Discovery and validation of COX2 as a target of flavonoids in Apocyni Veneti Folium: Implications for the treatment of liver injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Apocyni Veneti Folium (AVF), a popular traditional Chinese medicine (TCM), is known for its effects in soothing the liver and nerves and eliminating heat and water. It is relevant from an ethnopharmacological perspective. Pharmacological research has confirmed its benefits on antihypertension, antihyperlipidemia, antidepression, liver protection, immune system boosting, antiaging, and diabetic vascular lesions. Previous studies have shown that flavonoids, the active ingredients, have a hepatoprotective effect. However, the exact mechanism has not been clarified. AIM OF THE STUDY: This study aimed to identify the active flavonoids in AVF and their corresponding targets for liver injury. Multiple methods were introduced to confirm the targets. MATERIAL AND METHODS: AVF compounds were analyzed using liquid chromatography-mass spectrometry (LC-MS). Then, network pharmacology was utilized to screen potential hepatoprotection targets of the compounds. An enzyme activity assay was performed to determine the effect of the compounds on the targets. Biolayer interferometry (BLI) was applied to confirm the direct interaction between the compounds and the targets. RESULTS: A total of 71 compounds were identified by LC-MS and 19 compounds and 112 shared targets were screened using network pharmacology. These common targets were primarily involved in the TNF signaling pathway, cancer pathways, hepatitis B, drug responses, and negative regulation of the apoptotic process. Flavonoids were the primary pharmacological substance basis of AVF. The cyclooxygenase 2 (COX2) protein was one of the direct targets of flavonoids in AVF. The enzyme activity assay and BLI-based intermolecular interactions demonstrated that the compounds astragalin, isoquercitrin, and hyperoside exhibited stronger inhibition of enzyme activity and a higher affinity with COX2 compared to epigallocatechin, quercetin, and catechin. CONCLUSIONS: COX2 was preliminarily identified as a target of flavonoids, and the mechanism of the hepatoprotective effect of AVF might be linked to flavonoids inhibiting the activity of COX2. The findings can establish the foundation for future research on the traditional hepatoprotective effect of AVF on the liver and for clinical studies on liver disorders.

Integrating serum pharmacochemistry and network pharmacology to identify chemical markers for quality control of Apocyni Veneti Folium.

INTRODUCTION: Apocyni Veneti Folium (AVF) is a commonly used traditional Chinese medicinal herb for the treatment of hypertension. Chemical markers are crucial for the quality control of herbal medicines; however, the therapeutic components of AVF remain to be well elucidated. OBJECTIVES: This study was intended to integrate serum pharmacochemistry and network pharmacology to identify chemical markers of AVF and establish an efficacy-related quality control method of AVF. MATERIAL AND METHODS: Ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS/MS) was applied to identify the absorbed AVF constituents in rat serum. Network pharmacology was further used to identify anti-hypertension-related chemical markers. Subsequently, a quantitative method was established using UPLC with diode array detection (DAD) and applied for quality evaluation of commercial AVF samples. RESULTS: Thirteen prototype constituents were unequivocally or tentatively characterized in serum samples, among which quercetin, kaempferol, hyperoside, isoquercitrin, chlorogenic acid, cryptochlorogenic acid, and neochlorogenic acid were identified as dominant chemicals related to anti-hypertensive efficacy. The quantitative data showed that the total contents of seven marker components even showed 2-fold variation among 14 batches of commercial AVF samples with RSD values ranging from 12.15% to 75.61%. Hierarchical cluster analysis and heatmap analysis showed that 14 batches of commercial AVF samples could be divided into three main groups. CONCLUSION: The chemical markers obtained from this study could be applicable for efficacy-related quality control of AVF.

Metabolomics characterizes metabolic changes of Apocyni Veneti Folium in response to salt stress.

Apocyni Veneti Folium (AVF) has been raised great interest in the antioxidant properties recently for the preservation of human health. However, little research was found on the integrate metabolites except our previous investigation on the variations of the bioactive constituents. To understand the salt-tolerant mechanisms of the halophyte, metabolomic platform based on ultra-fast liquid chromatography tandem triple time-of-flight mass/mass spectrometer was applied in this study. The results showed that metabolic profiles were separated and differentiated among groups based on multivariate statistical analysis; different metabolites belonged to various chemical classes. Besides, phenylpropanoid pathway and terpenoid biosynthesis were disturbed in all salt-stressed AVF and low salt-treated group appeared to be better than other samples in terms of relative contents (peak areas) of the wide variety of bioactive components and physiological variations of photosynthetic pigments, osmotic homeostasis, lipid peroxidation product and antioxidative enzymes. This study may provide additional insight into the salt-tolerant mechanisms and the quality assessment of AVF in a holistic level based on the plant metabolomics.

Variations in Physiology and Multiple Bioactive Constituents under Salt Stress Provide Insight into the Quality Evaluation of Apocyni Veneti Folium.

As one of the major abiotic stresses, salinity stress may affect the physiology and biochemical components of Apocynum venetum L. To systematically evaluate the quality of Apocyni Veneti Folium (AVF) from the perspective of physiological and the wide variety of bioactive components response to various concentrations of salt stress, this experiment was arranged on the basis of ultra-fast liquid chromatography tandem triple quadrupole mass spectrometry (UFLC-QTRAP-MS/MS) technology and multivariate statistical analysis. Physiological characteristics of photosynthetic pigments, osmotic homeostasis, lipid peroxidation product, and antioxidative enzymes were introduced to investigate the salt tolerance mechanism of AVF under salinity treatments of four concentrations (0, 100, 200, and 300 mM NaCl, respectively). Furthermore, a total of 43 bioactive constituents, including 14 amino acids, nine nucleosides, six organic acids, and 14 flavonoids were quantified in AVF under salt stress. In addition, multivariate statistical analysis, including hierarchical clustering analysis, principal component analysis (PCA), and gray relational analysis (GRA) was employed to systematically cluster, distinguish, and evaluate the samples, respectively. Compared with the control, the results demonstrated that 200 mM and 100 mM salt stress contributed to maintain high quality of photosynthesis, osmotic balance, antioxidant enzyme activity, and the accumulation of metabolites, except for total organic acids, and the quality of AVF obtained by these two groups was better than others; however, under severe stress, the accumulation of the oxidative damage and the reduction of metabolite caused by inefficiently scavenging reactive oxygen species (ROS) lead to lower quality. In summary, the proposed method may provide integrated information for the quality evaluation of AVF and other salt-tolerant Chinese medicines.

Quality Evaluation of Apocyni Veneti Folium from Different Habitats and Commercial Herbs Based on Simultaneous Determination of Multiple Bioactive Constituents Combined with Multivariate Statistical Analysis.

Apocyni Veneti Folium (AVF) is a kind of staple traditional Chinese medicine with vast clinical consumption because of its positive effects. However, due to the habitats and adulterants, its quality is uneven. To control the quality of this medicinal herb, in this study, the quality of AVF was evaluated based on simultaneous determination of multiple bioactive constituents combined with multivariate statistical analysis. A reliable method based on ultra-fast liquid chromatography tandem triple quadrupole mass spectrometry (UFLC-QTRAP-MS/MS) was developed for the simultaneous determination of a total of 43 constituents, including 15 flavonoids, 6 organic acids, 13 amino acids, and 9 nucleosides in 41 Luobumaye samples from different habitats and commercial herbs. Furthermore, according to the contents of these 43 constituents, principal component analysis (PCA) was employed to classify and distinguish between AVF and its adulterants, leaves of Poacynum hendersonii (PHF), and gray relational analysis (GRA) was performed to evaluate the quality of the samples. The proposed method was successfully applied to the comprehensive quality evaluation of AVF, and all results demonstrated that the quality of AVF was higher than the PHF. This study will provide comprehensive information necessary for the quality control of AVF.

Site-specific accumulation and dynamic change of flavonoids in Apocyni Veneti Folium.

Site-specific accumulation of flavonoids in Apocyni Veneti Folium was determined by laser scanning confocal microscope (LSCM) and the localization of catechins also was observed via vanillin-HCl staining under the conventional optical microscope. The contents of five flavonoids in Apocyni Veneti Folium from different harvest times and growth parts were measured using HPLC method. LSCM observation showed that flavonoids are accumulated in cuticle of epidermal cells and vessel walls, especially in protoplasts and nucleolus of the collenchyma cells and the epidermal cells. Catechins are localized in the palisade parenchyma cells and vessel walls, particularly in the laticifers found in the phloem. On the basis of the difference of the maximal emission wavelength between quercetin and kaempferol derivatives which have fluorescence behavior by appropriate treatment, kaempferol and its derivatives are localized exclusively in the cuticle. Results showed that the content of astragalin in Apocyni Veneti Folium from different parts revealed the decreasing trend, while hyperin and isoquercitrin were higher in June and July analyzed by HPLC. In summary, the site-specific accumulation of flavonoids in Apocyni Veneti Folium can be determined by LSCM and vanillin-HCl staining. The contents of flavonoids in Apocyni Veneti Folium are correlated with harvest times and growth parts.