Chemoproteomic strategy identifies PfUCHL3 as the target of halofuginone.

The human malaria parasite Plasmodium falciparum (P. falciparum) continues to pose a significant public health challenge, leading to millions of fatalities globally. Halofuginone (HF) has shown a significant anti-P. falciparum effect, suggesting its potential as a therapeutic agent for malaria treatment. In this study, we synthesized a photoaffinity labeling probe of HF to identify its direct target in P. falciparum. Our results reveal that ubiquitin carboxyl-terminal hydrolase 3 (PfUCHL3) acts as a crucial target protein of HF, which modulates parasite growth in the intraerythrocytic cycle. In particular, we discovered that HF potentially forms hydrogen bonds with the Leu10, Glu11, and Arg217 sites of PfUCHL3, thereby inducing an allosteric effect by promoting the embedding of the helix 6' region on the protein surface. Furthermore, HF disrupts the expression of multiple functional proteins mediated by PfUCHL3, specifically those that play crucial roles in amino acid biosynthesis and metabolism in P. falciparum. Taken together, this study highlights PfUCHL3 as a previously undisclosed druggable target of HF, which contributes to the development of novel anti-malarial agents in the future.

Anemoside B4, a new pyruvate carboxylase inhibitor, alleviates colitis by reprogramming macrophage function.

OBJECTIVES: Colitis is a global disease usually accompanied by intestinal epithelial damage and intestinal inflammation, and an increasing number of studies have found natural products to be highly effective in treating colitis. Anemoside B4 (AB4), an abundant saponin isolated from Pulsatilla chinensis (Bunge), which was found to have strong anti-inflammatory activity. However, the exact molecular mechanisms and direct targets of AB4 in the treatment of colitis remain to be discovered. METHODS: The anti-inflammatory activities of AB4 were verified in LPS-induced cell models and 2, 4, 6-trinitrobenzene sulfonic (TNBS) or dextran sulfate sodium (DSS)-induced colitis mice and rat models. The molecular target of AB4 was identified by affinity chromatography analysis using chemical probes derived from AB4. Experiments including proteomics, molecular docking, biotin pull-down, surface plasmon resonance (SPR), and cellular thermal shift assay (CETSA) were used to confirm the binding of AB4 to its molecular target. Overexpression of pyruvate carboxylase (PC) and PC agonist were used to study the effects of PC on the anti-inflammatory and metabolic regulation of AB4 in vitro and in vivo. RESULTS: AB4 not only significantly inhibited LPS-induced NF-κB activation and increased ROS levels in THP-1 cells, but also suppressed TNBS/DSS-induced colonic inflammation in mice and rats. The molecular target of AB4 was identified as PC, a key enzyme related to fatty acid, amino acid and tricarboxylic acid (TCA) cycle. We next demonstrated that AB4 specifically bound to the His879 site of PC and altered the protein's spatial conformation, thereby affecting the enzymatic activity of PC. LPS activated NF-κB pathway and increased PC activity, which caused metabolic reprogramming, while AB4 reversed this phenomenon by inhibiting the PC activity. In vivo studies showed that diisopropylamine dichloroacetate (DADA), a PC agonist, eliminated the therapeutic effects of AB4 by changing the metabolic rearrangement of intestinal tissues in colitis mice. CONCLUSION: We identified PC as a direct cellular target of AB4 in the modulation of inflammation, especially colitis. Moreover, PC/pyruvate metabolism/NF-κB is crucial for LPS-driven inflammation and oxidative stress. These findings shed more light on the possibilities of PC as a potential new target for treating colitis.

Triterpenoids from the hook-bearing stems of Uncaria rhynchophylla.

An unprescribed nortriterpenoid with an aromatic E ring, uncanortriterpenoid A (1), together with fourteen known triterpenoids (2-15), were isolated from the hook-bearing stems of Uncaria rhynchophylla Miq. Based on extensive spectroscopic analyses, the NMR data of 2, 5, and 10 in CD3OD were assigned for the first time, and the wrongly assigned δC of C-27 and C-29 of 2 were revised. Among the known compounds, 7, 13, and 15 were isolated from this species for the first time, and 15 represents the first lanostane triterpenoid bearing an extra methylidene at C-24 for the Rubiaceae family. Additionally, compounds 6 and 14 exhibited moderate ferroptosis inhibitory activity, with an EC50 value of 14.74 ± 0.20 µM for 6 and 23.11 ± 1.31 µM for 14.

Inherent Capability of Self-Assembling Nanostructures in Specific Proteasome Activation for Cancer Cell Pyroptosis.

Understanding the direct interaction of nanostructures per se with biological systems is important for biomedical applications. However, whether nanostructures regulate biological systems by targeting specific cellular proteins remains largely unknown. In the present work, self-assembling nanomicelles are constructed using small-molecule oleanolic acid (OA) as a molecular template. Unexpectedly, without modifications by functional ligands, OA nanomicelles significantly activate cellular proteasome function by directly binding to 20S proteasome subunit alpha 6 (PSMA6). Mechanism study reveals that OA nanomicelles interact with PSMA6 to dynamically modulate its N-terminal domain conformation change, thereby controlling the entry of proteins into 20S proteasome. Subsequently, OA nanomicelles accelerate the degradation of several crucial proteins, thus potently driving cancer cell pyroptosis. For translational medicine, OA nanomicelles exhibit a significant anticancer potential in tumor-bearing mouse models and stimulate immune cell infiltration. Collectively, this proof-of-concept study advances the mechanical understanding of nanostructure-guided biological effects via their inherent capacity to activate proteasome.

Gastroprotective 2-(2-phenylethyl)chromone-sesquiterpene hybrids from the resinous wood of Aquilaria sinensis (Lour.) Gilg.

Six previously unprecedented 2-(2-phenylethyl)chromone-sesquiterpene hybrids, aquisinenins A-F (1 - 6), were isolated from the resinous wood of Aquilaria sinensis by a LC-MS-guided fractionation procedure. Their structures were determined by extensive spectroscopic analysis (1D and 2D NMR, UV, IR, and HRMS) and experimental and computed ECD data. Compounds 1 - 6 were rare dimeric 2-(2-phenylethyl)chromone-sesquiterpene derivatives featuring 5,6,7,8-tetrahydro-2-(2-phenylethyl)chromone hybridized with different sesquiterpene (eudesmane/guaiane type) moieties via ester bond. Furthermore, all the isolated compounds were evaluated for their protective effects on taurocholic acid (TCA)-induced GES-1 cell injury. The most effective aquisinenin F (6) was used to elucidate the involved mechanism on protection against TCA-induced gastric mucosal damage. Our results indicated that 6 protected against gastric mucosal cell insult by downregulation of the ER stress triggered by TCA.

[Rapid flavonoid-focused sub-chemome characterization of Draconis Sanguis using UPLC-Q-TOF-MS in combination with molecular weight imprinting and mass defect filtering].

Draconis Sanguis is a precious Chinese medicinal material for activating blood and resolving stasis, and its effective components are flavonoids. However, the structural diversity of flavonoids in Draconis Sanguis brings great challenges to the in-depth chara-cterization of its chemical composition profiles. To clarify the substance basis of Draconis Sanguis, ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was used in this study to acquire MS data of Draconis Sanguis. The molecular weight imprinting(MWI) and mass defect filtering(MDF) were developed for rapid screening of flavonoids in Draconis Sanguis. Full-scan MS and MS~2 were recorded within the mass range m/z 100-1 000 in positive ion mode. Accor-ding to previous literature, MWI was employed to hunt for reported flavonoids in Draconis Sanguis, and the mass tolerance range of [M+H]~+ was set as ±10×10~(-3). A five-point MDF screening frame was further constructed to narrow the screening range of flavonoids from Draconis Sanguis. Combined with diagnostic fragment ions(DFI) and neutral loss(NL) as well as mass fragmentation pathways, 70 compounds were preliminarily identified from the extract of Draconis Sanguis, including 5 flavan oxidized congeners, 12 flavans, 1 dihydrochalcones, 49 flavonoids dimers, 1 flavonoids trimer and 2 flavonoid derivatives. This study clarified the chemical composition of flavonoids in Draconis Sanguis. Moreover, it also showed that high-resolution MS combined with data post-processing methods such as MWI and MDF could achieve rapid characterization of the chemical composition in Chinese medicinal materials.

[Research progress on application of multi-enzyme-catalyzed cascade reactions in enzymatic synthesis of natural products].

As a biocatalyst, enzyme has the advantages of high catalytic efficiency, strong reaction selectivity, specific target products, mild reaction conditions, and environmental friendliness, and serves as an important tool for the synthesis of complex organic molecules. With the continuous development of gene sequencing technology, molecular biology, genetic manipulation, and other technologies, the diversity of enzymes increases steadily and the reactions that can be catalyzed are also gradually diversified. In the process of enzyme-catalyzed synthesis, the majority of common enzymatic reactions can be achieved by single enzyme catalysis, while many complex reactions often require the participation of two or more enzymes. Therefore, the combination of multiple enzymes together to construct the multi-enzyme cascade reactions has become a research hotspot in the field of biochemistry. Nowadays, the biosynthetic pathways of more natural products with complex structures have been clarified, and secondary metabolic enzymes with novel catalytic activities have been identified, discovered, and combined in enzymatic synthesis of natural/unnatural molecules with diverse structures. This study summarized a series of examples of multi-enzyme-catalyzed cascades and highlighted the application of cascade catalysis methods in the synthesis of carbohydrates, nucleosides, flavonoids, terpenes, alkaloids, and chiral molecules. Furthermore, the existing problems and solutions of multi-enzyme-catalyzed cascade method were discussed, and the future development direction was prospected.

[Anti-depression targets and mechanism study of Kaixin San].

This study identified the anti-depression targets of Kaixin San(KXS) in the brain tissue with "target fishing" strategy, and explored the target-associated pharmacological signaling pathways to reveal the anti-depression molecular mechanism of KXS. The Balb/c mouse model of depression was established by chronic unpredictable mild stress(CUMS) and the anti-depression effect of KXS was evaluated by forced swimming test and sucrose preference test. KXS active components were bonded to the benzophenone-modified magnetic nanoparticles by photocrosslinking reaction for capturing target proteins from cortex, thalamus and hippocampus of depressive mice. The target proteins were identified by liquid chromatography-mass spectrometry/mass spectrometry(LC-MS/MS). The enrichment analysis on signaling pathways was performed by Cytoscape. The potential biological functions of targets were verified by immunohistochemistry and Western blot assay. The results showed that KXS significantly improved the behavioral indexes. There were 64, 91, and 44 potential targets of KXS identified in cortex, thalamus, and hippocampus, respectively, according to the target identification experiment. The functions of these targets were mainly associated with vasopressin-regulated water reabsorption, salmonella infection, thyroid hormone synthesis, and other signaling pathways. Besides, the results of immunohistochemistry and Western blot showed that KXS up-regulated the expressions of argipressine(AVP) in the cortex, heat shock protein 60(HSP60), cytochrome C oxidase 4(COX4), and thyrotropin-releasing hormone(TRH) in the thalamus, and down-regulated the expressions of tumor necrosis factor-α(TNF-α) and nuclear factor kappa B(NF-κB) p65 in the thalamus. Therefore, KXS may exert anti-depression effect through regulating vasopressin signaling pathway in the cortex and inflammation, energy metabolism, and thyroid hormone signaling pathways in the thalamus, and the effect of KXS on hippocampus is not significant.

[Potentiating effect and mechanism of extract of Jingfang Granules on activation of macrophages].

This study aimed to explore the potentiating effect and mechanism of the extract of Jingfang Granules(JFG) on the activation of macrophages. The RAW264.7 cells were treated with JFG extract and then stimulated by multiple agents. Subsequently, mRNA was extracted, and reverse transcription-polymerase chain reaction(RT-PCR) was used to measure the mRNA transcription of multiple cytokines in RAW264.7 cells. The levels of cytokines in the cell supernatant were detected by enzyme-linked immunosorbent assay(ELISA). In addition, the intracellular proteins were extracted and the activation of signaling pathways was determined by Western blot. The results showed that JFG extract alone could not promote or slightly promote the mRNA transcription of TNF-α, IL-6, IL-1ß, MIP-1α, MCP-1, CCL5, IP-10, and IFN-ß, and significantly enhance the mRNA transcription of these cytokines in RAW264.7 cells induced by R848 and CpG in a dose-dependent manner. Furthermore, JFG extract also potentiated the secretion of TNF-α, IL-6, MCP-1, and IFN-ß by RAW264.7 cells stimulated with R848 and CpG. As revealed by mechanism analysis, JFG extract enhanced the phosphorylation of p38, ERK1/2, IRF3, STAT1, and STAT3 in RAW264.7 cells induced by CpG. The findings of this study indicate that JFG extract can selectively potentiate the activation of macrophages induced by R848 and CpG, which may be attributed to the promotion of the activation of MAPKs, IRF3, and STAT1/3 signaling pathways.

[Eleven new sesquiterpenoids from peeled stems of Syringa pinnatifolia].

The peeled stems of Syringa pinnatifolia(SP) is a representative Mongolian folk medicine with the effects of anti-depression, heat clearance, pain relief, and respiration improvement. It has been clinically used for the treatment of coronary heart disease, insomnia, asthma, and other cardiopulmonary diseases. As part of the systematic study on pharmacological substances of SP, 11 new sesquiterpenoids were isolated from the terpene-containing fractions of the ethanol extract of SP by liquid chromatography-mass spectrometry(LC-MS) and proton nuclear magnetic resonance(~1H-NMR) guided isolation methods. The planar structures of the sesquiterpenoids were identified by MS, 1D NMR, and 2D NMR data analysis, and were named pinnatanoids C and D(1 and 2), and alashanoids T-ZI(3-11), respectively. The structure types of the sesquiterpenoids included pinnatane, humulane, seco-humulane, guaiane, carryophyllane, seco-erimolphane, isodaucane, and other types. However, limited to the low content of compounds, the existence of multiple chiral centers, the flexibility of the structure, or lack of ultraviolet absorption, the stereoscopic configuration remained unresolved. The discovery of various sesquiterpenoids enriches the understanding of the chemical composition of the genus and species and provides references for further analysis of pharmacological substances of SP.

Gold Nanoparticle-Based Photo-Cross-Linking Strategy for Cellular Target Identification of Supercomplex Molecular Systems.

Cellular target identification plays an essential role in innovative drug development and pharmacological mechanism elucidation. However, very few practical experimental methodologies have been developed for identifying target proteins for supercomplex molecular systems such as biologically active phytochemicals or pharmaceutical compositions. To overcome this limitation, we synthesized gold nanoparticles (AuNPs) as solid scaffolds, which were bound with 4,4'-dihydroxybenzophenone (DHBP) as a photo-cross-linking group on the surface. Then, DHBP-modified AuNPs cross-linked various organic compounds from phytochemicals under ultraviolet radiation via carbene reactions, H-C bond insertion, for catalytic C-C bond formation. We next used the phytochemical-cross-linked AuNPs (phytoAuNPs) to pull down potential binding proteins from brain tissue lysate and identified 13 neuroprotective targets by mass spectrometry analysis. As an exemplary study, we selected Hsp60 as a crucial cellular target to further screen 14 target-binding compounds from phytochemicals through surface plasmon resonance (SPR) analysis, followed by Hsp60 activity detection and neuroprotective effect assay in cells. Collectively, this gold nanoparticle-based photo-cross-linking strategy can serve as a useful platform for discovering novel cellular targets for supercomplex molecular systems and help to explore pharmacological mechanisms and active substances.

Photoaffinity Labeling-Based Chemoproteomic Strategy Reveals RBBP4 as a Cellular Target of Protopanaxadiol against Colorectal Cancer Cells.

Protopanaxadiol (PPD), a main ginseng metabolite, exerts powerful anticancer effects against multiple types of cancer; however, its cellular targets remain elusive. Here, we synthesized a cell-permeable PPD probe via introducing a bifunctional alkyne-containing diazirine photo-crosslinker and performed a photoaffinity labeling-based chemoproteomic study. We identified retinoblastoma binding protein 4 (RBBP4), a chromatin remodeling factor, as an essential cellular target of PPD in HCT116 colorectal cancer cells. PPD significantly decreased RBBP4-dependent trimethylation at lysine 27 of histone H3 (H3K27me3), a crucial epigenetic marker that correlates with histologic signs of colorectal cancer aggressiveness, and PPD inhibition of proliferation and migration of HCT116 cells was antagonized by RBBP4 RNA silencing. Collectively, our study highlights a previously undisclosed anti-colorectal cancer cellular target of the ginseng metabolite and advances the fundamental understanding of RBBP4 functions via a chemical biology strategy.

Epoxymicheliolide directly targets histone H2B to inhibit neuroinflammation via recruiting E3 ligase RNF20.

Monoubiquitination plays a critical role as one of the largest histone post-translational modifications (PTMs). Recent study has revealed that histone H2B monoubiquitination (H2Bub1) at a unique lysine 120 (K120) is widely involved in the development of inflammation progression. However, small-molecules directly targeting H2B to exert anti-inflammation effects via editing monoubiquitination have not been hitherto reported. In this study, we first discover a natural small-molecule epoxymicheliolide (ECL), which directly binds to H2B to inhibit microglia-mediated neuroinflammation in vitro and in vivo. Mechanism study suggests that ECL covalently modifies a previously undisclosed lysine 46 (K46) in H2B, and recruits E3 ubiquitin ligase RNF20 to promote H2Bub1 at K120. ChIP-seq and transcriptomics further reveal that ECL-mediated H2Bub1 markedly disrupts the AP-1 recruitment to proinflammatory gene promoters for microglia inactivation. Collectively, our findings suggests that K46 of H2B serves as a promising pharmacological target to develop small-molecule drugs against microglia-mediated neuroinflammation, and ECL represents a valuable lead compound for neuroinflammation via regulating histone monoubiquitination.

Deoxyhypusine hydroxylase as a novel pharmacological target for ischemic stroke via inducing a unique post-translational hypusination modification.

Ischemic stroke remains one of the leading causes of death worldwide, thereby highlighting the urgent necessary to identify new therapeutic targets. Deoxyhypusine hydroxylase (DOHH) is a fundamental enzyme catalyzing a unique posttranslational hypusination modification of eukaryotic translation initiation factor 5A (eIF5A) and is highly involved in the progression of several human diseases, including HIV-1 infection, cancer, malaria, and diabetes. However, the potential therapeutic role of pharmacological regulation of DOHH in ischemic stroke is still poorly understood. Our study first discovered a natural small-molecule brazilin (BZ) with an obvious neuroprotective effect against oxygen-glucose deprivation/reperfusion insult. Then, DOHH was identified as a crucial cellular target of BZ using HuProt™ human proteome microarray. By selectively binding to the Cys232 residue, BZ induced a previously undisclosed allosteric effect to significantly increase DOHH catalytic activity. Furthermore, BZ-mediated DOHH activation amplified mitophagy for mitochondrial function and morphology maintenance via DOHH/eIF5A hypusination signaling pathway, thereby protecting against ischemic neuronal injury in vitro and in vivo. Collectively, our study first identified DOHH as a previously unreported therapeutic target for ischemic stroke, and provided a future drug design direction for DOHH allosteric activators using BZ as a novel molecular template.

Natural carbazole alkaloid murrayafoline A displays potent anti-neuroinflammatory effect by directly targeting transcription factor Sp1 in LPS-induced microglial cells.

Neuroinflammation is a leading cause for neurological disorders. Carbazole alkaloids, isolated from the medicinal plants of Murraya species (Rutaceae), have exhibited wide pharmacological activities particularly for neuroinflammation. However, its underlying cellular targets and molecular mechanisms still remain unclear. In current study, we found that murrayafoline A (MA), a carbazole alkaloid obtained from Murraya tetramera, potently inhibited the production of neuroinflammation mediators, such as nitric oxide (NO), TNF-α, IL-6 and IL-1ß in LPS-induced BV-2 microglial cells. Then, we performed thermal proteome profiling (TPP) strategy to identify Specificity protein 1 (Sp1) as a potential cellular target of MA. Moreover, we performed surface plasmon resonance (SPR), cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DRATS) assays to confirm the direct interaction between MA and Sp1. Furthermore, we downregulated Sp1 expression in BV2 cells using siRNA transfection, and observed that Sp1 knockdown significantly antagonized MA-mediated inhibition of neuroinflammation mediator production. Meanwhile, Sp1 knockdown also markedly reversed MA-mediated inactivation of IKKß/NF-κB and p38/JNK MAPKs pathways. Finally, in vivo studies revealed that MA significantly suppressed the expression of Iba-1, TNF-α, and IL-6, while increased the number of Nissl bodies in the brains of LPS-induced mice. Taken together, our study demonstrated that MA exerted obvious anti-neuroinflammation effect by directly targeting Sp1, thereby inhibiting NF-κB and MAPK signaling pathways. Our findings also provided a promising direction of pharmacological targeting Sp1 for anti-neuroinflammation therapeutics as well as novel agent development.

Comparative study on the main active components of Baoyuan decoction in plasma and urine of normal and heart failure rats.

The global morbidity and mortality of heart failure has been increasing in recent years. Traditional Chinese medicine (TCM) was increasingly used to treat cardiovascular diseases. Baoyuan decoction (BYD) was a famous classical prescription in China. Modern pharmacological studies showed that it had obvious therapeutic effects on cardiovascular diseases, but its pathological pharmacokinetic studies were unclear. In this research, the absorption of 16 bioactive components in plasma and the excretion of 9 representative components in urine of control rats and isoproterenol (ISO)-induced heart failure rats were studied using the large-volume direct-injection LC-MS method established by our research group. The results indicated that flavonoid constituents exhibited quicker absorption and elimination than saponin constituents after oral administration of BYD. The half-life period of some bioactive compounds in the model group was increased, which contributed to the longer therapeutic effect. The cumulative excretion rate of major flavonoid components of BYD decreased significantly in the ISO-induced heart failure rats.

Anti-Neuroinflammatory Components from Clausena lenis Drake.

Clausena lenis Drake (C. lenis) is a folk medicinal herb to treat influenza, colds, bronchitis, and malaria. The 95% and 50% ethanol extract of C. lenis showed significant nitric oxide (NO) inhibition activity in BV-2 microglial cells stimulated by lipopolysaccharide (LPS). Bio-guided isolation of the active extract afforded five new compounds, including a chlorine-containing furoquinoline racemate, (±)-claulenine A (1), an amide alkaloid, claulenine B (2), a prenylated coumarin, claulenin A (3), a furocoumarin glucoside, clauleside A (4), and a multi-prenylated p-hydroxybenzaldehyde, claulenin B (5), along with 33 known ones. Their structures were determined via spectroscopic methods, and the absolute configurations of new compounds were assigned via the electronic circular dichroism (ECD) calculations and single-crystal X-ray diffraction analysis. Compounds 2, 23, 27, 28, 33, and 34 showed potent anti-neuroinflammatory effects on LPS-induced NO production in BV-2 microglial cells, with IC50 values in the range of 17.6-40.9 µM. The possible mechanism was deduced to interact with iNOS through molecular docking.

[Update classical literature research on Cistanches Herba].

Cistanches Herba(CH), a valuable medicinal material which has long been used, originated from Shennong's Classic of Materia Medica. It has a wealth of names, such as Rousongrong, Heisiming, Dijing, and Dayun. The definition on the original plants which are parasitic and distributed in the unique environments in remote areas has been confusing, resulting in the emergence of various counterfeits and substitutes. Moreover, the records on the property, flavor, tropism, and indications of CH are also different. In order to further explore the cultural connotation and medicinal value of CH and further clarify its source and harvesting and processing methods, this study conducted further classical literature research on its name, harvesting and processing, property and flavor, meridian tropism, efficacy and clinical use, and textual research on its source and habitats, providing a reference for research, clinical medication, development and utilization, and industry development of CH.

[Steroids from Uncaria rhynchophylla].

Thirteen steroids(1-13) were isolated from the non-alkaloid constituents of Uncaria rhynchophylla by column chromatography on silica gel, ODS, Sephadex LH-20, and preparative HPLC chromatography, and their structures were elucidated by analyses of the MS and NMR spectral data. All the compounds were isolated from the genus Uncaria for the first time, and 1 was a new compound. The ~1H-NMR and ~(13)C-NMR data of two compounds(12 and 13) in deuteron-chloroform were completely assigned. This study enriched the steroid constituents of U. rhynchophylla and provided scientific references for the elucidation of active constituents and further development and utilization of U. rhynchophylla.

[Rapid chemome characterization of Pien-Tze-Huang by DI-MS/MS~(ALL)].

Pien-Tze-Huang, one of the most famous and widely used Chinese medicinal prescriptions in China, consists of Notoginseng Radix et Rhizoma, Bovis Calculus, Fel Serpentis, and Moschus.The prescription can clear heat and remove toxin, cool blood and resolve blood stasis, and relieve swelling and pain.Characterizing the chemical composition can facilitate the construction of the quality standard and the research on the effective compounds and action mechanism of Pien-Tze-Huang.Therefore, this study used direct infusion(DI)-MS/MS~(ALL) method to rapidly and accurately reveal the chemical composition of Pien-Tze-Huang.The principle of chemical composition profiling of Chinese medicinal prescriptions lies in the MS~1-MS~2 dataset construction, followed by structural annotation based on MS/MS spectra and summarizing of mass fragmentation pathways.MS/MS~(ALL) owns unique mass spectrometric separation ability via applying gas phase fractionation which enables MS~1 ion cohort successively enter the collision cell and acquire MS~2 spectrum for each precursor ion current with a width of m/z=1.Because DI can provide desired measurement time, MS/MS~(ALL) is able to acquire MS~2 spectrum for each compound individually except for the compounds which share identical nominal molecular weight, even isomers.A total of 52 compounds were identified in Pien-Tze-Huang, including 16 saponins, 24 bile acids, 9 fatty acids, 2 saccharides, and 1 other compound.DI-MS/MS~(ALL) can simultaneously identify the compounds with different polarities in a short time, which is superior to LC-MS.This study provides a powerful tool for the rapid chemome profiling of Chinese medicinal prescriptions.