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/lp/hindawi-publishing-corporation/rapid-characterization-and-action-mechanism-of-the-antidiabetic-effect-BVZ5gRljTH
- Publisher
- Hindawi Publishing Corporation
- ISSN
- 2090-8865
- eISSN
- 2090-8873
- DOI
- 10.1155/2022/8000126
- Publisher site
- See Article on Publisher Site
Abstract
Hindawi Journal of Analytical Methods in Chemistry Volume 2022, Article ID 8000126, 19 pages https://doi.org/10.1155/2022/8000126 Research Article Rapid Characterization and Action Mechanism of the Antidiabetic Effect of Diospyros lotus L Using UHPLC-Q-Exactive Orbitrap MS and Network Pharmacology 1,2 1 1 1,2 1 Shihan Qin , Mingjuan Liu , Sunv Tang , E. Shuai , Ziming Wang , 1 1 Kaiquan Yu , and Wei Cai School of Pharmaceutical Sciences, Sino-Pakistan Center on Traditional Chinese Medicine, Hunan University of Medicine, Huaihua 418000, China School of Pharmacy, Weifang Medical University, Weifang 261000, China Correspondence should be addressed to Wei Cai; 20120941161@bucm.edu.cn Received 9 August 2022; Revised 27 November 2022; Accepted 15 December 2022; Published 31 December 2022 Academic Editor: Luca Campone Copyright © 2022 Shihan Qin et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 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 profles remain to be elucidated. Tis 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. Te results showed that a total of 159 compounds were identifed from Diospyros lotus L, among which, 140 were reported for the frst 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 efect 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. Diabetes is a chronic, progressive, and complex meta- 1. Introduction bolic disease characterized by hyperglycemia, which is Diospyros lotus L, a genus of the family Ebenaceae, is an caused by insufcient insulin secretion, insufcient function, edible fruit that is widely distributed in China and other or the simultaneous occurrence of both [5, 6]. Owing to the long-term efects of hyperglycemia, various diabetic com- Asian countries. Diospyros lotus L fruit extract has anti- diabetic, antitumor, antinociceptive, and anti- plications can occur. Tese complications not only cause infammatory efects [1–4] and is used for treating vari- great harm to the physiological and psychological status of ous diseases, such as hypertension, diarrhea, and dry cough. the patients but also put enormous pressure on society [7]. However, to date, few studies have investigated the Extracts of Diospyros lotus L and its compounds have chemical composition and mechanism of the antidiabetic hypoglycemic efects [8–10]. However, the pharmacological efect of Diospyros lotus L. mechanisms and bioactive components of Diospyros lotus L 2 Journal of Analytical Methods in Chemistry remain unknown. Network pharmacology is based on the 20% B; 5–10 min, 75%–25% B; 10–12 min, 45%–5% B; chemical components of traditional Chinese medicine in the 12–20 min, 20%–80% B; 20–25 min, 5%–95% B; 25–30 min, existing database to explore its mechanism from multiple 95%–5% B. perspectives, such as target gene identifcation and function Mass detection was performed on a Q-Exactive Orbitrap prediction [11, 12]. MS equipped with an electrospray ionization source oper- In this study, a UHPLC-Orbitrap-MS combined with ating in negative mode with the following operating pa- PRM was developed for component identifcation of Dio- rameters: spray voltage at −3.0 kV; sheath gas fow rate at 30 spyros lotus L. Te bioactive ingredients and mechanism of arbs; auxiliary gas fow rate at 10 arbs; capillary temperature ° ° action of Diospyros lotus L on the targets of diabetes were at 320 C; heater temperature at 350 C; S-lens RF level at 50; investigated by network pharmacology, which is of great and normalized collision energies at 30%. Te MS spectra signifcance for further research on Diospyros lotus L. were recorded over an m/z range of 80–1000. All data were acquired and processed using Xcalibur software version 4.2. 2. Methodology 2.4. Candidate Ingredient Screening. To select the compo- 2.1. Materials and Chemicals. HPLC-grade acetonitrile and nents that have better biological availability in vivo, the methanol were obtained from Merck Company Inc. components were fltered using the principle of “drug-like (Darmstadt, Germany), and formic acid was obtained from soft” in FAFDrugs4. Screening parameters included re- Fisher Chemicals (Fairlawn, NJ, USA). Purifed water was striction to molecular weight, logP, and hydrogen bond purchased from the A.S. Watson Group Ltd. (Hong Kong). acceptors (HBA). Details of the physicochemical property Other reagents and chemicals were of analytical grade and flters are listed in Table 1. 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. 2.5. Targets of Diospyros lotus L and Diseases. Te targets of Reference standards, including neochlorogenic acid, the fltered components were obtained from the traditional chlorogenic acid, 1,3-dicafeoylquinic acid, isochlorogenic Chinese medicine systems pharmacology database and acid A, isochlorogenic acid B, and isochlorogenic acid C, analysis Platform (TCMSP) and predicted using Swiss were obtained from Chengdu Herbpurify Co., Ltd. Pro- TargetPrediction (STP). Setting the organism “Homo sa- cyanidin, phlorizin, trilobatin, and phloretin were acquired piens” and targets with a probability value greater than 0.1 from Sichuan Weikeqi Biological Technology Co., Ltd. were considered as potential efective targets for these Quinic acid, ferulic acid, catechin, quercetin, quercitrin, compounds in the STP database. quercetin 3-O-rutinoside, myricitrin, isoquercitrin, nicoti- Diabetes-related targets were searched in the Online forin, myricetin, eriodictyol, luteolin, naringenin, and Mendelian Inheritance in Man and GeneCards platform kaempferol were purchased from Chengdu Purechem with “diabetes” as the keyword. Te collected targets were Standard Co., Ltd. Te purity of all standard compounds was amalgamated and duplicated. Potential target genes of no less than 98% based on LC-UV. Diospyros lotus L therapy for diabetes were obtained through the jvenn intersection. 2.2. Standard and Sample Preparation. Diospyros lotus L (1 g) was extracted with 70% methanol (20 mL) by sonication 2.6. Construction of Protein-Protein Interaction (PPI) (1 h). Ten, the extract was centrifuged (15 min, 10 C, Network. Te PPI network between target proteins of the 12000 rpm) to obtain the supernatant. Finally, 2 μL of the related ingredients in Diospyros lotus L and diabetes was supernatant was injected into the LC-MS system for analysis. obtained by STRING and then imported into Cytoscape All the reference standards were accurately weighed software version 3.8.2 to construct and validate a visual using an electronic analytical balance (1 mg) and dissolved in network. Te species was set as “Homo sapiens,” and the methanol (1 mL). Ten, 10 μL of each standard solution was protein interaction was obtained with a medium confdence added to a 1-mL volumetric fask to prepare a mixed score of 0.4 to ensure the reliability of our analysis. In the PPI standard solution. Te obtained standard solution was network, topology parameters were calculated to obtain stored below 4 C before analysis. promising candidate targets that were visually characterized by the colors of nodes and to screen remarkable targets. 2.3. Chromatography and MS Conditions. Chromatographic separation was performed on a Dionex 2.7. Enrichment Analysis. Gene Ontology (GO) and KEGG Ultimate 3000 UHPLC (Termo Fisher Scientifc, San Jose, pathway enrichment analyses were performed using DAVID CA, USA), using a Termo Scientifc Hypersil GOLDTM aQ software. Subsequently, correlated “histograms” and “bubble (100 mm × 2.1 mm, 1.9 μm). graphs” were established. Te column compartment was maintained at 40 C, and the fow rate was set at 0.3 mL/min. Water containing 0.1% formic acid (solvent system A) and acetonitrile (solvent 2.8. Construction of Active Component-KeyGene-Pathway system B) served as the mobile phase. Te gradient elution Interaction Network. To further explore the mechanism of program was as follows: 0–2 min, 95%–5% B; 2–5 min, 80%– the antidiabetic efect of Diospyros lotus L, an active Journal of Analytical Methods in Chemistry 3 Table 1: Te range of the parameters of the “drug-like soft” UHPLC-Q-Exactive Orbitrap MS, most of the components principle. in the Diospyros lotus L showed efcient separation and parent/daughter ion pairs with high responses. Property Range Property Range MW 100–600 logP −3–6 HBA ≤12 HBD ≤7 3.4. Characterization of Diospyros lotus L. A total of 159 tPSA ≤180 Rotatable bonds ≤11 compounds, including 88 favonoids, 24 phenyl- Rigid bonds ≤30 Rings ≤6 propanoids, and 47 organic acids, were tentatively iden- Max size system ring ≤18 Carbons 3–35 tifed by UHPLC-Q-Exactive Orbitrap mass spectrometry; Hetero atoms 1–15 H/C ratio 0.1–1 among them, 140 were reported for the frst time in Charges ≤4 Total charge −4–4 Diospyros lotus L. Te chromatographic and mass data for the detected constituents are presented in Table 2. Te extracted ion chromatogram in negative ion mode is component-keygene-pathway interaction network was shown in Figure 1. constructed using Cytoscape 3.9.0 software. In the network, nodes with diferent shapes represented the active com- pounds, key genes, and related pathways, and an “edge” was 3.4.1. Identifcation of the Flavonoids in Diospyros lotus L. an association between the nodes. 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, 3. Results and Discussion 12.58, 12.82, 13.19, and 14.46 min, respectively. Tey were 3.1. Establishment of Qualitative Analysis Strategy. In this accurately identifed as catechin, quercetin 3-O-rutinoside, study, an analytical method of UHPLC-Q-Exactive Orbitrap isoquercitrin, myricitrin, nicotiforin, quercitrin, phlorizin, MS combined with the acquisition mode of the PRM mode myricetin, trilobatin, eriodictyol, quercetin, luteolin, nar- was used to identify the chemical components of Diospyros ingenin, phloretin, kaempferol, and procyanidin, re- lotus L. First, the extraction method and UHPLC-MS spectively, by comparing the data with those of authentic conditions of Diospyros lotus L were optimized. Second, the standards. sample was injected into the UHPLC-Q-Exactive Orbitrap Compounds 64 and 88 possessed the same quasi- MS to obtain high-resolution mass data, including MS and molecular ions and characteristic fragment ions as MS . Tird, the compounds were predicted using the compound 97; thus, they were characterized as quercetin Compound Discover version 3.0 workstation with the aid of 3-O-rutinoside isomers. Similarly, compounds 84, 114, the metabolism workfow template by adjusting relevant and 139 were isoquercitrin isomers, and compounds 109, parameters. Finally, the compounds were characterized 140, 146, and 154 were assigned as isomers of nicotiforin, based on full-scan MS and MS , retention times, standards, quercetin, myricetin, and luteolin, respectively. Com- and literature. pounds 110 and 119 were tentatively presumed to be phlorizin isomers. Compound 51, with the deprotonated ion [M-H] at 3.2. Optimization of the Extraction Method. To obtain the m/z 625.1413, was eluted at 6.08 min, with the main maximum extraction yield, the extraction method for characteristic fragment ion at m/z 463.0877, owing to the Diospyros lotus L was optimized with respect to time (0.5, 1, loss of a glucose residue (162 Da), which further gave rise to and 2 h); solvent type (methanol and ethanol); solvent product ions at m/z 301.0351. It was tentatively charac- concentration (60%, 70%, and 80%); and liquid-to-solid terized as a quercetin 3,4′-diglucoside [13]. Likewise, ratio (10 :1, 15 :1, and 20 :1). Te optimal extraction compounds 74, 92, 124, and 148 were deduced to be method was ultrasonic extraction with 70% methanol quercetin derivatives; compound 78 was quercetin 3- (20 ml) for 1 h. rutinoside 7-rhamnoside [14]; and compounds 100 and 108 were quercetin 3-O-(6″-galloyl)-β-D-glucopyranoside 3.3. Optimization of UHPLC-MS Conditions. To achieve isomers. Compound 105 was characterized as quercitrin 3- good chromatographic separation, UHPLC parameters O-glucuronide, and compounds 116 and 122 were quer- were optimized, including the mobile phase (methanol/ citrin 3-O-arabinoside isomers [15–17]. water and acetonitrile/water); type and content of acid Compounds 53, 69, and 89 exhibited quasi-molecular (acetic acid and formic acid, 0.05%, 0.1%, and 0.2%); ions [M-H] at m/z 303.0510, and fragment ions at m/z column (Waters ACQUITY BEH C18 column, 125.0232, 151.0026, and 177.0187 were tentatively charac- 100 mm × 2.1 mm, 1.7 μm, and HYPERSIL GOLD C18 terized as taxifolin isomers, as previously reported [16]. column, 100 mm × 2.1 mm, 1.9 µm); column temperature Compound 57 was found at 6.23 min, yielded a parent (30, 35, and 40 C); fow rate of the mobile phase (0.2, 0.3, ion [M-H] at m/z 609.1461 consisting of kaempferol and 0.4 mL/min); and the diference gradient of mobile (285 Da) and two glucose moieties (324 Da), and was phase.. Te MS parameters, including the fow rate of the identifed as kaempferol 3,7-diglucoside [13]. sheath gas and auxiliary, temperature of the capillary and Similarly, compound 141 was kaempferol-7-O-rham- auxiliary, heater temperature, spray voltage, and collision noside, and compounds 35, 45, 70, and 76 were suggested to energies were examined. In the optimized conditions of be kaempferol derivatives [18]. 4 Journal of Analytical Methods in Chemistry Table 2: Te chromatographic and mass data of detected components from Diospyros lotus L through UHPLC-Q-Exactive Orbitrap MS. Teoretical Experimental Error MS/MS Peak Peak t mass mass Formula Identifcation (ppm) fragment area (m/z) (m/z) 1 0.83 193.0347 193.0346 −4.23 C H O MS [193]: 71.0124(100), 101.0230(5), 113.0231(2) Glucuronic acid isomer 423409833 6 10 7 2 0.86 191.0561 191.0554 −3.72 C H O MS [191]:85.0282(100),111.0076(76), 87.0074(34) Quinic acid isomer 30408553 7 12 6 3 0.90 191.0197 191.0190 −3.90 C H O MS [191]: 111.0075(100) Citric acid isomer 2270126001 6 8 7 4 0.94 193.0347 193.0347 −3.76 C H O MS [193]: 71.0125(100), 113.0232(82), 101.0232(40) Glucuronic acid isomer 465384616 6 10 7 5 0.94 191.0561 191.0544 −9.06 C H O MS [191]: 111.0076(100), 87.0074(53), 85.0282(29) Quinic acid 65213188 7 12 6 6 1.05 191.0197 191.0189 −4.21 C H O MS [191]: 111.0076(100) Citric acid isomer 14530674970 6 8 7 7 1.07 331.0671 331.0671 −0.06 C H O MS [331]: 169.0132(100), 125.0231(71) 6-O-galloylglucose isomer 3462772 13 16 10 8 1.13 191.0197 191.0190 −3.90 C H O MS [191]: 111.0076(100) Citric acid isomer 26089906774 6 8 7 9 1.30 493.1199 493.1200 0.22 C H O MS [493]: 169.0132(100), 331.0669(93) 6-O-galloylsucrose 105557866 19 26 15 10 1.34 191.0561 191.0552 −5.03 C H O MS [191]: 111.0076(100), 87.0075(49), 85.0282(27) Quinic acid isomer 49104868 7 12 6 11 1.35 145.0506 145.0495 −7.81 C H O MS [145]: 57.0332(100), 71.0488(24), 83.0489(23) 3-Methylglutaric acid isomer 55463923 6 10 4 12 1.36 331.0671 331.0672 0.30 C H O MS [331]: 169.0132(100), 125.0231(65) 6-O-galloylglucose isomer 109327970 13 16 10 13 1.41 331.0671 331.0671 0.03 C H O MS [331]: 169.0132(100), 125.0231(64) 6-O-galloylglucose isomer 105149835 13 16 10 14 1.48 169.0142 169.0134 −4.83 C H O MS2[169]: 125.0233(100) Gallic acid 663649505 7 6 5 15 1.65 153.0193 153.0184 −6.35 C H O MS [153]: 108.0203(100), 109.0282(74), 123.0439(6) 2,3-Dihydroxybenzoic acid isomer 88754053 7 6 4 MS [151]: 108.0204(100), 124.0153(51), 123.0438(31), 16 1.67 151.0401 151.0391 −6.67 C H O Vanillin isomer 14956041 8 8 3 136.0155(16) 17 1.69 218.1030 218.1029 −2.18 C H NO MS [218]: 88.0391(100), 146.0813(53) Pantothenic acid isomer 692269068 9 17 5 18 1.76 218.1030 218.1029 −2.18 C H NO MS [218]: 88.0391(100), 146.0813(53) Pantothenic acid isomer 559182584 9 17 5 MS [151]: 108.0204(100), 124.0154(45), 123.0439(34), 19 1.84 151.0401 151.0391 −6.54 C H O Vanillin isomer 15928418 8 8 3 136.0154(11) MS [315]: 108.0203(100), 109.0281(43), 153.0186(18), 2,3-Dihydroxybenzoic acid 3-O-glucoside 20 1.84 315.0722 315.0724 0.65 C H O 13894787 13 16 9 123.0439(9) isomer 21 1.91 331.0671 331.0673 0.67 C H O MS [331]: 125.0231(100), 169.0132(94) 6-O-galloylglucose isomer 6714540 13 16 10 MS [145]: 83.0489(30), 101.0594(10), 57.0332(7), 22 2.31 145.0506 145.0497 −6.77 C H O 3-Methylglutaric acid isomer 37732233 6 10 4 71.0124(6) 23 2.65 299.0772 299.0770 −0.70 C H O MS [137]: 137.0233(100), 93.0332(78) P-hydroxybenzoic acid-O-glucoside isomer 5442116 13 16 8 24 2.85 299.0772 299.0773 0.30 C H O MS [137]: 137.0233(100), 93.0332(78) P-hydroxybenzoic acid-O-glucoside isomer 5284965 13 16 8 25 2.89 299.0772 299.0773 0.10 C H O MS [137]: 137.0233(100), 93.0332(78) P-hydroxybenzoic acid-O-glucoside isomer 5801580 13 16 8 26 3.00 299.0772 299.0775 0.80 C H O MS [137]: 137.0233(100), 93.0332(66) P-hydroxybenzoic acid-O-glucoside isomer 8954168 13 16 8 MS [359]: 138.0311(100), 182.0211(86), 197.0447(44), 27 3.00 359.0984 359.0989 1.59 C H O Syringic acid glucoside 16332217 15 20 10 153.0546(38) MS [151]: 108.0232(100), 124.0154(37), 123.0441(14), 28 3.09 151.0401 151.0391 −6.14 C H O Vanillin isomer 27511009 8 8 3 136.0153(11) MS [151]:108.0202(100), 124.0153(31), 123.0439(23), 29 3.19 151.0401 151.0391 −6.33 C H O Vanillin isomer 23855779 8 8 3 136.0153(23) 30 3.33 353.0878 353.0880 0.67 C H O MS [353]: 191.0551(100), 135.0443(10) Neochlorogenic acid 371210 16 18 9 31 3.53 299.0772 299.0770 −0.70 C H O MS [137]: 137.0232(100), 93.0332(90) P-hydroxybenzoic acid-O-glucoside isomer 6350760 13 16 8 32 3.75 299.0772 299.0771 −0.60 C H O MS [137]: 93.0332(100), 137.0233(85) P-hydroxybenzoic acid-O-glucoside isomer 4495564 13 16 8 MS [183]: 140.0103(100), 124.0153(78), 111.0074(65), 33 4.07 183.0299 183.0295 −3.86 C H O Methyl gallate 54964007 8 8 5 139.0025(53) 34 4.93 475.1457 475.1455 −0.49 C H O MS [475]: 167.0340(100), 123.0439(56) Vanillic acid-O-rutinoside 5156267 20 28 13 Journal of Analytical Methods in Chemistry 5 Table 2: Continued. Teoretical Experimental Error MS/MS Peak Peak t mass mass Formula Identifcation (ppm) fragment area (m/z) (m/z) 35 4.96 465.1038 465.1035 −0.86 C H O MS [465]: 285.0403(100), 125.0232(67), 151.0028(11) Kaempferol derivative 2220696 21 22 12 MS [289]:109.0282(100), 123.0433(70), 97.0288(32), 36 4.99 289.0718 289.0726 3.01 C H O Catechin 1491579 15 14 6 125.0231(30) 37 5.30 457.1351 457.1354 0.64 C H O MS [457]: 119.0491(100), 163.0391(58) P-coumaric acid-O-glucoside-rhamnoside 12791866 20 26 12 MS [177]: 133.0284(100), 105.0334(64), 89.0383(31), 38 5.36 177.0193 177.0186 −4.19 C H O Esculetin isomer 68123540 9 6 4 149.0234(16) 39 5.37 153.0193 153.0184 −6.16 C H O MS [153]: 109.0282(100), 108.0202(19) 2,3-Dihydroxybenzoic acid isomer 263921011 7 6 4 40 5.42 353.0878 353.0880 0.58 C H O MS [353]: 191.0554(100), 135.0441(12) Chlorogenic acid 36538266 16 18 9 MS [151]: 108.0204(100), 123.0439(35), 124.0154(32), 41 5.44 151.0401 151.0391 −6.27 C H O Vanillin isomer 16864944 8 8 3 136.0152(23) 42 5.46 179.0350 179.0345 −2.86 C H O MS [179]: 135.04401(100), 179.03407(87) Cafeic acid 12320409 9 8 4 43 5.49 457.1351 457.1356 1.03 C H O MS [457]: 119.0491(100), 163.0392(54) P-coumaric acid-O-glucoside-rhamnoside 25886930 20 26 12 44 5.60 457.1351 457.1354 0.64 C H O MS [457]: 119.0491(100), 163.0392(50) P-coumaric acid-O-glucoside-rhamnoside 6494575 20 26 12 MS [465]: 285.0403(100), 125.0233(54), 178.9976(17), 45 5.64 465.1038 465.1036 −0.58 C H O Kaempferol derivative 2286572 21 22 12 151.0029(9) 46 5.73 457.1351 457.1353 0.35 C H O MS [457]: 119.0490(100), 163.0391(48) P-coumaric acid-O-glucoside-rhamnoside 14719551 20 26 12 MS [595]: 355.0822(100), 385.0927(91), 415.1031(25), 47 5.86 595.1663 595.1680 1.37 C H O Naringenin-6,8-di-C-glucoside 3936541 27 32 15 475.1259(16) 48 5.95 641.1359 641.1370 1.60 C H O MS [641]: 479.0816(100), 178.9978(21), 151.0026(13) Myricetin 3,3’-digalactoside isomer 907936 27 30 18 49 5.95 329.0878 329.0880 0.53 C H O MS [329]: 167.0341(100), 123.0440(45) Vanillic acid glucoside 184241354 14 18 9 MS [319]: 193.0136(100), 125.0233(77), 151.0028(17), 50 6.07 319.0459 319.0461 0.34 C H O Dihydromyricetin isomer 42196138 15 12 8 165.0184(15) 51 6.08 625.1410 625.1413 0.44 C H O MS [625]: 463.0877(100), 301.0351(60), 151.0025(15) Quercetin 3,4’-Diglucoside 3023028 27 30 17 MS [449]: 259.0611(100), 269.0457(86), 151.0027(47), 52 6.09 449.1089 449.1091 0.37 C H O Maesopsin 4-O-glucoside isomer 36415310 21 22 11 287.0287(17) 53 6.16 303.0510 303.0510 −0.12 C H O MS [303]: 125.0232(100), 151.0026(10), 177.0187(5) Taxifolin isomer 4492484 15 12 7 54 6.17 475.1457 475.1459 0.39 C H O MS [475]: 167.0339(100), 123.0439(4) Vanillic acid-O-rutinoside 21026003 20 28 13 55 6.21 281.1396 281.1396 0.62 C H O MS [281]: 123.0803(100), 171.1169(83), 189.1278(22) Dihydrophaseic acid 13845836 15 22 5 MS [355]: 134.0363(100), 193.0500(93), 149.0598(26), 56 6.22 355.1035 355.1037 0.60 C H O Ferulic acid acyl-β-D-glucoside isomer 155512403 16 20 9 178.0265(13) 57 6.23 609.1461 609.1467 1.02 C H O MS [609]: 447.0926(100), 285.0401(34) Kaempferol 3,7-diglucoside 1288267 27 30 16 58 6.38 151.0401 151.0391 −6.14 C H O MS [151]: 108.0203(100), 136.0154(23), 123.0437(6) Vanillin isomer 34876373 8 8 3 59 6.39 177.0193 177.0186 −3.97 C H O MS [177]: 129.0183(100), 133.0284(21), 89.0385(8) Esculetin isomer 5548229 9 6 4 MS [449]: 259.0610(100), 269.0455(75), 287.0561(34), 60 6.44 449.1089 449.1091 0.30 C H O Maesopsin 4-O-glucoside isomer 12513053 21 22 11 151.0026(8) 61 6.48 179.0350 179.0343 −3.64 C H O MS [179]: 135.04428(100) Cafeic acid isomer 10375972 9 8 4 62 6.50 287.0561 287.0561 −0.18 C H O MS [287]:125.0232(100), 151.0026(13),161.0230(3) (2S)-5,7,2’,6′-tetrahydroxyfavanone isomer 7395938 15 12 6 MS [355]: 134.0363(100), 193.0500(89), 149.0598(30), 63 6.52 355.1035 355.1036 0.52 C H O Ferulic acid acyl-β-D-glucoside isomer 194391098 16 20 9 178.0266(18) 64 6.52 609.1461 609.1473 1.92 C H O MS [609]: 301.0354(100), 447.0933(43), 300.0265(5) Quercetin 3-O-rutinoside isomer 7578929 27 30 16 MS [319]: 125.0232(100), 193.0134(75), 151.0026(20), 65 6.53 319.0459 319.0459 −0.13 C H O Dihydromyricetin isomer 3940259 15 12 8 165.0184(20) 6 Journal of Analytical Methods in Chemistry Table 2: Continued. Teoretical Experimental Error MS/MS Peak Peak t mass mass Formula Identifcation (ppm) fragment area (m/z) (m/z) 66 6.58 433.1140 433.1144 0.76 C H O MS [433]: 271.0611(100), 165.0183(30), 113.0231(12) Naringenin 7-O-glucoside isomer 1556330 21 22 10 MS [515]: 173.0447(100), 179.0342(88), 191.0554(38), 67 6.65 515.1195 515.1184 −2.12 C H O Isochlorogenic acid B 672858 25 24 12 135.0441(15) MS [771]: 316.0225(100), 271.0247(47), 151.0026(8), 68 6.67 771.1989 771.1996 0.84 C H O Myricetin 3-rutinoside 7-rhamnoside 37547695 33 40 21 317.0232(4) MS [303]: 125.0233(100), 285.0408(47), 177.0183(11), 69 6.67 303.0510 303.0511 0.31 C H O Taxifolin isomer 1258429 15 12 7 151.0027(8) MS [465]: 285.0404(100), 125.0232(57), 178.9976(18), 70 6.68 465.1038 465.1030 −1.76 C H O Kaempferol derivative 3746637 21 22 12 151.0027(6) MS [641]: 479.0829(100), 317.0301(17), 316.0219(16), 71 6.69 641.1359 641.1365 0.94 C H O Myricetin 3,3′-digalactoside isomer 8628387 27 30 18 151.0026(2) 72 6.72 167.0350 167.0341 −5.34 C H O MS [167]: 123.0440(100), 108.0204(14) Vanillic acid isomer 22418279 8 8 4 73 6.81 625.1410 625.1408 −0.44 C H O MS [625]: 316.0224(100), 317.0266(19), 463.0877(8) Myricetin 3-O-rutinoside isomer 1981287 27 30 17 C H 14 16 2 74 6.88 423.0416 423.0393 −5.51 MS [423]: 151.0026(100), 178.9978(76), 301.0353(41) Quercitrin derivative 41312151 MS [433]: 313.0718(100), 343.0821(24), 271.0613(20), 75 6.96 433.1140 433.1137 −0.72 C H O Naringenin 6-C-glucoside isomer 2813357 21 22 10 151.0026(12) 76 6.96 465.1038 465.1032 −1.44 C H O MS [465]: 161.0446(100), 125.0232(57), 285.0406(29) Kaempferol derivative 1790280 21 22 12 MS [613]: 373.0929(100), 403.1037(56), 239.0556(27), 77 7.01 613.1779 613.1779 0.74 C H O Catechin di-C-hexoside 18384963 27 34 16 433.1139(17), 493.1359(17) MS [755]: 300.0274(100), 301.0337(16), 178.9976(9), 78 7.03 755.2040 755.2045 0.60 C H O Quercetin 3-rutinoside 7-rhamnoside 7686510 33 40 20 271.0247(4) MS [625]: 316.0244(100), 271.0250(48), 317.0293(12), 79 7.03 625.1410 625.1413 0.44 C H O Myricetin 3-O-rutinoside isomer 117356236 27 30 17 151.0027(9) 80 7.07 579.2083 579.2109 4.43 C H O MS [579]: 417.1554(100), 181.0496(84), 402.1320(20) Syringaresinol-O-β-D-glucoside isomer 1274845 28 36 13 MS [433]: 313.0717(100), 271.0611(82), 343.0820(22), 81 7.07 433.1140 433.1140 −0.02 C H O Naringenin 6-C-glucoside isomer 5777367 21 22 10 151.0025(14) MS [493]: 317.0302(100), 151.0027(45), 109.0284(11), 82 7.08 493.0624 493.0625 0.25 C H O Myricetin 3-O-glucuronide 61411051 21 18 14 271.0252(8) MS [479]:316.0255(100), 271.0249(41), 317.0301(13), 83 7.13 479.0831 479.0831 −0.09 C H O Myricetin 3-O-galactoside 179167814 21 20 13 151.0026(7) 84 7.16 463.0882 463.0885 0.65 C H O MS [463]: 301.0350(100), 300.0274(61) Isoquercitrin isomer 10171047 21 20 12 85 7.17 641.1359 641.1353 −1.07 C H O MS [641]: 479.0831(100), 317.0301(58), 316.0222(17), Myricetin 3,3′-digalactoside isomer 2349023 27 30 18 MS [167]: 123.0440(100), 167.0341(85), 108.0206(5), 86 7.23 167.0350 167.0341 −5.28 C H O Vanillic acid isomer 272148953 8 8 4 152.0109(4) 87 7.29 193.0506 193.050 −3.48 C H O MS [193]: 134.0362(100), 178.0266(48), 149.0598(21) Ferulic acid 13558093 10 10 4 88 7.40 609.1461 609.1458 −0.59 C H O MS [609]: 300.0273(100), 301.0328(16), 151.0025(7) Quercetin 3-O-rutinoside isomer 368931 27 30 16 89 7.40 303.0510 303.0511 0.11 C H O MS [303]: 125.0232(100), 151.0026(12), 177.0184(8) Taxifolin isomer 18082158 15 12 7 90 7.41 579.2083 579.2114 5.38 C H O MS [579]: 417.1553(100), 181.0497(79), 402.1321(13) Syringaresinol-O-β-D-glucoside isomer 1680286 28 36 13 91 7.44 433.1140 433.1141 0.21 C H O MS [433]: 271.0611(100), 151.0026(64), 119.0490(11) Naringenin 7-O-glucoside isomer 3568902 21 22 10 92 7.48 435.1297 435.1300 0.85 C H O MS [435]: 301.0307(100), 300.0271(5), 151.0027(3) Quercitrin derivative 3058494 21 24 10 Journal of Analytical Methods in Chemistry 7 Table 2: Continued. Teoretical Experimental Error MS/MS Peak Peak t mass mass Formula Identifcation (ppm) fragment area (m/z) (m/z) C H MS [423]: 317.0126(100), 125.0233(40), 151.0026(15), 14 16 93 7.50 423.0416 423.0394 −5.30 Myricetin derivative 461694539 O 285.0401(10) 94 7.58 137.0244 137.0234 −7.50 C H O MS [137]: 93.0333(100) P-hydroxybenzoic acid 1954942346 7 6 3 95 7.60 193.0506 193.0499 −3.64 C H O MS [193]: 134.0363(100), 149.0600(62), 178.0264(60) Ferulic acid isomer 943379 10 10 4 96 7.60 177.0193 177.0186 −4.30 C H O MS [177]: 129.0182(100), 89.0231(65), 133.0283(16) Esculetin isomer 8590955 9 6 4 97 7.60 609.1461 609.1466 0.73 C H O MS [609]: 300.0275(100), 301.0351(94), 151.0026(3) Quercetin 3-O-rutinoside 60494749 27 30 16 MS [463]: 316.0224(100), 271.0248(46), 287.0198(26), 98 7.60 463.0882 463.0886 0.84 C H O Myricitrin 47320337 21 20 12 151.0028(12), 317.0302(3) MS [597]: 357.0983(100), 387.1088(77), 167.0340(44), 99 7.62 597.1825 597.1829 0.71 C H O 3′, 5′-Di-C-β-D-glucosylphloretin 279295311 27 34 15 209.0451(40), 417.1192(18) Quercetin 3-O-(6″-galloyl)-β- 100 7.64 615.0992 615.0997 0.83 C H O MS [615]: 463.0883(100), 301.0352(27), 300.0278(1) 16017622 28 24 16 D-glucopyranoside isomer 101 7.70 641.1359 641.1364 0.74 C H O MS [641]: 479.0830(100), 317.0301(23), 316.0200(3) Myricetin 3,3′-digalactoside isomer 308863 27 30 18 102 7.72 579.1719 579.1717 −0.41 C H O MS [579]: 253.0504(100), 271.0613(48), 417.1552(17) Naringenin-O-glucoside-rhamnoside 1975268 27 32 14 103 7.73 433.1140 433.1139 −0.23 C H O MS [433]: 227.0709(100), 271.0611(54) Naringenin 7-O-glucoside isomer 3090379 21 22 10 104 7.76 193.0506 193.0499 −3.90 C H O MS [193]: 134.0363(100), 178.0265(61), 149.0599(49) Ferulic acid isomer 10013718 10 10 4 MS [477]: 301.0352(100), 151.0026(12), 255.0296(1), 105 7.76 477.0675 477.0678 0.66 C H O Quercitrin 3-O-glucuronide 71847819 21 18 13 300.0270(1) MS [625]: 463.0882(100), 301.0350(29), 316.0223(11), 106 7.78 625.1410 625.1418 1.21 C H O Myricetin 3-O-rutinoside isomer 3973219 27 30 17 317.0289(6) MS [463]: 300.0276(100), 271.0250(68), 301.0353(47), 107 7.82 463.0882 463.0888 1.25 C H O Isoquercitrin 193168329 21 20 12 255.0299(31), 151.0027(16) Quercetin 3-O-(6″-galloyl)-β- 108 7.92 615.0992 615.0994 0.35 C H O MS [615]: 463.0883(100), 301.0353(26) 2311932 28 24 16 D-glucopyranoside isomer 109 8.00 593.1512 593.1518 1.01 C H O MS [593]: 284.0325(100), 285.0400(62), 151.0025(6) Nicotiforin isomer 6641091 27 30 15 MS [435]: 167.0340(100), 273.0768(56), 125.0233(18), 110 8.00 435.1297 435.1299 0.55 C H O Phlorizin isomer 7020357 21 24 10 123.0439(9) 111 8.04 515.1195 515.1201 1.21 C H O MS [515]: 191.0553(100), 179.0341(92), 135.0440(14) 1,3-Dicafeoylquinic acid 1538798 25 24 12 MS [477]: 315.0510(100), 301.0352(95), 314.0434(15), 112 8.10 477.1038 477.1038 −0.12 C H O 3-Methylquercetin 7-O-glucoside isomer 6541397 22 22 12 299.0197(14), 300.0272(10) MS [151]: 108.0204(100), 124.0125(24), 123.0440(16), 113 8.10 151.0401 151.0392 −5.54 C H O Vanillin isomer 11922688 8 8 3 136.0153(13) 114 8.21 463.0882 463.0884 0.46 C H O MS [463]: 301.0352(100), 300.0277(2) Isoquercitrin isomer 7397726 21 20 12 MS [515]:191.0552(100), 179.0340(67), 353.0878(17), 115 8.28 515.1195 515.1184 −2.12 C H O Isochlorogenic acid A 3535566 25 24 12 135.0439(12) MS [433]: 300.0273(100), 271.0610(77), 151.0026(32), 116 8.31 433.1140 433.1142 0.42 C H O Quercitrin 3-O-arabinoside isomer 12897978 21 22 10 301.0332(26) MS [593]: 285.0405(100), 284.0327(85), 255.0299(67), 117 8.33 593.1512 593.1516 0.70 C H O Nicotiforin 53519420 27 30 15 227.0346(46) MS [579]: 417.1554(100), 271.0607(28), 178.9978(12), 118 8.35 579.1719 579.1707 −2.09 C H O Naringenin-O-glucoside-rhamnoside 2699074 27 32 14 151.0025(14) 119 8.40 435.1297 435.1299 0.41 C H O MS [435]: 167.0339(100), 273.0765(40), 125.0231(17) Phlorizin isomer 6540475 21 24 10 8 Journal of Analytical Methods in Chemistry Table 2: Continued. Teoretical Experimental Error MS/MS Peak Peak t mass mass Formula Identifcation (ppm) fragment area (m/z) (m/z) 120 8.40 187.0974 187.0967 −4.98 C H O MS [187]: 125.0958(100), 97.0644(13), 169.0857(2) Azelaic acid 1506853530 9 16 4 121 8.42 579.2083 579.2096 2.20 C H O MS [579]: 417.1554(100), 181.0497(93), 402.1317(13) Syringaresinol O-β-D-glucoside isomer 11293465 28 36 13 MS [433]: 271.0612(100), 300.0274(34), 151.0027(30), 122 8.53 433.1140 433.1140 −0.09 C H O Quercitrin 3-O-arabinoside isomer 4688398 21 22 10 301.0331(11) 123 8.56 287.0561 287.0562 0.45 C H O MS [287]: 125.0233(100), 151.0027(14) (2S)-5,7,2′,6′-tetrahydroxyfavanone isomer 113424998 15 12 6 MS [461]:301.0353(100),178.9977(36), 151.0025(29), 124 8.59 461.0725 461.0737 2.43 C H O Quercitrin derivative 5302276 21 18 12 300.0268(6) MS [447]: 300.0276(100), 301.0353(71), 271.0249(64), 125 8.67 447.0933 447.0935 0.50 C H O Quercitrin 314040631 21 20 11 255.0298(44), 151.0027(25) MS [477]: 314.0433(100), 315.0487(25), 301.0354(15), 126 8.72 477.1038 477.1036 −0.63 C H O 3-Methylquercetin 7-O-glucoside isomer 2529387 22 22 12 300.0277(5), 299.0193(4) MS [477]: 314.0432(100), 315.0489(23), 301.0352(6), 127 8.87 477.1038 477.1039 0.19 C H O 3-Methylquercetin 7-O-glucoside isomer 10332087 22 22 12 299.0194(5), 300.0273(3) ∗ 2 128 8.93 435.1297 435.1301 0.92 C H O MS [435]:167.0341(100), 273.0760(39) Phlorizin 715650 21 24 10 129 9.03 193.0506 193.0499 −3.79 C H O MS [193]: 149.0598(100), 134.0361(25), 178.0264(16) Ferulic acid isomer 6477816 10 10 4 130 9.13 263.1283 263.1289 0.22 C H O MS [263]: 191.0343(100), 203.1072(5) Abscisic acid isomer 3103377 15 20 4 131 9.18 151.0401 151.0392 −5.94 C H O MS [151]: 108.0204(1000, 123.0439(17), 136.0156(14) Vanillin isomer 11991089 8 8 3 132 9.20 507.1144 507.1150 1.08 C H O MS [507]: 345.0611(100), 330.0367(25) Viscidulin III 6′-O-β-D-glucoside isomer 2178417 23 24 13 133 9.29 507.1144 507.1144 0.05 C H O MS [507]: 345.0610(100), 330.0367(31) Viscidulin III 6′-O-β-D-glucoside isomer 573815 23 24 13 MS [317]: 151.0026(100), 137.0233(73), 107.0125(33), 134 9.36 317.0303 317.0301 −0.63 C H O Myricetin 2396594 15 10 8 178.9977(31) MS [491]: 313.0356(100), 271.0244(54), 299.0199(44), 135 9.44 491.1195 491.1198 0.63 C H O 5,2′6′-dihydroxy-7,8-dimethoxyfavone isomer 15215791 23 24 12 329.0678(9) MS [515]: 179.0340(100), 191.0552(79), 353.0879(22), 136 9.45 515.1195 515.1200 0.97 C H O Isochlorogenic acid C 3154984 25 24 12 135.0439(10) 137 9.58 477.1038 477.1040 0.32 C H O MS [477]: 315.0514(100), 300.0273(26), 301.0351(21) 3-Methylquercetin 7-O-glucoside isomer 1113920 22 22 12 MS [615]: 317.0302(100), 463.0877(5), 316.0222(3), 138 9.63 615.0992 615.0998 1.03 C H O Myricetin 3-O-(6″-galloyl)-β-D-rhamnoside 3665642 28 24 16 178.9975(1) 139 9.67 463.0882 463.0885 0.59 C H O MS [463]: 301.0354(100), 300.0278(2), 255.0298(1) Isoquercitrin isomer 31498699 21 20 12 MS [301]: 149.0234(100), 151.028(24), 107.0127(11), 140 9.82 301.0354 301.0355 0.28 C H O Quercetin isomer 60769559 15 10 7 121.0283(4) MS [431]: 285.0405(100), 255.0298(84), 284.0328(74), 141 9.99 431.0983 431.0983 −0.23 C H O Kaempferol-7-O-rhamnoside 104050627 21 20 10 227.0346(64) MS [477]: 315.0510(100), 300.0275(36), 301.0347(21), 142 10.00 477.1038 477.1030 −1.78 C H O 3-Methylquercetin 7-O-glucoside isomer 1327295 22 22 12 314.0753(11) MS [491]: 328.0587(100), 329.0660(86), 313.0354(37), 143 10.11 491.1195 491.1197 0.39 C H O 5,2′6′-dihydroxy-7,8-dimethoxyfavone isomer 6215669 23 24 12 299.0196(2) MS [435]: 273.0768(100), 167.0340(95), 125.0233(7), 144 10.13 435.1297 435.1299 0.41 C H O Trilobatin 1544931 21 24 10 123.0438(5) MS [263]: 203.1071(100), 191.0342(94), 151.0754(65), 145 10.14 263.1283 263.1290 0.45 C H O Abscisic acid isomer 43256699 15 20 4 152.0835(32) Journal of Analytical Methods in Chemistry 9 Table 2: Continued. Teoretical Experimental Error MS/MS Peak Peak t mass mass Formula Identifcation (ppm) fragment area (m/z) (m/z) MS [317]: 151.0026(100), 178.9976(33), 146 10.20 317.0303 317.0303 −0.65 C H O Myricetin isomer 6262287 15 10 8 137.0233(14), 107.0126(7) 147 10.33 287.0561 287.0561 −0.07 C H O MS [287]: 135.0440(100), 151.0026(17) Eriodictyol 6677796 15 12 6 148 11.00 505.0987 505.0993 1.00 C H O MS [431]:301.0355(100), 151.0027(5), 300.0273(2) Quercetin derivative 17504734 23 22 13 149 11.87 271.0612 271.0613 0.42 C H O MS [271]: 143.0491(100), 253.0505(85), 209.0603(78) Chrysin derivative 12485860 15 12 5 MS [301]: 149.0233(100), 151.027(30), 107.0127(11), 150 11.90 301.0354 301.0356 0.578 C H O Quercetin 18626868 15 10 7 121.0286(5) MS [285]: 133.0284(100), 151.0027(40), 175.0392(25), 151 12.11 285.0405 285.0409 1.40 C H O Luteolin 1581682 15 10 6 199.0392(14) 152 12.34 315.0505 315.0511 0.30 C H O MS [285]: 300.0275(100), 301.0309(16), 151.0028(1) Isorhamnetinisomer 16370381 16 12 7 ∗ 2 153 12.58 271.0612 271.0613 0.53 C H O MS [271]: 119.0491(100), 151.0027(70), 177.0185(11), Naringenin 129718757 15 12 5 154 12.59 285.0405 285.0410 1.86 C H O MS [285]: 133.0283(100), 199.0396(48), 151.0027(19) Luteolin isomer 3384989 15 10 6 ∗ 2 155 12.82 273.0768 273.0773 1.55 C H O MS [273]: 167.0342(100), 123.0456(39) Phloretin 2148980 15 14 5 156 12.97 315.0505 315.0528 5.73 C H O MS [285]: 300.0275(100), 301.0309(15) Isorhamnetinisomer 3605778 16 12 7 MS [285]: 285.0405(100), 178.9917(43), 151.0027(16), 157 13.19 285.0405 285.0407 0.98 C H O Kaempferol 9479859 15 10 6 185.0602(16), 229.0503(13) MS [299]: 285.0325(100), 255.0298(43), 239.0347(40), 158 13.51 299.0561 299.0564 0.87 C H O Chrysoeriol 3009700 16 12 6 227.0344(17) 159 14.46 593.1301 593.1305 0.72 C H O MS [593]: 209.0449(100), 121.0283(70) Procyanidin 3914568 30 26 13 identifed by comparison with standard. 10 Journal of Analytical Methods in Chemistry 80 14 60 98 69 108 22 43 117 139 142 30 74 -5 01 23 45 67 89 10 11 12 13 14 15 Time (min) (a) 3 90 114 137 20 50 57 128 64 67 8 150 -5 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Time (min) (b) Figure 1: Continued. Relative Abundance Relative Abundance Journal of Analytical Methods in Chemistry 11 100 121 10 92 24 147 28 40 60 32 157 62 138145 35 45 -5 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Time (min) (c) 65 113 54 143 72 109 34 126 20 33 36 11 111 15 127 23 131 10 156 31 159 -5 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Time (min) (d) Figure 1: Te high-resolution extraction ion chromatography of Diospyros lotus L in negative ion mode (a) 167.0350, 169.0142, 191.0561, 193.0350, 218.1030, 271.0612, 287.0561, 331.0671, 355.1035, 423.0416, 431.0983, 447.0933, 463.0882, 479.0831, 597.1825, 641.1359, 755.2040; (b) 151.0401, 167.0350, 177.0193, 191.0561, 271.0612, 287.0561, 303.0510, 319.0459, 331.0671, 423.0417, 433.1140, 435.1297, 463.0882, 465.1038, 477.1038, 507.1144, 579.2083, 609.146, 613.1779, 625.1410, 641.1359, 755.2040; (c) 145.0506, 151.0401, 153.0193, 167.0350, 177.01933, 179.0350, 187.0974, 193.0506, 263.1283, 273.0768, 285.0405, 287.0561, 299.0772, 303.0510, 315.0505, 317.0303, 319.0460, 329.0878, 331.0671, 353.0878, 433.11402, 435.1297, 449.1089, 457.1351, 461.0725, 463.0890, 465.1038, 475.1457, 477.0675, 491.1195, 493.0624, 507.1144, 515.1195, 579.1719, 579.2083, 609.1461, 613.1779, 615.0992, 625.1410, 641.1359; (d) 145.0506, 151.0401, 153.0193, 167.0350, 177.0193, 179.0350, 183.0299, 193.0506, 263.1283, 281.1396, 301.0354, 303.0510, 315.0722, 319.0459, 353.0878, 359.0984, 433.1140, 435.1297, 449.1089, 457.1351, 475.1457, 477.0675, 477.1038, 491.1195, 493.0624, 493.1199, 505.0987, 579.2083, 593.1512, 609.1461, 613.1779, 615.0992, 771.1989. Relative Abundance Relative Abundance 12 Journal of Analytical Methods in Chemistry Compounds 48, 71, 85, and 101 were detected at 5.95, 3.4.2. Identifcation of Phenylpropanoids in Diospyros lotus L. 6.69, 7.17, and 7.70 min, respectively, and possessed the same Compounds 30, 40, 42, 67, 87, 111, 115, and 136 were eluted quasi-molecular ions [M-H] at m/z 641.1359, MS/MS at 3.33, 5.42, 5.46, 6.65, 7.29, 8.04, 8.28, and 9.45 min, re- fragment ions at m/z 317.0301, and 316.0200 owing to the spectively. Tey were characterized as neochlorogenic acid, loss of two galactoside residues (324 Da), indicating the chlorogenic acid, cafeic acid, isochlorogenic acid B, ferulic presence of a myricetin group. Terefore, they have been acid, 1,3-dicafeoylquinic acid, isochlorogenic acid A, and characterized as myricetin 3,3′-digalactoside isomers. isochlorogenic acid C, respectively, by comparison to Likewise, compounds 68, 82, 83, 93, and 138 were assigned commercial reference standards. as myricetin 3-rutinoside-7-rhamnoside, myricetin 3-O- Compound 61 possessed the same quasi-molecular ions, glucuronide, myricetin 3-O-galactoside, myricetin de- and the characteristic fragment ion of compound 42 was rivative, and myricetin 3-O-(6″-galloyl)-β-D-rhamnoside, characterized as a cafeic acid isomer. Similarly, compounds respectively, and compounds 73, 79, and 106 were myricetin 95, 104, and 129 were ferulic acid isomers. 3-O-rutinoside isomers [19–21]. Compounds (56, t 6.22 min, and 63, t 6.52 min) had R R Compounds 50 and 65 were eluted at 6.07 and 6.53 min, the same quasi-molecular ions [M-H] at m/z 355.1035 and respectively, and possessed the same quasi-molecular ion the fragment ion at m/z 193.0500, corresponding to the [M-H] neutral loss of the glucose group (162 Da) and further at m/z 319.0459 and fragment ions at m/z 125.0232, 193.0134, and 151.0026. Tey were tentatively assigned as generation of the fragment ions of compound 87. Terefore, dihydromyricetin isomers by referring to the literature [22]. they were tentatively assigned as ferulic acid acyl-β-D- Compounds 52 and 60 yielded a quasi-molecular ion glucoside isomers [17]. [M-H] at m/z 449.1089, which was tentatively identifed as Compounds 37, 43, 44, and 46 were eluted at 5.30, 5.49, the maesopsin 4-O-glucoside isomer according to a pre- 5.60, and 5.73 min, respectively, and showed a deprotonated viously published paper [21]. Likewise, compounds 77, 99, molecular ion [M-H] at m/z 457.1351. Tey were tentatively 149, and 158 were catechin di-C-hexoside, 3′, 5′-di-C-β-d- inferred to be p-coumaric acid-O-glucoside-rhamnoside glucosylphloretin [23], chrysin derivatives, and chrysoeriol, based on the base peak ion in the MS spectrum. respectively. Compounds 135 and 143 were 5,2′,6′-dihy- Compounds 80, 90, and 121 were eluted at 7.07, 7.41, and droxy-7,8-dimethoxyfavone isomers [24], and compounds 8.42 min, respectively, yielding a deprotonated ion [M-H] 132 and 133 were viscidulin III 6′-o-β-d-glucoside at m/z 579.2083 and fragment ions at m/z 417.1554, isomers [24]. 181.0497, and 402.1317, which were suggested as syringar- Compounds 62 and 123 showed a deprotonated ion [M- esinol O-β-D-glucoside isomers in comparison with the H] at m/z 287.0561. Te appearance of fragment ions at m/z literature [28]. 125.0233 and 151.0027 in the MS spectrum of those Compounds 38, 59, and 96 were eluted at 5.36, 6.39, and compounds indicated that they were (2S)-5,7,2′,6′- 7.60 min, respectively, and yielded the same parent ion [M- tetrahydroxyfavanone isomers [24]. H] at m/z 177.0193. Tey were deduced as esculetin isomers Compounds 66, 91, and 103 yielded a quasi-molecular according to the MS and MS/MS spectra [25]. 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). 3.4.3. Identifcation of Organic Acids in Diospyros lotus L. Tus, they were considered to be naringenin 7-O-glucoside Compounds 2, 5, and 10 were found at 0.86, 0.94, and isomers [25]. Similarly, compounds 102 and 118 were 1.34 min, respectively, and possessed the same parent ion naringenin-O-glucoside-rhamnosides. [M-H] at m/z 191.0561. Compound 5 was identifed as Compounds 75 and 81 appeared at a retention time (t ) quinic acid by comparison with the reference substances. of 6.96 and 7.07 min, respectively, possessing the quasi- Tus, compounds 2 and 10 were identifed as isomers of molecular ions [M-H] at m/z 433.1140 and their frag- quinic acid. ment ions at m/z 313.0717 ([M-H-120] ) and 343.0820 ([M- Compounds 1 and 4 were observed at 0.83 and 0.94 min, H-90] ), which were identifed as naringenin 6-C-glucosidei respectively, and possessed the same quasi-molecular ions somer [26]. Similarly, compound 47 was deduced as [M-H] at m/z 93.0347 and MS/MS fragment ions at m/z naringenin-6,8-di-C-glucoside [27]. 71.0124, 101.0230, and 113.0231. Tey were tentatively Compounds 152 and 156 were found at 12.34 and assigned as glucuronic acid isomers by comparison to the 12.97 min, respectively, which show the common precursor literature. Similarly, compounds 3, 6, and 8 were citric acid ion [M-H] at m/z 315.0505, and the major fragment ion at isomers [20], compounds 11 and 22 were 3-methylglutaric m/z 300.0275 due to loss of a CH reside (15 Da), then they acid isomers, compound 27 was syringic acid glucoside [29], were tentatively characterized as isorhamnetinisomer [17]. compounds 17 and 18 were pantothenic acid isomers, Compounds 112, 126, 127, 137, and 142 appeared at re- compound 55 was dihydrophaseic acid, compound 120 was tention times (t ) of 8.10, 8.72, 8.87, 9.58, and 10.00 min, azelaic acid, and compounds 130 and 145 were abscisic acid respectively, which were tentatively identifed as 3-meth- isomers [16, 27]. ylquercetin-7-O-glucoside isomers. Te parent ions at m/z Compound 14 exhibited a quasi-molecular ion [M-H] 477.1038 were due to the loss of glucose moieties (162 Da) at m/z 169.0142 and generated the main characteristic and generated the characteristic fragment ions at m/z fragments ion at m/z 125.0233 ([M-CO -H] ), which was 315.0505 [17]. identifed as gallic acid by the MS and MS/MS spectra [30]. Journal of Analytical Methods in Chemistry 13 Compounds 7, 12, 13, and 21, with the same deprotonated compounds diabetes ions [M-H] at m/z 331.0671, were eluted at 1.07, 1.36, 1.41, and 1.91 min, respectively. Te 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 identifed as 6-O- 353 92 429 galloylsucrose. Compound 33 was eluted at 4.07 min, possessing a quasi- molecular ion [M-H] at m/z 183.0298, showing charac- teristic 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. Tey showed the same deprotonated ion [M-H] at m/z 153.0193 and the fragment ions at m/z Figure 2: Overlapping genes of diabetes and compound targets. 108.0203, 109.0282, and 123.0439, suggesting that they were 2,3-dihydroxybenzoic acid isomers [27, 31]. Compound 20 database. Finally, 92 overlapping genes of compound targets yielded a deprotonated ion [M-H] at m/z 315.0722, which and diabetes-related targets were regarded as potential showed a fragment ion at m/z 153.0186 by losing the glucose targets of Diospyros lotus L for the treatment of diabetes moiety (162 Da) in the MS fragment ions; therefore, it was (Figure 2). tentatively identifed as a 2,3-dihydroxybenzoic acid 3-O- glucoside isomer [31]. Compounds 16, 19, 28, 29, 41, 58, 113, and 131 were 3.6. PPI Network of Overlapping Genes. Te PPI network detected at 1.67, 1.84, 3.09, 3.19, 5.44, 6.38, 8.10, and graph was obtained by importing 92 overlapping targets into 9.18 min, respectively, and possessed the same quasi- STRING and removing one disconnected point. Tere were molecular ions [M-H] at m/z 151.0401. Te characteristic 91 nodes and 1488 edges; the average number of nodes was fragment ions at m/z 108.0204, 123.0439, and 136.0154 were 32.8, and the average local clustering coefcient was 0.703. identifed as vanillin isomers according to the base peaks and TSV data were downloaded and imported into Cytoscape retention times. Compounds 72 and 86 were found at 6.72 3.9.0 software to show the protein interaction network. and 7.23 min, respectively, and yielded the parent ions [M- Te results are shown in Figure 3, where the node size is H] at m/z 167.0350. Tey were identifed as vanillic acid positively correlated with the degree value and the lines isomers based on the MS and MS/MS spectra [16]. Com- represent interactions. As betweenness centrality increases, pound 49 appeared at a t of 5.95 min, possessing quasi- the color of the node changes from yellow to turquoise. molecular ions at m/z 329.0878 and the main fragment ion at Degree and betweenness centrality indicate the importance m/z 167.0341 owing to the loss of a glucose residue (162 Da), of the targets. Te target whose degree value was greater than which was characterized as vanillic acid glucoside [32]. the average value was considered the key target. Similarly, compounds 34 and 54 were confrmed as vanillic acid-O-rutinosides. Compound 94 at m/z 137.0244 with the molecular 3.7. Enrichment Analysis. Te key targets were further an- formula C H O and appearing at a t of 7.58 min was alyzed by functional association clustering to integrate 7 6 3 R suggested to be p-hydroxybenzoic acid based on the MS functional genomics annotations of the most important data [21]. Compounds 23, 24, 25, 26, 31, and 32 (t 2.65, 2.85, cluster of targets and pathways, which facilitates further 2.89, 3.00, 3.53, and 3.75 min, respectively) had the same understanding of the mechanism of the antidiabetic efect of quasi-molecular ion [M-H] at m/z 299.0772 and the Diospyros lotus L. characteristic fragment ion at m/z 137.0233 based on the As shown in Figure 4, the most representative GO-BP neutral loss of a glucose residue (162 Da). Tey were ten- terms were “positive regulation of transcription from RNA tatively characterized as p-hydroxybenzoic acid-O-glucoside polymerase II promoter” and “infammatory response,” isomers [33]. whereas the most representative GO-CC terms were “ex- tracellular space,” “extracellular region,” and “plasma membrane.” Te most representative GO-MF terms were “protein binding” and “enzyme binding.” 3.5. Active Components and Related Targets. Te active compounds were selected by using the “drug-like soft” in Te two most representative KEGG pathways (Figure 5) FAFDrugs4 with the criteria of 100≤ MW≤ 600, were the “MAPK signaling pathway” and “AGE-RAGE −3≤ logP≤ 6, and HBA≤ 12. Eventually, a total of 40 signaling pathway.” After exclusion of broad pathways, 47 compounds were screened (Supplementary Table 1). core common target genes were mainly related to the TNF, Combined with TCMSP and STP database search and PI3K-Akt, HIF-1, NAFLD, toll-like receptor, and other prediction, 445 component targets were obtained after re- multiple signaling pathways. Tis suggests that the efect of Diospyros lotus L on diabetes may involve multiple pathways moving duplicate targets. Furthermore, 521 diabetes-related targets were identifed by screening the disease-target as well as complex interactions among these pathways. 14 Journal of Analytical Methods in Chemistry Figure 3: PPI network graph. 3.8. Active Component-KeyGene-Pathway Interaction Net- insulin resistance [34]. Luteolin can play an antioxidant work Analysis. As shown in Figure 6, the active component- role by enhancing the activity of superoxide dismutase in keygene-pathway interaction network contained 104 nodes microvascular lesions in diabetes [35]. Kaempferol is (47 key genes, 37 active components, and 20 KEGG pathways a favonoid compound that plays an active role in the (top 20)) and 410 edges. In the network, the diamond, oval, prevention and treatment of diabetes and has anti- and elliptical nodes correspond to diferent active com- infammatory and antioxidant properties. It can reduce pounds, pathways, and targets, respectively. Te degrees of oxidative stress and infammation through the MAPK quercetin, luteolin, kaempferol, TNF signaling pathway, pathway to alleviate myocardial ischemia-reperfusion PI3K-Akt signaling pathway, HIF-1 signaling pathway, injury in diabetic rats [36]. Myricetin can enhance the PTGS2, AKT1, IL6, and TNF were 34, 20, 18, 12, 11, 10, 25, 21, antioxidant defense system in mice, increase insulin se- 20, and 17, respectively. Te average degrees of the diamond cretion, substantially reduce blood glucose levels, and ° ° and elliptical nodes were 5.93 and 8.72 , respectively. In efectively protect the liver and kidney from oxidative addition, at least nine genes were potentially involved in each damage in diabetic mice [37, 38]. IL-6 interferes with the diabetes-related pathway, suggesting that one active com- insulin signaling pathway and promotes apoptosis of ponent can potentially target multiple genes and have the pancreatic β-cells, which promotes insulin resistance in action characteristics of multiple active compounds, targets, multiple organs through a variety of infammatory sig- and pathways of Diospyros lotus L in the treatment of diabetes. naling pathways [39]. TNF is one of the cytokines con- Quercetin has many antihyperglycemic efects, such as stituting the acute infammatory response, which can enhancing insulin sensitivity, promoting glycogen syn- trigger the MAPK and NF-κB pathways, leading to insulin resistance [40]. thesis, inhibiting α-glucosidase activity, and improving Journal of Analytical Methods in Chemistry 15 ft Biological process Cellular component Molecular function BP CC MF Figure 4: Top 10 in GO analysis. Count positive regulation of transcription from RNA polymerase ll promoter 23.00 inflammatory response 15.00 positive regulation of transcription, DNA-templated 15.00 positive regulation of gene expression 14.00 negative regulation of apoptotic process 14.00 response to drug 12.00 negative regulation of cell proliferation 12.00 negative regulation of transcription from RNA polymerase ll promoter 12.00 cellular response to lipopolysaccharide 11.00 response to hypoxia 11.00 extracellular space 30.00 extracellular region 24.00 plasma membrane 23.00 cytosol 17.00 extracellular exosome 14.00 membrane 12.00 perinuclear region of cytoplasm 11.00 external side of plasma membrane 10.00 endoplasmic reticulum 7.00 membrane ra 6.00 protein binding 38.00 enzyme binding 13.00 cytokine activity 12.00 Identical protein binding 10.00 growth factor activity 8.00 receptor binding 8.00 transcription factor activity, sequence-specific DNA binding 8.00 heparin binding 7.00 transcription factor binding 7.00 protein homodimerization activity 7.00 16 Journal of Analytical Methods in Chemistry Malaria chagas disease (American trypanosomiasis) Infammatory bowel disease (IBD) TNF signaling pathway Pathways in cancer count Proteoglycans in cancer Leishmaniasis 12 Infuenza A Rheumatoid arthritis HIF - 1 signaling pathway Measles - log10 (pvalue) Tuberculosis Hepatitis B 12 Cytokine - cytokine receptor interaction Toll - like receptor signaling pathway Insulin resistance Non - alcoholic fatty liver disease (NAFLD) Toxoplasmosis HTLV - I infection PI3K - Akt signaling pathway 10 20 30 Figure 5: Signifcant pathway enrichment bubble diagram (top 20). Journal of Analytical Methods in Chemistry 17 Figure 6: Te active component-keygene-pathway interaction network. As the betweenness centrality increases, the color of the node changes from yellow to turquoise, and the larger the node, the greater the degree value. 4. Conclusion Authors’ Contributions In this study, an integrated approach combining UHPLC-Q- Shihan Qin and Mingjuan Liu contributed equally to Exactive Orbitrap MS and network pharmacology analysis was this work. adopted to explore the potential active ingredients and ame- liorative mechanisms of Diospyros lotus L against hyperglyce- Acknowledgments mia. Eventually, 159 compounds were identifed in Diospyros lotus L (140 of which were reported for the frst time). According Tis work was fnancially supported by the Science and to the results of the active components and key gene-pathway Technology Innovation Program of Hunan Province (no. interaction network, the antihyperglycemic efect of Diospyros 2022RC1228), the Scientifc Research Fund of Hunan lotus L is attributed to quercetin, luteolin, kaempferol, myr- Provincial Education Department (no. 19A353) and the icetin, and dihydromyricetin, which act on PTGS2, AKT1, IL6, Hunan University of Medicine High-Level Talent In- TNF, and MMP9 and participate in the TNF, PI3K-Akt, and troduction Startup Funds (no. 15001). HIF-1 signaling pathways, as well as NAFLD. In conclusion, the integrated approach combining UHPLC-Q-Exactive Orbitrap Supplementary Materials MS and network pharmacology analysis provided insights into the potential active ingredients and ameliorative mechanism of Supplementary Table 1: Results of screening compounds by Diospyros lotus L on hyperglycemia. “drug-likesoft” in FAFDrugs4. 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Published: Dec 31, 2022