention time, theoretical and experimental molecular ions ([M−H]−)
and the major MS2 fragment ions, as well as the MS data in literatures
(Lai et al., 2007; Lin and Harnly, 2010; Wang, Hao, et al., 2015; Yang
et al., 2016). For instance, peak 12, 13, 14 and 20 had a same molecular
ion ([M−H]−) at m/z 515.1191 and the major fragments at 353.0867
(loss of caffeoyl, 162 amu), 191.0553 ([M−H]− of quinic acid),
179.0330 ([M−H]− of caffeic acid) (Fig. S3), suggesting that they are
dicaffeoylquinic acid isomers (Table 1). The 4 peaks were all listed as
dicaffeoylquinic acid isomers since the linkages between the two caf-
feoyl and a quinic acid moieties were not clear from the MS data and
there have been 6 reported ways to link the three structural components
together to form 6 dicaffeoylquinic acid isomers (Beninger et al., 2004;
Clifford, Wu, Kirkpatrick, & Kuhnert, 2007; Kim & Lee, 2005; Lin and
Harnly, 2010). A total of 26 compounds including 21 flavonoids and 5
phenolic acids were tentatively identified from the hot-H2O extracts,
and a total of 31 chemical components including 25 flavonoids and 6
phenolic acids were detected and tentatively identified from the 75%
methanol extracts (Table 1). Among these compounds, 6,8-C,C-diglu-
cosylapigenin and eriodicyol-7-O-glucoside were reported in the hot-
water and 75% methanol extracts of the Snow chrysanthemum for the
first time (Table 1), though the two compounds have been previously
reported in HangJu variety (Lin and Harnly, 2010; Wang, Hao, et al.,
2015; Wang et al., 2014). Acetylmarein was detected for the first time
 Table 1
Chemical components in chrysanthemum samples.
Y. Li, et al.

ID Rt (min) Exptl. [M−H]− Theor. Fragments Chemical Tentative Identification Family (subclass) Occurrence
[M−H]− formula
Hot-H2O‡ 75% Methanol‡

1 1.85 465.1036 465.1033 303.0500, 285.0394 C21H22O12 Taxifolin-3′-glucoside Flavonoid (Flavanonol) KLX1-3, KLM1-3 KLX1-3, KLM1-3
2 2.02 353.0867 353.0873 191.0542 C16H18O9 Chlorogenic acid* Phenolic acid (Cinnamic All samples All samples
acid)
3 2.16 353.0871 353.0873 191.0544, 179.0334, C16H18O9 Caffeoylquinic acid Phenolic acid (Cinnamic HB1-3, HT1-3, G1-2, HJ, HB1-3, HT1-3, G1-2, HJ, FBJ1,
173.0435, 135.0435 acid) FBJ1, FBJ2 FBJ2
4 3.01 593.1506 593.1506 473.1100, 191.0543 C27H30O15 6,8-C,C-diglucosylapigenin§ Flavonoid (Flavone) HB1-3, HT1-3, G1-2, KLX1-3, HB1-3, HT1-3, G1-2, FBJ1,
KLM1-3, FBJ1, FBJ2 KLX1-3, KLM1-3, FBJ2
5 3.31 449.1085 449.1084 287.0544, 151.0019, C21H22O11 Eriodicyol-7-O-glucoside§ Flavonoid (Flavanone) KLX1-3, KLM1-3 KLX1-3, KLM1-3
135.0433
6 4.03 479.0823 479.0826 317.029 C21H20O13 Quercetagetin-7-O-β- glucoside Flavonoid (Flavonol) KLX1-3, KLM1-3 KLX1-3, KLM1-3
7 5.86 289.0552 449.1087 151.0020, 135.0437 C21H22O11 Flavanomarein Flavonoid (Flavanone) KLX1-3, KLM1-3 KLX1-3, KLM1-3
8 5.97 593.1501 593.1506 285.0403 C27H30O15 Luteolin-7-rutinoside Flavonoid (Flavone) HB1-3, HT1-3, G1-2, KLX1-3, HB1-3, HT1-3, G1-2, KLX1-3,
KLM1-3, FBJ1, FBJ2 KLM1-3, FBJ1, FBJ2
9 6.09 449.1078 449.1078 287.0548, 151.0018 C21H22O11 Marein Flavonoid (Chalcone) KLX1-3, KLM1-3 KLX1-3, KLM1-3
10 6.18 461.0723 461.0720 285.0394 C21H18O12 Luteolin-7-O-glucuronide Flavonoid (Flavone) HB1-3, HT1-3, G1-2, HJ, HB1-3, HT1-3, G1-2, HJ, FBJ1,
FBJ1, FBJ2 FBJ2
11 6.23 447.0923 447.0927 285.0394 C21H20O11 Luteolin-7-O-glucoside* Flavonoid (Flavone) HB1-3, HT1-3, G1-2, HJ, HB1-3, HT1-3, G1-2, HJ, FBJ1,
FBJ1, FBJ2 FBJ2
12 7.11 515.1187 515.1191 353.0867, 191.0553, C25H24O12 Dicaffeoylquinic acid Phenolic acid (Cinnamic All samples All samples
179.0330 acid)
13 7.22 515.1186 515.1191 353.0868, 191.0541 C25H24O12 Dicaffeoylquinic acid isomer Phenolic acid (Cinnamic All samples All samples
acid)
14 7.31 515.1191 515.1191 353.0869, 191.0543, C25H24O12 Dicaffeoylquinic acid isomer Phenolic acid (Cinnamic All samples All samples

11
179.0339, 135.0438 acid)
15 7.66 577.1554 577.1557 269.0441 C27H30O14 Apigenin-7-O-ruinoside Flavonoid (Flavone) HB1-3, HT1-3, FBJ1, FBJ2 HB1-3, HT1-3, FBJ1, FBJ2
16 7.86 285.039 285.0399 135.0432 C15H10O6 Kaempferol Flavonoid (Flavone) KLX1-3, KLM1-3 KLX1-3, KLM1-3
17 8.06 431.0968 431.0978 269.0432 C21H20O10 Apigenin-7-O-glucoside Flavonoid (Flavone) HB1-3, HT1-3, G1-2, HJ, HB1-3, HT1-3, G1-2, HJ, FBJ1,
FBJ1, FBJ2 FBJ2
18 8.48 491.1192 491.1190 287.0552, 151.0020, C23H24O12 Acetylmarein# Flavonoid (Chalcone) HB1-3, HT1-3, G1-2, KLX1-3, All samples
135.0436 KLM1-3, FBJ1, FBJ2
19 8.60 489.1042 489.1033 285.0392 C23H22O12 Luteolin-7-O-6′'-acetylglucuronide Flavonoid (Flavone) HB1-3, HT1-3, G1-2, HJ, All samples
FBJ1, FBJ2
20 8.70 515.1185 515.1191 353.0868, 191.0543, C25H24O12 Dicaffeoylquinic acid isomer Phenolic acid (Cinnamic All samples All samples
179.0331 acid)
21 9.05 287.0554 287.0556 151.0018, 135.0432 C15H12O6 Okanin Flavonoid (Chalcone) KLX1-3, KLM1-3 KLX1-3, KLM1-3
22 9.21 475.0885 475.0877 299.0549, 284.0310 C22H20O12 Diosmetin-7-O-glucuronide Flavonoid (Flavone) HB1-3, HT1-3, G1-2, FBJ1, HB1-3, HT1-3, G1-2, FBJ1, FBJ2
FBJ2
23 9.38 601.1196 601.1193 395.0971, 233.0652, C28H26O15 1,5-dicaffeoyl-3-methoxyoxaloylquinic Phenolic acid (Cinnamic N\A HB1-3, HT1-3, G1-2, FBJ1, FBJ2
173.0432 acid acid)
24 9.98 473.1082 473.1084 269.0439 C23H22O11 Apigenin-7-O-acetylglucoside Flavonoid (Flavone) HB1-3, HT1-3, G1-2, FBJ1, HB1-3, HT1-3, G1-2, FBJ1, FBJ2
FBJ2
25 10.21 473.1082 473.1084 269.0439 C23H22O11 Apigenin-7-O-acetylglucoside isomer Flavonoid (Flavone) N\A HB1-3, HT1-3, G1-2, FBJ1, FBJ2
26 10.70 473.1084 473.1084 269.0437 C23H22O11 Apigenin-7-O-acetylglucoside isomer Flavonoid (Flavone) HB1-3, HT1-3, G1-2, FBJ1, HB1-3, HT1-3, G1-2, FBJ1, FBJ2
FBJ2
27 11.56 503.1213 503.1190 299.0549 C24H24O12 Diosmetin-7-O-6′'-acetylglucoside Flavonoid (Flavone) HB1-3, HT1-3, G1-2, FBJ1, HB1-3, HT1-3, G1-2, FBJ1, FBJ2
FBJ2
28 11.63 285.0393 285.0399 C15H10O6 Luteolin* Flavonoid (Flavone) N\A All samples
29 13.13 591.1718 591.1714 283.0599, 268.0363 C28H32O14 Acacetin-7-O-D-rutinoside Flavonoid (Flavone) HB1-3, HT1-3, FBJ1, FBJ2 HB1-3, HT1-3, FBJ1, FBJ2
30 13.56 473.1081 473.1084 269.0433 C23H22O11 Apigenin-7-O-acetylglucoside isomer Flavonoid (Flavone) N\A HB1-3, HT1-3, G1-2
31 14.10 271.0596 271.0606 135.0455 C15H12O5 Butein Flavonoid (Flavanone) KLX1-3, KLM1-3 KLX1-3, KLM1-3
32 14.12 283.0599 283.0606 268.0363 C16H12O5 Acacetin Flavonoid (Flavone) N\A HB1-3, HT1-3, G1-2, HJ, FBJ1,
FBJ2
(continued on next page)
Food Chemistry 286 (2019) 8–16
Y. Li, et al. Food Chemistry 286 (2019) 8–16

Samples were extracted by soaking in 75% methanol or hot-H2O overnight and detailed procedures were described in sample preparation in Materials and Methods. HB1-3 represents Hangbaiju 1–3; HT1-3 represents
Rt represtents retention time; Theor. [M−H]− and Exptl. [M−H]− were theoretical and experimental m/z of molecular ions, respectively; the formulas were calculated using Masslynx 4.1 software by achieving the
in the 75% methanol extracts of the HangJu, GongJu and HuaiJu
chrysanthemum samples (Table 1), but it has been reported in Snow

HB1-3, HT1-3, FBJ1, FBJ2

HB1-3, HT1-3, FBJ1, FBJ2
chrysanthemum (Yang et al., 2016). Furthermore, the FubaiJu chry-
santhemum was investigated for its chemical composition and biolo-
gical activities for the first time.
75% Methanol‡

In addition, these 17 chrysanthemum samples differed in their

chemical compositions (Table 1). The five major compounds including
dicaffeoylquinic acid (peak 14), luteolin-7-O-6″-acetylglucuronide and
dicaffeoylquinic acid (peaks 19 and 20), marein (peak 9) and okanin
(peak 21) were quantified to further compare the chrysanthemum
samples (Table 2). Considering that peak 19 and 20 were shown as a
HB1-3, HT1-3, FBJ1, FBJ2

wide peak that could not be completely separated, the concentrations of

luteolin-7-O-6″-acetylglucuronide and dicaffeoylquinic acid are re-
ported in a single value. The greatest amounts of marein and okanin in
hot-H2O extracts were 3126.6 and 1654.3 μg rutin equivalents (RE)/g
Hangtaiju 1–3; G1-2 represents Gongju 1–2; KLX1-3 represents Kunlunxueju1-3; KLM1-3 represents Kunlunmiju1-3; HJ represents Huaiju; FBJ1-2 represents Fubaiju1-2.
* Compound was positively identified by comparing the retention time, high-resolution molecular ions ([M−H]−) and fragment ions with the authentic standards.
Occurrence

in KLM1, but the concentrations of those two compounds were 14566.1

Hot-H2O‡

and 22249.0 μg RE/g in the 75% methanol extract of KLX3, respec-

N\A

tively. Moreover, the greatest level of dicaffeoylquinic acid (peak 14) in

hot-H2O and 75% methanol extracts were 706.3 and 1933.7 μg
chlorogenic acid equivalent/g in FBJ1, respectively. The greatest
smallest mass error between theoretical and experimental m/z, taking into account of the fragment ions in the first and second mass spectral channel.
Flavonoid (Flavone)
Flavonoid (Flavone)

amount of total luteolin-7-O-6″-acetylglucuronide and dicaffeoylquinic

Family (subclass)

acid (peaks 19 and 20) were 2806.0 and 5077.9 μg luteolin-7-O-glu-

coside equivalent/g in the in hot-H2O and 75% methanol extracts of
FBJ1. In general, the hot-water extracted less flavonoids and phenolic
acids compared with 75% methanol (Table 2). Agreeing to previous
studies, the results from present study indicated that individual chry-
santhemum samples might significantly differ in the quality and
quantity of flavonoid and phenolic compositions (Wang, Hao, et al.,
2015; Xie et al., 2012).
Acacetin-7-O-glucuronide

3.2. Total phenolic contents (TPC) of chrysanthemum samples

Tentative Identification

The TPC values of the hot-H2O extracts were 1.06–12.72 mg GAE/g

(Fig. 1), which was lower than that of 32.3–45.7 mg GAE/g in the hot-
H2O extracts of C. morifolium chrysanthemum reported by Duh et al.
Acacetin

(1999). The TPC values of the hot-water extracts were comparable to

that of 5.31–35.60 mg GAE/g for the 75% methanol extracts (Fig. 1).
Moreover, the TPC values for hot-water and 75% methanol extracts
from the present study were greater than that of 0.25–0.37 mg GAE/g in
60% methanol extracts of C. morifolium chrysanthemum observed by
C22H20O11
C16H12O5
Chemical

Yang et al. (2011). Taking together, these results indicated that com-
formula

mercial chrysanthemum tea products might significantly differ in their

total phenolic content, and hot-water is an effective solvent for ex-
tracting phenolics from chrysanthemums.

3.3. Effects on IL-6, IL-1β and COX-2 mRNA expressions

Compound was first reported in HangJu, GongJu, HuaiJu.

Chronic inflammation has been associated with the development of

Compound was first reported in Snow chrysanthemum.
Fragments

several chronic diseases such as autoimmune and cardiovascular dis-

283.0601
268.0363

eases, and dietary anti-inflammatory components may reduce the risk

of these diseases (Abou-Raya & Abou-Raya, 2006; Chung et al., 2009).
Hot-water extracts of the 17 chrysanthemum samples were able to
suppress the LPS-induced IL-6, IL-1β and COX-2 mRNA expressions
under the experimental conditions (Fig. 2). Furthermore, the hot-water
[M−H]−

459.0527
283.0606
Theor.

extract was not necessary to have a weaker inhibitory effect on mRNA

expression for IL-6, IL-1β and COX-2 genes than its counterpart 75%
methanol extract (Fig. 2). Importantly, the 75% methanol extracts of
Exptl. [M−H]−

HB2, HB3, HT1, HT3, KLX3, KLM1, KLM3 and FBJ2 could suppress
these pro-inflammatory cytokines (Fig. 2), but some of the 75% me-
459.0925
283.0599

thanol extracts stimulated the LPS-induced IL-6, IL-1β and COX-2

Table 1 (continued)

mRNA expressions under the same experimental conditions (Fig. 2).

The 75% methanol extracts of HB1, HT2, G1, KLX1, HJ and FBJ1 sti-
Rt (min)

mulated the LPS-induced IL-6 mRNA expression (Fig. 2A), that of HB1,
14.23
16.44

HT2, G1, KLX1, KLX2, KLM2, HJ and FBJ1 increased the IL-1β mRNA
expression (Fig. 2B), and that of HT2, G1, G2 and KLX1 enhanced the
33
34
ID

#
§

COX-2 mRNA expression (Fig. 2C). These results indicated that hot-H2O

12
Y. Li, et al. Food Chemistry 286 (2019) 8–16

Table 2
Concentration of the selected compounds in chrysanthemum samples.
Sample Marein* (Peak 9) (μg rutin equivalent/g) Dicaffeoylquinic acid§ (Peak 14) (μg L & D # (Peak 19 and 20) (μg luteolin-7-O- Okanin* (Peak 21)
chlorogenic acid equivalent/g) glucoside equivalent/g) (μg rutin equivalent/
g)
Hot-H2O‡ 75% Methanol‡ Hot-H2O 75% Methanol Hot-H2O 75% Methanol Hot-H2O 75% Methanol

HB1 N\A N\A 290.8j ± 6.4 794.4 g ± 14.2 1761.8e ± 31.3 3620.4i ± 4.8 N\A N\A
HB2 N\A N\A 285.7j ± 4.0 887.4i ± 9.4 1767.8e ± 20.1 3775.9 k ± 4.7 N\A N\A
HB3 N\A N\A 373.8 m ± 3.9 637.3e ± 13.9 1913.9f ± 22.5 2411.6f ± 2.3 N\A N\A
HT1 N\A N\A 358.2 l ± 2.3 1256.3 m ± 10.4 2197.9 h ± 8.4 5067.5 m ± 11.6 N\A N\A
HT2 N\A N\A 273.7i ± 2.5 831.6 h ± 5.6 1774.6e ± 18.0 3520.4j ± 7.1 N\A N\A
HT3 N\A N\A 399.5n ± 3.2 845.4 h ± 9.1 2588.6i ± 5.0 2544.0 g ± 2.5 N\A N\A
G1 N\A N\A 56.7c ± 2.4 987.7 k ± 16.7 482.4b ± 6.9 3199.2i ± 3.5 N\A N\A
G2 N\A N\A 109.7e ± 0.3 989.1 k ± 7.2 675.7d ± 5.7 3147.5 h ± 45.3 N\A N\A
KLX1 1691.8e ± 85.3 7848.0d ± 14.4 123.1f ± 6.3 962.6j ± 9.9 38.6a ± 2.0 316.0d ± 1.7 220.2a ± 11.6 11027.7d ± 9.4
KLX 2 131.7a ± 1.2 6297.1b ± 9.3 140.6 g ± 5.1 709.2f ± 11.6 44.4a ± 2.6 90.3bc ± 0.6 1512.0e ± 37.5 16374.5e ± 3.5
KLX 3 1082.6d ± 54.1 14566.1f ± 36.7 60.0c ± 1.1 1075.0 l ± 13.4 9.4a ± 1.1 152.6c ± 0.8 424.4b ± 17.5 22249.0f ± 37.8
KLM1 3126.6f ± 12.8 9909.8e ± 24.6 73.4d ± 1.2 271.0d ± 8.2 30.6a ± 0.5 92.0bc ± 1.0 1654.3f ± 29.7 6825.9b ± 13.6
KLM 2 392.0c ± 14.0 7825.5c ± 13.8 42.9b ± 0.9 172.5b ± 4.8 40.9a ± 0.9 52.7ab ± 0.4 1201.9d ± 52.2 8011.2c ± 10.6
KLM 3 263.7b ± 6.9 493.6a ± 8.0 23.0a ± 0.4 21.1a ± 0.9 18.1a ± 0.8 13.2ab ± 0.4 822.9c ± 25.5 1579.2a ± 25.0
HJ N\A N\A 222.5 h ± 4.3 218.7c ± 10.9 541.4c ± 13.0 491.9e ± 0.5 N\A N\A
FBJ1 N\A N\A 706.3o ± 8.6 1933.7n ± 32.6 2806.0j ± 12.2 5077.9n ± 117.7 N\A N\A
FBJ2 N\A N\A 342.9 k ± 7.0 1054.6 l ± 10.3 1968.9 g ± 16.5 4076.7 l ± 12.4 N\A N\A

* The concentrations of marein or okanin were reported as μg rutin equivalent/g.

§
The concentrations of dicaffeoylquinic acid were reported as μg chlorogenic acid equivalent/g. l represents luteolin-7-O-6′'-acetylglucuronide as peak 19; D
represents dicaffeoylquinic acid as peak 20.
#
The total concentrations of peak 19 and 20 were reported as μg luteolin-7-O-glucoside equivalent/g.
‡
Samples were extracted by soaking in 75% methanol or hot-H2O overnight and detailed procedures were described in sample preparation in Materials and
Methods. HB1-3 represents Hangbaiju 1–3; HT1-3 represents Hangtaiju 1–3; G1-2 represents Gongju 1–2; KLX1-3 represents Kunlunxueju1-3; KLM1-3 represents
Kunlunmiju1-3; HJ represents Huaiju; FBJ1-2 represents Fubaiju1-2. N\A stands for not applicable. Data are represented as mean ± SD (n = 3). Values in the same
column with different letters are significantly different (P < 0.05). All measurements were carried out in triplicate.

benefits, supporting the development of functional drinks/foods using

chrysanthemum tea materials.

3.4. Effects on relative DPPH and oxygen radical scavenging capacities

The relative DPPH% scavenging capacity (RDSC) and oxygen radical

Fig. 1. Total phenolic content (TPC) of chrysanthemum samples extracted with
75% methanol and hot-H2O. GAE stands for gallic acid equivalent. HB1-3
presents Hangbaiju 1–3; HT1-3 presents Hangtaiju 1–3; G1-2 presents Gongju
1–2; KLX1-3 presents Kunlunxueju1-3; KLM1-3 presents Kunlunmiju1-3; HJ
presents Huaiju; FBJ1-2 presents Fubaiju1-2. The open columns were marked
with letters ordered alphabetically, while the solid columns were marked with
letters ordered in the reverse alphabetical way. Open or solid columns marked
with different letters are significantly different from each other (P < 0.05).
*indicates a difference between the two solvent groups (P < 0.05). Each
column represents the mean ± SD (n = 3). All experiments were carried out in
triplicate.
extracts of chrysanthemum may contain more inhibitor(s) for IL-6, IL-
1β and COX-2 mRNA expressions under the experimental conditions,
although the 75% methanol extracts had greater levels of flavonoid and
phenolic compounds. It is interesting that the individual compounds
with potential anti-inflammatory effects, especially the non-phenolic
compounds. Also noted was that hot-H2O infusion might be an excellent
way to consume chrysanthemum flowers for their anti-inflammatory
absorbance capacities (ORAC) were examined for the hot-H2O and 75%
methanol extracts of the 17 chrysanthemum samples (Fig. 3). The RDSC
values of the hot-H2O extracts ranged from 15.68 to 105.48 μmol TE/g,
which were comparable to that of 27.79–202.89 μmol TE/g for 75%
methanol extracts (Fig. 3A). The greatest RDSC value for the hot-H2O
extract was 105.48 μmol TE/g for KLM1, which was 7-fold of the lowest
RDSC value of 15.68 μmol TE/g in G2 (Fig. 3A). These values are
greater than that of 1.07–2.29 μmol TE/g in carrot juice, and compar-
able to that of 15.16–20.58 and 34.43–44.85 μmol TE/g in blueberry
juice and pomegranate juice, which were normally well-known natural
antioxidant products (Plank et al., 2012). Furthermore, a chry-
santhemum had a greater RDSC value for its hot-water extract might
not necessarily have a greater RDSC for its 75% methanol extract
(Fig. 3A). In another word, the order of RDSC for the hot-water extracts
differed to that for the 75% ethanol extracts, suggesting the radical
scavenging components have more diverse structures. In addition,
DPPH radical scavenging capacities were reported for HangJu, GongJu
and Snow chrysanthemums as IC50 values in μg/mL or mg/mL, which
were dependent on the DPPH radical concentrations in the testing re-
actions and the duration of DPPH radical and antioxidants (Wang et al.,
2014; Wang, Xi et al., 2015; Yang et al., 2011). DPPH radical scaven-
ging capacity was reported for the first time for FubaiJu chry-
santhemum.
The ORAC values of the hot-H2O extracts ranged from 266.45 to
1273.38 μmol TE/g (Fig. 3B), which were comparable to that of
800–880 μmol TE/g for C. morifolium chrysanthemum with methanol as
extraction solvent (Tsai, Tsai, Chien, Lee, & Tsai, 2008). The ORAC
values of the hot-water extracts were also comparable to that of
873.67–2132.58 μmol TE/g for the 75% methanol extracts (Fig. 3B).
Moreover, the ORAC values for the hot-H2O and 75% methanol extracts

13
Y. Li, et al.
Fig. 2. Relative A) IL-6, B) IL-1β and C) COX-2 mRNA expression of chry-
santhemum samples extracted with 75% methanol and hot-H2O. HB1-3 pre-
sents Hangbaiju 1–3; HT1-3 presents Hangtaiju 1–3; G1-2 presents Gongju 1–2;
KLX1-3 presents Kunlunxueju1-3; KLM1-3 presents Kunlunmiju1-3; HJ presents
Huaiju; FBJ1-2 presents Fubaiju1-2. The open columns were marked with let-
ters ordered alphabetically, while the solid columns were marked with letters
ordered in the reverse alphabetical way. Open or solid columns marked with
different letters are significantly different from each other (P < 0.05). *in-
dicates a difference between the two solvent groups (P < 0.05). Each column
represents the mean ± SD (n = 3). All experiments were carried out in tripli-
cate.
14
Food Chemistry 286 (2019) 8–16
Fig. 3. A) Relative DPPH% scavenging capacity (RDSC) and B) Oxygen radical
absorbance capacity (ORAC) of chrysanthemum samples extracted with 75%
methanol and hot-H2O. TE stands for trolox equivalents. HB1-3 presents
Hangbaiju 1–3; HT1-3 presents Hangtaiju 1–3; G1-2 presents Gongju 1–2;
KLX1-3 presents Kunlunxueju1-3; KLM1-3 presents Kunlunmiju1-3; HJ presents
Huaiju; FBJ1-2 presents Fubaiju1-2. The open columns were marked with let-
ters ordered alphabetically, while the solid columns were marked with letters
ordered in the reverse alphabetical way. Open or solid columns marked with
different letters are significantly different from each other (P < 0.05). *in-
dicates a difference between the two solvent groups (P < 0.05). Each column
represents the mean ± SD (n = 3). All experiments were carried out in tripli-
cate.
were greater than that of 57.28–76.76 μmol TE/g in the water extracts
of C. indicum and C. morifolium chrysanthemum reported recently
(Wang et al., 2017). Over production of free radicals may cause oxi-
dative stress and are closely related to the development of chronic in-
flammation and degenerative ailments such as cardiovascular diseases,
cancer, autoimmune disorders, and dietary antioxidants may reduce the
risk of these diseases (Halliwell, 1994; Pham-Huy, He, & Pham-Huy,
2008). The results from the present study indicated that hot-H2O ex-
tracts of chrysanthemum may serve as natural dietary anti-oxidants for
health promotion, and natural commercial chrysanthemum tea pro-
ducts may significantly differ in their radical scavenging properties.
3.5. Inhibitory effects on H2O2-induced oxidative stress in ARPE-19 cells
Dietary antioxidant may prevent the excessive production of in-
tracellular ROS that may cause severe oxidative stress and contribute to
the pathogenesis of several chronic degenerative and ocular diseases
(Iloki-Assanga et al., 2015). The 17 chrysanthemum extracts were
Y. Li, et al.
Fig. 4. Inhibitory effects on H2O2-induced increment in intracellular reactive
oxygen species (ROS) of chrysanthemum samples extracted with 75% methanol
and hot-H2O. The intracellular ROS levels were normalized as pmol DCF/μg
DNA. The content of 2′,7′- dichlorofluorescein (DCF) were monitored using a
fluorescence spectrophotometer with excitation and emission wavelengths of
485 and 520 nm, respectively. The DNA content were calculated using a
fluorescence spectrophotometer with excitation and emission wavelengths of
356 and 458 nm, respectively. HB1-3 presents Hangbaiju 1–3; HT1-3 presents
Hangtaiju 1–3; G1-2 presents Gongju 1–2; KLX1-3 presents Kunlunxueju1-3;
KLM1-3 presents Kunlunmiju1-3; HJ presents Huaiju; FBJ1-2 presents Fubaiju1-
2. The open columns were marked with letters ordered alphabetically, while the
solid columns were marked with letters ordered in the reverse alphabetical
way. Open or solid columns marked with different letters are significantly
different from each other (P < 0.05). *indicates a difference between the two
solvent groups (P < 0.05). Each column represents the mean ± SD (n = 6).
All experiments were carried out in triplicate.
measured for their inhibition on H2O2-induced ROS production in the
cultured ARPE-19 cells. The expression of intracellular ROS was sig-
nificantly greater after pre-treatment with 200 μM H2O2 as compared
with the control ARPE-19 cells. The hot-H2O extracts of the 17 chry-
santhemum samples significantly reversed the over expressions of in-
tracellular ROS, whereas not all the tested 75% methanol extracts were
able to suppress the intracellular ROS expression under the same con-
dition (Fig. 4). Only the 75% methanol extracts of HT1, HT2, HT3, G1,
KLX1, KLX2, KLX3, KLM1, KLM2, KLM3, HJ, FBJ1 and FBJ2 chry-
santhemum samples, 13 out of 17, were able to suppress the ROS
production (Fig. 4). Interestingly, the hot-H2O extracts of HB1, HB2,
HB3, HT1, HT2, HT3, KLX2 and KLX3 showed significantly greater
inhibition on ROS production than their counterpart 75% methanol
extracts (Fig. 4), indicating that hot-water is an excellent solvent for
extracting natural antioxidants capable of inhibiting intracellular ROS
production.
Chrysanthemum tea has been associated with eye health and eye-
sight improvement for a long time but without clear mechanism. Recent
studies have reported that chrysanthemum flavonoids including lu-
teolin and diosmetin might contribute to the eye health improvement
(Hytti et al., 2015; Matsuda, Morikawa, Toguchida, Harima, &
Yoshikawa, 2002; Shen et al., 2016). For example, luteolin has been
reported to protect human RPE cells from oxidative stress-induced cell
death and inflammatory damages, and to suppress the activity of rat
lens aldose reductase (Hytti et al., 2015; Matsuda et al., 2002). Dios-
metin has been observed to inhibit the apoptosis of retinal cells in
Sprague-Dawley rats and in the cultured ARPE-19 cells (Shen et al.,
2016). However, luteolin is a minor component in the hot-water and
75% methanol extract of the 17 chrysanthemum samples in the present
study, and luteolin and diosmetin were also reported as the minor
components in chrysanthemum (Sun, Hua, Ye, Zheng, & Liang, 2010;
Wang, Hao, et al., 2015; Xie et al., 2012). Their levels in the
15
Food Chemistry 286 (2019) 8–16
chrysanthemum extracts cannot completely explain the retinal protec-
tion effects of chrysanthemum. Considering that the hot-H2O extracts
showed potent suppression of ROS expression (Fig. 4) and greater in-
hibition on the IL-6, IL-1β and COX-2 mRNA expressions (Fig. 2) than
their counterpart 75% methanol extracts, the hot-H2O extracts of
chrysanthemum might be interesting key targets for future studies of
their eye health promotion properties.
4. Conclusions
In conclusion, this study investigated the chemical compositions
and the anti-inflammatory and antioxidant properties of chry-
santhemum hot-water extracts, and compared to that of the 75% me-
thanol extracts. The results from the present study showed for the first
time that hot-water extracts of chrysanthemum had comparable if not
greater anti-inflammatory, free radical scavenging, and cellular anti-
oxidant activities as their counterpart 75% methanol extracts, along
with greater or comparable level of total phenolics and less flavonoids
on a per chrysanthemum flower weight basis. In addition, FubaiJu
chrysanthemum was investigated for its chemical composition and
biological activities for the first time. These data support the utilization
of chrysanthemums in tea and other potential food products for eye
health and lower risk of inflammation. These data also warrant addi-
tional research to further investigate the anti-inflammation and anti-
oxidant compounds in chrysanthemum tea, and the biochemical me-
chanisms behind their bioactivities.
Acknowledgements
This research was partially supported by a grant from the National
Key Research and Development Program of China (Grant No.
2017YFC1600500); a grant from National Natural Science Foundation
of China (Grant No. 31501479); funds from the Beijing Advanced
Innovation Center for Food Nutrition and Human Health, Beijing
Technology & Business University (BTBU).
Conflict of interest
The authors declare that there are no conflicts of interest.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.foodchem.2019.02.013.
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