Simultaneous determination of eleven volatile components in Cinnamomi Oleum by GC-MS / 中国中药杂志

A gas chromatography-triple quadrupole mass spectrometry(GC-MS) method was established for the simultaneous determination of eleven volatile components in Cinnamomi Oleum and the chemical pattern recognition was utilized to evaluate the quality of essential oil obtained from Cinnamomi Fructus medicinal materials in various habitats. The Cinnamomi Fructus medicinal materials were treated by water distillation, analyzed using GC-MS, and detected by selective ion monitoring(SIM), and the internal standards were used for quantification. The content results of Cinnamomi Oleum from various batches were analyzed by hierarchical clustering analysis(HCA), principal component analysis(PCA), and orthogonal partial least squares-discriminant analysis(OPLS-DA) for the statistic analysis. Eleven components showed good linear relationships within their respective concentration ranges(R~2>0.999 7), with average recoveries of 92.41%-102.1% and RSD of 1.2%-3.2%(n=6). The samples were classified into three categories by HCA and PCA, and 2-nonanone was screened as a marker of variability between batches in combination with OPLS-DA. This method is specific, sensitive, simple, and accurate, and the screened components can be utilized as a basis for the quality control of Cinnamomi Oleum.

Content determination of seven active components of Eucommiae Cortex in aortic vascular endothelial cells of spontaneously hypertensive rats by UPLC-MS/MS / 中国中药杂志

In the present study, the contents of seven active components [genipinic acid(GA), protocatechuic acid(PCA), neochlorogenic acid(NCA), chlorogenic acid(CA), cryptochlorogenic acid(CCA),(+)-pinoresinol di-O-β-D-glucopyranosid(PDG), and(+)-pinoresinol 4'-O-β-D-glucopyranoside(PG)] of Eucommiae Cortex in aortic vascular endothelial cells of spontaneously hypertensive rats(SHR) were simultaneously determined by ultra-high liquid chromatography-triple quadrupole mass spectrometry(UPLC-MS/MS). The qualified SHR models were selected. The primary aortic endothelial cells(VECs) of rats were separated and cultured by ligation and adherence, followed by subculture. After successful identification, an UPLC-MS/MS method for simultaneously determining the contents of GA, PCA, NCA, CA, CCA, PDG, PG in seven components of Eucommiae Cortex in VECs was established, including specificity, linearity, matrix effect, recovery, accuracy, precision and stability. The established method had the lo-west limit of quantification of 0.97-4.95 μg·L~(-1), accuracy of 87.26%-109.6%, extraction recovery of 89.23%-105.3%, matrix effect of 85.86%-106.2%, and stability of 86.00%-112.5%. Therefore, the established accurate UPLC-MS/MS method could rapidly and simultaneously determine the contents of the seven active components of Eucommiae Cortex in VECs of SHRs, which provided a refe-rence for the study of cellular pharmacokinetics of active components of Eucommiae Cortex extract.

Simultaneous determination of content of eight components in Caesalpinia decapetala by UPLC-MS/MS / 中国中药杂志

The present study established the ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) method for simultaneous determination of the content of eight major active components in Caesalpinia decapetala and performed the quality evaluation of C. decapetala from different habitats with the chemical pattern recognition. The analysis was carried out on a Waters BEH C_(18) column(2.1 mm×100 mm, 1.7 μm) at 40 ℃, with the mobile phase of water containing 0.1% formic acid(A) and acetonitrile containing 0.1% formic acid under gradient elution, the flow rate of 0.3 mL·min~(-1), and the injection volume of 1 μL. The electrospray ionization(ESI) source in the negative mode and multiple reaction monitoring(MRM) were used for MS quantitative analysis. The content results were analyzed by the hierarchical cluster analysis(HCA) and principal component analysis(PCA) for the evaluation of the quality difference. Eight components showed good linear relationships within their respective concentration ranges(r>0.999), with the average recoveries of 96.85%-103.4% and RSD of 0.52%-2.8%. The analysis results showed that the quality of samples from different batches was different. The samples were classified into three clusters by HCA and PCA. The method is simple, sensitive, accurate, and efficient, and can be used for the quality evaluation of C. decapetala.

Advances in high-resolution mass spectrometric-based data-mining technologies for detecting and characterizing the components and metabolites of Chinese materia medica / 药学学报

Research on the material basis of Chinese materia medica (CMM) is the basis for modernization of CMM. High-resolution mass spectrometry (HRMS) has become a powerful tool for the qualitative analysis of the components of CMM. Some data-mining strategies based on high-resolution mass spectrometry, such as full-information tandem mass spectrometry scanning acquisition strategy, sequential windowed acquisition of all theoretical fragment ions, mass detect filter, characteristic ion filter, mass spectral tree similarity filter, etc. have greatly promoted the elucidation of the qualitative basis of CMM. In order to provide methods for the rapid discovery and structural characterization of components and metabolites of CMM, this review summarized the advances in HRMS-based data-mining technologies for detecting and characterizing the compounds and metabolites of CMM, which includes class compounds, all compounds and metabolites.

Research on in vivo metabolites of Longxue Tongluo Capsules by liquid chromatography coupled with hybrid ion trap/time-of-flight mass spectrometry / 中国中药杂志

Longxue Tongluo Capsules(LTC) has good efficacy against blood stasis syndrome during the recovery period of ischemic stroke. Its main active ingredient is the phenolic extract of Chinese dragon's blood. In our previous study, the primary mass fragmentation pathways of phenolic derivatives from LTC were clarified. Herein, the metabolites in rat plasma were characterized following the oral administration of loureirin A and loureirin C using liquid chromatography coupled with hybrid ion trap/time-of-flight mass spectro-metry(LC-IT-TOF-MS), with 18 and 55 metabolites identified, respectively. On this basis, with the help of the obtained accurate molecular weight, characteristic fragment ions, reference comparison, combined with LTC database and natural products database self-created in our group, 18 prototypes and 106 metabolites were tentatively identified in rat plasma after oral gavage of LTC at a dose of 500 mg·kg~(-1). Glucuronidation, sulfonation, and methylation were major biotransformation pathways of LTC. This study preliminarily clarified the LTC constituents absorbed into blood and laid the foundation for clarifying the effective substances of LTC.

Simultaneous determination of four components of Shuganning Injection in rat plasma by UPLC-MS/MS and its application to a pharmacokinetic study / 中国中药杂志

This study aims to reveal the pharmacokinetics of Shuganning Injection in normal rats. In this experiment,ultra-high performance liquid chromatography-electrospray-tandem mass spectrometry( UPLC-ESI-MS/MS) was used to establish an analytical method for simultaneous determination of chlorogenic acid,gardenioside,oroxylin A and baicalin in rat plasma. Then,the non-compartmental model( NCA) in Phoenix WinN onL in 6. 4 software was used to fit pharmacokinetic parameters. The methodological validation showed that the linear relationship of the components in rat plasma samples were good( r>0. 995). The recovery rate and matrix effect of plasma samples with low,middle and high concentration were 79. 14%-101. 4%. The intra-day and inter-day precision,accuracy and stability meet the requirements of biological sample analysis. The half-life( t1/2) of chlorogenic acid,gardenioside,oroxylin A did not change significantly and the area under blood concentration-time curve( AUC0-t) is proportional to the dose,which suggested that three components showed a linear kinetic characteristics,but baicalin showed nonlinear kinetic characteristics. Moreover,the retention time of each component in rats was short. The established UPLC-MS/MS quantitative analysis method is rapid,sensitive and accurate,which can be used for the determination of chlorogenic acid,gardenioside,oroxylin A and baicalin in rat plasma and pharmacokinetic study of Shuganning Injection.

Quantification of aristolochic acids and their DNA adducts in mice kidney and liver by HPLC-MS/MS / 中国中药杂志

This study aims to establish a quantitative method of 4 aristolochic acids-DNA adducts in mice kidney and liver based on high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS) for monitoring the content changes of aristolochic acids-DNA adducts. A Shiseido Capcellpak AQ C_(18) column(3 mm×100 mm, 3 μm) was used, with a mixture of 0.2% acetic acid-5 mmol·L~(-1) ammonium acetate as the aqueous phase and methanol as the organic phase for gradient elution. The multiple reaction monitoring(MRM) scanning method under positive mode by electrospray ionization(ESI) was performed for the detection of the aristolochic acids-DNA adducts which formed by combining aristolochic acid Ⅰ/Ⅱ with deoxyadenosine, deoxyguanosine, and deoxycytidine, respectively. Balb/c mice were given Guanmutong extract by gavage, and the relative content of aristolochic acids-DNA adducts in liver and kidney samples were analyzed within 60 days. It was found that the concentration of 4 aristolochic acids-DNA adducts in the kidney was significantly higher than that in the liver, and there were about 15.87 adducts in per 1×10~6 normal deoxynucleosides, which was 4.5-7.5 times than that of the liver. What's more, some adducts can still be detected on the 30 th day after administration. The concentration of the adducts in the liver was highest on the first day after administration, and a second peak appeared during the 7 th to 14 th days. The results indicated that aristolochic acids-DNA adducts are difficult to eliminate in vivo, and it is of great significance to study the mechanism of liver and kidney injury of aristolochic acid.

Study on metabolites of Laportea bulbifera extract in rat feces based on UHPLC-Q-TOF-MS~E technique / 中国中药杂志

This project is to study the metabolites of Laportea bulbifera extract in rat feces. After the SD rats were gavaged with the extract(136 g·kg~(-1), according to the crude drug dose), the metabolites in their feces were detected by UHPLC-Q-TOF-MS~E technique, and the obtained mass spectrometry data was combined with UNIFI software for prediction. The prototype components and metabolites in rat feces were identified with reference materials and related literature. A total of 43 metabolites were identified(including 8 prototype components and 35 metabolites). The metabolic pathways mainly include monocaffeoylquinic acid(hydrogenation reduction, ring-opening cracking, sulfation, hydroxylation, glucuronidation), quercetin(O-C2 bond ring-opening cleavage, C2-C3 double bond reduction, rutin carbonylation) and so on. The metabolites and metabolic process of L. bulbifera extract in rat feces were clarified, which provided a basis for the study of the active substances and its mechanism of action.

Determination of plasma protein binding rates of nine compounds of Inula cappa extraction based on method of equilibrium dialysis / 中国中药杂志

To determine the plasma protein binding rate of the nine compounds in Inula cappa extraction by the method of equilibrium dialysis. The proteins in plasma samples were precipitated by methanol, and the ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) was developed for determination of the concentrations of the nine active compounds, namely chlorogenic acid, scopolin, neochlorogenic acid, cryptochlorogenic acid, 1,3-O-dicaffeoylquinic acid, galuteolin, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, with the internal standard of puerarin. We found that all components have a good linearity(r≥0.999), and accuracy, precision, extraction recovery and stability conformed to the requirements of determination, without endogenous compounds disturbing within the range of optimum concentration. This suggested that the method was stable and reliable, and could be used for the determination of the plasma protein binding rates of the nine active compounds in rat and human plasma of I. cappa. The plasma protein binding rates of the nine active compounds in rat and human plasma respectively were(41.07±0.046)%-(94.95±0.008)%, and(37.66±0.043)%-(97.46±0.013)%. According to the results, there were differences in the plasma protein binding rates of the nine compounds in I. cappa extraction between rat and human.

Simultaneous quantification and consistency analysis of 19 representative components in Longshengzhi Capsules by HPLC-QQQ/MS / 中国中药杂志

Longshengzhi capsule consisting of 12 herbs is widely used in clinically treating cerebral ischemia during recovery period.In this study,in order to investigate the consistency of different batches of Longshengzhi capsules,a high performance liquid chromatography coupled to triple quadrupole mass spectrometry method(HPLC-QQQ/MS) was developed for the determination of 19 representative components in Longshengzhi Capsules within 9 min. Methodology validation indicated this method was simple,rapid,accurate,highly sensitive and reproducible,and it could be used for the content determination of components in Longshengzhi Capsules. The consistency analysis results showed that paeoniflorin and calycosin-7-glucoside in Longshengzhi Capsules had the highest content; RSD value of total content of 19 compounds was 5. 2% and the RSD value of main compounds such as astragaloside and calycosin-7-glucoside was all less than 15%,reflecting good consistency among different batches. This study has provided a scientific method and basis for the quality control and consistency evaluation of Longshengzhi Capsules.

UPLC-Q-TOF-MS/MS analysis on prototypes components and metabolites of effective fractions of Polygonum orientale flower in rat serum and urine / 中国中药杂志

Ultra performance liquid chromatography coupled with time-of-flight mass spectrometry( UPLC-Q-TOF-MS/MS) method was applied to analyze the prototypes and metabolites of the effective components of Polygonum orientale in SD rat serum and urine. The separation was performed on Agilent Eclipse Plus C_(18) column( 2. 1 mm×100 mm,1. 8 μm),with 0. 1% formic acid solution( A)-acetonitrile( B) as the mobile phase for gradient elution. Mass spectrometry data of biological samples were obtained under positive and negative electrospray ion mode. By comparing chromatogram differences between blank samples and drug treatment samples,prototype components and metabolites of the effective components of P. orientale extract were identified. The results showed that 12 metabolites were detected in serum and 26 metabolites in urine( including cross-components) of rats. The main metabolic pathways included hydrogenation,hydroxylation,glucuronidation,sulfation reaction,and methylation-glucuronidation,etc. The method established in this study was reliable and effective for studying the metabolic characteristics of the effective components of P. orientale in rats,and it can provide a reference for further studies on therapeutic material basis of this herb.

Effect of Intestinal Flora in Rats on Metabolism and Transformation of Effective Components in Polygonum orientale Inflorescences / 中国实验方剂学杂志

Objective:To observe the metabolic characteristics of effective components from Polygonum orientale inflorescences in intestinal flora of rats. Method:The incubating samples of effective components from P. orientale inflorescences in rat intestinal flora in vitro were detected by UPLC-ESI-Q-TOF-MS/MS, the mobile phase was consisted of 0.1%formic acid solution-0.1%formic acid acetonitrile solution and eluted in gradient mode at a flow rate of 0.3 mL·min-1.The mass spectral analysis was detected with electrospray ionization under positive ion mode and negative ion mode.The metabolites and possible biotransformation pathways of effective components form P. orientale inflorescences in rat intestinal flora in vitro was analyzed by Metabolite ToolsTM, mass defect filtration(MDF) and other metabolite analysis techniques and combined with the accurate relative molecular weight of the compounds, the fragment ion information and the literature data. Result:Eighteen metabolites were detected after incubation of effective components from P. orientale inflorescences in rat intestinal flora.The main biotransformation pathways were reduction, oxidation, hydrolysis in Ⅰ phase reaction and methylation in Ⅱ phase reaction. Conclusion:The effective components of P. orientale inflorescences can be transformed into a variety of metabolites under the action of intestinal flora in rats.It is suggested that whether the metabolites are bioactive components should be considered when P. orientale inflorescences is used as medicine.

Serum Pharmacochemical Analysis of Periploca forrestii Rhizomes Based on UPLC-Q-TOF-MS / 中国实验方剂学杂志

Objective:To study the serum pharmacochemistry of Periploca forrestii rhizomes,and to investigate the pharmacological material basis of extract of P. forrestii rhizomes in rats. Method:Rapid identification of constituents absorbed into blood was carried out by UPLC-Q-TOF-MS,according to retention time,accurate relative molecular mass and standard substance comparison,these constituents were identified and speculated by Data Analysis,Metabolite Detect and other softwares,then preliminary determination of constituents absorbed into blood of rats after oral administration of extract of P. forrestii rhizomes was investigated. Result:Totally 17 constituents absorbed into blood were detected in serum,ten of them were prototype constituents and the other were metabolites.Seven of the prototypes were identified as 5-O-caffeoylquinic acid,4-O-caffeoylquinic acid,3-O-caffeoylquinic acid,3,4-di-O-caffeoylquinic acid,3,5-di-O-caffeoylquinic acid,4,5-di-O-caffeoylquinic acid and periplocin. Conclusion:These constituents absorbed into blood may be substances that act directly in vivo of P. forrestii rhizomes,and it is helpful to clarify pharmacological material basis and mechanism of this herb.

Kinetic Analysis of Enzymatic Reaction of Active Components from Xinshao Formula in Liver Microsomes of Sham-Operated and Cerebral Ischemia-reperfusion Injury Rats / 中国实验方剂学杂志

Objective: To investigate and compare enzymatic kinetics of scutellarin,apigenin-7-O-glucronide and paeoniflorin from Xinshao fomula in liver microsomes of sham-operated rats and middle cerebral artery occlusion(MCAO) rats with focal cerebral ischemia-reperfusion injury. Method: Xinshao fomula were incubated respectively with liver microsomes of sham-operated rats and MCAO rats,UPLC-MS and substrate elimination method was employed,Michaelis constant(Km),maximum velocity of enzymatic reaction(Vmax) and intrinsic clearance(CLint) of these three components from Xinshao fomula in liver microsomes of sham-operated rats and MCAO rats were calculated,these parameters between different groups were evaluated by statistical analysis. Result: The Km values of scutellarin,apigenin-7-O-glucronide and paeoniflorin in liver microsomes of MCAO rats were (0.798±0.031),(0.213±0.017),(0.499±0.029) μmol·L-1,which were quite different to these in liver microsomes of sham-operated rats.Compared with the sham-operated group,Vmax and CLint values of scutellarin and paeoniflorin in liver microsomes of MCAO rats were significantly reduced(PPVmax of apigenin-7-O-glucronide in liver microsomes of MCAO rat was also significantly reduced(PConclusion: Metabolic rates of these three active components from Xinshao fomula in liver microsomes of MCAO rats with focal cerebral ischemia-reperfusion injury decrease with low elimination rate.

Absorption of Inula cappa extract based on everted intestinal sac method / 中国中药杂志

To investigate the absorptive characteristics of Inula cappa extract based on the rat everted intestinal sac method . Nine representative ingredients in I. cappa extract were selected as the study objects. An UPLC-MS/MS method was established to determine and detect their cumulative absorption amount for expounding the absorptive characteristics of ingredients in different intestinal sections. According to the results， the transport mechanism of 8 compounds showed passive diffusion by the reverted gut sac method. And scopolin was actively transported in the intestine. The best absorption site of chlorogenic acid was duodenum. The best absorption site of cryptochlorogenic acid， 1，3--dicaffeoylquinic acid， luteolin-7-glucoside and 3，4--dicaffeoylquinic acid were jejunum. The best absorption site of neochlorogenic acid， scopolin， 4，5--dicaffeoylquinic acid and 3，5--dicaffeoylquinic acid was ileum. The absorption of all the compounds was affected by pH and bile. All of the nine ingredients in I. cappa extract could be absorbed in intestines， but with differences in the absorption rate， the best absorptive site and mechanism， indicating that the intestinal absorption of I. cappa extract was selective.

Study on absorption activity of five components of Polygonum orientale L. Flower in Caco-2 cell monolayer / 中国药理学通报

Aim To observe the absorption activity of five components of Polygonum orientale L. Flower ex-tract in Caco-2 cell monolayer. Methods The effects of different concentrations, time, temperature, pH and P-glycoprotein inhibitors on Caco-2 cells were investi-gated by UPLC-MS/MS. Results The absorption of five components of Polygonum orientale L. Flower ex-tract presented a concentration-and time-dependent manner, and the uptake of quercetin was reduced in Caco-2 cells after 90 min. There was a highest intake at 37℃,and the uptake of four components was best in acid environment except for the quercetin at pH 6 , which was best at pH5 . The uptake of quercetin and kaempferol was significantly improved after the addition of P-glycoprotein inhibitors of verapamil. Conclusions The cellular uptake mechanisms of the five compo-nents of Polygonum orientale L. Flower extract is man-inly through passive diffusion. P-glycoprotein is in-volved in the uptake of quercetin and kaempferol.

Protective effect of Polygonum orientale flower extract on H₂O₂-induced oxidative damage of HUVEC cells / 中国中药杂志

To investigate the protective effects and mechanism of Polygonum orientale flower extract on H₂O₂-induced oxidative damage of human umbilical vein endothelial cells (HUVEC), H₂O₂ was used to induce the oxidativestress damage on HUVEC cells and efforts were made to screen the low, medium and high drug concentrations of P.orientale flower extract. Cell viability was detected by the MTS assay. The content of lactate dehydrogenase (LDH) and malondialdehyde (MDA), and the activities of superoxidedimutase (SOD) and catalase (CAT) were detected by biochemical kits. The mRNA and protein levels of Bax, Bcl-2 were detected respectively by quantitative real time polymerase chain reaction (qRT-PCR) and Western blot. The protein level of cleaved caspase-3 was detected by Western blot. According to the results, the viability of HUVEC cells was reduced to around 55% after being treated with 120 μmol·L⁻¹ H₂O₂ for 0.5 h. Treatment of H₂O₂ also could increase LDH leakage rate and MDA content and attenuate the activities of SOD and CAT, up-regulate the expression level of Bax and cleaved caspase-3, and down-regulate the expression level of Bcl-2. As compared with H₂O₂ model group, P.orientale flower extract of 50-200 mg·L⁻¹ could increase the viability of HUVEC cells, reduce LDH release and MDA content, enhance the activities of SOD and CAT, down-regulate pro-apoptotic protein cleaved caspase-3 and Bax, and up-regulate apoptosis inhibitory protein Bcl-2. In summary, P.orientale flower extract showed a protective effect on H₂O₂-induced HUVEC cells injury, which may result from enhancing the cell capability of clearing the oxygen free radial, decreasing the production of lipid peroxidation and inhibiting apoptosis.

Characterization of metabolites in rats after gavage of two lignans from fruits of Vitex negundo var. cannabifolia by UFLC-IT-TOF-MS / 中草药

Objective: To investigate in vivo metabolic profiles of two lignans, 6-hydroxy-4-(4-hydroxy-3-methoxyphenyl)-3- hydroxymethyl-7-methoxy-3,4-dihydro-2-naphthaldehyde (VB-1) and vitedoin A (VB-2) in the rats. Methods: A UFLC-IT- TOF-MS method was applied to characterize the prototypes and metabolites of VB-1 and VB-2 in rat feces, urine, bile, and plasma after oral administration. Results: Eleven metabolites of the two parent compounds were detected and two prototypes were identified unambiguously by comparing with references. Analysis of metabolites revealed that glucuronidation, sulfation, and hydroxylation were major biotransformation pathways of two lignans. Conclusion: In this study, under the analysis of metabolites of two lignans, its in vivo metabolic process is basically clarified. The results could be helpful for the further pharmacokinetics and pharmacological evaluations of VB-1 and VB-2.

Absorption characteristics of 5 components of Xinshao extract in Caco-2 cells / 中国中药杂志

To study the absorption characteristics of Xinshao extracts in Caco-2 cells. In this paper, human colon adenocarcinoma cell line Caco-2 cell model was established, and UPLC-MS method was applied to determinate the contents of five components of Xinshao extracts(albiflorin, gallic acid, caffeic acid, scutellarin and apigenin-7-O-glucronide) in cell lysates. This model was also used to study the effect of different drug concentrations, pH, time and temperature on the absorption of five components, investigate the transport of the five components of Xinshao extracts under the conditions with or without P-glycoprotein inhibitors, and predict the absorption mechanism of these five components in Caco-2 cells. The experimental results showed that the absorption of five components of Xinshao extracts in Caco-2 cells was time-dependent at 37 ℃, and concentration-dependent in the range of 0.5-12.5 g•L⁻¹, with a passive diffusion mechanism. At the pH of 4-7.4, the absorption of caffeic acid, scutellarin and apigenin-7-O-glucronide was significantly declined with the increase of pH(P<0.05). At the temperature of 4 to 37 ℃, the absorption of caffeic acid was declined with the increase of temperature, while the absorption of other four components was increased with the increase of temperature. Compared with the control group, caffeic acid and scutellarin cell absorption was significantly higher(P<0.05) after treatment with P-glycoprotein inhibitors(verapamil and cyclosporine A). The results indicated that, the absorption mechanism of five components in Xinshao extracts may be of passive diffusion, and the caffeic acid and scutellarin may be the substrates of P-glycoprotein.

Chemical constituents from the fruits of Vitex negundo var. cannabifolia and their biological activities in vitro / 中国中药杂志

Chemical constituents from the fruits of Vitex negundo var. cannabifolia and their nitric oxide (NO) inhibitory and cytotoxic activities were investigated. The compounds were isolated and purified by various column chromatography, and their structures were identified by physiochemical properties and spectroscopic data. Thirteen lignans and six phenolic compounds were isolated from the CH2Cl2 extract of the fruits of V. negundo var. cannabifolia, respectively. Their structures were elucidated as 6-hydroxy-4-(4-hydroxy-3-methoxyphenyl)-3-hydroxymethyl-7-methoxy-3,4-dihydro-2-naphthaldehyde (1), vitedoin A (2), vitexdoin F (3), detetrahydroconidendrin (4), vitexdoin E (5), 4-oxosesamin (6), L-sesamin (7), (+)-beechenol (8), ligballinol (9), 2-(4-hydroxyphenyl)-6-(3-methoxy-4-hydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane (10), (-)-pinoresinol (11), balanophonin (12), thero-guaiacylglycerol-β-coniferyl aldehyde ether (13), trans-p-coumaryl aldehyde (14), coniferyl aldehyde (15), 5,7-dihydroxychromone (16), trans-3,5-dimethoxy-4-hydroxy-cinnamic aldehyde (17), frambinone (18), and alternariol 4-methyl ether (19). Compounds 8-10,14,18,19 were firstly isolated from Verbenaceae family, compound 13 was obtained from Vitex species, and 6,7,12,15-17 from V. negundo var. cannabifolia for the first time, respectively. The isolated compounds were evaluated for their anti-inflammatory and cytotoxic effects in vitro. Eight compounds (3,5,7,10,11,14,15,17) showed inhibition against NO production in LPS-stimulated RAW 267.4 cells (IC₅₀ in the range of 7.8-81.1 μmol•L⁻¹) and four compounds (1-4) showed cytotoxicity on HepG-2 cells (IC₅₀ in the range of 5.2-24.2 μmol•L⁻¹).