Insufficient catalytic efficiency of flavonoid 6-hydroxylases in the fermentative production of scutellarin leads to the formation of at least about 18% of by-products. Here, the catalytic mechanisms of two flavonoid 6-hydroxylases, CYP82D4 and CYP706X, were investigated by molecular dynamics simulations and quantum chemical calculations. Our results show that CYP82D4 and CYP706X have almost identical energy barriers at the rate-determining step and thus similar reaction rates, while the relatively low substrate binding energy of CYP82D4 may facilitate product release, which is directly responsible for its higher catalytic efficiency. Based on the study of substrate entry and release processes, the catalytic efficiency of the L540A mutation of CYP82D4 increased by 1.37-fold, demonstrating the feasibility of theoretical calculations-guided engineering of flavonoid 6-hydroxylase. Overall, this study reveals the catalytic mechanism of flavonoid 6-hydroxylases, which may facilitate the modification and optimization of flavonoid 6-hydroxylases for efficient fermentative production of scutellarin.
Cytochrome P-450 Enzyme System/metabolism* ; Apigenin ; Glucuronates

This study explores the effect of apigenin(APG), oxymatrine(OMT), and APG+OMT on the proliferation of non-small cell lung cancer cell lines and the underlying mechanisms. Cell counting kit-8(CCK-8) assay was used to detect the vitality of A549 and NCI-H1975 cells, and colony formation assay to evaluate the colony formation ability of the cells. EdU assay was employed to examine the proliferation of NCI-H1975 cells. RT-qPCR and Western blot were performed to detect the mRNA and protein expression of PLOD2. Molecular docking was carried out to explore the direct action ability and action sites between APG/OMT and PLOD2/EGFR. Western blot was used to study the expression of related proteins in EGFR pathway. The viability of A549 and NCI-H1975 cells was inhibited by APG and APG+OMT at 20, 40, and 80 μmol·L~(-1) in a dose-dependent manner. The colony formation ability of NCI-H1975 cells was significantly suppressed by APG and APG+OMT. The mRNA and protein expression of PLOD2 was significantly inhibited by APG and APG+OMT. In addition, APG and OMT had strong binding activity with PLOD2 and EGFR. In APG and APG+OMT groups, the expression of EGFR and proteins in its downstream signaling pathways was significantly down-regulated. It is concluded that APG in combination with OMT could inhibit non-small lung cancer, and the mechanism may be related to EGFR and its downstream signaling pathways. This study lays a new theoretical basis for the clinical treatment of non-small cell lung cancer with APG in combination with OMT and provides a reference for further research on the anti-tumor mechanism of APG in combination with OMT.
Humans ; Carcinoma, Non-Small-Cell Lung ; Lung Neoplasms ; Apigenin ; Molecular Docking Simulation ; Alkaloids ; Quinolizines ; RNA, Messenger ; ErbB Receptors

This study aimed to evaluate the biological effect and mechanism of Vernonia anthelmintica Injection(VAI) on melanin accumulation. The in vivo depigmentation model was induced by propylthiouracil(PTU) in zebrafish, and the effect of VAI on melanin accumulation was evaluated based on the in vitro B16F10 cell model. The chemical composition of VAI was identified according to the high-performance liquid chromatography quadrupole-time-of-flight tandem mass spectrometry(UPLC-Q-TOF-MS). Network pharmaco-logy was applied to predict potential targets and pathways of VAI. A "VAI component-target-pathway" network was established, and the pharmacodynamic molecules were screened out based on the topological characteristics of the network. The binding of active molecules to key targets was verified by molecular docking. The results showed that VAI promoted tyrosinase activity and melanin production in B16F10 cells in a dose-and time-dependent manner and could restore the melanin in the body of the zebrafish model. Fifty-six compounds were identified from VAI, including flavonoids(15/56), terpenoids(10/56), phenolic acids(9/56), fatty acids(9/56), steroids(6/56), and others(7/56). Network pharmacological analysis screened four potential quality markers, including apigenin, chrysoeriol, syringaresinol, and butein, involving 61 targets and 65 pathways, and molecular docking verified their binding to TYR, NFE2L2, CASP3, MAPK1, MAPK8, and MAPK14. It was found that the mRNA expression of MITF, TYR, TYRP1, and DCT in B16F10 cells was promoted. By UPLC-Q-TOF-MS and network pharmacology, this study determined the material basis of VAI against vitiligo, screened apigenin, chrysoeriol, syringaresinol, and butein as the quality markers of VAI, and verified the efficacy and internal mechanism of melanogenesis, providing a basis for quality control and further clinical research.
Animals ; Vernonia/chemistry* ; Melanins/metabolism* ; Zebrafish/metabolism* ; Network Pharmacology ; Molecular Docking Simulation ; Apigenin/pharmacology* ; Drugs, Chinese Herbal/pharmacology* ; Chromatography, High Pressure Liquid

Glechomae Herba, the dried aerial part of Glechoma longituba(Labiatae), has the effects of promoting urination, draining dampness, and relieving stranguria. It has received wide attention in recent years owing to the satisfactory efficacy on lithiasis. Amid the in-depth chemical and pharmacological research, it has been found that Glechomae Herba has antibacterial, anti-inflammatory, antioxidant, antithrombotic, hepatoprotective, cholagogic, antitumor, hypoglycemic, and lipid-lowering effects. The main chemical constituents are volatile oils, flavonoids, terpenoids, phenylpropanoids, and organic acids. This paper summarized the chemical constituents and pharmacological effects of Glechomae Herba. Based on genetic relationship of plants, the characteristics, efficacy, and pharmacokinetics of the chemical constituents, and the potential of these constituents as quality markers(Q-markers), it was summed up that ursolic acid, caffeic acid, rosmarinic acid, luteolin-7-O-diglucuronide, apigenin, apigenin-7-O-diglucuronide, apigetrin, and glechone can be the candidate Q-markers of Glechomae Herba.
Apigenin ; Plant Extracts/pharmacology* ; Lamiaceae ; Drugs, Chinese Herbal/pharmacology* ; Flavonoids/pharmacology*

Drinking culture has high significance in both China and the world, whether in the entertainment sector or in social occasions; according to the World Health Organization's 2018 Global Alcohol and Health Report, about 3 million people died from excessive drinking in 2016, accounting for 5.3% of the total global deaths that year. Oxidative stress and inflammation are the most common pathological phenomena caused by alcohol abuse (Snyder et al., 2017). Scutellarin, a kind of flavonoid, is one of the main active ingredients extracted from breviscapine. It exerts anti-inflammatory, antioxidant, and vasodilation effects, and has been used to treat cardiovascular diseases and alcoholic liver injury. Although scutellarin can effectively alleviate multi-target organ injury induced by different forms of stimulation, its protective effect on alcoholic brain injury has not been well-defined. Therefore, the present study established an acute alcohol mice brain injury model to explore the effect of scutellarin on acute alcoholic brain injury. The study was carried out based on the targets of oxidative stress and inflammation, which is of great significance for the targeted therapy of clinical alcohol diseases.
Animals ; Apigenin/therapeutic use* ; Brain Injuries/drug therapy* ; Glucuronates/therapeutic use* ; Humans ; Mice ; Oxidative Stress

Clinopodium chinense, a traditional folk medicinal herb, has been used to treat abnormal uterine bleeding(AUB) for many years. Saponins and flavonoids are the main active components in C. chinense. To study the pharmacokine-tics of multiple components from the total extract of C. chinense(TEC), we established a sensitive and rapid method of ultra-perfor-mance liquid chromatography coupled with tandem mass spectrometry(UPLC-MS/MS) for simultaneous determination of five compounds in the plasma of AUB rats. After validation, the AUB model was established with SD female rats which got pregnant on the same day by gavage with mifepristone(12.4 mg·kg~(-1)) and misoprostol(130 μg·kg~(-1)). The established method was applied to the detection of hesperidin, naringenin, apigenin, saikosaponin a, and buddlejasaponin Ⅳb in AUB rats after the administration of TEC. The pharmacokinetic parameters were calculated by DAS 2.0. The five compounds showed good linear relationship within the detection range. The specificity, accuracy, precision, recovery, matrix effect, and stability of the method all matched the requirements of biolo-gical sample detection. The above 5 compounds were detected in the plasma of AUB rats after the administration of TEC. The C_(max) va-lues of hesperidin, naringenin, apigenin, saikosaponin a, and clinoposide A were 701.6, 429.5, 860.7, 75.1, and 304.1 ng·mL~(-1), respectively. All the compounds owned short half-life and quick elimination rate in vivo, and the large apparent volume of distribution indicated that they were widely distributed in tissues. Being rapid, accurate, and sensitive, this method is suitable for the pharmacokinetic study of extracts of Chinese herbal medicines and provides a reference for the study of pharmacodynamic material basis of C. chinense in treating AUB.
Administration, Oral ; Animals ; Apigenin/analysis* ; Chromatography, High Pressure Liquid/methods* ; Chromatography, Liquid ; Drugs, Chinese Herbal/chemistry* ; Female ; Flavonoids/analysis* ; Hesperidin ; Lamiaceae ; Mifepristone ; Misoprostol ; Oleanolic Acid/analogs & derivatives* ; Plant Extracts/chemistry* ; Rats ; Saponins ; Tandem Mass Spectrometry/methods* ; Uterine Hemorrhage

This study investigated the quality markers(Q-markers) of Euphorbiae Humifusae Herba based on the analytic hierarchy process(AHP)-criteria importance through intercriteria correlation(CRITIC) comprehensive weighting method. The Q-markers evaluation system was constructed based on the AHP-CRITIC comprehensive weighting method with quantitative identification of Q-markers of Euphorbiae Humifusae Herba as the target layer. The index weights of the factor layer and the control layer were integrated based on the weights of three indicators(effectiveness, testability, and specificity) in the factor layer calculated by the AHP method and weights of eight indicators(anti-inflammatory inhibitory rate, coagulation shortening rate, anti-cancer inhibition rate, component degree value, component test batch, component average content, content variation coefficient, and number of medicinal materials retrieved according to components) in the control layer calculated by the CRITIC method. The comprehensive score of the chemical components of Euphorbiae Humifusae Herba was weighted and ranked to identify the Q-markers of Euphorbiae Humifusae Herba. In terms of comprehensive scores, top 10 potential Q-markers of Euphorbiae Humifusae Herba were ranked as cynaroside > quercetin > gallic acid > apigenin > luteolin > apigenin-7-O-glucoside > quercetin-7-O-glucoside > ellagic acid > astragalin > ethyl gallate. This study provides a reference for the quality control of Euphorbiae Humifusae Herba and a methodological reference for the quantitative identification of Q-markers of Chinese medicine.
Chromatography, High Pressure Liquid/methods* ; Quercetin ; Apigenin ; Quality Control ; Glucosides ; Drugs, Chinese Herbal/chemistry*

This study explored the correlation between color and chemical components of Chrysanthemi Indici Flos(CIF), aiming at providing a reference for its procurement, evaluation, and breeding. Colorimeter and ultra-performance liquid chromatograph(UPLC) were used to determine the color(lightness-shade chromaticity value L~*, red-green chromaticity value a~*, yellow-blue chromati-city value b~*) and chemical components(cynaroside, linarin, luteolin, apigenin, and chlorogenic acid) of 84 CIF germplasms, respectively. Diversity analysis, correlation analysis, regression analysis, and cluster analysis were performed. The results showed that the color and chemical components of CIF were diversified. Chlorogenic acid was in significantly positive correlation with L~* and b~* and significantly negative correlation with a~*. Cynaroside and grey relational grade γ_i of chemical components were in significantly po-sitive correlation with b~* and L~*, respectively, whereas linarin, luteolin, and apigenin had no significant correlation with L~*, a~*, or b~*. The 84 CIF germplasms were clustered into 4 clades. In addition, germplasms in clade Ⅲ had higher γ_i and total color value(E~*_(ab)) than those in other clades, with the best quality and color, and a germplasm with the highest quality, bright yellow color, and highest content of linarin was screened out in this clade. Thus, CIF with bright yellow color had high content of cymaroside and chlorogenic acid and thereby high quality. In summary, the color can be used to quickly predict the quality of CIF. Our results provided data for the evaluation of CIF quality by color and a reference for its procurement and breeding.
Chrysanthemum/chemistry* ; Luteolin/analysis* ; Chlorogenic Acid/analysis* ; Apigenin/analysis* ; Plant Breeding ; Excipients ; Chromatography, High Pressure Liquid/methods*

Oxidative stress plays an important role in the pathogenesis of aminoglycoside-induced hearing loss and represents a promising target for treatment. We tested the potential effect of apigenin, a natural flavonoid with anticancer, anti-inflammatory, and antioxidant activities, on neomycin-induced ototoxicity in cochlear hair cells in vitro. Results showed that apigenin significantly ameliorated the loss of hair cells and the accumulation of reactive oxygen species upon neomycin injury. Further evidence suggested that the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway was activated by apigenin treatment. Disruption of the Nrf2 axis abolished the effects of apigenin on the alleviation of oxidative stress and subsequent apoptosis of hair cells. This study provided evidence of the protective effect of apigenin on cochlear hair cells and its underlying mechanism.
Apigenin/pharmacology* ; Apoptosis ; Hair Cells, Auditory/metabolism* ; Humans ; NF-E2-Related Factor 2/pharmacology* ; Neomycin ; Oxidative Stress ; Reactive Oxygen Species/metabolism* ; Signal Transduction

Chemical constituents were isolated and purified from the water extract of Artemisia annua by column chromatography of HP-20 macroporous resin, silica gel, ODS, Sephadex LH-20, HW-40, and semi-preparative RP-HPLC. Their structures were elucidated by physicochemical properties and spectral analyses. As a result, Fifteen compounds were isolated and identified as vitexnegheteroin M(1), sibricose A5(2), securoside A(3), citrusin D(4), annphenone(5), E-melilotoside(6), esculetin(7), scopoletin-7-O-β-D-glucoside(8), eleutheroside B_1(9), chrysosplenol D(10), patuletin-3-O-β-D-glucopyranoside(11), quercetin-7-O-β-D-glucoside(12), rutin(13), apigenin 6,8-di-C-β-D-glucopyranoside(14), isoschaftoside(15), among them, compounds 1-4 were identified from Artemisia for the first time. Additionally, the isolates were evaluated for their inhibitory effects on the production of PGE_2 in LPS-simulated RAW264.7 macrophages. The results showed that compounds 1, 2, 8, and 10-15 could reduce PGE_2 levels, to a certain extent.
Apigenin ; Artemisia annua ; Quercetin ; Rutin