•Scutellarin had a wide range of benefits to brain injury.•Eight methylated scutellarein derivatives were synthesized based on metabolism in vivo.•The thrombin inhibition activity, antioxidant activity and physicochemical properties of these derivatives were evaluated.•Preliminary SARs of these derivatives were analyzed.•6-O-methyl-scutellarein (5) showed good biological activity.Scutellarin (1) could be hydrolyzed into scutellarein (2) in vivo and then converted into methylated, sulfated and glucuronidated forms. In order to investigate the biological activities of these methylated metabolites, eight methylated analogs of scutellarein (2) were synthesized via semi-synthetic methods. The antithrombotic activities of these compounds were evaluated through the analyzation of prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and fibrinogen (FIB). Their antioxidant activities were assessed by measuring their scavenging capacities toward 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and the ability to protect PC12 cells against H2O2-induced cytotoxicity. Furthermore, the physicochemical properties of these compounds including aqueous solubility and lipophilicity were also investigated. The results showed that 6-O-methylscutellarein (5) demonstrated potent antithrombotic activity, stronger antioxidant activity and balanced solubility and permeability compared with scutellarin (1), which warrants further development of 5 as a promising lead for the treatment of ischemic cerebrovascular disease.6-O-methyl-scutellarein (5) demonstrated potent antithrombotic activity, stronger antioxidant activity and balanced solubility and permeability compared with scutellarin (1), which warrants further development for the treatment of ischemic cerebrovascular disease.

In order to improve the biological activity and water solubility of scutellarin (1), some derivatives of its main metabolite (scutellarein) were designed and synthesized. All the compounds were tested for their thrombin inhibition activity through the analyzation of thrombin time (TT), activated partial thromboplastin time (APTT), prothrombin time (PT) and fibrinogen (FIB). Their antioxidant activities were assessed by measuring their scavenging capacities toward 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and the ability to protect PC12 cells against H2O2-induced cytotoxicity, their water solubility were also assessed by ultraviolet (UV) spectrophotometer. The results showed that compound 8b demonstrated stronger anticoagulant and antioxidant activity, better water solubility compared with scutellarein (2), which warrants it as a promising agent for the treatment of ischemic cerebrovascular disease.

•Comprehensive metabolomics approach by UHPLC-QTOF/MS and pattern recognition methods.•Invigorate the circulation function of herb pair Gui-Hong.•Fourteen endogenous metabolites were tentatively identified and 16 metabolic pathways were found.•Danggui and Honghua can synergistically invigorate blood circulation.The compatibility of Angelicae Sinensis Radix (Danggui, DG) and Flos Carthami (Honghua, HH), a famous herb pair Gui-Hong (GH), can produce synergistic and promoting blood effects. Although some physiological and pathological function parameters of the acute blood stasis have been investigated, little information about the changes of small metabolites in biofluids has been reported. In present study, global metabolic profiling with ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS) combined with pattern recognition method was performed to discover the underlying blood-activating regulation mechanisms of DG, HH and GH on the acute blood stasis rats induced by subcutaneous injection of adrenaline hydrochloride and ice water bath. The total 14 metabolites (10 in urine and 4 in plasma), up regulated or down regulated (P < 0.05 or 0.01), were identified and contributed to the acute blood stasis progress. These promising identified biomarkers underpin the metabolic pathway including phenylalanine metabolism, sphingolipid metabolism, arachidonic acid metabolism and arginine and proline metabolism are disturbed in the acute blood stasis rats, which identified by using pathway analysis with MetPA. The altered metabolites and hemorheological indexes could be regulated closer to normal level after DG, HH and GH intervention. In term of activate blood circulation function, GH was the most effective as shown by the relative distance in PLS-DA score plots and relative intensity of metabolomics trategy, reflecting the synergic action between Danggui and Honghua. The results demonstrated that biofluids metabolomics was a powerful tool in clinical diagnosis and treatment of syndrome of blood stasis for providing information on changes in metabolites pathways.

•Four groups including 26 ginkgo characteristic compounds were simultaneously studied for the first time.•Response surface methodology was used to optimize the process variables.•UHPLC–MS/MS was the first determination of so many components in ginkgo.•The developed method may be applied for other ginkgo products.An optimized method of ultrasound-assisted extraction followed by ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UAE–UHPLC–TQ/MS2) was proposed for the simultaneous extraction and determination of 26 characteristic components covering four structure types (flavonoids, terpene lactones, ginkgolic acids and phenylpropanols) in ginkgo seeds (GSs). The UAE parameters (ultrasound power, time and solvent-to-material ratio) were optimized using a response surface methodology. This is the first report of the simultaneous analysis of 26 compounds in Ginkgo biloba using UHPLC–TQ/MS2; this analysis afforded good linearity, precision, repeatability and accuracy. UAE–UHPLC–TQ/MS2 was successfully applied to ginkgo seed samples, and the analysis showed that GSs are rich in terpene lactones and could be selected as a healthy food resource. The results suggest that UAE–UHPLC–TQ/MS2 might be able to be utilized as a tool for the quality assessment of samples from GSs or other related products using flavonoids, terpene lactones, ginkgolic acids and phenylpropanols as markers.

•Comprehensive metabolomics approach by UHPLC–QTOF/MS coupled with pattern recognition methods.•Hematopoietic functions of herb pair Gui-Xiong.•Thirteen endogenous metabolites were tentatively identified and 12 metabolic pathways were found.•Danggui and Chuanxiong had synergetic hematopoietic effect.The compatibility of Angelicae Sinensis Radix (Danggui, DG) and Chuanxiong Rhizoma (Chuanxiong, CX), a famous herb pair Gui-Xiong (GX), can produce synergistic and complementary hematopoiesis. In present study, global metabolic profiling with ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC–QTOF/MS) combined with pattern recognition method was performed to discover the underlying hematopoietic regulation mechanisms of DG, CX and GX on hemolytic and aplastic anemia rats (HAA) induced by acetyl phenylhydrazine (APH) and cyclophosphamide (CP). Thirteen endogenous metabolites contributing to the separation of model group and control group were tentatively identified. The levels of LPCs including lysoPC (18:0), lysoPC (20:4), lysoPC (16:0) and lysoPC (18:2), sphinganine, nicotinic acid, thiamine pyrophosphate, phytosphingosine, and glycerophosphocholine increased significantly (p < 0.05) in HAA, while the levels of oleic acid, 8,11,14-eicosatrienoic acid, ceramides (d18:1/14:0), and 17a-hydroxypregnenolone decreased significantly (p < 0.05) in comparison with control rats. Those endogenous metabolites were chiefly involved in thiamine metabolism and sphingolipid metabolism. The metabolic deviations could be regulated closer to normal level after DG, CX and GX intervention. In term of hematopoietic function, GX was the most effective as shown by the relative distance in PLS-DA score plots and relative intensity of metabolomic strategy, reflecting the synergic action between DG and CX. The relative distance calculation was firstly used in metabolomics for semi-quantization.

•UHPLC–TQ-MS/MS is used for the quantification of Foshousan and its combination herbs.•Three aromatic acids and six phthalides were determined.•The content changes of main components were compared.•The proposed method was fully validated.•The quantification results supported the previous synergistic effects.Fo-Shou-San (FSS) is an ancient and classic formula comprised of Angelicae Sinensis Radix (Danggui, DG) and Chuanxiong Rhizoma (Chuanxiong, CX) in a weight ratio of 3:2 with nourishing blood and dissipating blood stasis activities for the treatment of blood deficiency and blood stasis. In this study, a ultra-high-performance liquid chromatography coupled with a triple quadrupole electrospray tandem mass spectrometry (UHPLC–TQ-MS/MS) method was developed for simultaneous quantification of three aromatic acids (chlorogenic acid, caffeic acid, ferulic acid) and six phthalides (senkyunolide I, senkyunolide H, senkyunolide A, butylphthalide, ligustilide and butylidenephthalide) in DG, CX and FSS. The nine components were simultaneously determined within 10 min. The proposed method was fully validated in terms of linearity, sensitivity, precision, repeatability as well as recovery. The results showed that there were significant differences in their contents of DG and CX, and there were remarkable differences between the theorized content and observed content in FSS. The content of each component in formulae was not just the simple addition among its content in the single herbs. These research results might be helpful to illustrate the drug interactions during decocting process of herb pair according to the quantity changes of these marker compounds, which would lay foundation to further reveal the compatibility rule of the herb pair and other related formulae.

•UPLC-TQ/MS is used for DBD tissue distribution investigation.•The method was simple, rapid, reliable and sensitive.•Blood deficiency rats were conducted by bleeding.•DBD tissue distribution profiles were altered in blood deficiency rats.Astragali Radix (AR) and Angelicae Sinensis Radix (ASR) were frequently combined and used in China as herbal pair called as Danggui Buxue Decoction (DBD) for treatment of blood deficiency syndrome, such as women's ailments. This study is to investigate the tissue distribution profiles of five major bio-active constituents (ferulic acid, caffeic acid, calycosin-7-O-β-glucoside, ononin and astragaloside IV) in DBD after oral administration of DBD in blood deficiency rats, and to compare the difference between normal and blood deficiency rats. The blood deficiency rats were induced by bleeding from orbit at the dosages of 5.0 mL kg−1 every day, and the experimental period was 12 days. At the finally day of experimental period, both normal and blood deficiency rats were orally administrated with DBD, and then the tissues samples were collected at different time points. Ferulic acid, caffeic acid, calycosin-7-O-β-glucoside, ononin and astragaloside IV in different tissues were detected simultaneously by UPLC-TQ/MS, and the histograms were drawn. The results showed that the overall trend was CLiver > CKidney > CHeart > CSpleen > CLung, CC-30 min > CM-30 min > CM-60 min > CC-5 min > CM-5 min > CC-60 min > CM-240 min > CC-240 min. The contents of the detected compounds in liver were more than that in other tissues no matter in normal or blood deficiency rats. Compared to normal rats, partial contents of the compounds in blood deficiency rats’ tissues at different time points had significant difference (P < 0.05). This study was the first report about tissue distribution investigation in blood deficiency animals which is conducted by bleeding. And the results demonstrated that the five DBD components in normal and blood deficiency rats had obvious differences in some organs and time points, suggesting that the blood flow and perfusion rate of the organ were altered in blood deficiency animals.

Scutellarein, which is the main metabolite of scutellarin in vivo, has better neuroprotective effects than scutellarin against brain ischemia. However, there are few studies on the metabolic profile of scutellarein in vivo. In this study, a method based on ultra performance liquid chromatography/quadrupole-time-of-flight mass spectrometry was applied to identify the scutellarein and its metabolites in rat plasma, urine and feces after oral administration of scutellarein. Using MSE and mass defect filter techniques, scutellarein (M1), the glucuronide conjugate of scutellarein (M2, M3), scutellarin (M4), 6,7-di-O-β-D-glucuronide scutellarein (M5), the glucuronide conjugate of apigenin (M6), 4′-O-sulfate scutellarein (M7), the methylated conjugate of scutellarein (M8), the methylated and glucuronide conjugate of scutellarein (M9), and the acetylated conjugate of scutellarein (M10) were detected in rat urine. M1, M2, M4, and M10 were detected in rat plasma and only M10 was found in feces. Our studies indicated that scutellarein can be metabolised by glucuronide conjugation, dehydroxylation, sulfation, methylation and acetylation in vivo after oral administration.

O-Alkylated quercetin analogs were synthesized and their anticancer activities were assessed by a high-throughout screening (HTS) method. The structure–activity relationships (SAR) showed that introduction of long alkyl chain such as propyl group at the C-3 OH position or short alkyl chain such as ethyl group at the C-4′ OH position were very important for keeping inhibitory activities against the 16 cancer cell lines. Furthermore, when the two n-butyl groups were introduced into the C-3, C-7 or C-4′, C-7 positions, the anticancer activity was enhanced.

•Interaction analysis of multi-constituents in medicinal herbs during decocting in vitro.•Chemical fuzzy identification strategy was used for herbal constituent classification.•The approach is based on UPLC–TQ-MS, chemical fuzzy identification strategy and poly-proportion design.•The approach has been successfully applied to the interaction analysis of multi-constituents in kansui and licorice.A novel and generally applicable approach was established for the herb–herb interaction analysis when decocting together by using ultra-high-performance liquid chromatography coupled with a triple quadrupole electrospray tandem mass spectrometer and fuzzy chemical identification with poly-proportion design. A simple programme was originally developed for the rapid identification and classification of herbal constituents on the basis of the establishment of herbal constituent databases, recognition of the reference compound peaks, selection of the diagnostic ions or fragmentation pathways, classification of chemical groups and formation of group networks. In this study, the exact structures of the chemical constituents did not need to be determined, and only the constituents attributed to different groups were further considered for quantitative analysis. Such a novel approach was successfully applied to kansui–licorice interaction analysis when decocting together. A total of 26 constituents from kansui and 45 constituents from licorice were classified into different chemical groups, and they were further quantitatively analyzed on the basis of semi-symmetric proportion design. The results showed that kansui could significantly promote the concentration of most triterpenoid saponins, phenylpropanoids and their glycosides (the constituents from licorice) in solution when co-decocting, and licorice could clearly promote the concentration of most diterpenes and triterpenes (the constituents from kansui) in solution, potentially explaining the incompatibility of kansui and licorice. Overall, the presently developed strategy should be useful for the interaction analysis for complex mixtures containing various complicated constituents, such as herbal, environmental, agricultural and biological samples.

Four series of acid amides were synthesized, and through measurement using a fluorogenic substrate assay with human recombinant MMP-1, MMP-2 and MMP-9, compound 3f showed considerable inhibitory activities against MMP-2, MMP-9 and the best selectivity over MMP-1. Preliminary structure–activity relationship analysis indicated that caffeic acid amides with electron-donating groups at para-position of amino phenyl group showed better inhibitory activities and selectivity than those with electron-withdrawing groups, and the presence of adjacent dihydroxy in the caffeoyl group was very important for the MMP-2 and MMP-9 inhibitory activities.Four series of acid amides were synthesized, preliminary structure–activity relationship analysis indicated that caffeic acid amides with electron-donating groups at para-position of amino phenyl group showed better inhibitory activities and selectivity than those with electron-withdrawing groups, and the presence of adjacent dihydroxy in the caffeoyl group was very important for the MMP-2 and MMP-9 inhibitory activities.

Based on metabolic mechanism of scutellarin in vivo that scutellarin could be hydrolyzed into scutellarein by β-glucuronide enzyme, some glucose-containing scutellarein derivatives were designed and synthesized through the introduction of glucose moiety at C-7 position of scutellarein via a glucosidic bond. Biological activity evaluation showed that these glucose-containing scutellarein derivatives exhibited potent DPPH radical scavenging activities. Furthermore, the improvement of physicochemical properties such as anticoagulant and neuroprotective activities alongside with the water solubility was achieved by introducing glucose. These findings suggest that the introduction of the glucose moiety to scutellarein wattants further development of this kind of compounds as neuroprotective agents.Based on metabolic mechanism of scutellarin in vivo that scutellarin could be hydrolyzed into scutellarein by β-glucuronide enzyme, some glucose-containing scutellarein derivatives were designed and synthesized through the introduction of glucose moiety at C-7 position of scutellarein via a glucosidic bond.

This work presented a new analytical methodology based on hydrophilic interaction ultra-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry in multiple-reaction monitoring mode for analysis of 24 underivatized free amino acids (FAAs) in functional foods. The proposed method was first reported and validated by assessing the matrix effects, linearity, limit of detections and limit of quantifications, precision, repeatability, stability and recovery of all target compounds, and it was used to determine the nutritional substances of FAAs in ginkgo seeds and further elucidate the nutritional value of this functional food. The result showed that ginkgo seed turned out to be a good source of FAAs with high levels of several essential FAAs and to have a good nutritional value. Furthermore, the principal component analysis was performed to classify the ginkgo seed samples on the basis of 24 FAAs. As a result, the samples could be mainly clustered into three groups, which were similar to areas classification. Overall, the presented method would be useful for the investigation of amino acids in edible plants and agricultural products.

In blood, quercetin is mainly found in metabolized forms. In order to study the activities of these quercetin metabolites in cardiovascular disease, 17 methylquercetin derivatives were synthesized based on metabolism in vivo, their thrombin inhibition activity were evaluated through the analyzation of prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and fibrinogen (FIB). The results showed that 6 methylquercetin derivatives had stronger inhibitory activities than that of quercetin. Preliminary SARs analysis showed that hydroxyl groups at C-3′ and C-4′ position in the B-ring and hydroxyl group at C-3 position in the C-ring played key roles in the thrombin inhibitory activity. The findings of this study would provide information for the exploitation and utilization of quercetin as thrombin inhibitor for thrombotic disease treatment.Graphical abstract17 methylquercetin derivatives were synthesized based on metabolism in vivo, their thrombin inhibition activity were evaluated and preliminary SARs of these derivatives as thrombin inhibitors were analyzed.Highlights► Flavonoids maybe promising lead compounds as thrombin inhibitors. ► 17 methylquercetin derivatives were synthesized based on metabolism in vivo. ► The thrombin inhibition activity of 17 methylquercetin derivatives were evaluated. ► Preliminary SARs of these derivatives as thrombin inhibitors were analyzed.

Two new fatty alcohols named as (7S,8R,11S)-nonacosanetriol (1) and (10R,12R,15S)-nonacosanetriol (2), along with eight known compounds including ginkgolic acid (3), hydroginkgolic acid (4), sciadopitysin (5), ginkgetin (6), isoginkgetin (7), ginkgolide A (8), ginkgolide B (9) and ginkgolide C (10) have been isolated from the petroleum ether extract of Ginkgo biloba sarcotesta. Their structures were elucidated by means of chemical and extensive spectroscopic analysis. The absolute stereochemistry of compounds 1 and 2 was elucidated on the spectroscopic analysis of the R- and S-MTPA esters. Compounds 1 and 2 exhibited slight activity of antithrombin and moderate activity of antiplatelet aggregation in vitro. This was the first report regarding the anticoagulative activities of biflavonoids in G. biloba, and isoginkgetin (7) showed significant antithrombin and antiplatelet aggregation activity.Highlights► Two new nonacosanetriols from Ginkgo biloba. ► It was the first report about the anticoagulative activities of biflavonoids. ► The stereochemistry of 1 and 2 was elucidated by analysis of Mosher esters.

The anticancer activities of alkyl esters and NO-donors of ferulic acid (FA) and caffeic acid (CA) were assessed by a high-throughout screening (HTS) method, and the structure–activity relationships were described. CA alkyl esters had better anticancer activities than FA alkyl esters with the same alkyl substituent. Mono-nitrates and phenylfuroxan nitrates were more potent than the dual nitrates. Phenylsulfonylfuroxan nitrates of FA, especially compounds 8b–8d, exhibited more potent activities in anticancer.

Synthetic studies on the construction of 7-O-methylquercetin from quercetin in 36% yield in four steps is reported, this strategy relies on one hand on the selective protection of the catechol group with dichlorodiphenylmethane using dephenyl ether as a solvent, on the other hand, on the selective protection of the 3-hydroxyl group with benzyl bromide, these two different protecting groups can be removed under the same hydrogenation.

Two new isoflavone tetraglycosides (1 and 2) and six known compounds were isolated from the leaves of Sophora japonica. The new glycosides are genistein 7-O-β-d-glucopyranoside-4′-O-(6′′′-O-α-l-rhamnopyranosyl)-β-sophoroside (1) and genistein 7-O-α-l-rhamnopyranoside-4′-O-(6′′′-O-α-l-rhamnopyranosyl)-β-sophoroside (2). The structures of compounds 1 and 2 were established primarily by NMR experiments and chemical methods, and they are the first reported naturally occurring isoflavone glycosides with four attached sugar residues.

•This is the first reporting the dose-effect relationship of scutellarin, scutellarein.•An HILIC-MS/MS method is established for amino acids determination of brain tissue.•This is the first reporting the protective mechanisms of scutellarin, scutellarein.•The result provided useful information for further study of scutellarin, scutellarein.Scutellarin had protective effects against neuronal injury, however, there are few studies on the protective effect of scutellarein, which is the main metabolite of scutellarin in vivo. This study investigated whether the neural injury by ischemia/reperfusion would be influenced by different doses of scutellarin and scutellarein. Male Wistar rats were orally administered with scutellarin and scutellarein at the doses of 0.09, 0.17, 0.35, 0.70, 1.40 mmol/kg, respectively; then after six consecutive days, they were subjected to global ischemia by occlusion of the bilateral common carotid arteries (BCCAO). After reperfusion for about 21 h, neurological and histological examinations were performed. The present results showed that scutellarein attenuated neuronal cell damage, reduced cerebral water content, regulated the expression of glutamic acid (Glu), aspartic acid (Asp), glycine (Gly), γ-aminobutyric acid (GABA) and taurine (Tau), and improved the Ca2 +-ATPase and Na+,K+-ATPase activity. Meanwhile, significant difference was found among various doses of scutellarin and scutellarein. Our studies indicated that scutellarin and scutellarein could improve neuronal injury, and scutellarein had better protective effect than scutellarin in rat cerebral ischemia.

Our previous studies showed that after oral administration of an Huang-Lian-Jie-Du-Tang (HLJDT) decoction, there is a higher concentration of the pure components, berberine, baicalin and gardenoside in the plasma of Middle cerebral artery occlusion (MCAO) rats than in sham-operated rats, The aim of the present study was to determine whether these components could be reliably measured in MCAO rat tissues. First, the plasma concentration–time profiles of berberine, palmatine, baicalin, baicalein and gardenoside were characterised in MCAO rats after oral administration of the aqueous extract of HLJDT. Subsequently, liver, lung and kidney tissues were obtained from sudden death MCAO rats in the absorption phase (0.25 h), the distribution phase (1.0 h) and the elimination phase (8.0 h) after administration of the HLJDT aqueous extract. An HPLC method was developed and validated for the determination of the distribution characteristics of berberine, palmatine, baicalin, baicalein and gardenoside simultaneously from the above-mentioned rat tissues. The results indicated that berberine, palmatine, baicalin and baicalein distributed rapidly and accumulated at high levels in the lung, while gardenoside distributed widely in the lung and the kidney. To the best of our knowledge, this is the first report to describe the distribution of the active ingredients derived from HLJDT in MCAO rat tissues. The tissue distribution results provide a biopharmaceutical basis for the design of the clinic application of HLJDT in cerebrovascular disease.

Scutellarein, which is the main metabolite of scutellarin in vivo, has better neuroprotective effects than scutellarin against brain ischemia. However, there are few studies on the metabolic profile of scutellarein in vivo. In this study, a method based on ultra performance liquid chromatography/quadrupole-time-of-flight mass spectrometry was applied to identify the scutellarein and its metabolites in rat plasma, urine and feces after oral administration of scutellarein. Using MSE and mass defect filter techniques, scutellarein (M1), the glucuronide conjugate of scutellarein (M2, M3), scutellarin (M4), 6,7-di-O-β-D-glucuronide scutellarein (M5), the glucuronide conjugate of apigenin (M6), 4′-O-sulfate scutellarein (M7), the methylated conjugate of scutellarein (M8), the methylated and glucuronide conjugate of scutellarein (M9), and the acetylated conjugate of scutellarein (M10) were detected in rat urine. M1, M2, M4, and M10 were detected in rat plasma and only M10 was found in feces. Our studies indicated that scutellarein can be metabolised by glucuronide conjugation, dehydroxylation, sulfation, methylation and acetylation in vivo after oral administration.