Access to this full-text is provided by MDPI.
Content available from Nutrients
This content is subject to copyright.
Citation: Abdo, A.; Zhang, C.;
Al-Dalali, S.; Hou, Y.; Gao, J.;
Yahya, M.A.; Saleh, A.; Aleryani, H.;
Al-Zamani, Z.; Sang, Y. Marine
Chitosan-Oligosaccharide
Ameliorated Plasma Cholesterol in
Hypercholesterolemic Hamsters by
Modifying the Gut Microflora, Bile
Acids, and Short-Chain Fatty Acids.
Nutrients 2023,15, 2923. https://
doi.org/10.3390/nu15132923
Academic Editor: Arrigo Cicero
Received: 9 May 2023
Revised: 13 June 2023
Accepted: 14 June 2023
Published: 27 June 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
nutrients
Article
Marine Chitosan-Oligosaccharide Ameliorated Plasma
Cholesterol in Hypercholesterolemic Hamsters by Modifying
the Gut Microflora, Bile Acids, and Short-Chain Fatty Acids
Abdullah Abdo 1,2 , Chengnan Zhang 3, Sam Al-Dalali 2, Yakun Hou 1, Jie Gao 1, Mohammed Abdo Yahya 4,
Ali Saleh 2,4, Hamzah Aleryani 1,2, Zakarya Al-Zamani 2and Yaxin Sang 1,*
1College of Food Science and Technology, Hebei Agricultural University, Baoding 071001, China;
drabdoabdullah2021@gmail.com (A.A.); yakunhou86@hotmail.com (Y.H.); gaojiehbu@163.com (J.G.)
2Department of Food Sciences and Technology, Faculty of Agriculture and Food Sciences, Ibb University,
Ibb 70270, Yemen; salihsam4@gmail.com (S.A.-D.); aliqashaab@gmail.com (A.S.);
keminmao@outlook.com (Z.A.-Z.)
3School of Food Science and Health, Beijing Technology and Business University, Beijing 100048, China;
zhangcn@btbu.edu.cn
4
Department of Food Science and Nutrition, College of Food and Agriculture Sciences, King Saud University,
Riyadh 11451, Saudi Arabia; mabdo@ksu.edu.sa
*Correspondence: yxsang1418@163.com
Abstract:
This study evaluated the cholesterol-alleviating effect and underlying mechanisms of
chitosan-oligosaccharide (COS) in hypercholesterolemic hamsters. Male hamsters (n= 24) were
divided into three groups in a random fashion, and each group was fed one particular diet, namely a
non-cholesterol diet (NCD), a high-cholesterol diet (HCD), and an HCD diet substituting 5% of the
COS diet for six weeks. Subsequently, alterations in fecal bile acids (BAs), short-chain fatty acids
(SCFAs), and gut microflora (GM) were investigated. COS intervention significantly reduced and
increased the plasma total cholesterol (TC) and high-density lipoprotein-cholesterol (HDL-C) levels in
hypercholesteremic hamsters. Furthermore, Non-HDL-C and total triacylglycerols (TG) levels were
also reduced by COS supplementation. Additionally, COS could reduce and increase food intake and
fecal SCFAs (acetate), respectively. Moreover, COS had beneficial effects on levels of BAs and GM
related to cholesterol metabolism. This study provides novel evidence for the cholesterol-lowering
activity of COS.
Keywords:
chitosan-oligosaccharide; plasma cholesterol; gut microflora; bile acids; short-chain
fatty acids
1. Introduction
A high level of blood cholesterol is usually considered the key risk factor for heart
disease [
1
]. Therefore, the successful control of blood cholesterol is considered an effective
approach to ameliorating heart disease occurrence [
2
]. Currently, there is an increasing
demand to develop new drugs that can maintain low plasma TC levels [
3
]. Though the
TC-lowering drugs available in the market are quite effective, their synthetic chemical
nature can pose serious negative effects on consumers, which can result in adverse con-
sequences
[4,5]
. Given the side effects of the existing TC medicines, natural bioactive
compounds are considered an alternative approach to TC-lowering agents, which remains
an urgent necessity.
As a natural bioactive compound, it was stated that marine chitosan-oligosaccharide
(COS) has the ability to alleviate liver diseases by improving dyslipidemia in obese
rats
[3,6,7]
. A recent report showed that dietary chitin oligosaccharides, a chitosan deriva-
tive, could markedly lower blood TC levels in atherosclerosis-prone apolipoprotein E-
deficient (Apoe
−/−
) mice [
8
]. In diet-induced obese rats, supplementation of COS into a
Nutrients 2023,15, 2923. https://doi.org/10.3390/nu15132923 https://www.mdpi.com/journal/nutrients
Nutrients 2023,15, 2923 2 of 13
diet could improve dyslipidemia via reducing TC and TG levels [
6
]. However, the results
from other animal and human experiments were shown to be inconsistent. It was found
that a diet with a COS supplement could not affect LDL-C in rats fed a high-fat diet [
9
].
In patients with hypercholesterolemia, the HDL-C, LDL-C, and TG levels remained un-
changed by COS consumption [
10
]. Therefore, further studies are needed to establish the
anti-hypercholesterolemia activity of COS. Although the mechanism of COS’s effect on
blood cholesterol is not yet fully understood, a few studies have proposed that COS’s effect
on blood cholesterol is likely mediated by regulating BA-related enzymes and genes [7].
In cholesterol homeostasis, GM participates in fermenting indigestible food compo-
nents and producing SCFAs, which are effective in lowering blood cholesterol [
11
–
13
]. In
addition, GM plays a crucial role in BAs’ metabolism. Approximately 90% of conjugated
and a small percentage of unconjugated BAs will return to the liver via enterohepatic
circulation after being reabsorbed in the distal ileum. The GM deconjugates a small fraction
of conjugated BAs as they travel toward the ileum [
14
]. Diminishing the conjugated BAs
and increasing the unconjugated BAs will decrease reabsorbed BAs and blood TC [
13
].
Therefore, fecal BAs should be summed up to reflect BA metabolism. In this regard, COS
had beneficial action on altering the GM and SCFAs production in mice [
15
,
16
]. Neverthe-
less, how COS prevents a high cholesterol diet-induced hypercholesterolemia, especially
the improvements in BAs and GM, remains largely unclear.
In cholesterol studies, hamsters are usually utilized as human surrogates as they closely
mimic the metabolic characteristics of human cholesterol. To the best of our knowledge,
the effect of COS in hypercholesterolemic hamsters is still unclear. Thus, this research was
conducted to evaluate the cholesterol-alleviating action of marine chitosan-oligosaccharide
in hypercholesterolemic hamsters and investigate the bile acids, SCFAs, and intestinal
microbiota alteration in relation to COS’s cholesterol-alleviating action. In this study, the
effect of COS on GM and the BA profiles of hamsters fed high cholesterol time were
evaluated for the first time.
2. Materials and Methods
2.1. Materials
Chitosan-oligosaccharide was given by Qingdao BZ Oligo Biotech Co., Ltd. (Qingdao,
China). COS (91.2% degree of deacetylation, average molecular weight of 1231 Da) with
92.3% purity was produced by enzymatic hydrolysis from the chitosan of crab shells. The
standards, chemicals, and kits were purchased from Sigma-Aldrich (St. Louis, MO, USA).
2.2. Diets
NCD, HCD, and COS diets were produced by Trophic Animal Feed High-tech Co.,
Ltd. (Nantong, China). An NCD was produced by mixing the components, as shown in
Table 1. An HCD was prepared by mixing the NCD diet with cholesterol (0.2%). The 0.2%
cholesterol used in this study to induce hypercholesterolemia in hamsters was based on a
previous report [17] A COS was prepared by mixing the HCD diet with 5% COS (Table 1).
The quantity of COS used in this work was equivalent to 19 g/day daily consumption for
human based on humans consume 2000 kcal/day and physically achievable for adults [
18
].
2.3. Hamsters
Twenty-four male hamsters (8 weeks old) were purchased from Beijing Vital River
Laboratory Animal Technology Co., Ltd. (Beijing, China) and housed in a room (n= 2 per
cage) at 23
◦
C with changing light for twelve hours. The hamsters were acclimated for
2 weeks before being randomly divided into three groups (n= 8 each) and fed for 6 weeks
on one of the three diets (NCD, HCD, and COS). The hamster’s blood was collected at
weeks 0 and 6. Food intake and body weight were recorded every two days and one week,
respectively. After 6 weeks, the hamsters were sacrificed, and the organs were collected,
washed, and stored at
−
80
◦
C until analysis. Hamsters have been cared for according to
the guidelines of the national health institutes (NIH Publications No. 8023, revised 1978)
Nutrients 2023,15, 2923 3 of 13
and institutional protocols (serial No. UJS-LAER-2018042301). The experimental guidelines
were approved by the Animal Experimental Ethical Committee, Beijing Sport University
(Reference number: 2019008A).
Table 1.
Compositions of three diets: NCD, non-cholesterol diet; HCD, high-cholesterol diet; COS,
HCD with addition of 5% chitosan-oligosaccharide (COS).
NCD HCD COS
Ingredients
Corn starch 50.9 50.9 50.9
Sucrose 11.9 11.9 11.9
Lard 5 5 5
AIN 93 M Mineral Mix 4 4 4
AIN 93 Vitamin Mix 2 2 2
Gelatin 2 2 2
Cholesterol 0 0.2 0.2
Casein 24.2 24.2 24.2
Chitosan-oligosaccharide 0 0 5
Total (g) 100 100.2 105.2
Energy %
Protein 26 26 26
Carbohydrates 63 63 63
Fat 11 11 11
Total (kcal) 401 401 401
2.4. Plasma Lipid Measurements
TC, high-density lipoprotein-cholesterol (HDL-C), and total triacylglycerols (TG) in
hamsters’ plasma were detected using a biochemical analyzer (AU5800, Beckman Coulter
Co., Ltd., Brea, CA, USA) by following the procedure of commercial kits (Zhongsheng-
beikong Co., Ltd., Beijing, China). Non-HDL-C was determined by deducting HDL-C
from TC.
2.5. BAs Measurements
Briefly, BAs in 100 mg of fecal samples were extracted using 1 mL of methanol.
BAs in the optioned supernatant after 30 min of centrifugation at 12,000 rpm were ana-
lyzed. BAs were separated using a Dionex
™
UltiMate
™
3000 Rapid Separation LC (RSLC)
system (Thermo Scientific, Waltham, MA, USA) equipped with an HSS T3C18 column
(
2.1 mm ×100 mm
, 1.8
µ
m, Waters, Milford, CT, USA). The injection volume, temperature,
and flow rate were set to 50
◦
C, 10
µ
L, and 300
µ
L/min, respectively. Methanol (A) and
2 mmol/L of ammonium acetate (B) were used as the mobile phases. Furthermore, the
mass spectrometry was performed using a Thermo Scientific TM Q Exactive TM hybrid
quadrupole Orbitrap mass spectrometer equipped with a HESI-II probe. The negative
HESI-II spray voltage was 3.5 kV. The heated capillary temperature, sheath gas pressure,
auxiliary gas setting, and heated vaporizer temperature were 320
◦
C, 30 psi, 10 psi, and
300
◦
C, respectively. The auxiliary gas, sheath gas, and collision gas were all nitrogen at
1.5 m Torr. The full mass scan parameters were set as follows: an auto gain control target
under 1
×
10
6
, a resolution of 70,000, an m/zrange of 150–1500, and a maximum isolation
time of 50 ms [19].
2.6. Measurement of Fecal’s SCFAs and pH Values
The SCFAs in freeze-dried fecal samples were extracted by 0.1 mol/L of PBS buffer
(pH 7.4) and analyzed using Agilent 1260 UV High-Performance Liquid Chromatogra-
phy equipped with a UV detector and Aminex HPX-87H column (Bio-Rad Laboratories,
Hercules, CA, USA), respectively. SCFAs were separated using 0.005 N of sulfuric acid.
pH values in fecal samples were measured using a pH meter (Mettler Toledo, Zurich,
Switzerland) after dilution with distilled water [19,20].
Nutrients 2023,15, 2923 4 of 13
2.7. Measurement of Intestinal Microflora
Total bacteria’s DNA in fresh feces was extracted and measured using the QIAamp
®
DNA
Mini Kit (Qiagen, Valencia, CA, USA) and the Nano-Drop 1000 spectrophotometer (Nano-Drop
Technologies, Wilmington, DE, USA), respectively. The regions of the 16S rRNA gene in V3-V4
were amplified using commercial forward primer 338 F (5
0
-ACTCCTACGGGAGGCAGCA-3
0
)
and the reverse primer 806 R (5
0
-GGACTACHVG GGTWTCTAAT-3
0
). After purification of
amplicons using the AxyPrep DNA gel extraction kit (Axygen Biosciences, Union City, CA, USA)
and quantification using QuantiFluor
™
-ST (Promega, Madison, WI, USA), the sequencing was
performed at Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China) on a MiSeq
platform (Illumina, San Diego, CA, USA) [19].
2.8. Data Analysis
SPSS software (version 25.0, SPSS Inc., Chicago, IL, USA) was used to perform a one-
way analysis of variance (ANOVA) on the data. The significant changes were determined
as the p-value was less than 0.05. Spearman’s correlation was performed to find the
correlations between parameters of cholesterol metabolism and relative genus abundances.
A free online platform of Majorbio’s I-Sanger (www.i-sanger.com) was used to analyze
the microbiome data after normalization on 15 September 2022. Alpha diversity was
assessed by computing Simpson and Shannon indexes. The rationality and efficiency of
the sequencing depth were determined using rarefaction analysis. Beta diversity was
estimated by partial least squares discriminant analysis (PLS-DA) and by hierarchical
cluster analysis. The data were analyzed on the free online platform of Majorbio I-Sanger
Cloud Platform (www.i-sanger.com) on 15 September 2022. Values were expressed as an
average ±standard deviation (SD).
3. Results
3.1. Food Intake, Energy Intake, and Body and Organ Weights
The energy and food intake of the COS hamsters were significantly (p< 0.05) slower
than those of the HCD hamsters, suggesting that COS may decrease the hamster’s appetite.
At week 6, COS reduced the body weight of hypercholesteremic hamsters, but COS did not
significantly change the body weight. COS also had no significant effect on organ weights,
except that the weight of epididymal fat in COS hamsters was significantly (
p< 0.05
) smaller
than that of the HCD and NCD hamsters (Table 2).
Table 2.
Food intake, energy intake, and body and organ weights in hamsters fed one of the three
diets: NCD, non-cholesterol diet; HCD, high-cholesterol diet; COS, HCD with addition of 5% chitosan-
oligosaccharide (COS).
NCD HCD COS pValue
Food intake
(g/hamster/day) 10.481 ±0.174 c10.924 ±0.145 a10.451 ±0.254 bc 0.031
Energy intake 40.877 ±0.681 b43.698 ±0.581 a39.714 ±0.965 b0.001
(kcal/hamster/day)
Body weights (g)
week 0 142.712 ±10.302 137.887 ±12.922 141.375 ±7.448 0.771
week 6 147.725 ±18.378 149.5 ±13.318 139.425 ±14.41 0.233
Organ weights (g)
Liver 5.5453 ±1.030 b7.707 ±1.189 a7.336 ±1.973 a0.005
Heart 0.65 ±0.068 0.66 ±0.064 0.635 ±0.076 0.932
Kidney 1.216 ±0.244 1.27 ±0.097 1.37 ±0.09 0.343
Testis 4.046 ±0.595 4.511 ±0.686 4.136 ±0.434 0.472
Epididymal fat 2.882 ±0.713 ab 3.807 ±0.806 a2.158 ±1.192 b0.006
Perirenal fat 1.205 ±0.485 1.43 ±0.386 1.001 ±0.513 0.12
Values were expressed as mean
±
SD (n= 8).
a,b,c
Means at the same row with different letters differed significantly
at p< 0.05.
Nutrients 2023,15, 2923 5 of 13
3.2. Plasma Lipid Profile
As revealed in Table 3, the hamsters fed a 0.2% cholesterol diet for 6 weeks significantly
elevated plasma TC and non-HDL-C compared with those fed an NCD diet, indicating
that the hamster’s experimental hypercholesterolemia model was successful. The elevated
plasma TC in HCD-diet-fed hamsters was successfully (p< 0.05) reversed by 5% COS
supplementation. Non-HDL-C and TG levels were also decreased by COS. Similarly, plasma
HDL-C in the NCD and COS groups was higher (p< 0.05) than that of the HCD group.
Table 3.
Plasma total cholesterol (TC), triacylglycerol (TG), high-density lipoprotein cholesterol
(HDL-C), and non-HDL-C in hamsters fed one of three diets: NCD, non-cholesterol diet; HCD,
high-cholesterol diet; COS, HCD with addition of 5% chitosan-oligosaccharide (COS).
NCD HCD COS pValue
Week 0
TC (mmol/L) 4.047 ±1.816 4.142 ±0.749 4.103 ±0.443 0.992
HDL-C (mmol/L) 3.081 ±0.448 3.057 ±0.549 3.007 ±0.365 0.192
Non-HDL-C (mmol/L) 0.966 ±0.120 1.582 ±0.328 1.598 ±0.175 0.597
TG (mmol/L) 1.476 ±0.734 1.598 ±0.593 1.512 ±0.709 0.192
Week 6
TC (mmol/L) 5.076 ±0.396 c7.897 ±0.910 a6.901 ±0.601 b<0.001
HDL-C (mmol/L) 3.430 ±0.258 a2.503 ±0.733 b3.245 ±0.735 a<0.001
Non-HDL-C (mmol/L) 1.646 ±0.405 c5.393 ±0.607 a3.656 ±1.063 b<0.001
TG (mmol/L) 1.578 ±0.257 b2.602 ±0.421 a1.793 ±0.594 b<0.001
Values were expressed as mean
±
SD (n= 8).
a,b,c
Means at the same row with different letters differed significantly
at p< 0.05.
3.3. Fecal BAs
Briefly, The COS diet could reverse the high-cholesterol-induced BA alteration. Com-
pared with HCD, COS supplementation led to an increase in the excretion of unconjugated
fecal BAs, including allocholic acid (AlloCA), cholic acid (CA), gamma-muricholic acid
(
γ
-MCA), and deoxycholic acid (DCA). Excretion of other unconjugated BAs was also
increased by COS feeding, but there was no significant alteration in comparison with the
HCD group. In contrast, COS supplementation significantly decreased conjugated BAs,
including glycolithocholic acid (GLCA), glycohyodeoxycholic acid (GHDCA), taurolitho-
cholic acid (TLCA), taurochenodeoxycholic acid (TCDCA), and taurodeoxycholic acid
(TDCA), which were significantly (p< 0.05) decreased by COS supplementation, indicating
that conjugated BAs were converted into unconjugated BAs (Table 4).
3.4. Fecal SCFA Contents and pH Value
The fecal SCFA analysis found the major SCFAs were butyrate and acetate
(
10.283 ±1.776 mg/day/hamster
), followed by the minor propionate in fecal NCD ham-
sters (Table 5). On the one hand, COS could significantly (p< 0.05) enhance acetate
production in HCD hamsters. On the other hand, the generation of propionate and bu-
tyrate, as well as total SCFAs, were also increased, but they were not significantly (p< 0.05)
change by the addition of COS (Table 5). Results displayed that pH values of feces in the
hamster groups were in the order of HCD > COS > NCD (Table 5). In short, hamsters fed
with COS had a markedly (p< 0.05) lower fecal pH value than the HCD group.
Nutrients 2023,15, 2923 6 of 13
Table 4.
Fecal bile acids quantity at week 6 in hamsters fed one of three diets: NCD, non-cholesterol
diet; HCD, high-cholesterol diet; COS, HCD with addition of 5% chitosan-oligosaccharide (COS).
Bile Acids (µg/Day/Hamster) NCD HCD COS pValue
Unconjugated
Allocholic Acid 85.915 ±7.709 a0.605 ±0.092 b72.168 ±2.520 a<0.001
Apocholic acid 140.871 ±23.196 89.621 ±1.84 110.233 ±8.352 0.126
Cholic acid 20.578 ±1.550 a5.892 ±2.461 b18.372 ±3.245 a<0.001
Alpha-muricholic acid 1.838 ±0.085 b2.765 ±0.567 a8.238 ±0.698 a<0.001
Gamma -muricholic Acid 3.094 ±0.748 b7.269 ±2.367 b21.244 ±4.853 a<0.001
Omega -muricholic acid 496.639 ±24.622 a282.820±8.018 b384.075 ±16.634 ab <0.001
Chenodeoxycholic acid 97.371 ±4.579 121.613 ±8.430 157.724 ±21.487 0.465
Deoxycholic acid 1200.654 ±89.140 a651.065 ±80.756 b916.169 ±75.202 a<0.001
Hyodeoxycholic acid 27.534 ±1.795 a9.880 ±0.606 b19.203 ±1.705 a<0.001
Isoalblithocholic acid 683.351 ±31.117 453.915 ±25.199 683.726 ±20.337 0.136
7-ketodeoxycholic acid 33.260 ±5.170 32.864 ±3.13 47.268 ±9.721 0.159
7-ketolithocholic acid 7.972 ±1.827 11.809 ±2.694 14.670 ±4.548 0.179
12-ketolithocholic acid 428. 92 ±40.582 255.830 ±60.334 318.718 ±17.268 0.064
Lithocholic acid 1158.628 ±107.725 1081.634 ±101.04 1333.827 ±103.588 0.133
Murocholic acid 13.782 ±4.008 12.644 ±1.145 36.296 ±4.898 0.095
Ursodeoxycholic acid 56.366 ±5.591 47.326 ±5.895 61.695 ±3.170 0.269
Glycine-conjugated
Glycocholic acid 2.741 ±0.534 a0.29 ±0.0351 b0.393 ±0.042 b<0.001
Glycochenodeoxycholic acid 2.217 ±0.740 a0.1571 ±0.0359 b0.190 ±0.064 b<0.001
Glycodeoxycholic acid 4.089 ±0.726 a1.882 ±0.346 b2.109 ±0.662 b0.019
Glycohyodeoxycholic acid 0.296 ±0.034 b0.782 ±0.140 a0.277 ±0.060 b<0.001
Glycolithocholic acid 3.336 ±0.776 b7.927 ±1.55 a4.1414 ±1.050 b<0.001
Taurine-conjugated
Taurocholic acid 0.903 ±0.010 a0.340 ±0.0186 b0.435 ±0.097 b<0.001
Taurochenodeoxycholic acid 0.276 ±0.056 ab 0.351 ±0.055 a0.227 ±0.010 ab 0.107
Taurodeoxycholic acid 0.358 ±0.017 ab 0.412 ±0.053 a0.287 ±0.053 b<0.001
Taurohyodeoxycholic acid 0.254 ±0.011 c1.244 ±0.025 a1.211 ±0.019 a<0.001
Taurolithocholic acid 0.129 ±0.012 b0.320 ±0.073 a0.124 ±0.025 b<0.001
Values were expressed as mean
±
SD (n= 8).
a,b,c
Means at the same row with different letters differed significantly
at p< 0.05.
Table 5.
Fecal short-chain fatty acid (SCFA) quantity and PH at week 6 in hamsters fed one of
three diets: NCD, non-cholesterol diet; HCD, high-cholesterol diet; COS, HCD with addition of 5%
chitosan-oligosaccharide (COS).
NCD HCD COS pValue
SCFAs (mg/day/hamster)
Acetate 16.731 ±2.750 ab 10.283 ±1.776 c32.554 ±7.933 a<0.001
Propionate 2.715 ±0.271 ab 1.215 ±0.271 b2.179 ±2.179 ab 0.07
Butyrate 347.207 ±20.788 327.517 ±33.893 343.712 ±23.534 0.785
Total SCFAs 366.654 ±21.094 339.016 ±34.119 378.446 ±20.860 0.928
PH
Fecal PH value 5.740 ±0.176 c6.427 ±0.056 a6.185 ±0.190 b<0.001
Values were expressed as mean
±
SD (n= 8).
a,b,c
Means at the same row with different letters differed significantly
at p< 0.05.
3.5. Overall Structure and Composition of GM
To evaluate the influence of dietary COS on GM composition, the GM in fresh fecal
hamsters was determined. The GM by the administration of COS was revealed by analyzing
the structure and composition of gut bacteria.
Nutrients 2023,15, 2923 7 of 13
GM diversity was decreased by COS addition, including the increased Simpson index
and the decreased Shannon index at the OUT level (Figure 1A,B). Furthermore, the PLS-
DA analysis showed a distinct divergence in GM composition between the three groups
(Figure 1C). Hierarchical clustering tree results showed that COS was separated from HCD
(Figure 1D).
Nutrients 2023, 15, x FOR PEER REVIEW 7 of 14
Table 5. Fecal short-chain fatty acid (SCFA) quantity and PH at week 6 in hamsters fed one of three
diets: NCD, non-cholesterol diet; HCD, high-cholesterol diet; COS, HCD with addition of 5% chi-
tosan-oligosaccharide (COS).
NCD HCD COS p Value
SCFAs (mg/day/hamster)
Acetate 16.731 ±2.750
ab 10.283 ± 1.776 c 32.554 ± 7.933 a <0.001
Propionate 2.715 ± 0.271 ab 1.215 ± 0.271 b 2.179 ± 2.179 ab 0.07
Butyrate 347.207 ± 20.788 327.517 ± 33.893 343.712 ± 23.534 0.785
Total SCFAs 366.654 ± 21.094 339.016 ± 34.119 378.446 ± 20.860 0.928
PH
Fecal PH value 5.740 ± 0.176 c 6.427 ± 0.056 a 6.185 ± 0.190 b <0.001
Values were expressed as mean ± SD (n = 8). a,b,c Means at the same row with different letters differed
significantly at p < 0.05.
3.5. Overall Structure and Composition of GM
To evaluate the influence of dietary COS on GM composition, the GM in fresh fecal
hamsters was determined. The GM by the administration of COS was revealed by analyz-
ing the structure and composition of gut bacteria.
GM diversity was decreased by COS addition, including the increased Simpson in-
dex and the decreased Shannon index at the OUT level (Figure 1A,B). Furthermore, the
PLS-DA analysis showed a distinct divergence in GM composition between the three
groups (Figure 1C). Hierarchical clustering tree results showed that COS was separated
from HCD (Figure 1D).
Figure 1.
Effect of COS supplementation on gut flora diversity in hamsters. Alpha diversity assessed
by using a Simpson index (
A
) and Shannon index (
B
) Beta diversity assessed by using PLS-DA
analysis (
C
) and hierarchical clustering tree (
D
) on OTU level. NCD, non-cholesterol diet; HCD,
high-cholesterol diet; COS, HCD with addition of 5% chitosan-oligosaccharide (COS). Values were
expressed as mean
±
SD.
a
and
b
Means at the same row with different letters differed significantly at
p< 0.05 by one-way ANOVA analysis.
According to the Simpson and Shannon indexes, the rarefaction curve has flattened
over time, indicating that most of the flora diversity has been retrieved from a sufficient
volume (Figure S1).
The compositions of gut bacteria are shown in Figures 2and 3. At the phylum level,
hamsters fed with COS had a significant increase in the number of Bacteroidetes, whereas
they had a significant decrease in the number of Firmicutes and Firmicutes/Bacteroidetes ratio
(F/B) in comparison with HCD hamsters (Figure 2A,D).
Nutrients 2023,15, 2923 8 of 13
Nutrients 2023, 15, x FOR PEER REVIEW 8 of 14
Figure 1. Effect of COS supplementation on gut flora diversity in hamsters. Alpha diversity assessed
by using a Simpson index (A) and Shannon index (B) Beta diversity assessed by using PLS-DA anal-
ysis (C) and hierarchical clustering tree (D) on OTU level. NCD, non-cholesterol diet; HCD, high-
cholesterol diet; COS, HCD with addition of 5% chitosan-oligosaccharide (COS). Values were ex-
pressed as mean ± SD.
a
and
b
Means at the same row with different letters differed significantly at
p < 0.05 by one-way ANOVA analysis.
According to the Simpson and Shannon indexes, the rarefaction curve has flattened
over time, indicating that most of the flora diversity has been retrieved from a sufficient
volume (Figure S1).
The compositions of gut bacteria are shown in Figures 2 and 3. At the phylum level,
hamsters fed with COS had a significant increase in the number of Bacteroidetes, whereas
they had a significant decrease in the number of Firmicutes and Firmicutes/Bacteroidetes ra-
tio (F/B) in comparison with HCD hamsters (Figure 2A,D).
At the family level, Eubacteriaceae, Muribaculaceae, Erysipelotrichaceae, Rumino-
coccaceae, Bacteroidaceae, Lachnospiraceae, and Tannerellaceae represented the most
abundant families (Figure 2B). The numbers of Muribaculaceae and Bacteroidaceae in
COS were higher than that of the HCD group, while the concentrations of Erysipelotri-
chacea, Eubacteriaceae, and Ruminococcaceae were alleviated in the COS group (Figure
2B).
Figure 2. Effect of COS supplementation on gut flora composition in hamsters at phylum and family
levels. Composition of gut flora at phylum level (A), family level (B), the relative abundance of Fir-
micutes and Bacteroidetes (C), and the ratio of Firmicutes/Bacteroidetes (D) NCD, non-cholesterol
diet; HCD, high-cholesterol diet; COS, HCD with addition of 5% chitosan-oligosaccharide (COS).
Values were expressed as mean ± SD.
a
and
b
Means at the same row with different letters differed
significantly at p < 0.05 by one-way ANOVA analysis.
Figure 2.
Effect of COS supplementation on gut flora composition in hamsters at phylum and family
levels. Composition of gut flora at phylum level (
A
), family level (
B
), the relative abundance of
Firmicutes and Bacteroidetes (
C
), and the ratio of Firmicutes/Bacteroidetes (
D
) NCD, non-cholesterol
diet; HCD, high-cholesterol diet; COS, HCD with addition of 5% chitosan-oligosaccharide (COS).
Values were expressed as mean
±
SD.
a
and
b
Means at the same row with different letters differed
significantly at p< 0.05 by one-way ANOVA analysis.
Nutrients 2023, 15, x FOR PEER REVIEW 9 of 14
At the genus level, it was found that norank_f_Eubacteriaceae, norank_f_Muribaculaceae,
norank_f_Erysipelotrichaceae, Ruminococcus, Bacteroides, and Parabacteroides were the most
abundant genera. In particular, the levels of SCFA-producing bacteria in COS, including
norank_f_Muribaculaceae, Bacteroides, Parabacteroides, and Parasutterella, were significantly
higher than those in the HCD group, while COS significantly reduced the phylum of Fir-
micutes, including norank_f_Eubacteriaceae, norank_f_Erysipelotrichaceae, and Ruminococcus
(Figure 3).
Figure 3. Effect of COS supplementation on gut flora composition in hamsters at genus level. NCD,
non-cholesterol diet; HCD, high-cholesterol diet; COS, HCD with addition of 5% chitosan-oligosac-
charide (COS).
3.6. Spearman Correlation
Spearman’s correlation analysis examined the association between the modification
of GM and cholesterol-related markers. In comparison between the most abundant gen-
era, norank_f_Erysipelotrichaceae showed a negative correlation with acetate and unconju-
gated γ-MCA, while Bacteroides and Parabacteroides displayed a positive association with
acetate, and unconjugated γ-MCA and only Bacteroides presented a negative correlation
with conjugated glycocholic acid and glycochenodeoxycholic acid (GCDCA) and tau-
rocholic acid (TCA). In this study, we found that norank_f_Muribaculaceae was correlated
positively with unconjugated γ-MCA and negatively with conjugated GCDCA, while nor-
ank_f_Eubacteriaceae was correlated negatively with CA, Alpha-CA, GLCA, Taurohyo-
deoxycholic acid (THCA), and TLCA and positively with AlloCA, apocholic acid, HDCA,
and TCA, as presented in Figure 4.
Figure 3.
Effect of COS supplementation on gut flora composition in hamsters at genus level.
NCD, non-cholesterol diet; HCD, high-cholesterol diet; COS, HCD with addition of 5% chitosan-
oligosaccharide (COS).
Nutrients 2023,15, 2923 9 of 13
At the family level, Eubacteriaceae, Muribaculaceae, Erysipelotrichaceae, Ruminococ-
caceae, Bacteroidaceae, Lachnospiraceae, and Tannerellaceae represented the most abun-
dant families (Figure 2B). The numbers of Muribaculaceae and Bacteroidaceae in COS
were higher than that of the HCD group, while the concentrations of Erysipelotrichacea,
Eubacteriaceae, and Ruminococcaceae were alleviated in the COS group (Figure 2B).
At the genus level, it was found that norank_f_Eubacteriaceae,norank_f_Muribaculaceae,
norank_f_Erysipelotrichaceae, Ruminococcus, Bacteroides, and Parabacteroides were the most
abundant genera. In particular, the levels of SCFA-producing bacteria in COS, including
norank_f_Muribaculaceae, Bacteroides, Parabacteroides, and Parasutterella, were significantly
higher than those in the HCD group, while COS significantly reduced the phylum of
Firmicutes, including norank_f_Eubacteriaceae,norank_f_Erysipelotrichaceae, and Ruminococcus
(Figure 3).
3.6. Spearman Correlation
Spearman’s correlation analysis examined the association between the modification of
GM and cholesterol-related markers. In comparison between the most abundant genera,
norank_f_Erysipelotrichaceae showed a negative correlation with acetate and unconjugated
γ
-
MCA, while Bacteroides and Parabacteroides displayed a positive association with acetate, and
unconjugated
γ
-MCA and only Bacteroides presented a negative correlation with conjugated
glycocholic acid and glycochenodeoxycholic acid (GCDCA) and taurocholic acid (TCA). In
this study, we found that norank_f_Muribaculaceae was correlated positively with uncon-
jugated
γ
-MCA and negatively with conjugated GCDCA, while norank_f_Eubacteriaceae
was correlated negatively with CA, Alpha-CA, GLCA, Taurohyodeoxycholic acid (THCA),
and TLCA and positively with AlloCA, apocholic acid, HDCA, and TCA, as presented in
Figure 4.
Nutrients 2023, 15, x FOR PEER REVIEW 10 of 14
Figure 4. Spearman’s correlation analysis between 21 key genera and SCFAs and bile acids in ham-
sters fed one of three diets: NCD, non-cholesterol diet; HCD, high-cholesterol diet; COS, HCD with
addition of 5% chitosan-oligosaccharide (COS). The red to blue in the heatmap represented the R
value of spearman’s correlation changed from greater to lower. AlloCA, allocholic Acid; ApoCA,
apocholic acid; CA, cholic acid; Alpha-MA, α muricholic acid; Gamma-MA; γ-muricholic Acid;
Omega—MA, ω-muricholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; HDCA,
hyodeoxycholic acid; IsoLCA, isoalblithocholic acid; 7-KDCA,7-ketodeoxycholic acid; 7-KLCA, 7-
ketolithocholic acid; 12-KLCA, 12-ketolithocholic acid; LCA, lithocholic acid; MoCA, murocholic
acid; UDCA, ursodeoxycholic acid; GCA, glycocholic acid; GCDCA, glycochenodeoxycholic acid;
GDCA, glycodeoxycholic acid; GHDCA, glycohyodeoxycholic acid; GLCA, glycolithocholic acid;
TCA, taurocholic acid; TCDCA, taurochenodeoxycholic acid; TDCA, taurodeoxycholic acid; THCA,
taurohyodeoxycholic acid; TLCA, taurolithocholic acid.
4. Discussion
Although dietary cholesterol has a controversial effect on plasma TC, researchers
found a close correlation between high TC levels and heart disease [21–23]. In this study,
the dietary addition of COS at 5% was significantly effective in ameliorating TC in plasma
hypercholesteremic hamsters (Table 3). The amount of COS used in present study was
equivalent to 19 g/day daily based on human consumption of 2000 kcal/day which is phys-
ically achievable for an adult [18]. Our result was in agreement with the result of a previ-
ous study [7]. In their study, plasma cholesterol was lowered in rats fed a high-fat diet
with an added 5% COS [7]. In hyperlipidemic rats, COS remarkably lowered cholesterol
by promoting BA-related enzymes [3]. To our knowledge, alleviation of TC in hamsters
fed high cholesterol diet by COS was reported for the first time in our study. However,
COS in our study had a significant effect on improvement of plasma lipids, which was
consistent with previously reports [6,7].
In line, we found that the food intake in the COS group was significantly less than
that of the HCD group, suggesting the TC-lowering effect of COS is in part mediated by
reducing cholesterol intake (Table 2). Similarly, COS could reduce food intake in obese
mice [24]. Such a result may be related to the ability of COS to modulate appetite-related
hormones. It was reported that chitosan (source of COS) could successfully modulate ap-
petite-related hormones such as adiponectin and leptin [25].
Figure 4.
Spearman’s correlation analysis between 21 key genera and SCFAs and bile acids in
hamsters fed one of three diets: NCD, non-cholesterol diet; HCD, high-cholesterol diet; COS, HCD
with addition of 5% chitosan-oligosaccharide (COS). The red to blue in the heatmap represented the
R value of spearman’s correlation changed from greater to lower. AlloCA, allocholic Acid; ApoCA,
apocholic acid; CA, cholic acid; Alpha-MA,
α
muricholic acid; Gamma-MA;
γ
-muricholic Acid;
Omega—MA,
ω
-muricholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; HDCA,
hyodeoxycholic acid; IsoLCA, isoalblithocholic acid; 7-KDCA,7-ketodeoxycholic acid; 7-KLCA, 7-
ketolithocholic acid; 12-KLCA, 12-ketolithocholic acid; LCA, lithocholic acid; MoCA, murocholic
acid; UDCA, ursodeoxycholic acid; GCA, glycocholic acid; GCDCA, glycochenodeoxycholic acid;
GDCA, glycodeoxycholic acid; GHDCA, glycohyodeoxycholic acid; GLCA, glycolithocholic acid;
TCA, taurocholic acid; TCDCA, taurochenodeoxycholic acid; TDCA, taurodeoxycholic acid; THCA,
taurohyodeoxycholic acid; TLCA, taurolithocholic acid.
Nutrients 2023,15, 2923 10 of 13
4. Discussion
Although dietary cholesterol has a controversial effect on plasma TC, researchers
found a close correlation between high TC levels and heart disease [
21
–
23
]. In this study,
the dietary addition of COS at 5% was significantly effective in ameliorating TC in plasma
hypercholesteremic hamsters (Table 3). The amount of COS used in present study was
equivalent to 19 g/day daily based on human consumption of 2000 kcal/day which is
physically achievable for an adult [
18
]. Our result was in agreement with the result of a
previous study [
7
]. In their study, plasma cholesterol was lowered in rats fed a high-fat diet
with an added 5% COS [
7
]. In hyperlipidemic rats, COS remarkably lowered cholesterol by
promoting BA-related enzymes [
3
]. To our knowledge, alleviation of TC in hamsters fed
high cholesterol diet by COS was reported for the first time in our study. However, COS in
our study had a significant effect on improvement of plasma lipids, which was consistent
with previously reports [6,7].
In line, we found that the food intake in the COS group was significantly less than
that of the HCD group, suggesting the TC-lowering effect of COS is in part mediated by
reducing cholesterol intake (Table 2). Similarly, COS could reduce food intake in obese
mice [
24
]. Such a result may be related to the ability of COS to modulate appetite-related
hormones. It was reported that chitosan (source of COS) could successfully modulate
appetite-related hormones such as adiponectin and leptin [25].
The major pathway for reducing blood cholesterol is by enhancing its conversion
to BAs [
19
,
26
,
27
]. Therefore, we compared the excretion of fecal BAs among the three
groups. We found that the HCD diet had altered levels of several individuals of BAs in
feces compared with the NCD diet, whereas the COS diet could moderately reverse the
high cholesterol-induced alteration (Table 5), indicating that the cholesterol-alleviating
effect of COS was partially regulated via improving BA synthesis. In particular, COS could
reduce levels of several conjugated BAs, including T-DCA, THCA, TLCA, TCDCA, GLCA,
and GHDCA, while increasing the number of unconjugated BAs, such as CA,
γ
-MCA,
AlloCA, and DCA (Table 4). These results were in agreement with earlier findings stating
that COS could regulate BA synthesis-related enzymes [
3
]. It was reported that COS could
reduce plasma cholesterol in rats via upregulating the activity of cholesterol 7
α
-hydroxylase
(CYP7A1), which assists in conversion of cholesterol into bile acids [
3
]. This enzyme is
produced by gut flora, especially Bacteroides and Eubacterium [
28
]. What is more, a small
fraction of conjugated bile acids is deconjugated by gut microbiota as they travel down
toward the ileum [
14
]. In this regards, reducing the ratio of conjugated BAs to unconjugated
BAs will result in a decrease in the reabsorption of BAs as well as plasma TC [
13
]. Thus,
the enhanced excretion of unconjugated BAs in feces could be related to hydrolysis of
conjugated BAs via bile salt hydrolases (BSHs). This enzyme is produced by gut bacteria
such as Bacteroides spp., Listeria, and Brucella.Bacteroides spp. especially plays a major
role in deconjugation of conjugated BAs [
29
]. TC conversion into BAs can be promoted
by SCFAs [
13
,
30
]. In our study, COS could enhance SCFA-generating bacteria and BA
synthesis bacteria. Thus, the gut microbiota is essential for synthesis and excretion of BAs.
In connection with this finding, reducing the intestine pH may also boost the hydrolysis of
conjugated BAs to unconjugated BAs and improve the excretion of unconjugated BAs [
13
].
COS could obviously reduce the fecal pH value of HCD hamsters, leading to enhanced
conjugated BA hydrolysis into unconjugated BAs. Secondary BAs are less well absorbed
and mostly secreted in the feces, leading to loss of plasma TC [
31
]. Thus, BA synthesis
and deconjugation are an effective way to regulate of cholesterol in the body, by taking
cholesterol out of the circulation to be used for the synthesis of new BAs, replacing those
lost in the feces [
31
]. To our knowledge, it is the first report that COS is able to influence
the BA profile in hamsters fed with a high-cholesterol diet.
Another mechanism connected to the TC-ameliorating ability of COS was partially
attributable to its effect on SCFA generation by GM. SCFAs are capable of diminishing blood
TC by promoting the conversion of cholesterol into BAs [
13
,
30
]. Dietary supplementation
with COS markedly enhanced acetate production (Table 5). The ability of COS to promote
Nutrients 2023,15, 2923 11 of 13
SCFA production has previously been reported, which is consistent with our findings [
8
].
This suggests that COS’s TC-ameliorating ability was accompanied by promoting SCFA
production by increasing the levels of SCFA-producing bacteria. Thus, the population of
microflora in the gut was investigated to prove this hypothesis. The present work showed
that COS diets could alter the composition of the GM in HCD hamsters by lowering and
boosting Firmicutes and Bacteroidetes, respectively, leading to a notable reduction of the
F/B ratio (Figure 2D). The F/B ratio is positively linked with cardiovascular diseases
and obesity [
27
]. Similarly, COS could increase Bacteroidetes abundance at the expense of
Firmicutes and lower the F/B ratio in colitis mice [32].
Muribaculaceae and Bacteroidaceae enrichments in the COS groups were observed at
the family level (Figure 2B), which are SCFA-producing bacteria [
33
–
35
]. Accompanied
by this observation, Erysipelotrichaceae in HCD hamsters was reduced by COS. Numerous
studies have shown that the Erysipelotrichaceae level was positively and negatively cor-
related with increased liver cholesterol and fecal cholesterol excretion, respectively [
36
].
At the genus level, COS could elevate the relative concentration of SCFA-generating bac-
teria, including norank_f_Muribaculaceae,Bacteroides, and Parabacteroides [
15
,
37
]. Previ-
ously, norank_f_Muribaculaceae,Bacteroides, and Parabacteroides were increased by COS
treatment [
32
,
38
,
39
], which was consistent with our study. In connection with this, SCFA-
generating bacteria, especially Bacteroides, play a crucial role in BA synthesis and ex-
cretion as mentioned above. COS could reduce norank_f_Erysipelotrichaceae, a bacteria
linked to fat (cholesterol) accumulation in the human liver [
40
]. On this point, a reduction
in norank_f_Erysipelotrichaceae connected with feeding COS diets may contribute to the
cholesterol-ameliorating ability of COS detected in the plasma.
In addition, results from Spearman’s correlation analysis showed that improvement in
acetate generation was positively associated with a rise in relative abundance of Bacteroides
and Parabacteroides and negatively associated with norank_f_Erysipelotrichaceae (Figure 4).
These results indicate the hypocholesterolemic activity of COS was in part mediated by
shifting GM composition and promoting SCFAs production. GM also plays a vital role
in regulating the type and quantity of BAs via promoting and modulating BA synthesis
excretion [
2
]. In the correlation analysis, the alterations in several BAs were associated with
modifications in the key GM genera (Figure 4). However, the effect of COS on the gut flora
of hypercholesterolemia hamsters was studied for the first time in this work. In short, COS
induced beneficial BA modification, which was associated positively with the abundance
of norank_f_Muribaculaceae,Bacteroides, and Parabacteroides in the gut. Our results suggested
that the alteration in the GM was attributable to the cholesterol-lowering effect of COS via
improved SCFA and BA generation.
5. Conclusions
This study revealed that dietary supplementation with 5% COS-alleviated hyper-
chloremia in hamsters via reducing food intake, SCFA generation, and alteration of GM
and BAs. The favorable GM-regulatory activity was characterized by reducing Firmicutes
and increasing Bacteroidetes and SCFA-producing bacteria. Dietary supplementation with
COS could reduce plasma TC via enhancement of fecal excretion of bile acids. Such benefit
was accompanied by improving SCFA generation and remodeling gut flora. This work may
provide valuable evidence for future clinical trials of COS for hypercholesterolemic subjects.
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/nu15132923/s1, Figure S1: Rarefaction curves of Sobs index (A)
and Shannon index (B) in hamsters fed one of the three diets: NCD, Non-cholesterol diet; HCD, high
cholesterol diet; COS, HCD with addition of 5% chitosan-oligosaccharides (COS) (n= 6).
Author Contributions:
Methodology, A.A., C.Z. and S.A.-D.; Software, H.A.; Formal analysis,
Z.A.-Z.
; Investigation, Y.H. and J.G.; Writing—original draft, A.A.; Writing—review & editing,
A.A., M.Y. and A.S.; Project administration, Y.S. All authors have read and agreed to the published
version of the manuscript.
Nutrients 2023,15, 2923 12 of 13
Funding:
This study was supported financially by the Hebei Province High Level Talent Funding
Project (B2022005013) and National Key R D Program of China (2019YFD0902003).
Institutional Review Board Statement:
All the experimental protocols were approved and con-
ducted in accordance to the procedures of the Animal Experimental Ethical Committee, Beijing Sport
University (Reference number: 2019008A). Institutional guidelines (serial No. UJS-LAER-2018042301)
and the National Institutes of Health guide (NIH Publications No. 8023, revised1978) for animal care
and use were followed.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Wu, T.; Trevisan, M.; Genco, R.J.; Falkner, K.L.; Dorn, J.P.; Sempos, C.T. Examination of the Relation between Periodontal Health
Status and Cardiovascular Risk Factors: Serum Total and High Density Lipoprotein Cholesterol, C-Reactive Protein, and Plasma
Fibrinogen. Am. J. Epidemiol. 2000,151, 273–282. [CrossRef] [PubMed]
2.
Hao, W.; He, Z.; Zhu, H.; Liu, J.; Kwek, E.; Zhao, Y.; Ma, K.Y.; He, W.S.; Chen, Z.Y. Sea Buckthorn Seed Oil Reduces Blood
Cholesterol and Modulates Gut Microbiota. Food Funct. 2019,10, 5669–5681. [CrossRef] [PubMed]
3.
Jiang, Y.; Fu, C.; Liu, G.; Guo, J.; Su, Z. Cholesterol-Lowering Effects and Potential Mechanisms of Chitooligosaccharide Capsules
in Hyperlipidemic Rats. Food Nutr. Res. 2018,62, 1–15. [CrossRef] [PubMed]
4.
Campins, L.; Camps, M.; Riera, A.; Pleguezuelos, E.; Yebenes, J.C.; Serra-Prat, M. Oral Drugs Related with Muscle Wasting and
Sarcopenia. A Review. Pharmacology 2017,99, 1–8. [CrossRef] [PubMed]
5.
Wiggers, J.K.; Van Golen, R.F.; Verheij, J.; Dekker, A.M.; Van Gulik, T.M.; Heger, M. Atorvastatin Does Not Protect against
Ischemia-Reperfusion Damage in Cholestatic Rat Livers. BMC Surg. 2017,17, 35. [CrossRef]
6.
Deng, X.; Ye, Z.; Cao, H.; Bai, Y.; Che, Q.; Guo, J.; Su, Z. Chitosan Oligosaccharide Ameliorated Obesity by Reducing Endoplasmic
Reticulum Stress in Diet-Induced Obese Rats. Food Funct. 2020,11, 6285–6296. [CrossRef]
7.
Chiu, C.Y.; Yen, T.E.; Liu, S.H.; Chiang, M.T. Comparative Effects and Mechanisms of Chitosan and Its Derivatives on Hyperc-
holesterolemia in High-Fat Diet-Fed Rats. Int. J. Mol. Sci. 2020,21, 92. [CrossRef]
8.
Zhen, H.; Yan, Q.; Liu, Y.; Li, Y.; Yang, S.; Jiang, Z. Food Science and Human Wellness Chitin Oligosaccharides Alleviate
Atherosclerosis Progress in ApoE-/-Mice by Regulating Lipid Metabolism and Inhibiting Infl Ammation. Food Sci. Hum. Wellness
2022,11, 999–1009. [CrossRef]
9.
Bahijri, S.M.; Alsheikh, L.; Ajabnoor, G.; Borai, A. Effect of Supplementation with Chitosan on Weight, Cardiometabolic, and
Other Risk Indices in Wistar Rats Fed Normal and High-Fat/High-Cholesterol Diets Ad Libitum. Nutr. Metab. Insights 2017,10,
1178638817710666. [CrossRef]
10.
Metso, S.; Ylitalo, R.; Nikkilä, M.; Wuolijoki, E.; Ylitalo, P.; Lehtimäki, T. The Effect of Long-Term Microcrystalline Chitosan
Therapy on Plasma Lipids and Glucose Concentrations in Subjects with Increased Plasma Total Cholesterol: A Randomised
Placebo-Controlled Double-Blind Crossover Trial in Healthy Men and Women. Eur. J. Clin. Pharmacol.
2003
,59, 741–746.
[CrossRef]
11.
Ghaffarzadegan, T.; Essén, S.; Verbrugghe, P.; Marungruang, N.; Hållenius, F.F.; Nyman, M.; Sandahl, M. Determination of Free
and Conjugated Bile Acids in Serum of Apoe(
−
/
−
) Mice Fed Different Lingonberry Fractions by UHPLC-MS. Sci. Rep.
2019
,9,
3800. [CrossRef]
12. Soliman, G.A. Dietary Fiber, Atherosclerosis, and Cardiovascular Disease. Nutrients 2019,11, 1155. [CrossRef]
13.
Zhao, Y.; Liu, J.; Hao, W.; Zhu, H.; Liang, N.; He, Z.; Ma, K.Y.; Chen, Z.Y. Structure-Specific Effects of Short-Chain Fatty Acids on
Plasma Cholesterol Concentration in Male Syrian Hamsters. J. Agric. Food Chem. 2017,65, 10984–10992. [CrossRef]
14.
Zwicker, B.L.; Agellon, L.B. Transport and Biological Activities of Bile Acids. Int. J. Biochem. Cell Biol.
2013
,45, 1389–1398.
[CrossRef]
15.
Wang, Y.; Liu, S.; Tang, D.; Dong, R.; Feng, Q. Chitosan Oligosaccharide Ameliorates Metabolic Syndrome Induced by Overnutri-
tion via Altering Intestinal Microbiota. Front. Nutr. 2021,8, 743492. [CrossRef]
16.
Wu, M.; Li, J.; An, Y.; Li, P.; Xiong, W.; Li, J.; Yan, D.; Wang, M.; Zhong, G. Chitooligosaccharides Prevents the Development of
Colitis-Associated Colorectal Cancer by Modulating the Intestinal Microbiota and Mycobiota. Front. Microbiol.
2019
,10, 2101.
[CrossRef]
17.
Zhang, Z.; Wang, H.; Jiao, R.; Peng, C.; Wong, Y.M.; Yeung, V.S.Y.; Huang, Y.; Chen, Z. Choosing Hamsters but Not Rats as a
Model for Studying Plasma Cholesterol-lowering Activity of Functional Foods. Mol. Nutr. Food Res.
2009
,53, 921–930. [CrossRef]
18.
Schaafsma, G.; Slavin, J.L. Significance of Inulin Fructans in the Human Diet. Compr. Rev. Food Sci. Food Saf.
2015
,14, 37–47.
[CrossRef]
19.
Abdo, A.A.A.; Zhang, C.; Lin, Y.; Liang, X.; Kaddour, B.; Wu, Q.; Li, X.; Fan, G.; Yang, R.; Teng, C. Xylo-Oligosaccharides
Ameliorate High Cholesterol Diet Induced Hypercholesterolemia and Modulate Sterol Excretion and Gut Microbiota in Hamsters.
J. Funct. Foods 2021,77, 104334. [CrossRef]
Nutrients 2023,15, 2923 13 of 13
20.
Zhang, C.; Abdulaziz Abbod Abdo, A.; Kaddour, B.; Wu, Q.; Xin, L.; Li, X.; Fan, G.; Teng, C. Xylan-Oligosaccharides Ameliorate
High Fat Diet Induced Obesity and Glucose Intolerance and Modulate Plasma Lipid Profile and Gut Microbiota in Mice. J. Funct.
Foods 2020,64, 103622. [CrossRef]
21.
Poli, A.; Marangoni, F.; Corsini, A.; Manzato, E.; Marrocco, W.; Martini, D.; Medea, G.; Visioli, F. Phytosterols, Cholesterol Control,
and Cardiovascular Disease. Nutrients 2021,13, 2810. [CrossRef] [PubMed]
22.
Jeong, S.; Choi, S.; Kim, K.; Kim, S.M.; Lee, G.; Park, S.Y.; Kim, Y.; Son, J.S.; Yun, J.; Park, S.M. Effect of Change in Total Cholesterol
Levels on Cardiovascular Disease among Young Adults. J. Am. Heart Assoc. 2018,7, e008819. [CrossRef] [PubMed]
23.
Verschuren, W.M.M.; Jacobs, D.R.; Bloemberg, B.P.M.; Kromhout, D.; Menotti, A.; Aravanis, C.; Blackburn, H.; Buzina, R.;
Dontas, A.S.; Fidanza, F. Serum Total Cholesterol and Long-Term Coronary Heart Disease Mortality in Different Cultures:
Twenty-Five—Year Follow-up of the Seven Countries Study. JAMA 1995,274, 131–136. [CrossRef] [PubMed]
24.
Tang, D.; Wang, Y.; Kang, W.; Zhou, J.; Dong, R.; Feng, Q. Chitosan Attenuates Obesity by Modifying the Intestinal Microbiota
and Increasing Serum Leptin Levels in Mice. J. Funct. Foods 2020,64, 103659. [CrossRef]
25.
Fatahi, S.; Sayyari, A.A.; Salehi, M.; Safa, M.; Sohouli, M.; Shidfar, F.; Santos, H.O. The Effects of Chitosan Supplementation on
Anthropometric Indicators of Obesity, Lipid and Glycemic Profiles, and Appetite-Regulated Hormones in Adolescents with
Overweight or Obesity: A Randomized, Double-Blind Clinical Trial. BMC Pediatr. 2022,22, 527. [CrossRef]
26.
Groen, A.K.; Bloks, V.W.; Verkade, H.; Kuipers, F. Cross-Talk between Liver and Intestine in Control of Cholesterol and Energy
Homeostasis. Mol. Aspects Med. 2014,37, 77–88. [CrossRef]
27.
Hao, W.; Zhu, H.; Chen, J.; Kwek, E.; He, Z.; Liu, J.; Ma, N.; Ma, K.Y.; Chen, Z.Y. Wild Melon Seed Oil Reduces Plasma Cholesterol
and Modulates Gut Microbiota in Hypercholesterolemic Hamsters. J. Agric. Food Chem. 2020,68, 2071–2081. [CrossRef]
28.
Caliceti, C.; Punzo, A.; Silla, A.; Simoni, P.; Roda, G.; Hrelia, S. New Insights into Bile Acids Related Signaling Pathways in the
Onset of Colorectal Cancer. Nutrients 2022,14, 2964. [CrossRef]
29. Guzior, D.V.; Quinn, R.A. Review: Microbial Transformations of Human Bile Acids. Microbiome 2021,9, 140. [CrossRef]
30.
Jiao, A.R.; Diao, H.; Yu, B.; He, J.; Yu, J.; Zheng, P.; Huang, Z.Q.; Luo, Y.H.; Luo, J.Q.; Mao, X.B.; et al. Oral Administration of Short
Chain Fatty Acids Could Attenuate Fat Deposition of Pigs. PLoS ONE 2018,13, e0196867. [CrossRef]
31.
Pushpass, R.A.G.; Alzoufairi, S.; Jackson, K.G.; Lovegrove, J.A. Circulating Bile Acids as a Link between the Gut Microbiota and
Cardiovascular Health: Impact of Prebiotics, Probiotics and Polyphenol-Rich Foods. Nutr. Res. Rev.
2022
,35, 161–180. [CrossRef]
32.
Guo, C.; Zhang, Y.; Ling, T.; Zhao, C.; Li, Y.; Geng, M.; Gai, S.; Qi, W.; Luo, X.; Chen, L.; et al. Chitosan Oligosaccharides Alleviate
Colitis by Regulating Intestinal Microbiota and PPARγ/SIRT1-Mediated NF-KB Pathway. Mar. Drugs 2022,20, 96. [CrossRef]
33.
Hao, W.; Kwek, E.; He, Z.; Zhu, H.; Liu, J.; Zhao, Y.; Ma, K.Y.; He, W.S.; Chen, Z.Y. Ursolic Acid Alleviates Hypercholesterolemia
and Modulates the Gut Microbiota in Hamsters. Food Funct. 2020,11, 6091–6103. [CrossRef]
34.
Hu, W.; Lu, W.; Li, L.; Zhang, H. Both Living and Dead Faecalibacterium prausnitzii Alleviate House Dust Mite-Induced Allergic
Asthma through the Modulation of Gut Microbiota and Short-Chain Fatty Acid Production. J. Sci. Food Agric.
2021
,101, 5563–5573.
[CrossRef]
35.
Opeyemi, O.M.; Rogers, M.B.; Firek, B.A.; Janesko-Feldman, K.; Vagni, V.; Mullett, S.J.; Wendell, S.G.; Nelson, B.P.; New, L.A.;
Mariño, E.; et al. Sustained Dysbiosis and Decreased Fecal Short-Chain Fatty Acids after Traumatic Brain Injury and Impact on
Neurologic Outcome. J. Neurotrauma 2021,38, 2610–2621. [CrossRef]
36.
Sun, N.X.; Tong, L.T.; Liang, T.T.; Wang, L.L.; Liu, L.Y.; Zhou, X.R.; Zhou, S.M. Effect of Oat and Tartary Buckwheat—Based Food
on Cholesterol—Lowering and Gut Microbiota in Hypercholesterolemic Hamsters. J. Oleo Sci. 2019,68, 251–259. [CrossRef]
37.
Xiong, Y.; Ji, L.; Zhao, Y.; Liu, A.; Wu, D.; Qian, J. Sodium Butyrate Attenuates Taurocholate-Induced Acute Pancreatitis by
Maintaining Colonic Barrier and Regulating Gut Microorganisms in Mice. Front. Physiol. 2022,13, 813735. [CrossRef]
38.
Liu, W.; Li, X.; Zhao, Z.; Pi, X.; Meng, Y.; Fei, D.; Liu, D.; Wang, X. Effect of Chitooligosaccharides on Human Gut Microbiota and
Antiglycation. Carbohydr. Polym. 2020,242, 116413. [CrossRef]
39.
Zhang, C.; Jiao, S.; Wang, Z.A.; Du, Y. Exploring Effects of Chitosan Oligosaccharides on Mice Gut Microbiota in in Vitro
Fermentation and Animal Model. Front. Microbiol. 2018,9, 2388. [CrossRef]
40.
Kwek, E.; Zhu, H.; Ding, H.; He, Z.; Hao, W.; Liu, J.; Ma, K.Y.; Chen, Z.Y. Peony Seed Oil Decreases Plasma Cholesterol and
Favorably Modulates Gut Microbiota in Hypercholesterolemic Hamsters. Eur. J. Nutr. 2022,61, 2341–2356. [CrossRef]
Disclaimer/Publisher’s Note:
The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.
Content uploaded by Abdullah Abdulaziz Abbod Abdo
Author content
All content in this area was uploaded by Abdullah Abdulaziz Abbod Abdo on Jul 02, 2023
Content may be subject to copyright.
Content uploaded by Hamzah Aleryani
Author content
All content in this area was uploaded by Hamzah Aleryani on Jul 02, 2023
Content may be subject to copyright.
