Yajie Wang

Comparative pharmacokinetics of active alkaloids after oral administration of Rhizoma Coptidis extract and Wuji Wan formulas in rat using a UPLC–MS/MS method

Abstract Wuji Wan (WJW), containing Rhizoma Coptidis (Huanglian in Chinese, HL), Frutus Evodiae Rutaecarpae (Wuzhuyu, WZY) and Radix Paeoniae Alba (Baishao, BS), is a classical traditional Chinese medical formula employed in treating intestinal disorders. Berberine (BBR) and palmatine (PMT) are the major active alkaloids in HL and have analgesic and anti-microbial effects. A sensitive, specific and validated ultra-performance liquid chromatography–tandem mass spectrometric method was developed to investigate the pharmacokinetic profiles of BBR and PMT in rat plasma and in situ intestinal perfusion solution. In comparison with the pharmacokinetic parameters of BBR and PMT, t1/2, Cmax, Tmax, AUC, CL and MRTxa0after intragastric (i.g.) administration with HL extract alone, those remarkably changed after i.g. administration with WJW formulasxa01 and 2 (herb proportions are 12:2:3 and 12:1:12). Particularly, the oral bioavailability of PMT in WJW formulaxa01 was significantly increased. In rat intestinal perfusion experiments, the apparent permeability coefficient value of PMT was (1.45xa0±xa00.72)xa0×xa010−5 cm/s when perfusion with HL was performed, and the value was significantly increased to (3.92xa0±xa00.52)xa0×xa010−5 cm/s on perfusion with WJW formulaxa01. These results indicate that the pharmacokinetic parameters and absorption of BBR and PMT are affected by the other herbs or ingredients from WJW formulas.

Flavonoids casticin and chrysosplenol D from Artemisia annua L. inhibit inflammation in vitro and in vivo.

BACKGROUNDnThe aim of our experiments was to investigate the anti-inflammatory properties of casticin and chrysosplenol D, two flavonoids present in Artemisia annua L.nnnMETHODSnTopical inflammation was induced in ICR mice using croton oil. Mice were then treated with casticin or chrysosplenol D. Cutaneous histological changes and edema were assessed. ICR mice were intragastrically administrated with casticin or chrysosplenol D followed by intraperitoneal injection of lipopolysaccharide (LPS). Mouse Raw264.7 macrophage cells were incubated with casticin or chrysosplenol D. Intracellular phosphorylation was detected, and migration was assessed by trans-well assay. HT-29/NFκB-luc cells were incubated with casticin or chrysosplenol D in the presence or absence of LPS, and NF-κB activation was quantified.nnnRESULTSnIn mice, administration of casticin (0.5, 1 and 1.5μmol/cm(2)) and chrysosplenol D (1 and 1.5μmol/cm(2)) inhibited croton oil-induced ear edema (casticin: 29.39-64.95%; chrysosplenol D: 37.76-65.89%, all P<0.05) in a manner similar to indomethacin (0.5, 1 and 1.5μmol/cm(2); 55.63-84.58%). Casticin (0.07, 0.13 and 0.27mmol/kg) and chrysosplenol D (0.07, 0.14 and 0.28mmol/kg) protected against LPS-induced systemic inflammatory response syndrome (SIRS) in mice (all P<0.05), in a manner similar to dexamethasone (0.03mmol/kg). Casticin and chrysosplenol D suppressed LPS-induced release of IL-1 beta, IL-6 and MCP-1, inhibited cell migration, and reduced LPS-induced IκB and c-JUN phosphorylation in Raw264.7 cells. JNK inhibitor SP600125 blocked the inhibitory effect of chrysosplenol D on cytokine release.nnnCONCLUSIONSnThe flavonoids casticin and chrysosplenol D from A. annua L. inhibited inflammation in vitro and in vivo.

Establishment of an interleukin‑1β‑induced inflammation‑activated endothelial cell‑smooth muscle cell‑mononuclear cell co‑culture model and evaluation of the anti‑inflammatory effects of tanshinone IIA on atherosclerosis

Increasing evidence supports the hypothesis that inflammatory reactions serves an important function in the formation, progression and plaque rupture of atherosclerosis. Interleukin (IL)-1 primarily induces inflammation and is closely associated with the inflammatory environment and the formation of atherosclerosis. The present study aimed to establish an in vitro model for the evaluation of drug efficacy in the intervention of atherosclerosis from the inflammatory perspective, and to observe the anti-inflammatory effects of tanshinone IIA and andrographolide on atherosclerosis. The IL-1β-induced inflammation-activated endothelial cell (EC)-smooth muscle cell (SMC)-mononuclear cell (MC) co-culture model was established, based on the changes in a series of atherosclerosis-associated inflammatory markers secreted by ECs and SMCs. The expression of connexin in ECs, adhesion of MCs and changes in inflammatory signalling molecules were selected as evaluation indices for the inflammatory microenvironment of atherosclerosis. The use of this model revealed that tanshinone IIA exhibited significant efficacy against atherosclerosis and its inflammatory reactions. Inflammatory reactions were regarded as the primary mechanism underlying atherosclerosis. The established model simulated a series of relevant changes in the arterial wall under the inflammatory cytokines with oxidized low-density lipoprotein during the atherosclerotic process. The present study presented a reliable method for the identification of drugs with potential anti-inflammatory activity in atherosclerosis, for investigating the mechanisms of action, considering the improvement of the inflammatory state and the increase in plaque stability observed.

Lonicerae Japonicae Flos and Lonicerae Flos: A Systematic Pharmacology Review

Lonicerae japonicae flos, a widely used traditional Chinese medicine (TCM), has been used for several thousand years in China. Chinese Pharmacopeia once included Lonicerae japonicae flos of Caprifoliaceae family and plants of the same species named Lonicerae flos in general in the same group. Chinese Pharmacopeia (2005 Edition) lists Lonicerae japonicae flos and Lonicerae flos under different categories, although they have the similar history of efficacy. In this study, we research ancient books of TCM, 4 main databases of Chinese academic journals, and MEDLINE/PubMed to verify the origins and effects of Lonicerae japonicae flos and Lonicerae flos in traditional medicine and systematically summarized the research data in light of modern pharmacology and toxicology. Our results show that Lonicerae japonicae flos and Lonicerae flos are similar pharmacologically, but they also differ significantly in certain aspects. A comprehensive systematic review and a standard comparative pharmacological study of Lonicerae japonicae flos and Lonicerae flos as well as other species of Lonicerae flos support their clinical safety and application. Our study provides evidence supporting separate listing of Lonicerae japonicae flos and Lonicerae flos in Chinese Pharmacopeia as well as references for revision of relevant pharmacopeial records dealing with traditional efficacy of Lonicerae japonicae flos and Lonicerae flos.

Anti-tumor pharmacological evaluation of extracts from stellera chamaejasme L based on hollow fiber assay

BackgroundStellera chamaejasme L, a traditional Chinese herb, has long been used for treatment of various tumors in the Chinese population. In our previous study, we paid an attention to the cytotoxic and proapoptotic effects of Stellera chamaejasme L extracts (ESC, ESC-1 and ESC-2, the latter two were isolated from ESC) on 4 various tumor cells (NCI-H157, NCI-H460, BEL-7402 and SK-HEP-1) in vitro. ESCs showed significantly inhibitory effects on the 4 tumor cells. ESC-2 had the strongest inhibitory effect and the broadest sensitive cell spectrum. ESC-2 and ESC acted in a similar way against tumor cells, which suggested anti-tumor active fraction of ESC might exist in ESC-2. Here, we further observe the inhibitory and proapoptotic effects of Stellera chamaejasme L extracts in vivo.MethodsIn this study, we used hollow fiber tumor model to evaluate the inhibitory and proapoptotic effects of Stellera chamaejasme L extracts. Apoptotic rates of the cancer cells retrieved from the hollow fibers were measured with flow cytometric analysis, caspase 3, 8, 9 enzyme activities were detected by colorimetric assay, Fas, Fas-L, TNF-R1 and TNF-α expression were determined with elisa assay and radioimmunoassay respectively.ResultsThe results showed that ESC, ESC-2 all had inhibitory effects on 4 tumor cells. According to the effect strength, dose and antitumor spectrum, the order of antitumor effects of ESCs was: ESC-2u2009>u2009ESCu2009>u2009ESC-1. NCI-H460 cells were the most sensitive to ESCs. ESC, ESC-2 increased greatly the apoptotic rate and caspase 3, 8 enzyme activities in NCI-H460. ESCs had no significant effects on expression of Fas, Fas-L protein, but TNF-α/TNFR1 protein expression in NCI-H460 cells changed significantly after ESC and ESC-2 treatment.ConclusionESC-2 had the similar antitumor effect to that of ESC in vivo and further confirmed that ESC-2 may be the main antitumor active fraction of ESC, which was consistent with our previous results in vitro.

Chamaejasmenin B, a novel candidate, inhibits breast tumor metastasis by rebalancing TGF-beta paradox

Metastasis is the leading lethal factor severely restraining the effectiveness of clinical treatment. TGF-beta is the key regulator for metastasis and influences paradoxically on cancer progression. The known TGF-beta blockers exert little selectivity on its functions, indiscriminately causing the anti-metastatic and pro-growth effects. Under such circumstances, specifically rebalancing the oncological function of TGF-beta provides a crucial oncotarget against metastasis. In our study, we established the screening platform targeting cell motility and identified a potential flavonoid, Chamaejasmenin B (ICJ), extracted from Stellera chamaejasme L. It suppressed the migration and invasion in breast cancer cells in vitro. Moreover, by dynamical quantification of breast cancer progression in small-animal imaging system, ICJ was proved to be a potent inhibitor of metastasis with minimal toxic side effects. Mechanism study further revealed that ICJ efficiently blocked TGF-beta induced EMT, disrupted the interaction between β3 integrin-TβRII complex and, consequently, resulted in the selective inhibition of FAK:Src:p38 pathway. Meanwhile, specific blockage of this pathway largely attenuated the anti-metastatic function of ICJ. Importantly, in contrast with the antagonistic effects on TGF-beta induced metastasis, ICJ obviously sensitized its cytostatic activity, suggesting that it was not a pan-blocker but a rebalancer for the functional output of TGF-beta. Collectively, by targeting TGF-beta Paradox, we experimentally provided a promising candidate for metastatic intervention.

Cytotoxic biflavones from Stellera chamaejasme.

Bioassay-guided phytochemical studies on Stellera chamaejasme led to the isolation of two new biflavones, chamaejasmenin E (1) and chamaejasmin D (2), together with ten known compounds. The structures of new compounds were elucidated by extensive spectroscopic analyses and their absolute configurations on 2, 3, 2″ and 3″ were confirmed by TDDFT quantum chemical calculated ECD spectra combined with experimental ECD spectra. All isolated biflavones were evaluated for their cytotoxic activities against Bel-7402 and A549 tumor cell lines, and sikokianin D (3) was found to possess the most potential cytotoxic activities against both the two cell lines with IC50 values of 1.29 ± 0.21 and 0.75 ± 0.25 μM, respectively. Moreover, some structure-function relationships of these bioflavones for cytotoxic activities were explored and summarized.

Pharmacokinetics of Two Alkaloids after Oral Administration of Rhizoma Coptidis Extract in Normal Rats and Irritable Bowel Syndrome Rats

A comparative pharmacokinetic study of berberine and palmatine after oral administration of Rhizoma Coptidis extract (96u2009mg/kg, containing berberine 22u2009mg/kg and palmatine 5u2009mg/kg based on body weight) was performed in normal and postinflammation irritable bowel syndrome (PI-IBS) rats, induced by intracolonic instillation of acetic acid and restraint stress. Quantification of berberine and palmatine in rat plasma was achieved by using a sensitive and rapid UPLC-MS/MS method. Plasma samples were collected at 13 different time points and the pharmacokinetic parameters were analyzed by WinNonlin software. The significant differences in the pharmacokinetic behaviors, such as C maxu2061, AUC(0–t), V d/F, and CL/F, of berberine and palmatine were found between normal and PI-IBS model rats. The results indicated that PI-IBS pathological conditions in rats could alter the pharmacokinetic behavior of drug. Preclinical pharmacokinetic studies are usually carried out on healthy animals. However, we should pay more attention to the fact that the change of pharmacokinetic behavior plays an important role on efficacy. It is essential to investigate the pharmacokinetics of the drug in disease status.

Pharmacokinetic difference of berberine between normal and chronic visceral hypersensitivity irritable bowel syndrome rats and its mechanism

Berberine is one of active alkaloids from Rhizoma coptidis in traditional Chinese medicine. The pharmacokinetics of berberine in rat plasma were compared between normal and chronic visceral hypersensitivity irritable bowel syndrome rats (CVH-IBS) established by mechanical colon irritation using angioplasty balloons for 2xa0weeks after oral administration of berberine hydrochloride (25xa0mg/kg) with the equivalent dose of 22xa0mg/kg for berberine according to body weight. Immunohistochemical analysis of c-fos and myosin light chain kinase (MLCK) and immunofluorescence analysis of MLCK in rat colon were conducted. Quantification of berberine in rat plasma was achieved by using a sensitive and rapid UPLC-MS/MS method. Plasma samples were collected at 15 different points in time and the pharmacokinetic parameters were analyzed by WinNonlin software. The great different pharmacokinetic behavior of berberine was observed between normal and CVH-IBS model rats. Compared with normal group, T1/2 and AUC(0–t) of berberine in the model group were significantly increased, respectively (573.21xa0±xa0127.53 vs 948.22xa0±xa0388.57xa0min; 8,657.19xa0±xa01,562.54 vs 11,415.12xa0±xa01,670.72xa0min.ng/ml). Cl/F of berberine in the model group significantly decreased, respectively (13.89xa0±xa01.69 vs 9.19xa0±xa02.91xa0L/h/kg). Additionally, the expressions of c-fos and MLCK in model group were higher than those in normal group. The pharmacokinetic behavior of berberine was significantly altered in CVH-IBS pathological conditions, which indicated the dosage modification of berberine hydrochloride in CVH-IBS were necessary. Especially, improved exposure to berberine in rat plasma in CVH-IBS model rats was attributed to increased the expression of MLCK.

Studies on Efficacy Mechanism of Chinese Herbal Combination Based on Drug metabolizing Enzyme

Mixed Formula is the main form and method of Traditional Chinese Medicine (TCM) treatment. It can produce different therapeutic effects by changing the combination of TCM Formula. Wuji Wan is a formula of TCM and was composed of Rhizoma Coptidis (Huanglian in Chinese, HL), Fructus Evodiae Rutaecarpae (Wuzhuyu, WZY) and Radix Paeoniae Alba (Baishao, BS). Wuji Wan is mainly used for the treatment of intestinal diseases. This paper took Wuji Wan as example study on efficacy Mechanism of TCM Combination Based on drug metabolizing Enzyme. Inhibition of cytochrome P450 (CYP) is regarded as the most clinically important pharmacokinetic causes among the various possible factors for drug-drug or herb-herb/herb-drug interactions. In this study, the in vitro inhibitory effects of Wuji Wan with different combination within HL, WZY and BS on six major rat CYPs (CYP1A2, CYP2A6, CYP2C19, CYP2D6, CYP2E1 and CYP3A1/3A2) activities were examined by using HPLC and LC-MS. Wuji Wan with different combination were designed as 9 Formulae according to orthogonal table L9 (34); meanwhile the inhibitory effects of the single herb HL, WZY and BS also were done and compared with 9 Wuji Wan Formulae with different combination. Results demonstrated that BS showed negligible inhibitory effects on the six major CYP isoenzymes in rat liver microsomes, but HL showed strong inhibitory effects on 6 CYPs with almost all of the IC50 values below 200 μg (crude drug)•mL-1, and WZY showed a little bit inhibitory effects on 6 CYPs with the IC50 values between 870-2000 μg (crude drug)•mL-1; moreover, 9 Wuji Wan Formulae showed different inhibitory characteristic following with the dose levels of HL, WZY and BS in L9(34) design. In conclusion, this study demonstrates that Wuji Wan is likely to cause significant herb-drug interactions in humans when co-administered with substrates of the six CYPs (CYP1A2, CYP2A6, CYP2C19, CYP2D6, CYP2E1 and CYP3A)and also the different inhibitory effects on cytochrome P450 of Wuji Wan within different combination could be one of the reasons for explaining the different combination of TCM Formula producing different therapeutic effects, because the chemical composition of TCM in vivo (prototype or metabolites) is the Material basis of TCM effects.