Network pharmacology-guided and TCM theory-supported in vitro and in vivo component identification of Naoluoxintong

Abstract

Naoluoxintong (NLXT) has been used to treat ischemic stroke (IS) in China for more than two hundred years. However, the pharmacodynamic material basis of NLXT has not been fully studied. Under the guidance of the former network pharmacological analysis, a rapid and reliable method combining UPLC-Q-TOF-MS^E with the novel informatics UNIFI™ platform was established which was used to study the composition of NLXT and its prototype components and metabolites in vivo. A total of 102 compounds were identified. 13 compounds were sourced from “Monarch herb”, mainly involving flavonoids and their glycosides. 54 compounds were sourced from “Minister herb”, mainly involving triterpenoid saponins, organic acids and lactones. 11 compounds were from the “Assistant herb”, mostly containing citric acid and esters of citric acid. 24 compounds were from the “Guide herb”, mostly including flavonoids and their glycosides, organic acids and lactones. Moreover, 24 prototype components and 30 metabolites were detected, and in vivo transformation pathways for different types of chemical components were provided. This is a comprehensive report on the identification of major chemical components in NLXT and metabolic components in rats by UPLC-Q-TOF-MS combined with UNIFI platform under the guidance of network pharmacology, which is helpful for the quality control of NLXT and the study of quality markers.

Graphical abstract

1. Introduction

Traditional Chinese medicine prescriptions (TCMPs) have received increasing attention over the years due to their unique therapeutic and synergistic effects [1]. TCMPs with multi-sources, multi-components and multi-targets characteristics, such as Naoluoxintong (NLXT) [2] and Buyang Huanwu decoction (BYHWD) [3], play an important role in the prevention and control of ischemic stroke (IS), a global challenge [4]. NLXT, a classic herbal prescription of Xin'an medicine, has been used to treat IS for two hundred years [5]. It consists of six herbs and one animal medicine (Table 1): Astragalus mongholicus Bunge, Angelica sinensis (Oliv.) Diels, Carthamus tinctorius L., Panax notoginseng (Burkill) F.H. Chen, Ligusticum chuanxiong Hort. and Gastrodia elata Bl. [6]. Astragali radix acts as the “Monarch herb”, Chuanxiong rhizome and Notoginseng radix et rhizome are regarded as “Minister herb”, Gastrodiae rhizome and Scolopendra play an assistant role, while Carthami Flos and Angelicae sinensis radix are regarded as “Guide herb”. Based on TCM theory, recent studies can show the multifaceted effects of NLXT in the treatment of IS, such as improving the brain blood circulation, anti-apoptosis, pro-angiogenesis and nerve regeneration as well as promoting proliferation and differentiation of neural stem cells (NSCs) [[7], [8], [9], [10]]. It exerts neuroprotection targeting angiogenesis by upregulating the HIF-1α/VEGF signaling pathway [11], and it inhibits the expression of NogoA/RhoA/ROCK pathway to help regenerate axons on middle cerebral artery occlusion (MCAO) rats [6].

Table 1.

A list of the Latin, English, and Chinese pinyin names of herbs.

Number	Latin	English	Chinese pinyin
1	Astragalus mongholicus Bunge	Astragali radix	Huangqi
2	Angelica sinensis (Oliv.) Diels	Angelicae sinensis radix	Danggui
3	Carthamus tinctorius L.	Carthami flos	Honghua
4	Panax notoginseng (Burkill) F.H. Chen	Notoginseng radix et rhizoma	Sanqi
5	Ligusticum chuanxiong Hort.	Chuanxiong rhizoma	Chuanxiong
6	Gastrodia elata Bl.	Gastrodiae rhizoma	Tianma

Apparently, the efficacy of compounding is clear, but the material basis is vague, which includes not only the phytochemicals in TCMPs, but also those absorbed and metabolized in animals [12], seriously hampers the modernization of NLXT. We combined ultra-performance liquid chromatography (UPLC) with a diode array detector (DAD) and a new validation method for establishing fingerprints to perform semi-qualitative analysis of NLXT [13], establishing quality control standards. The network of drugs and disease targets such as CASP3, NOS3, VEGFA, TNF, PTGS2, and TP53 of NLXT for the treatment of IS was constructed by Network analysis and molecular docking techniques [14]. Gene ontology and Kyoto Gene and Genome Encyclopedia pathway analyses were performed for building protein-protein interaction networks. The effective components for the treatment of IS were screened by targeted network pharmacological analysis, including kaempferol, anthocyanin and forskolin. They were all shown in Table 2, and the targeting of NLXT was further validated in Fig. 1 (A, B), which contained the “Component-target-pathway” network and the “neuroinflammatory-pathway-target” sub-network. Under the guidance of network pharmacology, a method combining UPLC and Q-TOF-MS for the determination of chemical composition in UNIFI™ platform was established. UNIFI is an efficient data mining platform from Waters that incorporates its own library of scientific herbal medicines into a streamlined workflow designed to rapidly and qualitatively identify multiple components such as TCMPs and their metabolites in complex samples [[15], [16], [17]].

Table 2.

Molecular docking results of CASP3, NOS3, VEGFA, TNF, PTGS2, TP53 and their reverse screened chemical components.

MOL ID	Ingredient name	Target	Binding energy/kcal·mol−1
MOL000422	kaempferol	PTGS2	−9.4
MOL002714	baicalein	PTGS2	−9.4
MOL000417	Calycosin	PTGS2	−9.4
MOL001792	DFV	PTGS2	−9.2
MOL000392	formononetin	PTGS2	−9.1
MOL002712	6-Hydroxykaempferol	PTGS2	−9.1
MOL000239	Jaranol	PTGS2	−9.1
MOL002721	quercetagetin	PTGS2	−9.1
MOL000006	luteolin	PTGS2	−8.8
MOL000358	beta-sitosterol	PTGS2	−8.6
MOL002757	7,8-dimethyl-1H-pyrimido [5,6-g] quinoxaline-2,4-dione	PTGS2	−8.6
MOL000379	9,10-dimethoxypterocarpan-3-O-β-D-glucoside	PTGS2	−8.5
MOL005344	ginsenoside rh2	PTGS2	−8.3
MOL000442	1,7-Dihydroxy-3,9-dimethoxy pterocarpene	PTGS2	−8.1
MOL000371	3,9-di-O-methylnissolin	PTGS2	−8.1
MOL002140	Perlolyrine	PTGS2	−8.1
MOL000380	(6aR,11aR)-9,10-dimethoxy-6a, 11a-dihydro-6H-benzofurano [3,2-c] chromen-3-ol	PTGS2	−8.0
MOL002135	Myricanone	PTGS2	−8.0
MOL002694	4-[(E)-4-(3,5-dimethoxy-4-oxo-1-cyclohexa-2,5-dienylidene) but-2-enylidene]-2,6-dimethoxycyclohexa-2,5-dien-1-one	PTGS2	−7.4
MOL000098	quercetin	PTGS2	−7.3
MOL000296	hederagenin	PTGS2	−7.0
MOL002157	wallichilide	PTGS2	−6.9
MOL000449	Stigmasterol	PTGS2	−6.6
MOL000378	7-O-methylisomucronulatol	PTGS2	−6.6
MOL002717	qt_carthamone	PTGS2	−6.4
MOL001494	Mandenol	PTGS2	−5.6
MOL000098	quercetin	NOS3	−9.7
MOL000358	beta-sitosterol	CASP3	−9.6
MOL000006	luteolin	CASP3	−9.0
MOL005344	ginsenoside rh2	CASP3	−7.9
MOL002773	beta-carotene	CASP3	−7.6
MOL000422	kaempferol	CASP3	−7.4
MOL000098	quercetin	CASP3	−7.2
MOL000006	luteolin	TP53	−7.1
MOL002714	baicalein	TP53	−7.1
MOL000098	quercetin	TP53	−6.3
MOL000098	quercetin	TNF	−6.7
MOL000006	luteolin	TNF	−6.6
MOL000422	kaempferol	TNF	−6.4
MOL005344	ginsenoside rh2	TNF	−6.3
MOL000006	luteolin	VEGFA	−7.9
MOL002773	beta-carotene	VEGFA	−7.0
MOL000098	quercetin	VEGFA	−6.3

Fig. 1.

(A, B) “Component-target-pathway” network (A) and “neuroinflammatory-pathway-target” sub-network (B) analysis of NLXT.

102 compounds in vitro were detected. 13 compounds were sourced from “Monarch herb”, mainly involving flavonoids and their glycosides. 57 compounds were sourced from “Minister herb”, mainly involving triterpenoid saponins, organic acids and lactones. 54 compounds were sourced from “Minister herb”, mainly involving triterpenoid saponins, organic acids and lactones. 11 compounds were from the “Assistant herb”, mostly containing citric acid and esters of citric acid. 24 compounds were from the “Guide herb”, mostly including flavonoids and their glycosides, organic acids and lactones. And 24 prototype components and 30 metabolites were then identified in vivo. 5 compounds were sourced from “Monarch herb”. 9 compounds were sourced from “Minister herb”. 1 compound was from the “Assistant herb”. 5 compounds were from the “Guide herb”. Using the blood components as a guide, the in vivo pharmacodynamic basis of NLXT for the treatment of IS was initially elucidated. As for pharmacological effects, firstly, modern pharmacological studies have shown that Calycosin can relieve oxidative stress reaction of patients with cerebral hemorrhage through blood-brain barrier, protect neurons and play an antioxidant role in treating IS [18]. Formononetin has antioxidant effect on nerve cells [19]. Astragaloside IV can alleviate oxidative stress, inhibit apoptosis, promote cerebral vascular regeneration, and protect neurons [20]. Secondly, hydroxysafflor yellow A (HSYA) and syringin are flavonoids in Honghua. HSYA has protective effect on mitochondrial damage in brain, and has research value [21]. Next, Sanqi has the effects of dispelling blood stasis, banning bleeding, reducing swelling and relieving pain. The total saponins contained in Sanqi is beneficial to IS, and the effective components are Panax notoginseng saponins and ginsenosides. Ferulic acid is an organic acid chemical component in Huangqi, Chuanxiong and Danggui. Studies showed that ferulic acid can pass the blood-brain barrier, dilate cerebral vessels and protect nerves, and has certain effects on the treatment of IS [22].

Using blood components as a guide, the in vivo pharmacodynamic basis of NLXT for IS was initially elucidated.

2. Material and methods

2.1. Materials, chemicals and reagents

The crude slices of each herb were purchased from Guang Yintang Co., Ltd. (Bozhou, Anhui) and identified by Prof. Yu Nianjun from Anhui university of Chinese medicine, according to the Chinese Pharmacopoeia 2020.

The reference standards of Formononetin, Ferulic acid [110773–201614] and Ginsenoside Re [110704–201223] were purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Calycosin [Y14J8Y39707], Calycosin-7-O-β-D-glucoside (CG) [Y27F9H54731], Formononetin [111703–201504], Syringin [111574–201605], Ginsenoside Rb1 [Z20S9X70603], Ginsenoside Rg1 [G22D9Y77957], Notoginsenoside R1 [G27A10Y86799] and HSYA [R25A9F59977] were purchased from Shanghai Yuanye Biological Technology Co., Ltd. (Shanghai, China). The structures of these chemical standards are provided in Fig. 2, and the purity of all standard compounds were over 98.0%.

Fig. 2.

The total ion chromatograms (TIC) of NLXT.

Acetonitrile and methanol (UPLC-MS grade) were purchased from Oceanpak Corporation (European, Sweden). Formic acid was obtained from Sigma Corporation (USA). Water was purified by a Millipore Milli Q-Plus system (Millipore, Bedford, USA).

2.2. Preparation of sample and reference compound solutions

Firstly, the herbs of NLXT were weight in proportion, soaked for 30 min and boiled for twice for a total of 2 h to make the decoction. Next, added the Scolopendra powder immediately. Then, centrifugal evaporation gave the NLXT (1 mL containing 1.0 g of crude drug) [6]. Finally, the decoction diluted with methanol was filtered through 0.22 μm filter membrane subjected to UPLC-Q-TOF-MS^E analysis.

Ten reference standards were dissolved in appropriate concentration of methanol according to the Chinese Pharmacopoeia 2020. Before UPLC-Q-TOF-MS^E analysis, standard solutions were mixed together and filtered through 0.22 μm filter membrane.

2.3. Animals handling

Sixteen Male Sprague-Dawley rats (280–300 g, 8 weeks old) were obtained from the Experimental Animal Center of Anhui Medical University (Permit Number: SCXK-wan 2017–0007). These rats were then randomly divided into three groups: the blank group and the NLXT group (n = 8). All groups of rats were housed in individual cage systems (Kang Wei IR 60) at (22 ± 1) °C, 60% relative humidity, and a 12 h light/dark cycle, while having free access to food and water. Animals were observed for 1 week of acclimatization prior to the experiments. All animal experiments were performed in compliance with relevant national regulations and local guidelines.

All the rats underwent MCAO surgery, which was carried out according to the improved Longa EZ method (Hong et al., 2021; Longa et al., 1989). After neurological deficit test [23], all the rats were randomly divided into the blank group, the NLXT group and the model-NLXT group.

According to the clinical equivalent dose of NLXT, rats of NLXT group and model-NLXT were administered by gavage at 8.54 g/(kg·d) for 3 days, twice a day. The blank group was administered proper volume of saline according to the weight (1 mL/100 g). Their serum was all taken after the last administration on the third day.

2.4. Biological samples’ collection and pretreatment

2.4.1. Collection of serum samples

After continuing the administration for 3 d, rats were anesthetized intraperitoneally with urethane (15 mg/kg i.p.). Blood was taken from the rat hepatic portal vein 20 min after administration and left for more than half an hour. Then the blood samples were centrifuged at low temperature (4 °C, 3500 r/min, 10 min). Finally, the supernatant was collected, packed separately and stored at −80 °C.

2.4.2. Pretreatment of serum samples

All the rat plasma samples of each group were thawed and mixed, and 300 μL of biological samples were added appropriate volume of acetonitrile solution to precipitate the protein, vortexed for 5 min, centrifuged at 13000 r/min for 10 min, and fixed the volume to get the supernatant. Next, the above supernatant was concentrated and dried under nitrogen in water bath at 37 °C. And the residue was reconstituted by adding 200 μL acetonitrile. Next, repeat the procedure of vortexing and centrifuging to get the supernatant. Finally, an appropriate amount of supernatant was taken for UPLC-Q-TOF-MS injection analysis.

2.5. Chromatography and mass spectrometry conditions

The chromatography separation was performed on Waters Acquity™ UPLC system (Waters Corporation, Milford, USA). The analysis of samples was carried on Waters ACQUITY–CSH–C₁₈ (2.1 mm × 100 mm, 1.7 μm) column with a gradient elution using 0.1% formic acid (solvent A) and Acetonitrile (solvent B). The gradient elution was set as follows: 0–5 min, 10%-15%B; 5–15 min, 15%-20%B; 15–30 min, 20%-30%B; 30–40 min, 30%-40%B; 40–45 min, 40%-60%B; 45–47 min, 60%-10%B. In addition, the flow rate, column temperature and injection volume were set to 0.2 mL/min, 30 °C and 2 μL, respectively.

Waters Xevo G2 Q-TOF mass spectrometer (Waters Corporation, Milford, USA) equipped with an ESI source was used as mass spectrometric detection. And all samples were detected in negative mode. The full scan data were acquired from 50 to 1200 Da. The capillary voltage in negative ion mode was −2.5 kV, the sampling cone voltage in negative ion mode was 50 V, the extraction cone voltage was 4.0 V, the source temperature was 110 °C (ESI-), the cone gas flow rate was 50 L/h, the inert gas (N2) flow rate was 600 L/h, and the inert gas temperature was 350 °C. The collision voltage was set as 6.0 eV for low-energy scan and 20–80 eV for high-energy scan. To ensure the mass accuracy and spectral reproducibility under the MS condition, leucineenk ephalin ([M+H]+(m/z 556.2771) in positive ion mode, [M−H]^- (m/z 554.2615) in negative ion mode), consisting of a 200 pg/mL solution, was set as an external reference (Lock-Spray™) at a flow rate of 10 μL/min via a lock-spray interface.

2.6. Strategy for data processing

2.6.1. Self-built library establishment of NLXT in vitro and in vivo

Firstly, we established a self-built library of NLXT in vitro. Detailed information on chemical components of each herb were established and collected by retrieving on-line databases such as literature database (China Journals of Full-text database (CNKI), PubMed and Medline) and Chemical information database (Pubchem and ChemSpider). Finally, a self-built database of NLXT chemical components was successfully established, including item names, formulations, chemical structures and accurate molecular masses, which contains information on more than 800 components.

Next, in order to maintain the continuity of the research, the chemical components detected in vitro should be used as a basis to determine the prototype components and metabolites of NLXT in vivo. Therefore, a self-built library of chemical components of NLXT in vivo was successfully established, including item name, formula, chemical structure and accurate molecular mass.

2.6.2. Data analysis by UNIFI™ platform and Masslynx 4.1 software

The overall data analysis included the following steps: the first step was in vitro data analysis, which was carried on UNIFI™ platform, including peak finding, peak screening and peak identification of the raw data. Next, the prototype components and in vivo metabolites of NLXT were identified using the UNIFI^TM platform and Masslynx 4.1 software. The chromatographic behavior of the mixture standards in vivo was firstly used to qualitatively detect the cleavage patterns of the major prototype components in NLXT, so as to further determine the cleavage mode of the compounds represented by different types of prototype components. Then, the possible phase I and phase II metabolic pathways were selected in the working method setting, thus qualitatively detecting the metabolites of NLXT in vivo. Finally, the relevant parameters of the data processing method were set as follows: we selected negative adducts including [M − H]^- and [M + COOH]^-. 10 ppm was the upper error limit to identify and match chemical components. The range of mass defect loss was less than 80 mDa. In addition, the binary samples mode was selected as comparative analysis method for samples and blanks.

3. Results and discussion

3.1. Screening and identification of chemical components in NLXT by UPLC-Q-TOF-MS^E and UNIFI™ platform

The total ion chromatograms (TIC) of NLXT in negative ion mode are shown in Fig. 2. The UPLC-Q-TOF-MS^E analysis method established in this study can quickly obtain MS data with high sensitivity in a short time. Due to the complexity of the MS raw data, UNIFI™ platform was used to further process the information contained in the original data.

102 compounds were identified through auto-comparison, standard comparison and manual re-verification process (Table .3, Fig.S1). Among those in vitro components, 13 compounds were derived from “Monarch herb”, mainly including flavonoids and their glycosides. 54 compounds were sourced from “Minister herb”, mainly involving triterpenoid saponins, organic acids and lactones. 11 compounds were from the “Assistant herb”, mostly containing citric acid and esters of citric acid. 24 compounds were from the “Guide herb”, mostly including flavonoids and their glycosides, organic acids and lactones. In addition, some of the ingredients were common components in herbs, such as ferulic acid and chlorogenic acid.

Table 3.

Identification of chemical composition from NLXT by UPLC-Q/TOF-MS.^E.

No	tR (min)	Identification	Experimental mass (m/z)	Error (ppm)	Adducts	Formula	MSE fragment ions (m/z)	Source
1	1.70	Citric acid	191.0192	−2.5	[M − H]-	C6H8O7	133.0137 [M-H–CH2–COO]-	C
2	2.52	4- (β-D-Glucopyran- osyloxy)benzaldehyde	329.0870	−2.5	[M + HCOO]-	C13H16O7	150.0415 [M-H-C6H5O6]-	C
3	3.03	3,5-dimethoxybenzoic acid-4-O-β-D-glucopy ranoside	359.0977	−2.0	[M − H]-	C15H20O10	197.0448[M-H-Glc]- 135.0447[M-H-Glc-2CH3O]-	C
4	3.16	Protocatechuate	153.0187	−4.2	[M − H]-	C7H6O4	135.0447[M-H-H2O]- 119.0500[M-H–H2O–OH]-	D
5	3.48	Parishin B/ Parishin C	773.2123	−3.0	[M + HCOO]-	C32H40O19	459.1134[M-H-Glc-C7H6O]-	C
6	3.51	Parishin E	459.1144	0.0	[M − H]-	C19H24O13	210.0869[M-H-Glc-2COO]- 173.0084[M-H-Glc–C7H6O–H2O]-	C
7	3.93	Dimethyl phthalate	193.0503	−3.9	[M − H]-	C10H10O4	134.0368[M-H–CH3–COO]-	C
8	4.15	3-Hydroxybenzoic acid/ 4-Hydroxybenzoic acid	137.0239	−4.1	[M − H]-	C7H6O3	119.0500 [M-H-H2O]-	C
9	4.84	Chlorogenic acid	353.0877	−0.4	[M − H]-	C16H18O9	191.0555[M-H-C9H6O3]-	B/D
10	5.00	3-Hydroxybenzoic acid/ 4-Hydroxybenzoic acid	137.0239	−3.5	[M − H]-	C7H6O3	93.0345[M-H-COO]-	C
11	5.24	l-Phenylalanine	210.0766	−2.6	[M + HCOO]-	C9H11NO2	119.0504[M-H-HCOO]-	D
12	5.74	Caffeic acid	179.0343	−3.6	[M − H]-	C9H8O4	161.0450[M-H-H2O]-	B/D
13	5.82	Parishin B/ Parishin C	727.2093	0.3	[M − H]-	C32H40O19	/	C
14	6.43	HSYA/iso	611.1609	−1.3	[M − H]-	C27H32O16	449.1080[M-H-Glc]- 287.05535[M-H-2Glc]- 161.0449	D
15	6.58	Scytalone	239.0555	−2.4	[M + HCOO]-	C10H10O4	193.0342[M − H]-	D
16	7.43	Vanillic acid	167.0344	−3.6	[M − H]-	C8H8O4	123.0447[M-H-COO]-	B
17	8.45	HSYA*/iso	611.1608	−1.6	[M − H]-	C27H32O16	449.1084[M-H-Glc]- 287.0555[M-H-2Glc]-	D
18	8.63	Rutin	609.1455	−1.0	[M − H]-	C27H30O16	463.0873[M-H-Rha]- 301.0348[M-H-Rha-Glc]-	D
19	8.98	β-coumaric acid	163.0397	−2.4	[M − H]-	C9H8O3	119.0500[M-H-COO]-	D
20	8.99	Phenylacetaldehyde	119.0499	−2.6	[M − H]-	C8H8O	/	D
21	9.04	Calycosin-7-O-β-D-glucoside*	491.1198	0.5	[M + HCOO]-	C22H22O10	283.0610[M-H-Glc]- 268.0374[M-H-Glc-CH3]- 239.0347[M + HCOO-Glc-C6H2O]- 211.0395[M + HCOO-Glc-C7H2O2]-	A
22	9.69	Parishin A	1041.3129	3.5	[M + HCOO]-	C45H56O25	995.3064[M − H]- 727.2084[M-H-Glc-C7H6O]- 459.1074[M-H-2Glc-2C7H6O]-	C
23	9.89	d(+)-Sucrose/iso	387.1135	−2.3	[M + HCOO]-	C12H22O11	341.1077[M − H]- 179.0563[M-H-Glc]-	B
24	10.25	4-vinyl guaiacol	149.0603	−3.2	[M − H]-	C9H10O2	134.0368[M-H-CH3]-	D
25	10.26	Ferulic acid	193.0450	−1.7	[M − H]-	C10H10O4	134.0368[M-H–COO–CH3]-	B/D
26	10.26	2(4H)-Benzofuranone	133.0291	−3.1	[M − H]-	C8H6O2	/	D
27	11.37	Kaempferol-3-O-rutinoside/iso	593.1510	−0.3	[M − H]-	C27H30O15	447.1118[M-H-Rha]-	D
28	10.32	D (+)-Sucrose/iso	387.1134	−2.6	[M + HCOO]-	C12H22O11	341.1077[M − H]- 179.0556153[M-H-Glc]-	B
29	12.13	Hyperoside/ Isoquercitrin	463.0871	−2.4	[M − H]-	C21H20O12	301.0336[M-H-Glc]-	D
30	13.68	Kaempferol-3-O-rutinoside/iso	593.1514	0.3	[M − H]-	C27H30O15	447.1040[M-H-Rha]-	D
31	14.22	Kaempferol-3-O-rutinoside/iso	593.1516	0.7	[M − H]-	C27H30O15	285.0402[M-H-Rha-Glc]-	D
32	15.22	Hyperoside/ Isoquercitrin	463.0878	−0.9	[M − H]-	C21H20O12	301.0353[M-H-Glc]- 283.0611[M-H-Glc-H2O]-	D
33	15.35	Luteoloside	447.0926	−1.5	[M − H]-	C21H20O11	284.0318[M-H-Glc]- 256.0343[M-H-Glc-CO]-	D
34	15.62	5,7-Dihydrox-4′-methoxyisoflavone	283.0610	−0.9	[M − H]-	C16H12O5	268.0372[M-H-CH3]- 239.0343[M + HCOO–C6H2O]- 211.0393[M + HCOO–C7H2O2]-	A
35	15.96	D (+)-Sucrose/iso	387.1141	−0.9	[M + HCOO]-	C12H22O11	341.1083[M − H]- 179.0554[M-H-Glc]-	B
36	16.04	Notoginsenoside R3/ Notoginsenoside R6/ Notoginsenoside N/ 20-glucoginsenoside Rf/ Floralginsenoside Lb/iso	1007.5446	1.3	[M + HCOO]-	C48H82O19	961.5375[M − H]- 799.4825[M-H-Glc]-	B
37	16.84	Notoginsenoside R3/ Notoginsenoside R6/ Notoginsenoside N/ 20-glucoginsenoside Rf/ Floralginsenoside Lb/iso	1007.5460	2.8	[M + HCOO]-	C48H82O19	961.5390[M − H]- 799.4828[M-H-Glc]-	B
38	17.04	Anhydrosafflor yellow B	1043.2670	2.4	[M − H]-	C48H52O26	1025.2576[M-H-H2O]- 923.2235[M-H-C4H8O4]- 593.1504[M-H-C21H22O11]- 449.1083[M-H-C27H30O15]-	D
39	17.13	Baicalin	445.0767	−2.0	[M − H]-	C21H18O11	269.0450[M-H-C6H8O6]-	D
40	17.70	Ononin*	475.1241	−0.9	[M + HCOO]-	C22H22O9	267.0659[M-H-Glc]- 252.0421[M-H-Glc-CH3]- 223.0378[M + HCOO-Glc-C6H2O]- 195.0446[M + HCOO-Glc-C7H2O2]-	A
41	18.27	Notoginsenoside R3/ Notoginsenoside R6/ Notoginsenoside N/ 20-glucoginsenoside Rf/ Floralginsenoside Lb/iso	1007.5457	2.5	[M + HCOO]-	C48H82O19	961.5390[M − H]- 799.4815[M-H-Glc]- 637.4297[M-H-2Glc]-	B
42	18.48	20 S-Sanchirhinosides A6/ Gypenoside LXIX	1139.5877	−0.4	[M + HCOO]-	C53H90O23	1093.5817[M − H]-	B
43	19.32	Notoginsenoside R1*	977.5357	3.1	[M + HCOO]-	C47H80O18	931.5289[M − H]- 799.4840[M-H-Xyl]- 637.4313[M-H-Xyl-Glc]- 475.3783[M-H-Xyl-2Glc]-	B
44	19.47	Notoginsenoside R3/ Notoginsenoside R6/ Notoginsenoside N/ 20-glucoginsenoside Rf/ Floralginsenoside Lb/iso	1007.5449	1.6	[M + HCOO]-	C48H82O19	961.5379[M − H]-	B
45	19.87	Notoginsenoside R3/ Notoginsenoside R6/ Notoginsenoside N/ 20-glucoginsenoside Rf/ Floralginsenoside Lb/iso	1007.5453	2.1	[M + HCOO]-	C48H82O19	961.5382[M − H]-	B
46	20.47	(6aR,11aR)-3-hydroxy-9,10-dimethoxypterocar- pan-3-O-β-D-Glglucoside	507.1504	−0.8	[M + HCOO]-	C23H26O10	299.0918[M-H-Glc]- 269.0448[M-H-Glc-2CH3]-	A
47	20.56	Notoginsenoside R3/ Notoginsenoside R6/ Notoginsenoside N/ 20-glucoginsenoside Rf/ Floralginsenoside Lb/iso	1007.5448	1.6	[M + HCOO]-	C48H82O19	961.5376[M − H]- 799.4809[M-H-Glc]-	B
48	21.16	Ginsenoside Rg1*	845.4913	−2.9	[M + HCOO]-	C42H72O14	799.4850[M − H]- 637.4315[M-H-Glc]- 475.3788[M-H-2Glc]-	B
49	21.43	Ginsenoside Re*/ Notoginsenoside Re	991.5511	1.5	[M + HCOO]-	C48H82O18	945.5439[M − H]- 783.4856[M-H-Glc]-	B
50	22.40	4,7-dihydroxy-3-butylphthalide	221.0809	−4.8	[M − H]-	C12H14O4	177.0913[M-H-COO]-	C
51	22.61	7,3′-dihydroxy-2′,4′-dimethoxyisoflavan-7-O-β-D-glucoside	509.1657	−1.6	[M + HCOO]-	C23H28O10	463.1601[M − H]- 301.1078[M-H-Glc]- 286.0839[M-H-Glc-CH3]-	A
52	22.62	(3R)-7,2′-Dihydroxy-3′,4′-dimethoxyisoflavan	301.1073	−2.7	[M − H]-	C17H18O5	286.0839[M-H-CH3]-	A
53	22.99	Calycosin/iso*	283.0611	−0.3	[M − H]-	C16H12O5	268.0374[M-H-CH3]- 239.0346[M + HCOO–C6H2O]- 211.0394[M + HCOO–C7H2O2]-	A
54	23.30	dihydroactinidiolide	225.1128	−1.9	[M + HCOO]-	C11H16O2	/	B
55	23.42	Notoginsenoside A/iso	1169.5962	1.0	[M + HCOO]-	C54H92O24	1123.5908[M − H]- 961.5343[M-H-Glc]-	B
56	23.84	Sanchirhinoside B/iso	827.4785	−1.6	[M + HCOO]-	C42H70O13	781.4729[M − H]- 619.4191[M-H-Glc]-	B
57	25.10	Notoginsenoside G	1005.5281	0.6	[M + HCOO]-	C48H80O19	959.5216[M − H]- 797.4643[M-H-Glc]-	B
58	25.62	Senkyunolide D	219.0655	−3.6	[M − H]-	C12H12O4	/	B/D
59	25.87	Notoginsenoside A/iso	1169.5974	0.1	[M + HCOO]-	C54H92O24	1123.5899[M − H]- 961.5350[M-H-Glc]-	B
60	25.92	(6aR,11aR)-10-hydroxy-3,9-dimethoxyribane	299.0922	−0.9	[M − H]-	C17H16O5	269.0451[M-H-2CH3]-	A
61	26.57	20(S)-NotoginsenosideR2/ 20 S-SanchirhinosidesA3/ 20 S-Sanchirhinosides A4/ Ginsenoside F3	815.4781	−1.0	[M + HCOO]-	C41H70O13	769.4708[M − H]- 637.4299[M-H-Rha]-	B
62	30.69	Astragaloside Ⅴ/ Astragaloside Ⅵ/ AstragalosideⅦ	991.5132	1.2	[M + HCOO]-	C47H78O19	945.5070[M − H]- 783.4807[M-H-Glc]-	A
63	31.13	Senkyunolide F	205.0864	−3.0	[M − H]-	C12H14O3	/	B/D
64	31.68	Ginsenoside Rf/ Ginsenoside Rg1*	845.4894	−0.9	[M + HCOO]-	C42H72O14	799.4832[M − H]- 637.4281[M-H-Glc]-	B
65	31.83	Notoginsenoside H	993.5269	−0.7	[M + HCOO]-	C47H80O19	947.5207[M − H]- 785.3529[M-H-Glc]-	B
66	32.37	Kinobeon A	355.1179	−2.4	[M − H]-	C20H20O6	296.1042[M-H-OCH3]-	D
67	32.50	Astragaloside Ⅳ	815.4805	−1.9	[M + HCOO]-	C41H68O14	769.4747[M − H]- 637.4314[M-H-Xyl]- 475.3789[M-H-Xyl-Glc]-	A
68	33.26	Notoginsenoside I	1137.6073	0.9	[M + HCOO]-	C54H92O22	1091.6018[M − H]- 929.5434[M-H-Rha]-	B
69	33.72	Formononetin*	267.0658	−1.4	[M − H]-	C16H12O4	252.0421[M-H-CH3]- 223.0388[M + HCOO–C6H2O]- 195.0446[M + HCOO–C7H2O2]-	A
70	33.74	20(S)-NotoginsenosideR2/ 20 S-sanchirhinosides A3/ 20 S-sanchirhinosides A4/ Ginsenoside F3	815.4797	−0.2	[M + HCOO]-	C41H70O13	769.4737[M − H]- 637.4307[M-H-Rha]- 475.3782[M-H-Rha-Glc]-	B
71	33.95	Ginsenoside Rh1/ 20(S)-ginsenoside F1/iso	683.4375	−0.5	[M + HCOO]-	C36H62O9	637.4307[M − H]- 475.3782[M-H-Glc]-	B
72	34.01	Gypenoside Y	829.4588	−2.1	[M + HCOO]-	C42H72O13	783.4527[M − H]-	B
73	34.33	Ginsenoside Rb1*	1153.6052	0.0	[M + HCOO]-	C54H92O23	1107.5984[M − H]- 945.5434[M-H-Glc]- 783.4887[M-H-2Glc]- 621.4347[M-H-3Glc]-	B
74	34.47	Ginsenoside Rg2	829.4936	−2.3	[M + HCOO]-	C42H72O13	637.4290[M-H-Rha]- 475.3771[M-H-Rha-Glc]-	B
75	34.82	Astragaloside Ⅴ/ Astragaloside Ⅵ/ AstragalosideⅦ	991.5130	1.0	[M + HCOO]-	C47H78O19	945.5063[M − H]-	A
76	34.93	Ginsenoside Rh1/ 20(S)-ginsenoside F1/iso	683.4370	−0.9	[M + HCOO]-	C36H62O9	637.4298[M − H]- 475.3776[M-H-Glc]-	B
77	35.45	Malonyl-ginsenoside Rb1/iso	1193.5991	2.5	[M − H]-	C57H94O26	1149.6088[M-H-CO2]- 1107.5966[M-H–CO2–C2H2O]- 945.5362[M-H–CO2–C2H2O-Glc]-	B
78	35.81	Malonyl-ginsenoside Rb1/iso	1193.5992	2.6	[M − H]-	C57H94O26	1149.6089[M-H-CO2]-	B
79	36.04	Malonyl-ginsenoside Rb1/iso	1193.5985	2.0	[M − H]-	C57H94O26	1149.6078[M-H-CO2]- 1107.5957[M-H–CO2–C2H2O]- 945.5268[M-H–CO2–C2H2O-Glc]-	B
80	36.46	Ginsenoside Rb2/ Ginsenoside Rb3/ Ginsenoside Rc/ Vina-Ginsenoside R7/ Notoginsenoside L	1123.5931	1.6	[M + HCOO]-	C53H90O22	1077.5865[M − H]-	B
81	37.22	Ginsenoside Rh1/ 20(S)-ginsenoside F1/iso	683.4356	−2.9	[M + HCOO]-	C36H62O9	637.4300[M − H]- 475.3777[M-H-Glc]-	B
82	37.40	Quinquenoside L1	989.5321	−0.6	[M + HCOO]-	C48H80O18	943.5250[M − H]-	B
83	38.42	5-hydroxy-3-butylidenephthalide	203.0707	−3.1	[M − H]-	C12H12O3	160.0158[M-H-C3H7]-	B
84	38.46	20 S-Sanchirhinosides A2	857.4874	−3.6	[M + HCOO]-	C43H72O14	811.4817[M − H]- 769.4689[M-H-C2H2O]-	B
85	38.59	Ginsenoside Rd/ Gypenoside XVII	991.5518	3.5	[M + HCOO]-	C48H82O18	945.5450[M − H]- 783.4877[M-H-Glc]- 621.4354[M-H-2Glc]-	B
86	39.58	Malonyl-ginsenoside Rd/iso	1031.5443	1.0	[M − H]-	C51H84O21	945.5395[M-H–CO2–C2H2O]- 927.5248[M-H–CO2–C2H2O–H2O]-	B
87	39.99	Cyclocephaloside II	871.4709	1.5	[M + HCOO]-	C43H70O15	825.46212[M − H]-	A
88	40.13	Malonyl-ginsenoside Rd/iso	1031.5435	0.2	[M − H]-	C51H84O21	987.5529[M-H-CO2]-	B
89	40.25	Ginsenoside Rd/ Gypenoside XVII	991.5498	2.8	[M + HCOO]-	C48H82O18	945.5429[M − H]-	B
90	40.96	Notoginsenoside Fe/ Notoginsenoside Fd/ Notoginsenoside Ft1/ Gypenoside IX/ 20 S-sanchirhinosides A5/ Ginsenosides Rd2	961.5365	−1.3	[M + HCOO]-	C47H80O17	915.5285[M − H]-	B
91	42.19	Sanchirhinoside B/iso	827.4770	−3.4	[M + HCOO]-	C42H70O13	781.4711[M − H]- 739.4225[M-H-C2H2O]-	B
92	42.62	Notoginsenoside T5/iso	797.4679	−1.7	[M + HCOO]-	C41H68O12	751.4621[M − H]- 619.41984[M-H-Xyl]-	B
93	42.98	Ginsenoside Rg6/ Ginsenoside F4/ Ginsenoside Rk1/ Ginsenoside Rg5	811.4806	−5.3	[M + HCOO]-	C42H70O12	765.4760[M − H]-	B
94	43.11	Notoginsenoside T5/iso	797.4677	−2.0	[M + HCOO]-	C41H68O12	751.4619[M − H]- 619.4195[M-H-Xyl]- 161.0448	B
95	43.40	Ginsenoside Rg6/ Ginsenoside F4/ Ginsenoside Rk1/ Ginsenoside Rg5	811.3741	−3.4	[M + HCOO]-	C42H70O12	765.4767[M − H]-	B
96	43.54	Ginsenoside Rk3/ Ginsenoside Rh4	665.4261	−4.4	[M + HCOO]-	C36H60O8	619.4199[M − H]-	B
97	43.76	Ginsenoside F2	829.4931	−2.9	[M + HCOO]-	C42H72O13	783.4846[M − H]- 621.4338[M-H-Glc]-	B
98	43.98	Ginsenoside Rk3/ Ginsenoside Rh4	665.4261	−2.1	[M + HCOO]-	C36H60O8	619.418[M − H]-	B
99	44.16	AstragalosideⅠ/IsoastragalosideⅠ	913.4820	2.4	[M + HCOO]-	C45H72O16	867.4731[M − H]- 825.4616[M-H-C2H2O]- 807.4470[M-H–C2H2O–H2O]- 765.4405[M-H-2C2H2O–H2O]-	B
100	44.83	AstragalosideⅠ/ IsoastragalosideⅠ	913.4808	1.9	[M + HCOO]-	C45H72O16	867.4738 M − H]-	A
101	45.14	20 S-ginsenoside Rg3/ 20R-ginsenoside Rg3	829.4956	0.2	[M + HCOO]-	C42H72O13	783.4895[M − H]- 621.4348[M-H-Glc]-	B
102	45.75	AstragalosideⅠ/IsoastragalosideⅠ	913.4825	0.6	[M + HCOO]-	C45H72O16	867.4736[M − H]- 825. [M-H-C2H2O]- 807. [M-H–C2H2O–H2O]- 765.4489[M-H-2C2H2O–H2O]-	A

Glc: glucose; Rha: rhamnose; Xyl: xylose; A: Monarch herb; B: Minister herb; C: Assistant herb; D: Guide herb.

Next, the cleavage rules of flavonoids and their glycosides, triterpenoid saponins, organic acids were initially explored by representative chemical constituents, so as to further detect the potential chemical constituents in NLXT. The mass fragmentation patterns of the above-mentioned types of components are presented in categories.

3.1.1. Flavonoids

Flavonoids are the main compounds of NLXT. In this study, a total of 18 flavonoid chemical constituents were identified, and they were from Astragali radix of “Monarch herb” and Carthami Flos of “Guide herb”. And 5 flavonoids or aglycones and 13 flavonoid glycosides were identified among them. It is well known that the main cleavage modes of flavonoids or aglycones are neutral loss of CO₂, CO, CH₃ and the Retro Diels-Alder (RDA) pathway of C-ring. And the main cleavage rules of flavonoid glycosides are similar to flavonoids, the first reaction is the deglycosylation, such as glucosyl (Glc, 162 Da) and rhamidosyl (Rha, 146 Da), followed by neutral loss and RDA cleavage [24]. Among them, peaks 14, 21, 40, 53, 69 were identified as HSYA, CG, Ononin, Calycosin and formononetin by comparing their retention time and mass fragmentation patterns of reference standards. This section used peaks 14, 21 as an example to illustrate the cleavage of flavonoids. HSYA belongs to chalcone glycosides, and the main cleavage way is deglycosylation. The deprotonated precursor ion at m/z 611.1608 at low energy collisions, the product ions of m/z 449.1084 and 287.0555 were generated at high energy collision, which were obtained after removing one glucosyl group and two glucosyl groups from HSYA (Fig. 3). CG appears isoflavone glycoside compound, and it is also one of the representative compounds of Astragali radix from “Monarch herb”. CG displayed precursor ion m/z 491.1198 [M + HCOO]^-, fragment ions m/z 283.0610 [M-H-Glc]^- and 268.0374 [M-H-Glc-CH₃]^- were produced by successive neutral loss of glucosyl (Calycosin) and methyl (formononetin), and fragment ions m/z 239.0347 and 211.0395 were produced by RDA cleavage at the O, 4 and O, 3 positions of the C ring of Calycosin (Fig. 4). In vivo studies showed that HSYA treatment significantly reduced infarct size, neurological scores and cerebrovascular permeability in MCAO rats [25], while CG treatment significantly inhibited the expression and activity of MMPs and ensured the expression of cav-1 and tight junction protein in micro vessels isolated from the ischemic rat cortex [26].

Fig. 3.

MS/MS spectrum and possible fragmentation pathway of HSYA.

Fig. 4.

MS/MS spectrum and possible fragmentation pathway of Calycosin-7-O-β-D-glucoside.

As is shown in Fig. 5, peak 38 displayed a deprotonated precursor ion at m/z 1043.2670, and produced fragment ion at m/z 1025.2576 [M-H-H₂O]^-, 923.2235 [M-H-C₈H₈O]^-, 593.1504 [M-H-C₂₁H₂₂O₁₁]^-, 449.1083 [M-H-C₂₇H₃₀O₁₅]^- from its secondary ion mass spectrum, and it was consistent with the study of reference [27]. Therefore, peak 38 was identified as anhydrosafflor yellow B of Carthami Flos from “Assistant herb”. Peak 40 produced an adduct ion at m/z 475.1241 [M + HCOO]^-, which was 16 Da less than Calycosin-7-O-β-D-glucoside. In addition, according to secondary mass spectrometry, fragment ion at m/z 267.0659 [M-H-Glc]^-, 252.0421 [M-H-Glc-CH₃]^-, 223.0378 [M + HCOO-Glc-C₆H₂O]^- and 195.0446 [M + HCOO-Glc-C₇H₂O₂]^- indicated that the B ring of peak 40 had one less hydroxyl substituent than that of Calycosin-7-O-β-D-glucoside Fig. 6. Similarly, peaks 18, 29, and 30 were identified as rutin, isoquercitrin and hyperoside. Hyperoside and isoquercitrin were isomers of each other and need further identification. In addition, in improving cerebral ischemia-reperfusion injury, rutin treatment significantly increased the activities of glutathione peroxidase (GPx), glutathione reductase (GR), catalase (CAT) and superoxide dismutase (SOD) and increased glutathione (GSH) levels in the ischemic brain [28], while down-regulating MMP-9 expression and activity [29]. Thus, rutin has both anti-oxidative stress and anti-inflammatory properties.

Fig. 5.

From its secondary ion mass spectrum, peak 38 was identified as anhydrosafflor yellow B because it displayed a deprotonated precursor ion at m/z 1043.2670, and produced fragment ion at m/z 1025.2576 [M-H-H₂O]^-, 923.2235 [M-H-C₈H₈O]^-, 593.1504 [M-H-C₂₁H₂₂O₁₁]^-, 449.1083 [M-H-C₂₇H₃₀O₁₅]^-.

Fig. 6.

From its secondary ion mass spectrum, B ring of peak 40 had one less hydroxyl substituent than that of Calycosin-7-O-β-D-glucoside.

3.1.2. Triterpenoid saponins

Being one of the main compound species of NLXT, a total of 53 triterpenoid saponins were identified. These triterpenoid saponins were also the major bioactive components of Notoginseng radix et rhizome from “Minister herb” and Astragali radix from “Monarch herb”. It is well known that saponins isomers are common because of its complex and diverse substitution relationships. References indicated that the precursor ions of the triterpenoid saponins exist mainly as [M + HCOO]^-. And the main cleavage mode was continuous loss of C-3, C-6, C-20 substituted sugar groups, such as glucosyl (Glc, 162 Da), xylose (Xyl, 132 Da) and rhamnosyl (Rha, 146 Da), etc [30,31]. Among them, peaks 43, 73, 48 and 49 were identified as Notoginsenoside R1, Ginsenoside Rb1, Ginsenoside Rg1 and Ginsenoside Re by comparing retention time and mass fragmentation patterns of reference standards. In this part, the Notoginsnoside R1 and Ginsenoside Rg1 were used as examples to illustrate the cleavage pattern of triterpenoid saponins, and the potential saponin chemical components can be detected through this mass fragmentation pattern. Notoginsenoside R1 displayed parent ion at low collision energy at m/z 977.5357 [M + HCOO]^-, and the major fragment ions in the secondary mass spectrum at m/z 931.5289 [M − H]^-, 799.4840 [M-H-Xyl]^-, 637.4313[M-H-Xyl-Glc]^- and 475.3783 [M-H-Xyl-2Glc]^- through the deglycosylation reaction (Fig. 7). Take Ginsenoside Rg1 as an example. First, it showed that m/z 845.4913 [M + HCOO]^- as the precursor ion, and it is known from the glycosyl substitution that the product ions mainly consisted of 799.4850 [M − H]^-, 637.4315 [M-H-Glc]^-, 475.3788 [M-H-2Glc]^- (Fig. 8). Studies showed that Notoginsenoside R1 improves the permeability of the blood-brain barrier after acute IS [32]. Ginsenoside Rb1 improves facilitation of motor recovery and axonal regeneration in mice after IS [33]. Ginsenoside Rg1 protects against ischemic/reperfusion-induced neuronal injury [34].

Fig. 7.

MS/MS spectrum and possible fragmentation pathway of Notoginsenoside R1.

Fig. 8.

Ginsenoside Rg1 showed that m/z 845.4913 [M + HCOO]^- as the precursor ion and its product ions mainly consisted of 799.4850 [M − H]^-, 637.4315 [M-H-Glc]^-, 475.3788 [M-H-2Glc]^-.

Compound 75 showed m/z 829.4936 [M + HCOO]^- as its precursor ion, followed by the major fragment ions in the secondary mass spectrum at m/z 637.4290 [M-H-Rha]^- and 475.3771 [M-H-Rha-Glc]^-. The molecular weight comparison of database, structural comparison and literature review [31] revealed that the precursor ion at m/z 829.4936 [M + HCOO]^- had multiple isomers. However, according to the type and position of the glycosyl substitution, peak 75 was identified as Ginsenoside Rg2, and the cleavage mode was shown in Fig. 9. In addition, Astragaloside refers to the main chemical constituent Astragali radix from “Monarch herb”. Peak 101 indicated parent ion at m/z 913.4808 [M + HCOO]^-, and its main fragment ions included 867.4473 [M − H]^-, 825.4616 [M-H-C₂H₂O]^-, 807.4470 [M-H–C₂H₂O–H₂O]^- and 765.4405 [M-H-2C₂H₂O–H₂O]^-. There was an isomeric situation, so the peak 101 could only be initially identified as Astragaloside I or Isoastragaloside I. 53 potential triterpenoid saponin components were identified through similar Mass fragmentation patterns.

Fig. 9.

Peak 75 was identified as Ginsenoside Rg2, and the cleavage mode was shown.

3.1.3. Organic acids

Organic acids are also the main compounds of NLXT, and the main cleavage rule was neutral loss of small molecules [35]. A total of seven organic acids derived from Chuanxiong, Angelica and safflower were identified in this study. Peak 25 was determined as ferulic acid by comparing the retention time and MS data of standard compound. Therefore, our group used ferulic acid as an example to illustrate the fragmentation patterns of organic acids. As shown in Fig. 10, ferulic acid showed that its deprotonated ion was used as precursor ions at m/z 193.0450, and the fragment ions were mainly continuous neutral loss, such as fragment ions at m/z 134.0368 [M-H–COO–CH₃]^-. The precursor ions of peaks 8 and 10 were both displayed at m/z 137.0239 [M − H]^-. In the second-order mass spectrometry, the fragment ion of peak 8 is m/z 119.0500 [M-H-H₂O]^-, and the fragment ion of peak 10 was m/z 93.0345 [M-H-COO]^-. Combined with self-built database compounds and structural screening, it was preliminarily identified that peaks 8 and 10 are isomers, namely the compounds 3-Hydroxybenzoic acid and 4-Hydroxybenzoic acid (Fig. 11). Additionally, other detailed information involving other elements was shown in Table 3 and Fig.S1.

Fig. 10.

MS/MS spectrum and possible fragmentation pathway of Ferulic acid.

Fig. 11.

The precursor ions of peaks 8 and 10 were both displayed at m/z 137.0239 [M − H]^-. In the second-order mass spectrometry, the fragment ion of peak 8 is m/z 119.0500 [M-H-H₂O]^-, and the fragment ion of peak 10 was m/z 93.0345 [M-H-COO]^-.

3.1.4. Exploring the contribution of “monarch, minister, assistant, guide” compatibility of NLXT

From the discussion and analysis above, we were able to find that more than 70% compounds from “Monarch herb” and “Minister herb” among the 102 compounds detected in vitro. And the TIC of “Monarch, Minister, Assistant, Guide” herbs were shown in Fig. 12 (A-D). As we all know, pharmacological study classified TCMFs into “Monarch, Minister, Assistant, Guide” in line with the role of the Chinese medicines [36]. Therefore, an advanced science and technology UPLC-Q-TOF-MS was employed to briefly explain the rationality of the theory of TCM compatibility. TCM has the characteristics of multi-component, multi-target and multi-mechanism, so the importance of “Assistant herb” and “Guide herb” cannot be ignored. It also explained that the significance of compatibility of “Monarch, Minister, Assistant, Guide”.

Fig. 12.

(A–D). The total ion chromatograms (TIC) of “Monarch, Minister, Assistant, Guide” among the 102 compounds detected in vitro in NLXT (A: Monarch herb; B: Minister herb; C: Assistant herb; D: Guide herb).

3.2. Mass fragmentation patterns for seven major prototype components in vivo

In this study, the chromatographic behavior and mass spectrometric fragmentation of 7 representative components of NLXT were tested in vivo. The TIC of 7 major components in vivo were displayed in Fig. 13, and the cleavage modes of major compounds can be found in Fig. 14. It can be concluded that the main cleavages of free flavonoids prototype in vivo are the neutral loss of CO₂, CO, CH₃ and the RDA pathway of C-ring. And the main cleavage rules of flavonoid glycosides in vivo are similar to flavonoids, the first reaction is the deglycosylation, such as glucosyl (Glc, 162 Da) and rhamidosyl (Rha, 146 Da), followed by neutral loss and RDA cleavage [24]. The main cleavage mode of triterpenoid saponin prototype in vivo is continuous loss of C-3, C-6, C-20 substituted sugar groups, such as glucosyl (Glc, 162 Da), xylose (Xyl, 132 Da) and rhamnosyl (Rha, 146 Da), etc [30]. The cleavage of the chemical components of the organic acid prototype in vivo is mainly the loss of neutral molecules, especially the carboxyl groups loss [35]. Therefore, the results in this section not only provided the cleavage of different types of prototype components in vivo, but also a more realistic picture of the existence of 7 prototype components of NLXT in vivo.

Fig. 13.

The TIC of 7 major components in vivo.

Fig. 14.

The cleavage modes of major compounds of NLXT in vivo. (A: Ononin and Formononetin; B: Ginsenoside Rg1; C: Ferulic acid; D: HSYA).

3.3. Analysis of prototype components of NLXT in vivo

Based on the summary of the cleavage rules of different types of prototype compounds, verification of references, self-built library and MS^E data, a total of 24 prototype compounds were identified in vivo, mainly including flavonoids and their glycosides, triterpenoid saponins, organic acids, lactones and citrate esters. The TIC of prototypes of NLXT in vivo was displayed in Fig. 15. In addition, the prototype fractions and their associated MS data can be found in Table .4.

Fig. 15.

The total ion chromatogram (TIC) of prototypes of NLXT in vivo.

Table 4.

Identification of prototype components from NLXT in rats by UPLC-Q-TOF-MS.^E.

No	tR (min)	Identification	Experimental mass (m/z)	Error (ppm)	Adducts	Formula	MSE fragment ions (m/z)	Source
1	5.52	3-Hydroxybenzoic acid/ 4-Hydroxybenzoic acid	137.0236	−6.1	[M − H]-	C7H6O3	/	B
2	6.33	Parishin B/Parishin C	727.2093	0.3	[M − H]-	C32H40O19	/	C
3	7.86	HSYAa/iso	611.1608	0.3	[M − H]-	C27H32O16	504.1103[M-H- C7H5O]-	D
4	9.47	3-Hydroxybenzoic acid/ 4-Hydroxybenzoic acid	137.0236	−5.7	[M − H]-	C7H6O3	93.0341 [M-H-COO]-	D
5	10.12	Phenylacetaldehyde	119.0493	−7.8	[M − H]-	C8H8O	/
6	10.12	β-coumaric acid	163.0391	−5.7	[M − H]-	C9H8O3	119.0493[M-H-COO]-
7	11.35	Ferulic acid	193.0494	−6.6	[M − H]-	C10H10O4	134.0368[M-H–COO–CH3]-	D
8	12.32	5,7-Dihydrox-4′-methoxyisoflavone	329.0680	3.9	[M + HCOO]-	C16H12O5	/	A
9	14.27	Ferulic acid iso	193.0495	−6.0	[M − H]-	C10H10O4	178.0261 M-H- CH3]- 149.0598[M-H-COO]- 134.0368[M-H–COO–CH3]-	D
10	21.36	Ginsenoside Rg1a	845.4943	4.6	[M + HCOO]-	C42H72O14	799.4857[M − H]- 637.4313[M-H-Glc]-	B
11	21.71	Ginsenoside Re/ Notoginsenoside Re	991.5538	5.5	[M + HCOO]-	C48H82O18	945.5453[M − H]-	B
12	21.88	Dihydroactinidiolide	225.1117	−6.8	[M + HCOO]-	C11H16O2	/
13	23.36	(6aR,11aR)-3-hydroxy-9,10-dimethoxypterocar- pan-3-O-β-D-Glglucoside	507.1492	−3.1	[M + HCOO]-	C23H26O10	417.1175[M + HCOO–3CH3]- 273.0388 164.0463
14	24.41	Calycosina	283.0593	−6.4	[M − H]	C16H12O5	268.0356[M-H-CH3]-	A
15	29.66	4,7-dihydroxy-3-butylphthalide	221.0803	−7.6	[M − H]-	C12H14O4	177.0894[M-H-COO]-	B
16	32.05	Kinobeon A	355.1208	5.7	[M − H]-	C20H20O6	205.0851[M-H- C8H6O3]-	D
17	32.57	Senkyunolide F	205.0859	−5.6	[M − H]-	C12H14O3	161.0959[M-H-COO]-	B
18	34.54	Ginsenoside Rb1	1107.6018	5.6	[M + HCOO]-	C54H92O23	945.5424[M-H-Glc]- 783.4897[M-H-2Glc]- 621.4331[M-H-3Glc]-	B
19	34.56	Astragaloside Ⅴ/ Astragaloside Ⅵ/ AstragalosideⅦ	991.5530	4.7	[M + HCOO]-	C47H78O19	945.5424[M − H]- 783.4877[M-H-Glc]-	A
20	35.22	Formononetina	267.0655	−3.0	[M − H]-	C16H12O4	252.0411[M-H-CH3]- 223.0378[M + HCOO–C6H2O]- 195.0428[M + HCOO–C7H2O2]-	A
21	36.46	Ginsenoside Rb2/Ginsenoside Rb3/Ginsenoside Rc/ Vina-Ginsenoside R7/ Notoginsenoside L	1123.5929	2.1	[M + HCOO]-	C53H90O22	1077.5865[M − H]- 829.4590	B
22	37.16	Ginsenoside Rb2/ Ginsenoside Rb3/ Ginsenoside Rc/ Vina-Ginsenoside R7/ Notoginsenoside L	1123.5944	3.4	[M + HCOO]-	C53H90O22	1077.5865[M − H]-	B
23	38.37	Ginsenoside Rd/ Gypenoside XVII	991.5556	7.3	[M + HCOO]-	C48H82O18	945.5485[M − H]-	B
24	39.75	Cyclocephaloside II	871.4709	1.4	[M + HCOO]-	C43H70O15	530.3220 494.3231[M–HCOO–H-C15H23O8]-	A

^aIdentifcations confrmed with standard compound;/: Not detected.

3.4. Analysis of metabolites of NLXT in vivo

Research of this part was divided into three main steps. Firstly, the high-responders of different prototype components in vivo were set as the parent structures, and the possible metabolites were screened after using the UNIFI™ platform and Masslynx 4.1 software to process the main phase I and phase II metabolic pathways of parent structure. Then, examples are given to summarize the metabolic patterns carried out for different prototype components. Finally, more possible metabolites were screened according to the metabolic rules of different prototype components. Therefore, a total of 30 metabolites were identified in vivo, and the TIC of metabolisms of NLXT in vivo is displayed in Fig. 16. In addition, metabolites of NLXT in vivo and their associated MS data can be found in Table .5.

Fig. 16.

The total ion chromatogram (TIC) of metabolisms of NLXT in vivo.

Table 5.

Identification of metabolites from NLXT in rats by UPLC-Q-TOF-MS.^E.

No	tR (min)	Identification	Experimental mass (m/z)	Error (ppm)	Adducts	Formula	MSE fragment ions (m/z)
M1	3.75	Vanillic acid + CH2	181.0492	−8.1	[M − H]-	C9H10O4	163.0378[M-H-H2O]- 119.0498[M-H–H2O–COO]-
M2	5.99	3-Hydroxybenzoic acid-O + H2+ SO3	203.0020	0.0	[M − H]-	C7H8O5S	123.0442[M-H-SO3]-
M3	9.03	Ferulic acid + H2	195.0650	−6.8	[M − H]-	C10H12O4	107.0496 [M-H-H2-C3H2O3]- 93.0345 [M-H- H2–C4H4O3]-
M4	9.44	Betaine + SO3	216.9801	−5.3	[M − H]-	C7H6O6S	137.0238[M-H-SO3]- 93.0345[M-H–SO3–COO]-
M5	10.06	Vanillic acid + SO3	246.9910	−3.2	[M − H]-	C8H8O7S	167.0337[M-H-SO3]- 152.0103[M-H–SO3–CH3]- 123.0442[M-H–SO3–COO]- 108.0211[M-H–SO3–COO-CH3]-
M6	12.31	Ferulic acid-O + SO3	303.0155	−8.2	[M + HCOO]-	C10H10O6S	/
M7	14.68	β-coumaric acid + H2+SO3	245.0106	−7.9	[M − H]-	C9H10O6S	149.0598[M-H–SO3–O]-
M8	14.73	Ferulic acid + SO3	273.0075	0.2	[M − H]-	C10H10O7S	229.0160 [M-H-COO]- 178.0261[M-H–SO3–CH3]- 149.0598 [M-H–SO3–COO]- 134.0365[M-H- SO3–COO–CH3]-
M9	15.74	Ferulic acid+2O	271.0483	2.4	[M + HCOO]-	C10H10O6	178.0255 [M-H-2O–CH3]- 149.0590 [M-H-2O–COO]- 134.0361 [M-H-2O–COO–CH3]-
M10	15.91	7,3′-dihydroxy-2′,4′-dimethoxyisoflavan-7-O-β-D-glucoside + C6H8O6	639.1925	−0.8	[M − H]-	C29H36O16	463.1602[M-H-C6H8O6]- 301.1061[M-H- C6H8O6-Glc]-
M11	16.67	Calycosin + C6H8O6	459.0925	−1.7	[M − H]-	C22H20O11	283.0597[M-H-C6H8O6]- 268.0361[M-H–SO3–CH3]-
M12	17.30	Malonyl-ginsenosideRb1 -C12H20O11	899.5034	2.7	[M + HCOO]-	C45H74O15	605.2741[M-H–CO2–C2H2O-Glc]-
M13	19.49	4,7-dihydroxy-3-butylphthalide-O + C6H8O6	381.1179	−3.2	[M − H]-	C18H22O9	205.0857[M-H-O-C6H8O6]- 161.0951
M14	19.49	Senkyunolide F + C6H8O6	381.1179	−3.2	[M − H]-	C18H22O9	205.0857[M-H-C6H8O6]- 161.0951[M-H–C6H8O6–COO]-
M15	22.58	Parishin E-C6H10O5+SO3	377.0170	−3.6	[M − H]-	C13H14O11S	244.0284[M-H-SO3-3OH]- 116.0492[M-H–SO3–C7H4O2–COO–OH]-
M16	23.36	dihydroactinidiolide + C6H8O6	355.1378	−5.8	[M − H]-	C17H24O8	164.0822[M-H–C6H8O6–CH3]-
M17	23.84	4,7-dihydroxy-3-butylphthalide + O + CH2	251.0908	−6.9	[M − H]-	C13H16O5	192.0774[M-H-CH2-COO]- 177.0903[M-H-O-CH2-COO]- 163.0386[M-H-O-2CH2–COO]-
M18	23.96	(6aR,11aR)-10-hydroxy-3,9-dimethoxyribane + SO3	475.1239	−1.4	[M − H]-	C17H16O8S	299.0903[M-H-SO3]- 284.0674[M-H–SO3–CH3]- 269.0432[M-H–SO3–2CH3]-
M19	24.54	(3R)-7,2′-Dihydroxy-3′,4′-dimethoxyisoflavan + C6H8O6	477.1392	−2.3	[M − H]-	C23H26O11	301.1064[M-H-C6H8O6]- 286.0828[M-H–C6H8O6–CH3]- 269.0436[M-H–C6H8O6–CH3–OH]-
M20	25.89	Notoginsenoside I-Rha	975.5089	−8.2	[M + HCOO]-	C48H80O20	929.5034[M − H]-
M21	26.82	Formononetin-CH2	253.0491	−6.4	[M − H]-	C15H10O4	119.0492
M22	26.77	Formononetin-CH2+SO3	333.0059	−4.6	[M − H]-	C16H12O7S	253.0491[M-H–SO3–CH2]- 199.0057 119.0492
M23	28.06	Caffeic acid + SO3	259.9905	−5.0	[M − H]-	C9H8O7S	242.9947[M-H-OH]- 179.0326[M-H-SO3]- 135.0433[M-H–SO3–COO]-
M24	28.22	Calycosin + SO3	363.0166	−3.9	[M − H]-	C16H12O8S	283.0597[M-H-SO3]- 268.0359[M-H–SO3–CH3]-
M25	29.33	Senkyunolide D + C6H8O6	395.0965	−4.8	[M − H]-	C18H20O10	219.0634[M-H-C6H8O6]-
M26	29.89	(6aR,11aR)-3-hydroxy-9,10-dimethoxypterocar-pan-3-O-β-D-Glglucoside-CH2+SO3	527.0850	−2.7	[M − H]-	C22H24O13S	447.1302[M-H-SO3]- 351.0529 113.0238
M27	32.50	4,7-dihydroxy-3-butylphthalide + SO3	301.0377	−3.3	[M − H]-	C12H14O7S	221.0805[M-H-SO3]- 177.0909[M-H–SO3–COO]- 161.0959
M28	34.58	5-hydroxy-3-butylidenephthalide + SO3	283.0267	−5.3	[M − H]-	C12H12O6S	203.0702[M-H-SO3]-
M29	39.79	Formononetin + SO3	347.0214	−4.8	[M − H]-	C16H12O7S	267.0646[M-H-SO3]- 252.0411[M-H–SO3–CH3]- 195.0430[M + HCOO– SO3–C7H2O2]-
M30	45.73	20(S)-NotoginsenosideR2- C11H18O9	521.3831	−3.1	[M + HCOO]-	C30H52O4	/

3.4.1. Flavonoid-related metabolites

Flavonoid-related metabolites are the major metabolites of Astragali radix from “Monarch herb” and Carthami Flos in “Guide herb”. Based on accurate mass and expected biotransformation of phase I and phase II metabolism with neutral loss, a total of 9 flavonoid-related metabolites were identified in vivo. M11, M21, M22, M24, M29 were set as examples to explain the metabolism pathways of flavonoids in vivo. M11 showed the precursor ion of [M − H]^- at m/z 459.0925 at low energy collision. After the high-energy collision, the fragment ions of m/z 283.0597 and m/z 268.0361 were generated in the MS spectra. Among them, m/z 283.0597 differed from the precursor ion by 176 Da, and m/z 268.0361 lost a molecule of methyl on the basis of m/z 283.0597. Therefore, M11 could be initially identified as a glucuronidation metabolite of Calycosin. As above, M24 and M29 show the precursor ion at m/z 363.0166 and m/z 347.0214 at low energy collision. After high energy collision, the fragment ions at m/z 283.0597 and m/z 268.0359 was generated. Among them, the fragment ion at m/z 283.0597 differed from the precursor ion by 80 Da. Therefore, M24 is a sulfated metabolite of Calycosin. M29 showed fragment ions at m/z 267.0646 and 252.0411 at high energy. Among them, m/z 267.0646 differed from its precursor ion by 80 Da. Therefore, M29 is the sulfated metabolite of formononetin. From this we can further speculate that M21 and M22 are formononetin demethylated metabolites and its demethylated sulfated metabolites. The specific cleavage pathway is shown in Fig. 17 (A). Therefore, glucuronidation and sulfation in the phase II metabolism are the main metabolic pathways of flavonoid-related metabolites, and demethylation reaction in the phase I metabolism is its metabolic pathway, which is similar to previous studies [12,37].

Fig. 17.

The proposed metabolic pathways for flavonoid-related metabolites (A); triterpenoid saponins-related metabolites (B); organic acids-related metabolites (C); lactone-related metabolites (D) and citrate esters-related metabolites (E) of NLXT in vivo.

3.4.2. Triterpenoid saponins-related metabolites

Triterpenoid saponins-related metabolites are the major metabolites of Notoginseng radix et rhizome from “Minister herb” and Astragali radix from “Monarch herb”. Based on accurate mass and expected neutral loss of phase I and phase II metabolism by biotransformation, a total of 3 flavonoid-related metabolites were identified in vivo. Deglycosylation reaction in phase I metabolism is the main reaction [38]. M12 was set as example to explain the metabolism pathways of triterpenoid saponins in vivo. M12 showed the precursor ion at m/z 899.5034 at low energy, which is 340 Da (−178 Da–162 Da) smaller than the molecular weight of precursor ion of Malonyl-ginsenoside Rb, that is, Malonyl-ginsenoside Rb consecutively loses two glucose groups. After high-energy collisions, the fragment ion at m/z 605.2741 is generated in the secondary mass spectrum, which is 248 Da away from its precursor ion. It loses a malonyl and glucose group, generating the fragment ion at m/z 605.2741. Therefore, M12 could be initially identified as a deglycosylated metabolite of Malonyl-ginsenoside Rb and the specific cleavage pathway is shown in Fig. 17 (B). It can be inferred that M20 and M30 are deglycosylated metabolites of Notoginsenoside I and 20 (S)-Notoginsenoside R2 respectively. In the experiment, notoginsenoside-related metabolites were few and single. On the one hand, due to comprehensively consider the responsiveness and sensitivity of different types of compounds, the possible experimental method has a low sensitivity to saponin-related metabolites. On the other hand, it may be related to the way biological samples are collected and different affinity of the organs. Several studies indicate that deglycosylation is the main role of metabolism in rat blood, and that other metabolic pathways may exist in rat organs or other body fluids [[39], [40], [41]].

3.4.3. Organic acids-related metabolites

Organic acids-related metabolites are the major metabolites derived from Chuanxiong, Angelica and safflower. Based on accurate mass and expected neutral loss of biotransformation phase I and phase II metabolism, a total of 10 organic acids-related metabolites were identified in vivo. M1, M3, M5, M6, M8, M23 were set as examples to explain the metabolism pathways of organic acids in vivo. In the low-energy mass spectrum, M5, M8, and M23 show precursor ions at m/z 246.9910, 273.0075, and 259.9905, which were 80 Da smaller than those of ferulic acid, vanillic acid, and caffeic acid. In addition, fragment ions at m/z 167.0337 and m/z 179.0326 were obtained in the MS spectra of M5 and M23 at high energy, which are 80 Da different from the precursor ions of M5 and M23. Fragment ions at m/z 178.0261 and m/z 149.0598 were shown in the MS spectrum of M8, that is, one molecule of methyl group and one molecule of carboxyl group continued to be lost with a difference of 80 Da. That is, one molecule of methyl and one molecule of carboxyl were also lost based on the loss of 80 Da. Therefore, M5, M8, and M23 can be identified as sulfated metabolites of ferulic acid, vanillic acid and caffeic acid. M6 showed a precursor ion at m/z 195.0650, which was 2 Da different from the precursor ion of ferulic acid parent. The structure of ferulic acid contains unsaturated double bonds, so M3 can be initially identified as a reduced metabolite of ferulic acid. M1 showed a precursor ion at m/z 181.0492, which differed from precursor ion of vanillic acid by 14 Da. Vanillic acid structure contains hydroxyl groups, so M1 can be initially identified as a methylation metabolite of vanillic acid, and the specific cleavage pathway is shown in Fig. 17 (C). In short, for organic acid-related metabolites, reduction, dehydroxylation and demethylation reactions in the phase I metabolism, and methylation and the sulfation reactions in the phase II metabolism were the main metabolic pathways, which is similar to previous research [42]. What's more, the role of ferulic acid included anti-thrombosis [43]. Pretreatment with vanillic acid significantly restored the spatial memory, decreased the levels of IL-6, TNF-α and TUNEL positive cells and also increased the IL-10 levels in the IS model rats [44]. Caffeic acid exerts neuroprotective effects on whole brain ischemia-reperfusion injury in rats through inhibition of 5-LO [45].

3.4.4. Others-related metabolites

Other-related metabolites mainly include lactone-related metabolites and citrate esters-related metabolites. Lactone-related metabolites are the major metabolites in “Minister herb” and “Guide herb”. Citrate esters-related metabolites are the major metabolites in “Adjuvant herb”. Based on accurate mass and expected neutral loss of biotransformation phase I and phase II metabolism, a total of 7 lactone-related metabolites and 1 citrate esters-related metabolite were identified in vivo. M14 and M15 were set as examples to explain the metabolism pathways of lactone and citrate esters in vivo. The precursor ion at m/z 381.1179 of M15 was generated in the low-energy mass spectrum, which was 176 Da differed from the precursor ion of the Senkyunolide F, and a fragment ion at m/z 205.0857 was also shown in the secondary mass spectrum at high energy. Therefore, M14 can be initially identified as the glucuronidation metabolite of Senkyunolide F, and the specific metabolic pathway was shown in Fig. 17 (D). The precursor ion at m/z 377.0170 of M14 was obtained at low energy, 82 Da differed from the precursor ion of the Parishin E, which was sulfated after losing a molecule of glucose residue. Therefore, M14 can be initially identified as the deglycosylated and sulfated metabolite of Parishin E. The specific cleavage rule was shown in Fig. 17 (E). In summary, for lactone-related metabolites, glucuronidation in the phase II metabolism was the main metabolic pathway, which was similar to previous studies [46]. For the citrate esters-related metabolite, deglycosylation in the phase I metabolism and sulfation in the phase II metabolism were the main metabolic pathways [47]. Investigation suggested that Senkyunolide F was reported in the potent anticoagulative fraction of Angelicae Sinensis Radix, which suggested that they might also be anticoagulant active constituents in Angelicae Sinensis Radix [48].

3.5. Sample acquisition and LC-MS conditions

The collection method, time, and processing methods of biological sample were investigated in animal pre-treatment respectively. Firstly, blood collected from the hepatic portal vein contains not only the most primitive components that are absorbed, but also those contained in systemic circulating blood, providing a conducive way to analyze the migration components. Therefore, blood collection method of hepatic portal vein was the best choice. Next, blood was acquired at 5 min, 20 min, 50 min, 90 min after the last administration. By comparing the number and area of chromatographic peaks at different time points, 20 min after administration was selected as the optimal blood collection time, as shown in Fig. 18. Finally, biological samples at the same time point were treated with methanol precipitation, acetonitrile precipitation, and ethyl acetate extraction. By comparing the number and area of chromatographic peaks in different processing methods, the acetonitrile protein precipitation method was finally selected to process biological samples, as shown in Fig. 19.

Fig. 18.

The number and area of chromatographic peaks at different blood sampling time points.

Fig. 19.

The number and area of chromatographic peaks treated with different organic solvent extraction methods.

To better compare NLXT compounds in vivo and in vitro, LC-MS method used for the identification in vivo should be consistent with its in vitro counterpart. Based on our previous research, LC-MS condition for the identification of chemical components and metabolites in NLXT were further optimized. Therefore, we selected 0.1% formic acid water (A)-acetonitrile (B) as the mobile phase for the gradient separation of the chemical components in NLXT. As an organic phase, acetonitrile has excellent elution ability and retention behavior for different components. Additionally, add an appropriate proportion of acids can improve peak shape and avoid tailing. The results indicated many components in NLXT performed better in the negative ion mode than in the positive ion mode.

As for in vivo pharmacological effects, firstly, 5,7-Dihydrox-4′-methoxyisoflavone (Biochanin A) can alleviate cerebral I/R-induced damage partly via suppressing apoptosis, ER stress, and p38 MAPK signaling [49], thereby serving as a potent neuroprotective agent. Post-stroke Calycosin therapy increased the cerebral expression of BDNF/TrkB, ameliorated the neurological injury and switched the microglia from the activated amoeboid state to the resting ramified state in IS rats [50]. Formononetin has antioxidant effect on nerve cells [19]. Cyclocephaloside II [51] and Astragaloside Ⅴ [52] could help quality control. Astragaloside VI could effectively activate EGFR/MAPK signaling cascades, promote NSCs proliferation and neurogenesis, and improve the repair of neurological functions in post-ischemic stroke rats [53], reflecting its monarch role in NLXT. Secondly, Senkyunolide F [54] 4,7-dihydroxy-3-butylphthalide [55], 3-Hydroxybenzoic acid and 4-Hydroxybenzoic acid, which is helpful to the quality control of Chuanxiong. Thirdly, as quality markers of Tianma, Parishin B and C were detected in vivo, further confirmed its assistant role in the compound. Fourthly, HSYA is flavonoid in Honghua, which has protective effect on mitochondrial damage [21]. Kinobeon A, another component detected in this experiment that is absorbed from Honghua, has antioxidant stress effects and may be a useful cytoprotective reagent [56]. Next, 6 components of Sanqi were detected in vivo. Gypenoside XVII prevents atherosclerosis by attenuating endothelial apoptosis and oxidative stress [57]. Ginsenoside Rg1 [58] has protective effects on cerebral injury induced by ischeamia/reperfusion, which related to the increased in the expression of BDNF in the hippocampal CA1 region, the down-regulation of the expression of IL-1β, IL-6 and TNF-α. Ginsenoside Re suppressed the production of pro-inflammatory mediators by blocking CAMK/ERK/JNK/NF-κB signaling in microglial cells and protected hippocampal cells and could have therapeutic potential for various neurodegenerative diseases [59]. Ginsenoside Rb2 alleviated MI/R injury in rats by inhibiting oxidative stress and inflammatory response through SIRT1 activation [60]. Ginsenoside Rb3 attenuates oxidative stress via activating the antioxidation signaling pathway of PERK/Nrf2/HMOX1 [61]. Finally, Ferulic acid, is an organic acid chemical component in Huangqi, Chuanxiong and Danggui, detected both in vivo and in vitro.

As is well known, pairing two drugs together tend to show stronger medicinal effects than single drugs. Astragali Radix and Angelicae Sinensis Radix pairs, for example, have the effect of tonifying Qi and promoting blood, but there is a difference in the components. When the two were combined, the representative components of Astragalus and Angelicae were both reduced. A comparison of the high performance liquid chromatography (HPLC) fingerprints of the Astragalus-Angelicae pair revealed that the content of calycosin and fermononetin in Astragalus and the content of ferulic acid and Z-ligustilide in Angelicae were also reduced after the combination [62]. Z-ligustilide, which wasn't found in this paper, a volatile oil constituent of Angelicae, may have been lost due to due to the decoction process of NLXT. The difference in findings between drug pairs and formulations was mainly due to the content rather than the type of ingredient. Therefore, it is necessary to quantify these components. This will provide a theoretical basis for the study of the quality markers of NLXT, while understanding the effect of variations on the efficacies.

In this study, the high intensity, sensitivity and distribution of components in NLXT were investigated in both positive and negative ion modes. Based on the elucidation of the in vitro chemical composition set and the constructed local chemical composition database, relevant metabolite information will be added to the local chemical composition database of the in vivo drug composition to complete the database information. Based on this information, it is possible to elucidate the in vivo chemome for the drug-derived components in blood. Biological validation of the predicted key proteins had been performed to elucidate the in vivo pharmacological basis of TCM and their molecular mechanisms. To some extent, NLXT appears to have anti-thrombotic, anti-oxidative stress and anti-inflammatory properties, reduce infarct size, neurological scores and cerebrovascular permeability and exert neuroprotective effects against ischemia-reperfusion injury, which would largely restore IS.

4. Conclusion

This is a comprehensive report on the identification of components in NLXT both in vivo and in vitro by UPLC-Q-TOF-MS combined with UNIFI platform under the guidance of network pharmacology, which is helpful for the quality control of NLXT and the study of quality markers. 102 compounds were detected herein, including flavonoids, saponins, organic acids, lactones, citrate esters and others. 24 prototype components and 30 metabolites were identified in vivo. The metabolic reactions mainly involved reduction, hydroxylation, demethylation and deglycosylation in phase I metabolism and methylation, sulfation and glucuronidation in phase II metabolism. Guided by network pharmacology and pharmacodynamics, we have refined the NLXT in vivo and in vitro component analysis to provide new ideas for compounding studies.

Limitation: While the metabolic patterns of the components in vivo had been identified, pharmacokinetic parameters such as bioavailability as well as in vivo retention time, which are directly related to efficacy, have not yet been investigated. This deficiency will be investigated in subsequent experiments to achieve a basis for refining the efficacy of NLXT. Also, further tests will be carried out on the animal drug Scolopendra as no relevant components were detected here.

Author contribution statement

Ling He, Weidong Chen: Conceived and designed the experiments.

Lu Hong, Yutong Zhao: Performed the experiments; Wrote the article.

Xiaoqian Shi: Performed the experiments.

Huihui Jiang, Mingming Liu, Hanzhi Zhang: Analyzed and interpreted the data.

Huan Wu, Lei Wang, Guodong Zhao: Contributed reagents, materials, analysis tools or data.

Data availability statement

Data included in article/supp. material/referenced in article.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper

Abbreviations

NLXT

Naoluoxintong

IS

ischemic stroke

TCMPs

Traditional Chinese medicine prescriptions

BYHWD

Buyang Huanwu decoction

NSCs

neural stem cells

MCAO

middle cerebral artery occlusion

UPLC

ultra-performance liquid chromatography

HPLC

high performance liquid chromatography

DAD

diode array detector

Glc

glucosyl

Xyl

xylose

Rha

rhamnosyl

HSYA

hydroxysafflor yellow A

CG

Calycosin-7-O-β-D-glucoside

TIC

total ion chromatograms

RDA

Retro Diels-Alder

GPx

glutathione peroxidase

GR

glutathione reductase

CAT

catalase

SOD

superoxide dismutase

GSH

increased glutathione

Appendix A. Supplementary data

The following is the Supplementary data to this article: