Effects of Host Plants on Development and Immunity of a Generalist Insect Herbivore.

Secondary plant chemistry mediates a variety of communication signals among species, playing a fundamental role in the evolutionary diversification of communities and ecosystems. Herein, we explored diet-mediated host plant effects on development and immune response of a generalist insect herbivore. Vanessa cardui (Nymphalidae) caterpillars were reared on leaves of three host plants that vary in secondary metabolites, Plantago lanceolata (Plantaginaceae), Taraxacum officinale (Asteraceae) and Tithonia diversifolia (Asteraceae). Insect development was evaluated by larval and pupal viabilities, survivorship, and development rate. Immune response was measured as phenoloxidase (PO) activity. Additionally, chemical profiles of the host plants were obtained by liquid chromatograph-mass spectrometry (LC-MS) and the discriminant metabolites were determined using a metabolomic approach. Caterpillars reared on P. lanceolata exhibited the highest larval and pupal viabilities, as well as PO activity, and P. lanceolata leaves were chemically characterized by the presence of iridoid glycosides, phenylpropanoids and flavonoids. Taraxacum officinale leaves were characterized mainly by the presence of phenylpropanoids, flavones O-glycoside and germacranolide-type sesquiterpene lactones; caterpillars reared on this host plant fully developed to the adult stage, however they exhibited lower larval and pupal viabilities compared to individuals reared on P. lanceolata. Conversely, caterpillars reared on T. diversifolia leaves, which contain phenylpropanoids, flavones and diverse furanoheliangolide-type sesquiterpene lactones, were not able to complete larval development and exhibited the lowest PO activity. These findings suggested that V. cardui have adapted to tolerate potentially toxic metabolites occurring in P. lanceolata (iridoid glycosides), however caterpillars were not able to cope with potentially detrimental metabolites occurring in T. diversifolia (furanoheliangolides). Therefore, we suggest that furanoheliangolide-type sesquiterpene lactones were responsible for the poor development and immune response observed for caterpillars reared on T. diversifolia.

The preventive effect of loganin on oxidative stress-induced cellular damage in human keratinocyte HaCaT cells.

Loganin is a type of iridoid glycosides isolated from Corni fructus and is known to have various pharmacological properties, but studies on its antioxidant activity are still lacking. Therefore, in this study, the preventive effect of loganin on oxidative stress-mediated cellular damage in human keratinocyte HaCaT cells was investigated. Our results show that loganin pretreatment in a non-toxic concentration range significantly improved cell survival in hydrogen peroxide (H2O2)-treated HaCaT cells, which was associated with inhibition of cell cycle arrest at the G2/M phase and induction of apoptosis. H2O2-induced DNA damage and reactive oxygen species (ROS) generation were also greatly reduced in the presence of loganin. Moreover, H2O2 treatment enhanced the cytoplasmic release of cytochrome c, upregulation of the Bax/Bcl-2 ratio and degradation of cleavage of poly (ADP-ribose) polymerase, whereas loganin remarkably suppressed these changes. In addition, loganin obviously attenuated H2O2-induced autophagy while inhibiting the increased accumulation of autophagosome proteins, including as microtubule-associated protein 1 light chain 3-II and Beclin-1, and p62, an autophagy substrate protein, in H2O2-treated cells. In conclusion, our current results suggests that loganin could protect HaCaT keratinocytes from H2O2-induced cellular injury by inhibiting mitochondrial dysfunction, autophagy and apoptosis. This finding indicates the applicability of loganin in the prevention and treatment of skin diseases caused by oxidative damage.

Transcriptome profiling reveal key hub genes in co-expression networks involved in Iridoid glycosides biosynthetic machinery in Picrorhiza kurroa.

Picrorhiza kurroa is a medicinal herb rich in hepatoprotective iridoid glycosides, picroside-I (P-I) and picroside-II (P-II). The biosynthetic machinery of picrosides is poorly understood, therefore, 'no-direction' gene co-expression networks were used to extract linked/closed and separated interactions in terpenoid glycosides-specific sub-networks. Transcriptomes generated from different organs, varying for P-I and P-II contents such as shoots grown at 15 and 25 °C and nursery-grown shoots, stolons, and roots resulted in 47,726, 44,958, 40,117, 66,979, and 55,578 annotated transcripts, respectively. Occurrence of 2810 ± 136 nodes and 15,626 ± 696 edges in these networks indicated intense, co-expressed, closed loop interactions. Either deregulation/inhibition of abscisic acid (ABA) biosynthesis/signaling or constitutive degradation of ABA resulted in organ-specific accumulation of P-I and P-II. Biosynthesis, condensation and glucosylation of isoprene units may occur in shoots, roots or stolons; but addition of phenylpropanoid moiety and further modification/s of the iridoid backbone occurs mainly inside vacuoles in roots.

Complexity of gene paralogues resolved in biosynthetic pathway of hepatoprotective iridoid glycosides in a medicinal herb, Picrorhiza kurroa through differential NGS transcriptomes.

Picrorhiza kurroa is a medicinal herb with diverse pharmacological applications due to the presence of iridoid glycosides, picroside-I (P-I), and picroside-II (P-II), among others. Any genetic improvement in this medicinal herb can only be undertaken if the biosynthetic pathway genes are correctly identified. Our previous studies have deciphered biosynthetic pathways for P-I and P-II, however, the occurrence of multiple copies of genes has been a stumbling block in their usage. Therefore, a methodological strategy was designed to identify and prioritize paralogues of pathway genes associated with contents of P-I and P-II. We used differential transcriptomes varying for P-I and P-II contents in different tissues of P. kurroa. All transcripts for a particular pathway gene were identified, clustered based on multiple sequence alignment to notify as a representative of the same gene (≥ 99% sequence identity) or a paralogue of the same gene. Further, individual paralogues were tested for their expression level via qRT-PCR in tissue-specific manner. In total 44 paralogues in 14 key genes have been identified out of which 19 gene paralogues showed the highest expression pattern via qRT-PCR. Overall analysis shortlisted 6 gene paralogues, PKHMGR3, PKPAL2, PKDXPS1, PK4CL2, PKG10H2 and PKIS2 that might be playing role in the biosynthesis of P-I and P-II, however, their functional analysis need to be further validated either through gene silencing or over-expression. The usefulness of this approach can be expanded to other non-model plant species for which transcriptome resources have been generated.

Preference, performance, and chemical defense in an endangered butterfly using novel and ancestral host plants.

Adoption of novel host plants by herbivorous insects can require new adaptations and may entail loss of adaptation to ancestral hosts. We examined relationships between an endangered subspecies of the butterfly Euphydryas editha (Taylor's checkerspot) and three host plant species. Two of the hosts (Castilleja hispida, Castilleja levisecta) were used ancestrally while the other, Plantago lanceolata, is exotic and was adopted more recently. We measured oviposition preference, neonate preference, larval growth, and secondary chemical uptake on all three hosts. Adult females readily laid eggs on all hosts but favored Plantago and tended to avoid C. levisecta. Oviposition preference changed over time. Neonates had no preference among host species, but consistently chose bracts over leaves within both Castilleja species. Larvae developed successfully on all species and grew to similar size on all of them unless they ate only Castilleja leaves (rather than bracts) which limited their growth. Diet strongly influenced secondary chemical uptake by larvae. Larvae that ate Plantago or C. hispida leaves contained the highest concentrations of iridoid glycosides, and iridoid glycoside composition varied with host species and tissue type. Despite having largely switched to a novel exotic host and generally performing better on it, this population has retained breadth in preference and ability to use other hosts.

Microbial Biotransformation of Iridoid Glycosides from Gentiana Rigescens by Penicillium Brasilianum.

This study aimed to investigate the metabolic effects of endophytic fungi in Gentiana rigescens. From the 100 selected morphospecies, strain 7-2 (Penicillium brasilianum) showed a remarkable biocatalytic activity for gentiopicroside and swertiamarin, yielding seven products, including one new compound, 5-ethylidene-8-hydroxy-4,5,6,8-tetrahydropyrano[3,4-c]pyran-1-one (M04), alongside six known compounds. Gentianine (M01) was the only metabolite of swertiamarin in this study, while the remaining ones were all gentiopicroside metabolites. Among these, five compounds: gentianine (M01), (5S,6S)-5-(hydroxymethyl)-6-methyl-5,6-dihydro-1H,3H-pyrano[3,4-c]pyran-1-one (M02), (5R,6S)-5-(hydroxymethyl)-6-methyl-5,6-dihydro-1H,3H-pyrano[3,4-c]pyran-1-one (M03), 2-(3-formyl-2-oxo-3,6-dihydro-2H-pyran-4-yl)but-3-enoic acid (M06), and 2-oxo-4-(1-oxobut-3-en-2-yl)-3,6-dihydro-2H-pyran-3-carboxylic acid (M07) were similar to gentiopicroside metabolites in humans. Screening the metabolic potential of endophytic fungi in Gentiana rigescens provides an outstanding source for assessing the bioactive metabolites of iridoid glycosides. The above findings suggested that the endophytic fungi of G. rigescens possess multi-enzyme systems that mimic metabolic reactions in mammalian organisms.

Dynamic analysis of secondary metabolites in various parts of Scrophularia ningpoensis by liquid chromatography tandem mass spectrometry.

The roots of Scrophularia ningpoensis are used as traditional medicines for thousands of years in China, nevertheless the stems and leaves were discarded as non-medicinal parts. Modern research have indicated the chemical constituents in the stems and leaves are similar to the identified in the roots, and the therapeutic effects of stems and leaves are superior to roots for some disease. In the study, the chemical constituents in roots, stems and leaves of S. ningpoensis were analyzed qualitatively by HPLC-Q-TOF-MS/MS. 40 compounds including 17 iridoid glycosides, 15 phenylpropanoids and 8 flavonoids were identified. Meantime, the dynamic accumulations of six index constituents in various parts were measured by HPLC-DAD. The results indicated the S. ningpoensis stems contained high content of aucubin (30.09 mg/g) and harpagide (28.4 mg/g) in August, and the leaves contained high content of harpagoside (12.02 mg/g) in July. The study provides the basis for the full development and utilization of the resource of stems and leaves from S. ningpoensis.

Critical Phenological Events Affect Chemical Defense of Plant Tissues: Iridoid Glycosides in a Woody Shrub.

Plants are chemically-complex organisms; each individual contains diverse tissue-types, has the ability to differentially allocate secondary metabolites to these tissues and can change this allocation through time. The interaction of variation in chemical defense of different tissue types and variation in chemical defense through time, however, is rarely examined and has not been studied for iridoid glycoside-producing woody plants. In this study, we quantified allocation of iridoid glycosides (IGs) to the leaves, flowers, fruits, and seeds of 25 individuals of a long-lived shrub (Lonicera x bella Zabel, Caprifoliaceae), at five important phenological timepoints (leaf-out, flowering, fruit appearance, fruit ripening, and fruit dispersal) throughout a growing season. We found that leaves had 2x higher IG concentrations during flowering and fruiting than earlier in the season (after leaf-out), and later in the season (after fruit dispersal). The individual IG driving this increase in leaves during reproduction, secologanin, was also the most abundant IG in semiripe fruits. Flowers and seeds were composed of different proportions of individual IGs than fruits or leaves, but did not change across time and had overall low concentrations of IGs. In L. x bella, phenological events such as flowering and fruiting lead to an increase in leaf chemical defense that is likely to influence interactions with leaf-feeders. Our results stress the importance of considering phenology when sampling plants for the quantification of chemical defenses.

[Isolation and identification of endophytic fungi producing harpagoside and harpagide from Scrophularia ningpoensis].

The endophytic fungi from root,main stem,branch and leaf of Scrophularia ningpoensis were isolated from Zhejiang,whether these strains could yield harpagide or harpagoside were tested by HPLC and LC-MS. According to the morphological characteristic and the similarity of the nucleotide sequence of internal transcribed spacer( ITS) between r DNAs,the strains producing harpagide or harpagoside were identified. The results showed that 210 strains were isolated from the samples,which were classified into 9 orders,13 families and 17 genera by morphological study. Harpagide was detected in endogenous fungi ZJ17 and harpagoside was detected in endogenous fungi ZJ25 by HPLC coupled with LC-MS. ZJ17 was identified as Alternaria alternate and ZJ25 was identified as A.gaisen by its morphology and authenticated by ITS( ITS4 and ITS5 regions and the intervening 5. 8 S rDNA region).

Harpagide inhibits neuronal apoptosis and promotes axonal regeneration after spinal cord injury in rats by activating the Wnt/ß-catenin signaling pathway.

The neuronal apoptosis program associated with spinal cord injury (SCI) has a severe impact on spinal cord function, which leads to further secondary and permanent neuronal damage that may cause irreparable damage to the central nervous system. Activation of the Wnt/ß-catenin signaling pathway is effective in reducing apoptosis and preventing SCI. Harpagide is one of the main active constituents of the iridoid class of molecules, which have neuroprotective effects after SCI. In this study, we demonstrated that harpagide attenuated neuronal apoptosis via activation of the Wnt/ß-catenin signaling pathway. This resulted in a promotion of axonal regeneration and an inhibition of glial scar formation, which ultimately improved functional behavioral recovery after SCI in rats. Specifically, the administration of harpagide after SCI increased the expression levels of ß-catenin, c-myc and cyclin D1 proteins in spinal cord neurons, as well as increased the number of motor neurons and reduced the size of the SCI lesion area. In addition, the administration of harpagide after SCI also decreased the protein expression levels as well as the number of cells immuno-stained for the pro-apoptotic proteins Bax and cleaved-caspase 3. The expression level of the anti-apoptotic protein Bcl-2 was also increased. When the Wnt /ß-catenin signaling pathway was inhibited, a weakened anti-apoptotic effect of harpagide was observed. Additionally, the application of harpagide led to an increase in NF200 staining and a reduction in GFAP staining in the SCI injury site. In summary, our study suggested that harpagide may be a promising drug for the treatment of SCI.

Identification of the absorbed components and their metabolites of Tianma-Gouteng granule in rat plasma and bile using ultra-high-performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry.

Tianma-Gouteng granule (TGG), a Chinese herbal formula preparation, is clinically used for the treatment of cardio-cerebrovascular diseases such as hypertension, cerebral ischaemia, acute ischaemic stroke and Parkinson's disease. Although few reports have been published concerning the absorbed prototype components of TGG, the possible metabolic pathways of TGG in vivo remain largely unclear. In this study, a method using UPLC-Q/TOF MS was established for the detection and identification of the absorbed prototype components and related metabolites in rat plasma and bile after oral administration of TGG at high and normal clinical dosages. A total of 68 components were identified or tentatively identified in plasma and bile samples, including absorbed prototypes and their metabolites. The major absorbed components were gastrodin, isorhynchophylline, rhynchophylline, isocorynoxeine, corynoxeine, geissoschizine methyl ether baicalin, baicalein, wogonoside, wogonin, geniposidic acid, leonurine, 2,3,5,4'-tetrahydroxystilbene-2-O-ß-d-glucoside and emodin. The main metabolic pathways of these components involved phase I (isomerization, hydrolysis and reduction) and phase II (glucuronidation and sulfation) reaction, and the phase II biotransformation pathway was predominant. The present study provides rich information on the in vivo absorption and metabolism of TGG, and the results will be helpful for further studies on the pharmacokinetics and pharmacodynamics of TGG.

Enhanced accumulation of harpagide and 8-O-acetyl-harpagide in Melittis melissophyllum L. agitated shoot cultures analyzed by UPLC-MS/MS.

Harpagide and its derivatives have valuable medicinal properties, such as anti-inflammatory, analgesic and potential antirheumatic effects. There is the demand for searching plant species containing these iridoids or developing biotechnological methods to obtain the compounds. The present study investigated the effects of methyl jasmonate (MeJa, 50 µM), ethephon (Eth, 50 µM) and L-phenylalanine (L-Phe, 2.4 g/L of medium), added to previously selected variant of Murashige and Skoog medium (supplemented with plant growth regulators: 6-benzylaminopurine 1.0 mg/L, α-naphthaleneacetic acid 0.5 mg/L, gibberellic acid 0.25 mg/L) on the accumulation of harpagide and 8-O-acetyl-harpagide in Melittis melissophyllum L. agitated shoot cultures. Plant material was harvested 2 and 8 days after the supplementation. Iridoids were quantitatively analyzed by the UPLC-MS/MS method in extracts from the biomass and the culture medium. It was found that all of the variants caused an increase in the accumulation of harpagide. In the biomass harvested after 2 days, the highest harpagide content of 247.3 mg/100 g DW was found for variant F (L-Phe and Eth), and the highest 8-O-acetyl-harpagide content of 138 mg/100 g DW for variant E (L-Phe and MeJa). After 8 days, in some variants, a portion of the metabolites was released into the culture medium. Considering the total amount of the compounds (in the biomass and medium), the highest accumulation of harpagide, amounting to 619 mg/100 g DW, was found in variant F, and the highest amount of 8-O-acetyl-harpagide, of 255.4 mg/100 g DW, was found in variant H (L-Phe, MeJa, Eth) when harvested on the 8th day. These amounts were, respectively, 24.7 and 4.8 times higher than in the control culture, and were, respectively, 15 and 6.7 times higher than in the leaves of the soil-grown plant. The total amount of the two iridoids was highest for variant F (0.78% DW) and variant H (0.68% DW) when harvested on the 8th day. The results indicate that the agitated shoot cultures of M. melissophyllum can be a rich source of harpagide and 8-O-acetyl-harpagide, having a potential practical application. To the best of our knowledge we present for the first time the results of the quantitative UPLC-MS/MS analysis of harpagide and 8-O-acetyl-harpagide in M. melissophyllum shoot cultures and the enhancement of their accumulation by means of medium supplementation with elicitors and precursor.

Tentative characterization of precursor compounds and co-factors of pigment formation in production of 'wu mi' from Vaccinium bracteatum Thunb. Leaves.

Vaccinium bracteatum leaves (VBTL) are traditionally used in China to dye rice grains, which assume a deep blue color, named 'Wu mi'. Information on the mechanism of pigment formation is limited. In this study, CIELAB color space parameters were used to represent the color of 'Wu mi'. Precursor compounds of pigments formed during the dyeing process were identified by UPLC Q-TOF MS analysis. The changes in co-factors for pigment formation in VBTL were measured at different growth stages. The L∗ and b∗ values of dyed rice increased as the leaves aged, whereas a∗ values showed irregular changes. Six compounds were tentatively identified as pigment precursors by UPLC Q-TOF MS analysis. The pH and ß-glucosidase activity at different growth stages of VBTL were indicated to be crucial co-factors for pigment formation. A tentative hypothesis is presented that iridoid glycosides are hydrolyzed by acids and ß-glucosidases to form a dialdehyde structure that binds covalently with amino residues of lysine side chains in rice protein molecules.

[Research progress on metabolism and pharmacological activities of several iridoid glycosides].

As a large category of natural products widely present in traditional Chinese medicine, iridoid glycosides have multiple pharmacological activities. Recent researches suggest that iridoid glycosides mainly exist in vivo in the forms of original form, aglycone and a series of their Ⅰ and Ⅱ metabolites under the biotransformation effect, and their metabolites have been proved to have multiple pharmacological activities. The research progress on in vivo metabolism and metabolite activities of several iridoid glycosides would be reviewed in this article, to provide a theoretical basis for the further development and utilization of iridoid compounds and their metabolites.

Loganic Acid Methyltransferase: Insights into the Specificity of Methylation on an Iridoid Glycoside.

Loganin is an iridoid glycoside of interest as both an intermediate in the biosynthesis of indole alkaloids in plants and as a bioactive compound itself. Loganic acid methyltransferase catalyzes the methylation of a monoterpenoid glycoside precursor to produce loganin and demonstrates stereospecificity for the (6S,7R) substrate. We have biochemically characterized this biocatalyst and elucidated the basis for its strict substrate specificity. These studies could help facilitate the design of new classes of monoterpenoid indole alkaloids of pharmaceutical interest.

The Perennial Penstemon: Variation in Defensive Chemistry Across Years, Populations, and Tissues.

Plants produce a variety of secondary metabolites that function as a defense against their natural enemies. Production of these secondary metabolites is genetically controlled, but is also phenotypically plastic and varies in response to both biotic and abiotic factors. Therefore, plant species may vary widely in their chemical defenses and such variation can be evident at temporal, spatial and tissue levels. Focusing on the chemical defenses of a native Colorado wildflower, Penstemon virgatus, we assessed the variation in iridoid glycoside (IG) content across two non-consecutive growing seasons, six natural populations and three tissue types: leaves, stems and flowers. Our results indicate that P. virgatus plants contain high concentrations of IGs (mean = 23.36% dry weight of leaves) and that IGs were differentially allocated among tissue types. Leaves contained the highest concentration of IGs, which varied quantitatively between sampling years, among plant populations, and plant parts. We also quantified leaf herbivore damage at all six populations but we found very little herbivore damage. Our study indicates that the IG concentrations of P. virgatus plants are both spatially and temporally variable. Furthermore, the high concentrations of secondary metabolites combined with the low levels of damage suggest that these plants are well defended against generalist herbivores.

Screening and identification of multiple constituents and their metabolites of Zhi-zi-chi decoction in rat urine and bile by ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry.

Zhi-zi-chi decoction (ZZCD) is a classical formula widely used in Chinese clinical application. In the present study, a novel and efficient strategy has been developed for screening and identification of multiple constituents and their metabolites of ZZCD using ultra-high-performance liquid chromatography combined with triple time-of-flight mass spectrometry. The novel approach of an online data acquisition method dependent on multiple mass defect filter and dynamic background subtraction is combined with multiple data processing techniques. First, a total of 109 potential bioactive compounds were detected in ZZCD. Based on the same instrumental conditions, 100 compounds were found in rat biofluids after oral administration of ZZCD, including 61 original compounds of ZZCD as well as 39 metabolites. Conjugations with sulfate, glucuronate and amino acids were found as the predominant metabolic reaction of ZZCD. As more xenobiotics were detected in urine than those in bile were, it demonstrated that multiple components of ZZCD have undergone comprehensive renal excretion. This study reported the urinary and biliary excretion in rats after oral administration of ZZCD for the first time. The present study expands our knowledge about the constituents and metabolism of ZZCD, which could be very useful for further pharmacological and clinical studies of ZZCD.

Genome sequence and genetic diversity of European ash trees.

Ash trees (genus Fraxinus, family Oleaceae) are widespread throughout the Northern Hemisphere, but are being devastated in Europe by the fungus Hymenoscyphus fraxineus, causing ash dieback, and in North America by the herbivorous beetle Agrilus planipennis. Here we sequence the genome of a low-heterozygosity Fraxinus excelsior tree from Gloucestershire, UK, annotating 38,852 protein-coding genes of which 25% appear ash specific when compared with the genomes of ten other plant species. Analyses of paralogous genes suggest a whole-genome duplication shared with olive (Olea europaea, Oleaceae). We also re-sequence 37 F. excelsior trees from Europe, finding evidence for apparent long-term decline in effective population size. Using our reference sequence, we re-analyse association transcriptomic data, yielding improved markers for reduced susceptibility to ash dieback. Surveys of these markers in British populations suggest that reduced susceptibility to ash dieback may be more widespread in Great Britain than in Denmark. We also present evidence that susceptibility of trees to H. fraxineus is associated with their iridoid glycoside levels. This rapid, integrated, multidisciplinary research response to an emerging health threat in a non-model organism opens the way for mitigation of the epidemic.

Three New Iridoid Glycosides from the Aerial Parts of Asperula involucrata.

Three new iridoid glycosides, named involucratosides A - C (1 - 3), were isolated from the H2 O subextract of crude MeOH extract prepared from the aerial parts of Asperula involucrata along with a known iridoid glycoside (adoxoside), three flavone glycosides (apigenin 7-O-ß-glucopyranoside, luteolin 7-O-ß-glucopyranoside, apigenin 7-O-rutinoside) as well as two phenolic acid derivatives (chlorogenic acid and ferulic acid 4-O-ß-glucopyranoside). Their chemical structures were established by UV, IR, 1D- (1 H, 13 C and JMOD) and 2D- (COSY, HSQC, HMBC and NOESY) NMR experiments and HR-ESI-MS. In addition, the crude extract, subextracts and isolates were evaluated for their xanthine oxidase inhibitory and antioxidant activities in in vitro tests. This is the first report on the chemical composition and bioactivities of A. involucrata.

Identification of absorbed components and metabolites of Zhi-Zi-Hou-Po decoction in rat plasma after oral administration by an untargeted metabolomics-driven strategy based on LC-MS.

In this study, a novel untargeted metabolomics-driven strategy based on LC-MS was used to rapidly screen and identify the absorbed components and metabolites of Zhi-Zi-Hou-Po decoction (ZZHPD) in rat plasma. The plasma sample was obtained from orbital venous of rats after oral administration and pretreated by protein precipitation before analysis. All sample data from total ion chromatograms (TICs) of LC-TOF/MS were aligned and peak picked by XCMS and MetAlign combined to extract three-dimensional datasets (peak code, t R -m/z pairs and ion intensity). Xenobiotics in rat plasma were differentiated from endogenous components by multivariate statistical analysis and then divided into prototype compounds and metabolites by comparing t R -m/z with the chemical compounds of ZZHPD. Combined with fragment ions and structure information of LC-TSQ/MS, a total of 61 compounds, including 35 prototype compounds and 26 metabolites, were rapidly identified or tentatively characterized in rat plasma. Results indicated that iridoid glycosides, monoterpenoids, flavonoids, and lignans were the main absorbed chemical components of ZZHPD. Glucuronidation and sulfation were the main metabolic pathways of ZZHPD compounds in vivo. In addition, there were ring-opening reactions and reduction reactions for iridoid glycosides, hydrolysis for flavonoids, as well as hydroxylation and stereoscopic conversion reactions for lignans. This study offers a systematically applicable approach for rapid screening and identification of xenobiotics and metabolites derived from multi-herb prescription in vivo, and provides useful information for ascertaining bioactive ingredients and action mechanisms of ZZHPD. Graphical Abstract Diagram of untargeted metabolomics-driven strategy for ZZHPD in rat plasma.