Ci a ion: Han, X.; Song, Y.; Huang,
R.; Zhu, M.; Li, M.; Requena, T.;
Wang, H. An i-In lamma o y and Gu
Mic obio a Modula ion Po en ials o
Fla onoids Ex ac ed om Passi lo a
oe ida F ui s. Foods 2023,12, 2889.
h ps://doi.o g/10.3390/
oods12152889
Academic Edi o : Vic o Rodo
Recei ed: 7 July 2023
Re ised: 25 July 2023
Accep ed: 25 July 2023
Published: 29 July 2023
Copy igh : © 2023 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
oods
A icle
An i-In lamma o y and Gu Mic obio a Modula ion Po en ials
o Fla onoids Ex ac ed om Passi lo a oe ida F ui s
Xiangpeng Han 1,2,† , Ya Song 1,2,†, Riming Huang 1,2 , Minqian Zhu 1,2, Meiying Li 1,2, Te esa Requena 3,*
and Hong Wang 1,2,*
1Guangdong P o incial Key Labo a o y o Food Quali y and Sa e y, College o Food Science, Sou h China
Ag icul u al Uni e si y, Guangzhou 510642, China; [email p o ec ed] (X.H.); [email p o ec ed] (Y.S.);
[email p o ec ed] (R.H.); [email p o ec ed] (M.Z.); [email p o ec ed] (M.L.)
2Guangdong Labo a o y o Lingnan Mo de n Ag icul u e, Guangzhou 510642, China
3Ins i u o de In es igación en Ciencias de la Alimen ación CIAL (CSIC), Campus UAM Can oblanco,
28049 Mad id, Spain
*Co espondence: [email p o ec ed] (T.R.); [email p o ec ed] (H.W.)
†These au ho s con ibu ed equally o his wo k.
Abs ac :
This s udy aimed o explo e he an i-in lamma o y and gu mic obio a modula ion po en-
ials o la onoid- ich ac ion (PFF) ex ac ed om Passi lo a oe ida ui s. The esul s showed ha
PFF ma kedly educed he p oduc ion o ni ic oxide (NO), umo nec osis ac o
α
(TNF-
α
), and
in e leukin 6 (IL-6) in LPS-s imula ed RAW 264.7 cells. Meanwhile, PFF ea men also e ec i ely
dec eased he phospho yla ion le els o MAPK, PI3K/Ak , and NF-
κ
B signaling-pa hway- ela ed
p o eins (ERK, JNK, p38, Ak , and p65). Mo eo e , PFF had an impac on mic obial composi ion
and me aboli es in a ou -s age dynamic simula o o human gu mic obio a (BFBL gu model).
Speci ically, PFF exhibi ed he g ow h-p omo ing abili y o se e al bene icial bac e ia, including
Bi idobac e ium,En e ococcus,Lac obacillus, and Rosebu ia, and sho -chain a y acid (SCFA) gene a ion
abili y in gu mic obio a. In addi ion, spec oscopic da a e ealed ha PFF mainly con ained i e
la onoid compounds, which may be bioac i e compounds wi h an i-in lamma o y and gu mic o-
bio a modula ion po en ials. The e o e, PFF could be u ilized as a na u al an i-in lamma o y agen o
supplemen o heal h p oduc s.
Keywo ds:
Passi lo a oe ida ui s; la onoid- ich ac ion; an i-in lamma o y ac i i y; gu mic obio a;
sho -chain a y acids
1. In oduc ion
In lamma ion is a pi o al de ensi e esponse o he hos immune sys em agains
inju ious s imuli, such as oxic compounds, inju ed cells, pa hogenic o ganisms, and i -
adia ion [
1
,
2
]. I can p e en in ec ion caused by ea ly cell damage, elimina e damaged
cells o issue, and p omo e issue healing in he human body [
2
]. Howe e , excessi e o
uncon olled in lamma ion may esul in mul iple heal h diseases, including in lamma-
o y bowel disease (IBD), alle gies, me abolic synd ome, diabe es, au oimmune diseases,
ca dio ascula diseases, and cance [
3
]. Cu en ly, mos epidemiological s udies ha e
demons a ed ha in lamma ion- ela ed diseases a e closely in e ela ed wi h changes
in gu mic obio a [
4
–
6
]. Fo ins ance, he gu mic obio a composi ion o IBD pa ien s
exhibi s speci ic cha ac e is ics in compa ison wi h ha o non-IBD pa ien s [
7
]. O no e,
ce ain mic oo ganisms play i al oles in hos homeos asis, such as de ending agains i al
and pa hogenic in asion, abso bing nu ien s and ene gy, boos ing he immune sys em,
and imp o ing me abolism [
8
,
9
]. To da e, a ious pu i ied o syn he ic d ugs ha e been
explo ed o egula e and alle ia e in lamma ion- ela ed diseases, including immunomod-
ula o s, s e oids, and non-s e oidal an i-in lamma o y d ugs [
2
,
10
], whe eas he abo e
in e en ion d ugs gene ally p o ide limi ed clinical e ec i eness and ha e a se ies o
Foods 2023,12, 2889. h ps://doi.o g/10.3390/ oods12152889 h ps://www.mdpi.com/jou nal/ oods
Foods 2023,12, 2889 2 o 11
ad e se e ec s [
5
]. The e o e, he cu en esea ch ocuses on explo ing and de eloping
na u al an i-in lamma o y agen s de i ed om medicine ood homology plan s o he bal
medicines o enhance hei e icacy and sa e y.
In ecen yea s, e idence has been moun ing ha na u al la onoid-en iched oods,
such as esh ui s, ege ables, and ea, a e help ul in dec easing he isk o in lamma ion-
ela ed diseases [
11
]. Gene ally, la onoids possess excellen an i-in lamma o y p ope ies
o e ec i ely inhibi he in lamma o y p ocesses by dis up ing in lamma o y cy okine
elease and egula ing in lamma ion-associa ed signaling pa hways wi hou ha m ul side
e ec s [
5
]. Mo eo e , many s udies ha e e ealed ha die a y in ake o la onoids is
in ima ely ele an o he a ie y and composi ion o gu mic obio a [
12
]. Signi ican ly,
imp o ing he dys egula ed gu mic obio a is a momen ous a ge o new he apies o
in es inal in lamma ions [
13
]. Fo ins ance, la onoids can a enua e he hos in lamma-
o y esponse and educe he incidence o se e i y o IBD h ough in e ac ion wi h he
speci ic mic obio a and hei me aboli es [
14
]. Mo eo e , la onoids can also modula e
gu mic obio a by educing pa hogens (such as S aphylococcus,Pseudomonas,P o eus, e c.)
and inc easing p obio ics (such as Lac obacillus and Bi idobac e ia, e c.) [
15
], which can alle-
ia e he in lamma o y esponses by educing endo oxin p oduc ion, he eby p omo ing
gu heal h [
16
]. Hence, g owing a en ion o die a y nu i ion and illness p e en ion has
inc eased he daily demands o la onoid-en iched oods.
Passi lo a oe ida ui (Passi lo aceae) is an edible be y commonly consumed in opical
a eas, such as B azil and Sou h China, o ea ing acu e edema, auma ic co nea, conjunc-
i i is, and elie ing as hma [
17
]. To da e, he biological unc ions o P. oe ida ui s, includ-
ing an ibac e ial, an i-os eopo o ic, an i-in lamma o y, and an ioxidan ac i i ies, ha e been
epo ed [
11
]. Ou p e ious s udies showed ha P. oe ida ui s a e mainly ich in amino
acids (1097 mg/100 g), mine als (595.75 mg/100 g), suga s (3.34 g/100 g), o ganic acids
(0.24 mg/100 g), and polyphenols (284.4 mg/100 g), especially la onoids (239 mg/100 g),
which a e he majo componen o polyphenols [
18
]. Mo eo e , he la onoids we e ano he
main componen besides he polysaccha ides in he aqueous ex ac o P. oe ida ui s,
and he polysaccha ides ha e been cha ac e ized and demons a ed o possess excellen
immune-enhancing ac i i y [
18
–
20
]. Howe e , he composi ions and bioac i e bene i s o
la onoids om P. oe ida ui s s ill need o be disco e ed. The e o e, his p oposed s udy
aims o explo e he
in i o
an i-in lamma o y ac i i y and gu mic obio a modula ion
po en ial o PFF isola ed om P. oe ida ui s. This wo k will p o ide scien i ic e idence o
new uses o P. oe ida ui s and p omo e i s de elopmen and applica ion as a unc ional
ood o human heal h.
2. Ma e ials and Me hods
2.1. Ma e ials and Reagen s
The ma u e ui s o P. oe ida we e ha es ed om Dong ang ci y, Hainan p o ince,
China, in May 2021. RAW264.7 cells we e pu chased om Ame ican Type Cul u e Col-
lec ion (ATCC, Manassas, VA, USA). Dulbecco’s Modi ied Eagle’s medium (DMEM) was
ob ained om Gibco BRL (Gai he sbu g, MD, USA). Penicillin, s ep omycin, and e al
bo ine se um (FBS) we e p oduc s o HyClone (Logan, UT, USA). Lipopolysaccha ide
(LPS), 3-(4,5-dime hyl hiazol-2-yl)-2,5-diphenyl e azolium b omide (MTT), sul u ic acid,
i luo oace ic acid (TFA), enhanced chemiluminescence (ECL) ki , AB-8 mac opo ous esin,
and polyamide ille we e p oduced by Yuanye Bio echnology Co., L d. (Shanghai, China).
Bicinchoninic acid (BCA) ki , ni ic oxide (NO) de ec ion ki , ELISA ki s (TNF-
α
and IL-6),
and 4
0
,6-diamidino-2-phenylindole (DAPI) we e pu chased om Beyo ime Bio echnology
Co., L d. (Shanghai, China). Sephadex LH-20 dex an gel was pu chased om Gene al
Elec ic Co. (GE, Fai ield, CT, USA). The me hanol and ace oni ile we e p oduced by
Sigma Chemical Co. (S . Louis, MO, USA).
Foods 2023,12, 2889 3 o 11
2.2. P epa a ion o PFF
P. oe ida ui s we e sepa a ed and washed manually wi h dis illed wa e , hen d ied
in an o en. The d ied samples we e g ound and il a ed o p epa e P. oe ida powde , which
was p ese ed in an oxygen ba ie bag a
−
20
◦
C un il u he use. PFF ex ac ion and
isola ion we e pe o med acco ding o a p e ious s udy [
21
]. B ie ly, he c ude aqueous
ex ac o P. oe ida (AEP) was ob ained ia in usion wi h wa e (65
◦
C, powde : sol en , 1:10,
w/ ) o 2 h. The concen a ed AEP was p ecipi a ed by adding e hanol (96%) o e nigh a
4
◦
C, hen loaded on AB-8 mac opo ous esin column, elu ed sequen ially h ough dis illed
wa e and ex ac ed wi h e hanol (80%). Subsequen ly, he ex ac was u he pu i ied by a
polyamide column o ob ain PFF.
2.3. Cell Cul u e and Viabili y Assay
Cell iabili y was de e mined using he MTT me hod [
2
]. RAW264.7 cells (200
µ
L)
we e inocula ed in o a 96-well mic opla e (1
×
10
5
cells/mL) and cul u ed in DMEM
(con aining 10% FBS and 1% penicillin-s ep omycin) a 37
◦
C o 24 h. The medium was
eplaced wi h esh DMEM con aining a ious concen a ions o PFF (0, 6.25, 12.5, 25, 50,
100, 200, 400, and 600
µ
g/mL). A e incuba ion, he medium was emo ed, and 200
µ
L
o MTT solu ion (0.5 mg/mL) was added o a 96-well pla e, which was cul u ed o 4 h
a 37
◦
C. Subsequen ly, he pla e was washed wi h s e ile PBS, and 150
µ
L o DMSO was
added o each well. A e shaking in he da k o 15 min, he abso bance was eco ded a
570 nm by a mic opla e analyze (Molecula De ice, San Jose, CA, USA).
2.4. De e mina ion o NO and Cy okines
The p oduc ion o NO, TNF-
α
, and IL-6 was de e mined based on p e iously epo ed
me hods [
9
]. In b ie , RAW264.7 cells we e inocula ed in o a 48-well pla e (1
×
10
5
cells/mL)
con aining LPS (1
µ
g/mL) and PFF (0, 50, 100, and 200
µ
g/mL). A e 24 h o incuba ion,
he supe na an was ha es ed o quan i y NO, TNF-
α
, and IL-6 le els. NO p oduc ion was
de e mined using G iess eagen , and equal olumes o cul u e supe na an s and G iess
eagen we e mixed and eac ed o 15 min. Then, OD
540
was de e mined by a mic opla e
analyze . Meanwhile, he p oduc ion o IL-6 and TNF-
α
was de e mined using ELISA ki s.
2.5. Wes e n Blo Analysis
Wes e n blo was pe o med based on a p e iously desc ibed me hod [
22
]. In sho ,
RAW 264.7 cells we e inocula ed in o a 6-well pla e (1
×
10
5
cells/mL) and ea ed wi h LPS
(1
µ
g/mL) and PFF (0, 50, 100, and 200
µ
g/mL) o 24 h. A e incuba ion, he cells we e
ha es ed and lysed in adioimmunop ecipi a ion assay (RIPA) bu e . A e cen i uga ion
(12,000
×
g, 4
◦
C, 20 min), he p o ein concen a ion o he supe na an was quan i ied by
BCA Ki . Meanwhile, he samples and
×
5 loading bu e we e mixed and hea ed (100
◦
C,
10 min) o dena u e he p o ein comple ely. Then, 50
µ
g o o al p o ein was sepa a ed
by SDS-PAGE gel and ans e ed o a poly inylidene di luo ide memb ane, which was
blocked by 5% BSA and incuba ed wi h he p ima y an ibodies. A e ha , memb anes
we e u he incuba ed wi h he seconda y an ibody. Finally, p o ein bands we e eac ed
wi h ECL solu ions and isualized using an imaging sys em (GE, Chicago, IL, USA).
2.6. Immuno luo escence Analysis
Con ocal mic oscopy was pe o med as desc ibed by Duan e al. [
23
]. RAW264.7 cells
(1
×
10
5
cells/mL) we e p e ea ed in a 6-well pla e wi h LPS (1
µ
g/mL) and PFF (0, 100,
and 200
µ
g/mL). A e ha , cells we e collec ed and washed gen ly using s e ile PBS,
sequen ially ixed wi h 4% o maldehyde o 30 min, and pe meabilized wi h 1% T i on
X-100 o 30 min. A e wa d, ixed cells we e blocked by 5% BSA, washed and incuba ed
wi h p65 an ibody o e nigh . Subsequen ly, he cells we e incuba ed wi h FITC-labeled
second an ibody in da kness o 2 h, hen s ained wi h DAPI in da kness o 30 min. Images
we e obse ed using con ocal lase scanning mic oscopy (Leica, We zla , Ge many).
Foods 2023,12, 2889 4 o 11
2.7. Dynamic Simula o o he Gu Mic obiome
The p epa a ion o ecal samples was ca ied ou acco ding o a me hod epo ed by
Tang e al. [
24
]. Speci ically, esh ecal samples we e collec ed om i e heal hy human
olun ee s (BMI be ween 18.5 and 24.9 kg/m
2
) who did no ha e in es inal diseases o
had no aken an ibio ics o he p e ious h ee mon hs and ollowed a la onoid- ee die
o h ee days be o e ecal collec ion. The ecal samples we e immedia ely s o ed in an
anae obic ank and hen dilu ed in sodium phospha e bu e (0.1 M, pH 7.0) wi h sodium
hioglycola e and homogenized o ob ain a 20% (w/ ) eces slu y.
The BFBL gu model (Figu e S1) is a ou -s age eac o sys em ha simula es he small
in es ine (SI), ascending colon (R
1
), ans e se colon (R
2
), and descending colon (R
3
) o
humans
in i o
[
25
]. B ie ly, h ee colon eac o s (R
1
, R
2
, and R
3
) we e i s illed wi h
nu i i e medium (pH 2.0) in a olume o 250, 400, and 300 mL, espec i ely. Subsequen ly,
each colon eac o was inocula ed wi h 20 mL o eces slu y, hen con inuously s i ed
(150 pm) wi h an Agima ic N magne ic s i e wi h hea ing (Selec a, Spain), and he
empe a u e was main ained a 37
◦
C. Meanwhile, he sys em was main ained as anae obic
by con inuously lushing N
2
, and pH alues o he colonic eac o s we e au oma ically
adjus ed o main ain 5.7 ±0.2 in he R1, 6.3 ±0.2 in he R2, and 6.8 ±0.2 in he R3.
To main ain gu homeos asis, he small in es ine (SI) was ed 75 mL o nu ien medium
(pH 2.0) and 40 mL o a i icial panc ea ic juice mix u e (6 g/L cholangium, 1.9 g/L
po cine panc ea ic ypsin, and 12.5 g/L NaHCO
3
) h ee imes pe day o wo weeks.
Small in es ine diges ion was conduc ed a 37
◦
C, and essel con en s we e au oma ically
ans e ed o he ollowing colonic compa men , which ac i a ed he anspo o colonic
con en be ween R
1
, R
2
, and R
3
eac o s egula ed by le el senso s o main ain a cons an
eac o olume. Du ing he eeding p ocess, all compa men s we e lushed wi h ni ogen
gas o main ain an anae obic s a e. A e a wo-week s abiliza ion pe iod (CK), he BFBL
gu model was subjec ed o a one-week in ake pe iod by adding PFF (4 g/L) o he nu i i e
medium, ollowed by a one-week washou pe iod (WO) wi h he addi ion o nu ien
medium. Finally, samples we e collec ed om h ee colon eac o s daily h oughou he
s udy pe iod and p ese ed a −20 ◦C un il use.
2.8. Mic obio a Composi ion Analysis
Genomic DNA o mic obial samples om R
1
, R
2
, and R
3
eac o s was ex ac ed as
p e iously epo ed by Requena e al. [
25
]. The gene-speci ic p ime s, s anda ds, amplicon
size, and ampli ica ion condi ions o he quan i ica ion o a ge ed mic obial g oups a e
lis ed in Table S1. The SYBR G een-based RT-qPCR assay was conduc ed o quan i y DNA
samples and s anda ds using a ViiA7 Real-Time PCR Sys em (The mo Fishe Scien i ic,
Wal ham, MA, USA).
2.9. SCFAs Analysis
The BFBL gu model samples (1 mL) we e collec ed daily om each eac o (R
1
, R
2
,
and R
3
) o SCFAs analysis du ing each pe iod. SCFAs we e quan i ied by a me hod
epo ed by Ba oso e al. [
26
]. In sho , he il e ed (0.22
µ
m) samples we e analyzed by
an HPLC sys em equipped wi h Rezex ROA-O ganic Acids column (300 mm
×
7.8 mm,
Phenomenex, Maccles ield, UK) and UV-975 de ec o (Jasco, Tokyo, Japan). The mobile
phase was 0.005 M sul u ic acid solu ion wi h a linea g adien o 0.6 mL/min, and elu ion
cu es we e moni o ed a 210 nm. Calib a ion cu es we e es ablished o SCFAs wi h
concen a ions anging om 1 o 100 mM.
2.10. Isola ion o he Main Fla onoids in PFF
The ex ac ion and sepa a ion o he main la onoids in PFF we e conduc ed based on
p e ious s udies [
27
,
28
], wi h sligh modi ica ions. Fi s ly, PFF was pu i ied by pe mea ion
on he Sephadex LH-20 column using me hanol as he mobile phase (0.5 mL/min). Subse-
quen ly, he collec ed ac ion was subjec ed o g adien p epa a i e liquid ch oma og aphy,
conduc ed using he P ep 150 LC se wi h a 2988 pho odiode a ay de ec o , and connec ed
Foods 2023,12, 2889 5 o 11
o a SunFi e C18 OBD P ep column (250 mm
×
19 mm, 5
µ
m, Wa e s, Mil o d, MA, USA)
using 80% me hanol as an eluen (5.0 mL/min).
2.11. Ini ial Analysis o he Main Fla onoids in PFF
The ini ial analysis o he main la onoids in PFF was conduc ed as p e iously de-
sc ibed by Cho e al. [
29
] and Chao e al. [
30
]. B ie ly, each pu i ied sample was dissol ed
in me hanol (1 mg/mL), hen sample ilms we e do ed on po assium b omide c ys al
able s by he capilla y. The comp essed able s we e scanned by Fou ie ans o m in a ed
spec ome y (B uke , Rheins e en, Ge many) in a equency ange o 4000–400 cm
−1
, and
he in a ed abso p ion spec a o each pu i ied sample we e eco ded. Simul aneously,
hey we e also dilu ed and scanned om 200 o 600 nm using UV-Vis spec opho ome y
(The mo Fishe Scien i ic, Wal ham, MA, USA). Fu he mo e, liquid ch oma og aphy–mass
spec ome y (LC-MS) analysis was pe o med using LTQ O bi ap mass spec ome y
(The mo Fishe Scien i ic, Wal ham, MA, USA).
2.12. S a is ical Analysis
One-way ANOVA was ca ied ou o assess s a is ical signi icance using SPSS 20.0
(IBM, A monk, NY, USA). All g aphical e alua ions we e conduc ed by G aphPad P ism
8.0 (G aphPad, San Diego, CA, USA).
3. Resul s and Discussion
3.1. E ec s o PFF on he Le el o NO and Cy okines P oduced in RAW264.7 Cells
Mac ophages a e he mos impo an immune cells in a ious immune esponses,
especially in he immune esponse; hey can ini ia e and egula e in lamma o y p ocesses by
sec e ing he in lamma o y media o such as NO, as well as he p o-in lamma o y cy okines
such as TNF-
α
and IL-6 [
2
,
11
], so he e ec s o PFF on le els o NO and cy okines p oduced
in RAW264.7 cell we e in es iga ed. Fi s ly, he cell iabili y unde he PFF in e en ion
was de e mined. As illus a ed in Figu e 1, PFF exhibi ed no cy o oxic e ec on RAW264.7
cells wi h a concen a ion lowe han 200
µ
g/mL, so h ee concen a ions o PFF (50, 100,
and 200
µ
g/mL) we e selec ed o he ollowing expe imen . Then, NO, as a momen ous
ee adical in ol ed in he in lamma ion p ocess [
23
], was de e mined. I was obse ed
ha PFF signi ican ly inhibi ed NO p oduc ion in LPS-induced RAW264.7 cells and lowe ed
i s le el om 42.60
µ
M down o 7.70–23.51
µ
M a a concen a ion ange o 50–200
µ
g/mL
(p< 0.01) (Figu e 2A). Meanwhile, as shown in Figu e 2B, C, PFF ea men (50–200
µ
g/mL)
e icien ly supp essed he p oduc ion o TNF-
α
and IL-6 in a dose-dependen manne
(p< 0.01), and he inhibi o y a es we e 21.86–79.51% o TNF-
α
and 26.76–82.06% o
IL-6. The e o e, om he abo e expe imen al esul s, i could be ound ha PFF exhibi ed
an i-in lamma ion po en ial by supp essing some in lamma o y ac o s.
Foods 2023, 12, x FOR PEER REVIEW 6 o 12
Figu e 1. Effec s o PFF on cell iabili y o RAW264.7 cells. Da a we e exhibi ed as mean ± SD. ** p <
0.01, s. he con ol g oup.
Figu e 2. Effec s o PFF on p oduc ion o NO (A), IL-6 (B), and TNF-α (C) in LPS-induced RAW264.7
cells. Da a we e exhibi ed as mean ± SD. ** p < 0.01, s. he LPS- ea ed g oup (wi hou PFF).
3.2. Effec s o PFF on MAPK, PIK/Ak , and NF-κB Signaling Pa hways in RAW264.7 Cells
I has been epo ed ha MAPK and PI3K/Ak pa hways a e closely associa ed wi h
in lamma o y eac ions [31]. In pa icula , he phospho yla ion o some ex acellula sig-
nal kinases such as ERK, p38, JNK, and Ak ela ed o hese wo pa hways a e he im-
po an indexes o in lamma ion [2]. By Wes e n blo esul s, i could be ound ha he
phospho yla ion le els o ERK, p38, JNK, and Ak we e signi ican ly up- egula ed in LPS-
induced RAW264.7 cells (p < 0.01), which was consis en wi h he in lamma o y indica-
ions. Howe e , a e in e en ion wi h PFF (50–200 µg/mL), he phospho yla ion le els
o p38, JNK, and Ak we e signi ican ly educed in a dose-dependen manne (p < 0.01),
and educ ion a es we e 21.35–61.47% o p38, 15.88–29.10% o JNK, and 5.18–50.24% o
Ak (Figu e 3B–D). Meanwhile, he phospho yla ion le el o ERK was no e iden ly
changed a e ea men wi h PFF in a concen a ion anging om 50 o 100 µg/mL, bu i
was dec eased signi ican ly by 10.96% a doses o 200 µg/mL PFF (p < 0.01) (Figu e 3A).
Mo eo e , conside ing ha NF-κB, a i al ansc ip ion ac o mainly composed o IκB
and p65, plays a signi ican ole in egula ing mul iple in lamma o y diseases [32], he
impac s o PFF on NF-κB/p65 ansloca ion we e also s udied. The esul s (Figu e 4)
showed ha p65 p o ein was o e exp essed in he nuclea in LPS-induced RAW264.7
cells; howe e , i s exp ession was signi ican ly e e sed by PFF ea men (100 and 200
µg/mL), indica ing ha PFF dis u bed he ansloca ion o p65 s imula ed by LPS. Collec-
i ely, he abo e esul s sugges ed ha he an i-in lamma o y ac i i y o PFF was co e-
la ed wi h he ac i a ion o MAPK, PIK/Ak , and NF-κB signaling pa hways.
Figu e 1.
E ec s o PFF on cell iabili y o RAW264.7 cells. Da a we e exhibi ed as mean
±
SD.
** p< 0.01, s. he con ol g oup.

Foods 2023,12, 2889 6 o 11
Foods 2023, 12, x FOR PEER REVIEW 6 o 12
Figu e 1. Effec s o PFF on cell iabili y o RAW264.7 cells. Da a we e exhibi ed as mean ± SD. ** p <
0.01, s. he con ol g oup.
Figu e 2. Effec s o PFF on p oduc ion o NO (A), IL-6 (B), and TNF-α (C) in LPS-induced RAW264.7
cells. Da a we e exhibi ed as mean ± SD. ** p < 0.01, s. he LPS- ea ed g oup (wi hou PFF).
3.2. Effec s o PFF on MAPK, PIK/Ak , and NF-κB Signaling Pa hways in RAW264.7 Cells
I has been epo ed ha MAPK and PI3K/Ak pa hways a e closely associa ed wi h
in lamma o y eac ions [31]. In pa icula , he phospho yla ion o some ex acellula sig-
nal kinases such as ERK, p38, JNK, and Ak ela ed o hese wo pa hways a e he im-
po an indexes o in lamma ion [2]. By Wes e n blo esul s, i could be ound ha he
phospho yla ion le els o ERK, p38, JNK, and Ak we e signi ican ly up- egula ed in LPS-
induced RAW264.7 cells (p < 0.01), which was consis en wi h he in lamma o y indica-
ions. Howe e , a e in e en ion wi h PFF (50–200 µg/mL), he phospho yla ion le els
o p38, JNK, and Ak we e signi ican ly educed in a dose-dependen manne (p < 0.01),
and educ ion a es we e 21.35–61.47% o p38, 15.88–29.10% o JNK, and 5.18–50.24% o
Ak (Figu e 3B–D). Meanwhile, he phospho yla ion le el o ERK was no e iden ly
changed a e ea men wi h PFF in a concen a ion anging om 50 o 100 µg/mL, bu i
was dec eased signi ican ly by 10.96% a doses o 200 µg/mL PFF (p < 0.01) (Figu e 3A).
Mo eo e , conside ing ha NF-κB, a i al ansc ip ion ac o mainly composed o IκB
and p65, plays a signi ican ole in egula ing mul iple in lamma o y diseases [32], he
impac s o PFF on NF-κB/p65 ansloca ion we e also s udied. The esul s (Figu e 4)
showed ha p65 p o ein was o e exp essed in he nuclea in LPS-induced RAW264.7
cells; howe e , i s exp ession was signi ican ly e e sed by PFF ea men (100 and 200
µg/mL), indica ing ha PFF dis u bed he ansloca ion o p65 s imula ed by LPS. Collec-
i ely, he abo e esul s sugges ed ha he an i-in lamma o y ac i i y o PFF was co e-
la ed wi h he ac i a ion o MAPK, PIK/Ak , and NF-κB signaling pa hways.
Figu e 2.
E ec s o PFF on p oduc ion o NO (
A
), IL-6 (
B
), and TNF-
α
(
C
) in LPS-induced RAW264.7
cells. Da a we e exhibi ed as mean ±SD. ** p< 0.01, s. he LPS- ea ed g oup (wi hou PFF).
3.2. E ec s o PFF on MAPK, PIK/Ak , and NF-κB Signaling Pa hways in RAW264.7 Cells
I has been epo ed ha MAPK and PI3K/Ak pa hways a e closely associa ed wi h
in lamma o y eac ions [
31
]. In pa icula , he phospho yla ion o some ex acellula
signal kinases such as ERK, p38, JNK, and Ak ela ed o hese wo pa hways a e he
impo an indexes o in lamma ion [
2
]. By Wes e n blo esul s, i could be ound ha he
phospho yla ion le els o ERK, p38, JNK, and Ak we e signi ican ly up- egula ed in LPS-
induced RAW264.7 cells (p< 0.01), which was consis en wi h he in lamma o y indica ions.
Howe e , a e in e en ion wi h PFF (50–200
µ
g/mL), he phospho yla ion le els o
p38, JNK, and Ak we e signi ican ly educed in a dose-dependen manne (
p< 0.01
),
and educ ion a es we e 21.35–61.47% o p38, 15.88–29.10% o JNK, and 5.18–50.24%
o Ak (Figu e 3B–D). Meanwhile, he phospho yla ion le el o ERK was no e iden ly
changed a e ea men wi h PFF in a concen a ion anging om 50 o 100
µ
g/mL, bu i
was dec eased signi ican ly by 10.96% a doses o 200
µ
g/mL PFF (p< 0.01) (Figu e 3A).
Mo eo e , conside ing ha NF-
κ
B, a i al ansc ip ion ac o mainly composed o I
κ
B
and p65, plays a signi ican ole in egula ing mul iple in lamma o y diseases [
32
], he
impac s o PFF on NF-
κ
B/p65 ansloca ion we e also s udied. The esul s (Figu e 4)
showed ha p65 p o ein was o e exp essed in he nuclea in LPS-induced RAW264.7 cells;
howe e , i s exp ession was signi ican ly e e sed by PFF ea men (100 and 200
µ
g/mL),
indica ing ha PFF dis u bed he ansloca ion o p65 s imula ed by LPS. Collec i ely, he
abo e esul s sugges ed ha he an i-in lamma o y ac i i y o PFF was co ela ed wi h he
ac i a ion o MAPK, PIK/Ak , and NF-κB signaling pa hways.
Foods 2023, 12, x FOR PEER REVIEW 7 o 12
Figu e 3. Effec s o PFF on he exp ession o majo immune p o eins in MAPK and PI3K/Ak signal-
ing pa hways by wes e n blo ing. Rep esen a i e immunoblo s o p-ERK (A), p-JNK (B), p-p38 (C),
and p-Ak (D) p o eins a e shown. Da a we e exhibi ed as mean ± SD. * p < 0.05, ** p < 0.01, s. he
LPS- ea ed g oup (wi hou PFF).
Figu e 4. Effec s o PFF on p65 nuclea ansloca ion o LPS-s imula ed RAW264.7 cells.
Figu e 3.
E ec s o PFF on he exp ession o majo immune p o eins in MAPK and PI3K/Ak
signaling pa hways by wes e n blo ing. Rep esen a i e immunoblo s o p-ERK (
A
), p-JNK (
B
), p-p38
(
C
), and p-Ak (
D
) p o eins a e shown. Da a we e exhibi ed as mean
±
SD. * p< 0.05, ** p< 0.01,
s. he LPS- ea ed g oup (wi hou PFF).
Foods 2023,12, 2889 7 o 11
Foods 2023, 12, x FOR PEER REVIEW 7 o 12
Figu e 3. Effec s o PFF on he exp ession o majo immune p o eins in MAPK and PI3K/Ak signal-
ing pa hways by wes e n blo ing. Rep esen a i e immunoblo s o p-ERK (A), p-JNK (B), p-p38 (C),
and p-Ak (D) p o eins a e shown. Da a we e exhibi ed as mean ± SD. * p < 0.05, ** p < 0.01, s. he
LPS- ea ed g oup (wi hou PFF).
Figu e 4. Effec s o PFF on p65 nuclea ansloca ion o LPS-s imula ed RAW264.7 cells.
Figu e 4. E ec s o PFF on p65 nuclea ansloca ion o LPS-s imula ed RAW264.7 cells.
3.3. In es inal Regula o y Ac i i y o PFF
3.3.1. E ec s o PFF on he Gu Mic obio a
The inc easing e idence showed ha gu mic obio a and hei me aboli es a e closely
co ela ed wi h in lamma o y eac ions in o ganisms [
5
,
21
]. In his wo k, an
in i o
sim-
ula ion e men a ion by human ecal bac e ia was used o assess he p obio ic e ec s o
PFF acco ding o he BFBL gu model and explo e he co ela ion be ween gu mic obio a
and in es inal in lamma ion. Thus, we de e mined he a ge ed bac e ial coun in h ee
colon eac o s (R
1
, R
2
, and R
3
) used o simula e he human colon (Table 1). Compa ed
wi h he s abiliza ion pe iod (CK), 7-day PFF consump ion could inhibi se e al po en ially
pa hogenic bac e ia, including Alis ipes,Bilophia,En e obac e iaceae, and so on, which a e
gene ally co ela ed wi h in es inal mucosal inju y and in lamma o y diseases [
33
–
35
].
Howe e , he abundance o En e ococcus,Lac obacillus, and Rosebu ia was ob iously p o-
mo ed (p< 0.01). In addi ion, Bi idobac e ium also ended o inc ease sligh ly. Fo una ely,
all hese bac e ia can s eng hen he in es inal mucosal ba ie and ha e a posi i e ole in
alle ia ing in lamma ion by p oducing SCFAs [35–38].
3.3.2. E ec s o PFF on he Me aboli es
Conside ing some SCFA-p oducing bac e ia we e signi ican ly p omo ed by PFF, we
u he assessed he SCFA p oduc ion du ing he PFF in ake pe iod (Figu e 5). The esul
showed ha , du ing he s abiliza ion pe iod (CK), ace ic acid anked i s in SCFA con en ,
ollowed by p opionic acid and bu y ic acid. A e wa d, 7-day PFF consump ion ma kedly
inc eased he SCFA con en in R
1
, R
2
, and R
3
compa ed wi h he s abiliza ion pe iod
(CK) (
p< 0.05
). Among hese, he con en o ace ic acid, as he main componen o SCFAs,
inc eased by 1.19-, 1.03-, and 1.17- old in R
1
, R
2
, and R
3
, espec i ely. These esul s indica ed
ha PFF consump ion was a good sou ce o p omo e o SCFA in gu mic obio a. In
addi ion, high le els o SCFAs a e desi able because hei co esponding pH educ ion can
inhibi he g ow h o ha m ul bac e ia, he eby p e en ing colonic in lamma ion [
39
,
40
]. On
he whole, ou da a sugges ed a po en ial ole o PFF in a enua ing in es inal in lamma ion,
such as inc easing he ichness o speci ic bene icial bac e ia and con en o SCFAs, as well
as inhibi ing he g ow h o ha m ul bac e ia.
Foods 2023,12, 2889 8 o 11
Table 1.
qPCR alues (mean log copy numbe /mL
±
SD) o a ge ed mic obial g oup analysis in
ascending colon (R
1
), ans e se colon (R
2
), and descending colon (R
3
) o h ee expe imen pe iods
(CK, PFF, and WO). (* p< 0.05, ** p< 0.01, s. he CK).
Bac e ial G oup Compa men CK PFF WO
Alis ipes R17.07 ±0.01 5.31 ±0.21 ** 5.38 ±0.06 **
R28.69 ±0.07 8.58 ±0.14 8.36 ±0.06 *
R38.75 ±0.03 8.45 ±0.09 * 8.46 ±0.14 *
Bi idobac e ium R18.84 ±0.31 8.65 ±0.12 8.36 ±0.09
R28.30 ±0.38 8.65 ±0.20 8.46 ±0.08
R38.30 ±0.38 8.65 ±0.20 8.12 ±0.18
Bilophia R18.37 ±0.01 8.27 ±0.10 8.22 ±0.27
R28.39 ±0.01 8.16 ±0.09 8.23 ±0.24
R38.38 ±0.20 7.99 ±0.20 8.30 ±0.18
En e bac e iaceae R18.60 ±0.00 8.26 ±0.02 ** 7.83 ±0.05 **
R28.49 ±0.01 8.12 ±0.05 ** 7.90 ±0.02 **
R38.48 ±0.05 7.91 ±0.13 ** 8.03 ±0.04 **
En e ococcus R16.48 ±0.08 8.56 ±0.21 ** 8.87 ±0.12 **
R26.42 ±0.13 8.84 ±0.13 ** 8.82 ±0.01 **
R37.09 ±0.04 8.44 ±0.21 ** 8.40 ±0.02 **
Lac obacillus R13.68 ±0.00 3.99 ±0.02 ** 5.54 ±0.04 **
R23.60 ±0.03 4.44 ±0.15 ** 5.41 ±0.00 **
R33.98 ±0.00 4.42 ±0.07 ** 5.28 ±0.05 **
Rosebu ia R15.64 ±0.09 6.47 ±0.19 ** 4.93 ±0.02 **
R26.23 ±0.07 6.58 ±0.06 ** 5.56 ±0.03 **
R36.01 ±0.03 6.47 ±0.10 ** 5.29 ±0.06 **
Foods 2023, 12, x FOR PEER REVIEW 9 o 12
can inhibi he g ow h o ha m ul bac e ia, he eby p e en ing colonic in lamma ion
[39,40]. On he whole, ou da a sugges ed a po en ial ole o PFF in a enua ing in es inal
in lamma ion, such as inc easing he ichness o speci ic bene icial bac e ia and con en o
SCFAs, as well as inhibi ing he g ow h o ha m ul bac e ia.
Figu e 5. Effec s o PFF on SCFAs con en in he BFBL gu model. SCFAs con en in he ascending
colon (R1), ans e se colon (R2), and descending colon (R3) o h ee expe imen pe iods (CK, PFF,
and WO). Da a we e exhibi ed as mean ± SD. * p < 0.05, ** p < 0.01, s. he CK.
3.4. Ini ial Analysis o he Main Fla onoids in PFF
To con i m he componen basis o PFF on he effec o in lamma ion o gu mic obi-
o a, he bioac i e componen s o PFF we e u he analyzed using spec oscopic me hods.
The esul showed ha PFF con ained i e main compounds isola ed and iden i ied as
la onoids by HPLC combined wi h UV-Vis and FT-IR spec a (Figu es S2–S4). Mo eo e ,
LC-MS da a also showed ha he excime ion peaks [M-H]- o he i e la onoid com-
pounds we e 609.14691, 593.15125, 563.14044, 609.14630, and 593.15112, espec i ely (Fig-
u e S5). Based on he abo e da a and ele an e e ences [27,32,41–45], he i e compounds
we e p elimina ily in e ed as o ien in-2″-O-galac oside, apigenin-6,8-di-C-glucoside, i-
exin-2″-O-xyloside, o ien in-2″-O-glucoside, and i exin-2″-O-glucoside, espec i ely.
Fla onoids and hei de i a i es can p e en and ea a ious diseases, including in lam-
ma o y diseases [46]. P e ious s udies epo ed ha o ien in could effec i ely alle ia e
in lamma o y eac ions in LPS-induced RAW 264.7 cells [47,48], i exin could down- eg-
ula e in lamma o y media o s and educe he mig a ion o neu ophils o he in lamma-
o y ocus [49], and apigenin could ligh en he LPS-s imula ed BV2 mic oglia by inhibi ing
he elease o p o-in lamma o y cy okines [50]. Hence, hese i e la onoid compounds
may play a key ole in he an i-in lamma o y ac i i y exe ed by PFF, bu hei s uc u es
and biological ac i i ies s ill equi e u he analysis and de e mina ion. O e all, his
s udy could p omo e he de elopmen o no el na u al an i-in lamma o y agen s based
on bioac i e la onoids.
4. Conclusions
In his s udy, PFF ex ac ed om P. oe ida ui s could effec i ely inhibi he biosyn-
hesis and p oduc ion o he in lamma o y media o (NO) and p o-in lamma o y cy o-
kines (TNF-α and IL-6) om elie ing he symp oms o in lamma o y esponses. Mo eo-
e , i could also ob iously down- egula e he phospho yla ion le els o p o-in lamma-
o y p o eins (ERK, JNK, p38, Ak , and p65) in ol ed in egula ing MAPK, PI3K/Ak , and
NF-κB signaling pa hways. Mo e impo an ly, PFF exhibi ed he g ow h-p omo ing abil-
i y o se e al bene icial bac e ia, including Bi idobac e ium, En e ococcus, Lac obacillus, and
Rosebu ia, and SCFA gene a ion abili y in gu mic obio a. These me abolism effec s could
be conside ed o alle ia e in lamma o y eac ions. In addi ion, PFF mainly consis s o i e
la onoid compounds, which migh be ac i e compounds wi h an i-in lamma o y
Figu e 5.
E ec s o PFF on SCFAs con en in he BFBL gu model. SCFAs con en in he ascending
colon (R
1
), ans e se colon (R
2
), and descending colon (R
3
) o h ee expe imen pe iods (CK, PFF,
and WO). Da a we e exhibi ed as mean ±SD. * p< 0.05, ** p< 0.01, s. he CK.
3.4. Ini ial Analysis o he Main Fla onoids in PFF
To con i m he componen basis o PFF on he e ec o in lamma ion o gu mi-
c obio a, he bioac i e componen s o PFF we e u he analyzed using spec oscopic
me hods. The esul showed ha PFF con ained i e main compounds isola ed and iden-
i ied as la onoids by HPLC combined wi h UV-Vis and FT-IR spec a (Figu es S2–S4).
Mo eo e , LC-MS da a also showed ha he excime ion peaks [M
−
H]
−
o he i e
la onoid compounds we e 609.14691, 593.15125, 563.14044, 609.14630, and 593.15112,
espec i ely (Figu e S5). Based on he abo e da a and ele an e e ences [
27
,
32
,
41
–
45
], he
i e compounds we e p elimina ily in e ed as o ien in-2
00
-O-galac oside, apigenin-6,8-di-
C-glucoside, i exin-2
00
-O-xyloside, o ien in-2
00
-O-glucoside, and i exin-2
00
-O-glucoside,
espec i ely. Fla onoids and hei de i a i es can p e en and ea a ious diseases,
including in lamma o y diseases [
46
]. P e ious s udies epo ed ha o ien in could e -
ec i ely alle ia e in lamma o y eac ions in LPS-induced RAW 264.7 cells [
47
,
48
], i exin
could down- egula e in lamma o y media o s and educe he mig a ion o neu ophils o
Foods 2023,12, 2889 9 o 11
he in lamma o y ocus [
49
], and apigenin could ligh en he LPS-s imula ed BV2 mic oglia
by inhibi ing he elease o p o-in lamma o y cy okines [
50
]. Hence, hese i e la onoid
compounds may play a key ole in he an i-in lamma o y ac i i y exe ed by PFF, bu hei
s uc u es and biological ac i i ies s ill equi e u he analysis and de e mina ion. O e all,
his s udy could p omo e he de elopmen o no el na u al an i-in lamma o y agen s based
on bioac i e la onoids.
4. Conclusions
In his s udy, PFF ex ac ed om P. oe ida ui s could e ec i ely inhibi he biosyn he-
sis and p oduc ion o he in lamma o y media o (NO) and p o-in lamma o y cy okines
(TNF-
α
and IL-6) om elie ing he symp oms o in lamma o y esponses. Mo eo e ,
i could also ob iously down- egula e he phospho yla ion le els o p o-in lamma o y
p o eins (ERK, JNK, p38, Ak , and p65) in ol ed in egula ing MAPK, PI3K/Ak , and
NF-
κ
B signaling pa hways. Mo e impo an ly, PFF exhibi ed he g ow h-p omo ing abil-
i y o se e al bene icial bac e ia, including Bi idobac e ium,En e ococcus,Lac obacillus, and
Rosebu ia, and SCFA gene a ion abili y in gu mic obio a. These me abolism e ec s could
be conside ed o alle ia e in lamma o y eac ions. In addi ion, PFF mainly consis s o
i e la onoid compounds, which migh be ac i e compounds wi h an i-in lamma o y
p ope ies. The e o e, la onoids de i ed om P. oe ida ui , as new p e en i e and he a-
peu ic subs ances, p o ide he possibili y o de elop unc ional componen s o alle ia e
in lamma o y diseases in he ood and pha maceu ical indus ies.
Supplemen a y Ma e ials:
The ollowing suppo ing in o ma ion can be downloaded a : h ps://
www.mdpi.com/a icle/10.3390/ oods12152889/s1, Table S1: P ime s and ampli ica ion condi ions
o eal- ime PCR assays. Figu e S1: Schema ic diag am o BFBL gu model; Figu e S2: HPLC
ch oma og aphy spec um o PFF; Figu e S3: The UV-Vis spec a o pu i ied compounds
1
–
5
; Figu e
S4: The FT-IR spec a o pu i ied compounds
1
–
5
; Figu e S5: The LC-ESI-MS spec a o pu i ied
compound 1(A), 2(B), 3(C), 4(D), and 5(E).
Au ho Con ibu ions:
Concep ualiza ion, H.W. and T.R.; me hodology, Y.S.; so wa e, X.H.; ali-
da ion, M.Z.; o mal analysis, X.H.; in es iga ion, Y.S.; da a cu a ion, Y.S.; w i ing—o iginal d a
p epa a ion, X.H. and Y.S.; w i ing— e iew and edi ing, R.H., M.L. and H.W.; supe ision, H.W. All
au ho s ha e ead and ag eed o he published e sion o he manusc ip .
Funding:
This esea ch was unded by he Na u al Science Founda ion o China (31972157), Ling-
nan Mode n Ag icul u al Science and Technology Guangdong Labo a o y Independen Scien-
i ic Resea ch P ojec (NZ2021032), Guangdong P o incial Key Labo a o y o Food Quali y and
Sa e y (2020B1212060059), Spanish Minis y o Science and Inno a ion (AGL2016-75951-R, PID2019-
106071RB-I00), Sou h China Ag icul u al Uni e si y Doc o al S uden s (o e seas) Join Educa ion
P og ams (2018LHPY003), Na u al Science Founda ion o Guizhou P o ince (ZK[2023]451).
Da a A ailabili y S a emen : All da a a e epo ed in he a icle.
Acknowledgmen s: The au ho s acknowledge echnical suppo o Es eban Sáez and Da id Galán.
Con lic s o In e es : The au ho s decla e no con lic o in e es .
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2022
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[C ossRe ] [PubMed]