Journal of Separation Science: Version of Record Doi: 10.1002/jssc.202000911

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LC–MS based plasma metabolomics study of the intervention effect of different polar parts of
Hawthorn on hyperlipidemia rats
Lu Zeng1,2,3,4† Lan Luo 1,2,3,4†, Qi Xue 1,2,3,4, Qiong He 1,2,3,4, Xingyu Chen 1,2,3,4, Jiang Meng
1,2,3,4
, Shumei Wang 1,2,3,4*, Shengwang Liang 1,2,3,4*
1
Guangdong Pharmaceutical University, Guangzhou, Guangdong, 510006, China
2
Key Laboratory of Digital Quality Evaluation of Chinese Materia Medica, Guangzhou,
Guangdong, 510006, China
3
Engineering ＆ Technology Research Center for Chinese Materia Medica Quality of the
Universities of Guangdong Province, Guangzhou, Guangdong, 510006, China
4
Engineering＆ Technology Research Center for Chinese Materia Medica Quality of
Guangdong Province, Guangzhou, Guangdong, 510006, China
† Lu Zeng and Lan Luo should be considered co-first authors
* Corresponding author
Prof. Dr. Shengwang Liang, Guangdong Pharmaceutical University, Guangzhou, Guangdong,
510006 China
E-mail: swliang371@126.com; Tel.: 020-39352172.
Shumei Wang: 2395903468@qq.com
Received: 30/08/2020; Revised: 30/08/2020; Accepted: 18/12/2020
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1002/jssc.202000911.
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Abbreviations: LC-MS: Liquid chromatography-mass spectrometry; LDL-C:
Low-density lipoprotein-cholesterol; OPLS-DA: Orthogonal partial least squares
discrimination analysis; PCA: Principal component analysis; QC: Quality control;
TCM: Traditional Chinese medicine; TC: Total cholesterol; TG: Triglycerides; VIP:
Variable importance of projection
Abstract
Hawthorn, a well-known traditional Chinese medicine is used for treatment
of dyspepsia syndrome, cardiovascular disease and hyperlipidemia. Hawthorn
has complex composition, therefore, the effective fraction and mechanisms of
action in alleviating hyperlipidemia are unknown. Therefore, the aim of this study
was to evaluate effects of four different polar components of hawthorn on hyperlipidemia
rats, and to explore underlying mechanisms of action through LC-MS based plasma
metabolomics. Hyperlipidemia rat model was established by feeding rats using
high-fat diet. High-fat model rats were then treated with 4 polar components of
hawthorn for 14 consecutive days. Plasma samples were collected and subjected
to biochemical and metabolomics analysis. Biochemical analysis showed that
hawthorn n-butanol and ethyl acetate extracts had the highest efficacy on
hyperlipidemia rats. Water fraction showed a partial effect, whereas petroleum
ether extract was not effective against hyperlipidemia rats. Furthermore, LC-MS
metabolomics analysis showed that the most effective fraction of hawthorn
reversed the metabolic disorder in plasma of hyperlipidemia rats . Metabolomics

analysis showed that hawthorn exerts its activity by modulating lipid
metabolism, energy metabolism, oxidative stress and amino acid metabolism.
Keywords: effective fraction, hawthorn, hyperlipidemia, metabolomics ,
traditional Chinese medicine, underlying mechanism
1. Introduction
Incidence of hyperlipidemia have increased exponentially due to unhealthy
eating habits and excessive energy intake. Hyperlipidemia is a common lipid
metabolism disorder characterized by high levels of total cholesterol (TC),
triglycerides (TG) and low-density lipoprotein-cholesterol (LDL-C) in the blood
[1]. Hyperlipidemia is implicated in pathogenesis of cardiovascular diseases,
such as essential hypertension, coronary heart disease and atherosclerosis, and
diabetes [2]. Previous studies report that treatment of hyperlipidemia reduces the
mortality rate of cardiovascular diseases. Currently, hyperlipidemia is managed
through lifestyle changes and use of lipid-lowering drugs [3]. However,
lipid-lowering drugs, such as statins and fibrates, are associated with adverse
effects, such as rhabdomyolysis, liver toxicity and sleep disorders [4,5].
Therefore, studies have explored traditional Chinese medicine (TCM) as an
alternative therapy, as it is safe and is associated with few toxic effects [6,7].
Hawthorn is an edible natural product with good medicinal effects. The main
chemical components in hawthorn include organic acids, flavonoids and

triterpenes. Therefore, it exerts a variety of drug effects, such as aiding digestion,
improving blood circulation and alleviating atherosclerosis [8,9]. Previous
studies report use of hawthorn as an alternative therapy for cardiovascular
diseases, such as hyperlipidem ia, hypertension and arrhythmia. Although the
lipid-lowering effect of hawthorn has been reported previously [10], effective
fraction and mechanisms of action of hawthorn in treatment of hyperlipidemia are
unknown. Advances in metabolomics provide a basis for study of small molecules
in living organisms.
Metabolomics is a powerful tool for qualitative and quantitative analysis of
small molecules and has been used widely in TCM studies . Changes of
metabolites after endogenous or exogenous interference in the body can be
determined through metabolomics [11]. Therefore, it is used for study of efficacy
and toxicity of TCM [12]. Currently, analysis techniques of metabolomics mainly
include liquid chromatography-mass spectrometry (LC-MS), gas
chromatography-mass spectrometry (GC-MS), and nuclear magnetic resonance
(NMR). LC-MS is characterized by high sensitivity and high robustness,
therefore, it is used to analyze a wide range of metabolites. LC-MS is the most
widely used method in metabolomics research [13,14]. This technology has
provided great convenience for our research [15].
The aim of this study was to explore the effective fraction and potential
mechanism of action of hawthorn for treatment of hyperlipidemia. A

hyperlipidemia model was established and treated with four fractions of hawthorn
(H-A, H-B, H-C and H-D fractions in descending polarity, corresponding to
water, n-butanol, ethyl acetate and petroleum ether extracts, respectively) for 14
consecutive days. Plasma samples were collected and analyzed through
biochemical assays and metabolomics. Results showed that H-B and H-C
fractions had significant effects on hyperlipidemia. The findings imply that
mechanism of action of hawthorn is through modulation of lipid metabolism,
energy metabolism, oxidative stress and amino acid metabolism.
2. Materials and Methods
2.1 Reagents and materials
Acetonitrile (HPLC grade) and formic acid (HPLC grade) were purchased
from Merck, Germany. All other chemicals and reagents were of analytical
grade. Ultrapure water was prepared using WP-UP-WF-10 ultrapure water
machine (Sichuan Waterpu Water Treatment Equipment Co., Ltd.).
2.2 Materials and extraction of different fractions of hawthorn
Hawthorn was purchased from Guangzhou Zhixin Pharmaceutical Co., Ltd.
(Guangzhou, China), identified by Associate Professor Hong - Yan Ma
(Guangdong Pharmaceutical University) and verified to have met standards
specified by Chinese Pharmacopoeia.

Hawthorn was crushed into crude powder, extracted 3 times with 12 times water for 100
minutes each time, and concentrated under reduced pressure to obtain a crude extract. The
crude extract was then extracted sequentially with petroleum ether, ethyl acetate and
n-butanol. Powders of different extract fractions (water (H-A), n-butanol (H-B), ethyl acetate
(H-C), petroleum ether (H-D)) were obtained through evaporation of the solvent and freeze
drying.
2.3 Animal experiment
Male and female Sprague-Dawley rats weighing 200-220g, were purchased
from Experimental Animal Center of Guangdong (Guangdong, China). All rats
were maintained under SPF standard laboratory conditions (temperature 24 ± 2
℃, humidity 60 ± 5%, dark/light for 12 hours cycle), giving appropriate food and
water daily throughout the experimental period. All experimental procedures
were approved by the Animal Care and Ethics Committee of Guangdong
Pharmaceutical University.
After one week of acclimation, animals were randomly divided into 7
groups (n=6). Rats in the control group (Con) were fed with common diet, while
the other groups were fed with high-fat diet (20% sucrose, 15% lard, 10%
casein, 1.2% cholesterol, 0.6% calcium hydrogen phosphate, 0.4% stone
powder, 0.4% premix, 0.2% sodium cholate and the remaining 52.2% of normal
diet) for six weeks to establish hyperlipidemia disorder animal models [16,17].

Blood lipid level of rats were determined in the sixth week t o determine whether
the model was successful. After developing the model, high-fat diet rats were
randomly divided into high -fat diet group (HFD, 0.7g/100g), simvastatin group
(SIM, 0.004g/100g) and hawthorn 4 fraction group (HA-D,0.7g/100g). All drugs
were dissolved in carboxymethylcellulose sodium solution (0.5%CMC-Na). Con
and HFD groups were given the same volume of CMC -Na solution, whereas
other groups were given corresponding treatments once a day for 14 consecutive
days.
2.4 Sample collection
Blood samples were collected at day 14 after administration of the different
treatments. Blood samples were centrifuged at 3000×g for 10 min at 4 ℃, and
plasma was collected and stored at -80℃.
2.5 Plasma biochemistry analysis
TC, TG, and LDL-C levels were determined at the First Affiliated Hospital
of Guangdong Pharmaceutical University (China, Guangzhou) using Hitachi 7180
automatic biochemical analyzer.
2.6 Metabolomics analysis
2.6.1 Sample preparation

Before analysis, plasma sample was thawed at 4℃, and 100 μL of the plasma sample
was diluted with 200 μL of pre-chilled acetonitrile. The diluted sample was then vortexed for
1 min and centrifuged at 4℃ at 13000 rpm for 15 min. The supernatant was passed through a
0.22 μM microporous membrane, and the filtrate was used for UPLC-MS analysis.
To verify the accuracy of this method, 5 μL of each plasma sample was drawn and
mixed as quality control samples (QC). One QC sample was run for every eight analysis
samples during the entire process to evaluate the accuracy of the analysis.
2.6.2 UPLC conditions
HPLC analysis was performed on a Hypersil GOLD (2.1 mm×100 mm, 1.9
μm, Thermo, USA) chromatographic column, with column temperature
maintained at 40℃. The mobile phase was 0.1% formic acid (A) and acetonitrile
(B). UPLC elution conditions were: 0-4min, 5-30% B; 4-6min, 30-40% B;
6-7min, 40-60% B; 7-12min, 60-95% B. The flow rate was maintained at 0.3
ml/min, and injection volume was set at 2 μL.
2.6.3 Mass spectrometry conditions
Mass spectrometry was performed using a Thermo Quadrupole -Exactive
Orbitrap mass spectrometer. Positive and negative electrospray ionization mode
was for detection. Mass spectrometry and mass spectrometry/mass spectrometry
scans were in the range of m/z 100-1000. Collision energy was set at 15, 35 and
55 eV, and ion transfer tube temperature was 320℃.

2.6.4 Multivariate data analysis and identification of potential biomarkers
MS raw data files were imported into Compound Discoverer software
(Thermo Scientific Company, USA) for peak identification, peak matching and
peak alignment. Normalized data were then imported into SIMCA-P 14.1
(Umetrics, Sweden) for multivariate statistical analysis, including unsupervised
principal component analysis (PCA) and supervised orthogonal partial least
squares discrimination analysis (OPLS-DA). Clustering effect was displayed as a
score map. Variable importance of projection (VIP) and S-plot based on
OPLS-DA analysis were used to identify difference in metabolites between
groups. Substances with VIP>1.0 were screened, and one-way analysis of
variance was carried out. When both VIP>1.0 and p<0.05 were met, they
substances were considered as candidate biomarkers. Metabolites were searched
based on ms/ms in the HMDB (www.hmdb.ca/) and METLIN
(https://metlin.scripps.edu/) databases to further determine differences in
metabolites between the two groups. Differential metabolites were then used in
KEGG (https://www.kegg.jp/) and MetaboAnalyst
(https://www.metaboanalyst.ca/) analysis for biological interpretation and
metabolic pathway construction.
2.7 Statistical analysis

Blood lipid level was expressed as mean ± standard deviation. One-way
analysis of variance was used for comparison between multiple groups . Values
with P<0.01 were statistically significant.
3.Results and discussion
3.1 Biochemical assay
Biochemical analysis results are showed in Figure 1. Plasma TC, TG and LDL-C levels
were significantly higher in HFD group compared with levels in the Con group, implying
hyperlipidemia rat model was successfully established. Plasma TC, TG and LDL-C levels
were significantly lower for SIM, H-B and H-C groups compared with FHD group. In
addition, plasma TG and LDL-C levels were significantly lower in H-A group compared with
FHD group. However, administration with H-D showed no significant effects on plasma TC,
TG and LDL-C levels of hyperlipidemia rats. These findings imply that H-B and H-C groups
are the most effective fractions of hawthorn, H-A group has a partial effect, and H-D group
has no effect on hyperlipidemia rats.
3.2 Metabolomic analysis
LC-MS analysis was performed to acquire plasma metabonomic profiles in positive and
negative ion modes. Total ion chromatogram (TIC) of plasma samples in Con and HFD
groups in positive and negative ion modes, which characterizes differences in metabolic
profiles between these two groups is shown in Figure S1. 12 representative ions were
extracted from QC samples, 6 ions (RT -m/z: 1.21-132.1009, 3.75-188.0692, 5.68-340.2571,

8.27-299.1268, 9.07-158.1528, and 10.81-496.3360) in positive ion mode and 6 ions (RT
-m/z: 1.01-215.0313, 4.24-723.4975, 7.06-407.2773, 9.51-339.2303, 10.53-116.9296,
11.48-116.9276) in negative ion mode. RSDs of retention times, m/z and peak areas of the
ten selected ions ranged between 0.05-0.67, 0.00005-0.00060 and 0.26–7.68%,
respectively. Results of QC samples met the requirements, indicating that the
sample analysis method had excellent reproducibility, high stability, and met the
requirements of metabolomics.
PCA, an unsupervised pattern recognition analysis method, was used to
detect outliers based on LC-MS data. No point falls outside the confidence
interval, indicating that there were no outliers (Figure S2). OPLS-DA modeling
was then used to further explore differences between groups. OPLS-DA score
chart (ESI+: R2X = 0.74, Q2 = 0.509, ESI-: R2X = 0.765, Q2 = 0.523) showed
that each group was clearly separated and had good predictive ability (Figure 2).
Con and HFD groups were significantly separated, indicating that the metabolic
pattern of rats in the HFD group was significantly different from that in the Con
group. Furthermore, H-B and H-C groups were significantly separated from HFD
group and close to the Con group. This finding implies that H-B and H-C groups
alleviate hyperlipidemia, which was consistent with biochemical analysis results.
3.3 Identification of potential biomarkers

OPLS-DA model was established based on Con and HFD groups to explore
potential biomarkers associated with hyperlipidemia . A complete separation was
observed between Con and HFD groups, and the parameters of OPLS -DA were
(ESI+: R2Y = 0.992, Q2 = 0.979; ESI−: R2Y = 0.995, Q2 = 0.972) (Figure 3).
R2Y and Q2 were greater than 0.90 indicating that the model has good
explanatory and predictive properties. A total of 200 permutation tests showed
that the models are reliable and there was no overfitting. R2 and Q2 values of the
random permutation experiment were lower than the corresponding original
values, therefore, the regression line of Q2 was a negative intercept (Table S1).
Potential plasma biomarkers associated with hyperlipidemia were screened
through variable importance for the projection values (VIP>1.0) and p<0.05. A
total of 22 potential plasma biomarkers mainly comprising lysophosphatidylcholines (LPC),
unsaturated fatty acids, amino acids and cholic acid were identified (Table 1). S-Plot was
used to display metabolites that significantly contributed to clustering. The
further the ion is from the origin, the greater the inf luence of metabolites on the
separation of the two groups, and the greater its VIP value.
The [M+H] + ion (m/z 220.1110) with a retention time of 3.015 min was used as
an example to illustrate identification process. Through the calculation and
comparison of the formula Finder and HMDB, it is inferred that its molecular
formula is C 9 H 17 NO 5 . In the positive ion spectrum, characteristic MS/MS
fragment ions of the markers were observed at m/z 202.1079, 174.1130 and

160.0974 which may be formed after the loss of H 2 O, -COOH and -CH 2 . The
molecular formulas of these fragments are C 6 H 5 N, C 3 H 3 NO 2 , CHNO 2 ,
respectively. Therefore, it can be inferred that the marker is Pantothenic acid.
Relative intensities of all plasma biomarkers were presented as heat maps,
and the degree of change was marked in red (up-regulation) and blue
(down-regulation). Levels of L-Valine, Racemethionine, L-Tyrosine, L-Isoleucine,
Cinnamic acid, Pantothenic acid, L-Tryptophan, Taurine, Citric acid, Eicosapentaenoic acid,
Arachidonic acid, and Adrenic acid were significantly lower in in the HFD group compared
with Con group (Figure 5). Levels of all other compounds were significantly higher in the
HFD group compared with the Con group. In addition, the level of all biomarkers were
similar to the levels in the Con group after administration of the most effective fraction of
hawthorn.
3.4 Analysis of metabolic pathway of potential biomarkers
A total of 22 biomarkers were identified in plasma and were subjected to
topology analysis using MetaboAnalyst 4.0. Topology analysis was used to
evaluate the role of metabolites in biological reactions based on the position of
metabolites in related pathways, and to explore important metabolic pathways.
Seven metabolic pathways (impact value >0.1) were acutely affected (Figure 5).
Affected metabolic pathways were tricarboxylic acid cycle (TCA cycle),
glycerophospholipid metabolism, phenylalanine, tyrosine and tryptophan

biosynthesis, arachidonic acid metabolism, tryptophan metabolism, taurine and
hypotaurine metabolism, and tyrosine metabolism. The main metabolism
pathways related to the biomarkers are shown in Figure 6.
3.4.1 Lipid metabolism disorder
Disorders of lipid metabolism are the main causes of hyperlipidemia [18].
LPC is formed by decomposition of phosphorylcholine [ 19]. Studies report that
LPC induces lipid peroxidation of cell membranes [2 0,21], causes endothelial cell
damage and promotes inflammation, which is implicated in pathogenesis of
atherosclerosis. High-fat diet results in high levels of LPC, accelerates
inflammatory process, and aggravates endothelial damage. In our study, LPC
content of rats in the HFD group increased significantly and normalized after
treatment.
Fatty acids are important metabolites of lipid metabolism and an important
part of cell membranes, as they play a role in stability and permeability of cell
membranes [22]. Fatty acids are divided into saturated fatty acids (SFA),
monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA)
based on differences between saturated and unsaturated hydrocarb on chains.
Studies report that increase in SFA content results in hyperlipidemia and
atherosclerosis, whereas MUFA and PUFA are beneficial to the human body. In
this study, eicosapentaenoic acid (EPA), arachidonic acid (ARA) and epinephrine

were identified as PUFAs, which are important physiologically active substances
in the human body [23]. EPA is an omega-3 fatty acid, which plays a role in
inhibiting HDL-C oxidation, promoting LDL-C clearance and
anti-atherosclerosis. Studies report that EPA prevents fat and cholesterol from
depositing on the arterial wall and promotes the health of the cardiovascular
system [24,25]. ARA is an important part of cell membrane structure. ARA
esterifies cholesterol, increases blood vessel elasticity and reduces plasma
viscosity, and effectively prevents cardiovascular diseases [26,27]. In this study,
levels of EPA and ARA in the HFD group were lower compared with the levels in
the Con group. Increase in PUFA levels after treatment indicates that hawthorn
fractions reversed the disorder of unsaturated fatty acid metabolism.
3.4.2 Oxidative stress
High-fat diet results in excessive fat intake in rats, and excess cholesterol is
converted into bile acids. Fatty acid metabolism pathway studies report that bile
acid is important for fatty acid metabolism and combines with taurine then it is
excreted from the intestine. Increase in bile acid level consumes a large amount
of taurine, leading to decrease in taurine level, which in turn induces oxidative
stress [28]. Oxidative stress is implicated in pathogenesis of vascular endothelial
injury and atherosclerosis [29]. Studies report that taurine improves pathological
damage caused by oxidative stress, and low levels of taurine induces oxidative
stress leading to atherosclerosis and hypertension [3 0,31]. In this study, bile acid
content increased and taurine content decreased in hyperlipidemia rat model.
However, after treatment with hawthorn extract, bile acid content decreased
significantly whereas taurine content increased. These findings imply that the
therapeutic effect of hawthorn extract on hyperlipidemia may be through
regulation of oxidative stress and regulation of cholesterol metabolism.
Tryptophan is one of the eight essential amino acids n ecessary for humans
and animals. Previous studies report that antioxidant capacity of tryptophan plays
an important role in diabetes, atherosclerosis and hyperlipidemia [3 2]. In
addition, tryptophan can be converted into melatonin and serotonin, and
melatonin has antioxidant activities [33]. Melatonin is an aromatic indole ring
rich in electrons, therefore, it can react with electrophilic free radicals to
scavenge a variety of free radicals, thus playing an antioxidant role. In addition,
metabolites formed after oxidation of melatonin have antioxidant activities. The
main mechanism of melatonin is to provide free electrons to scavenge active
oxygen and increase activity of SOD and GSH -Px to inhibit peroxidation reaction
in the body. In our study, the tryptophan content was low in rats in the HFD
group. After treatment, tryptophan levels significantly increased indicating that
hawthorn may improve hyperlipidemia by regulating oxidative stress.
3.4.3 Energy metabolism disorder

In the energy metabolism pathway, triacylglycerol (TC) stored in adipose
tissue is gradually hydrolyzed by lipase enzyme into free fatty acid (FFA) and
glycerol, which are then released into the blood. Fatty acids undergo β -oxidation
to generate acetyl-Coenzyme, which enters the tricarboxylic acid cycle, and are
completely oxidized to carbon dioxide and water. Acetyl -Coenzyme is an
important intermediate metabolite of the three m ajor nutrients of the body (sugar,
fat, protein), and is the center of human energy conversion. Pantothenic acid and
citric acid are important components in the tricarboxylic acid cycle . Changes in
levels of pantothenic acid and citric acid affect synthesis of acetyl-CoA, which in
turn affects tricarboxylic acid cycle. In this study, levels of pantothenic acid and
citric acid in the HFD group decreased, which may lead to obstruction of energy
metabolism, resulting in increased fat metabolism, thereby inducing
hyperlipidemia. Levels of pantothenic acid and citric acid significantly increase
in the group given hawthorn extract, implying that TCA cycle plays an important
role in regulation of hyperlipidemia.
3.4.4 Amino acid metabolism disorder
Amino acids serve as substrates for protein synthesis, ketone production and
gluconeogenesis, and are also the energy center for TCA cycle. A decrease in
amino acid content results in increased protein breakdown and disrupts the
balance of amino acids [34]. Valine and isoleucine are branched -chain amino
acids, which participate in synthesis and decomposition of proteins, and enhance

energy metabolism in the body [35]. The decomposition products enter the
tricarboxylic acid cycle and provide energy for the human body. Disorders of
amino acid metabolism result in shift in energy metabolism to lipid metabolism.
Tyrosine is an essential amino acid for the human body and is the precursor of
catecholamines (such as epinephrine, norepinephrine and d opamine). Epinephrine
plays an important role in phosphate metabolism and lipid distribution and
transport. Tyrosine is a potential biomarker for hyperlipidemia and
atherosclerosis [36]. In this study, levels of tryptophan, valine, isoleucine and
tyrosine in the HFD group decreased, which is consistent with previous studies.
Increase in amino acid levels after administration of hawthorn extract implies that
hawthorn may regulate hyperlipidemia through amino acid metabolism pathways.
4. Conclusion
In summary, this study is the first to explore therapeutic effects and
mechanisms of different fractions of hawthorn on hyperlipidemia rats. The results
show that H-B and H-C fractions have better therapeutic effects. The findings
show that the mechanism of action of hawthorn involves alleviating
hyperlipidemia by regulating blood lipids, energy and amino acid metabolism
disorders, and reducing oxidative stress. This study shows that metabolomics
technology is a reliable and accurate method in evaluating efficacy of TCM and
elucidating complex mechanisms. The findings of this study provide new ideas
and technical approaches for TCM studies.

Acknowledgments
This research work was supported by the National Natural Science Foundation of China
(Grant NO. 81773905, 81803728).
Conflict of interest statement
The authors declare no conflicts of interest.
References
[1] Yu, J. N., Cunningham, J. A., Thouin, S. R., Gurvich, T., Liu, D., Hyperlipidemia.
Primary care. 2000, 27, 541-87.
[2] Tietge, U. J., Hyperlipidemia and cardiovascular disease: inflammation, dyslipidemia, and
atherosclerosis. Curr. Opin. Lipidology. 2014, 25, 94-5.
[3] Berent, R., Berent, T., Sinzinger, H., Therapeutic options in hyperlipidemia. Wien Med
Wochenschr. 2018, 168, 7-15.
[4] Bhardwaj, S. S., Chalasani, N., Lipid-lowering agents that cause drug-induced
hepatotoxicity. Clin. Liver Dis. 2007, 11, 597-613.
[5] Demyen, M., Alkhalloufi, K., Pyrsopoulos, N. T., Lipid-lowering agents and
hepatotoxicity. Clin. Liver Dis. 2013, 17, 699-714.

[6] Bahmani, M., Mirhoseini, M., Shirzad, H., Sedighi, M., Shahinfard, N., Rafieian-Kopaei,
M., A review on promising natural agents effective on hyperlipidemia. J Evid Based
Complementary Altern Med. 2015, 20, 228-38.
[7] Hunter, P. M., Hegele, R. A., Functional foods and dietary supplements for the
management of dyslipidaemia. Nat. Rev. Endocrinol. 2017, 13, 278-88.
[8] Chang, W. T., Dao, J., Shao, Z. H., Hawthorn: potential roles in cardiovascular disease.
Am. J. Chin. Med. 2005, 33, 1-10.
[9] Pittler, M. H., Guo, R., Ernst, E., Hawthorn extract for treating chronic heart failure.
Cochrane Database Syst Rev. 2008, 23, 1-29.
[10] Dehghani, S., Mehri, S., Hosseinzadeh, H., The effects of crataegus pinnatifida (Chinese
hawthorn) on metabolic syndrome: a review. Iran. J. Basic Med. Sci. 2019, 22, 460-8.
[11] Wang, M., Chen, L., Liu, D., Chen, H., Tang, D. D., Zhao, Y. Y., Metabolomics
highlights pharmacological bioactivity and biochemical mechanism of traditional
Chinese medicine. Chem.Biol. Interact. 2017, 273, 133-41.
[12] Sun, H., Zhang, A., Wang, X., Potential role of metabolomic approaches for Chinese
medicine syndromes and herbal medicine. Phytother. Res: PTR. 2012, 26, 1466-71.
[13] Chaleckis, R., Meister, I., Zhang, P., Wheelock, C. E., Challenges, progress and
promises of metabolite annotation for LC-MS-based metabolomics. Curr. Opin.
Biotechnol. 2019, 55, 44-50.

[14] Zhou B, Xiao JF, Tuli L, Ressom HW., LC-MS-based metabolomics. Mol. Biosyst.
2012, 8, 470-81.
[15] Cheng, S., Shah, S. H., Corwin, E. J., Fiehn, O., Fitzgerald, R. L., Gerszten, R. E., Illig,
T., Rhee, E. P., Srinivas, P. R., Wang, T. J., Jain, M., Potential impact and study
considerations of metabolomics in cardiovascular health and disease: a scientific
statement from the American heart association. Circ.Cardiovasc. Genet. 2017, 10,
e1-13.
[16] Zeng, W., Huang, K. E., Luo, Y., Li, D. X., Chen, W., Yu, X. Q., Ke, X. H., Nontargeted
urine metabolomics analysis of the protective and therapeutic effects of citri reticulatae
chachiensis pericarpium on high-fat feed-induced hyperlipidemia in rats. Biomed.
Chromatogr. 2020, 34, e4795.
[17] Zhang, Y., Wang, Z., Jin, G., Yang, X., Zhou, H., Regulating dyslipidemia effect of
polysaccharides from pleurotus ostreatus on fat-emulsion-induced hyperlipidemia rats.
Int. J. Biol. Macromol. 2017, 101, 107-16.
[18] Cai, Y., Zhao, M., Guan, Z., Han, X., Wang, M., Zhao, C., Metabolomics analysis of the
therapeutic mechanism of semen descurainiae oil on hyperlipidemia rats using (1)
H-NMR and LC-MS. Biomed. Chromatogr. 2019, 33, e4536.
[19] Jang, G. J., Sung, M. J., Hur, H. J., Yoo, M., Choi, J. H., Hwang, I. K., Lee, S.,
Metabolomics analysis of the lipid-regulating effect of allium hookeri in a hamster

model of high-fat diet-induced hyperlipidemia by UPLC/ESI-Q-TOF mass
spectrometry. Evid Based Complement Alternat Med. 2018, 2018, 5659174.
[20] Yang, B., Xuan, S., Ruan, Q., Jiang, S., Cui, H., Zhu, L., Luo, X., Jin, J., Zhao, Z.,
UPLC/Q-TOF-MS/MS-based metabolomics revealed the lipid-lowering effect of ilicis
rotundae ortex on high-fat diet induced hyperlipidemia rats. J Ethnopharmacol. 2020,
256, 112784.
[21] Peng, J., Luo, F., Ruan, G., Peng, R., Li, X., Hypertriglyceridemia and atherosclerosis.
Lipids Health Dis. 2017, 16, 233.
[22] Yang, B., Xuan, S., Ruan, Q., Jiang, S., Cui, H., Zhu, L., Luo, X., Jin, J., Zhao, Z.,
Untargeted metabolite profiling of adipose tissue in hyperlipidemia rats exposed to
hawthorn ethanol extracts. J. Food Sci. 2019, 84, 717-25.
[23] Rathnayake, K. M., Weech, M., Jackson, K. G., Lovegrove, J. A., Impact of meal fatty
acid composition on postprandial lipaemia, vascular function and blood pressure in
postmenopausal women. Nutr. Res. Rev. 2018, 31, 193-203.
[24] Doggrell S. A., Clinical trials of eicosapentaenoic acid (EPA) prescription products for
the treatment of hypertriglyceridemia. Expert Opin. Pharmacother. 2019, 20, 1221-5.
[25] Nelson, J. R., True, W. S., Le, V., Mason, R. P., Can pleiotropic effects of
eicosapentaenoic acid (EPA) impact residual cardiovascular risk. Postgrad. Med. 2017,
129, 822-7.

[26] Bogatcheva, N. V., Sergeeva, M. G., Dudek, S. M., Verin, A. D., Verin AD.,
Arachidonic acid cascade in endothelial pathobiology. Microvasc. Res. 2005, 69,
107-27.
[27] Sonnweber, T., Pizzini, A., Nairz, M., Weiss, G., Tancevski, I., Arachidonic acid
metabolites in cardiovascular and metabolic diseases. Int. J. Mol. Sci. 2018, 19, 1-26.
[28] Fan, L., Qu, X., Yi, T., Peng, Y., Jiang, M., Miao, J., Xiao, P., Metabolomics of the
protective effect of ampelopsis grossedentata and its major active compound
dihydromyricetin on the liver of high-fat diet hamster. Evid Based Complement Alternat
Med. 2020, 156, 104-115.
[29] Song, X., Wang, J., Wang, P., Tian, N., Yang, M., Kong, L., ¹H NMR-based
metabolomics approach to evaluate the effect of Xue-Fu-Zhu-Yu decoction on
hyperlipidemia rats induced by high-fat diet. J. Pharm. Biomed. Anal. 2013, 78-79,
202-10.
[30] Ji, H., Liu, Y., He, F., An, R., Du, Z., LC-MS based urinary metabolomics study of the
intervention effect of aloe-emodin on hyperlipidemia rats. J. Pharm. Biomed. Anal.
2018, 156, 104-15.
[31] Murakami S., Taurine and atherosclerosis. Amino acids. 2014, 46, 73-80.
[32] Anderson G., Linking the biological underpinnings of depression: role of mitochondria
interactions with melatonin, inflammation, sirtuins, tryptophan catabolites, DNA repair

and oxidative and nitrosative stress, with consequences for classification and cognition.
Prog. Neuro-Psychopharmacol & Biol. Psychiatry. 2018, 80, 255-66.
[33] Roager, H. M., Licht, T. R., Microbial tryptophan catabolites in health and disease. Nat.
Commun. 2018, 9, 3294.
[34] Nitz, K., Lacy, M., Atzler, D., Amino acids and their metabolism in atherosclerosis.
Arterioscler. Thromb. Vasc. Biol. 2019, 39, 319-30.
[35] Nie, C., He, T., Zhang, W., Zhang, G., Ma, X., Branched chain amino acids: beyond
nutrition metabolism. Int. J. Mol. Sci. 2018, 19, 1-16.
[36] Miao, H., Chen, H., Zhang, X., Yin, L., Chen, D. Q., Cheng, X. L., Bai, X., Wei, F.,
Urinary metabolomics on the biochemical profiles in diet-induced hyperlipidemia rat
using ultraperformance liquid chromatography coupled with quadrupole time-of-flight
SYNAPT high-definition mass spectrometry. J. Anal. Methods Chem. 2014, 2014, 1-9.

Figure captions
Fig. 1. Effect of hawthorn on plasma lipid levels after treatment for 2 weeks. TC (A), TG (B),
LDL-C (C). Values are expressed as mean ± standard (n =6). (#p<0.01, versus the Con
group; *p<0.01, versus the HFD group).

Fig 2. OPLS-DA score scatter plots of plasma samples from Con, HFD, SIM, H-B and H-C
groups in the positive (A) and negative (B) ESI mode.

Fig 3. OPLS-DA score scatter plots (1), 200 permutations (2) and S-plots (3) for Con
group with HFD group in the positive (A) and negative (B) ESI mode. The meaning of the
numbers is shown in Table 1.

Fig 4. Hierarchical clustering heat map of the 22 differential metabolites, with the degree of
change marked in red (up-regulation) and blue (down-regulation).

Fig 5. Metabolic pathway analysis using MetaboAnalyst (a: Phenylalanine, tyrosine and
tryptophan biosynthesis; b: Taurine and hypotaurine metabolism; c: Arachidonic acid
metabolism; d: tricarboxylic acid cycle; e: Glycerophospholipid metabolism; f:
Tryptophan metabolism; g: Tyrosine metabolism).

Fig 6. Schematic diagram of modulated metabolites and potential disturbed metabolic
pathways.
Table 1 Potential biomarkers identified and variation trends of hyperlipidemia rats in
positive and negative mode.
[[M+H]
NO RT（ +/ VIP HF H- H-
. min） Mass [M-H]− Formula Identified value D B C
117.079
1.278 [M+H]+ C5H11NO2 L-Valine 8.579 ↓## ↓ ↓
1 1
L-Acetylcarniti 13.47
+ **
1.328 203.116 [M+H] C9H17NO4 ↑## ↑
**
2 ne 3 ↑

149.051 C5H11NO2
+ * *
1.336 [M+H] Racemethionine 6.985 ↓## ↓ ↓
3 2 S
181.074
*
1.352 [M+H]+ C9H11NO3 L-Tyrosine 7.981 ↓## ↓
**
4 2 ↓
131.094
*
1.754 [M+H]+ C6H13NO2 L-Isoleucine 9.377 ↓## ↓ ↓
5 8
148.052
6 2.728 [M+H]+ C9H8O2 Cinnamic acid 9.405 ↓##
** **
7 ↓ ↓
Pantothenic
+
3.015 219.111 [M+H] C9H17NO5 1.908 ↓##
** **
7 axid ↓ ↓
204.090 C11H12N2 13.95

+ *
3.877 [M+H] L-Tryptophan ↓## ↓
**
8 1 O2 8 ↓
10.32 543.332 C28H50NO 10.57

+ *
[M+H] LysoPC(20:4） ↑## ↑ ↑
9 1 9 7P 2
10.44 569.348 C30H52NO

+ * *
[M+H] LysoPC (22:5) 1.147 ↑## ↑ ↑
10 9 8 7P
10.64 545.348 C28H52NO

+
11 [M+H] LysoPC (20:3) 1.880 ↑## ↑ ↑
4 7 7P

10.79 495.332 C24H50NO 13.99

+
[M+H] LysoPC (16:0) ↑## ↑ ↑
12 8 8 7P 6
11.01 521.348 C26H52NO

+
[M+H] LysoPC(18:1） 7.144 ↑## ↑ ↑
13 4 6 7P
125.014
*
1.293 [M-H]− C2H7NO3S Taurine 2.150 ↓## ↓ ↓
14 4
134.021
*
1.406 [M-H]− C4H6O5 L-Malic acid 1.258 ↑## ↑
**
15 2 ↑
192.026
16 1.481 [M-H]− C6H8O7 Citric acid 2.827 ↓## ↓ ↓
5
408.287
** **
7.312 [M-H]− C24H40O5 Cholic acid 2.873 ↑## ↑ ↑
17 3
392.292 Chenodeoxycho
− *
8.051 [M-H] C24H40O4 2.058 ↑## ↑ ↑
18 3 lic acid
481.316 C23H48NO 10.90

− **
8.931 [M-H] LysoPE (18:0) ↑## ↑
**
19 4 7P 2 ↑
302.224 Eicosapentaeno
− * *
9.469 [M-H] C20H30O2 1.258 ↓## ↓ ↓
20 3 ic acid

304.239 Arachidonic
− * *
21 9.867 [M-H] C20H32O2 4.856 ↓## ↓ ↓
9 acid
10.41 332.271
*
[M-H]− C22H36O2 Adrenic acid 1.080 ↓# ↓ ↓
22 8 3
#p < 0.05, ##p < 0.01, HFD group vs Con group; * p < 0.05, ** p < 0.01, H-Band H-C
groups vs HFD group. ↓ expressed down-regulation, and ↑ expressed up-regulation.

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Hawthorn on hyperlipidemia rats

Guangdong, 510006, China

Universities of Guangdong Province, Guangzhou, Guangdong, 510006, China

Guangdong Province, Guangzhou, Guangdong, 510006, China

† Lu Zeng and Lan Luo should be considered co-first authors

Prof. Dr. Shengwang Liang, Guangdong Pharmaceutical University, Guangzhou, Guangdong,

E-mail: swliang371@126.com; Tel.: 020-39352172.

Shumei Wang: 2395903468@qq.com

Received: 30/08/2020; Revised: 30/08/2020; Accepted: 18/12/2020

This article is protected by copyright. All rights reserved.

Abbreviations: LC-MS: Liquid chromatography-mass spectrometry; LDL-C:

Low-density lipoprotein-cholesterol; OPLS-DA: Orthogonal partial least squares

discrimination analysis; PCA: Principal component analysis; QC: Quality control;

Variable importance of projection

Hawthorn, a well-known traditional Chinese medicine is used for treatment

of dyspepsia syndrome, cardiovascular disease and hyperlipidemia. Hawthorn

has complex composition, therefore, the effective fraction and mechanisms of

was to evaluate effects of four different polar components of hawthorn on hyperlipidemia

metabolomics. Hyperlipidemia rat model was established by feeding rats using

to biochemical and metabolomics analysis. Biochemical analysis showed that

hyperlipidemia rats. Water fraction showed a partial effect, whereas petroleum

metabolomics analysis showed that the most effective fraction of hawthorn

reversed the metabolic disorder in plasma of hyperlipidemia rats . Metabolomics

analysis showed that hawthorn exerts its activity by modulating lipid

metabolism, energy metabolism, oxidative stress and amino acid metabolism.

Keywords: effective fraction, hawthorn, hyperlipidemia, metabolomics ,

traditional Chinese medicine, underlying mechanism

Incidence of hyperlipidemia have increased exponentially due to unhealthy

eating habits and excessive energy intake. Hyperlipidemia is a common lipid

metabolism disorder characterized by high levels of total cholesterol (TC),

triglycerides (TG) and low-density lipoprotein-cholesterol (LDL-C) in the blood

[1]. Hyperlipidemia is implicated in pathogenesis of cardiovascular diseases,

such as essential hypertension, coronary heart disease and atherosclerosis, and

mortality rate of cardiovascular diseases. Currently, hyperlipidemia is managed

through lifestyle changes and use of lipid-lowering drugs [3]. However,

effects, such as rhabdomyolysis, liver toxicity and sleep disorders [4,5].

Therefore, studies have explored traditional Chinese medicine (TCM) as an

chemical components in hawthorn include organic acids, flavonoids and

This article is protected by copyright. All rights reserved.

triterpenes. Therefore, it exerts a variety of drug effects, such as aiding digestion,

improving blood circulation and alleviating atherosclerosis [8,9]. Previous

studies report use of hawthorn as an alternative therapy for cardiovascular

diseases, such as hyperlipidem ia, hypertension and arrhythmia. Although the

lipid-lowering effect of hawthorn has been reported previously [10], effective

fraction and mechanisms of action of hawthorn in treatment of hyperlipidemia are

unknown. Advances in metabolomics provide a basis for study of small molecules

Metabolomics is a powerful tool for qualitative and quantitative analysis of

metabolites after endogenous or exogenous interference in the body can be

determined through metabolomics [11]. Therefore, it is used for study of efficacy

and toxicity of TCM [12]. Currently, analysis techniques of metabolomics mainly

include liquid chromatography-mass spectrometry (LC-MS), gas

chromatography-mass spectrometry (GC-MS), and nuclear magnetic resonance

(NMR). LC-MS is characterized by high sensitivity and high robustness,

therefore, it is used to analyze a wide range of metabolites. LC-MS is the most

widely used method in metabolomics research [13,14]. This technology has

provided great convenience for our research [15].

mechanism of action of hawthorn for treatment of hyperlipidemia. A

(H-A, H-B, H-C and H-D fractions in descending polarity, corresponding to

consecutive days. Plasma samples were collected and analyzed through

fractions had significant effects on hyperlipidemia. The findings imply that

mechanism of action of hawthorn is through modulation of lipid metabolism,