Antitumor, antioxidant and anti-inflammatory activities of kaempferol and its corresponding glycosides and the enzymatic preparation of kaempferol

Article abstract of DOI:10.1371/journal.pone.0197563

Kaempferol (kae) and its glycosides are widely distributed in nature and show multiple bio-activities, yet few reports have compared them. In this paper, we report the antitumor, antioxidant and anti-inflammatory activity differences of kae, kae-7-O-gluco

Full text of DOI:10.1371/journal.pone.0197563

RESEARCH ARTICLE
Antitumor, antioxidant and anti-inflammatory
activities of kaempferol and its corresponding
glycosides and the enzymatic preparation of
kaempferol
1
,2
1,2
1,2
1,2
2,3
Jingqiu Wang , Xianying Fang , Lin Ge , Fuliang Cao , Linguo Zhao *,
4
4
Zhenzhong Wang , Wei Xiao
1
Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing,
a1111111111
a1111111111
a1111111111
a1111111111
a1111111111
China, 2 College of Chemical Engineering, Nanjing Forestry University, Nanjing, China, 3 Jiangsu Key Lab
for the Chemistry & Utilization of Agricultural and Forest Biomass, Nanjing, China, 4 Jiangsu Kanion
Pharmaceutical Co., Ltd., Lianyungang, China
* njfu2304@163.com
Abstract
OPEN ACCESS
Kaempferol (kae) and its glycosides are widely distributed in nature and show multiple bio-
activities, yet few reports have compared them. In this paper, we report the antitumor, anti-
oxidant and anti-inflammatory activity differences of kae, kae-7-O-glucoside (kae-7-O-glu),
kae-3-O-rhamnoside (kae-3-O-rha) and kae-3-O-rutinoside (kae-3-O-rut). Kae showed the
highest antiproliferation effect on the human hepatoma cell line HepG2, mouse colon cancer
cell line CT26 and mouse melanoma cell line B16F1. Kae also significantly inhibited AKT
phosphorylation and cleaved caspase-9, caspase-7, caspase-3 and PARP in HepG2 cells.
A kae-induced increase in DPPH and ABTS radical scavenging activity, inhibition of conca-
navalin A (Con A)-induced activation of T cell proliferation and NO or ROS production in
LPS-induced RAW 264.7 macrophage cells were also seen. Kae glycosides were used to
produce kae via environment-friendly enzymatic hydrolysis. Kae-7-O-glu and kae-3-O-rut
were hydrolyzed to kae by β-glucosidase and/or α-L-rhamnosidase. This paper demon-
strates the application of enzymatic catalysis to obtain highly biologically active kae. This
work provides a novel and efficient preparation of high-value flavone-related products.
Citation: Wang J, Fang X, Ge L, Cao F, Zhao L,
Wang Z, et al. (2018) Antitumor, antioxidant and
anti-inflammatory activities of kaempferol and its
corresponding glycosides and the enzymatic
preparation of kaempferol. PLoS ONE 13(5):
e0197563. https://doi.org/10.1371/journal.
pone.0197563
Editor: Salvatore V Pizzo, Duke University School
of Medicine, UNITED STATES
Received: November 15, 2017
Accepted: May 4, 2018
Published: May 17, 2018
Copyright: © 2018 Wang et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Introduction
Kae is a major flavonoid aglycone extract from the Zingiberaceae Kaempferia rhizome and
Funding: This work was supported by The National mainly exists in nature as the glycoside form [1]. Many beneficial functions of kae and its gly-
Key Research Development Program of China,
cosides have been reported, such as cardiovascular [2], antioxidant [3], antidiabetic [4], anti-
(
Grant # 2017YFD0600805 to LZ); The National
Natural Science Foundation of China (Grant No.
1600465 to XF); The National Natural Science
inflammatory [5], hepatoprotective [6] and neuroprotective effects [7, 8]. Kae is gaining atten-
tion due to its applications in cancer chemotherapy and its other various pharmacological
effects [9, 10]. Epidemiological evidence suggests that the consumption of kae-rich foods may
reduce the risk of developing some types of cancer, including liver cancer, colon cancer and
3
Foundation of China (Grant # 31570565 to LZ); The
Forestry Achievements of Science and Technology
PLOS ONE | https://doi.org/10.1371/journal.pone.0197563 May 17, 2018
1 / 12

Kaempferol aglycone and glycosides’ biological activities and enzymatic preparation
to Promote Projects ([2017] 10 to LZ); A Project
Funded by the Priority Academic Program
Development of Jiangsu Higher Education
Institutions (PAPD); and The Study on biocatalysis
and transformation of natural drugs (Grant No.
skin cancer [11, 12]. Inhibiting cancer proliferation and promoting cancer cell apoptosis are
the main chemical mechanisms for cancer prevention [13, 14]. Protein kinase B (PKB), also
known as AKT, plays an important role in cell survival and apoptosis. Inhibition of PI3K and
de-phosphorylation of Akt at Ser473 and Thr308 were observed in K562 and U937 cells after
kae treatment [15]. Caspases are a family of cysteine proteases involved in the initiation and
execution of apoptosis. Kae has been found to induce the activation of caspase-3, caspase-7,
caspase-9 and PARP [16]. In addition, accumulating evidence suggests that reactive oxygen
species (ROS) have an important role in cancer development [17]. ROS are byproducts of aer-
obic metabolism, such as oxygen ions, superoxide anions, peroxides, hydroxyl radicals, oxygen
free radicals, and nitric oxide (NO). They play a key role in carcinogenesis, as indicated by
increased ROS in cancer cells, ROS-induced malignant cell transformation, and reduced ROS
levels leading to malignant cancer cell phenotype reversal [18]. Numerous reports have shown
that kae, some kae glycosides, and several kae-containing plants can decrease superoxide
anion, hydroxyl radical and peroxynitrite levels [19]. Inflammation has also been suggested to
have a significant role in cancer [20]. Inflammatory cells, chemokines and cytokines are pres-
ent in all tumor microenvironments studied in experimental animal models and humans from
the earliest stages of development. Both in vitro and in vivo anti-inflammatory activity have
been reported for kae, kae glycosides and/or kae-containing plants [21]. The anti-inflamma-
tory activity of kae may be mediated by several mechanisms of action. Kae can inhibit LPS-
and ATP-induced phosphorylation of PI3K and AKT in cardiac fibroblasts, thereby protecting
cells from inflammatory injury [22]. Kae and some of its glycosides can also significantly
inhibit the production of NO and tumor necrosis factor-alpha (TNF-α) in RAW 264.7 cells
stimulated by LPS [23]. Although significant research has focused on the activity of kae agly-
cone and its glycosides, few studies have compared their activities.
028340002). Authors ZW and WX received
support in the form of salary from Jiangsu Kanion
Pharmaceutical Co., Ltd. The funders had no role in
study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
Competing interests: The authors declare the
following interests: Zhenzhong Wang and Wei Xiao
are employed by Jiangsu Kanion Pharmaceutical
Co., Ltd. This study was funded in part by Jiangsu
Kanion Pharmaceutical Co., Ltd. There are no
patents, products in development or marketed
products to declare. This does not alter our
adherence to all the PLOS ONE policies on sharing
data and materials. The authors confirm there are
no other interests to declare.
Due to the low concentration of kae aglycone and high concentrations of kae glycosides in
plants [24, 25], deglycosylation may provide a way to produce kae from kae glycosides. Modifi-
cation of flavonoids via glycosylation can be achieved using chemical or biological methods.
Compared to chemical methods, biological methods have attracted attention due to their abil-
ity to catalyze hydrolysis reactions under milder conditions yielding highly stereo- and regiose-
lective products. At present, common biological methods include enzyme- and microbe-
induced transformations. Enzymes have received the most attention due to their many advan-
tages, such as strong selectivity, mild reaction conditions, easy separation and purification, and
environmental friendliness. The enzymatic hydrolysis of flavone glycosides to prepare flavone
aglycones has been explored using β-glucosidase and α-L-rhamnosidase.
In this study, we investigated the antitumor, antioxidant and anti-inflammatory activities of
kae, kae-7-O-glu, kae-3-O-rha and kae-3-O-rut. We demonstrated that kae has better antitu-
mor activity, possibly because kae significantly inhibits AKT phosphorylation and caspase-3,
caspase-7, caspase-9 and PARP cleavage while the other kae glucosides do not. Kae also dem-
onstrated better antioxidant and anti-inflammatory activities. To explore and optimize kae
glucoside hydrolysis, α-L-rhamnosidase and β-glucosidase were chosen due to their selectivity
and previous use in our laboratory. Only β-glucosidase could hydrolyze kae-7-O-glu to kae,
but both β-glucosidase and α-L-rhamnosidase could hydrolyze kae-3-O-rut to kae. After opti-
mizing the reaction conditions, complete hydrolysis was achieved with both enzymes.
Materials and methods
Chemicals and reagents
Roswell Park Memorial Institute-1640 medium (RPMI-1640), Dulbecco’s modified Eagle’s
medium (DMEM) and fetal bovine serum (FBS) were purchased from HyClone (USA).
PLOS ONE | https://doi.org/10.1371/journal.pone.0197563 May 17, 2018
2 / 12

Kaempferol aglycone and glycosides’ biological activities and enzymatic preparation
Sodium chloride (NaCl), potassium chloride (KCl), potassium hydrogen phosphate trihydrate
(
K HPO Á3H O), and disodium hydrogen phosphate anhydrous (Na HPO ) were purchased
2
4
2
2
4
from Nanjing Chemical Reagent Co., Ltd. (Nanjing, China). Concanavalin A (Con A), MTT,
and propidium iodide (PI) were purchased from Sigma (USA). Trypsin digestion solution, the
annexin V-FITC apoptosis detection kit, and WB and IP cell lysates were purchased from
Beyotime Biotechnology (Shanghai, China). Kae, rutin, kae-7-O-glu, kae -3-O-rha and kae-
3-O-rut standards were purchased from Nanjing Jing Zhu Biotechnology Co., Ltd. (Nanjing,
China). The BCATM protein assay kit was purchased from Pierce (USA). N,N’-Methylenebi-
sacrylamide (Bis), sodium dodecyl sulfate (SDS), tris(hydroxymethyl)aminomethane (Tris)
and glycine were purchased from Amresco (USA). Ammonium persulfate (APS) was pur-
chased from Nanjing Sunshine Biotechnology Co. Ltd. (Nanjing, China). N,N,N,N’-Tetra-
methylethylenediamine (TEMED) was purchased from Wako Pure Chemical Industries, Ltd.
(Japan). A prestained protein ladder was purchased from Thermo Scientific (USA). Polyvinyli-
dene fluoride (PVDF) was purchased from Millipore (USA). The following antibodies were
purchased: anti-cPARP, anti-GAPDH, anti-cleaved-caspase-3, anti-cleaved-caspase-7, anti-
cleaved-caspase-9, and anti-p-AKT. 20X LumiGLO Reagent and 20X Peroxide was purchased
from Cell Signaling Technology (USA). HRP-coupled anti-mouse and anti-rabbit IgG second-
ary antibodies and substrates were purchased from KPL (USA). All reagents were of the high-
est purity commercially available.
Cell culture
Human hepatoma cell line HepG2, mouse colon cancer cell line CT26, mouse melanoma cell
line B16F1 and mouse peritoneal macrophage RAW 264.7 were purchased from the Cell Bank
of the Chinese Academy of Sciences. HepG2, CT26 and B16F1 cells were grown in DMEM
medium supplemented with 10% fetal bovine serum. RAW 264.7 cells were grown in RPMI-
1640 medium supplemented with 10% fetal bovine serum. All cells were kept in a humidified
incubator with 5% CO and 95% air at 37˚C. Cells were routinely collected by centrifugation at
2
800 g for five minutes.
Recombinant glycosidases
The following recombinant enzymes were previously generated in our laboratory: glucosidase
derived from Thermotoga petrophila DSM 13995 [26] and rhamnosidase derived from Aspergil-
lus terreus [27].
Assessment of cell proliferation with the MTT assay
3
After trypan blue staining, 3 x 10 tumor cells were inoculated into each well of a 96-well plate.
After 6 h, various concentrations of drugs were added to each well, and then, the plate was cul-
tured at 37˚C in a 5% CO atmosphere for 72 h. MTT solution (20 μl, 4 mg/ml) was added to
2
the culture medium. After 4 h, the plate was centrifuged at 1000 g for 5 min, and the superna-
tant was removed. DMSO (200 μl) was added to the wells and mixed until the precipitate
completely dissolved. The absorbance at 540 nm was measured and recorded. All experiments
were performed in at least four parallels and repeated three times. The percentage of cell prolif-
eration inhibition was calculated using the following formula:
ꢀ
ꢁ
ODsample
ODcontrol
Inhibition rate of cell proliferation ð%Þ ¼ 1 À
 100%
PLOS ONE | https://doi.org/10.1371/journal.pone.0197563 May 17, 2018
3 / 12

Kaempferol aglycone and glycosides’ biological activities and enzymatic preparation
Apoptosis assay
6
Cell solutions (1×10 cells) were transferred into a flow cell tube at 4˚C and centrifuged at
000 g for 5 min. The supernatant was removed, and 200 μl of binding buffer (10 mM HEPES,
pH 7.4, 140 mM NaCl, 2.5 mM CaCl ) was added. After mixing, 1.25 μl of annexin V solution
1
2
(1 μg/ml) and 2 μl of PI solution (100 μg/ml PI, HEPES 20 mM, pH 7.4) were added. Following
lucifugal incubation at room temperature for 20 min, binding buffer (300 μl) was added. The
fluorescence in the FL-1 and FL-3 channels was measured by flow cytometry. Data were col-
lected using CellQuest software (BD Immunocytometry Systems) and analyzed using FlowJo
software. All experiments were repeated three times.
Western blotting analysis
The cells were lysed using 150 μl of lysate buffer (10 mM HEPES, 2 mM EDTA, 0.1% CHAPS, 5
mM DTT and 1 mM PMSF). After being chilled in an ice bath for 0.5 h, cells were centrifuged
at 12000 g for 2 min at 4˚C. The supernatant was removed and the protein concentration was
measured using the BCATM protein quantification kit. The protein content of each 50 g sample
was electrophoresed in a 10% SDS-PAGE gel at a constant 20 mA current for 1 h at low temper-
ature (4˚C). After electrophoresis, the proteins were transferred onto a PVDF membrane by wet
rotation. The membranes were incubated with blocking buffer (2% free fat milk, 10 mM Tris-
Cl, 50 mM NaCl 0.1% Tween 20, pH 7.4) at room temperature for 2 h to saturate the PVDF
membrane. The membrane was cut according to the indicated position of the pre-dyed protein
ladder and incubated with primary antibody overnight on the converter. The next day, the
membrane was washed in the washing buffer (10 mM Tris-Cl, 50 mM NaCl, 0.1% Tween 20,
pH 7.4) three times, twice for 5 min and once for 10 min, to remove any nonspecific primary
antibody binding. Next, the membrane was incubated with an appropriate dilution of secondary
antibody at room temperature for 2 h. The substrate was diluted (1:20) and incubated with the
membrane for 1 minute (ECL). A photograph of the gel was taken, and the relative band density
was analyzed by optical densitometry using ImageJ. All experiments were repeated three times.
Assay for DPPH and ABTS radical scavenging
The DPPH scavenging capacity was measured as previously described [28] with slight modifi-
cations. In brief, the sample (100 μl) was added into 100 μl of DPPH solution (0.5 mM DDPH
solution diluted in 95% ethanol) and incubated in a 96-well plate at room temperature for 30
min. The sample absorbance (A), ethanol absorbance (B) or control absorbance (C) were mea-
sured at 517 nm. The DPPH scavenging capacity was calculated using the following formula:
ꢂ
ꢃ
A À B
Scavenging capacity ð%Þ ¼ 1 À
x 100%
C
The ABTS kit instructions were followed for the ABTS assay (Beyotime, China) [29].
Briefly, ABTS solution (200 μl) and sample (10 μl) were added to each well of a 96-well plate.
The plate was gently mixed and incubated at room temperature for 5 min. The sample absor-
bance (A), ethanol absorbance (B) and control absorbance (C) were measured at 734 nm. The
ABTS scavenging capacity was calculated using the above formula. All experiments were per-
formed in at least four parallels and repeated three times.
Lymphocyte transformation test
7
The lymphocyte suspension was diluted to 1 x 10 cells/ml. Cell solutions (1 x 10 cells) and
6
Con A (2.5 μg/ml) were added to each well at different concentrations, and the plate was
PLOS ONE | https://doi.org/10.1371/journal.pone.0197563 May 17, 2018
4 / 12

Kaempferol aglycone and glycosides’ biological activities and enzymatic preparation
placed in the CO incubator at 37˚C for 48 h. Then, MTT (20 μl) was added, and cells were
2
grown for 4 h. The plate was centrifuged at 1000 g for 5 min; then, the supernatant was
removed, and 200 μl DMSO was added to each well. The OD values at 540 nm were measured
and recorded. All experiments were performed in at least four parallels and repeated three
times.
Assay for NO production and intracellular reactive oxygen species
5
RAW 264.7 cells were grown to a density of 2 x 10 cells/ml in the logarithmic phase. The cells
(100 μl) were then added to each well of a 96-well plate. After 24 h, the cells were treated with
LPS (500 ng/ml) and various concentrations of kae, kae-7-O-glu, kae-3-O-rha and kae-3-O-
rut. Each concentration was tested twice in triplicate with the control being the culture
medium containing DMSO. Each sample (100 μl) was added to an enzyme-labeled plate and
the mixed Griess reagent (100 μl) was added. After 10 min, the absorbance was measured at
540 nm. The inhibitory effect of NO release was calculated using the following formula:
ODLPS À OD
LPSþsample
ODLPS À ODblank
NO inhibition rate ð%Þ ¼
 100%
Intracellular ROS were determined as as previously described [30] with slight modifications
by using the Reactive Oxygen Species Assay Kit (Beyotime Biotechnology, China). All experi-
ments were performed in at least four parallels and repeated three times.
Expression of recombinant protein in Escherichia coli
The genetically engineered recombinant strain was cultured on an LB + Amp plate at 37˚C for
one day. A single colony was isolated from the plate, inoculated in LB + Amp liquid media (4
ml), and shaken at 180 r/min for 8 h at 37˚C. The sample was then inoculated in LB + Amp liq-
uid media (150 ml) and shaken again at 180 r/min for 10 h at 37˚C. Bacteria were collected via
centrifugation at 8000 rpm.
Enzyme activity assay
After mixing and preheating at 35˚C for 10 min, the diluted enzyme solution (10 μl) was
added to the buffer solution (150 μl, 100 mM, pH 6.5) with pNPR/pNPG artificial substrate
(
40 μl, 5 mM). Then, a Na CO solution (600 μl, 1 M) was added to terminate the reaction,
2
3
and the absorbance was measured at 405 nm. Enzyme solutions that inactivated the substrate
were compared. All experiments were repeated three times.
Enzymatic hydrolysis of kae-3-O-rut and kae-7-O-glu with α-L-rhamnoside
and/or β-glycosidase
All enzymatic reactions were carried out in a temperature-controlled, heated water bath. In
this study, a disodium hydrogen phosphate-citrate buffer (pH 3.5–6.5) was used. The typical
reaction mixture (200 μl) contained disodium hydrogen phosphate-citrate buffer, substrate (2
mM) and distilled water. The reaction was initiated by combining buffered enzyme solutions.
The mixtures were incubated at various pH values, temperatures, enzyme concentrations and
times with other conditions remaining fixed. The reaction was stopped by adding 800 μl of
methanol. The crude hydrolysis products were then centrifuged at 10000 rpm for 10 min,
and the supernatant solutions were filtered through a 0.45 μm filter before injection into the
HPLC. All experiments were repeated three times.
PLOS ONE | https://doi.org/10.1371/journal.pone.0197563 May 17, 2018
5 / 12

Kaempferol aglycone and glycosides’ biological activities and enzymatic preparation
Results
Evaluation of cytotoxicity against HepG2, CT26 and B16F1 cells
In this paper, kae, kae-3-O-rha, kae-7-O-glu and kae-3-O-rut were evaluated for their antipro-
liferative activities against HepG2, CT26 and B16F1 cells using the MTT assay. Kae-3-O-rha
and kae-3-O-rut had no effects on human hepatocellular carcinoma cell line HepG2 prolifera-
tion, while kae and kae-7-O-glu (10–100 μM) caused decreased proliferation with kae showing
the strongest inhibitory effects (Fig 1). In both human colon cancer cells CT26 and mouse mel-
anoma cells B16F1, only kae had an inhibitory effect, while the other three compounds had no
effect.
Effect on HepG2 cell apoptosis
The ability of various compounds to promote HepG2 cell apoptosis was assessed by flow
cytometry. Increasing kae concentrations (0–100 μM) caused increasing apoptosis rates (1.78–
17.4%), indicating a proportional relationship between kae concentration and tumor cell apo-
ptosis (Fig 2). However, kae-3-O-rha, kae-7-O-glu and kae-3-O-rut (0–100 μM) showed no
significant effects on tumor cell apoptosis. To explore the differences in their apoptosis-pro-
moting abilities, we examined the effects of these four compounds on the expression of related
proteins in the apoptotic pathway using Western blotting. The expression of cell apoptosis
marker proteins, such as cleaved-PARP, cleaved caspase-3, cleaved caspase-7 and cleaved cas-
pase-9, were upregulated in kae-treated cells. This upregulation can significantly inhibit the
phosphorylation of the proliferation signal AKT. However, the other three compounds had no
significant effects on apoptotic protein expression.
Caspase-3, a cysteine protease, is the key protease in apoptosis. Caspases can transmit apo-
ptotic signals or directly act as apoptotic effector molecules causing apoptosis changes, such as
chromatin condensation and DNA fragmentation [31, 32]. Normally, caspase-3 is an inactive
3
Fig 1. Kae exhibits greater antitumor effects than its glycosides. Cells (3 x 10 /well) were added to wells in a
96-well plate and incubated with various concentrations of kae, kae-3-O-rha, kae-7-O-glu and kae-3-O-rut for 72
h. The inhibitory rate of kae, kae-7-O-glu, kae-3-O-rha and kae-7-O-rut on cell proliferation was determined by
the MTT assay. (A) HepG2 cells. (B) CT26 cells. (C) B16F1 cells. (D) IC50 values of kae, kae-3-O-rha, kae-7-O-glu and
Ã
ÃÃ
kae-3-O-rut. P < 0.05, P < 0.01. The data are shown as the mean ± SEM of three independent experiments.
https://doi.org/10.1371/journal.pone.0197563.g001
PLOS ONE | https://doi.org/10.1371/journal.pone.0197563 May 17, 2018
6 / 12

Kaempferol aglycone and glycosides’ biological activities and enzymatic preparation
Fig 2. Kae induces liver cancer cell apoptosis, inhibits AKT phosphorylation, and induces caspase-dependent apoptosis in liver cancer
cells. (A) HepG2 cells were added to a 96 -well plate and incubated with kae, kae-3-O-rha, kae-7-O-glu and kae-3-O-rut (0, 10 or 100 μM) for 24
h. HepG2 cell apoptosis was determined by annexin V/PI staining. (B) HepG2 cells were treated with kae, kae-3-O-rha, kae-7-O-glu and kae-
3
-O-rut (10, 30 or 100 μM) for 24 h. Cleaved caspase-3, 7, and 9, cleaved PARP, and p-AKT protein levels were determined by Western blotting.
(C) HepG2 cell apoptosis of three independent experiments were shown in the bar graph. (D)-(H) ImageJ software was used to analyze the levels
Ã
ÃÃ
of p-AKT, cleaved caspase-3, 7, 9 and cleaved PARP with GAPDH as the reference. P < 0.05, P < 0.01 The data are shown as the
mean ± SEM of three independent experiments.
https://doi.org/10.1371/journal.pone.0197563.g002
zymogen that exists in the cytoplasm. Stimulated by the apoptotic signal, caspase-3 is activated
by protease hydrolysis to become four dimers. The activated caspase-3 cleaves molecules in the
cytoplasm and cell nucleus substrates, eventually leading to cell apoptosis.
Antioxidant activity differences
DPPH and ABTS methods were used to compare the antioxidant activities of the four com-
pounds. Kae showed the strongest free radical scavenging capacity, followed by kea-7-glu, and
weaker activity from the other two compounds (Fig 3A–3C). Therefore, kae had the strongest
antioxidant activity.
Anti-inflammatory activity differences
First, we examined the effects of the four compounds on ConA-activated T cell proliferation
[33]. Kae (100 μM) showed inhibitory effects on activated T cell proliferation, with an inhibi-
tory rate of 53.62% after 24 h, 86.7% after 48 h, and background-level cell suppression at 72 h.
The most active kae glycoside, kae-7-O-glu, showed inhibition rates up to 25.5% after 24 h,
51.12% after 48 h and up to 72.05% after 72 h. In general, these four compounds inhibited the
proliferation of activated T cells in a time- and dose-dependent manner, with kae exerting the
strongest activity. Current research has demonstrated the participation of reactive oxygen spe-
cies in inflammation. Stimulation of LPS-induced RAW 264.7 cells [34, 35] led to overproduc-
tion of NO. Kae and kae-7-O-glu significantly inhibited NO release, while the other two
PLOS ONE | https://doi.org/10.1371/journal.pone.0197563 May 17, 2018
7 / 12

Kaempferol aglycone and glycosides’ biological activities and enzymatic preparation
Fig 3. Increased antioxidant and anti-inflammatory effects of kae compared to kae glycosides. (A) The DPPH radical scavenging assay. (B)
6
The ABTS radical scavenging assay. (C) IC50 values for DPPH and ABTS radical scavenging. (D) The T cells (1 x 10 /well) were added to a
9
6-well plate with ConA (2.5 g/ml). Various concentrations of kae, kae-7-O-glu, kae-3-O-rha and kae-3-O-rut (0, 10, 30 or 100 μM) co-cultured
with activated T cells were incubated for 24 h, 48 h and 72 h. The MTT assay was used to detect ConA-activated T cell proliferation. (E) RAW
5
264.7 cells (2 x 10 cells/ml) were added to a 96-well plate (100 μl/well). After 24 h, the cells were treated with LPS (500 ng/ml) and kae, kae-7-O-
glu, kae-3-O-rha or kae-3-O-rut at various concentrations. Each concentration was tested twice in triplicate with the culture medium containing
DMSO as the control. Each sample (100 μl) was added to an enzyme-labeled plate and the mixed Griess reagent (100 μl) was added. After 10
min, the absorbance was measured at 540 nm. The inhibitory effect of NO release on RAW 264.7 cells was calculated. (F) Steady state ROS
concentration in the culturing medium was determined using the Reactive Oxygen Species Assay Kit (Beyotime Biotechnology, China). (G) IC50
Ã
ÃÃ
values for the inhibition of T cells. P < 0.05, P < 0.01. The data are shown as the mean ± SEM of three independent experiments.
https://doi.org/10.1371/journal.pone.0197563.g003
compounds had no significant effect (Fig 3E). Besides, kae inhibited LPS induced ROS produc-
tion in a concentration-dependent manner, while the other three compounds had no signifi-
cant effect (Fig 3F).
Enzymatic hydrolysis of kae-3-O-rha, kae-7-O-glu and kae-3-O-rut
First, we chose to study the hydrolytic effects of α-L-rhamnoside and β-glycosidase, which
were used in previous studies. Kae-3-O-rut and kae-7-O-glu can be hydrolyzed to kae by α-L-
rhamnoside and/or β-glucosidase, while kae-3-O-rha could not be hydrolyzed. To improve the
hydrolysis rates of kae-3-O-rut and kae-7-O-glu, various pH values, temperatures, enzyme
concentrations and reaction times were examined. The optimal kae-3-O-rut hydrolysis condi-
tions were 0.05 U/ml of α-L-rhamnoside at pH 6.0 and 65˚C for 60 min (Fig 4). As α-L-rham-
noside only could hydrolyze kae-3-O-rut to kae-3-O-glu, we next used β-glycosidase to
hydrolyze kae-3-O-glu to kae. The optimal kae-3-O-glu hydrolysis conditions were 2 U/ml of
α-L-rhamnoside at pH 5.5 and 85˚C for 60 min. Overall, kae-3-O-rut could be completely
hydrolyzed to kae by α-L-rhamnoside and β-glucosidase under the optimized conditions.
Kae-7-O-glu was also completely hydrolyzed by β-glucosidase under optimal conditions.
PLOS ONE | https://doi.org/10.1371/journal.pone.0197563 May 17, 2018
8 / 12

Kaempferol aglycone and glycosides’ biological activities and enzymatic preparation
Fig 4. Hydrolysis of kae glycosides by α-L-rhamnoside and/or β-glycosidase. Effects of pH value, temperature, enzyme concentration and
time on the hydrolysis rate of kae-3-O-rut by α-L-rhamnoside (A) and kae-3-O-glu by β-glucosidase (B). (C) Hydrolysis rates of kae-3-O-rut
and kae-7-O-glu by α-L-rhamnoside and/or β-glucosidase under the optimal conditions. Each value represents the mean of three independent
measurements.
https://doi.org/10.1371/journal.pone.0197563.g004
Discussion
Kae showed the best antitumor, antioxidant and anti-inflammatory activities, whereas kae-
3-O-rha, kae-7-O-glu and kae-3-O-rut showed poor activities. Kae had a stronger antiproli-
feration effect on HepG2 cells (IC = 30.92 μM), CT26 cells (IC = 88.02 μM) and B16F1 cells
50
50
(
IC = 70.67 μM). Antitumor activity differences could be linked to kae’s ability to signifi-
50
cantly inhibit AKT phosphorylation and cleave caspase-9, caspase-7, caspase-3 and PARP. The
participation of ROS in cancer systems also plays an important role. Using the DPPH and
ABTS assay, we found that kae has the highest free radical scavenging activity, followed by
kae-7-O-glu, while the other two compounds showed no significant activity. ROS has also
been reported to participate in inflammation [34]. The antioxidant activity difference between
kae and its glycosides indicated that kae may have better anti-inflammatory activity. To verify
that, we studied the effect of these four compounds on T-cell proliferation and NO release
from LPS-induced RAW 264.7 cells. Compared to the other compounds, kae significantly
inhibited T-cell proliferation and NO release, which confirmed our hypothesis. Thus, kae agly-
cone has the strongest antitumor, antioxidant and anti-inflammation activities.
Kae mainly exists in the glycoside form in nature. Therefore, enzymatic hydrolysis of kae
glycosides to produce kae was examined. We chose enzymes which had been previously used
in our laboratory and optimize the hydrolysis conditions. Kae-7-O-glu and kae-3-O-rut could
be completely hydrolyzed to kae under the optimal hydrolysis conditions, while kae-3-O-rha
could not be hydrolyzed. Further studies are needed to identify a rhamnosidase that can effi-
ciently hydrolyze kae-3-O-rha. This work presents a novel and efficient preparation of high-
value flavone-related products.
Supporting information
S1 Fig. High-performance liquid chromatograms of kae-3-O-rut, kae-7-O-glu and their
hydrolysis product kae. (A) Kae-3-O-rut standard. (B) The kae-3-O-rut hydrolysis product
PLOS ONE | https://doi.org/10.1371/journal.pone.0197563 May 17, 2018
9 / 12

Kaempferol aglycone and glycosides’ biological activities and enzymatic preparation
kae. (C) Kae-7-O-glu standard. (D) The kae-7-O-glu hydrolysis product kae.
TIF)
(
Acknowledgments
We thank American Journal Experts (AJE) for revising this manuscript.
Author Contributions
Data curation: Jingqiu Wang.
Formal analysis: Jingqiu Wang.
Funding acquisition: Linguo Zhao, Zhenzhong Wang, Wei Xiao.
Investigation: Jingqiu Wang.
Methodology: Xianying Fang, Lin Ge.
Project administration: Fuliang Cao, Linguo Zhao.
Resources: Zhenzhong Wang, Wei Xiao.
Supervision: Linguo Zhao.
Writing – original draft: Jingqiu Wang.
Writing – review & editing: Linguo Zhao.
References
1.
Hua L., Ji H. S., Kang J. H., Dongha S., Hoyong P., & Myungsook C., et al. (2015). Soy leaf extract con-
taining kaempferol glycosides and pheophorbides improves glucose homeostasis by enhancing pan-
creatic β-cell function and suppressing hepatic lipid accumulation in db/db mice. Journal of Agricultural
&
Food Chemistry, 63(32), 7198–210.
2.
3.
4.
5.
6.
7.
Suchal K., Malik S., Khan S. I., Malhotra R. K., Goyal S. N., & Bhatia J., et al. (2017). Molecular path-
ways involved in the amelioration of myocardial injury in diabetic rats by kaempferol: International Jour-
nal of Molecular Sciences, 18(5), 1001.
Arif H., Sohail A., Farhan M., Rehman A. A., Ahmad A., & Hadi S. M. (2017). Flavonoids-induced redox
cycling of copper ions leads to generation of reactive oxygen species: a potential role in cancer chemo-
prevention. International Journal of Biological Macromolecules.
Li F., Zhang B., Chen G., & Fu X. (2017). The novel contributors of anti-diabetic potential in mulberry
polyphenols revealed by uhplc-hr-esi-tof-ms/ms. Food Research International, 100(Pt 1), 873. https://
doi.org/10.1016/j.foodres.2017.06.052 PMID: 28873762
Nascimento A. M., Maria-Ferreira D., Dal Lin F. T., Kimura A., de Santana-Filho A. P., & Mfp W., et al.
(2017). Phytochemical analysis and anti-inflammatory evaluation of compounds from an aqueous
extract of croton cajucara benth. Journal of Pharmaceutical & Biomedical Analysis, 145(3), 821.
Zhao J., Zhang S., You S., Liu T., Xu F., & Ji T., et al. (2017). Hepatoprotective effects of nicotiflorin
from nymphaea candida against concanavalin a-induced and d-galactosamine-induced liver injury in
mice. International Journal of Molecular Sciences, 18(3), 587.
Wu Y., Sun J., George J., Ye H., Cui Z., & Li Z., et al. (2016). Study of neuroprotective function of ginkgo
biloba extract (egb761) derived-flavonoid monomers using a three-dimensional stem cell-derived neural
model. Biotechnology Progress, 32(3), 735–744. https://doi.org/10.1002/btpr.2255 PMID: 26919031
8
.
.
Wang L., Tu Y. C., Lian T. W., Hung J. T., Yen J. H., & Wu M. J. (2006). Distinctive antioxidant and anti-
inflammatory effects of flavonols. Journal of Agricultural & Food Chemistry, 54(26), 9798–804.
9
Calder o´ n-Montaño J. M., Burgos-Mor o´ n E., P e´ rez-Guerrero C., & L o´ pez-L a´ zaro M. (2011). A review on
the dietary flavonoid kaempferol. Mini Reviews in Medicinal Chemistry, 11(4), 298. PMID: 21428901
1
0. Pei Jianjun, Chen Anna, Zhao Linguo, Cao Fuliang, Ding Gang, Xiao Wei, One-Pot Synthesis of
Hyperoside by a Three-Enzyme Cascade Using a UDP-Galactose Regeneration System. Journal of
Agricultural and Food Chemistry, 2017, 65: 6042–6048. https://doi.org/10.1021/acs.jafc.7b02320
PMID: 28660766
PLOS ONE | https://doi.org/10.1371/journal.pone.0197563 May 17, 2018
10 / 12

Kaempferol aglycone and glycosides’ biological activities and enzymatic preparation
1
1
1
1. Neuhouser M. L. (2004). Dietary flavonoids and cancer risk: evidence from human population studies.
Nutrition & Cancer, 50(1), 1.
2. Le M. L. (2002). Cancer preventive effects of flavonoids—a review. Biomedicine & pharmacotherapy =
Biomedecine & pharmacotherapie, 56(6), 296.
3. Yi X., Zuo J., Tan C., Xian S., Luo C., & Chen S., et al. (2016). Kaempferol, a flavonoid compound from-
gynura medicainduced apoptosis and growth inhibition in mcf-7 breast cancer cell. African Journal of
Traditional Complementary & Alternative Medicines, 13(4), 210–215.
14. Elsharkawy E. R. (2017). Isolation of phytoconstituents and evaluation of anticancer and antioxidant
potential of launaea mucronata (forssk.) muschl. subsp. Pakistan Journal of Pharmaceutical Sciences,
399–405. PMID: 28649063
1
5. Marfe G., Tafani M., Indelicato M., Sinibaldisalimei P., Reali V., & Pucci B., et al. (2009). Kaempferol
induces apoptosis in two different cell lines via akt inactivation, bax and sirt3 activation, and mitochon-
drial dysfunction. Journal of Cellular Biochemistry, 106(4), 643–650. https://doi.org/10.1002/jcb.22044
PMID: 19160423
1
6. Kim K. Y., Jang W. Y., Lee J. Y., Jun D. Y., Ko J. Y., & Yun Y. H., et al. (2015). Kaempferol activates g2-
checkpoint of the cell cycle resulting in g2-arrest and mitochondria-dependent apoptosis in human
acute leukemia jurkat t cells. Journal of Microbiology & Biotechnology, 26(2).
1
1
1
2
2
7. Kim B., Jung J. W., Jung J., Han Y., Dong H. S., & Kim H. S., et al. (2017). Pgc1α induced by reactive
oxygen species contributes to chemoresistance of ovarian cancer cells. Oncotarget.
8. L o´ pez-L a´ zaro M. (2010). A new view of carcinogenesis and an alternative approach to cancer therapy.
Molecular Medicine, 16(3–4), 144. https://doi.org/10.2119/molmed.2009.00162 PMID: 20062820
9. Verma A. R., Vijayakumar M., Mathela C. S., & Rao C. V. (2009). In vitro and in vivo antioxidant proper-
ties of different fractions of moringa oleifera leaves. Food & Chemical Toxicology, 47(9), 2196–201.
0. Mantovani A., Allavena P., Sica A., & Balkwill F. (2008). Cancer-related inflammation. Nature, 454
(7203), 436–44. https://doi.org/10.1038/nature07205 PMID: 18650914
1. Park M. J., Lee E. K., Heo H. S., Kim M. S., Sung B., & Kim M. K., et al. (2009). The anti-inflammatory
effect of kaempferol in aged kidney tissues: the involvement of nuclear factor-kappab via nuclear factor-
inducing kinase/ikappab kinase and mitogen-activated protein kinase pathways. Journal of Medicinal
Food, 12(2), 351–358. https://doi.org/10.1089/jmf.2008.0006 PMID: 19459737
2
2
2
2. Tang X. L., Liu J. X., Dong W., Li P., Li L., & Hou J. C., et al. (2015). Protective effect of kaempferol on
lps plus atp-induced inflammatory response in cardiac fibroblasts. Inflammation, 38(1), 94–101. https://
doi.org/10.1007/s10753-014-0011-2 PMID: 25189464
3. Tran M. H., Nguyen H. D., Kim J. C., Choi J. S., Lee H. K., & Min B. S. (2009). Phenolic glycosides from
alangium salviifolium leaves with inhibitory activity on lps-induced no, pge (2), and tnf-alpha production.
Bioorganic & Medicinal Chemistry Letters, 19(15), 4389–4393.
4. Muthukrishnan S. D., Kaliyaperumal A., & Subramaniyan A. (2015). Identification and determination
of flavonoids, carotenoids and chlorophyll concentration in cynodon dactylon (l.) by hplc analysis.
Natural Product Research, 29(8), 785. https://doi.org/10.1080/14786419.2014.986125 PMID:
2
5495959
2
5. Agar O. T., Dikmen M., Ozturk N., Yilmaz M. A., Temel H., & Turkmenoglu F. P. (2015). Comparative
studies on phenolic composition, antioxidant, wound healing and cytotoxic activities of selected achillea
l. species growing in turkey. Molecules, 20(10), 17976–8000. https://doi.org/10.3390/
molecules201017976 PMID: 26437391
2
+
2
2
2
6. Xie J, Zhao D, Zhao L, Pei J, Xiao W, & Ding G. (2015). Overexpression and characterization of a Ca
activated thermostable β-glucosidase with high ginsenoside Rb1 to ginsenoside 20(s)-Rg3 bioconver-
sion productivity. Journal of Industrial Microbiology, 42(6), 839–850.
7. Lin G, Chen A, Pei J, et al. Enhancing the thermostability of α-L-rhamnosidase from Aspergillus terreus,
and the enzymatic conversion of rutin to isoquercitrin by adding sorbitol[J]. Bmc Biotechnology, 2017,
17(1):21. https://doi.org/10.1186/s12896-017-0342-9 PMID: 28241810
8. Hou W, Zhang W, Chen G, et al. Optimization of Extraction Conditions for Maximal Phenolic,
Flavonoid and Antioxidant Activity from Melaleuca bracteata Leaves Using the Response Surface
Methodology[J]. Plos One, 2016, 11(9):e0162139. https://doi.org/10.1371/journal.pone.0162139
PMID: 27611576
2
9. Chen Y, Meng G, Bai W, et al. Aliskiren protects against myocardial ischemia-reperfusion injury via an
endothelial nitric oxide synthase dependent manner.[J]. Clinical & Experimental Pharmacology & Physi-
ology, 2017, 44(2).
30. Bognar E, Sarszegi Z, Szabo A, et al. Antioxidant and Anti-Inflammatory Effects in RAW264.7 Macro-
phages of Malvidin, a Major Red Wine Polyphenol[J]. Plos One, 2013, 8(6):e65355. https://doi.org/10.
1371/journal.pone.0065355 PMID: 23755222
PLOS ONE | https://doi.org/10.1371/journal.pone.0197563 May 17, 2018
11 / 12

Kaempferol aglycone and glycosides’ biological activities and enzymatic preparation
3
3
3
3
1. Pu X, Storr S J, Zhang Y, et al. Caspase-3 and caspase-8 expression in breast cancer: caspase-3 is
associated with survival[J]. Apoptosis An International Journal on Programmed Cell Death, 2017, 22
(3):357. https://doi.org/10.1007/s10495-016-1323-5 PMID: 27798717
2. Enari M., Sakahira H., Yokoyama H., Okawa K., Iwamatsu A., & Nagata S. (1998). A caspase-activated
dnase that degrades dna during apoptosis, and its inhibitor icad. Nature, 391(6662), 43. https://doi.org/
10.1038/34112 PMID: 9422506
3. Almasi E., Gharagozloo M., Eskandari N., Almasi A., & Sabzghabaee A. M. (2017). Inhibition of apopto-
sis and proliferation in t cells by immunosuppressive silymarine. Iranian Journal of Allergy Asthma &
Immunology, 16(2), 107.
4. Nam T. G., Lim T. G., Lee B. H., Lim S., Kang H., & Eom S. H., et al. (2017). Comparison of anti-inflam-
matory effects of flavonoid-rich common and tartary buckwheat sprout extracts in lipopolysaccharide-
stimulated raw 264.7 and peritoneal macrophages. Oxidative Medicine & Cellular Longevity, 2017(10),
1–12.
3
5. Xu Q., Liu M., Liu Q., Wang W., Du Y., & Yin H. (2017). The inhibition of lps-induced inflammation in
raw264.7 macrophages via the pi3k/akt pathway by highly n-acetylated chitooligosaccharide. Carbohy-
drate Polymers, 174.
PLOS ONE | https://doi.org/10.1371/journal.pone.0197563 May 17, 2018
12 / 12