Abstract
Diospyros lotus L, F. Ebenaceae, is an edible fruit that is widely distributed in China and other Asian countries. Presently, Diospyros lotus L can be used to treat patients with diabetes; however, its chemical composition and pharmacological profiles remain to be elucidated. This study investigated the potential bioactive compounds of Diospyros lotus L and their mechanisms of action using LC-MS and network pharmacology analysis. First, the components of Diospyros lotus L were identify using a reliable strategy for UHPLC-Q-Exactive Orbitrap mass spectrometry combined with parallel reaction monitoring (PRM) in the negative ion mode. Second, a network pharmacology study, including target gene prediction and functional enrichment, was applied to screen the main quality markers of Diospyros lotus L and explore its potential mechanism for the treatment of diabetes. The results showed that a total of 159 compounds were identified from Diospyros lotus L, among which, 140 were reported for the first time. Furthermore, 40 active components, such as quercetin, luteolin, and kaempferol, were proposed as active components of Diospyros lotus L for the treatment of diabetes based on network pharmacology analysis. In addition, 92 relevant antidiabetic targets were mainly related to positive regulation of transcription from the RNA polymerase II promoter, extracellular space, and protein binding, suggesting the involvement of TNF, PI3K-Akt, and HIF-1 signaling pathways in the antidiabetic effect of Diospyros lotus L. Our results may provide a useful approach to identify potential active components and molecular mechanisms of Diospyros lotus L for the treatment of diabetes.
1. Introduction
Diospyros lotus L, a genus of the family Ebenaceae, is an edible fruit that is widely distributed in China and other Asian countries. Diospyros lotus L fruit extract has antidiabetic, antitumor, antinociceptive, and anti-inflammatory effects [1–4] and is used for treating various diseases, such as hypertension, diarrhea, and dry cough. However, to date, few studies have investigated the chemical composition and mechanism of the antidiabetic effect of Diospyros lotus L.
Diabetes is a chronic, progressive, and complex metabolic disease characterized by hyperglycemia, which is caused by insufficient insulin secretion, insufficient function, or the simultaneous occurrence of both [5, 6]. Owing to the long-term effects of hyperglycemia, various diabetic complications can occur. These complications not only cause great harm to the physiological and psychological status of the patients but also put enormous pressure on society [7].
Extracts of Diospyros lotus L and its compounds have hypoglycemic effects [8–10]. However, the pharmacological mechanisms and bioactive components of Diospyros lotus L remain unknown. Network pharmacology is based on the chemical components of traditional Chinese medicine in the existing database to explore its mechanism from multiple perspectives, such as target gene identification and function prediction [11, 12].
In this study, a UHPLC-Orbitrap-MS combined with PRM was developed for component identification of Diospyros lotus L. The bioactive ingredients and mechanism of action of Diospyros lotus L on the targets of diabetes were investigated by network pharmacology, which is of great significance for further research on Diospyros lotus L.
2. Methodology
2.1. Materials and Chemicals
HPLC-grade acetonitrile and methanol were obtained from Merck Company Inc. (Darmstadt, Germany), and formic acid was obtained from Fisher Chemicals (Fairlawn, NJ, USA). Purified water was purchased from the A.S. Watson Group Ltd. (Hong Kong). Other reagents and chemicals were of analytical grade and were supplied by the Aladdin Industrial Corporation. Dried Diospyros lotus L samples were collected from Shexian County, Hebei Province, China, in November of each year.
Reference standards, including neochlorogenic acid, chlorogenic acid, 1,3-dicaffeoylquinic acid, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C, were obtained from Chengdu Herbpurify Co., Ltd. Procyanidin, phlorizin, trilobatin, and phloretin were acquired from Sichuan Weikeqi Biological Technology Co., Ltd. Quinic acid, ferulic acid, catechin, quercetin, quercitrin, quercetin 3-O-rutinoside, myricitrin, isoquercitrin, nicotiflorin, myricetin, eriodictyol, luteolin, naringenin, and kaempferol were purchased from Chengdu Purechem Standard Co., Ltd. The purity of all standard compounds was no less than 98% based on LC-UV.
2.2. Standard and Sample Preparation
Diospyros lotus L (1 g) was extracted with 70% methanol (20 mL) by sonication (1 h). Then, the extract was centrifuged (15 min, 10°C, 12000 rpm) to obtain the supernatant. Finally, 2 μL of the supernatant was injected into the LC-MS system for analysis.
All the reference standards were accurately weighed using an electronic analytical balance (1 mg) and dissolved in methanol (1 mL). Then, 10 μL of each standard solution was added to a 1-mL volumetric flask to prepare a mixed standard solution. The obtained standard solution was stored below 4°C before analysis.
2.3. Chromatography and MS Conditions
Chromatographic separation was performed on a Dionex Ultimate 3000 UHPLC (Thermo Fisher Scientific, San Jose, CA, USA), using a Thermo Scientific Hypersil GOLDTM aQ (100 mm × 2.1 mm, 1.9 μm).
The column compartment was maintained at 40°C, and the flow rate was set at 0.3 mL/min. Water containing 0.1% formic acid (solvent system A) and acetonitrile (solvent system B) served as the mobile phase. The gradient elution program was as follows: 0–2 min, 95%–5% B; 2–5 min, 80%–20% B; 5–10 min, 75%–25% B; 10–12 min, 45%–5% B; 12–20 min, 20%–80% B; 20–25 min, 5%–95% B; 25–30 min, 95%–5% B.
Mass detection was performed on a Q-Exactive Orbitrap MS equipped with an electrospray ionization source operating in negative mode with the following operating parameters: spray voltage at −3.0 kV; sheath gas flow rate at 30 arbs; auxiliary gas flow rate at 10 arbs; capillary temperature at 320°C; heater temperature at 350°C; S-lens RF level at 50; and normalized collision energies at 30%. The MS spectra were recorded over an m/z range of 80–1000. All data were acquired and processed using Xcalibur software version 4.2.
2.4. Candidate Ingredient Screening
To select the components that have better biological availability in vivo, the components were filtered using the principle of “drug-like soft” in FAFDrugs4. Screening parameters included restriction to molecular weight, logP, and hydrogen bond acceptors (HBA). Details of the physicochemical property filters are listed in Table 1.
2.5. Targets of Diospyros lotus L and Diseases
The targets of the filtered components were obtained from the traditional Chinese medicine systems pharmacology database and analysis Platform (TCMSP) and predicted using Swiss TargetPrediction (STP). Setting the organism “Homo sapiens” and targets with a probability value greater than 0.1 were considered as potential effective targets for these compounds in the STP database.
Diabetes-related targets were searched in the Online Mendelian Inheritance in Man and GeneCards platform with “diabetes” as the keyword. The collected targets were amalgamated and duplicated. Potential target genes of Diospyros lotus L therapy for diabetes were obtained through the jvenn intersection.
2.6. Construction of Protein-Protein Interaction (PPI) Network
The PPI network between target proteins of the related ingredients in Diospyros lotus L and diabetes was obtained by STRING and then imported into Cytoscape software version 3.8.2 to construct and validate a visual network. The species was set as “Homo sapiens,” and the protein interaction was obtained with a medium confidence score of 0.4 to ensure the reliability of our analysis. In the PPI network, topology parameters were calculated to obtain promising candidate targets that were visually characterized by the colors of nodes and to screen remarkable targets.
2.7. Enrichment Analysis
Gene Ontology (GO) and KEGG pathway enrichment analyses were performed using DAVID software. Subsequently, correlated “histograms” and “bubble graphs” were established.
2.8. Construction of Active Component-KeyGene-Pathway Interaction Network
To further explore the mechanism of the antidiabetic effect of Diospyros lotus L, an active component-keygene-pathway interaction network was constructed using Cytoscape 3.9.0 software. In the network, nodes with different shapes represented the active compounds, key genes, and related pathways, and an “edge” was an association between the nodes.
3. Results and Discussion
3.1. Establishment of Qualitative Analysis Strategy
In this study, an analytical method of UHPLC-Q-Exactive Orbitrap MS combined with the acquisition mode of the PRM mode was used to identify the chemical components of Diospyros lotus L. First, the extraction method and UHPLC-MS conditions of Diospyros lotus L were optimized. Second, the sample was injected into the UHPLC-Q-Exactive Orbitrap MS to obtain high-resolution mass data, including MS and MS2. Third, the compounds were predicted using the Compound Discover version 3.0 workstation with the aid of the metabolism workflow template by adjusting relevant parameters. Finally, the compounds were characterized based on full-scan MS and MS2, retention times, standards, and literature.
3.2. Optimization of the Extraction Method
To obtain the maximum extraction yield, the extraction method for Diospyros lotus L was optimized with respect to time (0.5, 1, and 2 h); solvent type (methanol and ethanol); solvent concentration (60%, 70%, and 80%); and liquid-to-solid ratio (10 : 1, 15 : 1, and 20 : 1). The optimal extraction method was ultrasonic extraction with 70% methanol (20 ml) for 1 h.
3.3. Optimization of UHPLC-MS Conditions
To achieve good chromatographic separation, UHPLC parameters were optimized, including the mobile phase (methanol/water and acetonitrile/water); type and content of acid (acetic acid and formic acid, 0.05%, 0.1%, and 0.2%); column (Waters ACQUITY BEH C18 column, 100 mm × 2.1 mm, 1.7 μm, and HYPERSIL GOLD C18 column, 100 mm × 2.1 mm, 1.9 µm); column temperature (30, 35, and 40°C); flow rate of the mobile phase (0.2, 0.3, and 0.4 mL/min); and the difference gradient of mobile phase.. The MS parameters, including the flow rate of the sheath gas and auxiliary, temperature of the capillary and auxiliary, heater temperature, spray voltage, and collision energies were examined. In the optimized conditions of UHPLC-Q-Exactive Orbitrap MS, most of the components in the Diospyros lotus L showed efficient separation and parent/daughter ion pairs with high responses.
3.4. Characterization of Diospyros lotus L
A total of 159 compounds, including 88 flavonoids, 24 phenylpropanoids, and 47 organic acids, were tentatively identified by UHPLC-Q-Exactive Orbitrap mass spectrometry; among them, 140 were reported for the first time in Diospyros lotus L. The chromatographic and mass data for the detected constituents are presented in Table 2. The extracted ion chromatogram in negative ion mode is shown in Figure 1.
(a)
(b)
(c)
(d)
3.4.1. Identification of the Flavonoids in Diospyros lotus L
Compounds 36, 97, 98, 107, 117, 125, 128, 134, 144, 147, 150, 151, 153, 155, 157, and 159 were found at 4.99, 7.60, 7.60, 7.82, 8.33, 8.67, 8.93, 9.36, 9.45, 10.13, 10.33, 11.90, 12.11, 12.58, 12.82, 13.19, and 14.46 min, respectively. They were accurately identified as catechin, quercetin 3-O-rutinoside, isoquercitrin, myricitrin, nicotiflorin, quercitrin, phlorizin, myricetin, trilobatin, eriodictyol, quercetin, luteolin, naringenin, phloretin, kaempferol, and procyanidin, respectively, by comparing the data with those of authentic standards.
Compounds 64 and 88 possessed the same quasi-molecular ions and characteristic fragment ions as compound 97; thus, they were characterized as quercetin 3-O-rutinoside isomers. Similarly, compounds 84, 114, and 139 were isoquercitrin isomers, and compounds 109, 140, 146, and 154 were assigned as isomers of nicotiflorin, quercetin, myricetin, and luteolin, respectively. Compounds 110 and 119 were tentatively presumed to be phlorizin isomers.
Compound 51, with the deprotonated ion [M-H]− at m/z 625.1413, was eluted at 6.08 min, with the main characteristic fragment ion at m/z 463.0877, owing to the loss of a glucose residue (162 Da), which further gave rise to product ions at m/z 301.0351. It was tentatively characterized as a quercetin 3,4′-diglucoside [13]. Likewise, compounds 74, 92, 124, and 148 were deduced to be quercetin derivatives; compound 78 was quercetin 3-rutinoside 7-rhamnoside [14]; and compounds 100 and 108 were quercetin 3-O-(6″-galloyl)-β-D-glucopyranoside isomers. Compound 105 was characterized as quercitrin 3-O-glucuronide, and compounds 116 and 122 were quercitrin 3-O-arabinoside isomers [15–17].
Compounds 53, 69, and 89 exhibited quasi-molecular ions [M-H]− at m/z 303.0510, and fragment ions at m/z 125.0232, 151.0026, and 177.0187 were tentatively characterized as taxifolin isomers, as previously reported [16].
Compound 57 was found at 6.23 min, yielded a parent ion [M-H]− at m/z 609.1461 consisting of kaempferol (285 Da) and two glucose moieties (324 Da), and was identified as kaempferol 3,7-diglucoside [13].
Similarly, compound 141 was kaempferol-7-O-rhamnoside, and compounds 35, 45, 70, and 76 were suggested to be kaempferol derivatives [18].
Compounds 48, 71, 85, and 101 were detected at 5.95, 6.69, 7.17, and 7.70 min, respectively, and possessed the same quasi-molecular ions [M-H]− at m/z 641.1359, MS/MS fragment ions at m/z 317.0301, and 316.0200 owing to the loss of two galactoside residues (324 Da), indicating the presence of a myricetin group. Therefore, they have been characterized as myricetin 3,3′-digalactoside isomers. Likewise, compounds 68, 82, 83, 93, and 138 were assigned as myricetin 3-rutinoside-7-rhamnoside, myricetin 3-O-glucuronide, myricetin 3-O-galactoside, myricetin derivative, and myricetin 3-O-(6″-galloyl)-β-D-rhamnoside, respectively, and compounds 73, 79, and 106 were myricetin 3-O-rutinoside isomers [19–21].
Compounds 50 and 65 were eluted at 6.07 and 6.53 min, respectively, and possessed the same quasi-molecular ion [M-H]− at m/z 319.0459 and fragment ions at m/z 125.0232, 193.0134, and 151.0026. They were tentatively assigned as dihydromyricetin isomers by referring to the literature [22].
Compounds 52 and 60 yielded a quasi-molecular ion [M-H]− at m/z 449.1089, which was tentatively identified as the maesopsin 4-O-glucoside isomer according to a previously published paper [21]. Likewise, compounds 77, 99, 149, and 158 were catechin di-C-hexoside, 3′, 5′-di-C-β-d-glucosylphloretin [23], chrysin derivatives, and chrysoeriol, respectively. Compounds 135 and 143 were 5,2′,6′-dihydroxy-7,8-dimethoxyflavone isomers [24], and compounds 132 and 133 were viscidulin III 6′-o-β-d-glucoside isomers [24].
Compounds 62 and 123 showed a deprotonated ion [M-H]− at m/z 287.0561. The appearance of fragment ions at m/z 125.0233 and 151.0027 in the MS2 spectrum of those compounds indicated that they were (2S)-5,7,2′,6′-tetrahydroxyflavanone isomers [24].
Compounds 66, 91, and 103 yielded a quasi-molecular ion [M-H]− at m/z 433.1140 and were eluted at 6.58, 7.44, and 7.73 min, respectively, which showed fragment ions at m/z 271.0611 by the neutral loss of glucose moieties (162 Da). Thus, they were considered to be naringenin 7-O-glucoside isomers [25]. Similarly, compounds 102 and 118 were naringenin-O-glucoside-rhamnosides.
Compounds 75 and 81 appeared at a retention time (tR) of 6.96 and 7.07 min, respectively, possessing the quasi-molecular ions [M-H]− at m/z 433.1140 and their fragment ions at m/z 313.0717 ([M-H-120]−) and 343.0820 ([M-H-90]−), which were identified as naringenin 6-C-glucosidei somer [26]. Similarly, compound 47 was deduced as naringenin-6,8-di-C-glucoside [27].
Compounds 152 and 156 were found at 12.34 and 12.97 min, respectively, which show the common precursor ion [M-H]− at m/z 315.0505, and the major fragment ion at m/z 300.0275 due to loss of a CH3 reside (15 Da), then they were tentatively characterized as isorhamnetinisomer [17]. Compounds 112, 126, 127, 137, and 142 appeared at retention times (tR) of 8.10, 8.72, 8.87, 9.58, and 10.00 min, respectively, which were tentatively identified as 3-methylquercetin-7-O-glucoside isomers. The parent ions at m/z 477.1038 were due to the loss of glucose moieties (162 Da) and generated the characteristic fragment ions at m/z 315.0505 [17].
3.4.2. Identification of Phenylpropanoids in Diospyros lotus L
Compounds 30, 40, 42, 67, 87, 111, 115, and 136 were eluted at 3.33, 5.42, 5.46, 6.65, 7.29, 8.04, 8.28, and 9.45 min, respectively. They were characterized as neochlorogenic acid, chlorogenic acid, caffeic acid, isochlorogenic acid B, ferulic acid, 1,3-dicaffeoylquinic acid, isochlorogenic acid A, and isochlorogenic acid C, respectively, by comparison to commercial reference standards.
Compound 61 possessed the same quasi-molecular ions, and the characteristic fragment ion of compound 42 was characterized as a caffeic acid isomer. Similarly, compounds 95, 104, and 129 were ferulic acid isomers.
Compounds (56, tR 6.22 min, and 63, tR 6.52 min) had the same quasi-molecular ions [M-H]− at m/z 355.1035 and the fragment ion at m/z 193.0500, corresponding to the neutral loss of the glucose group (162 Da) and further generation of the fragment ions of compound 87. Therefore, they were tentatively assigned as ferulic acid acyl-β-D-glucoside isomers [17].
Compounds 37, 43, 44, and 46 were eluted at 5.30, 5.49, 5.60, and 5.73 min, respectively, and showed a deprotonated molecular ion [M-H]− at m/z 457.1351. They were tentatively inferred to be p-coumaric acid-O-glucoside-rhamnoside based on the base peak ion in the MS2 spectrum.
Compounds 80, 90, and 121 were eluted at 7.07, 7.41, and 8.42 min, respectively, yielding a deprotonated ion [M-H]− at m/z 579.2083 and fragment ions at m/z 417.1554, 181.0497, and 402.1317, which were suggested as syringaresinol O-β-D-glucoside isomers in comparison with the literature [28].
Compounds 38, 59, and 96 were eluted at 5.36, 6.39, and 7.60 min, respectively, and yielded the same parent ion [M-H]− at m/z 177.0193. They were deduced as esculetin isomers according to the MS and MS/MS spectra [25].
3.4.3. Identification of Organic Acids in Diospyros lotus L
Compounds 2, 5, and 10 were found at 0.86, 0.94, and 1.34 min, respectively, and possessed the same parent ion [M-H]− at m/z 191.0561. Compound 5 was identified as quinic acid by comparison with the reference substances. Thus, compounds 2 and 10 were identified as isomers of quinic acid.
Compounds 1 and 4 were observed at 0.83 and 0.94 min, respectively, and possessed the same quasi-molecular ions [M-H]− at m/z 93.0347 and MS/MS fragment ions at m/z 71.0124, 101.0230, and 113.0231. They were tentatively assigned as glucuronic acid isomers by comparison to the literature. Similarly, compounds 3, 6, and 8 were citric acid isomers [20], compounds 11 and 22 were 3-methylglutaric acid isomers, compound 27 was syringic acid glucoside [29], compounds 17 and 18 were pantothenic acid isomers, compound 55 was dihydrophaseic acid, compound 120 was azelaic acid, and compounds 130 and 145 were abscisic acid isomers [16, 27].
Compound 14 exhibited a quasi-molecular ion [M-H]− at m/z 169.0142 and generated the main characteristic fragments ion at m/z 125.0233 ([M-CO2-H]−), which was identified as gallic acid by the MS and MS/MS spectra [30]. Compounds 7, 12, 13, and 21, with the same deprotonated ions [M-H]− at m/z 331.0671, were eluted at 1.07, 1.36, 1.41, and 1.91 min, respectively. The main fragment ions, at m/z 169.0132, were obtained by the loss of glucose moieties (162 Da) as well as characteristic fragment ions of gallic acid (m/z 125.0233), which were deduced as 6-O-galloylglucose isomers [14]. Similarly, compound 9 was identified as 6-O-galloylsucrose.
Compound 33 was eluted at 4.07 min, possessing a quasi-molecular ion [M-H]− at m/z 183.0298, showing characteristic fragment ions at (m/z 140.0103, 124.0153), and was characterized as methyl gallate [16].
Compounds 15 and 39 were detected at 1.65 and 5.37 min, respectively. They showed the same deprotonated ion [M-H]− at m/z 153.0193 and the fragment ions at m/z 108.0203, 109.0282, and 123.0439, suggesting that they were 2,3-dihydroxybenzoic acid isomers [27, 31]. Compound 20 yielded a deprotonated ion [M-H]− at m/z 315.0722, which showed a fragment ion at m/z 153.0186 by losing the glucose moiety (162 Da) in the MS2 fragment ions; therefore, it was tentatively identified as a 2,3-dihydroxybenzoic acid 3-O-glucoside isomer [31].
Compounds 16, 19, 28, 29, 41, 58, 113, and 131 were detected at 1.67, 1.84, 3.09, 3.19, 5.44, 6.38, 8.10, and 9.18 min, respectively, and possessed the same quasi-molecular ions [M-H]− at m/z 151.0401. The characteristic fragment ions at m/z 108.0204, 123.0439, and 136.0154 were identified as vanillin isomers according to the base peaks and retention times. Compounds 72 and 86 were found at 6.72 and 7.23 min, respectively, and yielded the parent ions [M-H]− at m/z 167.0350. They were identified as vanillic acid isomers based on the MS and MS/MS spectra [16]. Compound 49 appeared at a tR of 5.95 min, possessing quasi-molecular ions at m/z 329.0878 and the main fragment ion at m/z 167.0341 owing to the loss of a glucose residue (162 Da), which was characterized as vanillic acid glucoside [32]. Similarly, compounds 34 and 54 were confirmed as vanillic acid-O-rutinosides.
Compound 94 at m/z 137.0244 with the molecular formula C7H6O3 and appearing at a tR of 7.58 min was suggested to be p-hydroxybenzoic acid based on the MS2 data [21]. Compounds 23, 24, 25, 26, 31, and 32 (tR 2.65, 2.85, 2.89, 3.00, 3.53, and 3.75 min, respectively) had the same quasi-molecular ion [M-H]− at m/z 299.0772 and the characteristic fragment ion at m/z 137.0233 based on the neutral loss of a glucose residue (162 Da). They were tentatively characterized as p-hydroxybenzoic acid-O-glucoside isomers [33].
3.5. Active Components and Related Targets
The active compounds were selected by using the “drug-like soft” in FAFDrugs4 with the criteria of 100 ≤ MW ≤ 600, −3 ≤ logP ≤ 6, and HBA ≤ 12. Eventually, a total of 40 compounds were screened (Supplementary Table 1). Combined with TCMSP and STP database search and prediction, 445 component targets were obtained after removing duplicate targets. Furthermore, 521 diabetes-related targets were identified by screening the disease-target database. Finally, 92 overlapping genes of compound targets and diabetes-related targets were regarded as potential targets of Diospyros lotus L for the treatment of diabetes (Figure 2).
3.6. PPI Network of Overlapping Genes
The PPI network graph was obtained by importing 92 overlapping targets into STRING and removing one disconnected point. There were 91 nodes and 1488 edges; the average number of nodes was 32.8, and the average local clustering coefficient was 0.703. TSV data were downloaded and imported into Cytoscape 3.9.0 software to show the protein interaction network.
The results are shown in Figure 3, where the node size is positively correlated with the degree value and the lines represent interactions. As betweenness centrality increases, the color of the node changes from yellow to turquoise. Degree and betweenness centrality indicate the importance of the targets. The target whose degree value was greater than the average value was considered the key target.
3.7. Enrichment Analysis
The key targets were further analyzed by functional association clustering to integrate functional genomics annotations of the most important cluster of targets and pathways, which facilitates further understanding of the mechanism of the antidiabetic effect of Diospyros lotus L.
As shown in Figure 4, the most representative GO-BP terms were “positive regulation of transcription from RNA polymerase II promoter” and “inflammatory response,” whereas the most representative GO-CC terms were “extracellular space,” “extracellular region,” and “plasma membrane.” The most representative GO-MF terms were “protein binding” and “enzyme binding.”
The two most representative KEGG pathways (Figure 5) were the “MAPK signaling pathway” and “AGE-RAGE signaling pathway.” After exclusion of broad pathways, 47 core common target genes were mainly related to the TNF, PI3K-Akt, HIF-1, NAFLD, toll-like receptor, and other multiple signaling pathways. This suggests that the effect of Diospyros lotus L on diabetes may involve multiple pathways as well as complex interactions among these pathways.
3.8. Active Component-KeyGene-Pathway Interaction Network Analysis
As shown in Figure 6, the active component-keygene-pathway interaction network contained 104 nodes (47 key genes, 37 active components, and 20 KEGG pathways (top 20)) and 410 edges. In the network, the diamond, oval, and elliptical nodes correspond to different active compounds, pathways, and targets, respectively. The degrees of quercetin, luteolin, kaempferol, TNF signaling pathway, PI3K-Akt signaling pathway, HIF-1 signaling pathway, PTGS2, AKT1, IL6, and TNF were 34, 20, 18, 12, 11, 10, 25, 21, 20, and 17, respectively. The average degrees of the diamond and elliptical nodes were 5.93° and 8.72°, respectively. In addition, at least nine genes were potentially involved in each diabetes-related pathway, suggesting that one active component can potentially target multiple genes and have the action characteristics of multiple active compounds, targets, and pathways of Diospyros lotus L in the treatment of diabetes.
Quercetin has many antihyperglycemic effects, such as enhancing insulin sensitivity, promoting glycogen synthesis, inhibiting α-glucosidase activity, and improving insulin resistance [34]. Luteolin can play an antioxidant role by enhancing the activity of superoxide dismutase in microvascular lesions in diabetes [35]. Kaempferol is a flavonoid compound that plays an active role in the prevention and treatment of diabetes and has anti-inflammatory and antioxidant properties. It can reduce oxidative stress and inflammation through the MAPK pathway to alleviate myocardial ischemia-reperfusion injury in diabetic rats [36]. Myricetin can enhance the antioxidant defense system in mice, increase insulin secretion, substantially reduce blood glucose levels, and effectively protect the liver and kidney from oxidative damage in diabetic mice [37, 38]. IL-6 interferes with the insulin signaling pathway and promotes apoptosis of pancreatic β-cells, which promotes insulin resistance in multiple organs through a variety of inflammatory signaling pathways [39]. TNF is one of the cytokines constituting the acute inflammatory response, which can trigger the MAPK and NF-κB pathways, leading to insulin resistance [40].
4. Conclusion
In this study, an integrated approach combining UHPLC-Q-Exactive Orbitrap MS and network pharmacology analysis was adopted to explore the potential active ingredients and ameliorative mechanisms of Diospyros lotus L against hyperglycemia. Eventually, 159 compounds were identified in Diospyros lotus L (140 of which were reported for the first time). According to the results of the active components and key gene-pathway interaction network, the antihyperglycemic effect of Diospyros lotus L is attributed to quercetin, luteolin, kaempferol, myricetin, and dihydromyricetin, which act on PTGS2, AKT1, IL6, TNF, and MMP9 and participate in the TNF, PI3K-Akt, and HIF-1 signaling pathways, as well as NAFLD. In conclusion, the integrated approach combining UHPLC-Q-Exactive Orbitrap MS and network pharmacology analysis provided insights into the potential active ingredients and ameliorative mechanism of Diospyros lotus L on hyperglycemia.
Data Availability
The data used to support the finding of this study are available from the corresponding author upon request.
Disclosure
The funder has no role/influence in this study.
Conflicts of Interest
The authors declare that there are no conflicts of interest.
Authors’ Contributions
Shihan Qin and Mingjuan Liu contributed equally to this work.
Acknowledgments
This work was financially supported by the Science and Technology Innovation Program of Hunan Province (no. 2022RC1228), the Scientific Research Fund of Hunan Provincial Education Department (no. 19A353) and the Hunan University of Medicine High-Level Talent Introduction Startup Funds (no. 15001).
Supplementary Materials
Supplementary Table 1: Results of screening compounds by “drug-likesoft” in FAFDrugs4. (Supplementary Materials)