Microbial biocatalysis of quercetin-3-glucoside and isorhamnetin-3-glucoside in: Salicornia herbacea and their contribution to improved anti-inflammatory activity

Article abstract of DOI:10.1039/c9ra08059g

Salicornia herbacea (glasswort) is a traditional Asian medicinal plant which exhibits multiple nutraceutical and pharmaceutical properties. Quercetin-3-glucoside and isorhamnetin-3-glucoside are the major flavonoid glycosides found in S. herbacea. Multipl

Full text of DOI:10.1039/c9ra08059g

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Microbial biocatalysis of quercetin-3-glucoside and
isorhamnetin-3-glucoside in Salicornia herbacea
and their contribution to improved anti-
inflammatory activity
Cite this: RSC Adv., 2020, 10, 5339
a
b
c
a
d
Hyung Jin Ahn, Hyun Ju You, Myeong Soo Park, Zhipeng Li, Deokyeong Choe,
d
d
ac
Tony Vaughn Johnston, Seockmo Ku * and Geun Eog Ji*
Salicornia herbacea (glasswort) is a traditional Asian medicinal plant which exhibits multiple nutraceutical
and pharmaceutical properties. Quercetin-3-glucoside and isorhamnetin-3-glucoside are the major
flavonoid glycosides found in S. herbacea. Multiple researchers have shown that flavonoid glycosides can
be structurally transformed into minor aglycone molecules, which play a significant role in exerting
physiological responses in vivo. However, minor aglycone molecule levels in S. herbacea are very low. In
this study, Bifidobacterium animalis subsp. lactis AD011, isolated from infant feces, catalyzed >85% of
quercetin-3-glucoside and isorhamnetin-3-glucoside into quercetin and isorhamnetin, respectively, in
h, without breaking down flavonoid backbones. Functionality analysis demonstrated that the quercetin
and isorhamnetin produced showed improved anti-inflammatory activity vs. the original source
molecules against lipopolysaccharide induced RAW 264.7 macrophages. Our report highlights a novel
protocol for rapid quercetin and isorhamnetin production from S. herbacea flavonoids and the
applicability of quercetin and isorhamnetin as nutraceutical molecules with enhanced anti-inflammatory
properties.
2
Received 4th October 2019
Accepted 9th January 2020
DOI: 10.1039/c9ra08059g
rsc.li/rsc-advances
1,2,5,6,15–17

avonoids, minerals, and polysaccharides.
Among
Introduction
them, avonol glycosides, especially quercetin-3-glucoside
Salicornia herbacea L., also known as glasswort, is widely (Q3G) and isorhamnetin-3-glucoside (IR3G), have been regar-
distributed in saline soil areas such as the seashore, foreshore, ded as the principle substances responsible for the observed
1
8,19
and salt lakes in Korea, Japan, Iran, the United States, and biofunctional activities.
Q3G is a member of a group of
1–5
European countries. As an herbal halophyte, S. herbacea has avonoids that have a glucose moiety at position 3; the aglycone
been used as a folk medicine in Asian countries for the treat- form is known as quercetin (Q). Quercetin is a representative
ment of constipation, diabetes, nephropathy, hepatitis, and avonol and has a 2-phenyl chromen-4-one backbone and
Multiple scholars have reported that S. herbacea a double bond between carbons 2 and 3 with ve hydroxy
exhibits multiple bioactive functionalities, including antioxi- groups at positions 3, 5, 7, 3 , 4 . Isorhamnetin has the same
2,3,6
diarrhea.
0
0
20
dant, anticancer, anti-inammatory, osteoblastogenesis, anti- backbone as quercetin and isorhamnetin is the methylated
5
–14
0
hyperglycemic, and anti-hyperlipidemic effects.
herbacea has been widely cultivated and processed in various position 3 (Fig. 1).
forms as commercially available functional cosmetics, dietary
Research has shown that the specic makeup of each indi-
supplements and medicines due to its proven health benets vidual microbiome affects the range of catalysis occurring in
Recently, S. form of quercetin at position 3 . IR3G has a glucose moiety in
21
1,3
22
23
and marketing strengths. Previous phytochemical studies each individual. Hasegawa reported that the pathways
revealed that S. herbacea contains bioactive molecules such as employed in in vivo conversion of glycosides may be different
due to the diversity of microorganisms present in each host's
gut. Therefore, orally consumed avonoid molecules are cata-
lysed and structurally transformed into deglycosylated avo-
a
Department of Food and Nutrition, Research Institute of Human Ecology, Seoul
National University, Seoul 08826, Republic of Korea. E-mail: geji@snu.ac.kr
b
noid forms (aglycones) by gut microbiota and their
Center for Human and Environmental Microbiome, Institute of Health and
24
glycosidases. Flavonoids in the form of aglycones are known to
Environment, Seoul 08826, Republic of Korea
c
Research Center, BIFIDO Co., Ltd., Hongcheon 25117, Republic of Korea
be more efficiently transferred into the bloodstream from the
d
Fermentation Science Program, School of Agriculture, College of Basic and Applied intestinal tract and more effectively act as bioactive molecules
Sciences, Middle Tennessee State University, Murfreesboro, TN 37132, USA. E-mail:
25–27
than the avonol glycosides from which they are produced.
seockmo.ku@mtsu.edu
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shaking at 120 rpm for 4 h. S. herbacea particles were ltered out
using 0.45 mm microlters. The collected permeates were
evaporated and fractionated by dichloromethane, n-hexane,
and n-butanol (1 L each with the same volume of water) to
18
remove chlorophyll, lipids, proteins, and sugars. The insol-
uble residue of the n-butanol fraction was removed by ltration
(Whatman® Grade 3, Z240478, Maidstone, England) and the
collected ltrate was evaporated using an Eyela N-100 rotary
evaporator (Rikakikai Co. Ltd., Tokyo, Japan) and dissolved in
500 mL methanol. A portion of the methanol suspended n-
butanol residue was fractionated by loading 5 mL onto an
activated SPE® Vac C18 column (Sep-Pak, WAT054945, Waters
Corp, Milford, MA, USA) and eluting with a sequence of water,
methanol/water 40–60% (v/v), and 100% methanol. The 100%
methanol fraction (the avonoid-enriched fraction) was evapo-
rated and resuspended in water for biotransformation.
Fig. 1 Biotransformation pathways from quercetin-3-glucoside and
isorhamnetin-3-glucoside to quercetin and isorhamnetin by b-
glucosidase, respectively.
Preparation of microbial crude enzyme and b-D-glucosidase
assay
Glucose is the most common sugar moiety of avonol glyco-
sides in plants, and the b-glucosidic bonds of Q3G and IR3G are
The following probiotic strains were generously donated by
BIFIDO LTD (Hongcheon, Korea): Lactobacillus delbrueckii sp.
delbrueckii KCTC 1047 (LD1047), Lactobacillus delbrueckii subsp.
bulgaricus KCTC3188 (LD3188), Lactobacillus cacei KCTC 3109
24,28–31
possibly deconjugated via b-glucosidase catalysis.
This
catalysis would produce quercetin and isorhamnetin (Fig. 1).
3
2
33,34
Certain microbial strains, such as Bacteroides, Clostridium,
and Eubacterium,
35,36
are capable of structurally transforming
(
LC3109), Bidobacterium adolescentis Int57 (Int57), Leuconostoc
paramesenteroides KCTC 3531 (LP3531), Bidobacterium sp. SJ32
SJ32), Bidobacterium infantis KCTC 3249 (BI 3249), Bido-

avonol glycosides into functional aglycones. However, many of
these microbes are not suitable for food processing applications
due to safety and marketing concerns. Also, many of the
previously mentioned studies were limited to the biocatalysis of
soybean isoavones. The principle objective of this research
was, therefore, to develop effective quercetin and isorhamnetin
production using biotransformation of Q3G and IR3G in S.
herbacea via probiotic enzyme catalysis. We screened potential
probiotic strains, obtained b-glucosidase from selected cell
strains, and evaluated their catalytic substrate transformation
capabilities. Aer quercetin and isorhamnetin production, we
conducted qualitative (liquid chromatography/mass spectrom-
etry [LC/MS] and cell inammatory assays) and quantitative
(
bacterium sp. SH5 (SH5), Bidobacterium bidum BGN4 (BGN4),
and Bidobacterium animalis subsp. lactis AD011 (AD011). All
bacteria were grown in Lactobacilli MRS medium (Becton-
Dickinson Company, Detroit, MI, USA), supplemented with
ꢀ
0
.05% (w/v) L-cysteine$HCl at 37 C under anaerobic conditions
for 18 h, and subcultured in 50 mL of MRS medium. Cultured
probiotic cells were collected via centrifugation (3000 ꢂ g for
ꢀ
ꢁ1
3
0 min at 4 C). The collected cells (8 log CFU mL ) were
washed twice with 50 mM phosphate buffer (PB, pH 6.0) and
resuspended in 10 mL of phosphate buffer. To separate the
intracellular enzymes from the cytosol, all cells were disrupted
(high-performance liquid chromatography [HPLC]) analyses to
via sonication (Sonics and Materials, Inc., Newtown, CT, USA) at
demonstrate the practicality of our method, which is potentially
applicable to commercial quercetin and isorhamnetin produc-
tion. Commercial production of quercetin and isorhamnetin via
probiotic bacteria could lead to the development of treatments
for inammation-associated diseases.
ꢀ
4
C with 1 s burst pulse and 1 s cooling intervals at an ampli-
31
tude of 45 for 30 min, as in our previous study.
b-D-Glucosidase activity in the disrupted cell suspensions of
all ten probiotic strains were assayed by the degradation of
articial substrates to produce free p-nitrophenols. Para-nitro-
phenyl-b-D-glucopyranoside (Sigma-Aldrich, #N7006, St. Louis,
MO, USA) was used as an articial substrate. Twenty mL of crude
enzyme extract and 20 mL of 5 mM p-nitrophenyl-b-D-glucopyr-
anoside were combined with 60 mL of PB (0.02 M, pH 6.0) in
Experimental
Extraction of quercetin-3-glucoside and isorhamnetin-3-
glucoside from S. herbacea
each well of a 96 well plate (SPL, #32296, Pocheon, Korea). The
ꢀ
S. herbacea powder was generously donated by Daeshinhamcho mixtures were incubated at 37 C for 8 min, shaking at 100 rpm.
company (Sinan, Korea). S. herbacea was cultivated from March The enzyme reaction was stopped by adding 100 mL of 0.5 M
to July and harvested at the seashore of Sinan-gun in Korea. Na
2
CO
3
, and the released p-nitrophenol was measured with a 96
ꢀ
Harvested S. herbacea was heat-dried (60 C, 30 h) or freeze- well microplate reader (Bio-Rad Laboratories, Philadelphia, PA,
dried (ꢁ65 ꢀC, in vacuo, 30 h), and ground. To extract Q3G USA) at 405 nm. To obtain the specic activity, enzyme activity
and IR3G, water, 70% methanol, or 100% methanol were used was divided by mg of protein. The level of protein was evaluated
as solvents. One hundred grams of S. herbacea powder was via a BCA protein Assay Kit (Pierce™, CAT 23225, Waltham,
ꢀ
added to 1 L of each solvent and held at 80 C in a water bath USA).
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Biotransformation of quercetin-3-glucoside and
In order to obtain isorhamnetin and quercetin from S. her-
bacea extracts, enzyme-treated samples were subjected to
enzyme inactivation via heat treatment for 10 min at 100 C,
isorhamnetin-3-glucoside from S. herbacea using microbial
crude enzyme extract and preparation of isorhamnetin-3-
glucoside, quercetin-3-glucoside, isorhamnetin and quercetin
ꢀ
followed by freeze-drying. The dried samples were dissolved in
1
00% methanol and used to separate isorhamnetin and quer-
Biotransformation of Q3G and IR3G was conducted based on
24,28–30,37–39
cetin fractions via semi-preparative HPLC. The preparative
HPLC (Young Lin Acme 9000, Younglin Instrument Co., Ltd.,
Anyang, Korea), equipped with a semi-preparative ZORBOX SB-
C18 5 mm, 9.4 mm ꢂ 250 mm column (Agilent Technologies,
Santa Clara, CA, USA), was utilized. The mobile phase consisted
of solvent A (0.1% (v/v) triuoroacetic acid in HPLC grade water,
pH 2.5) and solvent B (methanol) with the following gradient: 0–
our previous study.
Crude S. herbacea extracts were
resuspended in a 60 mL of 50 mM PB (pH 6.0), mixed with 940 mL
ꢀ
of microbial crude enzyme extract, and incubated at 37 C in
a water bath shaking at 150 rpm (0, 0.5, 1, 1.5, 2, 8, 16, 36 h).
ꢀ
Aer biotransformation, mixtures were boiled (100 C) for ten
minutes to terminate enzyme reaction. Finally, mixtures were
freeze-dried and resuspended in MeOH for analysis. The
biotransformation percentage was calculated by the following
2
0 min, linear gradient from 65% to 75% B; 20–35 min, 75–80%
37,40
B, 35–50 min, 80–100% B. The injection volume of the samples
formula:
ꢁ
1
was 1 mL, the ow rate was 5 mL min , and the absorbance
was measured using a UV detector (UV VIS detector, Younglin
Instrument Co., Ltd., Anyang, Korea) at a wavelength of 254 and
Conversion percentage of quercetin (or isorhamnetin) content (%)
¼
(transformed content of quercetin (or isorhamnetin)
after biotransformation at given incubation time/
3
70 nm. Q3G and IR3G were extracted by the same method as
above but the S. herbacea extracts were not treated with micro-
bial enzyme. Each collected Q3G, quercetin, IR3G, and iso-
rhamnetin in HPLC solvent (water and methanol) were
evaporated using an Eyela rotary evaporator N-100 (Rikakikai
Co., Ltd., Tokyo, Japan) and used for evaluation of anti-
inammatory effects.
(
residual content of Q3G (or IR3G) + transformed
content of quercetin (or isorhamnetin) after
biotransformation at given incubation time)) ꢂ 100.
Analysis of quercetin-3-glucoside, isorhamnetin-3-glucoside,
quercetin, and isorhamnetin using chromatographic methods
Evaluation of anti-inammatory effects of quercetin-3-
glucoside, isorhamnetin-3-glucoside, quercetin, and
isorhamnetin
Q3G, quercetin, and isorhamnetin standards were purchased
from Sigma-Aldrich and an IR3G standard was purchased from
Extrasynthese (Lyon, France). Stock solutions of each standard The anti-inammatory effects of Q3G, quercetin, IR3G, and
compound and lyophilized samples were dissolved in meth- isorhamnetin were evaluated by measuring the production of
anol, ltered through 0.45 mm syringe lters (Pall, Ann Arbor, proinammatory cytokines TNF-a and IL-6 in LPS-induced RAW
MI, USA), and used for HPLC analysis. The separation and 264.7 cell line cells using commercially available enzyme-linked
measurement of avonoids were performed on a Dionex P680 immunosorbent assay (ELISA) kits. RAW 264.7 cells (KCLB
HPLC (Dionex Corporation, Sunnyvale, CA, USA) equipped with 40071) were obtained from the Korean Cell Line Bank (Seoul,
an ASI-100 auto sampler (Dionex Corporation, Sunnyvale, CA, Korea). These cells were maintained and subcultured according
USA) and a UVD 170 UV-vis detector (Dionex Corporation, to the distributor's instruction. Briey, these cells were cultured
Sunnyvale, CA, USA). A Waters Sunre C18 column (4.6 mm ꢂ in Dulbecco's modied Eagle's medium (GIBCO, 12491-023,
150 mm, 3.5 mm particle size, Waters Corporation, Milford, MA, Carlsbad, CA, USA) with 10% (v/v) fetal bovine serum (GIBCO,
USA) and a TCC-100 thermostatically controlled column 12483-020, Carlsbad, CA, USA) and 1% (v/v) antibiotic-
compartment (Dionex Corporation, Sunnyvale, CA, USA) were antimycotic solution (GIBCO, R25005, Carlsbad, CA, USA) at
ꢀ
ꢀ
37
used; the column was maintained at 30 C during the analysis. 37 C in a humidied atmosphere of 95% air and 5% CO
2
.
The mobile phase consisted of solvent A (0.1% [v/v] triuoro- To evaluate anti-inammatory effects, RAW 264.7 cells were
4
acetic acid [TFA] in water, pH 2.5) and solvent B (acetonitrile). seeded at 1 ꢂ 10 cells per well in a 96-well plate and incubated
ꢀ
The gradient program was: 0–5 min, 10% B; 5–45 min, linear at 37 C for 22 h. The cells were then treated and incubated with
gradient from 10% to 40% B. The injection volume of standards 1, 5, or 10 mM of Q3G, quercetin, IR3G, or isorhamnetin for 2 h.
ꢁ1
ꢁ1
and samples was 20 mL, and the ow rate was 0.8 mL min
Simultaneous detection at 254 nm and 370 nm was the plate again incubated for 24 h. Aer incubation, the levels of
accomplished. TNF-a and IL-6 in 100 mL of each cell supernatant were
.
LPS (0.1 mg mL ) was added to each cell in the 96-well plate and
Sample molecular weights were determined and compared measured using ELISA kits (BD OptEIA™ Mouse TNF ELISA Kit,
with standard compounds using an Agilent 6410A triple quad- 560478, BD Pharmingen, San Diego, Calif., USA and BD
rupole LC-MS system (Agilent Technologies, MA, USA) equipped OptEIA™ Mouse IL-6 ELISA Kit, 550950, BD Pharmingen, San
with a Waters (Milford, MA, USA) Sunre C18 column (150 mm Diego, Calif., USA) according to the manufacturer's protocols.
ꢂ 4.6 mm, 3.5 mm particle size). Mass spectrometric analysis
The cytotoxicity of Q3G, quercetin, IR3G, and isorhamnetin
was carried out at the Central Laboratory for Instrumental was evaluated by MTT assay. In brief, RAW 264.7 cells were
4
Analysis at Kyung Hee University's Global Campus (Yongin, seeded at 5 ꢂ 10 cells per well in a 96-well plate (Corning® 96
ꢀ
South Korea).
Well, #3596, Corning, NY, USA) and incubated at 37 C for 22 h.
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Either 1, 5, or 10 mM of Q3G, quercetin, IR3G or isorhamnetin bond, which makes it slightly more hydrophilic than iso-
was added to each cell and the plate was again incubated for 2 h. rhamnetin. Traditionally, various organic solvents (e.g. meth-
ꢁ1
LPS (0.1 mg mL ) (Sigma-Aldrich, L4516, St. Louis, MO, USA) anol, ethanol, butanol and chloroform) or water have been used
was added to each of the 96 cells and the plate was incubated for when extracting avonoids from plants. Among these organic
2
4 h. Aer this incubation, a 10% (v/v) MTT stock solution (5 mg solvents, methanol has a polarity of 6.6, which is known to be
ꢁ1
ꢀ
mL ) was added to each well, followed by incubation at 37 C higher than the other organic solvents and possibly has a high
for 2 h. Aer centrifugation at 100g for 5 min at 4 C, the affinity with quercetin or isorhamnetin. Due to the structural
ꢀ
supernatants were removed. The converted formazan product properties of Q3G and IR3G with their low-polarity, organic
was dissolved in 200 mL of dimethyl sulfoxide (DMSO) and the solvents or aqueous-based methanol solutions are normally
43,44
absorbance was measured at 540 nm using a microplate reader used for their extraction from plant materials.
Bio Rad Laboratories, Inc., Hercules, CA, USA). The percentage quercetin-3-glucose and isorhamnetin-3-glucose have a glucose
of viable cells was estimated compared with that of the moiety with hydrophilic properties in common. Therefore, we
Both
(
untreated control cells.
used methanol and aqueous-based solvents (water and 70%
methanol) to extract quercetin-3-glucose and isorhamnetin-3-
glucose from S. herbacea.
As a result, the extraction efficiency of Q3G using methanol
as a solvent was 3.9–4.2 times higher than that using water as
a solvent and 1.1–1.2 times higher than using 70% methanol as
a solvent. Additionally, the extraction efficiency of IR3G using
Results and discussion
The effect of drying methods and extraction conditions on the
quantity of quercetin-3-glucoside and isorhamnetin-3-
glucoside extracted from S. herbacea
19
18
0
Several kinds of avonoids (IR3G, Q3G, 2S-2 ,7-dihydroxy-6- methanol was 6.5–6.7 times higher than with water extraction
0
methoxyavanone, 2S-2 -hydroxy-6,7-dimethoxy-avanone, and and 1.2 times higher than 70% methanol extraction (Table 1).
0
2
2
S-5,2 -dihydroxy-6,7-methylenedioxyavanone ) have been re-
The drying of S. herbacea before extraction is the most
ported to exist in S. herbacea, and these avonoids have been important step for increasing the yield of Q3G and IR3G.
separated and identied using spectroscopic methods such as Traditionally, Korean people have used sunlight to dry a variety
HPLC, MS, and NMR. Among them, Q3G and IR3G have been of vegetables and plant medicines (e.g. red peppers, radish and
18,19
regarded as the key bioactive molecules of S. herbacea.
ginseng). For example, the quality and price of sun-dried red
However, the bioavailabilities of these two avonoid substances pepper is higher than red pepper dried via other techniques.
vary in vivo depending on the presence of a sugar residue. Many Korean food companies market red pepper and red
Flavonol aglycones do not have sugar residues and are known to pepper-containing products such as kimchi and pepper paste
have better functional properties than those of avonol glyco- using the term, “sun-dried” to highlight this processing tech-
41,42
sides.
glycosides are not yet standardized. Because the major goal of are exposed to UV radiation, their physical structures change.
our study was to obtain and assess biotransformed products Flavonoids may also be structurally changed by enzymes in
quercetin and isorhamnetin) via enzymatic catalysis, we plant cells, endophytic microorganisms, high temperatures,
Extraction and separation protocols for these avonol nique. However, it has been reported that when plant avonoids
45
(
needed to develop effective extraction and separation protocols and oxidative stress during the drying process. Therefore,
of mother molecules (Q3G and IR3G, respectively) in S. herba- natural drying is not the best technique for the preservation of
46–49
cea. We therefore compared the efficiency of using a combina- avonoid content in natural foods.
To compare the quantity
tion of two drying methods (heat and freeze-drying) and three of Q3G and IR3G in S. herbacea aer drying by heat or freeze-
solvents (water, 70% MeOH and 100% MeOH) for extraction. All drying, samples were dried using both methods and extrac-
extracted molecules were quantitatively and qualitatively ana- tion of the target avonoids was executed using the previously
lysed by HPLC and LC/MS.
described technique (water, 70% methanol and methanol at
ꢀ
Both quercetin and isorhamnetin have avonoid structures 80 C for 4 h incubation). The quantity of Q3G remaining aer
in which two phenyl groups are structurally linked by three freeze-drying was about 1.5 times higher than the quantity of
carbon bridges which form an aromatic ring in a closed struc- Q3G remaining aer heat-drying. Similarly, the quantity of IR3G
ture, and both molecules have double bonds on carbon aer freeze-drying was about 1.9 times higher than the quantity
numbers 2 and 3. The aromatic part of avonol molecules show of IR3G remaining aer heat-drying. In light of these observa-
hydrophobic properties. However, the avonols, quercetin and tions and using this general strategy, the effective Q3G and
isorhamnetin, also have hydroxyl groups at 3, 5, 7, 3 , 4 and IR3G extraction from S. herbacea may be optimized based on the
exhibit hydrophilic properties. Q3G is the conjugated form of relative polarities of target compounds.
carbon number 3 of quercetin and carbon number 1 of glucose
0
0
with a b-glycosidic linkage. The presence of glucose in Q3G
conferred more hydrophilic properties compared to quercetin.
b-Glucosidase screening from probiotic microorganisms
Isorhamnetin has a avonol backbone structurally similar to Probiotics have been widely used to produce glycosidases for
quercetin. However, isorhamnetin, unlike quercetin, has transforming certain phytochemical glycosides, including gin-
0
a methyl group instead of OH at the 3 carbon of the aromatic senosides, isoavones, anthocyanins, and avonols. Probiotic
ring and is therefore slightly more hydrophobic than quercetin. enzymatic biotransformation is widely used to transform
The glucose molecule in IR3G is also linked via a b-glucoside phytochemicals because of the many advantages it offers,
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Table 1 Comparison of quercetin-3-glucoside and IR3G content of S. herbacea extract after heat-drying, and freeze-drying, using different
a
solvents
Extraction solution
Contents
Molecules
Drying methods
Water
70% methanol
100% methanol
ꢁ
1
b
c
b
a
Concentration (mg mL
)
Q3G
Heat-drying
Freeze-drying
Heat-drying
Freeze-drying
10.4 ꢃ 0.31
33.1 ꢃ 0.8
40.5 ꢃ 1.4
c
b
a
14.3 ꢃ 0.2
56.6 ꢃ 0.8
60.4 ꢃ 0.8
c
c
b
a
IR3G
7.8 ꢃ 0.0
42.7 ꢃ 0.0
52.0 ꢃ 0.3
c
b
a
15.4 ꢃ 0.3
81.0 ꢃ 1.1
100.0 ꢃ 2.0
a
Extractions were replicated three times and all values are presented as mean ꢃ SD (n ¼ 3). Different superscripts within the same rows indicate
b
c
that values are signicantly different at p < 0.05 (Tukey HSD and Games-Howell tests). Q3G denotes quercetin-3-glucoside. IR3G denotes
isorhamnetin-3-glucoside.
ꢁ
1
ꢁ1
including low cost, easy reaction control and stereospecicity ꢃ 0.6 mmol pNP (min mL) (mg of protein) . The total b-D-
vs. the results generated using chemical or thermal processing glucosidase activities of ve probiotic extracts (LD1047, LD3188,
2
4,28–30,50,51
techniques.
Highly region-specic enzymatic trans- LC3109, SJ32, and BGN4) were shown to be less than 5 mmol
ꢁ1
formations may be promising, but preparing for the purica- pNP (min mL) , which is signicantly lower than other groups.
tion of one specic enzyme is expensive in terms of time and The total b-D-glucosidase activities of the other ve probiotic
money. To save both time and cost, crude enzyme extracts or strains (Int57, LP3531, BI3249, SH5, and AD011) ranged from
fermentation may be used. Although these methods have 21.7 ꢃ 0.5 to 88.4 ꢃ 1.1 mmol pNP (min mL) , with b-glucosi-
signicant advantages in creating target substances, the trans- dase activity levels higher than those of the other ve probiotics.
formation should be carefully controlled since several enzymes Thus, crude enzyme extracts of Int57, LP3531, BI3249, SH5, and
exist in crude enzyme extracts. Crude enzyme extracts can also AD011 were selected to transform Q3G and IR3G from S.
include enzymes that degrade target substance(s) or create herbacea.
other forms of the substance(s). Finally, if microorganisms are
ꢁ
1
20
used to supply the enzymes, suitable specic organisms must
52,53
be identied to accomplish the desired end product(s).
Biotransformation of quercetin-3-glucoside and isorhamnetin-
In order to nd a microorganism suitable for providing the 3-glucoside into quercetin and isorhamnetin
enzymes required to transform Q3G and IR3G, we screened 10
Qualitative analysis via HPLC was performed to evaluate the
food-grade microorganisms and evaluated their b-glucosidase
activities based on their ability to cleave p-nitrophenyl-b-D-glu-
copyranoside into p-nitrophenol and glucose. p-Nitrophenol
conversion percentage of natural substrates (Q3G and IR3G)
during biotransformation by b-glucosidase from Int57, LP3531,
BI3249, SH5, AD011. Specically, the overall level of substrate
and end product were enumerated so we could measure the
amount of substrate remaining that could be biotransformed to
aglycones. When the conversion percentage was predicted
using articial substrate (pNP), AD011 showed a conversion
(
4
(
pNP), an articial substrate, can be enumerated by detection at
50 nm, allowing for a measurement of b-glucosidase activity
Fig. 2).
The total activities of the b-D-glucosidase within each crude
enzyme extract ranged from 2.8 ꢃ 0.5 to 88.4 ꢃ 1.1 mmol pNP
th
percentage that ranked 4 among the 5 strains. In the experi-
ꢁ1
(
min mL) , and the specic activities of the b-D-glucosidase
ment with natural substrates, AD011 showed the highest
glycoside catalytic activities against natural substrates among
the ve selected microbial strains (Table 2).
within each crude enzyme extract ranged from 0.5 ꢃ 0.0 to 17.0
AD011 transformed 88.4 ꢃ 1.1% of Q3G to quercetin. The
conversion percentage from the other four samples ranged from
4
.5 ꢃ 0.8 to 38.1 ꢃ 0.8%. Also, the transformation percentage of
IR3G to isorhamnetin aer 8 h was 93.3 ꢃ 0.4% when the same
crude enzymes extracted from AD011 were applied, while the
transformation percentage of IR3G from the other four samples
ranged from 0.7 ꢃ 0.0 to 47.0 ꢃ 2.3% (n ¼ 3) aer 8 h incuba-
tion. In the case of Int57 and BI3249, the total b-glucosidase
activities of these microorganisms against pNP were more than
2
times higher than the total b-glucosidase activities of AD011.
However, the transformation percentages of Q3G and IR3G of
Int57 and BI3249 were signicantly lower than that of AD011.
Although p-nitrophenol, quercetin, and isorhamnetin are
structurally homologous (they are all linked to glucose by b-1,4
glucosidic linkages), different conformations of substrates
Fig. 2 Total and specific b-D-glucosidase activities of 10 probiotic cell
strains. One-way ANOVA followed by Games-Howell post hoc test
was performed. Treatments with different letters are significantly
different at p < 0.05 (n ¼ 3).
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Table 2 The residual flavonol glycoside (Q3G [quercetin-3-glucoside] and IR3G [isorhamnetin-3-glucoside]) and aglycone (Q [quercetin] and IR
a
[
isorhamnetin]) contents after 8 h of reaction with crude extracts from five lactic acid bacteria strains
Microorganisms
No.
Molecules (mM)
Control
Int57
LP3531
BI3249
SH5
AD011
b
a
d
a
a
e
a
d
c
d
d
c
e
c
b
d
c
bc
c
d
a
a
d
a
a
1
2
3
4
5
6
Q3G
Q
107.8 ꢃ 0.9
5.8 ꢃ 1.0
17.7 ꢃ 4.0
42.5 ꢃ 0.9
60.3 ꢃ 4.9
6.7 ꢃ 1.1
31.8 ꢃ 3.4
6.3 ꢃ 0.3
38.1 ꢃ 3.4
58.2 ꢃ 5.5
1.2 ꢃ 0.0
59.5 ꢃ 5.6
47.6 ꢃ 6.9
5.0 ꢃ 0.9
52.6 ꢃ 7.1
86.1 ꢃ 5.4
3.4 ꢃ 0.0
89.5 ꢃ 5.4
38.4 ꢃ 5.4
13.3 ꢃ 4.3
98.6 ꢃ 3.1
112.0 ꢃ 3.4
12.3 ꢃ 3.2
168.5 ꢃ 0.6
180.8 ꢃ 2.7
c
b
33.5 ꢃ 2.6
71.9 ꢃ 7.9
68.9 ꢃ 6.9
47.0 ꢃ 2.9
115.9 ꢃ 9.8
bc
d
b
Total (sum.)
113.7 ꢃ 1.9
179.2 ꢃ 1.2
1.1 ꢃ 0.0
d
b
d
b
bc
c
IR3G
e
b
IR
84.9 ꢃ 4.1
91.6 ꢃ 5.1
b
b
Total (sum.)
180.2 ꢃ 1.2
a
Values with different superscripts within the same rows are signicantly different at p < 0.05 (Tukey HSD and Games-Howell tests) and mean ꢃ SD
b
c
d
e
(
n ¼ 3). Q3G denotes quercetin-3-glucoside. IR3G denotes isorhamnetin-3-glucoside. Q denotes quercetin. IR denotes isorhamnetin.
might affect binding times and/or activation energies could be (52.2% and 23.5% of molar conversion yield), respectively. Our
different. b-glucosidase screening results indicate that crude enzyme
When S. herbacea extracts were treated with crude enzymes preparations from AD011 produced the best biotransformation
from Int57, LP3531, BI3249 and SH5, the overall level of avonol percentages and the lowest degradation percentages for trans-
molecules (i.e. Q3G, quercetin, IR3G and isorhamnetin) were forming Q3G and IR3G (Table 2). Crude enzyme extracts of
signicantly decreased. However, quantitative assessment aer AD011 were therefore chosen for further experimentation over
bioconversion showed that the total amount of Q3G and quer- longer exposure times. Before and aer transformation, Q3G,
cetin in the AD011-administered group was not statistically IR3G, quercetin, and isorhamnetin were quantied by HPLC
different from the total Q3G and quercetin amount in the and qualied by LC/MS in the crude enzyme extracts using
control group in which no microorganism was administered. Electrospray Ionization (ESI)-Triple Quadrupole LC/MS. A total
The total amount of IR3G and isorhamnetin in the AD011- of 89.4 ꢃ 0.8% of Q3G was converted into quercetin and 92.7 ꢃ
administered group was also not statistically changed aer 1.4% of isorhamnetin-3-glucoside was converted into iso-
biotransformation process. The LP3531 treatment group rhamnetin in 8 h with a crude enzyme extract of AD011.
showed the highest degradation percentage during trans-
formation of Q3G to quercetin and IR3G to isorhamnetin, with microbial biotransformation processes, it is important to
6.5 ꢃ 2.5% (n ¼ 3) and 67.1 ꢃ 3.2% (n ¼ 3), respectively. Int57, develop effective protocols to generate the target molecule(s)
BI3249 and SH5 treated groups showed 36.8 ꢃ 6.0 to 53.8 ꢃ without generating unwanted by-products, which complicate
When producing minor aglycones from herb extracts via
6
5
.5% (n ¼ 3) degradation percentage for Q3G transformation downstream processing. Crude enzymes can be dened as
and 35.7 ꢃ 5.5 to 50.3 ꢃ 3.0% (n ¼ 3) degradation percentage for complex organic mixtures containing enzymes and other
IR3G transformation. It is apparent that the crude enzyme from cellular materials produced via cell/microbial lysis and
AD011 selectively transformed Q3G and IR3G into quercetin breakage. When avonoid molecules are biotransformed using
and isorhamnetin, respectively. However, other enzymes from crude enzyme extracts from bacteria, these unfractionated
the other four microorganisms may transform and degrade all organic complexes may contain key enzymes that are essential
four S. herbacea avonoids into other compounds nonspeci- for avonoid bioconversion. However, some molecules in
54
cally by modifying their backbones or functional groups.
bacterial lysate can act as enzyme inhibitors and/or inhibitor
producers via glycosylation, oxidation, sulphation, methylation,
hydroxylation, and aromatic ring degradation due to the lack of
Evaluation of Bidobacterium animalis subsp. lactis AD011
biotransformation properties
54
enzyme purication. From the producers' point of view, these
unintended chemical reactions can contribute to the produc-
tion of unwanted by-products, leading to increased production
costs, reduced target product recovery, and difficult down-
stream processing.
For example, Xu et al. attempted to produce Q3G through
the microbial biotransformation of quercetin with Gliocladium
deliquescens NRRL 1086 and produced their target substance via
When producing target molecules via biotransformation, not
only the yield of the target molecule but also the conversion
55
time should be considered. Guan et al. reported that when
a crude enzyme extract of Rhodopseudomonas palustris was
utilized for rutin conversion, the conversion percentage was
only 2.68% and 81.09% aer 5 and 21 h of catalytic reaction,
respectively. According to Quan et al., 12 h of Microbacterium
esteraromaticum-derived b-glycosidase incubation was applied
to remove the glucose moiety of ginsenoside Rb2 and generate
compound Y. Compound K was then sequentially generated by
further removing the arabinose moiety of compound Y.
57
56
3-O-glycosylation. However, they reported that when the glyco-
sylation reaction occurred, an oxidation cleavage reaction of the
C-ring occurred simultaneously. As a result, unwanted by-
products (e.g. 2-protocatechuoly-phloroglucinol carboxylic
acid) were generated in addition to Q3G, and 2-protocatechuoly-
phloroglucinol carboxylic acids were chemically decomposed
ꢁ
ꢁ1
1
Specically, 0.74 mg mL of Rb2 was converted to 0.27 mg
mL and 0.1 mg mL of compound Y and compound K
ꢁ1
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into 2,4,6-trihydroxybenzoic acid and protocatechuic acid aer
biocatalysis. Because of these additional reactions, they were
only able to achieve 46% of the possible Q3G aer 12 h of
58
reaction. According to Krishnamurty et al., rutin, a precursor
of quercetin, was successfully converted to quercetin by
detachment of glucose and rhamnose molecules in rutin by
Butryrivibrio sp. crude enzymes. However, in their research
quercetin was further degraded to phloroglucinol, CO
2
, 3,4-
dihydroxybenzaldehyde due to additional enzyme reaction. Oka
59
and Simpson also reported that multiple unwanted by prod-
ucts, including carbon dioxide and 2-protocatechuoly-
phloroglucinol carboxylic acid, are produced by enzymatic
oxygenation and quercetinase in quercetin biosynthesis using
35
Aspergillus avus. According to Schneider et al., growing
Clostridium orbiscindens degraded 0.5 mM quercetin and
structurally converted it to 3,4-dihydroxyphenylacetic acid in
60
6
h. Braune et al. reported that Eubacterium ramulus and its
enzymes converted quercetin to 3,4-dihydroxyphenylacetic acid
through taxifolin and alphitonin as intermediates with the
reduction of the double bond at the 2,3-position and C-ring
Fig. 4 Concentrations of (A) quercetin-3-glucoside (:) and quercetin
(O) and (B) isorhamnetin-3-glucoside (A) and isorhamnetin (>) in S.
herbacea extracts during 36 h incubation with crude AD011 b-

ssion. In order to avoid unwanted enzyme reactions with glucosidase.
avonoid degradation and maximize quercetin and iso-

rhamnetin productivities, proper enzyme host selection is
crucial. Instead of host selection, separation of enzymes or transformation; Q3G reached 92.6 ꢃ 0.4% and IR3G reached
proteins which catalyze unwanted reactions can be conducted, 95.5 ꢃ 0.4% aer 36 h. No structural degradation of the four
but purication of specic enzymes is a time consuming and avonoids (Q3G, IR3G, quercetin, and isorhamnetin) was
expensive process. Therefore, using less puried crude enzyme observed during this time. Enzyme exposure was extended an
extracts that are more appropriate for the specic biotransfor- additional 20 h under the same conditions and again, no
mation desired is a better choice.
signicant changes to the avonoids and no degradation
To verify that there was no structural change in the aglycone products were observed.
products aer exposure to the AD011 crude enzyme over an
The ESI-MS were performed in the negative mode to deter-
extended time, the transformation reaction was conducted for mine the molecular weights of the compounds of interest. In
ꢁ
3
6 h. Aer about 16 h, Q3G and IR3G were converted to quer- the negative ion mode, the deprotonated ion [M–H] of Q3G,
cetin and isorhamnetin at percentage of 91.0 ꢃ 0.8% and 94.8 ꢃ IR3G, quercetin, and isorhamnetin was at m/z 463.1, 477.2,
0.4%, respectively (Fig. 3 and 4). The transformed quercetin and 301.1, 315.1, displaying a sharp and distinguished peak. These
isorhamnetin molecules had no further metabolization or results conrm that the crude enzyme extract of AD011
successfully transformed Q3G and IR3G into quercetin and
isorhamnetin with enzymatic hydrolysis of the b-1,4-glycosidic
linkage without further unwanted reactions (Fig. 5).
The anti-inammatory effects of quercetin and isorhamnetin
compared to quercetin-3-glucoside and isorhamnetin-3-
glucoside on LPS-induced TNF-a and IL-6 production in RAW
264.7 mouse macrophages
The anti-inammatory effects of S. herbacea are reported to be
due to the IR3G present. The anti-inammatory effect is a result
of IR3G suppressing lipopolysaccharide (LPS)-induced nitric
oxide production, inducible nitric oxide synthase (iNOS), tumor
necrosis factor-a (TNF-a), and interleukin-1b (IL-1b) in Raw
61
2
64.7 cells. Lipopolysaccharide (LPS) in the outer membrane
of Gram-negative bacteria is recognized by the toll-like receptor
(TLR4) of the macrophage, which in turn activates macro-
4
phages to serve as innate immune cells. TLR4 signaling causes
macrophage inammation signal transduction, which trans-
locates the transcription factor NF-kB in the cytosol into the
nucleus, resulting in the expression of macrophage
Fig. 3 HPLC chromatogram showing changes in molecular distribu-
tion of S. herbacea extracts before and after bioconversion using crude
AD011 enzyme.
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cultured aer quercetin and LPS were administered to Raw
2
64.7 cells. As a result, 50 mM quercetin inhibited the apoptosis
67
of L929 cells by >80%. Endale et al. reported that quercetin
blocked Src and Syk associated with PI3k, PDK1, and AKT
activation and blocked the association of P85 and TLR4/MyD88,
resulting in inhibition of downstream signalling pathways
IRAK1, TRAF6, activation of TAK and IKKa/NF-kB phosphory-
lation. These intracellular responses reduce the secretion and
mRNA expression of the proinammatory cytokines TNF-a and
IL-6.
64
Lee, et al. extracted quercetin, Q3G, and hyperin (forms
with galactose at quercetin carbon 3) from Acanthopanax chii-
sanensis, which has traditionally been used for the treatment of
inammation in Asia. They studied whether these three
substances inhibit the nitrite production of LPS-induced rat
peritoneal macrophages. In their work, the inhibition
percentage of quercetin was 66.1% at a concentration of 100
mM. In addition, quercetin inhibited the phosphorylation of
pp44/42 MAPK, p38 MAPK and JNK compared to the other two
Fig. 5 Mass spectrum of quercetin-3-glucoside (A) and isorhamnetin-
3
-glucoside (C) from S. herbacea and their transformed aglycones,
quercetin (B) and isorhamnetin (D).
65
substances. Wang et al. also tested whether anthocyanins and
avonoids inhibit TNF-a production of RAW 264.7 activated
inammatory genes. NF-kB and AP-1 are transcriptional regu-
lators that regulate the expression of inammatory mediators
and cytokines at the transcription level. LPS articially added to
the RAW 264.7 cell medium stimulates macrophages to produce
inammatory mediators (e.g. inducible nitric oxide synthase

with LPS and IFN-g. Quercetin was shown to inhibit TNF-
a production in a concentration-dependent manner at 125, 250
0
and 500 mM. Isorhamnetin is a 3 -methoxylated derivative of
66
quercetin. Jin et al. conrmed that IL-6 production of LPS-
induced RAW 264.7 was decreased in a dose-dependent
manner by administration of 12.5, 25, 50 mM isorhamnetin at
[
[
iNOS] and COX-2) or cytokines (e.g. TNF-a, IL-6, and interleukin
IL]-1b) as the result of intrinsic immune responses at high
concentrations. These inammatory responses play an impor-
tant role in the innate immunity of the human body, but
excessive production of inammatory cytokines, such as TNF-a,
IL-6, and IL-1b, may cause systemic inammatory response
disease syndrome (SIRS), severe tissue damage and/or sepsis.
When septic shock occurs in the human body, it causes
multiple organ (e.g. kidney, liver, and lung) failure and lethality
rates are high. In addition, chronic inammation has been re-
ported to be associated with cancer, neurological diseases, heart
disease, stroke, rheumatoid arthritis, and atopic dermatitis.
Flavanoids and their subclasses, avanones, avones, avonols,
anthocyanidins, and isoavones are known to have nutraceut-
ical activities, including anticancer, anti-inammatory, anti-
viral, and antimicrobial effects. Various groups have
demonstrated that avonol molecules have anti-inammatory
capacities through in vitro, in vivo and clinical research (Table
41
mRNA and protein levels. Lesjak et al. evaluated the IC s of
5
0
quercetin, isorhamnetin, IR3G that can reduce amount of
inammation mediators derived from arachidonic acid (e.g.
12(S)-hydroxy(5Z,8E,10E)-heptadecatrienoic acid (12-HHT),
thromboxane B2 (TXB2), and 12(S)-hydroxy-(5Z,8Z,10E,14Z)-
eicosatetraenoic acid (12-HETE) from human platelets). Among
the three avonoids, quercetin showed the highest anti-
inammatory activity, followed by isorhamnetin and IR3G. In
9
the case of IR3G, Kim et al. conrmed that IR3G extracted from
S. herbacea inhibited NO, iNOS, TNF-a, and IL-1b production in
LPS-induced RAW 264.7. However, they did not study the
inammation inhibitory effect of isorhamnetin.
In this study, to compare the anti-inammatory effects of S.
herbacea glycosides (Q3G and IR3G) and aglycones (quercetin
and isorhamnetin), we evaluated the LPS-induced TNF-a (Fig. 6)
and IL-6 levels (Fig. 7) in Raw 264.7 (KCLB 40071) mouse
macrophage cells aer Q3G, IR3G, quercetin and isorhamnetin
treatments into macrophage cell culture media.
3).
It has been reported that quercetin inhibits the activation of
IKK, NF-kB, STAT1, iNOS, NO, TNFa, MAPKs, Akt, Src, JAK-1 and
Tyk2. Inhibition of nuclear translocation of NF-kB p65 is known
to inhibit expression of IL-1b, TNF-a and IL-6. Specically,
quercetin inhibits the expression of IL-1b, TNF-a and IL-6 by
inhibition of nuclear translocation of NF-kB p65.
Raw 264.7 cells were incubated with various concentrations
of Q3G, quercetin, IR3G, and isorhamnetin (0, 1, 5, 10 mM) for
2
h. Then, except for the negative control group (no LPS), they
ꢁ
1
were treated with LPS (0.1 mg mL ) and incubated for 24 h.
Finally, TNF-a and IL-6 production levels from Raw 264.7 cells
63
Manjeet and Ghosh reported that the production of LPS-
induced nitric oxide and tumor necrosis factor-a (TNF-a) from
macrophage RAW 264.7 cells was inhibited by 1–50 mM of
quercetin in a dose-dependent manner. They also used L929
cells to demonstrate that the level of TNF-a produced by Raw
were determined using an ELISA assay and
a 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay was conducted to evaluate cell viability at the same time
(Fig. 8).
Our results showed that Q3G increased TNF-a production,
while quercetin decreased TNF-a production in a dose depen-
dent manner. IR3G decreased TNF-a production but
264.7 cells is reduced by quercetin. L929 cells are known to
induce apoptosis by TNF-a. In their work, L929 cells were
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Table 3 Anti-inflammatory effects of quercetin derivatives
Compounds
Quercetin
Quercetin-3-sulfate 10 mM
Quercetin
Concentration
Cell lines
Induced by
Inhibited inammatory mediators
Ref.
a
b
c
d
e
f
1–30 mM
Mouse BV-2 microglia LPS/IFN-g
Mouse BV-2 microglia LPS/IFN-g
NO , iNOS (mRNA), IKK , NF-kB , STAT1 , AP-1
61
a
NO (not inhibited)
a
g
h
i
j
k
1–10 mM
NO , phosphorylation of ERK , JNK , p38 , Akt , JAK-1 , 62
l
m
Tyk2 , and Src
a
u
Quercetin
Quercetin
Quercetin-3-
glucoside
1–50 mM
100 mM
RAW 264.7
Rat peritoneal
macrophages
LPS
LPS
NO , TNF-a
63
a
i
n
h
NO , phosphorylation of p44/42 MAPK, p38 MAPK , JNK 64
Hyperin
Quercetin
Quercetin-3-
glucoside
Isorhamnetin
Quercetin
Isorhamnetin
Isorhamnetin-3-
glucoside
Isorhamnetin-3-
glucoside
a
u
16–500 mM
RAW 264.7
LPS/IFN-g
NO , TNF-a
65
u
TNF-a (not inhibited)
o
p
d
e
12.5–50 mM
Different
concentrations
RAW 264.7
Human platelets
LPS
Calcium
ionophore
Ho-1 mRNA expression, IL-6 , NF-kB p50, STAT1
12-HHT , TXB2 , PGE2 , 12-HETE
66
41
q
r
s
t
ꢁ1
b
u
v
0.1–10 mg mL (0.2– RAW 264.7
20 mM)
LPS
iNOS (protein level), TNF-a , IL-1b
9
a
b
c
d
e
NO, nitric oxide. iNOS, inducible nitric oxide synthase. IKK, IkB kinase. NF-kB, nuclear factor-kappa B. STAT1, signal transducer and
f
g
h
i
activator of transcription-1. AP-1, activating protein-1. ERK, extracellular signal-regulated kinase. JNK, c-Jun N-terminal kinase. p38,
mitogen-activated protein kinase p38. Akt, protein kinase B. JAK-1, Janus kinase-1. Tyk2, tyrosine kinase 2. Src, proto-oncogene tyrosine-
protein kinase. MAPK, mitogen-activated protein kinase. Ho-1, heme oxygenase-1. IL-6, interleukin 6. 12-HHT, 12(S)-hydroxy(5Z,8E,10E)-
j
k
l
m
n
o
p
q
r
s
t
heptadecatrienoic acid. TXB2, thromboxane B2. PGE2, prostaglandin E2. 12-HETE, 12(S)-hydroxy-(5Z,8Z,10E,14Z)-eicosatetraenoic acid.
u
v
TNF-a, tumor necrosis factor alpha. IL-1b, interleukin 1 beta.
isorhamnetin decreased TNF-a production 1.5–4.6 times that of of IL-6 production compared to IR3G; isorhamnetin was about
IR3G with the 1–10 mM treatment. Regarding IL-6 production, 2.1 times higher at 1–10 mM. Cell viability was measured by MTT
quercetin-3-glucose did not decrease IL-6 production with LPS. assay, with and without LPS. Q3G showed a trend of slightly
In contrast, quercetin did decrease TNF-a production in a dose decreasing cell viability, while IR3G, quercetin, and iso-
dependent manner. Isorhamnetin exhibited greater inhibition rhamnetin did not affect cell viability at 1–10 mM. Thus, these
Fig. 6 (A) Comparison of TNF-a production levels with quercetin-3- Fig. 7 (A) Comparison of IL-6 production levels with quercetin-3-
glucoside and quercetin and (B) isorhamnetin-3-glucoside and iso- glucoside and quercetin and (B) isorhamnetin-3-glucoside and iso-
rhamnetin. Values are the mean ꢃ SD of three independent experi- rhamnetin. Values are the mean ꢃ SD of three independent experi-
ments. (*) p < 0.05, (**) p < 0.01, and (***) p < 0.001 indicate significant ments. (*) p < 0.05, (**) p < 0.01, and (***) p < 0.001 indicate significant
differences compared to the LPS-treated group.
differences compared to the LPS-treated group.
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herbacea-containing functional foods that produce anti-
inammatory effects.
Conflicts of interest
Hyung Jin Ahn, Hyun Ju You, Zhipeng Li, Deokyeong Choe,
Tony Vaughn Johnston, and Seockmo Ku declare no conict of
interests. Myeong Soo Park is directly employed at BIFIDO Ltd.
as CTO. Geun Eog Ji and Myeong Soo Park hold BIFIDO Ltd.
stocks.
Acknowledgements
This work was carried out with support from the National
Research Foundation of Korea (NRF) grant (No.
2
017R1A2B2012390) funded by the Korean government (MSIP),
High Value-added Food Technology Development Program (No.
17043-3), Korea Institute of Planning and Evaluation for
3
Technology in Food, Agriculture, Forestry and Fisheries (IPET),
Ministry of Agriculture, Food and Rural Affairs (MAFRA), and
the Bio & Medical Technology Development Program of the
National Research Foundation (NRF) funded by the Ministry of
Science, ICT & Future Planning (NRF-2017M3A9F3041747). This
work was also supported by the Basic Science Research Program
through the National Research Foundation of Korea (NRF)
funded by the Ministry of Education (2017R1A6A3A11029462)
and the Faculty Research and Creative Activity Committee
Fig. 8 (A) Cell viability comparison of quercetin-3-glucoside and
quercetin and (B) isorhamnetin-3-glucoside and isorhamnetin. Values
are the mean ꢃ SD of three independent experiments. (*) p < 0.05
indicates significant differences compared to the LPS-treated group.
results suggest that quercetin and isorhamnetin, produced by
biotransformation using crude enzyme extract of AD011, inhibit
inammatory marker production more effectively than their
precursors, Q3G and IR3G, respectively, in RAW 264.7 cells. The
anti-inammatory effect of aglycone molecules agrees with
(FRCAC) grant (no. 221745) and the Office of Research and
Sponsored Programs (ORSP) grant for the postdoc researcher
recruitment funded by Middle Tennessee State University
(MTSU).
41,66,68–73
previous data from other groups.
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