Integrated causal inference modeling uncovers novel causal factors and potential therapeutic targets of Qingjin Yiqi granules for chronic fatigue syndrome

Objective:Chronic fatigue syndrome(CFS)is a prevalent symptom of post-coronavirus disease 2019(COVID-19)and is associated with unclear disease mechanisms.The herbal medicine Qingjin Yiqi granules(QJYQ)constitute a clinically approved formula for treating post-COVID-19;however,its potential as a drug target for treating CFS remains largely unknown.This study aimed to identify novel causal factors for CFS and elucidate the potential targets and pharmacological mechanisms of action of QJYQ in treating CFS.Methods:This prospective cohort analysis included 4,212 adults aged≥65 years who were followed up for 7 years with 435 incident CFS cases.Causal modeling and multivariate logistic regression analysis were performed to identify the potential causal determinants of CFS.A proteome-wide,two-sample Mendelian randomization(MR)analysis was employed to explore the proteins associated with the identified causal factors of CFS,which may serve as potential drug targets.Furthermore,we performed a virtual screening analysis to assess the binding affinity between the bioactive compounds in QJYQ and CFS-associated proteins.Results:Among 4,212 participants(47.5%men)with a median age of 69 years(interquartile range:69–70 years)enrolled in 2004,435 developed CFS by 2011.Causal graph analysis with multivariate logistic regression identified frequent cough(odds ratio:1.74,95%confidence interval[CI]:1.15–2.63)and insomnia(odds ratio:2.59,95%CI:1.77–3.79)as novel causal factors of CFS.Proteome-wide MR analysis revealed that the upregulation of endothelial cell-selective adhesion molecule(ESAM)was causally linked to both chronic cough(odds ratio:1.019,95%CI:1.012–1.026,P=2.75 e^(−05))and insomnia(odds ratio:1.015,95%CI:1.008–1.022,P=4.40 e^(−08))in CFS.The major bioactive compounds of QJYQ,ginsenoside Rb2(docking score:−6.03)and RG4(docking score:−6.15),bound to ESAM with high affinity based on virtual screening.Conclusions:Our integrated analytical framework combining epidemiological,genetic,and in silico data provides a novel strategy for elucidating complex disease mechanisms,such as CFS,and informing models of action of traditional Chinese medicines,such as QJYQ.Further validation in animal models is warranted to confirm the potential pharmacological effects of QJYQ on ESAM and as a treatment for CFS.

Offline two-dimensional liquid chromatography coupled with ion mobility-quadrupole time-of-flight mass spectrometry enabling fourdimensional separation and characterization of the multicomponents from white ginseng and red ginseng

Inherent complexity of plant metabolites necessitates the use of multi-dimensional information to accomplish comprehensive profiling and confirmative identification.A dimension-enhanced strategy,by offline two-dimensional liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry(2 D-LC/IM-QTOF-MS)enabling four-dimensional separations(2 D-LC,IM,and MS),is proposed.In combination with in-house database-driven automated peak annotation,this strategy was utilized to characterize ginsenosides simultaneously from white ginseng(WG)and red ginseng(RG).An offline 2 DLC system configuring an Xbridge Amide column and an HSS T3 column showed orthogonality 0.76 in the resolution of ginsenosides.Ginsenoside analysis was performed by data-independent high-definition MSE(HDMSE)in the negative ESI mode on a Vion?IMS-QTOF hybrid high-resolution mass spectrometer,which could better resolve ginsenosides than MSEand directly give the CCS information.An in-house ginsenoside database recording 504 known ginsenosides and 58 reference compounds,was established to assist the identification of ginsenosides.Streamlined workflows,by applying UNIFI?to automatedly annotate the HDMSEdata,were proposed.We could separate and characterize 323 ginsenosides(including 286 from WG and 306 from RG),and 125 thereof may have not been isolated from the Panax genus.The established 2 D-LC/IM-QTOF-HDMSEapproach could also act as a magnifier to probe differentiated components between WG and RG.Compared with conventional approaches,this dimensionenhanced strategy could better resolve coeluting herbal components and more efficiently,more reliably identify the multicomponents,which,we believe,offers more possibilities for the systematic exposure and confirmative identification of plant metabolites.

The exciting and magical journey of components from compound formulae to where they fight

With its long-term empirical clinical practice and increasing number of health benefits reported,Chinese Materia Medica(CMM)is gaining increasing global acceptance.Importantly,the identification of chemical constituents in vitro and exposed forms in vivo is a prerequisite for understanding how CMM formulae prevent and treat diseases.This review systematically summarizes the exciting and magical journey of CMM components from compound formulae to where they fight,the possible structural transformation of CMM components in vitro and in vivo,and their pharmacological contribution.When a decoction is prepared,significant chemical reactions are observed,including degradation and production of polymers and self-assembling supramolecules,leading to the construction of a component library with diverse decoction structures.After ingestion,compounds pass through the intestinal and blood-brain barriers and undergo a more wonderful journey involving the gut microbiota,microbial enzymes,and endogenous drug-metabolizing enzymes(mainly liver enzymes).At this stage,they are modified and assembled into novel and complex compounds,such as newly generated metabolites,conjugates,and self-assembling superamolecules.This review might provide a strategic orientation to explore the active compounds of CMM formulae in vivo.

New Spirostanol Sulfonate from the Rhizomes of Helleborus thibetanus(Ranunculaceae)

A new spirostanol sulfonate, spirost-5,25（27）-dien-1β,3β-diol 1-sulfonate（1）, was isolated from the rhizomes of Helleborus thibetanus（Ranunculaceae）, and the structure was identified on the basis of a detailed spectroscopic analyses, including 2D NMR spectrometry, IR and HRESI-MS.

Dimension-Enhanced Ultra-High Performance Liquid Chromatography/Ion Mobility-Quadrupole Time-of-Flight Mass Spectrometry Combined with Intelligent Peak Annotation for the Rapid Characterization of the Multiple Components from Seeds of Descurainia sophia

The complex composition of herbal metabolites necessitates the development of powerful analytical techniques aimed to identify the bioactive components.The seeds of Descurainia sophia(SDS)are utilized in China as a cough and asthma relieving agent.Herein,a dimension-enhanced integral approach,by combining ultra-high performance liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry(UHPLC/IMQTOF-MS)and intelligent peak annotation,was developed to rapidly characterize the multicomponents from SDS.Good chromatographic separation was achieved within 38 min on a UPLC CSH C18(2.1×100 mm,1.7μm)column which was eluted by 0.1%formic acid in water(water phase)and acetonitrile(organic phase).Collision-induced dissociation-MS^(2)data were acquired by the data-independent high-definition MS^(E)(HDMS^(E))in both the negative and positive electrospray ionization modes.A major components knockout strategy was applied to improve the characterization of those minor ingredients by enhancing the injection volume.Moreover,a self-built chemistry library was established,which could be matched by the UNIFI software enabling automatic peak annotation of the obtained HDMS^(E)data.As a result of applying the intelligent peak annotation workflows and further confirmation process,a total of 53 compounds were identified or tentatively characterized from the SDS,including 29 flavonoids,one uridine derivative,four glucosides,one lignin,one phenolic compound,and 17 others.Notably,four-dimensional information related to the structure(e.g.,retention time,collision cross section,MS^(1)and MS^(2)data)was obtained for each component by the developed integral approach,and the results would greatly benefit the quality control of SDS.

Tea as a natural gift for discovering antiviral candidates

Coronavirus disease(COVID-19)remains rampant worldwide and poses a serious threat to human health.Tea is a medicinal and edible homologous plant that exhibits potential anti-SARS-CoV-2 properties via the prevention of virus entry into host cells,inhibition of virus replication,and enhancement of the innate and cellular immune responses.In this review,the properties of six major types of tea were systematically summarized,including green tea,yellow tea,white tea,oolong tea,black tea,and dark tea.We focused on the primary components of tea exhibiting antiviral activities,which included(–)-epigallocatechin-3-gallate,(–)-gallocatechin gallate,tannic acid,oolonghomobisflavan A,theaflavins,and white-tip silver needle flavonoids.Among them,(–)-epigallocatechin-3-gallate is proposed to be an antiviral compound that interferes with the entire life cycle of SARS-CoV-2 by balancing inflammation and immunity.Thus,this compound can serve as a promising lead structure for the development of SARS-CoV-2 inhibitors.

Identification of prototype compounds and their metabolites in rats’ serum from Xuefu Zhuyu Decoction by UPLC-Q-TOF/MS

Objective: As a classic prescription in traditional Chinese medicine, Xuefu Zhuyu Decoction(XFZYD) has been widely used in the clinical treatment of cardiovascular and cerebrovascular diseases. In order to unveil the potentially effective compounds, a rapid ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF/MS) method was established to identify prototype compounds and their metabolites from XFZYD in rats’ serum.Methods: The serum from rats after intragastric administration of XFZYD aqueous extract was analyzed by UPLC-Q-TOF/MS method. The prototype compounds and their metabolites were identified by comparison with the reference standards and tentatively characterized by comprehensively analyzing the retention time, MS data, characteristic MS fragmentation pattern and retrieving literatures.Results: A total of 175 compounds(24 prototype compounds and 151 metabolites) were identified and tentatively characterized. The metabolic pathways of prototype compounds in vivo were also summarized, including glucuronidation, hydrolyzation, sulfation, demethylation, and hydroxylation, and so on.Conclusion: In this study, a UPLC-Q-TOF/MS technique was developed to analyze prototype compounds and their metabolites from XFZYD in serum, which would provide the evidence for further studying the effective compounds of XFZYD.

Identification of Q-Markers from Hedan Tablet by employing “spiderweb”mode and taking compounds'hepatotoxicity into account

Objective Due to the complicated compounds and the synergistic effect of multi-compounds,the quality control and assessment of Chinese materia medica(CMM)encounters a great challenge about how to identify the key compounds,which are directly correlated with its efficacy and safety.On the guidance of study on quality marker(Q-Marker),identification of Q-Markers was performed from Hedan Tablet(HDT)by the aid of the“spider-web”mode and hepatotoxicity evaluation derived from our previous researches and literatures.Methods By the established ultra performance liquid chromatography with photodiode array detector(UPLC-PDA)method,online UPLC-DPPH·and offline antioxidant assay,21 candidate compounds of HDT were systematically investigated and comprehensively evaluated by the“spider-web”mode for them properties of Q-Marker based on“content-stability-activity”.In addition,the Q-Markers related with hepatotoxicity based on our previous researches and literatures were identified.Results Salvianolic acid B(SaB),quercetin-3-O-glucuronide(Qug),isoquercitrin(IQ)and hyperoside(Hyp)were adopted as the preferable Q-Markers of HDT according to the shaded area(A)of tested compounds in“spider-web”mode.Psoralen(Ps),isopsoralen(IP),psoralenoside(PO)and isopsoralenoside(IPO)were also strongly recommended as Q-Markers closely related with safety by considering hepatotoxicity of the accumulated Ps and IP and conversion between glycoside(PO and IPO)and aglycone(Ps and IP).Conclusion This study provided scientific evidence for quality control and assessment of HDT,and also provided a meaningful reference for application of Q-Markers in CMM.

Simultaneous Determination of Eight Constituents in Fruits of Rubus chingii by UPLC

Objective To develop a simple, efficient, and reliable method for routine quantitative analysis of main constituents presented in the fruits of Rubus chingii, which is widely used in Chinese materia medica （CMM）, known as Fupenzi （FPZ） in Chinese. Methods An ultra performance liquid chromatography-photo diode array （UPLC-PDA） system was employed for simultaneous quantification of eight compounds, i. e. adenosine, gallic acid, brevifolin carboxylic acid, ethyl gallate, ellagic acid, kaempferol-3-O-rutinoside, kaempferol-3-O-^-D-glucopyranoside, and tiliroside. The chromatographic analysis was performed on a C18 column using a gradient elution of acetonitrile -0.1% formic acid aqueous solution within a runtime of 25 min. Results All calibration curves were linear （R2〉 0.9997） over the tested ranges. The intra- and inter-day precisions as determined from sample solutions were both less than 2.45% and 2.78%, respectively. The average recoveries for the eight constituents ranged from 94.77% to 101.35% with RSD ≤ 4.41%. The newly-developed method was applied to the quality assessment of various R. chingii samples, including both ripe and unripe fruits of R. ching/i from different habitats. Conclusion The relative levels of the investigated compounds vary remarkably in the fruits of R. chingiicollected from different habitats. As only two of the eight compounds, adenosine and ellagic acid, are determined in the ripe fruits of R. chingii, the results may explain the reason why only the unripe fruits can be used in CMM.

Eight darmarane-type saponins isolated from the roots of Panax notoginseng

Eight darmarane-type saponins,named as sanchirhinoside D(1),3β,6 α-20(S)-6,20-bis(β-D-glucopyranosyloxy)-3-hydroxy dammar-24-en-12-one(2),20(R)-ginsenoside Rg3(3),ginsenosides F2(4)and Rd(5),gypenoside XVII(6),ginsenosides Rb1(7)and ginsenoside V(8)were isolated from the 70%ethanol extract of Panax notoginseng roots by silica gel,octadecyl silane(ODS)column chromatography and HPLC,and identified by chemical and physical methods,especially by spectral analysis.Among them,1 was a new compound and 2 was a new natural product.

Monitoring unbound warfarin in drug combination therapy by pharmacokinetics and fluorospectrometry

Objective: Monitoring the unbound drug concentration in blood in combination therapy is necessary,because its concentration determines the efficacy of drug therapy. This study was designed to explore the effect of Dan Hong Injection(DHI) on the unbound warfarin using two approaches including an in vivo pharmacokinetic and in vitro fluorescence studies.Methods: The effect of DHI on the pharmacokinetic properties of the unbound warfarin was investigated by a microdialysis sampling method coupled with LC–MS/MS. The effect of DHI and salvianolic acid B(Sa B) on warfarin binding with bovine serum albumin(BSA) was conducted by fluorescence spectrometry.Results: The AUC_(0-tn)of warfarin with DHI group was higher than that of warfarin alone group. The result showed that DHI could increase the concentration of unbound warfarin in rat blood, which may be due to the competition between warfarin and DHI as well as its components binding to serum albumin. The competition process was demonstrated by fluorescence study.Conclusion: Combination therapy of DHI with warfarin could enhance the release profile of warfarin from serum protein.

Bioactive xanthones from whole plants of Gentianella acuta

Objective： To study the constituents from the whole plants of Gentianella acuta and their biological activities.Methods： The compounds were isolated by multiple chromatographic methods and the structures of mentioned isolates were determined by routine NMR experiments and chemical methods.Results： A phytochemical investigation to obtain intestine motility inhibitor resulted in the isolation of one new xanthone glycoside,gentixanthonoside A（1）,along with nine tetrahydroxanthones,1,3,5R,8S-tetrahydroxy-5,6,7,8-tetrahydroxanthone（2）,1,3,5S,8S-tetrahydroxy-5,6,7,8-tetrahydroxanthone（3）,amarellin E（4）,amarellin F（5）,swertiachoside B（6）,amarellin D（7）,amarellin C（8）,amarellin A（9）,and amarellin B（10）from the whole plants of G.acuta.Conclusion： Compounds 2 –10 showed significant reduce effects on contraction tension at 40 μM.

Ultra-High Performance Liquid Chromatography/Ion Mobility-Quadrupole Time-of-Flight Mass Spectrometry and Database-Driven Automatic Peak Annotation for the Rapid Profiling and Characterization of the Multicomponents from Stephaniae Tetrandrae Radix(Fang-Ji)

Objective:Quality control of traditional Chinese medicine(TCM)begins with the chemical basis elucidation.The root of Stephania tetrandra has long been utilized as an antirheumatic,analgesic,and diuretic TCM,Stephaniae Tetrandrae Radix(STR;Fang-Ji).Powerful analytical strategies enabling its multicomponent characterization is still rare.Methods:A rapid,reliable,and enhanced profiling approach,by ultra-high performance liquid chromatography coupled with ion mobility/quadrupole time-of-flight mass spectrometry(UHPLC/IM-QTOF-MS)and automatic peak annotation facilitated by computational matching of in-house library,was established and utilized to characterize the multicomponents from STR.A knockout strategy was utilized by automated valve switching to overcome the interference of predominant peaks.Results:Good chromatographic separation was achieved within 17 min on a reversed-phase BEH C18 column eluted with acetonitrile/0.1%ammonium hydroxide in water,while data-independent high-definition MS^(E)(HDMS^(E))in positive mode was applied to acquire the MS^(2)data by using a Vion TM IM-QTOF instrument,which in theory,could cover all the profiled precursor ions.An in-house library of 163 compounds was established and incorporated into the UNIFITM platform.By feat of these efforts,we were able to identify or tentatively characterize 76 alkaloids from the methanolic extract of STR,including 14 aporphine-type,four morphine-type,48 bisbenzylisoquinoline-type,seven tetrahydroprotoberberine-type,one protopine-type,one benzylisoquinoline-type,and one other.Four-dimensional information,such as the retention time,collision cross section(CCS),high-accuracy MS1 and MS2 data,for each component was provided.Conclusions:The systematic multicomponent characterization of STR was accomplished with high coverage,high degree of automation,and high reliability.