Characterization of the Components and Metabolites of Achyranthes Bidentata in the Plasma and Brain Tissue of Rats Based on Ultrahigh Performance Liquid Chromatography–High-Resolution Mass Spectrometry (UHPLC–HR-MS)

Background: Achyranthes bidentata (AR) is a traditional Chinese herb used for the treatment of hypertension and cerebral ischemia, but its pharmacological effects are not known. Aim of study: We aimed to detect and accurately identify the components and metabolites of AR in the plasma and brain tissue of Sprague Dawley rats. Methods: We employed ultrahigh performance liquid chromatography–high-resolution mass spectrometry (UHPLC–HR-MS) to detect AR components in the plasma and brain tissue of rats. The absorption and metabolites in the plasma and brain tissue of normal control rats and rats that underwent middle cerebral artery occlusion (MCAO) were characterized and compared. Results: A total of 281 compounds, including alkaloids, flavonoids, terpenoids, phenylpropanes, sugars and glycosides, steroids, triterpenes, amino acids, and peptides, was identified in samples of Achyranthes bidentata (TCM-AR). Four types of absorbable prototype components and 48 kinds of metabolites were identified in rats in the normal control plasma group which were given AR (AR plasma group), and five kinds of metabolites were identified in rats of the normal control brain tissue group which were given AR (AR brain group). Three absorbed prototype components and 13 metabolites were identified in the plasma of rats which underwent MCAO and were given AR (MCAO + AR plasma group). Six absorbed prototype components and two metabolites were identified in the brain tissue of rats who underwent MCAO and were administered AR (MCAO + AR brain group). These results showed that, after the oral administration of AR, the number of identified components in plasma was more than that in brain tissue. The number of prototype components in the AR plasma group was higher than that in the MCAO + AR plasma group, which may indicate that metabolite absorption in rats undergoing MCAO was worse. The number of prototype components in the MCAO + AR brain group was higher than that in the AR brain group, indicating that the blood–brain barrier was destroyed after MCAO, resulting in more compounds entering brain tissue. Conclusions: UHPLC–HR-MS was used to rapidly analyze the components and metabolites of AR in the blood and brain of rats under normal and pathologic conditions, and to comprehensively characterize the components of TCM-AR. We also analyzed and compared the absorbable components and metabolites of normal rats under cerebral ischemia-reperfusion injury to explore the potential mechanism of action. This method could be applied to various Chinese herbs and disease models, which could promote TCM modernization.


Introduction
Stroke is the second leading cause of death and the third leading cause of disability worldwide, affecting one in four people [1].Stroke includes ischemic diseases and hemorrhagic cerebrovascular diseases, which are associated with high morbidity, disability, and mortality [2].Among them, ischemic stroke accounts for about 75-85% of the total number of patients with stroke [3].The incidence of ischemic stroke is increasing year-by-year, and up to 60-80% of patients who have suffered a stroke will die [4].Even if patients survive, they have serious sequelae, which seriously affects their quality of life [5].
In ischemic stroke, the cerebral blood supply is insufficient due to the stenosis/occlusion of the arteries which serve it, resulting in the necrosis of brain tissue.If ischemic brain tissue receives a blood supply again, ischemic injury will be aggravated, and even more serious consequences (e.g., neuron death, fatal brain edema) may result.This phenomenon is called "cerebral ischemia-reperfusion injury" (CIRI) [6].The cause of CIRI appears to be the sudden restoration of a blood supply of cerebral vessels in the ischemic state, which leads to a series of pathologic changes in the brain.
Achyranthes bidentata (AR) is the dried root of plants from the Amaranthaceae family.In traditional Chinese medicine (TCM) theory, AR has the role of tonifying the liver and kidney, promoting blood circulation, and removing blood stasis [7].It is often used in TCM formulations to treat hypertension and stroke [8,9].Ecdysterone and triterpenoids are the main components of AR species [10,11].Various active ingredients in AR have been shown to have antioxidant, anti-inflammatory, and neuroprotective effects [12].
A TCM formulation is a complex mixture of many ingredients [13].These components (and their metabolites) can interact in complex ways with various proteins in the body [14].Most ingredients of TCM formulations must reach specific concentrations after being metabolized in the blood after oral administration to be efficacious [15].Ultrahigh-performance liquid chromatography (UHPLC) combined with high-resolution mass spectrometry (HR-MS) is used to characterize the metabolites of biological samples based on accurate mass numbers and MS/MS(Mass Spectrometry) data [16,17].The pathologic state of stroke can further affect the absorption and distribution of drugs.
Herein, we identified the prototype and metabolites of AR absorption in plasma and brain tissue of Sprague Dawley (SD) rats under normal and pathologic conditions.Based on our comprehensive characterization and analyses, this study could aid further research on the targets and mechanism of action of AR for CIRI treatment.

Model Verification
The MCAO method was used to model rats.Two hours after ischemia, 2,3,5-triphenyltetrazolium chloride (TTC) staining was carried out on the brain tissues of three rats in a blank group, three rats in a model group, and three rats in the drug administration group (Figure 1).After TTC staining, the brain tissue of rats in the blank control group was red.Many white infarcts appeared on the right side of the brain tissue of rats immediately after modeling.Hence, modeling was successful.

Component Identification and Analyses of TCM-AR
The AR was processed to obtain the samples of Achyranthes bidentata tested on the machine, namely TCM-AR.UPLC-MS/MS was used to detect the components of TCM-AR.MS data were processed and analyzed according to the screening platform of Progenesis QI v3.0, combined with the LuMet-CM database and HERB database.Based on multidimensional matching (retention time, precise mass number, secondary fragments, and isotope distribution), the compounds and structures of TCM-AR were identified accurately and characterized using a literature review [10,18,19].

Component Identification and Analyses of TCM-AR
The AR was processed to obtain the samples of Achyranthes bidentata tested on the machine, namely TCM-AR.UPLC-MS/MS was used to detect the components of TCM-AR.MS data were processed and analyzed according to the screening platform of Progenesis QI v3.0, combined with the LuMet-CM database and HERB database.Based on multidimensional matching (retention time, precise mass number, secondary fragments, and isotope distribution), the compounds and structures of TCM-AR were identified accurately and characterized using a literature review [10,18,19].
A total of 281 compounds (142 compounds in positive ion mode and 139 compounds in negative ion mode) from Achyranthes bidentata for machine testing (TCM-AR) was identified.These were as follows: 43 sugars and glycosides; 40 amino acids and peptides; 31 alkaloids; 31 terpenoids; 30 phenylpropanes; 21 flavonoids; 21 fatty acylates; 12 organic acids and their derivatives; 11 steroids; 5 quinones; 5 phenols; 8 carboxylic acids and their derivatives; 3 nucleotides and their derivatives; 3 pyridines and their derivatives; and 17 other compounds (Table 1, Supplementary Table S1).Table 1 shows the compound adduction ion form, retention time, theoretical plastic-nucleus ratio, measured plastic-nucleus ratio, molecular weight deviation, characteristic fragment ion, molecular formula, name, peak area ratio, and InChIKey (International Chemical Identifier Key).The Extracted Ion Chromatogram (EIC)s and MS/MS spectra of identified compounds in comparison with databases are shown in Supplementary Figure S1. Figure 2 shows the UHPLC-MS/MS Base Peak Chromatogram (BPC) and Total Ion Chromatogram (TIC) of TCM-AR in positive and negative ion modes.A total of 281 compounds (142 compounds in positive ion mode and 139 compounds in negative ion mode) from Achyranthes bidentata for machine testing (TCM-AR) was identified.These were as follows: 43 sugars and glycosides; 40 amino acids and peptides; 31 alkaloids; 31 terpenoids; 30 phenylpropanes; 21 flavonoids; 21 fatty acylates; 12 organic acids and their derivatives; 11 steroids; 5 quinones; 5 phenols; 8 carboxylic acids and their derivatives; 3 nucleotides and their derivatives; 3 pyridines and their derivatives; and 17 other compounds (Table 1, Supplementary Table S1).Table 1 shows the compound adduction ion form, retention time, theoretical plastic-nucleus ratio, measured plasticnucleus ratio, molecular weight deviation, characteristic fragment ion, molecular formula, name, peak area ratio, and InChIKey (International Chemical Identifier Key).The Extracted Ion Chromatogram (EIC)s and MS/MS spectra of identified compounds in comparison with databases are shown in Supplementary Figure S1. Figure 2 shows the UHPLC-MS/MS Base Peak Chromatogram (BPC) and Total Ion Chromatogram (TIC) of TCM-AR in positive and negative ion modes.
Taking compound number 4 as an example, at a retention time of 9.33 min, the peak of the excimer ion was at m/z 304.1438 [M+H] + , the molecular formula was C 19 H 17 N 30 , and the main fragment ions were at m/z values of 134.0599, 171.0913, and 304.1442, according to the literature [20].The molecular weight and structure of the secondary fragments identified as evodiarine are shown in Figure 5e.[19].The compound was identified as achyranthoside E, and its EIC, molecular weight of secondary fragments, and annotated structure are shown in Figure 5g.
Taking compound number 34 as an example, at a retention time of 8.97 min, the excimer ion peak was m/z 343.1169 [M+H, M+Na] + in positive ion mode, the molecular formula was C 19 H 18 O 6 , and the main ion fragments were at m/z values of 157.0128, 313.0701, and 343.1171.The compound was inferred to be scutellarein tetramethyl ether after database comparison.The EIC, molecular weight of secondary fragments, and structure of scutellarein tetramethyl ether are shown in Figure 5k.

Analyses of AR Components Passing into Plasma and Brain after Administration
Rat blood samples were collected at different periods after the oral administration of AR for 3 days.After static centrifugation, we mixed the plasma samples collected at all time points to obtain a dose of plasma for analysis.To characterize the absorbable components of AR in rat plasma and brain tissue, we first established a UHPLC-HR-MS method to screen these components.The control plasma group and TCM-AR group were used as negative and positive controls, respectively.
The extracted ion peak appeared in the plasma group containing AR (AR plasma group), in the plasma group for rats that underwent MCAO + AR (MCAO + AR plasma group), and in the TCM-AR group, but did not appear in the control plasma group.This was identified as the prototype component of absorption.Extracted ion peaks were detected in the AR plasma group, but not in the control plasma group or in TCM-AR, and were determined to be metabolites.Based on BPC (Figures 6a,b and 7a,b), total ion chromatograms (TICs) are shown in Supplementary Figure S2.Compared with TCM-AR, 52 absorption components (four prototype components and 48 metabolites) were identified in the AR plasma group.Compared with TCM-AR, we identified 16 absorption components in the MCAO + AR plasma group (three prototype components and 13 metabolites).Compared with TCM-AR, the 52 absorptive components in the AR plasma group were 36 more than the 16 absorptive components in the MCAO + AR plasma group.We speculated that in CIRI, the metabolism of rats was disturbed, which led to a reduction in drug-absorption efficiency and a decrease in the number of absorbed components.
The brain tissues of rats were collected 1.5 h after the final administration of AR 3 days after oral administration.The extracted ion peak appeared in the brain tissue group given AR (AR brain group), the brain tissue group given AR after MCAO (MCAO+AR brain group), and the TCM-AR group, but did not appear in the control brain tissue group, which was identified as the prototype component.Extracted ion peaks were detected in the AR brain group and the MCAO + AR brain group, but not in the control brain tissue group or in the TCM-AR group, and were determined to be metabolites.Based on BPC (Figures 6c,d and 7c,d) and TICs (Supplementary Figure S2), compared with TCM-AR, five absorption components were identified in the AR brain group, five of which were metabolites.Compared with TCM-AR, we identified eight absorption components in the MCAO+AR brain group (six prototype components and two metabolites).Compared with TCM-AR, the eight absorbable components in the MCAO+AR brain group were three more than the five absorbable components in the brain tissue of the AR brain group.The main function of the blood-brain barrier (BBB) is to maintain the homeostasis of the brain, protect the brain from potential endogenous/exogenous injuries, and inhibit pathogens and toxic compounds from entering the brain [22,23].If the brain is damaged, the BBB breaks down and more compounds enter the brain tissue.Therefore, it is speculated that, after the blood-brain barrier is damaged, more AR absorption components enter the brain tissue, which is consistent with the above-mentioned MCAO+AR group, which absorbed more components than the AR group.

Analyses of the Prototype Components of AR into Plasma and Brain
According to the EICs and MS/MS spectra of reference compounds, compared with TCM-AR, four prototypes were identified in the AR plasma group and three prototypes were identified in the MCAO + AR plasma group as absorbable components.Compared with TCM-AR, no prototype components were identified in the AR brain group, whereas six prototypes were identified in the MCAO + AR brain group as absorbable components, and their absorbable components are shown in Table 2 (serial numbers 1-4 are the prototype absorbable components identified in the AR plasma group, 5-7 are the prototype absorbable components identified in the MCAO + AR plasma group, and 8-13 are the prototype absorbable components identified in the MCAO + AR brain group).The EICs of prototype component compounds and MS/MS spectra of reference compounds are shown in Supplementary Figure S3.Table 2 shows the form, retention time, theoretical mass-charge ratio, measured mass-charge ratio, molecular weight deviation, characteristic fragment ion, molecular formula, name, peak area ratio, and InChIKey.The identified components had an error of <5 ppm, in which very little of the prototype component was absorbed and most of the metabolites were metabolized.
metabolites.Compared with TCM-AR, we identified eight absorption components in the MCAO+AR brain group (six prototype components and two metabolites).Compared with TCM-AR, the eight absorbable components in the MCAO+AR brain group were three more than the five absorbable components in the brain tissue of the AR brain group.The main function of the blood-brain barrier (BBB) is to maintain the homeostasis of the brain, protect the brain from potential endogenous/exogenous injuries, and inhibit pathogens and toxic compounds from entering the brain [22,23].If the brain is damaged, the BBB breaks down and more compounds enter the brain tissue.Therefore, it is speculated that, after the blood-brain barrier is damaged, more AR absorption components enter the brain tissue, which is consistent with the above-mentioned MCAO+AR group, which absorbed more components than the AR group.

Analyses of the Metabolites of AR in the Plasma and Brain
According to the EICs and MS/MS spectra of reference compounds, 48 metabolites were identified in the AR plasma group and 13 metabolites were identified in the MCAO + AR plasma group.Five metabolites were identified in the AR brain group, and two metabolites were identified in the MCAO + AR brain group (Table 3; serial numbers 1-48 are from metabolites identified in the AR plasma group, 49-61 are from metabolites identified in the MCAO + AR plasma group, 62-66 are from metabolites identified in the AR brain group, and 67-68 are from metabolites identified in the MCAO + AR brain group).
The specific EICs of these compounds and MS/MS spectra of their reference compounds are shown in Supplementary Figure S4.Table 3 shows the adduct ion form, retention time, measured mass-charge ratio, molecular weight deviation, parent compound, molecular formula, name, and type of metabolite.General prototype products can be excreted directly by metabolites generated in the phase I metabolic reaction, or they can be excreted again through the phase II metabolic reaction [24,25].The identified components had an error of <5 ppm, in which very little of the prototype component was absorbed and most of the metabolites were metabolized.

Analyses of the Prototype Components of AR into Plasma and Brain
According to the EICs and MS/MS spectra of reference compounds, compared with TCM-AR, four prototypes were identified in the AR plasma group and three prototypes were identified in the MCAO + AR plasma group as absorbable components.Compared with TCM-AR, no prototype components were identified in the AR brain group, whereas six prototypes were identified in the MCAO + AR brain group as absorbable components, and their absorbable components are shown in Table 2 (serial numbers 1-4 are the prototype absorbable components identified in the AR plasma group, 5-7 are the prototype absorbable components identified in the MCAO + AR plasma group, and 8-13 are the prototype absorbable components identified in the MCAO + AR brain group).The EICs of prototype component compounds and MS/MS spectra of reference compounds are shown in Supplementary Figure S3.Table 2 shows the form, retention time, theoretical mass-charge ratio, measured mass-charge ratio, molecular weight deviation, characteristic fragment ion, molecular formula, name, peak area ratio, and InChIKey.The identified components had an error of <5 ppm, in which very little of the prototype component was Metabolite identification was carried out according to established databases (HERB, LuMet-CM) of metabolites.After sampling, Progenesis QI was used to carry out peak alignment and peak extraction on original data.Then, we searched the databases for analysis, and then determined the metabolites.
Compared with TCM-AR, 39 parent components in the AR plasma group were transformed into 48 metabolites.This process occurred mainly through phase I metabolic reactions such as oxidation, reduction, hydroxylation, carboxylation, and demethylation, as well as phase II metabolic reactions such as methylation and sulfate esterification.In addition, 12 parent components in the MCAO+AR plasma group were transformed into 13 metabolite products.These transformations occurred mainly through hydroxylation, carboxylation, hydrolysis, oxidation, reduction, acetyl oxidation, demethylation, decarboxylation, and other phase I metabolic reactions (e.g., methylation and sulfate esterification), as well as phase II metabolic reactions.Taking metabolite number 28 as an example in negative ion mode at a retention time of 7.85 min, the excimer ion peak was m/z 493.2447 [M-H] − .After searching a metabolic MS database, the score for the fragment ion was 60.7 and the molecular formula was C 26 H 38 O 9 .We concluded that metabolite number 28 may be derived from the intermediate product of M0 after a hydroxylation phase I reaction, and then a glucuronidation glycolaldehyde phase II reaction.The EICs, MS/MS spectra, and metabolic network diagram of its reference compounds are shown in Figure 8.For the remainder of the metabolites, please refer to Supplementary Figures S4 and S5.
Compared with TCM-AR, the five parent components in the AR brain group were transformed into five metabolites.These transformations mainly involved phase I metabolic reactions such as hydroxylation and hydrolysis, and phase II metabolic reactions such as glucuronidation glycolaldehyde acidification and methylation.In addition, the transformation of two parent components into two metabolites in the MCAO + AR brain group was analyzed.This transformation included phase-I metabolic reactions such as hydroxylation and phase-II metabolic reactions such as sulfate esterification.Taking metabolite number 68 as an example, in positive ion mode at a retention time of 4.71 min, the quasi-ion peak was m/z 260.0585 [M+NH4] + .A search of a metabolite MS database suggested the molecular formula to be C10H10O5S and the parent compound to be 5-Hydroxy-1-tetralone.We concluded that metabolite number 67 may be produced by M0 in the phase II metabolic reaction of sulfuric acid esterification.The EIC, MS/MS spectra, and metabolic network diagram of its reference compounds are shown in Figure 9.  Compared with TCM-AR, the five parent components in the AR brain group were transformed into five metabolites.These transformations mainly involved phase I metabolic reactions such as hydroxylation and hydrolysis, and phase II metabolic reactions such as glucuronidation glycolaldehyde acidification and methylation.In addition, the transformation of two parent components into two metabolites in the MCAO + AR brain group was analyzed.This transformation included phase-I metabolic reactions such as hydroxylation and phase-II metabolic reactions such as sulfate esterification.Taking metabolite number 68 as an example, in positive ion mode at a retention time of 4.71 min, the quasi-ion peak was m/z 260.0585 [M+NH 4 ] + .A search of a metabolite MS database suggested the molecular formula to be C 10 H 10 O 5 S and the parent compound to be 5-Hydroxy-1-tetralone.We concluded that metabolite number 67 may be produced by M0 in the phase II metabolic reaction of sulfuric acid esterification.The EIC, MS/MS spectra, and metabolic network diagram of its reference compounds are shown in Figure 9.

Discussion
We employed UHPLC-HR-MS to detect TCM-AR components in the plasma and brain tissue of rats.AR components are complex and varied, and are prone to change in the plasma and brain tissue of the body.Positive and negative ion modes were used to obtain the MS information of compounds in a sample with maximum intensity.
Compared with the reference and database, we identified 281 compounds in TCM-AR, including sugars and glycosides, amino acids and peptides, alkaloids, terpenoids, phenylpropanes, flavonoids, fatty acids, organic acids and their derivatives, steroids, quinones, phenols, carboxylic acids and their derivatives, nucleotides and their derivatives, pyridine and its derivatives, and other compounds.The quantities and classes of various components of TCM-AR were documented.TCM-AR contained high contents of sugars and glycosides, steroids, amino acids, and polypeptides, as well as organic acids and their derivatives; these data are consistent with results from other studies [10].The active ingredients of AR were identified by prototype absorption in rat plasma and brain tissue after oral administration of AR in normal conditions and in tandem with MCAO, respectively.Four prototype components and 48 metabolites were characterized preliminarily in the AR plasma group.Three prototype components and 13 metabolites were identified or characterized preliminarily in the MCAO + AR plasma group.Five metabolites were identified or characterized preliminarily in the AR brain group.Six prototype components and two metabolites were identified or characterized preliminarily in the MCAO + AR brain group.The main metabolic pathways of absorbable components were oxidation, reduction, hydrolysis, dehydrogenation, dehydration, hydroxylation, carboxylation, demethylation, and other phase I metabolic reactions.Glutathione binding, acetylation, methylation, sulfate esterification, glycination glycosylation, and other phase II reactions

Discussion
We employed UHPLC-HR-MS to detect TCM-AR components in the plasma and brain tissue of rats.AR components are complex and varied, and are prone to change in the plasma and brain tissue of the body.Positive and negative ion modes were used to obtain the MS information of compounds in a sample with maximum intensity.
Compared with the reference and database, we identified 281 compounds in TCM-AR, including sugars and glycosides, amino acids and peptides, alkaloids, terpenoids, phenylpropanes, flavonoids, fatty acids, organic acids and their derivatives, steroids, quinones, phenols, carboxylic acids and their derivatives, nucleotides and their derivatives, pyridine and its derivatives, and other compounds.The quantities and classes of various components of TCM-AR were documented.TCM-AR contained high contents of sugars and glycosides, steroids, amino acids, and polypeptides, as well as organic acids and their derivatives; these data are consistent with results from other studies [10].The active ingredients of AR were identified by prototype absorption in rat plasma and brain tissue after oral administration of AR in normal conditions and in tandem with MCAO, respectively.Four prototype components and 48 metabolites were characterized preliminarily in the AR plasma group.Three prototype components and 13 metabolites were identified or characterized preliminarily in the MCAO + AR plasma group.Five metabolites were identified or characterized preliminarily in the AR brain group.Six prototype components and two metabolites were identified or characterized preliminarily in the MCAO + AR brain group.The main metabolic pathways of absorbable components were oxidation, reduction, hydrolysis, dehydrogenation, dehydration, hydroxylation, carboxylation, demethylation, and other phase I metabolic reactions.Glutathione binding, acetylation, methylation, sulfate esterification, glycination glycosylation, and other phase II reactions were also possible.In addition, many previous studies have shown that many effective components of Achyranthes bidentata have neuroprotective [26,27], anti-inflammatory [28], and antioxidant effects [29], which can reduce the damage caused by focal cerebral ischemia-reperfusion [30].However, the results showed that some compounds of oxidative stress, such as allantoin (No. 8 in Table 2), were found in the brain tissue of rats in MCAO + AR group, indicating that the MCAO model successfully caused oxidative stress; on the other hand, subsequent experiments would further quantitatively analyze whether the change in allantoin content was due to the role of achyranthosa in cerebral ischemia-reperfusion injury.
Absorbable components were characterized or identified in plasma rather than in brain tissue.Rats in the AR plasma group absorbed more components than rats in the MCAO + AR plasma group.The metabolism of animals is disturbed during cerebral ischemia [31,32] and the absorption and the number of absorbed components may be reduced.The compounds hydroxycitric acid, ascorbyl, N-trans-sinapoyltyramine-M1, and nuciferine-M1 were identified in the AR plasma group and the MCAO+AR plasma group.Hence, these compounds could be absorbed and metabolized under cerebral ischemia.Among the components that entered the brain, rats in the AR brain group absorbed fewer than the MCAO+AR brain group.During cerebral ischemia-reperfusion, various proinflammatory factors are released by cells to increase BBB destruction, resulting in the entry of neurotoxic substances to worsen brain injury [33].Cerebral ischemia and hypoxia induce the dissolution of the basal layer of the endothelium and the destruction of tight-junction proteins, followed by an increase in BBB permeability [34], and reperfusion usually leads to more severe BBB damage and aggravated neuron damage [35,36].Hence, the BBB is broken, allowing more compounds to enter brain tissue.There were more absorbed components in the MCAO+AR brain group than in the AR brain group.Whether this phenomenon is due to the metabolic changes in sugars and other substances in brain tissue caused by CIRI merits further investigation.In addition, the changes in metabolites in blood and brain tissue caused by differences in animal physiological states, rather than the composition differences caused by mass spectrometry detection, need to be confirmed through further study and analysis of the blood and brain composition of model animals (MCAO) and control animals.

Preparation of Samples Achyranthes Bidentata (TCM-AR)
We took 100 mg of the radix hyssop of AR and added 1 mL of a methanol-water solution (3:1, v/v, containing 2-Chloro-L-phenylalanine (4 µg/mL)).This mixture was dissolved at −80 • C. Next, two small steel balls were added, followed by cooling at −40 • C for 2 min, and then the mixture was placed into a grinding machine (60 Hz, 2 min).Ultrasonic extraction in an ice water bath for 60 min was followed by standing at −40 • C for 30 min.After centrifugation (12,000 rpm, 30 min, 4 • C), the supernatant was diluted 10 times with a methanol-water mixture.Next, the mixture was passed through a 0.22 µm filter, and 200 µL was placed in an injection vial.
Samples of Achyranthes bidentata (TCM-AR) were obtained after the above treatment; that is, the positive control group.

Preparation of Reference Substances
The database of reference substances (LuMet-TCM) was established by Luming Biotech Co., Ltd., (Shanghai, China).

Animals
Specific pathogen-free-grade male Sprague Dawley rats (200 ± 20 g) were purchased from Shanghai Sipur-Bikai Experimental Animal (Experimental Animal License Number.SCXK (Shanghai, China) 2023-0009; Shanghai, China) and raised in the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine (Experimental Animal License Number SYXK (Shanghai, China) 2020-0009).This room was kept at 25 • C and a relative humidity of 45~55%.Rats were exposed to a 12 h light-dark cycle and had access to food and water.After adaptive feeding, 24 rats were randomly divided into four groups of six: a control group of rats, a model group of rats (middle cerebral artery occlusion (MCAO) group), an Achyranthes bidentata group of rats (AR group) and a model Achyranthes bidentata group of rats (MCAO + AR group).In addition, three more models were made and compared with other groups.The AR group and MCAO + AR group were administered (I.G.) 25 g/kg of TCM-AR twice a day for 3 days.Simultaneously, the control group was given the same dose of distilled water.The protocol for animal experiments was approved (PZSHUTCM2303240002) by the Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine (Shanghai, China).

Preparation of Rat Plasma and Brain Tissue
Blood samples were collected from the ocular orbit at 15 and 30 min, 1, 2, and 4 h after the last administration [37] in the AR group and MCAO + AR group, and plasma was collected using an EDTA (Ethylenediaminetetraacetic Acid) anticoagulant tube.After standing at room temperature for a short time, the tube was centrifuged (4000 rpm, 10 min, 4 • C) and the supernatant was removed to obtain plasma.Equal amounts of plasma at different time points were then mixed to obtain mixed plasma samples.Plasma samples were stored at −80 • C. Before loading, plasma (150 µL) was swirled with 450 µL of methanol-acetonitrile (2:1, v/v, containing 2-Chloro-L-phenylalanine (4 µg/mL)) for 1 min.After mixing and centrifugation (12,000 rpm, 10 min, 4 • C) and standing at −40 • C for 2 h, 500 µL of the supernatant was placed in a liquid chromatography-mass spectrometry (LC-MS) sample vial and dried.Then, 150 µL of methanol-acetonitrile-water (2:1:1, v/v/v) was added, followed by vortex-mixing for 1 min, and ultrasonic agitation for 3 min.After overnight storage at −40 • C and centrifugation (12,000 rpm, 10 min, 4 • C), 100 µL of supernatant was used for HR-MS.
After the last collection of blood, rats were killed.Brain tissue was removed, rinsed with pure water, drained, and stored at −80 • C.After thawing, a 100 mg sample was added to 400 µL of methanol-water (4:1, v/v, containing 2-Chloro-L-phenylalanine (4 µg/mL)).Two small steel balls were added, followed by cooling at −40 • C for 2 min.After grinding in a machine (60 Hz, 2 min), ultrasonic extraction was carried out in an ice water bath for 10 min.After standing at −40 • C for 30 min, we centrifuged the sample (12,000 rpm, 10 min, 4 • C).Next, we took 300 µL of the supernatant and placed it in a LC-MS sample vial to dry.Then, 300 µL of methanol-acetonitrile-water (2:1:1, v/v/v) was added, followed by vortex-mixing for 1 min, and ultrasonic agitation for 3 min.After overnight storage at −40 • C and centrifugation (12,000 rpm, 10 min, 4 • C), 100 µL of supernatant was used for HR-MS.

Staining with 2,3,5-Triphenyltetrazolium Chloride (TTC)
After the last administration of AR, three rats were selected randomly from each group for deep anesthesia.Their brain tissues were removed, and residual blood was washed out with physiologic (0.9%) saline.Then, the brain tissue was frozen at −20 • C for 20 min, and cut into 2 mm slices with a surgical blade (Shanghai Pudong Jinghuan Medical Products, Shanghai, China).Sections were prepared with 2% TTC (purity > 98.0%; Shanghai yuanye Biotechnology, Shanghai, China) at 37 • C for 30 min.The percentage of tissue affected by cerebral infarction was measured using ImageJ (US National Institutes of Health, Bethesda, MD, USA).
MS was carried out on a mass spectrometer (Q Exactive Orbitrap; Thermo Fisher Scientific) equipped with a heated electrospray ionization source.Positive and negative ion scanning modes and data-dependent acquisition (DDA) mode were used for data acquisition.The full scan range was 100-1200 m/z for full MS (resolution = 70,000) and MS/MS (resolution = 17,500).The MS parameters of positive ion mode were sheath gas flow = 35 Arb, auxiliary gas flow = 8 Arb, capillary temperature = 320 • C, and auxiliary gas heater temperature = 350 • C. Normalized collision energies were set to 10, 20, and 40 V.The spray voltage was 3.8 kV in positive ion mode and 3.0 kV in negative ion mode.

Data Processing and Analysis
Data preprocessing was undertaken for pattern recognition.Raw data obtained with software based on metabolomics analysis (Progenesis QI v3.0; Nonlinear Dynamics, Newcastle, UK) were used to identify the corrections for baseline filter, peak, integral, alignment, and retention time, as well as peak normalization.Compounds were identified based on precise mass numbers, secondary fragments, and isotopic distribution using the LuMet-CM database and HERB database on 28 September 2023 (http://herb.ac.cn/).
For substances detected qualitatively using Progenesis QI, substances whose total score was ≥40 points were retained as the original components of the TCM formulation.Substances whose fold change was ≥2.0 in the treatment group (administered plasma) and control group (blank plasma) were used as the components entering the plasma and passing into the brain.Substances in positive ion mode and negative ion mode were merged and de-weighted.The total content of the relative peak area of metabolites was set at 100% to obtain the qualitative and quantitative data matrix.This contained all the information extracted from the original data that could be used for subsequent analyses.Extracted ion chromatograms (EICs) and MS/MS spectra with annotations of the structures of secondary fragments were obtained for each identified original TCM formulation and its components entering the plasma and brain.Pie charts were drawn for all identified components of TCM formulations according to their type and quantity.

Conclusions
UHPLC-HR-MS was used to rapidly analyze the components and metabolites of AR in the plasma and brain of rats under normal and pathologic conditions and to comprehensively characterize the components of TCM-AR, which will be helpful to explore the material basis of pharmacological effects of AR in future.We also analyzed and compared the absorbable components and metabolites of normal rats under CIRI to explore the potential mechanism of action.However, the prototype components and metabolites in rats under normal and case conditions are significantly different, suggesting that cerebral ischemia-reperfusion may cause inflammation, nerve damage, and blood-brain barrier damage, etc.However, only qualitative characterization was performed in this paper,

Figure 3 .
Figure 3. Quantities of the components in Achyranthes bidentata.Figure 3. Quantities of the components in Achyranthes bidentata.

Figure 4 .
Figure 4. Contents of the components in Achyranthes bidentata.

Figure 6 .
Figure 6.Base Peak Chromatogram (BPC) of Achyranthes bidentata in plasma passing into the brain according to UHPLC-HR-MS.Control (control group); treatment (AR group); TCM (TCM-AR group).(a) Positive ion mode for the chromatogram of Achyranthes bidentata in plasma.(b) Negative ion mode for the chromatogram of Achyranthes bidentata in plasma.(c) Positive ion mode for the chromatogram of Achyranthes bidentata in brain tissue.(d) Negative ion mode for the chromatogram of Achyranthes bidentata in brain tissue.

Figure 6 .
Figure 6.Base Peak Chromatogram (BPC) of Achyranthes bidentata in plasma passing into the brain according to UHPLC-HR-MS.Control (control group); treatment (AR group); TCM (TCM-AR group).(a) Positive ion mode for the chromatogram of Achyranthes bidentata in plasma.(b) Negative ion mode for the chromatogram of Achyranthes bidentata in plasma.(c) Positive ion mode for the chromatogram of Achyranthes bidentata in brain tissue.(d) Negative ion mode for the chromatogram of Achyranthes bidentata in brain tissue.

Figure 7 .
Figure 7. Base peak intensity (BPC) in the chromatogram of Achyranthes bidentata in plasma passing into the brain according to UHPLC-HR-MS.Control (control group); treatment (MCAO+AR group); TCM (TCM-AR group).(a) Positive ion mode for the chromatogram of Achyranthes bidentata in plasma.(b) Negative ion mode for the chromatogram of Achyranthes bidentata in plasma.(c) Positive ion mode for the chromatogram of Achyranthes bidentata in brain tissue.(d) Negative ion mode for the chromatogram of Achyranthes bidentata in brain tissue.

Figure 7 .
Figure 7. Base peak intensity (BPC) in the chromatogram of Achyranthes bidentata in plasma passing into the brain according to UHPLC-HR-MS.Control (control group); treatment (MCAO+AR group); TCM (TCM-AR group).(a) Positive ion mode for the chromatogram of Achyranthes bidentata in plasma.(b) Negative ion mode for the chromatogram of Achyranthes bidentata in plasma.(c) Positive ion mode for the chromatogram of Achyranthes bidentata in brain tissue.(d) Negative ion mode for the chromatogram of Achyranthes bidentata in brain tissue.

Figure 8 .
Figure 8. Identification of metabolite number 28.(a) EICs of the metabolites and MS/MS spectra of reference compounds.(b) Network diagram for metabolites.

Figure 8 .
Figure 8. Identification of metabolite number 28.(a) EICs of the metabolites and MS/MS spectra of reference compounds.(b) Network diagram for metabolites.

Figure 9 .
Figure 9. Identification of metabolite number 67.(a) EICs of metabolites and MS/MS spectra of reference compounds.(b) Network diagram for metabolites.

Figure 9 .
Figure 9. Identification of metabolite number 67.(a) EICs of metabolites and MS/MS spectra of reference compounds.(b) Network diagram for metabolites.

Table 1 .
Identification of the chemical composition of TCM-AR based on UHPLC-HR-MS.
16,17-diol, calenduloside E, geniposide, ginsenoside Ro, gentiopicroside, Chikusetsusaponin Iva, and other compounds.Figure 5g,h show the EICs of achyranthoside E and roseoside and their MS/MS spectra compared with the databases, respectively.Using compound number 60 as an example, at a retention time of 7.84 min, the excimer ion peak was at m/z 949.4384 [M+NH 4 , M+Na] + , and the molecular formula was predicted to be C 46 H 70 O 19 .The molecular weights of secondary fragment ions were 189.1633, 191.1792, 201.1634, 203.1791, 205.1948, 247.1689, 309.0447, 393.3502, 439.3565, and 944.4835, which were consistent with the literature

Table 2 .
Prototype components absorbed by the AR plasma group, MCAO+AR plasma group, AR brain group, and MCAO+AR brain group.

Table 3 .
Absorbed metabolites in the AR plasma group, MCAO+AR plasma group, AR brain group, and MCAO+AR brain group.