Preliminary investigation on the analysis of the whole components of Pogejiuxin decoction and its formulation pattern based on ultrahigh-performance liquid chromatography with quadrupole time-of-flight mass spectrometry.

RATIONAL: Pogejiuxin decoction (PGJXD) is one of the most important formulas for the treatment of heart failure. However, there is a great lack of research on the material basis of this formula, especially research on its compatibility laws, which restricts its clinical use. Studying the complete ingredients and compatibility rules of PGJXD has great significance for guiding clinical medication. METHODS: The entire formula, the major single herbs, the drug pairs and the disassembled formula were analyzed by ultrahigh-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UHPLC/QTOFMS/MS), matching the chemical composition database and global natural product social molecular networking to explain the chemical composition as well as the combination pattern of PGJXD. RESULTS: A total of 1048 chemical constituents were fully analyzed from the major single herbs, the drug pairs and the disassembled formula and 188 chemical constituents, including 13 potential novel compounds, were firstly identified from the whole formula. We found that the chemical compositions were reduced after the single herbs were matched to the other herbs, especially the significant reduction of highly toxic diester alkaloids after compatibility, indicating that the medicines of PGJXD were interdependent and controlled by each other. CONCLUSION: This study innovatively researches and compares the compositional differences between the entire formula of PGJXD, the single, paired and separated formulas, greatly extending our understanding of the chemical substance basis of these compounds, and preliminarily explores the compatibility laws of PGJXD, providing some theoretical guidance for clinical medication.

[Determination of three plant growth regulators in Dendrobium officinale and Anoectochilus roxburghii by three-phase hollow fiber liquid phase microextraction- high performance liquid chromatography].

Dendrobium officinale (D. officinale) and Anoectochilus roxburghii (A. roxburghii) are precious raw materials for traditional Chinese medicine. The growing demand for D. officinale and A. roxburghii cannot be met by current production techniques. Hence, the widespread artificial cultivation of D. officinale and A. roxburghii using substantial amounts of plant growth regulators (PGRs) has emerged. The excessive use of PGRs not only affects the quality and efficacy of medicinal materials but also causes a series of safety issues. Therefore, expanding research on residual PGRs in valuable Chinese medicinal materials is important to avoid the health hazards caused by these substances. Unfortunately, the identification of PGRs is challenging because of their trace and complex matrices. High performance liquid chromatography (HPLC) has become one of the mainstream analytical methods for PGR determination. An important consideration in the application of this technique to the detection of trace acidic PGRs is how to improve its accuracy and sensitivity. Three-phase hollow fiber liquid phase microextraction (3P-HF-LPME) has the advantages of a high enrichment factor, complex sample purification ability, low reagent consumption, low cost, and easy integration with chromatographic systems. Thus, the 3P-HF-LPME method overcomes the many shortcomings of traditional sample pretreatment methods. In this study, a novel, simple, and effective analytical method based on 3P-HF-LPME combined with HPLC was developed to extract, purify, enrich, and detect three trace acidic PGRs (indole-3-acetic acid, naphthyl acetic acid and indolebutyric acid) in D. officinale and A. roxburghii. The chromatographic separation conditions and 3P-HF-LPME model parameters were systematically optimized for this purpose. First, the sample solution was prepared by ultrasonication and low-temperature standing, and then adjusted to pH 3.0 using dilute hydrochloric acid. The sample solution (10 mL) and NaCl (1.50 g) were stored in a 15 mL brown extraction bottle with a built-in magnetic stirrer. Next, 30 µL of NaOH solution (pH 11.0) as the inner phase solution was injected into the inner cavity of a hollow fiber tube, which was subsequently sealed at both ends. The hollow fiber tube was soaked in n-octanol for 5 min and dried naturally to remove excess extraction solvent from its surface. Finally, the fiber tube was placed in a brown extraction bottle and stirred using a thermostatic magnetic stirrer at 40 ℃ and 1600 r/min for 2 h. After extraction, the three target analytes were separated on a Welch Ultimate XB-C18 column (250 mm×4.6 mm, 5 µm) under isocratic elution conditions using acetic acid aqueous solution and methanol (45∶55, v/v) as the eluent. The results indicated that the three PGRs showed good linearity in the range of 0.5-100.0 µg/L (coefficients of determination (r2)=0.9999), with limits of detection (LODs) of 0.02-0.15 µg/L. The method recoveries were 88.5-102.2%, with relative standard deviations (RSDs) of less than 3.7% (n=3). The extraction efficiencies and enrichment factors of the three PGRs in 15 batches of fresh D. officinale and A. roxburghii products were found to be 42.0%-86.8% and 140-289. Full-scan mass spectrometry was used to further identify positive samples to avoid false-positive results and enhance the reliability of the experimental method. In summary, the proposed method is sensitive, accurate, reliable, environment friendly, and capable of high enrichment. It could be used to determine the residues of three acidic PGRs in D. officinale and A. roxburghii. Moreover, it can provide technical support for the residue detection of PGRs in other Chinese medicinal materials.

Determination of Multiple Neurotransmitters through LC-MS/MS to Confirm the Therapeutic Effects of Althaea rosea Flower on TTX-Intoxicated Rats.

Tetrodotoxin (TTX) inhibits neurotransmission in animals, and there is no specific antidote. In clinical practice in China, Althaea rosea (A. rosea flower) extract has been used to treat TTX poisoning. In this work, the efficacy of the ethyl acetate fraction extract of A. rosea flower in treating TTX poisoning in rats was investigated. A high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to determine nine neurotransmitters in rat brain tissue, including γ-aminobutyric acid (GABA), dopamine (DA), 5-hydroxytryptamine (5-HT), noradrenaline (NE), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxyindole-3-acetic acid (5-HIAA), epinephrine (E), and tyramine (Tyn). The detoxifying effect of A. rosea flower was verified by comparing the changes in neurotransmitters' content in brain tissue before and after poisoning in rats. The assay was performed in multiple reaction monitoring mode. The quantification method was performed by plotting an internal-standard working curve with good linearity (R2 > 0.9941) and sensitivity. Analyte recoveries were 94.04-107.53% (RSD < 4.21%). Results indicated that the levels of 5-HT, DA, E, and NE in the brains of TTX-intoxicated rats decreased, whereas the levels of GABA, Tyn, and 5-HIAA showed an opposite trend, and HVA and DOPAC were not detected. The levels of all seven neurotransmitters returned to normal after the gavage administration of ethyl acetate extract of A. rosea flower to prove that the ethyl acetate extract of A. rosea flower had a therapeutic effect on TTX poisoning. The work provided new ideas for studies on TTX detoxification.

Uncovering the protective mechanism of Pien-Tze-Huang in rat with alcoholic liver injury based on cytokines analysis and untargeted metabonomics.

Pien-Tze-Huang (PTH) is a well-known traditional Chinese patent medicine with excellent liver-protection effect. However, the mechanism of hepatoprotective action has not yet been entirely elucidated. The aim of this study was to investigate the mechanism of protective effect of PTH on alcohol-induced liver injury in rats using cytokine analysis and untargeted metabolomics approaches. An alcoholic liver disease (ALD) model with SD rats was established, and PTH was administered according to the prescribed dose. The hepatoprotective effect of PTH was evaluated by pathological observation of liver tissue and changes in biochemical index activity and cytokines in serum. Serum samples were analyzed by ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS), and differentially expressed metabolites were screened by multivariate statistical analysis. KEGG combined with metabolic pathway analysis were used to evaluate the underlying metabolic pathways. Results showed liver histopathology injury was attenuated. The levels of IL-6, TNF-α and NF-κB were significantly decreased in rats intervened with PTH groups, suggesting that it may alleviate inflammation via suppressing the inflammatory cytokines signaling pathway. Eighty differentially expressed metabolites were found and identified. Pathway analysis indicated that the hepatoprotective effects of PTH occurred through the regulation of inflammatory cytokines signaling pathway, primary bile acid biosynthesis, vitamin B6 metabolism pathway, cholesterol metabolism, and tyrosine metabolism. PTH showed favorable hepatoprotective effect through multiple pathways. This study has great importance in fully revealing the mechanism of hepatoprotective action and can help improve the clinical application of PTH.

Three-phase hollow fiber liquid-phase microextraction combined with HPLC for determination of three trace acidic plant growth regulators in Anoectochilus roxburghii (Wall.) Lindl.

The analysis of plant growth regulators presents a challenge due to their trace quantities and complex matrices. A novel, simple, and effective analytical method for the determination of three trace acidic plant growth regulators in Anoectochilus roxburghii (Wall.) Lindl was developed to address this issue. Three-phase hollow fiber liquid-phase microextraction combined with high-performance liquid chromatography was applied for the enrichment, purification, and determination of three acidic plant growth regulators, namely, indole-3-acetic-acid, indole-3-butyric-acid, and (+)-abscisic acid. The factors affecting extraction performance, including extractant species, pH of donor and acceptor phases, salt addition dosage, extraction time, temperature, and stirring rate, were investigated and optimized. Under optimum conditions, the proposed method provided good linearity (R2 , 0.9994-0.9999), low limit of detection (0.038-0.12 ng/mL), and acceptable relative recoveries (56.7-117.6%). The enrichment factors were between 153 and 328. The developed method was successfully applied to the enrichment and determination of plant growth regulators in Anoectochilus roxburghii (Wall.) Lindl and exhibited increased purification capacity, higher sensitivity, and decreased organic solvent consumption compared with conventional sample preparation methods. This method may provide a testing platform for the monitoring of plant growth regulator residues, ensuring the safe and effective use of traditional Chinese medicine.

Zhibaidihuang Decoction Ameliorates Cell Oxidative Stress by Regulating the Keap1-Nrf2-ARE Signalling Pathway.

Zhibaidihuang decoction (ZBDHD) is a Chinese herbal formula, which is used in Chinese traditional medicine to treat symptoms of Yinxuhuowang (Yin deficiency and high fire) syndrome. This study elucidates the mechanism of ZBDHD on oral ulcers, one Yinxuhuowang syndrome. Simultaneously, some ingredients in ZBDHD were found and identified by ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). A Ganjiangfuzirougui decoction- (GJD-) induced Yinxuhuowang syndrome SD rat model was used to demonstrate the efficiency of ZBDHD treatment. The oral mucosa of rat in the GJD group, stained with hematoxylin and eosin (H&E), showed epidermal shedding and inflammatory cell infiltration. And an alleviation efficiency of ZBDHD in GJD-induced pathological changes in the oral mucosa could be obtained. ZBDHD treatment restored the GJD-induced imbalance of metabolites, which were choline, glycocholic acid, and palmitoyl-L-carnitine (PALC). GJD stimulated the expression of NF-κB. And the overexpressed of NF-κB in mucosa of rat in the GJD group could be inhibited by ZBDHD treatment. Simultaneously, the optimal efficiency of ZBDHD treatment on the cellular ATP content, oxygen consumption rate (OCR), and superoxide dismutase (SOD) concentration was evaluated, in vitro assay. Compared to the control cells, the ATP content, OCR, and SOD activity in the ZBDHD-treated cells were significantly higher. For the mechanisms study, seven cytokines were screened with a Dual-Luciferase Reporter gene assay. In the ARE assay, the luciferase signal was stimulated significantly by ZBDHD. In cells, the transcription of nrf2, maf, and keap1, which were related to the ARE pathway, was elevated by ZBDHD treatment. Our study demonstrated that high-dose GJD could lead to Yinxuhuowang syndrome, such as oral ulcers, and the imbalance in serum metabolites. And ZBDHD can improve oral cell inflammation and the imbalance of metabolism by inhibiting NF-κB and enhancing the activity of the ARE signalling pathway to ameliorate oxidative stress in the cell. This study provides a theoretical basis for the clinical application of ZBDHD.

Three new methylated Δ8-pregnene steroids from the Polyalthia laui-derived fungus Stemphylium sp. AZGP4-2.

Three new methylated Δ8-pregnene steroids, stemphylisteroids A-C (1-3) were isolated from the medicinal plant Polyalthia laui-derived fungus Stemphylium sp. AZGP4-2. Their structures were elucidated by the detailed analysis of comprehensive spectroscopic data. The absolute configuration of 1 was determined by X-ray crystallographic analysis. Compound 1 show antibacterial activity against Escherichia coli with the MIC value of 6.25 µg/mL, and 2 exhibited a broad spectrum of antibacterial activities against six pathogenic bacteria with the MIC values ranging from 12.5 to 50 µg/mL. The discovery of three methylated Δ8-pregnene steroids 1-3 are a further addition to diverse and complex array of methylated steroids.

Jatrorrhizine inhibits mammary carcinoma cells by targeting TNIK mediated Wnt/ß-catenin signalling and epithelial-mesenchymal transition (EMT).

BACKGROUND: Traf2 and Nck interacting serine protein kinase (TNIK) is a tumour target protein which its high expression is closely related to the occurrence and development of mammary carcinoma cells. Molecular docking revealed that jatrorrhizine, a protoberberine alkaloid, exhibits good binding affinity and interaction with TNIK. However, the underlying mechanisms of jatrorrhizine targeting TNIK inhibits the proliferation and metastasis of breast cancer cells remain unclear. METHODS: To figure out the mechanisms in vitro and in vivo, the CRISPR/Cas9 technology was used to knockout TNIK gene and detected qualitatively by immunofluorescence and immunoblotting assay. The MTT cell viability assay for cytotoxicity test, the apoptosis were detected by flow cytometry, the migration and invasion were evaluated by colony formation, wound healing assay and cell invasion assay, respectively. Anticancer effects were further corroborated by 4T1/Luc homograft tumour model. RESULTS: The results showed that targeted knockout of TNIK that attenuated Wnt/ß-catenin signalling and epithelial-mesenchymal transition (EMT) expression, the effects were potentiated by the addition of jatrorrhizine. Moreover, jatrorrhizine distinctly inhibited the proliferation of MDA-MB-231, MCF-7 and 4T1 cells with IC50 values of 11.08 ± 1.19 µM, 17.11 ± 4.54 µM and 22.14 ± 2.87 µM, induced mitochondrial dysfunction and early apoptosis involving mitochondrial apoptotic pathway. These results were further corroborated by the 4T1 tumour-bearing mice, which showed that jatrorrhizine significantly suppressed the proliferation and metastasis of mammary carcinoma cells without obvious toxicity. CONCLUSION: These findings provide an overall perspective that jatrorrhizine potentially restrains TNIK regulating Wnt/ß-catenin signalling and EMT expression for mammary cancer targeted therapy.

The Role and Mechanism of miRNA-1224 in the Polygonatum sibiricum Polysaccharide Regulation of Bone Marrow-Derived Macrophages to Osteoclast Differentiation.

MicroRNAs (miRNAs) are endogenous noncoding small molecule RNAs that regulate cell proliferation, differentiation, fat metabolism, and hormone secretion. Studies have shown that miRNAs regulate the processes related to osteoporosis, including the differentiation of osteoblasts, osteoclasts, and chondrocytes, and are one of the important regulatory factors of some bone metabolic diseases. In our previous study, it has been revealed that natural compound Polygonatum sibiricum polysaccharide (PSP) can promote osteoblast formation and block osteoclastogenesis through Wnt/ß-catenin signaling pathway. This study was designed to investigate whether PSP can inhibit expression of osteoclast-related genes by Hippo signaling pathway, which was prevented by effectively blocking the expression of miR-1224. This study showed that there were 27 differentially expressed miRNAs when PSP inhibits osteoclastogenesis, the most notable of which was miR-1224. Furthermore, the study showed that PSP increased the level of Limd1, which was the target gene of miR-1224. In conclusion, these findings demonstrate that PSP suppressed osteoclastogenesis in vitro through the Hippo signaling pathway based on miR-1224. This study may aid in the development of a therapeutic approach utilizing PSP for the enhancement of bone health and prevention of osteoporosis.

Sulforaphane Induces Cell Death Through G2/M Phase Arrest and Triggers Apoptosis in HCT 116 Human Colon Cancer Cells.

Sulforaphane (SFN), an isothiocyanate, exists exclusively in cruciferous vegetables, and has been shown to possess potent antitumor and chemopreventive activity. However, there is no available information that shows SFN affecting human colon cancer HCT 116 cells. In the present study, we found that SFN induced cell morphological changes, which were photographed by contrast-phase microscopy, and decreased viability. SFN also induced G2/M phase arrest and cell apoptosis in HCT 116 cells, which were measured with flow cytometric assays. Western blotting indicated that SFN increased Cyclin A, cdk 2, Cyclin B and WEE1, but decreased Cdc 25C, cdk1 protein expressions that led to G2/M phase arrest. Apoptotic cell death was also confirmed by Annexin V/PI and DAPI staining and DNA gel electrophoresis in HCT 116 cells after exposure to SFN. The flow cytometric assay also showed that SFN induced the generation of reactive oxygen species (ROS) and Ca[Formula: see text] and decreased mitochondria membrane potential and increased caspase-8, -9 and -3 activities in HCT 116 cell. Western blotting also showed that SFN induced the release of cytochrome c, and AIF, which was confirmed by confocal microscopy examination. SFN induced ER stress-associated protein expression. Based on those observations, we suggest that SFN may be used as a novel anticancer agent for the treatment of human colon cancer in the future.

Bufalin Inhibits NCI-H460 Human Lung Cancer Cell Metastasis In Vitro by Inhibiting MAPKs, MMPs, and NF-κB Pathways.

Bufalin, a component of Chan Su (a traditional Chinese medicine), has been known to have antitumor effects for thousands of years. In this study, we investigated its anti-metastasis effects on NCI-H460 lung cancer cells. Under sub-lethal concentrations (from 25 up to 100 nM), bufalin significantly inhibits the invasion and migration nature of NCI-H460 cells that were measured by Matrigel Cell Migration Assay and Invasion System. Bufalin also suppressed the enzymatic activity of matrix metalloproteinase (MMP)-9, which was examined by gelatin zymography methods. Western blotting revealed that bufalin depressed several key metastasis-related proteins, such as NF-κB, MMP-2, MMP-9, protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3-K), phosphorylated Akt, growth factor receptor-bound protein 2 (GRB2), phosphorylated extracellular signal-regulated kinase (ERK), phosphorylated p38, and phosphorylated c-Jun NH2-terminal kinase (JNK). As evidenced by immunostaining and the electrophoretic mobility shift assay (EMSA), bufalin induced not only a decreased cytoplasmic NF-κB production, but also decreased its nuclear translocation. Several metastasis-related genes, including Rho-associated (Rho A), coiled-coil-containing protein kinase 1 (ROCK1), and focal adhesion kinase (FAK), were down-regulated after bufalin treatment. In conclusion, bufalin is effective in inhibiting the metastatic nature of NCI-H460 cells in low, sub-lethal concentrations. Such an effect involves many mechanisms including MMPs, mitogen-activated protein kinases (MAPKs) and NF-κB systems. Bufalin has a potential to evolve into an anti-metastasis drug for human lung cancer in the future.

Investigation of in vitro and in vivo antioxidant activities of flavonoids rich extract from the berries of Rhodomyrtus tomentosa(Ait.) Hassk.

This study investigated the in vitro and in vivo antioxidant activities of the flavonoids rich extract from Rhodomyrtus tomentosa Hassk (R. tomentosa) berries. The in vitro antioxidant assay demonstrated that the flavonoids rich extract (62.09% rutin equivalent) extracted by ethanol and purified by AB-8 macroporous resin was strong in reducing power, superoxide radical, hydroxyl radical and DPPH radical scavenging activity, as well as inhibiting lipid peroxidation. In the in vivo assays, the flavonoids rich extract significantly enhanced the activities of antioxidant enzymes in serums of mice after they were administered with the extract. The results suggested that the flavonoids rich extract from R. tomentosa fruits possesses potent antioxidant properties. In addition, the chemical compositions of flavonoids rich extract were identified by UPLC-TOF-MS/MS. Six flavonoids were tentatively identified as myricetin, quercetin, dihydromyricetin, kaempferol, quercetin 7,4'-diglucoside and vitexin. Therefore, R. tomentosa berries could be used as a new source of antioxidant ingredient.

The crude extract of Corni Fructus inhibits the migration and invasion of U-2 OS human osteosarcoma cells through the inhibition of matrix metalloproteinase-2/-9 by MAPK signaling.

Osteosarcoma is the most common primary malignancy of the bone cancers. In the Chinese population, the crude extract of Corni Fructus (CECF) has been used as Traditional Chinese medicine to treat several different diseases for hundreds of years. In the present study, effects of CECF on inhibition of migration and invasion in U-2 OS human osteosarcoma cells were examined. CECF significantly inhibited migration and invasion of U-2 OS human osteosarcoma cells. We also found that CECF inhibited activities of matrix metalloproteinases-2 (MMP-2) and matrix metalloproteinases-9 (MMP-9). CECF decreased protein levels of FAK, PKC, SOS1, MKK7, MEKK3, GRB2, NF-κB p65, COX-2, HIF-1α, PI3K, Rho A, ROCK-1, IRE-1α, p-JNK1/2, p-ERK1/2, p-p38, Ras, p-PERK, MMP-2, MMP-9, and VEGF in U-2 OS cells. Results of this study indicate that CECF may have potential as a novel anticancer agent for the treatment of osteosarcoma by inhibiting migration and invasion of cancer cells.

Alteration of the tongue manifestation reflects clinical outcomes of peptic ulcer disease.

OBJECTIVES: This study investigated whether the tongue inspection technique in Traditional Chinese Medicine (TCM) can be used as a noninvasive auxiliary diagnostic tool to differentiate the subtypes of peptic ulcer disease (PUD) and as an indicator of therapeutic efficacy. SUBJECTS AND METHODS: A total of 198 outpatients from the China Medical University Hospital were recruited. The control group comprised 50 healthy adults. The remaining 148 patients were diagnosed with gastric ulcer, duodenal ulcer, or Helicobacter pylori (Hp) infection using upper gastrointestinal (GI) endoscopy, biopsy, and Campylobacter-like organism test. Tongue appearance was evaluated by a physician experienced in clinical Chinese medicine. Images of the tongue were immediately recorded using a high-resolution digital camera system. RESULTS: The affected group of 148 patients received an 8-week course of ulcer therapy. Of these, 108 patients infected with Hp were subjected to triple therapy in the first week. Forty-nine of these 108 cases infected with Hp completed secondary examination of upper GI endoscopy and tongue inspection. Forty-one of 49 cases (83.7%) were fully cured of Hp infection. These results showed that the color of the tongue body did not change in the cured patients; however, tongue fur was markedly thinner with a color change to white (p<0.05), while sublingual veins with engorgement (p<0.05) and blood stasis (p<0.01) improved after the ulcer healed and Hp was eradicated. CONCLUSIONS: TCM tongue inspection can be potentially used as a noninvasive auxiliary diagnostic method and as an indicator for clinical outcomes for patients with PUD.

Memory defect induced by ß-amyloid plus glutamate receptor agonist is alleviated by catalpol and donepezil through different mechanisms.

Our previous studies demonstrate that a non-cholinesterase inhibitor (AChEI) compound catalpol, purified from a traditional Chinese medicinal herb Rehmannia glutinosa, could improve the symptoms and pathological changes in animal and cellular models of memory related neurodegenerative diseases. In this study, we compared catalpol with the most commonly used AChEI donepezil in respect to their mechanism of action on the neurodegenerative changes in an animal model induced by beta-amyloid (Aß) plus glutamate receptor agonist. It was found that the model mice showed significant deficit in the learning ability and memory in Y maze avoidance test, and meanwhile both donepezil and catalpol greatly improve the learning ability and memory after 2 to 3 months' administration. At the selected doses, donepezil only partially raised the declined brain muscarinic acetylcholine receptor (M receptor) density and choline acetyltransferase (ChAT) activity resulting in these levels still lower than normal control, while catalpol completely retrieved these two parameters. ELISA revealed that catalpol, instead of donepezil, possessed the capability of elevating the declined brain BDNF level of the animal model. The ELISA results on the BDNF protein level was confirmed by quantitative RT-PCR measurement of BDNF mRNA in Aß25₋35-treated primary culture of forebrain neurons. In combination with our previous work, we think the neuroprotective effects of donepezil and catalpol are mediated through different mechanisms. Since BDNF has been proved to be an important intrinsic factor in protecting neurodegenerative diseases, catalpol may be a hopefully effective compound against neurodegenerative changes induced by Aß and glutamate receptor agonist.

Quercetin and rutin reduced the bioavailability of cyclosporine from Neoral, an immunosuppressant, through activating P-glycoprotein and CYP 3A4.

Quercetin and rutin are popular flavonoids in plant foods, herbs, and dietary supplements. Cyclosporine (CSP), an immunosuppressant with a narrow therapeutic window, is a substrate of P-glycoprotein (P-gp) and cytochrome P-450 3A4 (CYP3A4). This study investigated the effects of quercetin and rutin on CSP pharmacokinetics from Neoral and relevant mechanisms. Rats were orally administered Neoral with and without quercetin or rutin. The blood CSP concentration was assayed by a specific monoclonal fluorescence polarization immunoassay. The results showed that quercetin and rutin significantly decreased the C(max) of CSP by 67.8 and 63.2% and reduced the AUC(0-540) by 43.3 and 57.2%, respectively. The in vitro studies indicated that the quercetin and rutin induced the functions of P-gp and CYP3A4. In conclusion, quercetin and rutin decreased the bioavailability of CSP through activating P-gp and CYP3A. Transplant patients treated with Neoral should avoid concurrent consumption of quercetin or rutin to minimize the risk of allograft rejection.

Diallyl trisulfide (DATS) inhibits mouse colon tumor in mouse CT-26 cells allograft model in vivo.

Our earlier studies showed that DATS induced apoptosis in human colon cancer HT29 and colo 205 cell lines in vitro. However, there is no report to show that DATS induced apoptosis in vitro and inhibited CT26 cancer cells in vivo on a murine allograft animal model. In vitro studies, the results indicated that DATS induced morphological changes and induction of apoptosis in CT26 cells. In vivo studies, CT26 cancer cells were implanted into BALB/c mice and groups of mice were treated with vehicle, DATS (10 and 50 mg/kg of body weight). DATS were injected once per four days intraperitoneally (i.p.), with treatment starting 4 weeks prior to cells inoculation. Treatment with vehicle or with 10 and 50 mg/kg of DATS resulted in a reduction in tumor volume and weight. Tumor volume and total hemoglobin in allograft mice treated with 50 mg/kg DATS were significantly smaller than that in the control group. These findings indicated that DATS inhibits tumor growth in an allograft animal model. Thus, DATS may represent a colon cancer preventive agent and can be used in the future.

Different influences on tacrolimus pharmacokinetics by coadministrations of zhi ke and zhi shi in rats.

Tacrolimus, an immunosuppressant with narrow therapeutic window, has been used widely in transplant patients. Grapefruit juice and pomelo have been reported to increase the blood levels of tacrolimus. Zhi Ke and Zhi Shi, the ripe peels and unripe fruits of Citrus aurantium which is chemotaxonomically related to grapefruit and pomelo, are in wide use in clinical Chinese medicine. To investigate the possible interaction of these two Citrus herbs with tacrolimus, male Sprague-Dawley rats were orally given tacrolimus (1.5 mg/kg) with and without Zhi Ke and Zhi Shi decoctions in a cross-over design. Blood samples were withdrawn via cardiopuncture at specific time and quantitated by a microparticle enzyme immunoassay. In addition, to explore the mechanism of interaction, LS 180 cell line was used for the transport study of rhodamine 123, a typical substrate of P-glycoprotein (P-gp). The results showed that Zhi Shi significantly decreased the C(max) and AUC(0-t) of tacrolimus by 72.4% and 72.0%, respectively, whereas Zhi Ke did not affect tacrolimus pharmacokinetics. LS 180 cell line study indicated that Zhi Shi increased the efflux activity of P-gp, enabling us to explain the decreased oral bioavailability of tacrolimus caused by Zhi Shi. Hence, we suggest that Zhi Shi be contraindicated for transplant patients treated with tacrolimus to reduce the risk of allograft rejection.

The roles of AIF and Endo G in the apoptotic effects of benzyl isothiocyanate on DU 145 human prostate cancer cells via the mitochondrial signaling pathway.

It is well known that the response of cancer cells to chemotherapeutic drugs involves the activation of apoptotic pathways. Benzyl isothiocyanate (BITC) is an important compound found in plant food and has been shown to have anti-cancer effects on human cancer cells, but its effect on prostate cancer cells in vitro remains unknown. The aim of the present study was to investigate the effects of BITC on DU 145 human prostate cancer cells in order to clarify whether a time/concentration range for optimal BITC-induced apoptosis exists and to find the associated signaling pathway. Cell morphological changes, percentage of cell viability, DNA damage and apoptosis in DU 145 cells were examined by phase-contrast microscopy, flow cytometric assay, 4',6-diamidine-20-phenylindole dihydrochloride staining, comet assay and Western blotting analysis. The results indicate that BITC induces cell morphological changes, decreases the percentage of viable cells (induction of cell cytotoxicity), and induces DNA damage and apoptosis in DU 145 cells in a time- and dose-dependent manner. Flow cytometric assays indicated that BITC promoted reactive oxygen species and Ca2+ productions and decreased the levels of mitochondrial membrane potential (ΤYm), while the pre-treatment with N-acetylcysteine caused an increase in the percentage of viable cells. BITC also promoted caspase-3, -8 and -9 activities. Furthermore, when cells were pre-treated with the caspase-3 inhibitor and then treated with BITC, this led to an increase in the percentage of viable cells. Confocal laser microscopy examination indicated that BITC promoted the expression of AIF and Endo G, which were released from the mitochondria in DU 145 cells. In conclusion, BITC induces apoptosis in DU 145 cells through the release of AIF and Endo G from the mitochondria and also promotes caspase-3 activation.

Triptolide induces apoptosis in human adrenal cancer NCI-H295 cells through a mitochondrial-dependent pathway.

Triptolide, the main active component obtained from Tripterygium wilfordii Hook. f, has been reported to present potent immunosuppressive and anti-inflammatory biological activities. It has been previously shown that due to the cytotoxicity of triptolide it has a limited use in the clinic. Although numerous studies have shown that triptolide induced apoptosis in many human cancer cells there is no report to show triptolide-induced apoptosis in human adrenal cancer cells. We treated the human adrenal cancer NCI-H295 cells with triptolide in vitro and investigated its cytotoxic effects. The cytotoxicity was examined and quantitated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and the viability of inhibition and apoptosis was determined by flow cytometric assay, using propidium iodide (PI) staining for apoptosis. Flow cytometric assay also used for the determination of reactive oxygen species (ROS) production and the levels of mitochondrial membrane potential (ΔΨm), and the caspase-3 and -9 activation in NCI-H295 cells. Western blotting was used for examining the changes of apoptotic associated proteins. The results indicated that triptolide induced cytotoxicity (decreased the percentage of viable cells) and induced sub-G1 phase (apoptosis) occurring in NCI-H295 cells and those effects are dose-dependent. Results also showed that triptolide promoted the production of ROS and decreased the levels of ΔΨm in examined NCI-H295 cells. The results showed that triptolide promoted the levels of cytochrome c, Apaf-1, AIF, Endo G, caspase-9 and -3 which were analyzed by Western blotting. These results suggest that triptolide is able to induce apoptosis on NCI-H295 cells through the mitochondrial-dependent signal pathway.