Monthly headache frequency directly correlates with personal/societal burden and impacts severity and preventive treatment decisions. This post hoc analysis identified shifts from higher to lower frequency headache categories over 6 months in patients with migraine participating in the PROMISE clinical trials receiving two eptinezumab doses.Headache frequency at baseline and over study months 1–6 was categorized into 4 groups: chronic migraine , high-frequency episodic migraine , low-frequency episodic migraine , and ≤ 3 MHDs. Outcomes included the percentage of patients within each MHD category, the percentage of patients improving by ≥ 1 MHD category, and the number of months with reduction of ≥ 1 MHD category. Data from patients who received approved eptinezumab doses (100 mg or 300 mg) or placebo were included.Mean headache frequency at baseline in PROMISE-1 was 10 MHDs; most patients were classified as having HFEM (48.6%) or LFEM (43.9%). At Month 1, 62/221 (28.1%), 75/222 (33.8%), and 45/222 (20.3%) patients who received eptinezumab 100 mg, 300 mg, and placebo had ≤ 3 MHDs, with 97/221 (43.9%), 108/222 (48.6%), and 84/222 (37.8%), respectively, falling below the diagnostic EM threshold at Month 6. More than one-third , had 6 months of reduction of ≥ 1 frequency category. At baseline in PROMISE-2, mean headache frequency was 20.5 MHDs. All patients (100%) in the eptinezumab 100 mg and placebo groups had CM, as did 99.4% of patients receiving eptinezumab 300 mg. At Month 1, 209/356 (58.7%), 216/350 (61.7%), and 167/366 (45.6%) patients treated with eptinezumab 100 mg, 300 mg, and placebo had ≤ 14 MHDs, with 240/356 (67.4%), 249/350 (71.1%), and 221/366 (60.4%), respectively, falling below CM threshold at Month 6. Additionally, 153/356 (43.0%), 169/350 (48.3%), and 116/366 (31.7%) patients in the eptinezumab 100 mg, 300 mg, and placebo groups, respectively, had 6 months of reduction of ≥ 1 frequency category.In the PROMISE studies, episodic and chronic migraine patients treated with eptinezumab were more likely to reduce their headache frequency versus placebo, which directly and in a sustained way improved their diagnostic category classification.ClinicalTrials.gov Identifier: NCT02559895, NCT02974153. Headache frequency varies widely across the migraine spectrum and, in individual patients, may increase or decrease as their migraine worsens or improves , 2. BecaP = 0.0182 vs placebo) and 4.3 (P = 0.0001 vs placebo) days, respectively [P < 0.0001 vs placebo) and 8.2 (P < 0.0001 vs placebo) days, respectively, and reduced monthly headache days (MHDs) over the same time period  [Eptinezumab  is a calcitonin gene-related peptide antagonist approved for migraine prevention in adults –15. The ctively) . The objData were from the randomized, double-blind, placebo-controlled PROMISE studies , 18. PROHeadache frequency at baseline and over study Months 1–6 was categorized into four groups for migraine category: CM (≥ 15 MHDs), high-frequency episodic migraine , low-frequency episodic migraine , and ≤ 3 MHDs , 25.Outcomes included the percentage of patients within each MHD category, the percentage of patients improving by ≥ 1 MHD category, and the sustained response with reduction of ≥ 1 MHD category. Data from patients who received approved eptinezumab doses (100 mg or 300 mg) or placebo were included. For PROMISE-2, outcomes in the subgroup of patients with medication-overuse headache (MOH) were also examined.n = 221; 300 mg, n = 222) and 222 received placebo [n = 356; 300 mg, n = 350) and 366 received placebo [A total of 443 adults received eptinezumab 100 mg or 300 mg in PROMISE-1 , 138/222 (62.2%), and 116/222 (52.3%) patients in the eptinezumab 100-mg, 300-mg, and placebo groups, respectively, had a reduction of ≥ 1 frequency category at Month 1 and 156/221 (70.6%), 163/222 (73.4%), and 138/222 (62.2%) at Month 6 , 216/350 (61.7%), and 167/366 (45.6%) patients in the eptinezumab 100-mg, 300-mg, and placebo groups, respectively, had a reduction of ≥ 1 frequency category at Month 1 and 240/356 (67.4%), 249/350 (71.1%), and 221/366 (60.4%) at Month 6 . The results reported here are in alignment with the responses observed in previous work , which sIn patients with CM (PROMISE-2), sustained reductions in headache frequency with eptinezumab were of sufficient magnitude and duration to permit many patients to fall within the range typically considered EM (≤ 14 MHDs) beginning the first month after treatment initiation. Specifically, more than half (60.2%) had ≤ 14 MHDs during the first month after eptinezumab initiation and 66.0%, 64.7%, 71.5%, 68.4%, and 69.3% had ≤ 14 MHDs during Months 2, 3, 4, 5, and 6, respectively. Some CM patients even improved to the point that they fell below the frequency threshold typically used for the indication of preventive treatment (≤ 4 MHDs), with 10.3% achieving this status during Month 1 and 15.3%, 15.3%, 23.4%, 22.5%, and 22.9% during Months 2, 3, 4, 5, and 6, respectively. While these reductions in MHD associated with eptinezumab use likely reduce migraine-related burden, it is important to note that these data should not be interpreted as indicating a patient should discontinue preventive migraine treatment. Based on previous analyses , we woulIn addition, whereas these findings are suggestive of clinically meaningful changes in headache frequency, they raise some interesting questions for future research. In both studies, marked differences between Months 3 and 4 suggest that the administration of the second dose may not only sustain improvements, but further reduce headache frequency, i.e., an additive effect. These results are supportive of the updated American Headache Society  and EuroFrequency-based classification of migraine is complex, and factors such as severity and associated disability are an important part of defining migraine. Analysis of data from the PROMISE-2 study indicated that reductions in headache frequency were associated with decreases in pain severity; common symptoms such as nausea, phonophobia, and photophobia; and activity limitations . The catChanges in headache frequency during the first 6 months of eptinezumab treatment in the PROMISE studies were frequently of sufficient magnitude and duration to permit a shift in frequency and reclassify to categories associated with better quality of life and reduced healthcare resource utilization.


Flavonoid aglycones possess biological activities, such as antioxidant and antidiabetic activities compared to glycosides. Taxifolin, a flavonoid aglycones, is detected only in trace amounts in nature and is not easily observed. Therefore, in this study, to investigate the hair tonic and hair loss inhibitors effect of taxifolin, high content of taxifolin aglycone extract was prepared by enzymatic hydrolysis. Taxifolin effectively regulates the apoptosis of dermal papilla cells, which is associated with hair loss, based on its strong antioxidant activities. However, inhibition of dihydrotestosterone (DHT), which is a major cause of male pattern hair loss, was significantly reduced with taxifolin treatment compared with minoxidil, as a positive control. It was also confirmed that a representative factor for promoting hair growth, IGF-1, was significantly increased, and that TGF-β1, a representative biomarker for hair loss, was significantly reduced with taxifolin treatment. These results suggest that taxifolin from enzymatic hydrolysis of RM is a potential treatment for hair loss and a hair growth enhancer. Rhododendron mucronulatum (RM) is a deciduous shrub with a height of approximately 2 m and is distributed in some parts of China and Japan and all over Korea  from the Seoul Yangnyeongsi Medicine Market, Republic of Korea, was soaked in 60% EtOH at room temperature for 1 week and then filtered. The filtrate was concentrated and freeze-dried . Then, 10 g of RM 60E was diluted in distilled water  and Pectinex XXL, Ultra SP-L   were added. The mixture was shaken aerobically at 300 rpm for 24 h at 40°C, heated for 5 min at 85°C to inactivate the enzyme, and centrifuged for 10 min at 10,000 rpm and filtered. Then, the filtrate was fractionated with ethyl acetate, and the ethyl acetate layer extract was concentrated and freeze-dried to obtain 5.38 g.To isolate taxifolin (aglycone), Yamazen AI-580S system Medium Pressure Liquid Chromatography  was used. The ethyl acetate layer (4.5 g), and taxifolin (aglycone) were isolated. Silica gel column  and ODS column  were used. Silica gel column was used with CM solution (chloroform: methanol/5:1) and applied as an isocratic condition. After that, the isolating layer was concentrated and freeze-dried to a powder. Then powder (210 mg) was applied to the ODS column and eluted with distilled water. MW solution (methanol: water) was applied as an gradient condition . These conditions were repeated to obtain a final 103 mg of single taxifolin. A flowchart of taxifolin isolation is presented in 254 plate  with chloroform and methanol  and chloroform, methanol and water . The spots were detected under UV radiation (254 nm) and by spraying with 2SO4, FeCl3 and 10% H2SO4 followed by heating. Compound 1 was identified by several instrumental analyses. 1H-(700 MHz) and 13C-(175 MHz) NMR experiments were recorded with Bruker Avance Ⅲ 700  at Gyeonggi bio center. Also, Negative LC-MS/MS was conducted with QTRAP 4500  at Central laboratory Kangwon national university.Thin layer chromatography (TLC) was carried out using a pre-coated silica gel 60 F50)  .2O at a concentration of 2.45 mM. The two solutions were mixed in a 1:1 ratio and left in the dark for 12–16 h to prepare a radical stock solution. The solution was diluted with phosphate-buffered saline, , and absorbance was measured with INNO Microplate Spectrophotometer  at 750 nm so that an absorbance value between 0.68 and 0.72 was obtained. In a 96 well plate , the sample and ABTS reaction ratio were performed to 1:9 (v/v), and the reaction was performed in the dark for 15 min at room temperature. After the reaction, absorbance was measured at 750 nm wavelength. The radical scavenging activity was indicated as half-maximal inhibitory concentration (IC50).ABTS radical scavenging assay was measured by modifying the method of Re et al., 1999. ABTS  reagent was dissolved in distilled water at a concentration of 7.0 mM, and potassium persulfate  was dissolved in H3CN (B) was used to run the separation. The elution program was set as follows: from 10% CH3CN (B) to 35% CH3CN (B) in 20 min, from 35% CH3CN (B) to 100% CH3CN (B) in 5 min, from 100% CH3CN (B) to 10% CH3CN (B) in 10 min, and 10% CH3CN (B) for 5 min; temperature, room temperature; flow rate, 1.0 ml/min; injection volume, 20 μl; UV detection, 280 nm. Taxifolin  was used for standard. Taxifolin, compound 1, enzymatic hydrolysis of RM and RM 60E were weighed to 1 mg and dissolved in 1 ml of methanol, respectively. Then, taxifolin was diluted to 500, 250, 125, 62.5, and 31.25 ppm and compound 1 was dilluted to 500 ppm.High-pressure liquid chromatography (HPLC) was analyzed using a Waters 2,695 system (United States) by modifying the method of 2 maintained at 37°C and different complete media were supplemented with 1% antibiotics and growth factors (ATCC). The cells were used for in vitro experiments, after at least 14 days of proliferation.Human follicle dermal papilla cells  and the appropriate media  and serum-free fibroblast growth kit , rh FGF β, rh EGF/TGF β-1 supplement, rh insulin and ascorbic acid were obtained from the American Type Culture Collection . Cells were maintained following by manufacturer’s instructions. The humidified incubator was supplied with 5% CO2O2 for 12 h to investigate the effects of each dose on oxidative-stress-induced cyotoxicity in HDFPC. After incubation, the MTT stock solution  of 3--2,5-diphenyltetrazolium bromide  was added to each well, and further incubated for 20 min in CO2 incubator. After removal of the supernatant, dimethylsulfoxide (100 μl)  were added to each well. The solubilized formazan after 20 min, the absorbance were measured at an 570 nm using a microplate reader .Cells were pre-incubated with the indicated doses of taxifolin and treated with HEnzyme-linked immunosorbent assays (ELISA) were performed for IGF-I , TGF-β  and DHT . The sample collection and assay protocols were adapted from the manufacturer’s instructions.t-test. SPSS for Windows software  was used to conduct the statistical analyses. Significance was set at p < 0.05.All the experiments were repeated three times, and the mean value and standard deviation were presented. To compare each group, a one-way analysis of variance (ANOVA) followed by Tukey’s multiple range test was used and Student’s −; 1H-NMR : δ 6.80–6.96 , 5.92 , 5.88 , 4.92 , 4.51 ; 13C-NMR : 198.47 (C-4), 168.74 (C-7), 165.35 (C-5), 164.55 (C-9), 147.18 (C-4′), 146.35 (C-3′), 120.94 (C-6′), 115.91 (C-5′), 116.11 (C-2′), 101.87 (C-10), 97.33 (C-6), 96.30 (C-8), 85.16 (C-2), 73.71 (C-3). The defined chemical structure of purified taxifolin , a peak presumed to have the ABX type 3,4-dihydroxylation pattern of the B ring appeared. The signals of H-8 and H-6, which are meta-coupled through δ 5.92 , and 5.88 , respectively, were analyzed. Therefore, it was confirmed that the structure of the A-ring was a 5,7-dihydroxylation pattern. In addition, through δ 4.92  and δ 4.51 , it was flavanonol structure that had no double bond in the C ring  was 4.95 ± 0.05 µg/ml.50 of vitamin C, compound 1, RM60E, and enzymatic hydrolysis of RM were 4.36 ± 0.12 µg/ml, 1.68 ± 0.07 µg/ml, 4.98 ± 0.08 µg/ml, and2.99 ± 0.06 µg/ml, respectively.ABTS radical scavenging activity of compound 1, RM60E and enzymatic hydrolysis of RM is shown in R2 value). The standard calibration curve was obtained with concentrations of five increments for taxifolin, Y = 79768x − 191089 (R2 = 0.9985) as shown in n = 3) by the above formula. However, taxifolin was not detected in RM 60E . The HDFPCs were treated with various concentrations of taxifolin, for 12 h. Cell viability was determined by the MTT assay. The endogenous cytotoxicity of taxifolin is presented in 2O2-induced cytotoxicity model with HDFPC (2O2 whereas the taxifolin from enzymatic hydrolysis of RM -treated HDFPCs showed a statistically significant increase in antioxidant activities in a dose-dependent manner compared with the control group.To investigate the effects of anti-oxidative stress by taxifolin, we examined recovering abilities of taxifolin from enzymatic hydrolysis of RM on Hth HDFPC . Cell vi2O2 compared normal control group on the intracellular region of HDFPC, whereas pretreatment of taxifolin from enzymatic hydrolysis of RM rescued IGF-1 level in a dose-dependent manner compared to H2O2 alone. These results suggested that taxifolin from enzymatic hydrolysis of RM was interrupt the oxidative stress-induced malfunction of hair growth cycles.Changes in IGF-1 level on oxidative stress-induced HDFPC damage and recovery effects of taxifolin from enzymatic hydrolysis of RM are shown in 2O2 significantly increased TGF-β1 expression compared to the normal controls. However, taxifolin from enzymatic hydrolysis of RM treatment abrogated these effects in a dose-dependent manner, resulting in a significant reduction in TGF-β1 expression compared to H2O2 treatment alone. These results suggested that taxifolin from enzymatic hydrolysis of RM was regulates the oxidative stress-induced breakdown of hair growth cycles.As TGF-β families are a well-known inhibitory factors on hair growth and we examined the effect of oxidative stress on the production of TGF-β1. The effect of taxifolin from enzymatic hydrolysis of RM on TGF-β1 expression in the intracellular region was determined . TreatmeIn hair follicle papillae, 5α-reductase, DHT producing enzyme, normally predominated and known androgenic hormone response oi hair follicles depends on an intrafollicular androgenic signaling. The inhibition of DHT was investigated with various concentrations of taxifolin, and 10 µM minoxidil was used in the positive control group . The posTaxifolin is known to have various pharmacological properties such as antioxidants, anticancer, cardiovascular, and effective inhibition of oxidative stress, cell apoptosis, malignancies, microbial infection, cardiovascular disease, and liver disease. And it has an anti-cancer activity with a little or no side effects to the normal cells . In this2O2-induced apoptosis, taxifolin showed statistically significant antioxidant activity. Therefore, it can be expected that taxifolin may modulate the cell death signaling and required further experiments.Classically, hair loss is caused by various and complex factors, and one of the causes is oxidative stress. Oxidative stress is known to induce apoptosis then stimulate various cell types within the scalp and hair components; these cell types are keratinocytes, hair follicle cells, papilla cells, and various immune cells. Particularly, apoptosis is mediated by various signal cascades including Bcl-2, Bax, caspase family, and PARP . In our IGF-1 is known to be an important growth factor that protects against oxidative stress-induced epithelial cell apoptosis and promotes hair growth . TherefoTGF-β inhibits the proliferation of hair follicles and it can regulate hair follicles as a negative growth factor. Androgen is related to ROS production, and ROS is involved in TGF-β secretion. Particularly, TGF-β1 secretion is closely related to androgenetic alopecia  and it fDHT is the representative factor in male pattern hair loss. When testosterone, a male hormone, is oxidized by 5α-reductase, it changes to DHT . The resIn this study, experiments were conducted to confirm that new molecules derived from natural sources can provide an objective basis for scientifically controlling hair loss and have a low risk of side effects. In general, flavonoid aglycone has stronger biological activities, such as antioxidant activity and antidiabetic activitiesthan glycosides. Taxifolin is detected only in trace amounts in nature and is not easily observed . Therefo


Lateolabrax maculatus), which were named LmPanx1, LmCx32.2, LmCx32.3, LmCx32.7, and LmCx43. Their similar topological structures were discovered by sequence analysis: a relatively unconserved C-terminal region and four highly conserved transmembrane (TM) domains, and so on. Each extracellular (ECL) region of Panx1 has two conserved cysteine residues. Unlike Panx1, each ECL region of Cx32 and Cx43 contains three conserved cysteine residues, forming two conserved motifs: CX6CX3C motif in ECL1 and CX4CX5C motif in ECL2. Furthermore, Panx1 and Cx43 share similar genomic organization and synteny with their counterparts in selected vertebrates. Cx32 and CX43 were located in the same locus in fish, but diverged into two loci from amphibian. Moreover, despite varying expression levels, the identified genes were constitutively expressed in all examined tissues. All genes were upregulated by PAMP [lipopolysaccharide and poly(I:C)] stimulation or bacterial infection in vivo and in vitro, but they were downregulated in the brain at 6 or 12 h after stimulation. Especially, the three LmCx32 isoforms and LmCx43 were upregulated by ATP stimulation in primary head kidney leukocytes; however, downregulation of LmCx32.3 and LmCx43 expression were noted at 12 h. Conversely, ATP treatment inhibited the expression of LmPanx1. Importantly, we showed that the spotted sea bass Panx1, Cx43, and Cx32 were localized on the cellular membrane and involved in inflammation-induced ATP release. Taken together, our results demonstrated that Panx1, Cx32, and Cx43 are important neuro-related immune response genes involved in inflammation-induced ATP release.Many immunological diseases can be treated by regulating neurobehavior, in which extracellular ATP is a vital member of endogenous danger-associated molecular pattern signaling molecule that plays a crucial part in innate neuro-related immunity. It is actively released through pannexin (Panx) and connexin (Cx) hemichannels from activated or stressed cells during inflammation, injury, or apoptosis. In addition to participating in ATP release, Panxs and Cxs also have crucial immune functions. In this study, pannexin1, three connexin32 isoforms and connexin43 were identified and characterized in spotted sea bass ( Regulating neurobehavior shows great promise for therapeutic application in a variety of immunological diseases and clinical conditions . For exaConnexins (Cxs) are the main members of gap junctions, which control several second messengers to diffuse between adjacent cells . In addiIn addition to Cxs, pannexin (Panx) hemichannels were also involved in ATP release in mammals . Panxs aParalichthys olivaceus). These results first showed that the Panx1, Cx32, and Cx43 genes are involved in innate immunity in fish. Panx1 is also an important immune response gene involved in bacterial infection-induced ATP release in tilapia (Oreochromis niloticus)   . BesidesLateolabrax maculatus) widely farmed in East Asia because of their high nutritional value and adaptation to various salinity waters   and poly(I:C)] stimulation or bacterial infection in vivo and in vitro using real-time quantitative polymerase chain reaction (qPCR). Importantly, we determined their subcellular localization and explored their function in inflammation-induced ATP release. Our findings will contribute to further understanding of the innate immune response mediated by extracellular ATP in fish and of neuroimmunity in vertebrates.As a vital commercial fishes, spotted sea bass  , 34. FurL. maculatus (100 ± 10 g) were farmed in a freshwater fish aquaculture system at 26°C ± 2°C for more than 2 weeks prior to experiments. Fish were sourced from a fish freshwater farm in Hangzhou city, Zhejiang province, China.LmPanx1, LmCx32.2, LmCx32.3, LmCx32.7, and LmCx43 were then cloned and verified by our previous methods  and Expasy website (http://www.expasy.org) were used to analyze nucleotide and protein sequences. Phylogenetic trees and multiple sequence alignment of Panx1, Cx32, and Cx43 were analyzed using the ClustalW, GeneDoc, and MEGA 5.1 program, according to the method described previously  were obtained from healthy eviously . All priL. maculatus were intraperitoneal (i.p.) injected with 500 µL Edwardsiella tarda [1 × 105 colony-forming units (CFU)/mL], LPS (1 mg/mL), poly(I:C) (1 mg/mL), or phosphate-buffered saline (PBS) (control) for the challenge experiments by our previous methods  were treated with LPS (100 µg/mL), poly(I:C) (50 µg/mL), and ATP (100 µM or 1 mM), respectively. Each condition was done in quadruplicate. Cell samples were collected at 6, 12, 24, and 48 h after stimulation, and total RNA was then reversed to cDNA for qPCR. ATP was purchased from Sigma–Aldrich, and all cell culture reagents were purchased from Gibco (USA).Primary head kidney leukocytes were isolated by using a discontinuous Percoll gradient as previously described . The leuLmPanx1, LmCx32s, or LmCx43 were constructed, that is, pEGFP-N1-LmPanx1, pEGFP-N1-LmCx32.2, pEGFP-N1-LmCx32.3, pEGFP-N1-LmCx32.7, and pEGFP-N1-LmCx43. We then transfected the recombinant pEGFP-N1 plasmids into HEK293 T cells by the same methods as before (The pEGFP-N1 expression plasmid containing the coding sequence (CDS) of s before . The tras before . Next, cL. maculatus, the leukocytes (1 × 105/well) were cultured in a 24-well plate  and stimulated with LPS (100 µg/mL) or PBS. The supernatants were collected at 15 and 30 min after stimulation and used to measure extracellular ATP levels. Each condition was done in quadruplicate. The ATP release level was measured with Enhanced ATP Assay Kit  in LumiPro . All cell culture reagents were purchased from Gibco.To examine the LPS-induced extracellular ATP release in primary head kidney leukocytes of LmPanx1, LmCx32, and LmCx43 in LPS-induced extracellular ATP release, the pcDNA3.1 expression plasmid containing the CDS of LmPanx1, LmCx32s, or LmCx43 was constructed, that is, pcDNA3.1-LmPanx1, pcDNA3.1-LmCx32.2, pcDNA3.1-LmCx32.3, pcDNA3.1-LmCx32.7, and pcDNA3.1-LmCx43. The same number of HEK293 T cells transfected with expression plasmids or empty plasmid (pcDNA3.1) was cultured in a 24-well plate at 24 h, and then were stimulated with LPS (100 µg/mL). Meanwhile, the mock transfected and empty plasmid transfected cells (negative controls) were cultured in another 24-well plate at 24 h, but were not stimulated with LPS. The supernatants were collected at 15 and 30 min after stimulation and used to measure extracellular ATP levels. Each condition was done in quadruplicate. The ATP levels were then measured as described previously.To explore the role of p < 0.05 or p < 0.01) between experimental groups and control groups were analyzed using analysis of variance as previously described . Significant differences (escribed .L. maculatus were submitted to the GenBank database: OM315303 (LmPanx1), OM315304 (LmCx32.2), OM315305 (LmCx32.3), OM315306 (LmCx32.7), and OM315307 (LmCx43).The Panx1, Cx32, and Cx43 sequences in LmPanx1 has been cloned, which contains 1,959 bp including a 161-bp 5′ untranslated region (UTR), an open reading frame (ORF) with 1,320 bp encoding 439 amino acids (aa), and a 478-bp 3′-UTR. Moreover, there is a polyadenylation signal (ATTAAA) at the 3′-UTR of the sequence     domain, and a connexin-ccc domain  at the 3′-UTR. By multiple sequence alignment, the LmCx43 protein exhibits a high degree of conservation and contains a CNX domain, a connexin-ccc domain, and a PDZ domain. Like Cx32s, three conserved cysteine residues in each of the ECL region form two conserved motifs: CX6CX3C motif in ECL1 and CX4CX5C motif in ECL2 (As shown in LmPanx1 and of Morone saxatilis formed one branch (88% bootstrap support) and then were clustered with homologs from other fish species. Corresponding to the clades formed by Cx32.2, Cx32.3, and Cx32.7 homologs from different fish, LmCx32s were clustered into three distinct branches , and LmCx43 were found in muscle, liver, and brain, respectively. In addition, the lowest expression levels of LmCx32 and LmCx43 were both found in head kidney. In contrast liver exhibited the lowest expression for LmPanx1, and the moderate expression levels of LmCx32 and LmCx43 were found in the intestine, muscle, and skin. Differently, the moderate expression levels of LmPanx1 were found in the brain, gill, and spleen.We analyzed the expression of LmPanx1, LmCx32, and LmCx43 were analyzed in tissues including the head kidney, spleen, gill, intestine, brain, and liver after PAMP [LPS and poly(I:C)] and E. tarda challenge (L. maculatus were i.p. injected with 500 µL E. tarda (1 × 105 CFU/mL), LPS (1 mg/mL), poly(I:C) (1 mg/mL), or PBS for the challenge experiments. Tissue samples were obtained at 6, 12, 24, and 48 h after injection, and total RNA was then reversed to cDNA for qPCR. In head kidney, gill, and intestine, the expression of LmPanx1 was upregulated to the different degrees after three kinds of stimulation. In spleen, the expression of LmPanx1 was upregulated after poly(I:C) (at 12 h) and E. tarda (except 24 h) stimulation, but there was no significant change after LPS stimulation; In the brain, LmPanx1 was downregulated at 6 h after LPS and E. tarda stimulation, but upregulated at 48 h after LPS and poly(I:C) stimulation; in the liver, LmPanx1 was upregulated only at 24 h after poly(I:C) and E. tarda stimulation , whereas the expression of LmCx32.2 and LmCx32.3 remained constant. Moreover, LmCx32.2 was upregulated in the spleen (at 12 h), brain (at 6 h), and liver (at 48 h), but downregulated in the gill (at 24 h) by poly(I:C) stimulation , and LPS  stimulation  or E. tarda stimulation ] and ATP stimulation (LmPanx1 expression was upregulated after LPS (at 6 h) and poly(I:C) (at 6 and 12 h) stimulation, whereas it remained unchanged at other stimulation conditions. Beyond that, all the genes except LmPanx1 were upregulated at 48 h by PAMP stimulation. Among the three LmCx32 isoforms, LmCx32.7 responded most strongly to stimulation, and the expression of LmCx32.7 was obviously upregulated at all times except 12 h after poly(I:C) stimulation. According to the LmCx32 isoforms and LmCx43 was upregulated by ATP stimulation; nevertheless, when cells were stimulated with 100 µM or 1 mM ATP, the significantly downregulated expression of LmCx32.3 and LmCx43 was noted at 12 h. Conversely, treatment of 100 µM or 1 mM ATP inhibited the LmPanx1 expression.Expression patterns of LmPanx1, LmCx32, and LmCx43, plasmids pEGFP-N1-LmPanx1, pEGFP-N1-LmCx32.2, pEGFP-N1-LmCx32.3, pEGFP-N1-LmCx32.7, and pEGFP-N1-LmCx43 were constructed to express the GFP-tagged LmPanx1, LmCx32.2, LmCx32.3, LmCx32.7, and LmCx43 fusion protein in the HEK293T cells, respectively. After 24-h culture, the cells were examined under a laser confocal microscope. In HEK293 T cells transfected with expression plasmids, GFP fluorescence was mainly located on the cell membrane, whereas in HEK293 T cells transfected with empty plasmids, GFP fluorescence was mainly located in the intracellular area  were stimulated with LPS, and the ATP levels were measured at 15 and 30 min after stimulation. In overexpression experiments, as negative controls, mock transfected and empty plasmid transfected cells were cultured in another 24-well plate at 24 h, but were not stimulated with LPS. As shown in LmPanx1, LmCx32, or LmCx43 in HEK293 T cells, compared with cells transfected with empty plasmids, resulted in a significant increase in extracellular ATP levels after LPS stimulation at 15 or 30 min  and LmCx43 were found in the liver and brain, respectively. Interestingly, in mouse, Cx32 has been shown to be the major Cx protein in the liver, protecting the liver against liver injury ,  tissues , 32, 41.r injury , whereasLmPanx1, LmCx32, and LmCx43 were upregulated by PAMP [LPS and poly(I:C)] stimulation or E. tarda infection in vivo and in vitro ] stimulation or The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/All fish experiments were conducted under the national regulations of laboratory animals of China and reviewed and approved by the ethics committee of laboratory animals of Shanghai Ocean University (SHOU-DW-2019-012).ZS, YC, CX, and DL performed laboratory experiments and analyzed the data. QG and ZS designed the experiments. QG, PW, and ZS wrote the manuscript. All authors have read and approved the manuscript.This work was financially supported by the National Key Research and Development Program of China (no. 2018YFD0900605).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


These water-soluble porphyrins were evaluated as PSs, and their photophysical and photochemical properties and photodynamic effects on E. coli were assessed. The presence of one CD unit and three positive charges on the Por structure (3a–5a and 7a) enhanced their aqueous solubility. The photoactivity of the cationic Pors 3a–5a and 6a–8a ensured their potential against the Gram-negative bacterium E. coli. Within each series of methoxypyridinium vs thiopyridinium dyes, the best PDI efficiency was achieved for 5a with a bacterial viability reduction of 3.5 log10  and for 8a with a total bacterial viability reduction . Here, the presence of the methoxypyridinium units is less effective against E. coli when compared with the thiopyridinium moieties. This study allows for the conclusion that the peripheral charge position, quaternized substituent type/CD unit, and affinity to the outer bacterial structures play an important role in the photoinactivation efficiency of E. coli, evidencing that these features should be further addressed in the pursuit for optimised PS for the antimicrobial PDI of pathogenic microorganisms.Photodynamic action has been used for diverse biomedical applications, such as treating a broad range of bacterial infections. Based on the combination of light, dioxygen, and photosensitizer (PS), the photodynamic inactivation (PDI) approach led to the formation of reactive oxygen species (ROS) and represented a non-invasive, non-toxic, repeatable procedure for pathogen photoinactivation. To this end, different tetrapyrrolic macrocycles, such as porphyrin (Por) dyes, have been used as PSs for PDI against microorganisms, mainly bacteria. Still, there is significant room for improvement, especially new PS molecules. Herein, unsymmetrical new pyridinone ( Escherichia coli belongs to the Enterobacteriaceae family, considered pathogenic Gram-negative bacteria. This bacterium is present in the intestinal microbiome of humans + and 601.0 [M+2H]2+.1  and α-, β-, or γ-cyclodextrin  was dissolved in 5 mL of DMF. The reaction was carried out for 72 h at 60 °C under an N2 atmosphere, allowing for the nucleophilic substitution, also confirmed by TLC. The solvent was removed under reduced pressure and the reactional crude was dissolved in MeOH, then precipitated with water, filtered, and washed with cool water. The obtained purple solids were dried under a vacuum system over 6 h at 80 °C. The desired porphyrin–cyclodextrin conjugates were obtained in moderate yields .In a 25 mL round-bottom flask, a mixture of Por H)-yl)phenyl]porphyrin, Por 3: 1H NMR : δ −3.08 , 6.28–6.54 , 7.96–8.30 , 9.14–9.65  ppm. 19F NMR : δ −145.98–−144.86 , −135.00–−133.69  ppm. ESI–MS m/z: 1076.3 [M+2H]2+ and 710.9 [M+3H]3+. UV–Vis (DMSO): λ (log ε) 417 (4.17); 510 (2.01); 583 (1.47) nm.5--10,15,20-trisporphyrin, Por 4: 1H NMR : δ −3.03 , 6.27–6.76 , 7.92–8.20 , 9.22–9.43  ppm. 19F NMR : δ −146.31–−145.17 , −135.57–−134.55  ppm. ESI–MS m/z: 1157.5 [M+2H]2+ and 773.3 [M+3H]3+. UV–Vis (DMSO): λ (log ε) 417 (3.59); 509 (2.14); 584 (1.83) nm.5--10,15,20-trisporphyrin, Por 5: 1H NMR : δ −3.09 , 6.39–6.66 , 7.83–8.40 , 9.17–9.75  ppm. 19F NMR : δ −146.92–−144.68 , −135.72–−133.87  ppm. ESI–MS m/z: 1239.5 [M+2H]2+ and 827.0 [M+3H]3+. UV–Vis (DMSO): λ (log ε) 418 (3.96); 510 (2.03); 584 (1.56) nm.5--10,15,20-tris-10,15,20-trisporphyrin, Por 4a: 1H NMR : δ −3.08 , 4.36 , 8.06–8.20 , 9.41–9.48  ppm. 19F NMR : δ −144.32–−143.38 , −133.95–−133.42  ppm. UV–Vis (DMSO): λ (log ε) 417 (3.37); 509 (2.14); 583 (1.61) nm.5--10,15,20-trisporphyrin, Por 5a: 1H NMR : δ −3.10 , 4.37 , 8.18–8.22 , 9.34–9.53  ppm. 19F NMR : δ −144.08–−143.48 , −133.83–−133.27  ppm. UV–Vis (DMSO): λ (log ε) 417 (3.10); 510 (1.82); 583 (1.43) nm.5--10,15,20-trisporphyrin, Por 2 2+. UV–Vis (DMSO): λ (log ε) 420 (3.13); 510 (2.00); 588 (1.65) nm.5--10,15,20-trisporphyrin, Por 7  was dissolved in DMF (3.0 mL) and was added to an excess of methyl iodide . The reaction was stirred overnight at 40 °C. After this period, the reactional mixture was cooled until room temperature and the positive charged product was precipitated in CH2Cl2, filtered, and washed with the same solvent. The obtained purple solid was dried under a vacuum system over 6 h at 80 °C and was recovered in excellent yield, obtaining the compound 7a .In a sealed tube, Por 7a: 1H NMR : δ −3.08 , 4.35 , 8.44–8.48 , 8.90–9.00 , 9.59–9.71  ppm. 19F NMR : δ −132.89–−132.76 , −128.00–−127.87  ppm. UV–Vis (DMSO): λ (log ε) 420 (3.09); 514 (1.95); 588 (1.62) nm.5--10,15,20-trisporphyrin triodide, Por 3a–8a (3 mL in quartz cuvettes) were irradiated under the same light conditions applied in the biological experiments. The UV–Vis spectrum was monitored, and the Soret band absorbance value (between 416 and 420 nm) of each PS (3a–8a) was displayed before (time t0 = Abs0) and after white light exposure for pre-defined intervals  (tAbs):The PBS solutions of Pors −2. The light irradiance was determined through a Power Meter Coherent FieldMaxII196 Top with a Coherent PowerSens PS19Q energy sensor.A white light LED system  was used for the irradiation of the Por dyes at different irradiation intensities of 25 and 50 mW cmTPP in DMF was used as a reference (ΦΔ = 0.65) [3a–8a, and the reference was registered in a first-order plot [Different solutions of each PS (Abs at 420 nm ≈ 0.2) were prepared in DMF with 9,10-dimethylanthracene  at a concentration of 50 µM. These solutions were irradiated in quartz cuvettes with blue light (λ = 420 nm). A solution of  = 0.65) ,45. The der plot ,47,48. TE. coli Top10 (by the luxCDABE genes of the marine bioluminescent bacterium Allivibrio fischeri) was grown in a Tryptic Soy Broth medium , enriched with 50 mg mL−1 of ampicillin (Amp) and 34 mg mL−1 of chloramphenicol (Cm). Before the assay, one colony was placed into a flask with Tryptic Soy Broth , enriched with Amp and Cm, and was grown at 25 °C overnight under shaking at 120 rpm. Then, an aliquot was transferred into 10 mL of TSB under the same growth conditions until the stationary growth phase was achieved. An optical density of 600 nm (OD600) of 1.6 ± 0.1 corresponded to 108 colony forming units per millilitre (CFU mL−1). The correlation of CFU mL−1 and the bioluminescence signal  of the bioluminescent E. coli is described in the literature [The genetically transformed bioluminescent terature .E. coli were grown overnight in TBS. After that, it was diluted in PBS (pH = 7.49) to a final concentration of ≈ 107 CFU mL−1, corresponding to ~107 RLU. This bacterial suspension was equivalently distributed in 6-well plates and a suitable volume of each PS was added in order to accomplish the final concentration of 5.0 µM. From our previous published binding studies [E. coli. At the end of the incubation process, all the samples were irradiated under agitation for 60 min at 20 °C. Aliquots of 1.0 mL were collected and the bacterial bioluminescence was measured in a luminometer . The detection limit of the equipment is ca. 2.3 log. Three independent experiments were performed.The bacteria of  studies , the besIn addition to measuring bioluminescence, aliquots of treated and control samples were taken from the first and last time points, serially diluted, and poured in triplicate onto a Tryptic Soy Agar (TSA) medium. The plates were incubated for 24 h at a controlled temperature of 37 °C, and the concentration of the viable cells was determined by counting the CFU. At least three independent experimental measurements were conducted with three replicates. The results are determined as the average of the three assays.At least three independent experiments, with three replicates per assay for each condition, were done. The statistical analysis was performed with GraphPadPrism 8. The Kolmogorov–Smironov test was used to check for normal distributions, and the homogeneity of variance was verified with the Brown–Forsythe test. ANOVA and Dunnet’s multiple comparison tests were applied to assess whether the samples were statistically significant.1 and 3–5, and the cationic Pors 3a–5a , the cationic derivatives 3a–5a were achieved in good yields . The substituted Pors were synthesized following experimental procedures established in our research group [2 , and were characterized by UV–Vis absorption and emission spectroscopy  corresponding to the resonance of 6 meta-protons and 6 ortho-protons on the 4-pyridinone groups, respectively. Additionally, the 19F NMR spectrum of Por 1 is compatible with a para substitution pattern of three pentafluorophenyl rings and one unsubstituted pentafluorophenyl ring. The 19F NMR spectra of Pors 3–5 revealed the disappearance of the signal at −149.75 ppm due to the para-fluorine replacement by the cyclodextrin and the appearance of two multiplets on the interval of δ −146.92–−144.68 ppm and δ −135.72–−133.69 ppm, corresponding these signals to the ortho- and meta-fluorine atoms. The cationization of the neutral compounds provided to the Pors 3a–5a were confirmed by the appearance of the singlet in the aliphatic region located at δ 4.36–4.37 ppm of the 1H NMR, which corresponds to the resonance of the -OCH3 protons.The m/z ratios.The ESI–MS analysis shows the observed species resulting from the characteristic fragmentation processes of these Pors with the formation of species with different overall 3a–8a were recorded in DMSO solutions (~10−5 M) at 298 K , and fluorescence quantum yield for Pors 1O2), one of the main ROS responsible for causing cell damage, leading to cell death. As previously mentioned, one of the key characteristics of a molecule that is considered a PS is its ability to generate singlet oxygen  was compared to the known cationic Pors 6a, 8a, and a TPP reference (ΦΔ = 0.65 in DMF) that is considered a good generator of 1O2 [1O2 after light exposure. The gamma-cyclodextrin Por derivatives 5a and 8a are the best producers of 1O2 among the three cyclodextrins  assessed. Comparing methoxypyridinium Por–CD dyes (3a–5a) with the corresponding thiopyridinium Por–CD dyes (6a–8a) [1O2 species.Thus, the production of r of 1O2 ,45. Acco (6a–8a) , the res3a–8a under light conditions to photoinactive E. coli, their photostability was studied by monitoring the absorption decay of the Soret band after irradiation with white light (400–800 nm) at an irradiance of 50 mW cm−2 for 60 min. The obtained results are summarized in Since the application purpose is the use of Por–CD derivatives 3a–8a showed excellent photostability in PBS solution, showing a robust behavior facing the irradiation used, with 86% to 94% of the porphyrin conjugates remaining unaltered after irradiation with white light at an irradiance of 50 mW cm−2 for 60 min  (methoxypyridinium (3a–5a) and thiopyridinium (6a–8a)) was compared for the conditions of 5.0 μM and 25 mW cm−2 . Nevertheless, when the light irradiance used was increased twice  after 30 and 60 min of light exposure, respectively, followed by Pors 4a and 3a  for the same light exposition time. The results point out the importance of the 1O2 generation by the PS. The photodynamic efficiency, to photoinactivate E. coli, of each PS 3a–5a (1O2 generation (1O2 production follows the ascending efficiency order (3a < 4a < 5a).The photodynamic efficiency of the cationic derivatives Por–CD was assessed against a bioluminescent  mW cm−2 A and 50 cm−2  in an external position. Additionally, the cationic substituent type/CD unit and affinity to the outer bacterial structures cannot be ruled out. Overall, within each series of quaternized dyes (methoxypyridinium vs. thiopyridinium), the best PSs candidates were the cationic Por–γ–CD dyes 5a and 8a at irradiances of 50 mW cm−2 and 25 mW cm−2, respectively.Comparing inverted methoxypyridinium Pors  mW cm−2 , it was adiation . Pors 3aE. coli bioluminescence with the reduction expressed in log10 CFU mL−1 accomplished with the pour-plate methodology at times 0 and 60 min. The obtained results are presented as the average of the three assays in 3a–5a confirmed the results obtained through the bioluminescence monitoring of the PDI treatment. Only the conjugate 5a was able to generate a decrease of ~1.5 log  in the viability of E. coli after 60 min of light irradiation. Besides, at the same irradiance (25 mW cm−2) and light exposure time (60 min), the Por–CD 7a caused a total photoinactivation of the E. coli, which corroborated the determined bioluminescence results, and is similar to the PDI results found in the literature for Por–CD 6a and 8a17). When the irradiance was increased to 50 mW cm−2, Pors 3a–5a caused a decrease of ~2 log10 to ~4 log10 in the viability of E. coli, and Por 7a reached a complete bacteria inactivation. These results are in agreement with the RLU results found for photoinactivation bioluminescent E. coli. All obtained results confirmed that the external positive charge and type of the cyclodextrin substituent of the Por–CD dyes considerably influences the PDI efficiency.For a better consistency in our study, it was related to the reduction in 1O2 for all cationic Por derivatives, the γ-cyclodextrin derivatives (5a and 8a) were revealed to be better 1O2 generators. This fact might justify the more efficient E. coli inactivation profile achieved for Por–CD conjugates 5a  and 8a , and it was more pronounced for 8a due to its highest 1O2 generation, good water solubility, and the maximized electrostatic interactions between their more exposed peripheral positive charges and the Gram-negative bacterium. Moreover, different solubility profiles in the physiological medium of Por–CD derivatives can also justify the distinct PS inactivation behavior of E. coli. It is widely known that γ-cyclodextrin is more water-soluble than α-cyclodextrin [5a and 8a (γ-cyclodextrin derivatives) tend to have better PDI performances compared with the others Por–CD conjugates.It is well-known that the PS efficacy on the PDI treatments is correlated with different aspects, such as the microbial adsorption behavior, the ability of PS to generate singlet oxygen, the photostability profile, the solubility/aggregation comportment, or cell localization, among others . Despiteodextrin ,52, whic3a–5a) can also influence the photoinactivaion efficiency. The substitution by the nitrogen, instead of the expected oxygen-bridge, as in a thio–bridge (6a–8a), probably makes the former less flexible. In addition, the position of the positive charge may also contribute to the aggregation behaviour in water once it is more protected from the aqueous environment. The position of the positive charge plays a key role in the physicochemical and biological features of the Por conjugates, evidencing that these features should be further addressed in the pursuit of optimized PS for antimicrobial photodynamic inactivation.It is also important to highlight that the structural manner in which hydroxypyridine reacts with the Por structure  showed that, only under white light at an irradiance of 50 mW cm−2, a significative E. coli photoinactivation occurred. The Por 5a proved to be the best PS, causing a decrease of 3.5 and 4.0 log10 of the bioluminescence signal after 30 and 60 min of light irradiation, respectively. Analyzing the new inverted methoxypyridinium Pors 3a–5a and the already-known thiopyridinium Pors 6a–8a, at 5.0 μM at an irradiance of 25 mW cm−2, a faster (in 30 min) and complete E. coli photoinactivation was confirmed, particularly for thiopyridinium Pors 6a and 8a. The obtained results show that the methoxypyridinium Por–CD conjugates (3a–5a) evidence less effectivity against the bacterial viability than the thiopyridinium Por–CD conjugates (6a–8a). According to the American Society of Microbiology, this is higher than the minimum required (reduction > 3 log CFU mL−1) for a new approach to be termed as antimicrobial. The Por 5a (50 mW cm−2) and Pors 6a–8a (25 mW cm−2) can be considered promising PS drugs for PDI.The new free-base Pors


Leishmania amazonensis, its cytotoxicity, and potential mechanisms of action. β-Ocimene presented direct activity against the parasite, with excellent growth inhibition of promastigotes (IC50 = 2.78 μM) and axenic amastigotes (EC50 = 1.12 μM) at concentrations non-toxic to RAW 264.7 macrophages (CC50 = 114.5 µM). The effect is related to changes in membrane permeability and resulting abnormalities in the parasitic cell shape. These were, respectively, observed in membrane integrity and atomic force microscopy assays. β-Ocimene was also shown to act indirectly, with greater activity against intra-macrophagic amastigotes (EC50 = 0.89 μM), increasing TNF-α, nitric oxide (NO), and reactive oxygen species (ROS), with lysosomal effects, as well as promoting decreases in IL-10 and IL-6. Against intra-macrophagic amastigote forms the selectivity index was higher than the reference drugs, being 469.52 times more selective than meglumine antimoniate, and 42.88 times more selective than amphotericin B. Our results suggest that β-ocimene possesses promising in vitro antileishmania activity and is a potential candidate for investigation in in vivo assays.Leishmaniasis is a group of infectious-parasitic diseases with high mortality rates, and endemic in many regions of the globe. The currently available drugs present serious problems such as high toxicity, costs, and the emergence of drug resistance. This has stimulated research into new antileishmania drugs based on natural products and their derivatives. β-Ocimene is a monoterpene found naturally in the essential oils of many plant species which presents antileishmanial activity, and which has not yet been evaluated for its potential to inhibit the etiological agent of leishmaniasis. The aim of this work was to evaluate the activity of β-ocimene against  Leishmania spp. are protozoan parasites of the Trypanosomatidae family and the etiological agents of leishmaniasis. They are transmitted during blood meals taken by infected female phlebotomine sandflies [andflies . Accordiandflies .Leishmania spp. strain and host immunity, clinical manifestations differ significantly [Depending on the ficantly . Two prificantly .Currently, there are no human vaccines, and leishmaniasis treatment is based on chemotherapy. Pentavalent antimonial drugs were introduced in the 1940s and are still the first-line treatment for leishmaniasis. They present many limitations such as high toxicity and cost, long treatment durations, and induction of resistance . Second-Due to these problems in leishmaniasis therapy, drugs with antileishmanial activity based on natural product derivatives are being sought. In this context, plant-derived volatile essential oils and their principal compounds (monoterpenes and sesquiterpenes) offer promising antileishmanial activity .cis-3-hexen-1-ol (a C6 volatile), Perring et al. [β-Ocimene  is an acyclic olefin monoterpene found in the essential oils of leaves and flowers of many plant species. It performs many biological functions, including use of its allopathic effect, in pesticides, and for defense against herbivores ,9. β-Ocig et al.  treated g et al. . β-ocimePistacia lentiscus L. [Piper marginatum Jacq. [Syzygium cumini (L.) Skeels. essential oil [Leishmania (Leishmania) amazonensis, its cytotoxicity against mammalian cells, and its possible mechanisms of action.β-ocimene is present in the compositions of many essential oils with proven antileishmanial activity such as iscus L.  and Pipeum Jacq. . β-ocimetial oil ,15, yet 50) of 2.78 μM  for promastigotes and axenic amastigotes  presented by β-ocimene was, respectively, 114.5 µM for RAW 264.7 macrophages, and 298.54 µM for Vero cells , 2 × IC50 (p < 0.001), and at 4 × IC50 (p < 0.001), similar to the positive control—promastigotes were treated with 0.1% Triton X-100  in promastigotes treated with β-ocimene at ICon X-100 .L. amazonensis was observed, morphological alterations were evaluated using AFM techniques. Using the AFM deflection image, it was observed that the untreated promastigotes presented normal membrane texture and were topographically vacuolated. The presence of a long, exteriorized flagellum confirmed cell normality  of 0.89 μM, while meglumine antimoniate and amphotericin B, respectively, generated EC50 values of 577.5 and 0.13 μM , an (S)-limonene derivative, has been reported against axenic amastigotes [The antileishmanial activity of β-ocimene was initially evaluated against i et al. , the antf 252 μM . As withstigotes .L. amazonensis promastigotes, 102.23 times more toxic to axenic amastigotes, and 128.65 times more toxic to intra-macrophage amastigotes than to macrophages. The selectivity index assesses how much more selective a compound is towards the parasite than to the cell. Substances presenting an SI greater than 20 are considered safe [In order to obtain a substance with selective antileishmanial activity for the parasite and with low toxicity to cells, a cytotoxicity assay was performed in RAW 264.7 macrophages and Vero cells. When the SI was calculated, the compound was 41.18 times more toxic to red safe . β-OcimeConsidering the antileishmania effectiveness of β-ocimene, its mechanisms of action against the parasite were evaluated. Membrane integrity assays, and immunomodulatory activity studies (in vitro) were performed.It was demonstrated that β-ocimene acts directly on the parasite. We found that β-ocimene alters plasma membrane permeability, as observed in an experiment using SYTOX green, a high-affinity nucleic acid stain that penetrates cells with compromised plasma membranes and (upon nucleic acid binding) augments their fluorescence by more than 500-fold . The altIn addition to membrane permeability, structural changes caused by β-ocimene in parasites at the nanometer scale were evaluated through atomic force microscopy (MFA). After treatment with β-ocimene, changes were observed in both the membranes and shapes of the parasites. Cell retraction, membrane alteration, and vesicle formation are typical characteristics of cells undergoing apoptosis . Such chLeishmania spp. infection, macrophages commence phagocytosis, and production of cytokines and microbicidal molecules (such as reactive oxygen and NO species) [In response to species) . Our stuViable and metabolically active cells are able to take up neutral red dye through lysosomal vesicles . In thisL. amazonensis, phagocytic capacity was analyzed using zymosan particle incorporation. Zymosan particles are readily phagocytized by viable macrophages and present a proven ability to stimulate a Th1-type response [In macrophages not stimulated with response . In thisL. amazonensis) increased production of Th1 cytokines (TNF-α), ROS, and NO, and reduced levels of the Th2 profile cytokines (IL-6 and IL-10). Previous studies have shown that increased production of Th1 cytokines and reduction of Th2 cytokines is associable with parasite control and is a target for new antileishmanial drugs [Investigating cellular mechanisms, our study demonstrated that β-ocimene  produce INF-γ; and secondly, activation of Toll-like receptors (TLRs) induces production of TNF-α (associated with the activation of macrophages to fight the parasite) [L. amazonensis, β-ocimene (at the highest concentrations tested) induced production of ROS and NO. Yet according to Vouldoukis et al. [There are two macrophage activation signals in the innate immune response to infection by arasite) . INF-γ iarasite) . In thiss et al. , an incrv/v) H3PO4 in Milli-Q water), and neutral red, were purchased from Merck Company . Fetal bovine serum (FBS) was obtained from ThermoFisher . Interferon gamma (IFN-γ) and lipopolysaccharide (LPS) were obtained from Life Technologies Corporation . (E)-β-ocimene  was obtained from Vertec Fine Chemistry .Cell culture medium (DMEM and Schneider), antibiotic solution (penicillin and streptomycin), 3- 2,5-bromide diphenyltetrazolium (MTT), zymosan particles, Griess reagent  amazonensis (IFLA/BR/67/PH8) (promastigote form) were cultivated in Schneider’s insect medium , at 26 °C, and at pH 7, in a biological oxygen demand (BOD) incubator. To transform the promastigote forms into axenic amastigotes, the temperature was increased to 32 °C and the pH of the medium was adjusted to 5.5 [Strains of d to 5.5 .2.RAW 264.7 lineage macrophages  were used, grown in sterile bottles with DMEM medium, pH 7.2 containing 10% FBS and 1% penicillin 100 U/mL and streptomycin 100 μg/mL, and kept in an oven at 37 °C and 5% CO6 parasites per well in 100 μL of supplemented Schneider medium. The parasites were treated with differing concentrations of β-ocimene (1.56–12.5 μM), and reference drugs, meglumine antimoniate  and amphotericin B (0.062–0.5 μM). The plates were incubated in a BOD incubator at 26 °C for 72 h. Afterwards, 10 µL of MTT (5 mg/mL) was added to each well, and the plate was incubated again for 4 h, when 50 µL of 10% SDS was added. Absorbances were then measured using an ELISA plate reader  at 540 nm. The negative control consisted of Schneider medium with 0.5% DMSO and was considered as 0% inhibition.Promastigote and axenic amastigote growth inhibition was tested as previously described by DIAS et al. . In a 96L. amazonensis treated with β-ocimene was evaluated using the SYTOX green dye method [50, 2 × IC50 and 4 × IC50 in the presence of SYTOX green dye (5 μM) for 7 h at 26 °C in a BOD incubator. The plates were then read in a fluorescence reader with an emission filter of 523 nm and excitation at 488 nm.Plasma membrane integrity in promastigote forms of e method . PromastL. amazonensis were grown in 96-well plates at a concentration of 106 per well in complete Schneider medium. β-ocimene was added at concentrations equivalent to IC50, 2 × IC50, and 4 × IC50, and incubated at 26 °C for 72 h in BOD, and the parasites were then washed twice with ice-cold PBS (under 1100× g for 15 min at room temperature), and then fixed with 2.5% (v/v) glutaraldehyde in PBS, pH 7.2, for 1 h. Morphological analysis using AFM , at a resonant frequency of approximately 242 kHz, was then performed [Promastigote forms of erformed .5 cells per well in 100 μL of supplemented DMEM medium. The plate was incubated for 3 h at 37 °C and 5% CO2 for cell adhesion. Afterwards, to remove non-adhered cells, washing with PBS  was performed. Furthermore, 100 μL of DMEM supplemented with β-ocimene (3.12–400 μM), and the reference drugs, meglumine antimoniate (250–2000 μM) and amphotericin B (0.062–0.5 μM) were then added to the wells, with oven incubation at 37 °C, and 5% CO2 for 72 h. Then, 10 μL of MTT (5 mg/mL) per well was then added, being kept for another 4 h in an oven at 37 °C and 5% CO2. The supernatant was removed, and 100 µL of DMSO was added to all wells. After 30 min of stirring, readings were taken at 540 nm. DMEM supplemented with 0.5% DMSO was used as a negative control and considered as 100% macrophage viability. The selectivity index for each treatment was calculated by dividing the CC50 by the IC50, or EC50 [In a 96-well plate, RAW 264.7 macrophages were plated at 10 or EC50 .5 cells to 1 mL of supplemented DMEM. For cell adhesion, the plates were incubated at 37 °C and 5% CO2 for 3 h. Three washes were then performed with PBS at 37 °C to discard non-adherent cells, after which 1 mL of DMEM supplemented (stationary phase) promastigote forms of L. amazonensis was added at the ratio of 10 promastigotes per macrophage. The plate was then incubated at 5% CO2 and 37 °C for 4 h. After infection, the medium was aspirated and the treatment was added using 1 mL of DMEM supplemented with β-ocimene (3.12–25 μM) with the reference drugs, meglumine antimoniate (1.56–200 μM) and amphotericin B (0.031–2 μM), this was subsequently incubated at 37 °C and 5% CO2 for 72 h. Finally, the coverslips were removed, fixed, stained, and mounted on permanent slides using Entellan. The number of infected macrophages and the number of parasites per macrophage were counted: 300 cells were evaluated on each slide at 1000× magnification. The infection index was calculated by multiplying the number of infected macrophages by the number of amastigotes per infected macrophage. The negative control was performed using DMEM with 0.5% DMSO. The supernatant was removed at the end of the experiment and kept at −20 °C for later use in nitric oxide dosing tests [In a 24-well plate with 13 mm round sterile cover slips, RAW 264.7 macrophages were added (at a concentration of 105 cells per well) to the 96-well plate in 100 μL of DMEM with different concentrations of β-ocimene (3.12–25 μM). The plate was incubated for 72 h at 37 °C and 5% CO2. Afterwards, 10 μL of neutral red 0.2% DMSO solution was added and incubated for 30 min. The supernatant was then removed, and the wells were washed with 0.9% saline at 37 °C, and 100 μL of extraction solution was added to dissolve the neutral red inside the lysosomal secretion vesicles. After 30 min on a Kline shaker, the plate was read at 540 nm [RAW 264.7 macrophages were added (10t 540 nm .5 cells per well in 100 μL of DMEM medium containing different concentrations of β-ocimene (3.12–25 μM). The plate was incubated for 72 h at 37 °C and 5% CO2; after which 10 μL of stained zymosan per well was added, and the plate conditioned for 30 min at 37 °C. Then, 100 μL of Baker’s fixative  was added to stop phagocytosis, and 30 min later, the plate was washed with 0.9% saline to remove the zymosan not phagocytized by macrophages. Afterwards, the supernatant was removed and 100 μL of extraction solution was added and solubilized in a Kline shaker. Absorbances were measured at 540 nm [RAW 264.7 macrophages were plated in a 96-well plate at 10t 540 nm .Modulation of Th1 , Th2 , and NO cytokine production was investigated in the supernatants of infected macrophages, whether treated or untreated with β-ocimene. Cytokine concentration (pg/mL) in macrophage supernatants was determined using the ELISA sandwich technique according to the manufacturer’s (eBioscience) instructions, using interpolation with a standard curve of recombinant cytokines. Optical density was read at 450 nm using a microplate spectrophotometer.2.Nitrite levels from the supernatants of the infected macrophages, whether treated or untreated with β-ocimene, were used indirectly to estimate NO production. The NO concentration (µM) was determined using the Griess reaction following a previously described methodology , by inteLPS (1 μg/mL) plus IFN-γ (1000 U/mL) were used as a positive control.L. amazonensis and treated with β-ocimene, were determined by the H2DCFDA test [2 DCFDA dye was added. The plate was incubated at 37 °C for 30 min (in the dark), and the fluorescence intensity was measured using a spectrofluorometer (FLx800) with excitation at 485 nm and emission at 528 nm.ROS levels, as produced by the macrophages infected with FDA test . Macroph50, EC50, and CC50 with a confidence limit of 95%, the probit regression model from SPSS 13.0 was used. For comparisons between groups, ANOVA analysis of variance was performed followed by Tukey’s test using GraphPad Prism version 8.0, and taking the value of p < 0.05 as the maximum level of statistical significance.To determine the CIL. amazonensis with low levels of cytotoxicity to host cells. The effects of β-ocimene against the parasite are associated with compromised plasma membrane integrity. Our study also revealed that the antileishmanial activity of β-ocimene involves indirect immunomodulation; evidenced by increases in lysosomal activity and levels of TNF-α, NO, and ROS, and decreases in IL-10 and IL-6. These results encourage further studies with this compound in in vivo models of tegumentary leishmaniasis and synergistic studies with reference drugs, towards development of new antileishmania agents.In conclusion, our results reveal that β-ocimene exhibits selective antileishmanial activity against both stages of


The selectivity, linearity, recovery, accuracy, precision, matrix effect and stability of UPLC-MS/MS assay were satisfied with the standards of United States Food and Drug Administration guidelines. In control group, AUC0-∞ and Cmax of CLZ were 2,169.5 ± 363.1 ng/ml*h and 258.9 ± 82.6 ng/ml, respectively. The corresponding results were 3,767.6 ± 1,049.8 ng/ml*h and 308.6 ± 87.9 ng/ml for 3, 4-CLZ, 728.8 ± 189.9 ng/ml*h and 100.3 ± 51.3 ng/ml for 4′-CLZ, respectively. After combination with baicalein, AUC0-∞ and Cmax of CLZ were 1.48, 1.38 times higher than the controls. Additionally, AUC0-∞ and Cmax were separately decreased by 36.12 and 19.54% for 3,4-CLZ, 13.11 and 44.37% for 4′-CLZ. Baicalein obviously alters the pharmacokinetic parameters of CLZ, 3,4-CLZ and 4′-CLZ in rats. These results suggested that there was a potential drug-drug interaction between baicalein and CLZ. Therefore, it must raise the awareness when concomitant use of CLZ with baicalein, the dosage regimen of CLZ should be taken into consideration, if this result is confirmed in clinical studies.This study aimed to explore the effect of baicalein on the pharmacokinetics of cilostazol (CLZ) and its two metabolites 3,4-dehydro cilostazol  and 4′-trans-hydroxy cilostazol (4′-CLZ) in rats using a newly established ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method. Ticagrelor was used as an internal standard (IS), then cilostazol and its two metabolites were separated by means of a UPLC BEH C18 column  using gradient elution method with 0.4 ml/min of flow rate. Acetonitrile as organic phase and water with 0.1% formic acid as aqueous phase constructed the mobile phase. Selective reaction monitoring (SRM) mode and positive ion mode were preferentially chosen to detect the analytes. Twelve SD rats were divided into two groups ( Scutellaria baicalensis, is widely used as a medicinal material in China for thousands of years. The roots of Scutellaria baicalensis called Huangqin were reported to have various pharmacological effects reviewed in a previous study , an anti-platelet drug, is commonly applied as a treatment intervention method in cardiovascular disease, ischemic stroke, diabetes, etc. . CLZ wasin vitro . ConsideIn the past decades, there have been reported several methods such as high performance liquid chromatography (HPLC), liquid chromatography tandem mass spectrometry (LC-MS/MS) and ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) for quantitative analysis of CLZ and its metabolites in different species and various biological samples. Previous studies have reported HPLC method of CLZ and its metabolites in various biological samples, including human plasma, human urine, human liver microsomal incubation mixtures . LC-MS/MIn this experiment, we attempted to develop a rapid, stable and sensitive UPLC-MS/MS method to simultaneously measure the concentrations of CLZ, 3,4-CLZ and 4′-CLZ in rat plasma. Moreover, we used this novel quantitative method for application of the investigation of the pharmacokinetic effect of baicalein on CLZ, 3,4-CLZ and 4′-CLZ in rats.All chemicals and reagents were showed at Usually, stock solutions were used as mother samples, which could be easily dissolved into a series of the lower concentrations of targets. In this study, we accurately prepared four kinds of stock solutions such as CLZ, 3,4-CLZ, 4′-CLZ and ticagrelor  into the final concentrations of 1.00 mg/ml by methanol. Subsequently, work solutions were prepared through the corresponding four stock solutions. Working solutions of CLZ, 3,4-CLZ, 4′-CLZ were dissolved into methanol to prepare the mixture standard working solutions contained three targets with the same final concentrations ranged from 1 to 500 ng/ml. The final concentration of IS working solution diluted with methanol was 200 ng/ml. Finally, the working solutions were used to construct calibrations curve with eight different concentrations of 1, 2, 5, 20, 50, 100, 200, and 500 ng/ml. In the similar method described above, mixture quality control samples (QCs) consisted of CLZ, 3,4-CLZ, 4′-CLZ with four different final concentrations of 1 , 2 , 80  and 400  ng/mL were prepared. All the solutions contained stocking solution and QCs were stored at a refrigerator kept 4°C.SD rats, weighted from 180 to 220 g, were provided from the Laboratory Animal Center of Wenzhou Medical University . Subsequently, all rats were stayed and kept without special treatments for seven continuous days in the proper environment, such as temperature 25–28°C, humidity 50–60% and 12 h light/12 h dark, etc. During this period, unlimited drinking water and barley-based diets were supplied.g for 8 min to obtain the supernatants as plasma samples.The Institutional Ethics Committee of Wenzhou Medical University  approved this animal experiments, which strictly adhered to the National Institute of Health (NIH) guidelines. The detailed experimental process was showed as follows: firstly, a total of 12 SD rats were equally divided into single group (CLZ) and combination group . Water was allowed before the first gavage, but a necessary restriction of food for 12 h was needed. Combination group of rats were orally given baicalein with the concentration of 80 mg/kg. Control ones (single group) were orally administrated with the equal volume of distilled water. Subsequently, 10 mg/kg CLZ was applied for all rats after half an hour. Caudal veins for blood collection were used at different time points of 0, 0.333, 0.667, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 48 h after the last oral administration. Heparin-containing tubes were used for temporarily storing blood samples, which were further centrifuged at 4,000 × A simple process of protein precipitation with acetonitrile was finally supplied for direct plasma preparation. A complete plasma preparation was consisted of three parts, including addition of IS, access to precipitant and centrifuge for the acquisition of the supernatants. Firstly, added 20 μl ticagrelor as IS into 100 μl of plasma samples, then 300 μl acetonitrile as precipitant was added into the mixture above. Subsequently, we centrifuged after adequately vortexing. Finally, 100 μl of the supernatants was obtained for the further UPLC-MS/MS quantitative analysis.CLZ and its metabolites were separated and detected by a Waters ACQUITY UPLC I-Class system and a Xevo TQ-S triple quadrupole tandem mass spectrometer equipped with an electro-spray ionization (ESI) source . The temperature of column and autosampler were 40 and 10°C, respectively. 2.0 µl of injection volume was pushed into UPLC-MS/MS system and 0.40 ml/min of total flow rate was employed for the further analysis. Gradient elution consisted of solvent A (acetonitrile) and solvent B (0.1% formic acid in water) was performed on a Acquity UPLC BEH C18 column . The detailed elution condition was as follows: 0–0.5 min, 90% B; 0.5–1 min, 90%–10% B; 1–1.5 min, 10% B; 1.5–1.6 min, 10%–90% B; 1.6–2.0 min, 90% B.m/z 370.30 → 125.02, m/z 368.20 → 286.12, and m/z 386.20 → 288.16, respectively. The detailed mass spectrometer conditions were showed at Selective reaction monitoring (SRM) mode and positive ion mode was used to determine the levels of three targets and IS. In this study, ion transitions of CLZ, 3,4-CLZ and 4′-CLZ for quantitative detection were https://www.fda.gov/regulatory-information/search-fda-guidance-documents/bioanalytical-method-validation-guidance-industry).UPLC-MS/MS method has been a homeliness and practical approach applied for the determination of biological specimens. In this research, a rapid, stable and sensitive UPLC-MS/MS method was established and verified for selectivity, sensitivity, linearity, LLOQ, matrix effect, recovery, accuracy, precision and stability. To accurately and legitimately detect three targets, the method validation strictly adhered to the United States Food and Drug Administration (FDA) guidelines , Center for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CVM), Bioanalytical Method Validation Guidance for Industry, Center for Drug Evaluation and Research, 2018, Selectivity was mainly to check the absence of interferences at the nearby retention times of three targets and IS using six replicates samples. Three kinds of analytic specimens such as blank rat plasma, the mixture standard solution of three targets at LLOQ concentrations and real rat plasma after oral administration of CLZ. Sensitivity was scientifically estimated by the value of the lowest quantitative point, namely LLOQ, which was finally investigated at the condition of 10 times of signal-to-noise ratio (S/N). In this study, calibration curves of the analytes were evaluated by eight values identified as the ratio of peak area  against the nominal concentrations of the analyte using a weighted (1/×) least square regression model.Precision, accuracy, recovery, and matrix effect were eventually estimated by six replicates specimens using QCs in three different levels  of CLZ, 3,4-CLZ and 4′-CLZ. In addition, 1 ng/ml of CLZ, 3,4-CLZ and 4′-CLZ was also used to assess precision and accuracy. Relative standard deviation (RSD%) and relative error (RE%) were separately used to evaluate accuracy and precision. Precisions divided into intra-precision and inter-precision were detected on the same day and three continuous days, respectively. Recovery was recognized as the peak area of CLZ or 3,4-CLZ or 4′-CLZ before and after plasma preparation. Matrix effect of CLZ or 3,4-CLZ or 4′-CLZ was investigated by comparative study between the peak area of the spike-after-extraction samples and the corresponding results of the pure solution.Stability was estimated by six replicates specimens using QCs in three different levels . The stability evaluation of CLZ or 3,4-CLZ or 4′-CLZ was performed under different environmental conditions composed of temperature and perservation time, including room temperature (2 h), −80°C (3 weeks), 10 °C in an autosampler (4 h after preparation) and three freeze/thaw cycles (−80°C to room temperature).via a non-compartmental method. Furthermore, unpaired t-test analysis applied for difference detection was performed in SPSS 17.0, where p < 0.05 was considered as the obvious criteria.Drug and Statistics (DAS) 3.0 software, produced by Mathematical Pharmacology Professional Committee of China, was eventually used to analyze and calculate the pharmacokinetic parameters of CLZ and its main metabolites m/z 370.30, 368.20, 386.20, respectively, and the corresponding ion of IS was m/z 523.05. The most abundant fragment ion at m/z 125.02, 286.12, 288.16, and 153.02 separately belonged to CLZ, 3,4-CLZ, 4′-CLZ and IS, which were chosen as the product ions for the bioanalytical method.To get the better analytic results, we optimized the MS parameters and acquired the better settings using CLZ, 3,4-CLZ, 4′-CLZ and IS. The bioanalytical assay was developed in several parts such as LLOQ, time consumption and injection volume compared with other quantitative methods in rat plasma. According to the previous study, the LLOQ of CLZ and 3,4-CLZ were 20 ng/ml . In thisThree kinds of biological samples such as blank plasma, blank plasma with standard solutions at LLOQ concentrations and plasma specimens with standard preparations after gavage administration of CLZ were applied for estimating selectivity. The final results were listed at In this research, we used eight different concentrations to construct calibration curves of CLZ and its two metabolites. Calibration curve of CLZ was Y = 3.27213 × X + 5.07792, Y = 1.80371 × X + 2.14749 for 3,4-CLZ and Y = 1.76848 × X + 1.13212 for 4′-CLZ. Correlation coefficient of three targets reached over 0.99, which represented an excellent linearity. LLOQ of three targets at the concentration of 1 ng/ml was finally validated and accepted in standard ranges.The parameters of precision and accuracy was described as RSD% and RE%, respectively. Stability testing results of CLZ and its two metabolites under different preparation and storage conditions in rat plasma were showed at 1/2), time to reach the peak plasma concentration (Tmax), and the peak plasma concentration (Cmax) after single CLZ or combinations. All the results were finally filled in The plasma concentration-time curves of CLZ in rat plasma was showed in 1/2 value of CLZ was 10.7 ± 3.4 h, which showed a long t1/2. The similar results found in 3,4-CLZ and 4′-CLZ. AUC(0-∞) and CLz/F, Vz/F were 2,169.5 ± 363.1 ng/ml*h, 4.8 ± 1.0 L/h/kg and 71.1 ± 18.6 L/kg for CLZ, 3,767.6 ± 1,049.8 ng/ml*h, 2.9 ± 1.1 L/h/kg, 60.1 ± 19.2 L/kg for 3,4-CLZ, 728.8 ± 189.9 ng/ml h, 14.4 ± 3.4 L/h/kg and 253.0 ± 118.0 L/kg for 4′-CLZ, respectively.Interestingly, results from 0-∞ (3221.1 ± 1088.0 ng/ml*h) resulted in 1.48-fold increase versus the control, Cmax (357.1 ± 64.7 ng/ml) resulted in 1.38-fold increase versus the control. In the presence of baicalein, the pharmacokinetic parameters of 3,4-CLZ and 4′-CLZ were changed: AUC0-∞, Cmax decreased by 36.1 and 19.5% for 3,4-CLZ, 13.1 and 44.4% for 4′-CLZ, respectively. In addition, CLz/F represented 2.24-fold increase for 3,4-CLZ and 1.77-fold increase for 4′-CLZ compared with those in the controls. These results described above indicated that an apparent herb-drug interaction existed after CLZ combination with baicalein.Baicalein obviously increased systemic exposure of CLZ: AUCScutellaria baicalensis  of single ciprofloxacin (Previous studies showed that baicalein could alter the pharmacokinetic parameters of many clinical proved drugs, when it was combination with other drugs. Hwang, Y. H. et al. found the co-administration of baicalein had an inhibitory effect on the pharmacokinetics of ciprofloxacin and significantly changed the AUCfloxacin . Data frfloxacin . This rein vivo contains four processes, including absorption, metabolism, distribution and excretion. Firstly, drugs are absorbed into the blood through the transports like P-glycoprotein, multidrug resistance proteins. CYP450s such as CYP3A4, CYP2C9, CYP2C19 mainly affected and controlled the metabolism of drugs. Transporters such as P-glycoprotein and the multidrug resistance proteins ABCC2 and ABCC3 significantly altered the pharmacokinetics of etoposide in mice (Commonly, physiological disposition of drugs  in mice . Tang pf in mice . Therefo0→∞ of 3,4-CLZ was almost 5-fold higher than this in 4′-CLZ before combination, which indicated CLZ was more metabolized into the 3,4-CLZ than 4′-CLZ. AUC of 3,4-CLZ and 4′-CLZ significantly decreased after combination implied that baicalein could inhibited the hepatic metabolism of CLZ. We also found that the AUC0→∞ ratio of 3,4-CLZ toward 4′-CLZ changed from 5.17 to 3.8, it seemed that metabolic pathway of CLZ metabolized into 3,4-CLZ was more easily to be affected by baicalein than 4′-CLZ. The changes in pharmacokinetic parameters of CLZ, 3,4-CLZ and 4′-CLZ mentioned above might due to the hepatic enzyme and P-glycoprotein. The former study showed that baicalein might suppress the function of CYP3A4 and P-glycoprotein to obviously alter the pharmacokinetics of tamoxifen and nimodipine in rats (max and AUC (In this study, significant changes of pharmacokinetic parameters such as AUC and CLz/F among CLZ, 3,4-CLZ and 4′-CLZ were observed. These results indicated and provided an evidence for the potential herb-drug interaction between baicalein and CLZ in rats. AUC of CLZ markedly increased after combination indicated that baicalein could enhance the oral bioavailability of CLZ. What’s more, AUC in rats . Moreove in rats . A CYP3A and AUC . These r and AUC . We specIn conclusion, we established a reliable UPLC-MS/MS method to simultaneously quantify the plasma concentrations of CLZ and its two main metabolites in rats. Meanwhile, a significant influence of baicalein on the pharmacokinetics of CLZ and its metabolites in rats was found in this study. What’s more, there should be a warning notice of the interaction between baicalein and CLZ. Further research should be illustrated the impact of baicalein on the CLZ in human. Preparations containing baicalein should be changed or paid more attentions due to the possibility of herb-drug interactions.