Abstract

   Coreopsis tinctoria (CT) improves energy metabolism. However, the role
   of CT in alleviating obesity-induced hyperglycemia by targeting the
   liver remains unknown. Therefore, this article aims to explore the
   mechanism by which CT improves energy metabolism and resists
   hyperglycemia. The water and ethanol extracts of CT were administered
   to high-fat diet-induced (HFD) obese C57BL/6J mice at a dose of
   4 g/kg.bw (low-dose water extract, WL; low-dose ethanol extract, EL) or
   10 g/kg.bw (high-dose water extract, WH; high-dose ethanol extract,
   EH). Mice that consumed a maintenance diet (LFD) were included as blank
   controls. Network pharmacology, liquid chromatography-mass spectrometry
   (LC-MS), L02 cell cultivation, and liver transcriptomics were used to
   examine the mechanism and functional components of CT against
   obesity-induced hyperglycemia. The results indicated that WL
   significantly (p < 0.05) alleviated glucose intolerance and insulin
   resistance in obesity-induced hyperglycemia. Kaempferol is the main
   active compound of CT, which demonstrated significant (p < 0.05)
   anti-hyperglycemic effects in obese mice and L02 cells. Finally,
   kaempferol significantly (p < 0.05; fold change >1.2) shifted the genes
   involved in carbon metabolism, glycolysis/gluconeogenesis, and the
   mitogen-activated protein kinase (MAPK) pathways toward the trend of
   LFD, indicating that it exerts an anti-hyperglycemic effect through
   these molecular mechanisms. Overall, oral intake of CT lowers blood
   glucose and improves insulin sensitivity in mice with obesity-induced
   hyperglycemia. Kaempferol is the primary functional component of CT.

   Keywords: Coreopsis tinctoria, Obesity, Hyperglycemia, Liver,
   Kaempferol

1. Introduction

   Obesity and metabolic diseases resulting from excessive energy intake
   are global public health issues. A meta-analysis showed that the global
   prevalence of central obesity is approximately 41.5% [[37]1]. Obesity
   alters lipids, hormones, inflammatory factors, and enzymes, leading to
   complications such as insulin resistance, type 2 diabetes,
   dyslipidemia, hyperglycemia, and inflammation [[38]2,[39]3]. Therapies
   for obesity and its complications have received significant attention
   in recent years. Some natural products derived from plants (such as
   viburnum stellato-tomentosum, ephedra alata, and poria cocos) alleviate
   obesity and hyperglycemia [[40][4], [41][5], [42][6]]. However, the
   current research on the impact and mechanism of Coreopsis tinctoria on
   improving hyperglycemia in obese individuals is insufficient.

   Coreopsis tinctoria (CT) is a plateau plant native to North America
   that is widely cultivated in Xinjiang and Yunnan, China [[43]7,[44]8].
   CT has a long history of healthcare utilization. Traditional medicine
   has applied CT to optimize energy metabolism and treat infections and
   diarrhea [[45]9]. Studies have found that the main active ingredient in
   CT is flavonoids, and it also contains more than 120 other ingredients,
   such as polyacetylenes, phenylpropanoids, polysaccharides, and volatile
   oils [[46]9,[47]10]. As the polarities of these components differ, the
   composition and content of natural products vary depending on the
   solvents used for extraction. In Vivo and in Vitro experiments have
   shown that CT has biological activities against oxidation, obesity,
   cancer, aging, hyperglycemia, and hyperlipidemia [[48][11], [49][12],
   [50][13], [51][14], [52][15], [53][16]]. In exploring the mechanism of
   CT regulation of glucose and lipid metabolism, existing studies have
   mainly focused on its down-regulation of proteins related to white
   adipose differentiation, restoration of pancreatic function, and
   improvement of intestinal microbes [[54][16], [55][17], [56][18]].
   However, limited research has focused on the regulatory effect of CT on
   liver energy metabolism.

   The liver is crucial for energy metabolism. It converts abundant
   circulating glucose into glycogen and lipids and fuels the body with
   glycogen and fatty acids during fasting [[57]19]. The glucose and lipid
   metabolism of the liver is affected by various factors, including
   nutritional status, cytokines, and hormones. Under conditions of
   diet-induced obesity and elevated blood glucose levels, accumulation of
   triglycerides in the liver and crosstalk between the liver and other
   tissues, result in a decreased ability of the liver to regulate energy
   balance [[58]20]. Therefore, restoring liver functions at the molecular
   level is crucial for alleviating metabolic diseases induced by a
   high-fat diet. Studies have found that CT protects against acute liver
   injury caused by acrylamide, carbon tetrachloride, and
   lipopolysaccharide [[59][21], [60][22], [61][23]]. Also, dietary
   supplementation with CT flower tea while exposed to high-fat diets
   (HFD) prevents hepatic energy metabolism disorders in rats [[62]24].
   Our previous studies found that CT reduces hyperglycemia in obese mice
   by affecting gut microbiota [[63]25]. Overall, CT can protect the
   liver, but its effects and mechanisms on the liver in
   obese-hyperglycemic individuals remain unproven.

   To explore the potential of CT in mitigating hyperglycemia and insulin
   resistance caused by obesity, we examined the effects of CT water
   extract and ethanol extract on energy metabolism regulation in obese
   hyperglycemia mice. Also, we studied the mechanism by which CT targets
   the liver to regulate energy metabolism, as well as the functional
   components of CT and their mechanisms for alleviating hyperglycemia.

2. Materials and methods

2.1. Extractions of the active ingredients in Coreopsis tinctoria

   The Coreopsis tinctoria (CT) was harvested in August 2019 from a
   planting base at an altitude of 3800 m in Saitula, Xinjiang, China.
   Firstly, it was ground using a high-speed universal grinder (Tianjin
   Taisite Instrument Co., Ltd). A 60-mesh sieve was used to collect
   powders with a diameter of less than 0.3 mm. Distilled water and 65%
   ethanol (E809061, Macklin Co., Ltd) were used as extractants,
   respectively. The extraction process included sonication, suction
   filtration, rotary evaporation, and lyophilization. The Coreopsis
   tinctoria powder was extracted three times at a temperature of 80 °C
   with a solid-liquid ratio of 1:10, and each extraction lasted 45 min
   [[64]25]. The instrument was purchased from Beijing Earth Long Science
   and Technology Co., Ltd, Wenling Yangyi Mechanical and Electrical
   Products Co., Ltd, Beijing Kewei Yongxing Instrument Co., Ltd, and
   Shanghai Hecheng Instrument Manufacturing Co., Ltd, respectively.

2.2. Explore the impact of Coreopsis tinctoria extract on obese mice

2.2.1. Construction of mouse model and administration of Coreopsis tinctoria
extract

   4-week-old male C57BL/6J mice, which were specific-pathogen-free, with
   an initial body weight ranging from 12 to 15 g, were purchased from
   Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice
   induced with obesity were fed a 60% high-fat diet ([65]H10060, Beijing
   HFK Bioscience Co., Ltd.), while the control group of mice was fed
   maintenance diets (1016706714625204224, Beijing Keao Xieli Feed Co.,
   Ltd.). Four mice were kept in each cage. The mice have free access to
   food and sterile water. This experiment was conducted in the SPF-grade
   laboratory of the Superslon & Test Center for Agriculture Product
   Quality, Ministry of Agriculture (SYXK (Beijing) 2015–0045). The air in
   the laboratory was exchanged 15 times per hour, and light and dark
   alternated every 12 h. The mice were observed once a day and weighed
   weekly.

   After one week of adaptive feeding, the mice were assigned to either
   60% high-fat diets (HFD) or maintenance diets for 11 weeks. The
   HFD-induced obese-hyperglycemic mice were selected based on a more than
   20% increase in body weight and significantly higher fasting blood
   glucose levels compared to mice on maintenance diets [[66]26]. The HFD
   mice were randomly divided into 5 groups (8 mice in each group), and
   the initial mean body weights were similar. 4 g or 10 g/kg body-weight
   (.bw) of CT ethanol or water extract was administered to 4 groups of
   HFD mice every day for 8 weeks, respectively. This dose has been shown
   to improve energy metabolism in obese mice [[67]25]. Blood biochemistry
   (liver function, kidney function, and energy metabolism indicators;
   detailed experimental methods are shown in Section [68]2.2.4) was used
   to test the safety at this dose, and the results showed that these
   doses were non-toxic to mice (supplementary material 1). One group of
   HFD mice (the HFD group) and the maintenance diet-treated mice (the LFD
   group) were gavaged with sterile water. This experiment was approved by
   the Animal Ethics Committee of China Agricultural University (Approval
   number: KY1700038).

2.2.2. Insulin and glucose tolerance test

   An insulin tolerance test (ITT) was conducted in the 4th week of
   gavage. The mice were fasted for 4 h and injected intraperitoneally
   with 0.7 U/kg.bw insulin (NovoRapid® Penfill®, Novo Nordisk Co., Ltd.)
   [[69]27,[70]28]. The mice had their plasma glucose (PG) levels measured
   at 0, 15, 30, 45, and 60 min after injection. A glucose tolerance test
   (GTT) was conducted in the fifth week. The mice were fasted for 16 h
   and injected intraperitoneally with 1.5 g/kg.bw glucose (50-99-7,
   Sigma-Aldrich Co., Ltd.) [[71][29], [72][30], [73][31]]. The blood
   glucose level of each mouse was measured at 0 min, 15 min, 30 min,
   45 min, 60 min, and 120 min after the injection. The ITT and GTT
   results were quantified by calculating the area under the curve (AUC)
   using the following formula: Duration * (PG [0 min] + PG [15 min])/2 +
   duration * (PG [n-1 min] + PG n [min])/2 [[74]32].

2.2.3. Detection of energy metabolism and obese hyperglycemia development

   After 7 weeks of gavage, the mice's respiratory oxygen consumption was
   measured for 24 h using a Panlab Harvard Apparatus 10 Cage Respiratory
   Metabolism System. The data was processed by Metabolism Version 3.0. In
   the final week of the experiment, the mice were subjected to cold
   stimulation at 4 °C for 4 h, and their core temperature was measured
   with a rectal thermometer. Finally, mice were weighed and sacrificed by
   cervical dislocation after a 16-h fasting period. Finally, the mice
   were sacrificed by cervical dislocation. Liver, subcutaneous fat (SUB),
   and epididymal fat (EP) were collected. The organ coefficient was
   calculated as the proportion of organ weight to fasting body weight.

2.2.4. Serum biochemical analysis

   Blood was collected when the mice were sacrificed and serum was
   obtained by centrifugation. Serum insulin levels were measured using an
   insulin ELISA kit (HY-10069, Beijing Huaying Biotechnology). Blood was
   collected when sacrificed the mice and serum was obtained by
   centrifugation. The Hitachi 7600–020 automatic biochemical analyzer was
   used to detect the levels of serum biochemical indicators. The
   high-density lipoprotein (HDL, 1000020238), low-density lipoprotein
   (LDL, 1000020248), triglycerides (TG, 1000020090), cholesterol (CHO,
   1000020080), alkaline phosphatase (ALP, 1000020020), aspartate
   aminotransferase (AST, 1000020010), alanine aminotransferase (ALT,
   1000020000), total protein (TP, 1000020140), albumin (ALB, 1000020150),
   uric acid (UA, 1000020070), creatinine (CREA, 1000020170), and glucose
   (GLU, 1000020100). The kits were purchased from Zhongsheng Beikong
   Biotechnology Co., Ltd.The blood urea nitrogen (BUN, BC1530) kits were
   purchased from Solarbio Life Science Co., Ltd. and all procedures were
   conducted following the instructions provided in the instructions in
   the manual.

2.3. Identification of active ingredients in Coreopsis tinctoria

2.3.1. Composition analysis

   Liquid chromatography-mass spectrometry (LC-MS, TripleTOF5600+, AB
   SCIEX™) was used to detect and compare the components of CT's water
   extract and ethanol extract. The water and ethanol extracts were
   pre-treated by ultrasonication in a water-ice bath and then centrifuged
   to obtain the supernatant for liquid-phase detection. In liquid
   chromatography-mass spectrometry, the chromatographic column used is
   the SHIMADZU InerSustain C18 (100 × 2.1 mm, 2 μm). The column
   temperature was 35 °C. The flow rate is 0.300 mL/min. The mobile phase
   is A.Equate = “acetonitrile”, B.Equate = “0.1% CH3COOH–H2O”. The
   chromatographic conditions are as follows: 0 min (A:0% B:100%), 10 min
   (A:50% B:50%), 13 min (A:95% B:5%), 14 min (A:0% B:100 %), and 15 min
   (A:0% B:100%); the mass spectrometry conditions include a scan range of
   m/z 100–1500, DIA for the scan mode, a capillary voltage of 5000 V
   (positive) and a 4500 V (negative), a capillary temperature of
   500 °C(DP: 60V, CE: 35V, CES: 15V). The LC-MS raw data was imported
   into MS-DIAL 3.82 software for preprocessing, and the extracted peak
   information was compared with the MassBank, Respect, and GNPS (14,951
   records in total) databases. Overall, the outputs include sample
   information, retention time, mass-to-nucleus ratio, and mass spectral
   response intensity (peak area).

2.3.2. Network pharmacology

   The bioactive substances in CT were identified by screening the
   compounds detected by LC-MS using the Traditional Chinese Medicine
   Systems Pharmacology Database and Analysis Platform (TCMSP) based on
   oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18. The
   protein targets in the human body affected by the bioactive substances
   in CT were distinguished using the PharmMapper database (norm fit
   ≥0.7). Subsequently, the target proteins were converted into gene names
   using the UniProt database. Obesity, hyperglycemia, insulin resistance,
   and diabetes-related targets were searched in the DisGeNET database.
   Bioinformatics was used to identify the intersection between CT and
   disease-associated targets.

   The protein-protein interaction (PPI) was constructed using the STRING
   11.0 database, and the Cytoscape 3.8.2 software was used to analyze the
   network. The critical target protein for CT to alleviate
   obesity-induced hyperglycemia is screened with the closeness centrality
   and betweenness centrality greater than the median and the degree value
   (degree) greater than 2 times the median. Bioinformatics was used for
   the enrichment analysis of biological processes (BP), cellular
   components (CC), and molecular functions (MF) in terms of GO (Gene
   Ontology) function and KEGG (Kyoto Encyclopedia of Genes and Genomes)
   pathway enrichment analysis. Finally, the Cytoscape 3.8.2 software was
   used to visually analyze the relationship between active ingredients
   and disease targets in CT. Molecular docking was performed using the
   CB-Dock online platform to identify combinations with a binding
   activity (affinity less than −7.0 kcal mol^−1) [[75]33].

2.3.3. In vitro verification of active ingredients against obese-induced
hyperglycemia

   Hepatic L02 cells were obtained from China Agricultural University. L02
   is a normal human hepatocyte cell line, which better reflects energy
   metabolism in humans [[76]34]. The cells were cultured at 37 °C with 5%
   CO[2]. 300 μM sodium palmitate (PA, P799157, Macklin Co., Ltd) was used
   to induce insulin resistance in L02 cells [[77]35,[78]36]. This
   concentration of PA significantly reduced glucose consumption in
   L02 cells, as measured by the Glucose Oxidase Assay Kit (E1011,
   Applygen Co., Ltd.). However, it did not affect cell viability, as
   measured by the Enhanced CCK-8 Kit (C0037, Beyotime Biotechnology Co.,
   Ltd.). The flavonoid substances of CT identified in 2.3.2 (kaempferol,
   taxifolin, eriodictyol, luteolin) were administered to
   insulin-resistant L02 cells at concentrations of 0, 1.5625, 3.125,
   6.25, 12.5, 25, 50, and 100 μM for 24 h, respectively. The purity of
   the flavonoid substances are greater than 98% (HPLC grade), and were
   purchased from Solarbio Life Science Co., Ltd (SK8030, SL8300, YZ1359S,
   amd SE8200). The Glucose Oxidase Assay Kit was used to determine the
   improvement in glucose consumption caused by the flavonoid compounds.
   These flavonoids were dissolved in a 0.05% Dimethyl Sulfoxide (DMSO,
   D8371, Solarbio Life Science Co., Ltd.) solution. To eliminate the
   influence of DMSO on the experiment, a PA control group was established
   in which the cells were treated with a 0.05% DMSO solution.

2.3.4. In vivo transcriptomics of verification anti-hyperglycemic mechanism

   4-week-old male C57BL/6J mice of SPF grade were used as experimental
   animals. The feeding method is the same as described in Section
   [79]2.2.1. Our previous results showed that WL and its main active
   ingredient (kaempferol, KL) showed promising hypoglycemic effects. We
   then constructed an obese hyperglycemic mouse model and provided them
   with WL and KL, respectively. The obese hyperglycemic mice were
   obtained by feeding a 45% high-fat and 35% carbohydrates diet
   ([80]H10045, Beijing HFK Bioscience Co., Ltd.) for 10 weeks
   [[81]37,[82]38]. The inclusion criteria for HFD-induced
   obese-hyperglycemic mice were identical to those described in section
   [83]2.2.1, ensuring the reliability and reproducibility of the findings
   [[84]26]. The obese hyperglycemic mice were treated with 4 g/kg.bw of
   CT water extract or 25 mg/kg.bw of kaempferol (IK0020, Solarbio Life
   Science Co., Ltd.) for 8 weeks. 5 mice were arranged into each group.

   After sacrificing mice, total RNA was extracted from liver tissue using
   the Trizol method. The RNA was sequenced on the Illumina platform, and
   the transcriptome mRNA sequencing mode was paired-end 150bp (PE50).
   Reference genome alignments were performed using Cutadapt (v1.10),
   Tophat2 (v2.0.13), Cufflinks (v2.2.1), Cuffnorm, and Cuffdiff.
   Differentially expressed gene analysis, GO functional enrichment
   analysis, and KEGG pathway analysis were performed [[85]39,[86]40].
   Finally, Venny 2.1.0 was used to visualize the significantly changed
   genes between WL and KL compared to HFD. Lingbo MicroClass was used to
   complete principal component analysis (PCA) and visualize the fold
   difference (log[2]) comparison between LFD and HFD versus KL and HFD.

2.4. Data analysis

   Office 2019 Excel software was used for data calculation, and the
   results were expressed as the mean ± standard deviation (SD). One-way
   analysis of variance was used to calculate the significant differences
   between groups in transcriptomic analysis, with Tukey analysis as a
   post-hoc test. The Student's t-test was used to calculate the
   significant difference between groups in other analyses. Graphing was
   performed using the GraphPad Prism 8 software package and
   BioMicroClass.

3. Results

3.1. Coreopsis tinctoria improves energy metabolism in HFD mice

   The low dose (4 g/kg.bw) water extract (WL), high dose (10 g/kg.bw)
   water extract (WH), low dose (4 g/kg.bw) ethanol extract (EL), and high
   dose (10 g/kg.bw) ethanol extract (EH) of Coreopsis tinctoria (CT)
   inhibited the body weight gain of HFD mice. Although no significant
   difference was observed between the mice treated with CT extract and
   those on an HFD in the first four weeks of gavage, there are downward
   trends that can be observed. In the fourth week, the average body
   weight of mice treated with WL was 76.51% lower than that of the HFD
   group, while the average body weight of mice treated with EH was 31.54%
   lower than that of the HFD group ([87]Fig. 1A–D). Also, although CT
   treatments did not significantly reduce the visceral coefficient of
   white adipose (SUB and EP) and liver organs in mice, WL and EH lowered
   these indicators by 32.50%，35.22%，6.42%, and 15.86%, 21.08%, 9.22%
   compared with those of the HFD mice, respectively. These results
   indicated that WL and EH had a slight reversing effect on the obesity
   of HFD mice ([88]Fig. 1E–F).

Fig. 1.

   [89]Fig. 1
   [90]Open in a new tab

   The Anti-obesity effect of Coreopsis tinctoria in HFD hyperglycemic
   mice. (A) Weight change after CT water extract treatment; (B) weight
   gain after CT water extract treatment; (C) weight change after CT
   ethanol extract treatment; (D) weight gain after CT ethanol extract
   treatment; (E) organ coefficient of CT water extract-treated mice;
   (F)organ coefficient of CT ethanol extract-treated mice; (G) oxygen
   consumption after WL treatment (H) oxygen consumption after EH
   treatment (I) core body temperature after cold stimulation.

   In contrast to WH and EL, WL and EH treatments showed better
   performance in improving obesity. Therefore, the oxygen consumption
   (which reflects metabolic rate) of these two groups of mice in both
   light and dark environments was measured and compared with that of
   HFD-treated mice. The results showed that WL treatment significantly
   (P < 0.05) increased the oxygen consumption and energy metabolism
   levels of HFD mice ([91]Fig. 1G). However, EH treatment did not
   significantly impact oxygen consumption in HFD mice ([92]Fig. 1H).
   Moreover, the rectal temperature test results showed that WL, WH, EL,
   and EH treatments prevented the decrease in core temperature among HFD
   mice during cold stimulation ([93]Fig. 1I), indicating that CT
   treatment increased energy utilization in the HFD mice.

   LFD represents the mice that ingested maintenance feed and were gavaged
   with sterile water; HFD represents the mice that ingested HFD and were
   gavaged with sterile water; WL represents the mice that ingested HFD
   and were gavaged with 4 g/kg.bw CT water extracts Mice; WH represents
   mice that ingested HFD and were gavaged with 10 g/kg.bw CT water
   extract; EL represents mice that ingested HFD and were gavaged with
   4 g/kg.bw CT ethanol extract; EH represents mice that ingested HFD and
   were gavaged with 10 g/kg.bw CT ethanol extract. The body weight and
   viscera coefficient of the mice were measured during and after the
   experiment, respectively (n = 7–8). Oxygen consumption was measured
   after 7 weeks of treatment (n = 4). Core body temperature (n = 4). The
   results were expressed as mean ± SD. The Student's t-test was used to
   calculate the significance of each group compared to the HFD group. *
   indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001.

3.2. CT improves glucose metabolism and insulin resistance in HFD mice

   As shown in [94]Fig. 2A–B, WL treatment significantly decreased the AUC
   of HFD-induced obesity in mice (P < 0.05), indicating that WL has the
   potential to restore impaired glucose tolerance. In contrast, the WH,
   EL, and EH treatments slightly decreased the AUC compared to the HFD
   group ([95]Fig. 2A–D). WL has also been found to improve insulin
   tolerance ([96]Supplementary Material 3). Also, WL, WH, EL, and EH
   significantly (P < 0.05) increased serum insulin levels ([97]Fig.
   2E–F).

Fig. 2.

   [98]Fig. 2
   [99]Open in a new tab

   Coreopsis tinctoria improves GTT and increased the insulin levels of
   HFD mice (A) water extract GTT (B) area under water extract GTT curve
   (C) ethanol extract GTT (D) area under ethanol extract GTT curve (E)
   plasma insulin levels of water extract-treated mice (F) plasma insulin
   levels of ethanol extract-treated mice. GTT was performed on the 5th
   week of the experiment. Serum insulin levels were measured when the
   mice were sacrificed in the 8th week. The GTT results were quantified
   by calculating the AUC. The results were expressed as mean ± SD
   (n = 7–8). The Student's t-test was used to calculate the significance
   of each group compared to the HFD group. The results were expressed as
   mean ± SD. * indicates p < 0.05; ** indicates p < 0.01; *** indicates
   p < 0.001.

3.3. Network pharmacology prediction of the mechanism of CT in reversing
obesity-induced hyperglycemia

   The LC-MS analysis revealed over 100 components in the water and
   ethanol extracts of CT. The lists of critical components and the
   profile results of total ion chromatograms are displayed in
   supplementary material 2 ([100]Table S2 and [101]Fig. S2,
   respectively). Out of these components, 6 active ones were identified
   after screening for OB ≥ 30% and DL ≥ 0.18 ([102]Table 1). These
   protein targets were converted into 91 corresponding genes ([103]Fig.
   3A).

Table 1.

   Six active compounds in Coreopsis tinctoria.
         Detected In       Active Component OB（%）  DL
   Water & Alcohol extract kaempferol       41.88 0.24
   Water & Alcohol extract taxifolin        57.84 0.27
   Water & Alcohol extract eriodictyol      71.79 0.24
   Water & Alcohol extract luteolin         36.16 0.25
   Alcohol extract         aloe-emodin      83.38 0.24
   Alcohol extract         quercetin        46.43 0.28
   [104]Open in a new tab

Fig. 3.

   [105]Fig. 3
   [106]Open in a new tab

   Prediction of the mechanism of Coreopsis tinctoria on alleviating
   hyperglycemia caused by obesity by Network pharmacology (A) Venn
   diagram of functional components in Coreopsis tinctoria and disease
   targets (B) PPI network diagram (C) KEGG enrichment (D) GO
   enrichment—biological process, cellular component, molecular function
   (E) correspondence between functional components and intervention
   targets.

   In addition to obesity and hyperglycemia, overnutrition is often
   accompanied by insulin resistance and diabetes, and these diseases
   further hinder cells from effectively utilizing glucose [[107]41].
   Therefore, the shared target genes of CT with obesity, diabetes,
   hyperglycemia, and insulin resistance were calculated. The results
   showed that CT has 57, 44, 27, and 6 shared genes with obesity (2.02%),
   diabetes (1.87%), hyperglycemia (2.46%), and insulin resistance
   (3.70%), respectively ([108]Fig. 3A). Prediction results suggest that
   CT has the potential to target these diseases.

   Cytoscape 3.8.2 was used to quantify the PPI network obtained from the
   String database. It reflects the interactions among the target proteins
   of CT shared with the four diseases ([109]Fig. 3B). The larger the node
   and the redder the color, the higher the association frequency between
   the gene and other nodes. Critical target genes were identified based
   on network topology parameters, which included betweenness centrality
   (>0.0247), closeness centrality (>0.5333), and degree (>16). We found
   that Albumin (ALB), epidermal growth factor receptor (EGFR), estrogen
   receptor 1 (ESR1), human heat shock protein 90 kDa alpha (HSP90AA1),
   SRC proto-oncogene, non-receptor tyrosine kinase (SRC), and kinase
   insert domain receptor (KDR) have a high node association frequency,
   association tightness, and communication function in protein
   interaction, which means they play critical roles in CT intervention of
   hyperglycemia. Furthermore, the KEGG enrichment showed that CT acts on
   the FoxO signaling pathway, endocrine resistance, and estrogen
   signaling pathway ([110]Fig. 3C). GO enrichment analysis showed that CT
   interfered with the cellular response to hormones and reactive oxygen
   species at the biological process level ([111]Fig. 3D). Also, it mainly
   regulates cellular components such as vesicle lumen, ficolin-1-rich
   granule, and ficolin-1-rich granule lumen. The active ingredients are
   predicted to regulate molecular functions through ATPase binding,
   nuclear receptor activity, and steroid binding. Based on the chemical
   properties of the active compound in CT, flavonoids are the primary
   functional substances ([112]Fig. 3E).

   Functional substances detected from CT extracts included kaempferol,
   taxifolin, eriodictyol, luteolin, aloe-emodin, and quercetin. The
   intersection targets of these substances with obesity, hyperglycemia,
   insulin resistance, and diabetes were calculated and analyzed.

3.4. Anti-hyperglycemic composition of Coreopsis tinctoria

   To identify the key flavonoids in CT that improve hepatic glucose
   metabolism in individuals with excess energy, in Vitro experiments were
   conducted using the L02 cell line. This experiment focused on the
   effects of functional flavonoids (kaempferol, taxifolin, eriodictyol,
   luteolin) in the water extract of CT on the glucose consumption and
   viability of PA-treated L02 cells. The results showed that kaempferol
   at the dose of 50–100 μM significantly (P < 0.05) improved impaired
   glucose consumption ([113]Fig. 4B) caused by PA treatment of L02 cells
   and maintained the growth of L02 cells ([114]Fig. 4J). However,
   taxifolin, eriodictyol, and luteolin did not improve the impaired
   glucose consumption caused by PA treatment ([115]Fig. 4). Thus,
   kaempferol is a promising anti-hyperglycemic substance in CT and is
   safe for liver cells. It was further verified in Vivo in the following
   experiment.

Fig. 4.

   [116]Fig. 4
   [117]Open in a new tab

   Effects of Coreopsis tinctoria flavonoids on glucose consumption and
   cell viability in L02 cells (A–D) Glucose consumption after flavonoids
   treatment (E–H) Cell viability after flavonoids treatment 0.05% DMSO
   solution was used as the solvent. The DMSO solution at this dose did
   not affect the glucose consumption and cell viability of L02 cells.
   0.05% DMSO was added to the control group to eliminate errors. The
   results (n = 6) were expressed as mean ± SD. The Student's t-test was
   used to calculate the significance of the difference between the groups
   treated with flavonoids and those that did not receive flavonoid
   treatment. The results were expressed as mean ± SD. * indicates
   p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001.

3.5. In vivo anti-hyperglycemic property of CT and kaempferol

   After being treated with 4 g/kg.bw of CT water extract (WL) or
   25 mg/kg.bw of kaempferol (KL) for 8 weeks, blood glucose levels in
   obese hyperglycemic mice were reduced (p = 0.06 and p < 0.01,
   respectively, [118]Fig. 5A). Compared to obese hyperglycemic mice, WL
   treatment significantly altered the expression of 1448 genes in the
   mouse liver, while KL treatment significantly altered the expression of
   977 genes ([119]Fig. 5B). 400 of these genes overlap and corroborate
   that KL is the critical active ingredient in the water extract of CT.
   Partial least squares-discriminant analysis (PLS-DA) showed that the
   RNA levels in the livers of mice treated with WL or KL deviated from
   those of mice on HFD and shifted towards the mice on LFD ([120]Fig.
   5C).

Fig. 5.

   [121]Fig. 5
   [122]Open in a new tab

   Validation of Coreopsis tinctoria and kaempferol in alleviating
   obese-hyperglycemia based on liver transcriptomics (A)Alleviating
   effects of WL and KL treatments on high-fat diet-induced hyperglycemia
   (B) Venn diagram of genes altered in WL and KL compared to HFD (C)
   Partial least squares-discriminant analysis (PLS-DA) analysis (D) WL VS
   HFD KEGG enrichment (E) KL VS HFD KEGG enrichment (F) Heat-map of
   KL-altered genes in carbon metabolism, glycolysis/gluconeogenesis, and
   MAPK signaling pathways (G) Protein interaction and molecular docking
   (with KL) of differential genes enriched in carbon metabolism,
   glycolysis/gluconeogenesis, and MAPK signaling pathways (H) The
   molecular docking of PKLR and SDSL proteins with KL.

   The KEGG enrichment analysis showed that WL impacted several signal
   pathways, including thermogenesis, non-alcoholic fatty liver disease
   (NAFLD), steroid hormone biosynthesis, retinol metabolism, PPAR
   signaling pathway, bile secretion, and primary bile acid biosynthesis
   ([123]Fig. 5D). Differential genes between HFD and KL-treated mice were
   found to be enriched in MAPK signaling pathway, steroid hormone
   biosynthesis, retinol metabolism, carbon metabolism, glucagon signaling
   pathway, PPAR signaling pathway, AGE-RAGE signaling pathway in
   diabetic, and glycolysis/gluconeogenesis signaling pathways ([124]Fig.
   5E). Among the pathways, the carbon metabolism,
   glycolysis/gluconeogenesis, and MAPK signaling pathways are directly
   related to liver glucose metabolism. Compared to the HFD group, the
   significantly altered genes enriched in these pathways by KL
   administration shifted towards those of the LFD group, indicating that
   KL improved hyperglycemia caused by HFD through these signaling
   pathways ([125]Fig. 5F).

   Using PPI and molecular docking, we simulated interactions between
   proteins identified in [126]Fig. 5F and the possibility of KL binding
   to these proteins at the protein level ([127]Fig. 5G). The size of the
   protein node in [128]Fig. 5G corresponds to their interactions with
   other proteins. Pyruvate kinase L/R (PKLR) is the most critical protein
   in the network and participates in two signaling pathways: carbon
   metabolism and glycolysis/gluconeogenesis. The results of molecular
   docking affinity analysis revealed that KL can bind to multiple target
   proteins. An affinity score that is below −7 kcal mol^−1 (marked in
   red) indicates that the binding potential is high, while a score
   greater than −7 kcal mol^−1 (marked in blue) suggests a low binding
   potential. The serine dehydratase like (SDSL) protein, which is
   specific to type 2 diabetes and mellitus, showed the best affinity
   score of −9.4 kcal mol^−1 among all the proteins analyzed [[129]42].
   The molecular docking diagram of PKLR and SDSL proteins with KL is
   shown in [130]Fig. 5H.

   For [131]Fig. 5A, the results were expressed as mean ± SD. * means a
   significant difference (p < 0.05) compared with the HFD group. In
   transcriptomics, significantly changed genes were identified using a
   p-value of less than 0.05 (determined through one-way ANOVA) and a fold
   change greater than 2. All pathways enriched by KEGG (WL and KL) and
   enriched by GO are presented in [132]Supplementary Material 4.

4. Discussion

   Obesity, which can be caused by excessive energy intake, is often
   accompanied by increased adipose tissue, abnormal chemokines,
   adipokines, and pro-inflammatory cytokines [[133]43]. These indicators
   lead to decreased insulin sensitivity, an inability to suppress glucose
   output from the liver, and impaired glucose utilization by cells
   [[134]44]. In individuals with insulin resistance, cellular glucose
   transport is reduced, and plasma fatty acids and glucose levels are
   elevated [[135]35,[136]45]. Our results found that the water and
   alcoholic extracts of CT slightly reduced the body weight and the
   proportion of white adipose in HFD mice. Furthermore, the water and
   ethanol extracts of CT improved GTT and ITT in HFD mice. Among them,
   the water extract of CT at a dose of 4 g/kg.bw (WL) showed the best
   recovery effect on insulin sensitivity with a significant difference
   compared to the HFD group. Studies have shown that insulin resistance,
   accompanied by insufficient insulin secretion caused by damage to
   pancreatic β cells, results in type 2 diabetes [[137]45]. The serum
   insulin levels of HFD mice were significantly increased after the
   administration of CT extract, suggesting that CT extract can prevent
   the development of type 2 diabetes in obese individuals. Literature has
   confirmed that CT regulates insulin production by improving pancreatic
   inflammation and reducing damage in obese individuals
   [[138]13,[139]46,[140]47]. Under a high-fat diet, the body takes in
   excess energy, which leads to a physiological imbalance in energy
   consumption [[141]48]. Previous studies have confirmed that increasing
   energy expenditure improves obesity and insulin sensitivity [[142][49],
   [143][50], [144][51]]. We found that administration of 4 g/kg.bw CT
   water extract increased the mice's oxygen consumption, indicating that
   CT increased metabolic expenditure compared to the HFD mice [[145]52].

   Network pharmacology aims to predict biologically active natural
   products in plants and their molecular mechanisms by analyzing the
   relationship among components, targets, and pathways [[146]53]. Our
   prediction results indicated that the bioactive natural products
   (kaempferol, taxifolin, eriodictyol, luteolin, aloe-emodin, and
   quercetin) in CT act on the FoxO signaling pathway, endocrine
   resistance, and estrogen signaling pathways. The PI3K/AKT signaling
   pathway negatively regulates FoxO transcription factors, and the
   activation of FoxO transcription factors in the liver triggers
   hyperglycemia [[147]54,[148]55]; endocrine resistance leads to an
   imbalance between insulin secretion and sensitivity in target tissues,
   causing metabolic dysregulation and elevated blood sugar [[149]56].
   Estrogen signaling pathways regulate glucose metabolism through
   downstream pathways, including glucose transporter receptor α and
   insulin release [[150]57,[151]58]. ALB, EGFR, ESR1, HSP90AA1, SRC, and
   KDR are critical targets of CT. The HSP90 family and SRC contribute to
   insulin resistance, which interacts with the endocrine resistance
   signaling pathway [[152]59,[153]60]. EGFR and ESR1 are closely related
   to the estrogen signaling pathway and regulate energy metabolism
   [[154]61]. Furthermore, CT water extracts regulated the steroid hormone
   biosynthesis pathway in the mouse life, which was consistent with the
   estrogen signaling pathway predicted by network pharmacology. In
   addition, CT water extract acts on the non-alcoholic fatty liver
   disease and PPAR signaling pathways. These pathways are related to
   insulin resistance and energy metabolism in response to excess
   nutrition [[155]62,[156]63]. These findings suggest that improved
   energy metabolism under overnutrition is a potential mechanism by which
   CT alleviates hyperglycemia.

   Network pharmacology showed that the main active ingredient of CT in
   lowering blood sugar is flavonoids. The CT water extract contains
   bioactive flavonoids such as kaempferol, taxifolin, eriodictyol, and
   luteolin. The impact of these flavonoids on cellular glucose uptake
   under nutrient overload was verified in the L02 cell line. Elevated
   glucose uptake increases the utilization of energy by liver cells,
   glycogen production, and insulin sensitivity, thereby reducing the
   excess of glucose in the circulation [[157]64,[158]65]. The results
   showed that the flavonoid kaempferol in CT significantly promoted
   glucose uptake in L02 cells under PA stimulation. It was shown that CT
   improved the homeostasis of liver glucose metabolism under conditions
   of excess energy, and kaempferol was identified as the main functional
   component in CT. Transcriptomics analysis revealed that treatment with
   KL (25 mg/kg.bw kaempferol) in HFD mice intervened in the clustering of
   genes in the liver. Furthermore, KL treatment restores carbon
   metabolism, glycolysis/gluconeogenesis, and MAPK signaling pathways in
   the liver of obese hyperglycemic mice to levels similar to mice on LFD.
   These pathways are directly related to liver glucose metabolism. Carbon
   metabolism and glycolysis/gluconeogenesis signaling pathways are
   responsible for regulating the glucose storage and release of the
   liver, playing a crucial role in maintaining blood glucose homeostasis
   [[159][66], [160][67], [161][68]]. The MAPK signaling pathway connects
   extracellular stimuli with intracellular signaling and is crucial in
   glucose homeostasis and thermogenesis [[162]69]. Among these signaling
   pathways, PKLR and SDSL are predicted to be potential protein targets
   for KL. Overall, oral administration of KL alleviates obesity-induced
   hyperglycemia by restoring the signaling pathways against HFD.

   This article explores the mechanism by which CT improves hyperglycemia
   and insulin sensitivity for the first time at the transcription level,
   using network pharmacology analysis and examining liver RNA
   expressions. However, further investigation is needed to address
   certain research gaps in this study. Firstly, we used water and ethanol
   as extraction solvents because dried CT is commonly consumed in food by
   brewing tea or infusing it in liquor. This study elucidates the
   mechanism of CT as a functional food against hyperglycemia caused by
   HFD. However, there may be more efficient methods from an industrial
   extraction perspective. Secondly, this article assesses HFD mice as
   obesity-mediated hyperglycemia, basing their increase in body weight
   and blood glucose criteria. It is important to note that there are
   differences between the two batches of mice (both were modeled as
   obesity-hyperglycemia) included in the experiment in terms of the HFD
   (either 45% for 10 weeks or 60% for 11 weeks) they were given. Finally,
   The scientific significance of this article is limited to the
   verification at the transcription level only, as it did not include the
   measurement of changes in protein levels. Future research could focus
   on determining the intervention effect of CT on hyperglycemia at the
   protein level, as well as its impact on mouse liver primary cells.

   In conclusion, obesity resulting from excessive energy intake is often
   accompanied by elevated blood glucose levels and insulin resistance.
   Based on in Vivo and in Vitro verification, we found that Coreopsis
   tinctoria alleviates obesity, promotes energy consumption, and improves
   insulin sensitivity in mice. The water extract of CT exhibited the most
   effective recovery effect on energy metabolism at a dose of 4 g/kg.bw.
   Kaempferol is the key active compound in CT. It significantly
   facilitates the impaired glucose consumption caused by PA treatment in
   L02 cells. Also, kaempferol affects carbon metabolism,
   glycolysis/gluconeogenesis, and MAPK signaling pathways in the liver,
   reducing blood sugar levels in obese mice.

Ethics statement

   68 SPF grade male C57BL/6J mice was used in this experiment. All animal
   experiments complied with the ARRIVE guidelines. This experiment was
   approved by the Animal Ethics Committee of China Agricultural
   University (Approval number: KY1700038).

Funding

   Not applicable: This experiment did not receive any funding from
   organization, enterprise or individual.

CRediT authorship contribution statement

   Bingxin Huangfu: Writing – original draft, Software, Formal analysis,
   Conceptualization. Minglan Yang: Writing – original draft, Formal
   analysis, Data curation, Conceptualization. Jia Xu: Methodology. Ruxin
   Gao: Methodology. Yanzhou Hu: Data curation. Yijia Zhao: Methodology.
   Kunlun Huang: Writing – review & editing, Supervision. Xiaoyun He:
   Writing – review & editing, Supervision, Resources.

Declaration of competing interest

   The authors declare that they have no conflict of interest or personal
   relationships related to the publication of this paper.

Footnotes

   ^Appendix A

   Supplementary data to this article can be found online at
   [163]https://doi.org/10.1016/j.heliyon.2024.e27449.

Abbreviation

Abbreviation Full Spelling

   ALB
          albumin

   ALP
          alkaline phosphatase

   ALT
          alanine aminotransferase

   AST
          aspartate aminotransferase

   BP
          biological process

   BUN
          blood urea nitrogen

   bw
          body-weight

   CC
          cellular component

   CHO
          cholesterol

   CREA
          creatinine

   CT
          Coreopsis tinctoria

   EGFR
          epidermal growth factor receptor

   EH
          high dose ethanol extract

   EL
          low dose ethanol extract

   EP
          epididymal fat

   ESR1
          estrogen receptor 1

   GLU
          glucose

   GO
          gene ontology

   gtt
          glucose tolerance test

   HDL
          high-density lipoprotein

   HFD
          high-fat diets

   PKLR
          Pyruvate kinase L/R

   HSP90AA1
          human heat shock protein 90 kDa alpha itt insulin tolerance test

   KDR
          kinase insert domain receptor

   KEGG
          Kyoto Encyclopedia of Genes and Genomes

   LC-MS
          liquid chromatography-mass spectrometry

   LDL
          low-density lipoprotein

   MF
          molecular function

   NAFLD
          non-alcoholic fatty liver disease

   PA
          sodium palmitate

   PLS-DA
          Partial least squares-discriminant analysis

   PPI
          protein-protein interaction

   SD
          square deviation

   SRC
          SRC proto-oncogene, non-receptor tyrosine kinase

   SUB
          subcutaneous fat

   TCMSP
          traditional Chinese medicine systems pharmacology database and
          analysis platform

   TG
          triglycerides

   TP
          total protein

   UA
          uric acid

   WH
          high dose water extract

   WL
          low dose water extract

   PG
          plasma glucose

   SDSL
          serine dehydratase like

Appendix A. Supplementary data

   The following is/are the supplementary data to this article:
   Multimedia component 1
   [164]mmc1.docx^ (2.1MB, docx)

Data availability

   Data will be made available on request from the authors.

References