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Abstract

Objective: Analyzing the chemical composition and blood-entry components of Nourishing Blood Diuretic Formula (NBDF) by Liquid Chromatography-Mass Spectrometry (LC-MS), and analyzing the mechanism of NBDF in the treatment of chronic renal failure by combining network pharmacology and molecular docking technology. Methods: The prototype components were obtained by LC-MS, and the targets of the prototype components and the targets for the treatment of chronic renal failure (CRF) were predicted by network pharmacology to obtain the common targets of the drug and the disease, which were then subjected to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, and molecular docking was carried out between the compounds of the prototype components and the key targets, so as to screen out the active ingredients, the targets and the signaling pathways that were closely related to the efficacy of NBDF. Results: Synthesizing the test information, 125 compounds were identified from the solution of NBDF, and 48 blood-entry components were identified from rat plasma containing the drug, including 6 prototypical components, and 102 potential targets of action, 515 GO entries, and 133 KEGG pathways were obtained from the web-based pharmacological analyses, and molecular docking was performed to obtain the binding of the 6 prototypical component compounds and the 9 key targets, and the formononetin, emodin, epicatechin, chlorogenic acid, sennoside A, and astragaloside III were hypothesized to be the active components of NBDF in the treatment of CRF. Conclusion: This study initially demonstrated that Nourishing Blood and Diuretic Formula exert its therapeutic effects on CRF through multicomponents, multitargets, and multimethods, and elucidated its pharmacological material basis and therapeutic mechanism.

Keywords

nourishing blood diuretic formula liquid chromatography-mass spectrometry network pharmacology serum pharmacochemistry pharmacological substance basis

Introduction

Chronic kidney disease (CKD) is a growing public health problem due to its high morbidity and mortality rates and greatly increased risk of chronic renal failure (CRF) and cardiovascular disease.¹ Recent data show that the global prevalence of CKD was estimated to be between 11% and 13% in 2016.² In addition, as CKD progresses, it can easily lead to cardiovascular disease.³ Renal fibrosis, characterized by tubulointerstitial fibrosis and glomerulosclerosis, is a common pathway and inevitable consequence of the various progression of CKD to ESRD, which requires renal replacement therapy such as dialysis and transplantation.^4–7 Therefore, it is of great medical and social value to study the treatment of CRF.

Nourishing Blood Diuretic Formula (NBDF) is an effective clinical experience prescription for treating CRF. It consists of 8 traditional Chinese medicines, including Abelmoschi corolla, Rhei Radix et Rhizoma, Astragali Radix, Angelicae Sinensis Radix, Poriae cutis, Dioscoreae Rhizoma, Hirudo, and Bombyx batryticatus. Clinical studies have shown that this formula can significantly improve the levels of blood creatinine and urea nitrogen in patients with renal failure, reduce urinary protein, protect kidney function, improve prognosis, and slow down the process of chronic kidney disease. However, due to the unclear substance basis of its efficacy and few literature reports on this prescription, it is necessary to further study the chemical components and blood components contained in this prescription. In this study, the chemical components and blood entry components of NBDF were analyzed by LC-MS technology, and the effective components of NBDF in the treatment of CRF were initially explored by combining network pharmacology and molecular docking technology, so as to clarify its pharmacodynamic material basis and provide theoretical basis and reference for clinical application research.

Materials and Methods

Instruments and Materials

In this study, we used U3000 Ultra High-Performance Liquid Chromatograph (Thermo Fisher Scientific Corporation); Q Exactive plus Orbitrap High-Resolution Mass Spectrometer (Thermo Fisher Scientific Corporation); ACQUITY UPLC I Class system Liquid Chromatographs (Waters Corporation); Synapt G2-Si Qtof Mass Spectrometer (Waters Corporation); Vortex-2 Genie Vortex Mixer (Scientific Industries Corporation); Model 5810R Cryogenic Centrifuge (Eppendorf Corporation, GER); Model WD-9415C Ultrasonic Cleaner (Liuyi Biotechnology Corporation); and Milli Q Advantage A10 Ultrapure Water Machine (Merck Corporation).

A. corolla (batch number: 210401), Rhei Radix et Rhizoma (batch number: 210501), B. batryticatus (batch number: 200701), and Hirudo (batch number: 210101) were purchased from Bozhou Jinshaotang Traditional Chinese Medicine Decoction Pieces Co., LD Astragali Radix (batch number: G03221006-07) and Angelicae Sinensis Radix (batch number: G01201117-21) were purchased from Gansu Jiuzhou Tianrun Traditional Chinese Medicine Industry Co., LD P. cutis (batch number: 211101) was purchased from Hubei Jurui Biotechnology Co., LD; Dioscoreae Rhizoma (batch number: D22090401) was purchased from Huanggang Jingui Traditional Chinese Medicine Industry Development Co., LTD; Heparin sodium (batch number: 003211009) was purchased from Chengdu Haitong Pharmaceutical Co., LTD; acetonitrile, these herbs were all characterization by Professor Duan Xueyun from Hubei University of Traditional Chinese Medicine (Wuhan, China), which conforms to the provisions of the 2020 edition of Chinese Pharmacopoeia. All storing samples were stored in the herbarium of the Hubei Institute of Traditional Chinese Medicine (Wuhan, China). Methanol and formic acid were chromatographically pure and purchased from Thermo Fisher Scientific; water was deionized water.

Animals

Fourteen SPF-grade healthy male SD rats, weighing 180∼220 g, were purchased from Wuhan Wanqian Jiaxing Biotechnology Co., LD, license No.: SCXK (Hubei) 2017-0013. Reared in the experimental animal center of Hubei Hospital of Traditional Chinese Medicine, experimental facility license: SYXK (Hubei) 2017-0095, room temperature: 22°C ± 3°C, relative humidity: 55%∼60%, no convection, artificial day and night (12 h day, 12 h night). The animal experiments involved in this study were approved by the experimental animal center of Hubei Provincial Hospital of Traditional Chinese Medicine, and the approval number is No. 2020003 of Hubei Provincial Hospital.

Chromatographic Conditions

The determination was performed on Waters ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.8 µm) with a mobile phase consisting of 0.1% formic acid aqueous solution (A) −0.1% acetonitrile formate solution (B). Condition 1 (chemical composition identification) is gradient elution (0∼10 min, 0%∼30% B;10∼25 min, 30%∼40% B; 25∼30 min, 40%∼50% B; 30∼40 min, 50%∼70% B; 40∼45 min, 70%∼100% B; 45 min∼60 min, 100% B; 60 min∼60.5 min, 100%∼0% B; 60.5 min∼70 min, 0% B). Condition 2 (identification of blood components) is gradient elution (0∼15 min, 0%∼20% B; 15∼50 min, 20%∼100% B; 50∼60 min, 100% B; 60 min to 70 min, 100% to 0% B). The flow rate was 0.3 mL/min, the sample size was 10 μL, and the column temperature was 35°C.

Mass Spectrometry Conditions

Condition 1 (chemical composition identification) is electrospray ion source, the detection mode was Full MS-dd MS2, the positive and negative ion modes were scanned respectively, the scanning range was m/z 100-1200, the resolution of MS1 was set to 70,000, the resolution of MS2 was set to 17,500, the voltage of ion source was set to 3.2 kv, and the temperature of ion transmission tube was set to 320°C. The heating temperature of the auxiliary device was 350°C, the sheath gas flow rate was 40 L/min, and the auxiliary gas flow rate was 15 L/min. The collision energy triggering the MS2 scan was set to 30, 40, and 50 eV using the stepped fracture voltage. Condition 2 (identification of blood components): an electrospray ion source, positive and negative ion mode scanning, the mass range is 50-1500 Da, capillary voltage is 3000 V (ESI+), 2500 V (ESI−), cone hole voltage is 40 V, collision energy is 10-50 eV, and ion source temperature is 125°C.

Preparation of NBDF

According to the prescription ratio of NBDF, weigh 8 herbs such as A. corolla, Rhei Radix et Rhizoma, Astragali Radix, Angelicae Sinensis Radix, P. cutis, Dioscoreae Rhizoma, Hirudo, and B. batryticatus, add 10 times the amount of water, soak for 30 min, boil for 1 h, filter, repeat 3 times, combine the filtrate, leave overnight, concentrate the superserum, and store in the refrigerator at 4°C for later use.

Carefully remove 2 mL of NBDF solution and place it in a 15 mL centrifuge tube, add 10 mL of 50% methanol aqueous solution, ultrasound for 30 min, take 1 mL of supernatant and place it in a centrifuge tube, centrifuge at 14,000 r/min for 5 min, take the supernatant through a 0.22 μm microporous filter membrane, and put it into the sample bottle. To be analyzed by UPLC-Q-Exactive Plus-Orbitrap-MS.

Collection and Preparation of Drug-Containing Plasma

After 1 week of adaptive feeding, 14 male SD rats were randomly divided into a blank group and a drug administration group, with 7 rats in each group. The blank group was given normal saline by intragastric administration every day, and the drug administration group was given NBDF solution by intragastric administration at the dose of 12.3 g/kg per day (referring to the equivalent dose conversion method, the dose given to rats was about 6.25 times of that given to adults). After the last administration, the rats were fasted without water for 12 h. On the 8th day, blood was taken from the orbital venous plexus of the rats 15 min, 30 min, 1 h, 2 h, and 4 h after gavage, about 0.5 mL of blood was collected each time and placed into a centrifugal tube coated with heparin sodium solution. Centrifuge at 3000 r/min at 4°C for 15 min, take the supernatant and then obtain the plasma sample and store it in the refrigerator at −80°C. The plasma samples were removed from the refrigerator at −80°C and thawed on ice. The plasma samples taken from each group at various time points were mixed well, accurately absorbed 150 μL, placed in a centrifugal tube, methanol was added according to the volume ratio of 1:4, swirled for 1 min, ultrasounded in an ice bath for 15 min, and then left for 1 h in the refrigerator at 4°C. Centrifuge at 3000 r/min for 10 min, take the supernatant, dry it with nitrogen at 37°C, redissolve the residue with methanol 400 μL, swirl for 1 min, ultrasound in an ice bath for 15 min, stand in the refrigerator at 4°C for 1 h, centrifuge at 12 000 r/min for 5 min, take 200 μL of the supernatant and put it into the sample bottle. To be analyzed by UPLC-Q-TOF-MS. The blank plasma samples were treated under the same conditions.^8–10

Data Processing

Compound Discover 3.2 software was used to extract characteristic peaks from raw mass spectrometry data, and the mass deviations of characteristic peak element matching, molecular formula prediction and isotope distribution matching were all set to less than 5 ppm. Characteristic peaks were identified using the mzcloud online database and mzVault database. The screening criteria for positive results were mass deviation <5 ppm, consistent isotope distribution, and matching score >70 points in mzVault best match database. The compounds were identified by referring to relevant literature and databases (Pub Chem, Mass Bank, Sci Finder, etc) and comparing them with the mass spectrum information of the screened target compounds.

Prediction of Prototype Component Targets and Disease Targets

The prototype component compounds were imported into SwissTargetPrediction (http://www.swisstargetprediction.ch/) in SMILES format to obtain the relevant targets, and the targets related to chronic renal failure disease were obtained through GeneCards (https://www.genecards.org/), and the blood component targets of NBDF as well as the disease targets of CRF were imported into Venny software to take the intersection of the intersection of the intersecting targets, which is the intersecting target of the NBDF for the treatment of CRF.

Construction of the PPI Network

The intersection targets were imported into the String (https://cn.string-db.org/) database to predict PPI, the species was set to human. The results were output in TSV format and imported into Cytoscape 3.8.2 software, and using the “cytoHubba” plugin in the Cytoscape 3.8.2 software to calculate the degree of interaction between nodes degree value, all nodes of this PPI network are presented according to the size of the degree value.

GO/KEGG Enrichment Analysis

GO includes the analysis of 3 modules: biological processes, cellular components, and molecular functions of targets, which is currently the most representative tool for describing systems biology. KEGG is a systematic database that analyzes gene functions and links genomic and functional information, which can be used not only for the discovery of differentially expressed genes but also for the discovery of network relationships and mechanisms between different biological processes. We imported the key targets into the DAVID (https://david.ncifcrf.gov/) database for analysis, with the species set to human and the P-value set to less than .05.

Molecular Docking

The 3D structures of the compounds of the prototypical components of NBDF were downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov/) and the compounds were imported into ChemDraw 20.0 for energy minimization, hydrogenation, and electric charge. The 3D structure of the screened target protein was downloaded from the PDB (http://www.rcsb.org) and imported into PyMOL 2.3.0 for the removal of nonprotein molecules from the ligand. Finally, the processed receptor and ligand were imported into AutoDockTools 1.5.7 for docking to obtain the binding energy between the ligand and the receptor, and the 6 sets of results with the best binding were plotted using PyMOL 2.3.0.

Results

Chemical Composition of NBDF

UPLC-Q-Exactive Plus-Orbitrap-MS technology was used to analyze the chemical composition of the NBDF solution. The total ion flow diagram of NBDF in positive and negative ion modes is shown in Figure 1(A) and (B). A total of 125 compounds were identified, including 39 flavonoids, 9 anthraquinones, 9 terpenoids, 16 organic acids, 22 phenylpropanoids, 4 polyphenols, 4 aldehydes, 4 alkaloids, 4 carbohydrates, 3 amino acids, and 3 phthalides. There were 2 saponins and 6 other compounds, of which hyperoside is the main component of A. corolla, emodin is the main component of Rhei Radix et Rhizoma, formononetin is a representative component of Astragali Radix, poricoic acid A is the important component of P. cutis, ferulic acid and caffeic acid are the basic ingredients of Angelicae Sinensis Radix, and the amino acids and carbohydrates are the main components of Dioscoreae Rhizoma, B. batryticatus, and Hirudo. The detailed results are shown in Table 1.

Figure 1.

Total ion flow diagram of NBDF solution in positive ion mode (A). Total ion flow diagram of NBDF solution in negative ion mode (B).

Table 1.

NBDF chemical composition information table.

No	Peak appearance time	Theoretical value	Value	Ion species	Ion fragment	Molecular formula	Ingredient name	Classification
1	1.50	182.0791	181.0719	[M-H]⁻	163.0612，101.0244，71.0138，59.0139	C₆H₁₄O₆	Mannitol	Other
2	1.51	147.0532	146.0459	[M-H]⁻	128.0354，102.0560，75.0087	C₅H₉NO₄	L-Glutamic acid	Amino acid
3	1.55	117.079	118.0863	[M + H]⁺	59.0733，58.0655	C₅H₁₁NO₂	Betaine	Alkaloid
4	1.63	137.0478	138.0551	[M + H]⁺	110.0601，94.0652	C₇H₇NO₂	Fenugreek hydrochloride	Alkaloid
5	1.63	550.1749	549.1676	[M-H]⁻	503.1644，221.0668，179.0561	C₁₈H₃₂O₁₆	Cottonseed sugar	Carbohydrate
6	1.64	342.1162	341.1089	[M-H]⁻	179.0562，101.0244，89.0244	C₁₂H₂₂O₁₁	Fructose	Carbohydrate
7	1.64	115.0634	116.0707	[M + H]⁺	98.0712，71.0628，70.0652	C₅H₉NO₂	L-Proline	Amino acid
8	2.53	504.1693	503.1622	[M-H]⁻	221.0667，113.0244，101.0244，89.0244	C₁₈H₃₂O₁₆	Mannitotriose	Carbohydrate
9	2.88	123.0322	124.0395	[M + H]⁺	96.0444，80.0495，53.0390	C₆H₅NO₂	Niacin	Organic acid
10	2.88	111.0433	112.0506	[M + H]⁺	95.0240，68.9972	C₄H₅N₃O	Cytosine	Other
11	2.89	151.0496	152.0569	[M + H]⁺	135.0302，134.0462，110.0350	CH₅N₅O	Guanine	Other
12	3.31	122.0481	123.0554	[M + H]⁺	96.0445，80.0495	C₆H₆N₂O	Niacinamide	Other
13	3.48	192.0271	191.0199	[M-H]⁻	173.0092，129.0195，111.0088	C₆H₈O₇	Citrate	Organic acid
14	4.22	162.1158	163.1231	[M + H]⁺	132.0809，117.0574，106.0652，95.0492	C₁₀H₁₄N₂	Nicotine	Alkaloid
15	4.53	666.2228	665.2156	[M-H]⁻	323.0988，179.0562，101.0244	C₂₄H₄₂O₂₁	Stachyos	Carbohydrate
16	5.34	164.0476	165.0549	[M + H]⁺	147.0442，123.0441，95.0492	C₉H₈O₃	p-Hydroxycinnamic acid	Phenylpropanoid
17	8.05	170.0216	169.0143	[M-H]⁻	125.0244，107.0139	C₇H₆O₅	Gallic acid	Organic acid
18	11.32	267.0969	268.104	[M + H]⁺	136.0618，57.0338	C₁₀H₁₃N₅	Adenosine	Other
19	11.34	135.0547	136.0619	[M + H]⁺	119.0353，94.0401	C₅H₅N₅	Adenine	Other
20	16.33	138.0317	137.0244	[M-H]⁻	93.0345	C₇H₆O₃	p-Hydroxybenzoic acid	Organic acid
21	16.47	154.0267	153.0194	[M-H]⁻	109.0294，91.0188	C₇H₆O₄	Protocatechuic acid	Organic acid
22	17.02	126.0318	127.0391	[M + H]⁺	109.0285，81.0335，69.0336	C₆H₆O₃	5-Hydroxymethylfurfural	Aldehyde
23	17.55	290.079	291.0866	[M + H]⁺	165.0548，147.0442，139.0391，123.0442	C₁₅H₁₄O₆	Catechin	Polyphenol
24	17.76	204.09	203.0828	[M-H]⁻	142.0661，116.0505	C₁₁H₁₂N₂O₂	L-Tryptophan	Amino acid
25	17.77	354.0952	353.088	[M-H]⁻	191.0562，179.0351，161.0244，135.0452	C₁₆H₁₈O₉	Chlorogenic acid	Phenylpropanoid
26	18.14	271.121	272.1283	[M + H]⁺	255.1017，161.0598，123.0442，107.0492	C₁₆H₁₇NO₃	Higenamine	Alkaloid
27	18.18	138.0317	137.0244	[M-H]⁻	108.0216，70.3051	C₇H₆O₃	Protocatechualdehyde	Aldehyde
28	18.42	328.0796	327.0723	[M-H]⁻	312.0491，234.0170，192.0066，165.0557	C₁₄H₁₆O₉	Bergeninum	Polyphenol
29	18.47	154.0267	153.0193	[M-H]⁻	109.0295	C₇H₆O₄	Gentianic acid	Organic acid
30	18.66	146.0368	147.0442	[M + H]⁺	119.0293，91.0543	C₉H₆O₂	Coumarin	Phenylpropanoid
31	19.00	192.0635	191.0562	[M-H]⁻	127.0400，85.0294	C₇H₁₂O₆	Quinic acid	Organic acid
32	19.01	290.0791	289.0718	[M-H]⁻	245.0819，203.0714，123.0452，109.0294	C₁₅H₁₄O₆	Epicatechin	Polyphenol
33	19.02	354.0952	353.0879	[M-H]⁻	191.0562，179.0349，135.0451	C₁₆H₁₈O₉	1-Caffeoylquinic acid	Phenylpropanoid
34	19.35	168.0422	167.035	[M-H]⁻	121.0659，79.0553	C₈H₈O₄	4-Methoxysalicylic acid	Organic acid
35	19.62	432.1056	433.113	[M + H]⁺	271.0602，225.0549，197.0599	C₂₁H₂₀O₁₀	Sophoricoside	Flavonoid
36	19.72	180.0424	179.0351	[M-H]⁻	135.0452	C₉H₈O₄	Caffeic acid	Phenylpropanoid
37	19.87	518.1636	517.1564	[M-H]⁻	193.0506，175.0400，160.0166，134.0374	C₂₂H₃₀O₁₄	Sibiricose A5	Phenylpropanoid
38	20.56	682.2116	681.2043	[M-H]⁻	137.0244	C₃₁H₃₈O₁₇	Tenuifoliside A	Phenylpropanoid
39	20.59	624.1693	625.1764	[M + H]⁺	317.0657，302.0420，301.0344	C₂₈H₃₂O₁₆	Narcissoside	Flavonoid
40	20.85	318.0373	319.0446	[M + H]⁺	273.0395，245.0447，179.0339，144.0810	C₁₅H₁₀O₈	Myricetin	Flavonoid
41	21.22	152.0474	153.0547	[M + H]⁺	134.0602，125.0599，111.0442，93.0336，65.0388	C₈H₈O₃	4-Methoxysalicylaldehyde	Aldehyde
42	21.22	198.0529	197.0456	[M-H]⁻	169.0143，140.0117，125.0244	C₉H₁₀O₅	Ethyl gallate	Organic acid
43	21.36	610.1534	609.1462	[M-H]⁻	300.0276，271.0249，151.0037	C₂₇H₃₀O₁₆	Rutin	Flavonoid
44	21.42	446.1212	447.1284	[M + H]⁺	285.0758，270.0522，225.0546，137.0235	C₂₂H₂₂O₁₀	Calycosin-7-glucoside	Flavonoid
45	21.74	464.0956	463.0884	[M-H]⁻	300.0276，271.0249，255.0300，151.0036	C₂₁H₂₀O₁₂	Isoquercitrin	Flavonoid
46	21.74	302.0425	303.0498	[M + H]⁺	257.0446，229.0497，153.0184，137.0235	C₁₅H₁₀O₇	Morin	Flavonoid
47	21.9	594.1591	593.1517	[M-H]⁻	284.0327，255.0300，227.0350	C₂₇H₃₀O₁₅	Kaempferol-3-O-glucorhamnoside	Flavonoid
48	22.01	194.058	195.0653	[M + H]⁺	178.0583，149.0600，134.0367	C₁₀H₁₀O₄	Ferulic acid	Phenylpropanoid
49	22.02	134.0368	193.0506	[M-H + HAc]⁻	176.0114，149.0244，105.0346，81.0345	C₁₀H₁₀O₄	Isoferulic acid	Phenylpropanoid
50	22.02	116.0263	117.0335	[M + H]⁺	134.0364，105.0337，79.0543	C₉H₈O₂	Cinnamic acid	Phenylpropanoid
51	22.03	174.0317	207.0653	[M + H + MeOH]⁺	175.0391，147.0442，119.0493，91.0543	C₁₁H₁₀O₄	Limettin	Phenylpropanoid
52	22.07	190.063	191.0704	[M + H]⁺	163.0754，135.0806，120.0809	C₁₁H₁₀O₃	7-Methoxy-4-methylcoumarin	Phenylpropanoid
53	22.07	712.2589	711.2516	[M-H]⁻	417.1555，181.0507，166.0271	C₃₃H₄₄O₁₇	(−)-Syringaresnol-4-O-β-D-apiofuranosyl-(1→2)-β-D-glucopyranoside	Phenylpropanoid
54	22.16	432.1059	431.0986	[M-H]⁻	271.0602，243.1018，215.0704，153.0183，107.0492	C₂₁H₂₀O₁₀	Genistin	Flavonoid
55	22.16	594.1595	595.1668	[M + H]⁺	287.0550	C₂₇H₃₀O₁₅	Kaempferol 3-rutinoside	Flavonoid
56	22.20	516.127	515.1198	[M-H]⁻	353.0822，191.0563，173.0456，135.0452	C₂₅H₂₄O₁₂	1,3-Dicaffeoylquinic acid	Phenylpropanoid
57	22.26	222.0527	223.06	[M + H]⁺	208.0366，190.0261，162.0313，107.0492	C₁₁H₁₀O₅	Isofraxidin	Phenylpropanoid
58	22.29	520.1948	519.1876	[M-H]⁻	357.1351，151.0401，136.0166，89.0243	C₂₆H₃₂O₁₁	(+)-pinoresinol-β-D-glucoside	Phenylpropanoid
59	22.38	862.1964	861.1892	[M-H]⁻	611.1539，449.1032，389.0879，227.0350	C₄₂H₃₈O₂₀	Sennoside A	Anthraquinone
60	22.44	258.0894	259.0967	[M + H]⁺	217.0497，189.0548	C₁₅H₁₄O₄	Murrayone	Phenylpropanoid
61	22.58	286.0475	287.0551	[M + H]⁺	241.0493，213.0547，165.0183，153.0183，121.0285	C₁₅H₁₀O₆	Kaempferol	Flavonoid
62	22.87	462.1164	463.1237	[M + H]⁺	301.0708，286.0473，241.0496	C₂₂H₂₂O₁₁	Tectoridin	Flavonoid
63	22.88	300.0633	301.0706	[M + H]⁺	286.0473，241.0496，153.0184	C₁₆H₁₂O₆	Diosmetin	Flavonoid
64	22.95	516.1271	515.1198	[M-H]⁻	353.0880，173.0455	C₂₅H₂₄O₁₂	Isochlorogenic acid C	Phenylpropanoid
65	22.98	302.0792	303.0865	[M + H]⁺	285.0761，177.0552，153.0184	C₁₆H₁₄O₆	Hesperetin	Flavonoid
66	23.05	448.1007	447.0937	[M-H]⁻	284.0327，255.0300	C₂₁H₂₀O₁₁	Astragalin	Flavonoid
67	23.21	188.105	187.0977	[M-H]⁻	125.0972	C₉H₁₆O₄	Azelaic Acid	Organic acid
68	23.36	464.0956	463.0884	[M-H]⁻	301.0354，257.0458，178.9987，151.0037	C₂₁H₂₀O₁₂	Hyperoside	Flavonoid
69	23.36	178.063	179.0704	[M + H]⁺	179.0704，161.0598，147.0442	C₁₀H₁₀O₃	Coniferol	Phenylpropanoid
70	23.37	302.0425	303.0498	[M + H]⁺	229.0498，137.0236	C₁₅H₁₀O₇	Quercetin	Flavonoid
71	23.54	138.0317	137.0244	[M-H]⁻	93.0345	C₇H₆O₃	Salicylic acid	Organic acid
72	23.60	294.1258	327.1592	[M + H + MeOH]⁺	163.0755，137.0599，103.0543	C₁₈H₃₀O₂	Dehydrodiisoeugenol	Phenylpropanoid
73	23.71	548.1538	547.1463	[M-H]⁻	269.0456，225.0561	C₂₇H₃₀O₁₅	Oroxin B	Flavonoid
74	23.77	488.1322	489.1395	[M + H]⁺	285.0759，225.0547，137.0235	C₂₄H₂₄O₁₁	6″-O-Acetylglycitin	Flavonoid
75	23.92	254.058	253.0502	[M-H]⁻	225.0557，209.0588	C₁₅H₁₀O₄	Daidzein	Flavonoid
76	23.96	430.1264	431.1336	[M + H]⁺	269.0810，226.0623	C₂₂H₂₂O₉	Ononin	Flavonoid
77	23.97	594.195	593.188	[M-H]⁻	417.1557，268.0378，181.0508，166.0271	C₂₈H₃₄O₁₄	Poncirin	Flavonoid
78	24.02	462.1166	461.1096	[M-H]⁻	271.0461，211.0249，169.0142，125.0245	C₂₂H₂₂O₁₁	Diosmetin-7-O-β-D-glucopyranoside	Flavonoid
79	24.45	168.0423	169.0496	[M + H]⁺	151.0391，138.0551，127.0391，85.0285	C₈H₈O₄	Orsellinic acid	Organic acid
80	24.71	462.1523	461.1453	[M-H]⁻	301.0708，167.0704，123.0442	C₂₃H₂₆O₁₀	9-O-Methylnissolin 3-O-glucoside	Flavonoid
81	24.71	166.0631	167.0703	[M + H]⁺	152.0469，134.0363，106.0415	C₉H₁₀O₃	2,3-Dimethoxybenzaldehyde	Aldehyde
82	24.81	418.1266	417.1193	[M-H]⁻	254.0586	C₂₁H₂₂O₉	Isoliquiritin	Flavonoid
83	25.08	464.1684	463.1612	[M-H]⁻	301.1083，121.0295	C₂₃H₂₈O₁₀	Isomucronulatol7-O-Glucoside	Flavonoid
84	25.19	256.0736	255.0664	[M-H]⁻	135.0088，119.0502，91.0189	C₁₅H₁₂O₄	Liquiritigenin	Flavonoid
85	25.34	408.1421	407.1349	[M-H]⁻	245.0819，230.0584，157.0507	C₂₀H₂₄O₉	Tinnevellin glucoside	Anthraquinone
86	25.49	432.1056	431.0985	[M-H]⁻	268.0377	C₂₁H₂₀O₁₀	Emodin-8-glucoside	Anthraquinone
87	25.62	284.0684	283.0611	[M-H]⁻	268.0377，239.0351，211.0402	C₁₆H₁₂O₅	Calycosin	Flavonoid
88	25.78	254.058	253.0507	[M-H]⁻	225.0557，182.0368	C₁₅H₁₀O₄	Rubiadin	Anthraquinone
89	25.79	416.111	415.1037	[M-H]⁻	277.0505，253.0507，239.0715，225.0553	C₂₁H₂₀O₉	Pulmatin	Anthraquinone
90	26.13	446.1214	447.1289	[M + H]⁺	285.0759，270.0521，242.0572	C₂₂H₂₂O₁₀	Glycitin	Flavonoid
91	26.66	448.1007	447.0934	[M-H]⁻	284.0326，256.0375，239.0344	C₂₁H₂₀O₁₁	Asiatica	Flavonoid
92	27.29	314.0428	313.0355	[M-H]⁻	269.0456，225.0557，161.0608	C₁₆H₁₀O₇	Wedelolactone	Phenylpropanoid
93	27.31	272.0685	271.0612	[M-H]⁻	177.0194，151.0037，119.0502，107.0138	C₁₅H₁₂O₅	Naringenin chalcone	Flavonoid
94	27.38	270.0892	271.0965	[M + H]⁺	153.0183，147.0442，121.0285	C₁₆H₁₄O₄	Echinatin	Flavonoid
95	27.5	270.0528	269.0456	[M-H]⁻	225.0544，201.0559，183.0452，159.0453	C₁₅H₁₀O₅	Genistein	Flavonoid
96	27.67	234.1622	235.1693	[M + H]⁺	217.1585，193.0862，133.1013	C₁₅H₂₂O₂	Curcumenol	Sesquiterpene
97	27.88	300.0634	299.0561	[M-H]⁻	284.0328，255.0300，227.0349	C₁₆H₁₂O₆	Tectorigenin	Flavonoid
98	27.97	234.1622	235.1693	[M + H]⁺	133.1013，121.1012，107.0856，93.0700	C₁₅H₂₂O₂	Artemisinic acid	Organic acid
99	28.23	316.0584	315.0512	[M-H]⁻	300.0276，271.0259，245.0814，151.0036	C₁₆H₁₂O₇	Isorhamnetin	Flavonoid
100	28.30	300.0634	299.0561	[M-H]⁻	284.0328，255.0301，227.0352	C₁₆H₁₂O₆	Hydroxygenkwanin	Flavonoid
101	29.10	830.4669	829.4597	[M-H]⁻	306.4576，113.0244，101.0246，71.0137	C₄₁H₆₈O₁₄	Astragaloside A	Triterpenoid saponin
102	29.28	300.027	299.0198	[M-H]⁻	255.0300，227.0351，211.0401	C₁₅H₈O₇	Demethylwedelolactone	Phenylpropanoid
103	29.38	784.4614	783.4528	[M + H]⁺	483.3595，161.0455，113.0244，101.0244，71.0138	C₄₁H₆₈O₁₄	Astragaloside III	Triterpenoid saponin
104	29.39	454.3449	455.352	[M + H]⁺	437.3420，225.1642，147.1170，123.1169，81.0700	C₃₀H₄₆O₃	Dehydrotrametenolic acid	Triterpene
105	29.48	188.0838	189.0911	[M + H]⁺	171.0805，143.0856，133.0285，117.0699	C₁₂H₁₂O₂	3-Butylidenephthalide	Phthalide
106	29.70	268.0736	267.0663	[M-H]⁻	252.0428，135.0886	C₁₆H₁₂O₄	Formononetin	Flavonoid
107	30.65	122.0367	123.0441	[M + H]⁺	121.0649，103.0543	C₇H₆O₂	Benzoic acid	Organic acid
108	30.76	270.0528	269.0456	[M-H]⁻	240.0428，223.0399，183.0449	C₁₅H₁₀O₅	Aloe emodin	Anthraquinone
109	31.18	440.3657	441.373	[M + H]⁺	423.3625，107.0856	C₃₀H₄₈O₂	roburicacid	Triterpene
110	31.54	284.0321	283.0248	[M-H]⁻	257.0457，239.0351，211.0400，183.0452	C₁₅H₈O₆	Rhein	Anthraquinone
111	31.54	256.0372	255.0299	[M-H]⁻	227.0348，211.0401，167.0503	C₁₄H₈O₅	Purpurin	Anthraquinone
112	31.89	454.3448	455.3522	[M + H]⁺	437.3413，197.1330，123.1169，95.0856，71.0493	C₃₀H₄₆O₃	Wilforlide A	Triterpene
113	33.06	294.1833	293.1761	[M-H]⁻	236.1056，220.1470，177.0922	C₁₇H₂₆O₄	6-Gingerol	Polyphenol
114	33.22	284.0684	285.0759	[M + H]⁺	270.0520，242.0574，213.0546，197.0600	C₁₆H₁₂O₅	Biochanin A	Flavonoid
115	33.86	192.1152	193.1225	[M + H]⁺	175.1119，147.1170，137.0598，105.0700，91.0543	C₁₂H₁₆O₂	3-N-Butyl-4,5-dihydrophthalide	Phthalide
116	35.03	868.4821	869.4894	[M + H]⁺	653.4047，437.3415，217.0707，139.0391	C₄₅H₇₂O₁₆	Isoastragaloside I	Triterpenoid saponin
117	35.27	470.3401	471.3474	[M + H]⁺	262.2462，175.1327，135.1011	C₃₀H₄₆O₄	Glycyrrhetinic acid	Triterpene
118	36.02	278.2247	279.232	[M + H]⁺	109.1013，95.0856，81.0700	C₁₈H₃₀O₂	α-Linolenic acid	Organic acid
119	36.13	270.0528	269.0456	[M-H]⁻	241.0510，225.0558，210.0319	C₁₅H₁₀O₅	Emodin	Anthraquinone
120	36.17	328.1886	327.1813	[M-H]⁻	265.1811，239.2018，124.0165	C₁₇H₂₈O₆	Spiculisporic acid	Organic acid
121	41.41	498.3348	497.3276	[M-H]⁻	423.2909，395.2965，297.2181	C₃₁H₄₆O₅	Poricoic acid A	Triterpene
122	42.36	284.0683	285.0759	[M + H]⁺	270.0526，242.0573，253.0487，211.0756	C₁₆H₁₂O₅	Glycitein	Flavonoid
123	44.01	190.0995	191.1068	[M + H]⁺	173.0963，163.1118，145.1034	C₁₂H₁₄O₂	Ligustilide	Phthalide
124	44.04	294.1257	295.1332	[M + H]⁺	277.1224，249.1276，206.1092	C₁₉H₁₈O₃	Tanshinone-A	Diterpene
125	45.85	496.3556	497.3628	[M + H]⁺	437.3413，320.0474，295.2420，173.1319，121.1012	C₃₀H₅₀O₄	Bryodulcosigenin	Triterpene

NBDF, Nourishing Blood Diuretic Formula.

Flavonoids

A total of 39 flavonoid compounds were identified in this study, which generally refers to a series of compounds made of 2 benzene rings interconnected by 3 carbon atoms, ie, a general term for a class of compounds with the C6-C3-C6 structure, which often lose neutral CH₃, H₂O, CO, CO₂, etc, plasma fragments to form fragment ions. Taking compound No. 68 as an example, its quasi-molecular ion peak was m/z 463.0884[M-H]⁻ with a retention time of 23.36 min, and its molecular formula was presumed to be C₂₁H₂₀O₁₂. Fragmentation ions such as m/z 301.0354[M-H-C₆H₁₀O₅] appeared in the secondary mass spectrum, and based on the cleavage pattern and the literature and database matching, it was hypothesized that the compound was hyperoside, an active ingredient in the A. corolla, which is hepatoprotective, cardiovascular and cerebrovascular protective, neuroprotective, antiinflammatory, analgesic, hypolipidemic, and enhances immune functions.^11,12 The cleavage process is shown in Figure 2A, and the fragment ion map is shown in Figure 3A.

Figure 2.

Cleavage pathway of hypericin (A), protocatechuic acid (B), caffeic acid (C), poricoic acid A (D), 6-gingerol (E), and emodin (F).

Figure 3.

Fragment ions of hypericin (A), protocatechuic acid (B), caffeic acid (C), poricoic acid A (D), 6-gingerol (E), and emodin (F).

Organic Acid Compounds

A total of 16 organic acid compounds were identified in this study, which contain carboxyl groups in their molecular structures, mostly phenol carboxyl-substituted aromatic rings, fatty acids, etc. According to their structural characteristics, they can be classified into aromatic, aliphatic and terpenoid organic acids. Taking compound No. 21 as an example, its quasi-molecular ion peak was 153.0194 [M-H]⁻ with a retention time of 16.47 min, and its molecular formula was presumed to be C₇H₆O₄, and the secondary mass spectra yielded the characteristic fragment ions, such as m/z 109.0294 [M-H-CO₂]⁻, m/z 91.0188 [M-H-CO₂-H₂O]⁻, etc, and based on the cleavage pattern and the literature and database matching, it was hypothesized that the compound was protocatechuic acid. The cleavage process is shown in Figure 2B, and the fragment ion map is shown in Figure 3B.

Phenylpropanoids

A total of 22 phenylpropanoid compounds were identified in this study, including caffeic acid, ferulic acid, chlorogenic acid, cinnamic acid, coumarin, and wedelolactone, most of which were coumarins. Taking compound No. 36 as an example, its quasi-molecular ion peak was 179.0351[M-H]⁻ with a retention time of 19.72 min, and its molecular formula was presumed to be C₉H₈O₄. The secondary mass spectra showed m/z 135.0452[M-H-CO₂]⁻ fragment ions, based on the cleavage pattern and the literature and database matching, the compound was hypothesized to be caffeic acid, the active ingredient in Angelicae Sinensis Radix, which is analgesic, antiinflammatory, antioxidant, microcirculation improvement, and antiplatelet aggregation effects.¹³ The cleavage process is shown in Figure 2C, and the fragment ion map is shown in Figure 3C.

Terpenoids

A total of 9 terpenoids were identified in this study, most of which were triterpenoids, including oak cherry acid, poriaxinic acid A, and glycyrrhizinic acid. Taking compound No. 121 as an example, its quasi-molecular ion peak was 497.3277[M-H]⁻, with a retention time of 41.41 min, and its molecular formula was presumed to be C₃₁H₄₆O₅. m/z 423.0909 [M-H-C₃H₆O₂]⁻, m/z 395.2965 [M-H-C₂H₄]⁻, m/z 297.2181 [M-H-C₇H₁₄]⁻ and other fragment ions, based on the cleavage pattern matched with the literature and database, it is presumed that the compound is poricoic acid A, an active ingredient in P. cutis, which has antiinflammatory, antioxidant, antitumor, hypolipidemic, and diuretic effects.^14–16 The process of cleavage is shown in Figure 2D, and the fragment ion map is shown in Figure 3D.

Polyphenols

A total of 4 polyphenolic compounds were identified in this study. Taking compound No. 113 as an example, its quasi-molecular ion peak was 293.1761[M-H]⁻ with a retention time of 33.06 min, and its molecular formula was presumed to be C₁₇H₂₆O₄. m/z 236.1056[M-H-C₄H₉]⁻, m/z 221.1544[M-H-C₄H₉-CH₃]⁻, m/z 177.0922 [M-H-C₇H₁₅O]⁻ and other fragment ions, and the compound was presumed to be 6-gingerolby matching the cleavage pattern with the literature and the database. The cleavage process is shown in Figure 2E, and the fragment ion map is shown in Figure 3E.

Anthraquinones

A total of 9 anthraquinones were identified in this study. Taking compound No.119 as an example, its quasi-molecular ion peak was 269.0456[M-H]⁻ with a retention time of 36.13 min, and its molecular formula was presumed to be C₁₅H₁₀O₅. In the secondary mass spectra, there were fragment ions, such as m/z 241.0510[M-H-CO]⁻, m/z 225.0558[M-H-CO₂]⁻, and m/z 210.0319 [M-H-CO₂-CH₃]⁻ and other fragment ions. By matching the cleavage pattern with the literature and the database, it was hypothesized that the compound was emodin, the active ingredient in Rhei Radix et Rhizoma, which has antiinflammatory, antiviral, antitumor, and other pharmacological effects.¹⁷ The cleavage process is shown in Figure 2F, and the fragment ion map is shown in Figure 3F.

In this study, 4 aldehydes, 4 alkaloids, 4 carbohydrates, 3 amino acids, 3 phthalides, 2 saponins, and 6 other compounds were also identified, which were analyzed and identified in the same way as the abovementioned compounds, and no more examples will be given.

Blood Entry Components of NBDF

Blank plasma from rats and plasma after gavage of NBDF solution were analyzed and identified by UPLC-Q-TOF-MS, and their total ion flow diagrams in positive and negative ion modes are shown in Figure 4(A)-(D). A total of 48 compounds were identified, among which 6 prototypical components were identified, including formononetin, emodin, epicatechin, chlorogenic acid, sennoside A, and astragaloside III. The detailed information is shown in Table 2.

Figure 4.

Total ion flow diagram of a blank plasma sample in positive ion mode (A). Total ion flow diagram of a blank plasma sample in negative ion mode (B). Total ion flow diagram of plasma sample after administration in positive ion mode (C). Total ion flow diagram of plasma sample after administration in negative ion mode (D).

Table 2.

Information table of blood component of NBDF.

No	Peak appearance time	Theoretical value	Value	Ion species	Ion fragment	Molecular formula	Ingredient name	Classification
1	1.00	694.4292	693.4198	[M-H]⁻	558.5372，268.7518	C₃₈H₆₂O₁₁	Mongholicoside II	Saponin
2	1.02	784.4609	783.4554	[M-H]⁻	560.5345，268.7519	C₄₁H₆₈O₁₄	Astragaloside III	Saponin
3	1.19	468.1056	469.1119	[M + H]⁺	386.1282，280.1479，162.1055	C₂₄H₂₀O₁₀	Theophyllin A	Polyphenol
4	1.24	946.5137	945.5057	[M-H]⁻	848.4246，322.8045，200.9930，146.9420	C₄₇H₇₈O₁₉	Astragaloside VI	Saponin
5	9.35	862.1956	861.1893	[M-H]⁻	610.9212，446.9386，211.9565	C₄₂H₃₈O₂₀	Sennoside A	Anthraquinone
6	9.94	261.1365	262.1430	[M + H]⁺	226.1222，188.1273	C₁₅H₁₉NO₃	Polycanthine	Alkaloid
7	10.34	432.1420	431.1369	[M-H]⁻	295.0798，211.9561，159.9985	C₂₂H₂₄O₉	Octenatide-7-O-β-D-glucoside	Flavonoid
8	10.36	650.4030	651.4114	[M + H]⁺	410.1745，181.1308，162.1107，144.0993	C₃₆H₅₈O₁₀	Astragalus membranaceus stem saponin A	Saponin
9	10.39	270.1216	271.1275	[M + H]⁺	162.1107，144.0993	C₁₂H₁₈N₂O₅	Astragaline F	Alkaloid
10	10.92	462.1162	461.1097	[M-H]⁻	242.9855，163.0342	C₂₂H₂₂O₁₁	Kaempferitrin-4′-methyl ether-3-glucoside	Flavonoid
11	11.28	376.1383	377.1451	[M + H]⁺	210.0540，192.0437，86.1450	C₁₇H₂₀N₄O₆	Vitamin B2	Other
12	12.88	304.0947	303.0859	[M-H]⁻	275.0540，245.0009，165.0498	C₁₆H₁₆O₆	Cassialactone	Naphtho-pyrone
13	14.08	174.1117	175.1193	[M + H]⁺	130.1191	C₆H₁₄N₄O₂	Arginine	Amino acid
14	14.80	314.0790	313.0710	[M-H]⁻	268.9962，239.9948，210.9941	C₁₇H₁₄O₆	8,3′-Dihydroxy-7,4′-dimethoxyisoflavone	Flavonoid
15	15.16	652.4186	653.4280	[M + H]⁺	521.1495，295.1162，86.1453	C₃₆H₆₀O₁₀	Astragalus membranaceus stem saponin B	Saponin
16	17.04	354.0951	355.1018	[M + H]⁺	297.1790，159.1724，126.0752	C₁₆H₁₈O₉	Chlorogenic Acid	Phenylpropanoid
17	17.04	1030.5349	1029.5236	[M-H]⁻	928.4722，514.2279，273.0276	C₅₁H₈₂O₂₁	Agroastragaloside Ⅲ	Saponin
18	19.64	254.0579	253.0496	[M-H]⁻	169.0439，150.9620，89.9905	C₁₅H₁₀O₄	Chrysophanic acid	Anthraquinone
19	20.22	452.1107	451.1046	[M-H]⁻	309.0753，96.9253	C₂₄H₂₀O₉	Cinchonain Ia	Polyphenol
20	21.51	296.3079	297.3145	[M + H]⁺	163.2041，135.1348，93.1195	C₂₀H₄₀O	Phytol	Terpene
21	23.53	912.5082	913.5131	[M + H]⁺	817.5923，355.3166，288.3457	C₄₇H₇₆O₁₇	Agroastragaloside Ⅰ	Saponin
22	23.62	491.3094	492.3156	[M + H]⁺	355.3166，288.3457	C₂₄H₄₅NO₉	Morusimic acid C	Organic acid
23	24.44	284.0685	283.0603	[M-H]⁻	238.1699	C₁₆H₁₂O₅	Obtusifolin	Anthraquinone
24	24.80	268.0736	267.0657	[M-H]⁻	148.9598	C₁₆H₁₂O₄	Formononetin	Flavonoid
25	27.11	622.4081	623.4163	[M + H]⁺	302.3610，264.3252，119.1382	C₃₅H₅₈O₉	Astramembrannin II	Saponin
26	27.30	270.0528	269.0443	[M-H]⁻	239.1172，149.0198，79.9246	C₁₅H₁₀O₅	Emodin	Anthraquinone
27	28.58	828.4871	829.4956	[M + H]⁺	357.3297，184.1288，119.1375	C₄₃H₇₂O₁₅	Agroastragaloside II	Saponin
28	28.86	642.2160	641.2068	[M-H]⁻	482.2688，389.2163，168.0001，78.9264	C₂₉H₃₈O₁₆	7-Hydroxy-3′,4′-dimethoxy-2′,5′-diglucose isoflavanoside	Flavonoid
29	32.11	652.4186	653.4232	[M + H]⁺	325.2007，184.1298	C₃₆H₆₀O₁₀	Astragalus membranaceus stem saponin C	Saponin
30	32.22	386.1941	387.2025	[M + H]⁺	325.2007，277.2905，91.1038	C₁₉H₃₀O₈	Theophylloside I	Triterpene
31	32.88	694.4292	693.4203	[M-H]⁻	558.5372，326.7080，268.7519，92.8929	C₃₈H₆₂O₁₁	Astragalus membranaceus stem saponin D	Saponin
32	33.05	284.2715	285.2274	[M + H]⁺	145.1560，131.1393，91.1036	C₁₈H₃₆O₂	Linolenic acid	Organic acid
33	33.05	256.2402	257.2469	[M + H]⁺	145.1560，131.1393，91.1036	C₁₆H₃₂O₂	Palmitic acid	Organic acid
34	33.80	446.1213	445.1144	[M-H]⁻	345.1912，168.0002	C₂₂H₂₂O₁₀	Calycosin-7-O-beta-D-glucoside	Flavonoid
35	34.92	288.0998	289.1084	[M + H]⁺	184.1290，105.1207	C₁₆H₁₆O₅	7,2′,3′-Trihydroxy-4′-methoxyisoflavane	Polyphenol
36	34.93	446.1213	445.1135	[M-H]⁻	345.1912，295.1775，205.0780	C₂₂H₂₂O₁₀	Gluco-obtusifolin	Anthraquinone
37	35.43	441.1397	440.1330	[M-H]⁻	311.1176，299.2092，182.9684	C₁₉H₁₉N₇O₆	Folic acid	Organic acid
38	38.37	448.1006	447.0926	[M-H]⁻	301.1666，285.1935，116.8906	C₂₁H₂₀O₁₁	Trifolin	Flavonoid
39	40.12	478.1111	477.1054	[M-H]⁻	441.1977，325.1329，182.9683，96.9246	C₂₂H₂₂O₁₂	Isorhamnetin 3-O-glucoside	Flavonoid
40	40.21	282.2559	283.2619	[M + H]⁺	184.1299，91.1036	C₁₈H₃₄O₂	Elaidic acid	Organic acid
41	42.49	406.1264	405.1197	[M-H]⁻	305.1974，144.8826，116.8906	C₂₀H₂₂O₉	Cassia Glycoside	Naphtho-pyrone
42	44.27	636.4237	637.4279	[M + H]⁺	583.4490，311.3654，219.2207	C₃₆H₆₀O₉	Mongholicoside Ⅰ	Steroid
43	44.69	610.1323	611.1398	[M + H]⁺	310.3657，196.1684，121.1543	C₃₀H₂₆O₁₄	Epigallocatechin (4β,8)-gallocatechin	Phenylpropanoid
44	46.90	576.4390	577.4452	[M + H]⁺	491.3563，433.3259，133.1396	C₃₅H₆₀O₆	β-Carotene	Saponin
45	47.06	272.0685	271.0600	[M-H]⁻	182.9681，116.8907	C₁₅H₁₂O₅	Toralactone	Phenylpropanoid
46	47.82	290.0790	289.0718	[M-H]⁻	253.0864，213.0578	C₁₅H₁₄O₆	Epicatechin	Polyphenol
47	47.82	300.0998	299.0911	[M-H]⁻	285.0747	C₁₇H₁₆O₅	Astrapterocarpan	Phenylpropanoid
48	48.40	491.3094	490.3014	[M-H]⁻	327.1815，202.9476，176.9532	C₂₄H₄₅NO₉	Morusimic acid A	Organic acid

A total of 6 prototypical components were identified in this study, and compounds No. 16 and No. 24 were analyzed as examples. The quasi-molecular ion peak of compound No. 16 was 355.1018[M + H]⁺ with a retention time of 17.04 min, and the molecular formula was presumed to be C₁₆H₁₈O₉, and the secondary fragment ions were m/z 297.1790[M-H-C₂H₂O₂]⁺, m/z 159.1724[M-H-C₇H₁₆O₆]⁺, m/z 126.0752[M-H-C₉H₉O₇]⁺, according to the cleavage pattern and related literature, the compound was presumed to be chlorogenic acid.^18–20 The cleavage process is shown in Figure 5A, and the fragment ion map is shown in Figure 6A.

Figure 5.

Cleavage pathway of chlorogenic acid (A) and formononetin (B).

Figure 6.

Fragment ions of chlorogenic acid (A) and formononetin (B).

The quasi-molecular ion peak of compound No. 24 was 267.0654 [M-H]⁻ with a retention time of 24.80 min, and the molecular formula was presumed to be C₁₆H₁₂O₄, and the secondary fragment ions was m/z 148.9598[M-H-C₈H₅O]⁻, based on the cleavage pattern and related literature, it was hypothesized that the compound is formononetin, the main component of Astragali Radix, which has antibacterial, cholesterol-lowering and hypolipidemic effects.^21,22 The cleavage process is shown in Figure 5B, and the fragment ion map is shown in Figure 6B.

Screening of Prototype Component Targets and Disease Targets

Three hundred and eleven prototype component targets were obtained from the SwissTargetPrediction database, 1054 disease targets related to CRF were obtained from the GenCards database, and 102 potential targets of NBDF related to the treatment of CRF were identified by importing the prototype component targets and disease targets into Venny software to take the intersections for making a Venn diagram, as shown in Figure 7A.

Figure 7.

Venn diagram of prototype component targets and disease targets (A). The PPI network diagram (B-C). Bubble diagram of GO enrichment analysis (D). Bubble diagram of KEGG pathway enrichment analysis (E).

PPI Network Construction

The 102 potential targets were imported into Cytoscape 3.8.2 to construct a PPI network, and the 25 core targets with the highest degree values in the network were obtained using the plug-in Mcode in Cytoscape 3.8.2, and by comparing and analyzing these 25 core targets, we found that targets such as ALB, AKT1, and EGFR occupied a central position in the PPI network, as shown in Figure 7(B)-(C), and thus we predicted that NBDF might affect protein expression by regulating targets such as ALB, AKT1, and EGFR.

GO/KEGG Enrichment Analysis

GO enrichment analysis yielded 515 GO entries, sorted by P-value in ascending order, including 379 biological process (BP) entries, 48 cellular component (CC) entries, 88 molecular function (MF) entries, and the top 20 entries were selected for annotation analysis. BP processes such as negative regulation of apoptotic process, positive regulation of cell migration, protein phosphorylation, and signal transduction. CC processes such as extracellular space and extracellular region. MF processes such as enzyme binding and protein kinase/trans-kinase activity.

KEGG enrichment analysis showed that 133 pathways were significant, and annotation analysis was performed for the top 20 pathways, which were associated with cancer, endocrinology, glucose-lipid metabolism, cellular proliferation and differentiation, and inflammatory response, as shown in Figure 7(D) and (E), such as lipid and atherosclerosis, FoxO signaling pathway, Ras signaling pathway, and Prolactin signaling pathway.

Molecular Docking

Based on the analysis results of the PPI network, nine targets with high degree composite values: ALB, ESR1, EGFR, CASP3, AKT1, HSP90AA1, MMP9, PPARG, and SRC, as well as six prototypical constituents: formononetin, emodin, epicatechin, chlorogenic acid, sennoside A, and astragaloside III were selected and molecularly docked by the AutoDockTools 1.5.7 was used for molecular docking to obtain the binding energy between the ligand and the receptor, as shown in Figure 8, the lower the binding energy between the ligand and the receptor indicates the more solid the intermolecular binding, the higher the possibility of interaction, and the binding is better when the binding energy is ≤−5.0 kcal/mol. The docking results of the six groups with the lowest binding energy are shown in Figure 9(A)-(F).

Figure 8.

Heat map of binding energy of composition and target docking.

Figure 9.

Docking results of ESR1 with emodin (A), ESR1 with formononetin (B), HSP90AA1 with formononetin (C), MMP9 with emodin (D), MMP9 with formononetin (E), and PPARG with formononetin (F).

Discussion

The composition of Chinese medicine compounds is complex, and the material basis of its efficacy is difficult to be clarified. Oral Chinese medicine prescriptions exert their medicinal effects by entering into the bloodstream, so we can elucidate the material basis of the medicinal effects of Chinese medicine prescriptions by studying the components that enter into the bloodstream.

In this study, the solution of NBDF and its components before and after blood entry were analyzed using LMS technology. The samples were prepared by mixing equal volumes of rat plasma samples collected at different time points, which was able to maximize the reflection of the components of the blood entry after the administration of the compound formula. Combining the effective information of compound mass number, retention time, molecular ion peaks, fragment ions, etc, a total of 125 chemical components in the compound formula and 48 blood-entry components were identified through the analysis of cleavage pattern and database comparison. Among them, there were 6 blood-entry prototypical components, including formononetin, emodin, epicatechin, chlorogenic acid, sennoside A, and astragaloside III. Formononetin is an isoflavone phytoestrogen of Astragali Radix, which has a variety of biological activities, such as antioxidant, antiinflammatory, antitumor, lowering blood pressure, and improving cardiovascular health^23–28; emodin is a natural compound taken from rhubarb, thuja and other herbs, with antiinflammatory, antiviral, antitumor, analgesic, and other pharmacological effects^29,30; astragaloside has a preventive and curative effect on nephritis, so that the renal pathology is reduced, improve renal function, but also regulate the immune function of the muscles and antiaging³¹; epicatechin has an antioxidant, lipid-lowering, antiinflammatory and antibacterial effects, etc, hypoglycemic, antiinflammatory, and antibacterial effects.^32,33 These components were basically consistent with the pharmacological effects of the NBDF, so it was hypothesized that they might be the pharmacological substances of the NBDF. Through network pharmacology, we obtained the common targets of components and diseases, and constructed protein interaction network maps for these targets to obtain the potential targets of NBDF for the treatment of CRF, and after the network topology analysis, the targets with high degree values included ALB, ESR1, EGFR, CASP3, AKT1, HSP90AA1, MMP9, PPARG, and SRC, indicating that these targets are closely related to the treatment of CRF with NBDF.

AKT1, an isoform of the AKT signaling pathway, is a serine/threonine protein kinase that participates in a variety of biological processes, including cell growth, metabolism, proliferation, and insulin signaling, mediated by phosphorylation, and whose activation is dependent on P13 K signaling. Studies have confirmed that AKT1 is activated during the progression of acute kidney injury (AKI) to CKD, and the absence of AKT1 attenuates renal interstitial fibrosis as well as apoptosis during the progression of AKI to CKD, and AKT1 can be used as a potential therapeutic target to inhibit the transition from AKI to CKD.³⁴ CASP3 disrupts cell structure and acts on regulatory proteins FAK, PAKα, etc, causing them to lose their functions and play an important role in the process of apoptosis. EGFR can be expressed in a variety of renal cells, including endothelial cells, tethered cells, etc. It can be involved in renal fibrosis, renal interstitial as well as glomerular injury, and plays an important role in many acute and chronic kidney injuries. Its mediated overexpression of HIPK2 is thought to be a key regulatory mechanism in the transition from vancomycin-induced AKI to CKD. Increasing evidence suggests that EGFR activation is associated with a variety of physiological and pathophysiological functions and that aberrant EGFR activation is a mediator of progressive kidney injury in diabetic nephropathy.^35–37

MMP9 is a member of the matrix metalloproteinase family that provides homeostasis between synthesis and degradation of extracellular matrix to maintain the structural and functional integrity of the glomerulus. It is widely expressed in renal tissues and is involved in the development of renal inflammatory responses and glomerular fibrosis. Renal tubular epithelial cells and macrophages can lead to the production of MMP9. Studies have confirmed that MMP9 is significantly elevated in urine, serum, and renal tissues of DKD patients.^38–40 It has been shown that MMP9 is a key pathological mediator of renal fibrosis in diabetic rats and that MMP9 deficiency is beneficial in preventing renal fibrosis.⁴¹ The molecular docking results showed that formononetin and MMP9 had the strongest binding ability and the lowest binding energy of −9.93 kcal/mol. Therefore, we speculate that the mechanism of NBDF for the treatment of CRF may be related to the binding of formononetin and MMP9, and through the inhibition of MMP9, thus affecting the synthesis and degradation of renal ECM, and ultimately inhibiting the formation of renal fibrosis.

In summary, using two techniques, this study predicted that the NBDF might act on the targets of ALB, ESR1, EGFR, CASP3, AKT1, HSP90AA1, MMP9, PPARG, and SRC through the key components, such as formononetin, emodin, epicatechin, chlorogenic acid, sennoside A, and astragaloside III, by reducing the inflammatory response and exerting therapeutic effects on CRF by reducing the inflammatory response. In this study, fewer prototypical components of NBDF were detected in the plasma of rats, which may be attributed to the faster metabolism of the components in the blood and the production of other substances. Therefore, the efficacy of NBDF needs to be further explored and explored. In addition, this study only explored the blood-entry components of NBDF and predicted the target and therapeutic mechanism of this formula through network pharmacology, but it was verified through experiments, and we will investigate the efficacy of different drug concentrations on chronic renal failure disease through cellular experiments and animal pharmacodynamic experiments, and investigate the mechanism of the NBDF in the treatment of CRF disease through the application of Western blotting.

Conclusion

In the present study, we analyzed the chemical composition and blood components of NBDF by LC-MS technology, and preliminarily predicted the efficacy of NBDF in the treatment of CRF by combining with the network pharmacology and molecular docking technology, so as to elucidate its efficacy material basis and therapeutic mechanism.

Footnotes

Authors’ Notes

Si-Cheng Yang, Xia Lei, and Si-Fang Xie have contributed equally to this work.

Author Contributions

Si-Cheng Yang, Xia Lei, and Si-Fang Xie: methodology, investigation, and formal analysis; Ju Huang, Feng-Qin Yue, Si-Di Chen, and Wei Peng: project administration and data curation; Si-Cheng Yang and Xia Lei: Writing-original draft; Heng Fan, Sen Li, Xue-Yun Duan, and Wan-Jin Sun: writing-review and editing.

Data Availability

Data will be made available on request.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical Approval

This study was approved by the Experimental Animal Committee of Hubei Provincial Hospital of Traditional Chinese Medicine.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the State Administration of Traditional Chinese Medicine of the People's Republic of China (Grant Nos: [2022] No. 76, [2023] No. 96).

ORCID iD

Si-Cheng Yang

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

Statement of Human and Animal Rights

The animal experimental procedures involved in this study were conducted in accordance with the guidelines for animal experiments of the Hubei Provincial Hospital of Traditional Chinese Medicine Laboratory Animal Center and approved by the Hubei Provincial Hospital of Traditional Chinese Medicine Laboratory Animal Committee.

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Study on the Chemical Composition and Blood-Entry Components of Nourishing Blood Diuretic Formula Based on Liquid Chromatography-Mass Spectrometry and Network Pharmacology Techniques

Abstract

Keywords

Introduction

Materials and Methods

Instruments and Materials

Animals

Chromatographic Conditions

Mass Spectrometry Conditions

Preparation of NBDF

Collection and Preparation of Drug-Containing Plasma

Data Processing

Prediction of Prototype Component Targets and Disease Targets

Construction of the PPI Network

GO/KEGG Enrichment Analysis

Molecular Docking

Results

Chemical Composition of NBDF

Flavonoids

Organic Acid Compounds

Phenylpropanoids

Terpenoids

Polyphenols

Anthraquinones

Blood Entry Components of NBDF

Screening of Prototype Component Targets and Disease Targets

PPI Network Construction

GO/KEGG Enrichment Analysis

Molecular Docking

Discussion

Conclusion

Footnotes

Authors’ Notes

Author Contributions

Data Availability

Declaration of Conflicting Interests

Ethical Approval

Funding

ORCID iD

Statement of Informed Consent

Statement of Human and Animal Rights

References