"UHPLC-Q-TOF/MS-chemometrics-network pharmacology" integrated strategy to discover quality markers of raw and stir-fried Fructus Tribuli and process optimization of stir-fried Fructus Tribuli.

INTRODUCTION: Fructus Tribuli, the dried ripe fruit of Tribulus terrestris L., has various beneficial effects, including liver-calming and depression-relieving effects. Raw Fructus Tribuli (RFT) and stir-fried Fructus Tribuli (SFT) are included in the Chinese Pharmacopoeia 2020 edition (Ch. P 2020). However, owing to the lack of specific regulations on SFT-processing parameters in Ch. P 2020, it is difficult to ensure the quality of commercially available SFT. OBJECTIVE: The present study aimed to screen the quality markers (Q-markers) of RFT and SFT and optimize the processing technology of SFT based on the identified Q-markers. METHODS: First, the ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) technology as well as multiple statistical analysis along with network pharmacology was used to comprehensively identify the Q-markers of RFT and SFT. Then, based on single-factor experiments, the Box-Behnken design (BBD) response surface methodology (RSM) was used to optimize the processing technology of SFT and perform process validation. RESULTS: A total of 63 components were identified in RFT and SFT extracts. Terrestrosin D and Terrestrosin K were initially considered the Q-markers of RFT and SFT, respectively. The optimum processing technology conditions were 208°C, 14 min, and 60 r·min-1 . Three batches of process validation were performed, and the mean composite score was 56.87, with a relative standard deviation (RSD) value of 1.13%. CONCLUSION: The content of steroidal saponin components in RFT was significantly different before and after stir-frying. Terrestrosin D and Terrestrosin K were validated as the Q-markers of RFT and SFT, respectively. The identification of Q-markers for RFT and SFT offered a clear index for optimizing the SFT-processing technology and provided a basis for the quality control of RFT and SFT decoction pieces.

Nanomedicines based on trace elements for intervention of diabetes mellitus.

Epidemiology shows that the incidence of diabetes mellitus (DM) is increasing year by year globally. Proper interventions are highly aspired for diabetics to improve the quality of life and prevent development of chronic complications. Trace elements, also known as microelements, are chemical substances that are present in our body in minute amounts. They are necessitated by the body for growth, development and functional metabolism. For the past few years, trace element nanoparticles have aroused considerable interest as a burgeoning form of nanomedicines in antidiabetic applications. These microelement-based nanomedicines can regulate glucose metabolism in several ways, showing great potential for diabetes management. Starting from the pathophysiology of diabetes, the state-of-the-art of diabetes treatment, the physiological roles of trace elements, various emerging trace element nanoparticles specific for diabetes were comprehensively reviewed in this work. Our findings disclose that trace element nanoparticles can fight against diabetes by lowering blood glucose, promoting insulin secretion, alleviating glucose intolerance, improving insulin sensitivity, ameliorating lipid profile, anti-inflammation and anti-oxidant stress, and other mechanisms. In conclusion, trace element nanoparticles can be applied as nanomedicines or dietary modifiers for effective intervention for diabetes.

Selenized Polymer-Lipid Hybrid Nanoparticles for Oral Delivery of Tripterine with Ameliorative Oral Anti-Enteritis Activity and Bioavailability.

The oral delivery of insoluble and enterotoxic drugs has been largely plagued by gastrointestinal irritation, side effects, and limited bioavailability. Tripterine (Tri) ranks as the hotspot of anti-inflammatory research other than inferior water-solubility and biocompatibility. This study was intended to develop selenized polymer-lipid hybrid nanoparticles loading Tri (Se@Tri-PLNs) for enteritis intervention by improving its cellular uptake and bioavailability. Se@Tri-PLNs were fabricated by a solvent diffusion-in situ reduction technique and characterized by particle size, ζ potential, morphology, and entrapment efficiency (EE). The cytotoxicity, cellular uptake, oral pharmacokinetics, and in vivo anti-inflammatory effect were evaluated. The resultant Se@Tri-PLNs were 123 nm around in particle size, with a PDI of 0.183, ζ potential of -29.70 mV, and EE of 98.95%. Se@Tri-PLNs exhibited retardant drug release and better stability in the digestive fluids compared with the unmodified counterpart (Tri-PLNs). Moreover, Se@Tri-PLNs manifested higher cellular uptake in Caco-2 cells as evidenced by flow cytometry and confocal microscopy. The oral bioavailability of Tri-PLNs and Se@Tri-PLNs was up to 280% and 397% relative to Tri suspensions, respectively. Furthermore, Se@Tri-PLNs demonstrated more potent in vivo anti-enteritis activity, which resulted in a marked resolution of ulcerative colitis. Polymer-lipid hybrid nanoparticles (PLNs) enabled drug supersaturation in the gut and the sustained release of Tri to facilitate absorption, while selenium surface engineering reinforced the formulation performance and in vivo anti-inflammatory efficacy. The present work provides a proof-of-concept for the combined therapy of inflammatory bowel disease (IBD) using phytomedicine and Se in an integrated nanosystem. Selenized PLNs loading anti-inflammatory phytomedicine may be valuable for the treatment of intractable inflammatory diseases.

Protective Effect of Hedyotis diffusa Willd. Ethanol Extract on Isoniazid-Induced Liver Injury in the Zebrafish Model.

Objective: This study aims to investigate the hepatoprotective effect and molecular mechanism of Hedyotis diffusa Willd. ethanol extract (HDWE) against isoniazid (INH)-induced liver injury in the zebrafish model. Methods: INH-induced liver injury model was established by adding an immersion bath of INH in 3 days post-fertilisation (dpf) healthy transgenic zebrafish with liver-specific fluorescence (L-FABP: EGFP). HDWE and INH were given to the zebrafish to observe liver morphology and pathology, fluorescence intensity, and the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and superoxide dismutase (SOD), as well as the content of glutathione (GSH). The chemical composition of HDWE was analysed using high-performance liquid chromatography coupled with a quadrupole-time-of flight hybrid mass spectrometer (HPLC-Q-TOF-MS). The bioactive compounds, molecular targets and signalling pathways of HDWE were predicted using network pharmacology. Subsequently, molecular docking was adopted to analyze the affinities between the bioactive components and targets by Autodock. Finally, in vitro experiments were conducted to further verify the findings. Results: Our findings showed that HDWE had a remarkable protective effect on INH-induced liver injury in zebrafish. Twenty compounds in HDWE were identified. Nineteen hub targets were identified as possible targets of HDWE, and a compound-target-pathway network was constructed. Nine bioactive compounds, ten molecular targets, and seven key signalling pathways were found to play a pivotal role in the hepatoprotective effect of HDWE against INH-induced liver injury. In vitro studies revealed that the important bioactive compound quercetin-3-O-sambubioside (QSA) could significantly reverse INH-induced cell viability decreases and had a significant effect on the associated targets predicted by network pharmacology and molecular docking. Conclusion: In this study, through the research of hepatoprotective effect of HDWE and bioinformatics analysis, the bioactive compounds, important pathways and key molecular targets were discovered. These findings could provide scientific evidence for the use of HDW in liver injury and prove to help explore its efficacy and the mechanism of action.

Topical astilbin ameliorates imiquimod-induced psoriasis-like skin lesions in SKH-1 mice via suppression dendritic cell-Th17 inflammation axis.

Astilbin, an essential component of Rhizoma smilacis glabrae, exerts significant antioxidant and anti-inflammatory effects against various autoimmune diseases. We have previously reported that astilbin decreases proliferation and improves differentiation of HaCaT keratinocytes in a psoriatic model. The present study was designed to evaluate the potential therapeutic effects of topical administration of astilbin on an imiquimod (IMQ)-induced psoriasis-like murine model and to reveal their underlying mechanisms. Topical administration of astilbin at a lower dose alleviated IMQ-induced psoriasis-like skin lesions by inducing the differentiation of epidermal keratinocytes in mice, and the therapeutic effect was even better than that of calcipotriol. Moreover, the inflammatory skin disorder was relieved by astilbin treatment characterized by a reduction in both IL-17-producing T cell accumulation and psoriasis-specific cytokine expression in skin lesions. Furthermore, we found that astilbin inhibited R837-induced maturation and activation of bone marrow-derived dendritic cells and decreased the expression of pro-inflammatory cytokines by downregulating myeloid differentiation factor 88. Our findings provide the convincing evidence that lower doses of astilbin might attenuate psoriasis by interfering with the abnormal activation and differentiation of keratinocytes and accumulation of IL-17-producing T cells in skin lesions. Our results strongly support the pre-clinical application of astilbin for psoriasis treatment.

Isolation, purification, structural characteristics, pharmacological activities, and combined action of Hedyotis diffusa polysaccharides: A review.

Hedyotis diffusa polysaccharides, as the main component and an important bioactive substance of Hedyotis diffusa, are effective immunomodulators with various pharmacological activities, including antitumour, anti-inflammatory, antioxidant, anti-fatigue and immunity-enhancing activities. The total polysaccharides extracted from Hedyotis diffusa and Scutellaria barbata have great effects in treating liver cancer, gastric cancer, rectal cancer, glioma and nasopharyngeal carcinoma. Moreover, different materials and extraction methods result in differences in the structure and bioactivity of Hedyotis diffusa polysaccharides. Therefore, this paper summarizes the isolation, purification, structural characteristics, pharmacological activities, and combined action of Hedyotis diffusa polysaccharides to provide a reference for further study.

Banxia Baizhu Tianma decoction attenuates obesity-related hypertension.

ETHNOPHARMACOLOGICAL RELEVANCE: Banxia Baizhu Tianma decoction (BBTD) is a classical representative prescription for expelling phlegm, extinguishing wind, strengthening the spleen and dissipating excessive fluid in traditional Chinese medicine (TCM). According to both TCM theory and about 300 years of clinical practice, BBTD is especially suitable for hypertensive patients of abdominal obesity and lacking physical activity. AIM OF THE STUDY: The present study tried to interpret the pharmacology of the ancient formula of BBTD. Herein, we focused on the plasma metabonomics of BBTD and evaluated the effect and targets of BBTD on endothelial protective effect. METHODS: Obesity-related hypertensive mice were induced by high-fat diet for 20 weeks. BBTD (17.8 g/kg) was administered intragastrically for 8 weeks, and telmisartan group (12.5 mg/kg) was used as positive drug. Body weight, blood pressure, triglyceride and cholesterol were recorded to evaluate the efficacy of BBTD in vivo. Lipid deposition in aortic roots was assessed by oil red O staining, while morphology of aortas was observed by HE staining. Ultra performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) was performed to study the plasma non-targeted metabonomics. According to the data of metabonomics, human aortic endothelial cells (HAECs) were treated by oxidized low-density lipoprotein (ox-LDL, 50 µg/mL) with/without BBTD (2, 1 or 0.5 mg/mL). Apoptosis rate (Annexin V-FITC/PI), migration (Transwell), cytoskeleton (Phalloidin) and density of VE-cadherin (Immunofluorescence staining) were used to investigate the effect of BBTD in vitro. Transcriptome sequencing was performed (2 mg/mL BBTD vs ox-LDL) to screen the possible targets of BBTD in endothelial protection against ox-LDL. RESULTS: BBTD effectively reduced the body weight and total cholesterol, and decreased 12.1 mmHg in SBP and 10.5 mmHg in DBP of obesity-related hypertensive mice (P < 0.05). BBTD attenuated lipid deposition in arterial roots and improved the morphology of aortas in vivo. Plasma metabolite profiles identified 94 differential metabolites and suggested BBTD mainly affected glycerophospholipids and fatty acyls. Bioinformatics analysis indicated sphingolipid metabolism and fluid shear stress and atherosclerosis were main pathways. Therefore, we focused on endothelial protective effect of BBTD against ox-LDL. In vitro, BBTD demonstrated endothelial protective effects, decreasing apoptosis rate, improving cell migration in dose-dependent manner and maintaining cell morphology. Transcriptome sequencing identified 251 downregulated and 603 upregulated mRNAs after 24h-BBTD treatment, which reversed 51.8% change in mRNAs (393 DE mRNAs) induced by ox-LDL. Bioinformatics analysis supported the potential of BBTD in hypertension and suggested that BBTD improved endothelial cells by targeting mainly on p53 and PPAR signaling pathways. CONCLUSIONS: BBTD attenuates obesity-related hypertension by regulating metabolism of glycerophospholipids and endothelial protection.

Paris polyphylla ethanol extract induces G2/M arrest and suppresses migration and invasion in bladder cancer.

BACKGROUND: Paris polyphylla is a traditional Chinese medicinal herb with multiple antitumor activities, but the role of P. polyphylla in bladder cancer (BC) is under investigation. This study aims to examine the antitumor activities of P. polyphylla ethanol extract (PPE) on BC cells and elucidate the underlying mechanisms. METHODS: Viable cells were counted using the trypan blue exclusion assay. The cell cycle was analyzed using flow cytometry, and scratch wound-healing and transwell assays were used to evaluate cell migration and invasion abilities, respectively. The protein expression levels were determined by western blotting. A xenograft model was used to assess the in vivo inhibitory effect of PPE on BC tumor growth. RESULTS: Our results showed that PPE inhibited the growth of BC cells in vivo and in vitro. Mechanistically, PPE regulated the levels of cell cycle-associated proteins, with PPE-induced G2/M phase arrest occurring through cyclin-dependent kinase inhibitor 1 (CDKN1A) accumulation and cyclin B1 (CCNB1)/cyclin-dependent kinase 1 (CDK1) inhibition. BC tumor growth was also inhibited by PPE treatment. Moreover, the migration and invasion abilities of J82 cells were suppressed through modulating epithelial-mesenchymal transition (EMT) regulatory factors with upregulation of cadherin-1 (CDH1) and downregulation of cadherin-2 (CDH2), snail family transcriptional repressor 2 (SNAI2), and twist family bHLH transcription factor 1 (TWIST1). CONCLUSIONS: PPE inhibited cell growth, induced G2/M arrest, and suppressed the migration and invasion of J82 cells. BC tumor growth in vivo was also inhibited by PPE. Our results lay the foundation for further studies on the antitumor mechanisms of PPE.

Polyphyllin I induces cell cycle arrest in prostate cancer cells via the upregulation of IL6 and P21 expression.

BACKGROUND: Polyphyllin I has been reported to possess anticancer properties in various cancer types, including prostate cancer. However, little is known about the potential of Polyphyllin I in induction of prostate cancer cell cycle arrest and its underlying mechanisms. METHODS: The anti-proliferation activity of Polyphyllin I was tested using cell counting kit-8 assay. The cell cycle arrest effects of Polyphyllin I were confirmed by flow cytometry. Western blot was used to test the effect of Polyphyllin I on cell cycle-related protein expression. Reverse transcription-polymerase chain reaction was used to test the expression of genes regulating P21 expression. RESULTS: Polyphyllin I induced prostate cancer cell cycle arrest in G0/G1 phase in concentration-dependent manner. Polyphyllin I induced cell cycle arrest by upregulating the expression of P21. Further studies showed that the upregulation of p21 expression induced by Polyphyllin I via the upregulation of IL6 expression. CONCLUSION: Polyphyllin I could induce cell cycle arrest in G0/G1 phase in prostate cancer cells by upregulating the expression of P21 and IL6.

Polysaccharides from Hedyotis diffusa enhance the antitumor activities of cytokine-induced killer cells.

Hedyotis diffusa is a well-known traditional Chinese herbal medicine. The polysaccharides extracted from H. diffusa (HDP) exhibit a range of pharmacological activities. Transfusion of cytokine-induced killer (CIK) cell is one type of adoptive cellular immunotherapy, which is becoming an important method of cancer immunotherapy. In this present study, we investigate the immunostimulatory effect of HDP on CIK cells. CIK cells were generated by culturing and stimulating peripheral blood monocytes of healthy volunteers. They were treated with HDP at three different concentrations (10, 50, and 100 µg/mL). The effect of HDP on CIK cell populations, intracellular cytokine production, and apoptosis was examined by flow cytometry. The antitumor effect of HDP on CIK cells was determined by cytotoxicity assay. Furthermore, the effect of HDP on the antitumor activity of CIK cells in a mouse model was investigated. HDP increased the percentage of CD3+CD56+ CIK cells but did not significantly change the percentage of CD4+, CD8+, or CD4+CD25+ CIK cells. The HDP-treated CIK cells showed a greater ability to kill tumor cells, as well as higher production of interferon-γ and tumor necrosis factor-α, compared with the no-HDP-treated CIK cells. The HDP-treated CIK cells also found a lower apoptosis level in vitro. Moreover, HDP combined with CIK cells had a stronger inhibitory effect on tumor growth in the mouse model compared with the CIK or HDP treatment alone. In conclusion, the results indicated that HDP enhanced the antitumor activity of CIK cells and could be used for cancer immunotherapy combined with CIK cell therapy.

Solutol®HS15+pluronicF127 and Solutol®HS15+pluronicL61 mixed micelle systems for oral delivery of genistein.

Purpose: We aimed to prepare two oral drug delivery systems consisting of polyoxyl 15 hydroxystearate (HS15) with pluronicF127 (F127) and HS15 with pluronicL61 (L61) to overcome the challenges of genistein's poor oral bioavailability. This provides a good strategy for enhancing the potential value of genistein. Methods: We designed two binary mixed micelle systems employing the organic solvent evaporation method using surfactants (HS15, L61, and F127). Formulations (GEN-F and GEN-L) were characterized by transmission electron microscopy. Drug content analysis, including entrapment efficiency (EE%), drug loading (DL%), and the cumulative amount of genistein released from the micelles, was performed using HPLC. The permeability of optimum formulation was measured in Caco-2 cell monolayers, and the oral bioavailability was evaluated in SD rats. Results: The solutions of GEN-F and GEN-L were observed to be transparent and colorless. GEN-F had a lower EE% of 80.79±0.55% and a DL% of 1.69±0.24% compared to GEN-L, which had an EE% 83.40±1.36% and a DL% 2.26±0.18%. TEM results showed that the morphology of GEN-F and GEN-L was homogeneous and resembled a spherical shape. The dilution and storage conditions had no significant effect on particle size and EE%. Genistein demonstrated a sustained release behavior when encapsulated in micelles. Pharmacokinetics study showed that the relative oral bioavailability of GEN-F and GEN-L increased by 2.23 and 3.46 fold while also enhancing the permeability of genistein across a Caco-2 cell monolayer compared to that of raw genistein. Conclusion: GEN-F and GEN-L as a drug delivery system provide an effective strategy for enhancing and further realizing the potential value of GEN.

[Research progress of Gastrodiae Rhizoma machining processing, composition analysis and in vivo metabolism].

Steaming method is a traditional processing method for Gastrodiae Rhizoma(GR). The current studies on the steaming method's mechanism of GR are mainly focused on facilitating softening slice, destroying the ß-glycosidic bond enzymes to reduce the decomposition of gastrodia glycosides (killing enzyme and protecting glycosides). The researches on the processing mechanism are still incomplete, while revealing and analyzing the active components in the body's metabolic process are important channels and new models to clarify the mechanism of traditional medicine processing. In order to provides a reference for the in-depth study of the processing mechanism of GR, we have reviewed the relevant literature at home and abroad in recent years and briefly summarized the processing, composition analysis and in vivo metabolism of GR in this study.

Paris Polyphylla-Derived Saponins Inhibit Growth of Bladder Cancer Cells by Inducing Mutant P53 Degradation While Up-Regulating CDKN1A Expression.

OBJECTIVES: Paris polyphylla var. yunnanensis (PPVY), a Chinese herb, has long been used for cancer treatment, and its steroidal saponins are suggested to exert an anti-tumor activity, however, the underlying mechanism is incompletely understood and their effect on bladder cancer (BC) remains unknown. The present study is thus designed to address these issues. MATERIAL AND METHODS: Total steroidal saponins were extracted with ethanol from PPVY and used to treat BC cells (HT1197 and J82 carrying mutant p53). Gene expression was determined using qPCR and immunoblotting and cell cycle analyzed using flow cytometry. DNA damage response activation was assessed using immunofluorescence staining. RESULTS: PPVY saponins treatment led to dose-dependent declines in the number of both HT1197 and J82 cells with IC50 approximately 1.2 µg/ml, which was coupled with strong growth arrest at G2/M phase and the activation of DNA damage response pathway. Moreover, the clonogenic potential of these cells was severely impaired even in the presence of low concentrations of PPVY saponins. Mechanistically, PPVY saponins induced the degradation of mutant p53 while stimulated CDKN1A gene transcription. Phosphorylated AKT was diminished in PPVY saponin-treated cells, but its specific inhibitor LY294002 exhibited significantly weaker efficacy in inducing CDKN1A expression than did PPVY saponins. CONCLUSION: PPVY saponins activate DNA damage response pathway, degrade mutant p53 and stimulate CDKN1A expression, thereby inhibiting BC cell growth. Given their poor absorption via oral administration, PPVY saponins may be applicable for intravesical instillations in BC treatment.

Anti-Cancer Effects of Paris Polyphylla Ethanol Extract by Inducing Cancer Cell Apoptosis and Cycle Arrest in Prostate Cancer Cells.

OBJECTIVE: To evaluate the potential anti-prostate cancer effects of Paris polyphylla ethanol extract (PPEE) and its underlying mechanisms. MATERIALS AND METHODS: The anti-proliferation activity of PPEE was tested on PC3 and DU145 cells using Cell Counting Kit-8 assay. The pro-apoptotic and cell cycle arrest effects of PPEE were confirmed by flow cytometry. Apoptosis of prostate cancer cells was induced by PPEE through endogenous and exogenous pathways. A mouse xenograft model was used to examine its anti-prostate cancer effects in vivo. RESULTS: We found that the IC50 of PPEE on PC3 cells was 3.98 µg/ml and the IC50 of PPEE on DU145 cells was 8 µg/ml. PPEE induced prostate cancer cell apoptosis in a concentration dependent manner, through endogenous and exogenous pathways. PPEE induced PC3 cell cycle arrest in G0/G1 and G2/M phases, while in DU145cell it induced cell arrest in the G0/G1 phase. PPEE inhibited the growth of prostate cancer cells in vivo. CONCLUSION: PPEE could inhibit prostate cancer growth in vitro and in vivo, induce apoptosis of prostate cancer cells, and cause cell cycle arrest, which laid the foundation for further research on the anti-tumor mechanism of PPEE.

Characterization of forsythoside A metabolites in rats by a combination of UHPLC-LTQ-Orbitrap mass spectrometer with multiple data processing techniques.

Forsythoside A (FTA), the main active constituent isolated from Fructus Forsythiae, has various biological functions including anti-oxidant, anti-viral and anti-microbial activities. However, while research on FTA has been mainly focused on the treatment of diseases on a material basis, FTA metabolites in vivo have not been comprehensively evaluated. Here, a rapid and sensitive method using a UHPLC-LTQ-Orbitrap mass spectrometer with multiple data processing techniques including high-resolution extracted ion chromatograms, multiple mass defect filters and diagnostic product ions was developed for the screening and identification of FTA metabolites in rats. As the result, a total of 43 metabolites were identified in biological samples including 42 metabolites in urine, 22 metabolites in plasma and 15 metabolites in feces. These results demonstrated that FTA underwent a series of in vivo metabolic reactions including methylation, dimethylation, sulfation, glucuronidation, diglucuronidation, cysteine conjugation and their composite reactions. The research enhanced our understanding of FTA metabolism and built a foundation for further toxicity and safety studies.

[Preparation and in vitro release evaluation of slow releasing intrauterine silicone rubber bar made of Panax notoginseng and Rubia cordifolia].

To prepare the intrauterine slow release silicone rubber bar made of Panax notoginseng and Rubia cordifolia, and finish its preliminary evaluation of in vitro releasing properties. The open mill method was used for plasticating of silicone rubber. The process parameters of the silicone rubber and drugs mixing were optimized by orthogonal test. The parameters of silicone rubber vulcanization was optimized by single factor test. The preliminary evaluation of in vitro release performance of the silicone rubber bar was conducted with ginsenoside Rg1, ginsenoside Rb1, notoginsenoside R1, purpurin and rubimaillin as the indexes. The results showed that optimum technologic parameters for silicone rubber and drugs mixing：the roller spacing 2 mm; speed ratio 1∶1.2; front roller temperature 55-60 ℃; rear roll temperature 50-55 ℃; and mixing time 20 min. The optimum parameters for silicone rubber vulcanization：temperature 90 ℃, and time 60 min. The studies on release process in vitro revealed that the release process of silicone rubber bar was in line with the Higuchi equations. After 90 days, the cumulative release of ginsenoside Rg1, ginsenoside Rb1 and notoginsenoside R1 was 46.7%, and the cumulative release of purpurin and rubimaillin was 51.9%. The preparation method can be applied to the preparation of silicone rubber bar, with slow release characteristics.

Identification of metabolites of gardenin A in rats by combination of high-performance liquid chromatography with linear ion trap-Orbitrap mass spectrometer based on multiple data processing techniques.

Gardenin A is one of the less abundant hydroxylated polymethoxyflavonoids (OH-PMFs) in nature, and has many potential significant health benefits. In the present study, an efficient strategy was established using high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometer to profile the in vivo metabolic fate of gardenin A in rat plasma and various tissues. First, an online LC-MS(n) data acquisition method was developed to trace all the probable metabolites. Second, a combination of offline data processing methods including extracted ion chromatography and multiple mass defect filters was employed to screen the common and uncommon metabolites from the background noise and endogenous components. Finally, structures of the metabolites were elucidated based on an accurate mass measurement, the diagnostic product ions of PMFs, and relevant drug biotransformation knowledge. Based on the proposed strategy, a total of 26 metabolites were observed and characterized. The results indicate that some biotransformations, such as methylation, demethoxylation, demethylation, glucuronide conjugation, sulfate conjugation and their composite reactions, have been discovered for OH-PMFs. Moreover, some diagnostic biotransformation pathways are summarized. Overall, this study gives us a first insight into the in vivo metabolism of gardenin A. The study also provides a practical strategy for rapidly screening and identifying metabolites, which can be widely applied for the other biotransformations.