Simultaneous determination of periplocin, periplocymarin, periplogenin, periplocoside M and periplocoside N of Cortex Periplocae in rat plasma and its application to a pharmacokinetic study.

A sensitive and specific ultra-performance liquid chromatographic-tandem mass spectrometric method was developed and validated to simultaneously determine periplocin, periplocymarin (PM), periplogenin (PG), periplocoside M (PSM) and periplocoside N (PSN) in rat plasma. Acetonitrile was employed to precipitate plasma with appropriate sensitivity and acceptable matrix effects. Chromatographic separation was performed using a Waters HSS T3 column with a gradient elution using water and acetonitrile both containing 0.1% formic acid and 0.1 mm ammonium formate within 8 min. Detection was performed in positive ionization mode using multiple reaction monitoring. The method was fully validated in terms of selectivity, linearity, accuracy, precision, recovery, matrix effects and stability. Using this method, the concentrations of periplocin, PM, PG, PSM and PSN were established after oral administration of Cortex Periplocae extract to rats, and the pharmacokinetic characteristics of periplocin, PM, PG, PSM and PSN were assessed. Generally, PM, PG, PSM and PSN were eliminated slowly and their half-lives were all >8 h. In addition, the systemic exposure of PSM showed significant differences between genders with more than 10 times higher area under the concentration-time curve in female rats than in male rats. The findings of this study provide useful information for further research on Cortex Periplocae.

Correlation between Quality and Geographical Origins of Cortex Periplocae, Based on the Qualitative and Quantitative Determination of Chemical Markers Combined with Chemical Pattern Recognition.

Quality assessment of Cortex Periplocae remains a challenge, due to its complex chemical profile. This study aims to investigate the chemical components of Cortex Periplocae, including its non-volatile and volatile constituents, via liquid chromatograph-mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) assays. The established strategy manifested that Cortex Periplocae from different producing areas was determined by identifying 27 chemical markers with ultra-high-performance liquid chromatography, coupled with quadrupole tandem time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS), including four main groups of cardiac glycosides, organic acids, aldehydes, and oligosaccharides. These groups' variable importance in the projection (VIP) were greater than 1. Simultaneously, the samples were divided into four categories, combined with multivariate statistical analysis. In addition, in order to further understand the difference in the content of samples from different producing areas, nine chemical markers of Cortex Periplocae from 14 different producing areas were determined by high performance liquid chromatography coupled with mass spectrometry (HPLC-MS/MS), and results indicated that the main effective constituents of Cortex Periplocae varied with places of origin. Furthermore, in GC-MS analysis, samples were divided into three groups with multivariate statistical analysis; in addition, 22 differential components whose VIP were greater than 1 were identified, which were principally volatile oils and fatty acids. Finally, the relative contents of seven main volatile constituents were obtained, which varied extremely with the producing areas. The results showed that the LC-MS/MS and GC-MS assays, combined with multivariate statistical analysis for Cortex Periplocae, provided a comprehensive and effective means for its quality evaluation.

Purification, Characterization, and Antioxidant Activity of Polysaccharides Isolated from Cortex Periplocae.

In this study, crude Cortex Periplocae polysaccharides (CCPPs) were extracted with water. CCPPs were decolored with AB-8 resin and deproteinated using papain-Sevage methods. Then, they were further purified and separated through DEAE-52 anion exchange chromatography and Sephadex G-100 gel filtration chromatography, respectively. Three main fractions-CPP1, CPP2, and CPP3, (CPPs)-were obtained. The average molecular weights, monosaccharide analysis, surface morphology, and chemical compositions of the CPPs were investigated by high-performance gel permeation chromatography (HPGPC), gas chromatography-mass spectrometry (GC/MS), UV-vis spectroscopy, Fourier transform infrared (FT-IR) spectrum, and nuclear magnetic resonance (NMR). In addition, the antioxidant activities of these three polysaccharides were investigated. The results indicated that all of the CPPs were composed of rhamnose, arabinose, mannose, glucose, and galactose. These three polysaccharides exhibited antioxidant activities in four assays including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, 2,2'-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS) radical, reducing power, and total antioxidant activity in vitro. The data indicated that these three polysaccharides could be utilized as potential natural sources of alternative additives in the functional food, cosmetics, and pharmaceutical industries.

A Review on Phytochemistry and Pharmacology of Cortex Periplocae.

Cortex Periplocae, as a traditional Chinese herbal medicine, has been widely used for autoimmune diseases, especially rheumatoid arthritis. Due to its potential pharmaceutical values, more studies about the biological activities of Cortex Periplocae have been conducted recently. Meanwhile, the adverse reaction of Cortex Periplocae is not a negligible problem in clinic. In this article, we reviewed a series of articles and summarized the recent studies of Cortex Periplocae in the areas of phytochemistry and pharmacology. More than 100 constituents have been isolated and identified from Cortex Periplocae, including steroids, cardiac glycosides, terpenoids, and fatty acid compounds. The crude extracts of Cortex Periplocae and its active compounds exhibit various biological activities, such as cardiotonic effect, anticancer action, and anti-inflammatory effect. This paper aims to provide an overall review on the bioactive ingredients, pharmacological effect, and toxicity of this plant. Furthermore, this review suggests investigating and developing new clinical usages according to the above pharmacological effects.

Screening and identification of reactive metabolic compounds of Cortex Periplocae based on glutathione capture-mass spectrometry.

As a traditional Chinese medicine (TCM), Cortex Periplocae (CP) has a wide range of pharmacological effects, as well as toxic side effects. The main toxic components of it are cardiac glycosides, which tend to cause cardiotoxicity. Currently, it has also been reported in studies to cause hepatotoxicity, but it is not clear whether the hepatotoxicity is related to the toxicity caused by the reactive metabolites. This study aims to investigate the target components of CP that generate reactive metabolic toxicity. The fluorescent probe method was used to detect glutathione (GSH)-trapped reactive metabolites in a co-incubation system of CP extract with rat liver microsomes. Identification of GSH conjugates was performed by LC-MS/MS and that of the possible precursor components that produce reactive metabolites was conducted by UPLC-Q-TOF/MS. Cell viability assays were performed on HepG2 and L02 cells to determine the cytotoxicity of the target components. The findings of our study demonstrate that the extract derived from CP has the ability to generate metabolites that exhaust the intracellular GSH levels, resulting in the formation of GSH conjugates and subsequent cytotoxic effects. Through the utilization of the UPLC-Q-TOF/MS technique, we were able to accurately determine the molecular weight of the precursor compound in CP to be 355.1023. The primary evidence to determining the GSH conjugetes relies on the appearance of characteristic product ions resulting from central neutral loss (CNL) scanning of 129 Da and product scanning of m/z 660 in the positive MS/MS spectrum. Through analysis, it was ultimately ascertained that the presence of chlorogenic acid (CGA) and its isomers, namely neochlorogenic acid (NCGA) and cryptochlorogenic acid (CCGA), could lead to the production of GSH conjugates, resulting in cytotoxicity at elevated levels. Taking these findings into consideration, the underlying cause for the potential hepatotoxicity of CP was initially determined.

Exploring the anti-glioma mechanism of the active components of Cortex Periplocae based on network pharmacology and iTRAQ proteomics in vitro.

BACKGROUND: The active components of Cortex Periplocae (CP) exert antitumor properties in many cancers. However, little is known about their effects on glioma or the related underlying mechanisms. OBJECTIVES: The study investigated the underlying mechanism of CP in treating glioma. MATERIAL AND METHODS: The U251 and TG905 cells were treated with an ethanol extract from CP. Cell proliferation was detected using Cell Counting Kit-8 (CCK-8) and a colony formation assay. The flow cytometric analysis was applied to explore the induction of cell cycle arrest and apoptosis. The expression levels of cell cycleand apoptosis-associated proteins were measured with western blot. A network pharmacology method was performed to predict the potential mechanism underlying the effects of the active components of CP on glioma. Then, isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics analysis was used to verify the differentially expressed proteins and pathways in order to reveal the underlying mechanisms. Furthermore, to determine the iTRAQ results, 6 candidate proteins were chosen for quantification using parallel reaction monitoring (PRM). RESULTS: The CP extract inhibited the proliferation of U251 and TG905 cells and induced cell cycle arrest and apoptosis. There are 16 active compounds of CP. The antitumor mechanism of CP may be related to the apoptosis pathway, p53 signaling pathway, PI3K-AKT pathway, or transcriptional misregulation in cancer pathway. Six proteins (HSP90AB1, TOP2A, ATP1A1, TGFß1, ATP1B1, and TYMS) were determined to be key factors involved in regulating CP in glioma. CONCLUSIONS: Our research revealed the underlying mechanism of CP in treating glioma using integrated network pharmacology and iTRAQ-based quantitative proteomics technology.

Antioxidant and DNA protecting activity of carboxymethylated polysaccharides from Cortex periplocae.

In this study, polysaccharide from Cortex periplocae (CPP) was modified and three carboxymethylation modification polysaccharides (CPPCs) were obtained, and their physicochemical characteristics and in vitro biological activities were investigated. Based on the ultraviolet-visible (UV-Vis) scan, CPPs (CPP and CPPCs) did not contain nucleic acids or proteins. However, the Fourier transform infrared (FTIR) spectrum showed a new absorption peak around 1731 cm-1. In addition, three absorption peaks near 1606, 1421, and 1326 cm-1 were enhanced after carboxymethylation modification. Based on UV-Vis scan, the maximum absorption wavelength of Congo Red + CPPs exhibited a red-shift compared to Congo Red meant CPPs had a triple helix conformation. Scanning electron microscopy (SEM) indicated that CPPCs exhibited more fragments and non-uniform-sized filiform than CPP. Thermal analysis showed that CPPCs degraded between the temperature 240 °C-350 °C and CPP in the 270 °C-350 °C. In addition, the antioxidant and DNA protecting activities of CPPCs were significantly enhanced compared to CPP. Overall, this study demonstrated the potential applications of CPPs in food and pharmaceutical industries.

A validated LC-MS/MS method for determination of periplogenin in rat plasma and its application in pharmacokinetic study.

A method coupling high performance liquid chromatography with tandem mass spectrometry has been developed and validated for quantifying periplogenin in rat plasma using psoralen as an internal standard (IS). Plasma samples were pretreated using a simple liquid-liquid extraction with ethyl acetate and the chromatographic separation of periplogenin and psoralen was achieved on a Waters XBridge™ BEH C18 column with 0.1% formic acid and acetonitrile as mobile phase at a flow rate of 0.4mL/min. The detection was performed on a positive ion mode with electrospray ionization (ESI) source. The optimized ion transition pairs for quantitation were m/z 391.3→m/z 337.2 for periplogenin and m/z 187.0→m/z 131.0 for IS. The total run time was 9.0min. The calibration curve was linear over the range of 0.2-250ng/mL (r>0.99) with the lower limit of quantitation (LLOQ) at 0.2ng/mL. The intra- and inter-day precision were below 9.85% and the mean accuracy were from -10.03% to 10.26%. The average recoveries of periplogenin in plasma ranged from 85.1% to 95.6%. The proposed method was successfully applied in evaluating the pharmacokinetics of periplogenin after an oral dose of 30mg/kg Cortex Periplocae extract in rats.

Chemical constituents of Cortex Periplocae Radicis and its main toxic ingredients:research advances / 国际药学研究杂志

Cortex Periplocae Radicis(CPR)is the dried root barks of Periploca spium Bge. It has been used in China for treat?ing rheumatoid arthritis and strengthening the bone and the musculature for thousands of years. Recently,an increasing number of bio?activities of CPR have been recognized,including tumor suppression and anti-chronic heart failure function. However,long-term use or large doses of CPR may produce adverse reactions,which constrains its applications in clinical therapy. Therefore,it is critical to know the chemical components of CPR in order to understand the toxicity mechanism. Nearly a hundred chemical compositions have been found,however,various classes,obfuscated names of different compounds,and disaccord between chemical structure and name were major obstacles to studying pharmacodynamics and toxicity of CPR. In this paper,94 chemical components of CPR are classified and reviewed,which is valuable for the comprehensive understanding of its biological functions and safe clinical use.

Chemical constituents of cortex periplocae radicis and its main toxic ingredients: Research advances / 国际药学研究杂志

Cortex Periplocae Radicis (CPR) is the dried root barks of Periploca spium Bge. It has been used in China for treat­ing rheumatoid arthritis and strengthening the bone and the musculature for thousands of years. Recently, an increasing number of bio­activities of CPR have been recognized, including tumor suppression and anti-chronic heart failure function. However, long-term use or large doses of CPR may produce adverse reactions, which constrains its applications in clinical therapy. Therefore, it is critical to know the chemical components of CPR in order to understand the toxicity mechanism. Nearly a hundred chemical compositions have been found, however, various classes, obfuscated names of different compounds, and disaccord between chemical structure and name were major obstacles to studying pharmacodynamics and toxicity of CPR. In this paper, 94 chemical components of CPR are classified and reviewed, which is valuable for the comprehensive understanding of its biological functions and safe clinical use.

Effects of Periplocin from Cortex Periplocae on Apoptosis of Human Lung Cancer A549 Cells and Expression of Survivin / 医药导报

Objective To investigate the effects of periplocin from Cortex Periplocae (CPP) on apoptosis of human lung cancer A549 cells and expression of survivin, and demonstrate its anti-tumor effect and the possible mechanism. Methods Inhibitory effect of CPP at different concentrations (1. 25, 2. 50, 5. 00, 10. 00, 20. 00 ng·mL-1 ) and for different time length (24, 48, 72 h) on A549 cell proliferation was tested by MTT method. Apoptosis rate of A549 cells treated with CPP at different concentrations (2. 50, 5. 00, 10. 00 ng·mL-1 ) were measured using flow cytometry (FCM) for 6, 12, 24, 48, 72 h, respectively. The morphological and ultrastructural changes of the apoptosis cells were observed by acridine orange/ ethidium bromide (AO/ EB) staining and transmission electron microscopy (TEM). The effects of CPP on mRNA and protein expression of apoptosis associated gene survivin were assessed by RT-PCR and Western blotting. Results CPP could significantly inhibit the growth of A549, and the inhibition rate reached (93. 46±2. 35)% . The results of FCM showed that the apoptosis rate of A549 cells treated with CPP was increased significantly as compared to the control group ( P<0. 05). Meanwhile, typical apoptotic peaks were detected. The characteristic morphological changes of apoptosis were observed in A549 exposed to CPP, including cell shrinkage, the nuclei became yellow-red by AO/ EB staining, and typical ultrastructural changes, including nuclear chromatin condensation along the nuclear membrane, vacuolar degeneration of cytoplasm observed by TEM. The result of RT-PCR indicated that survivin mRNA expression decreased obviously in A549 cells exposed to CPP. The protein expression of survivin in A549 cells treated with 10. 0 ng·mL-1 CPP(0. 251±0. 012)was weaker than that in control group(0. 928±0. 016). Conclusion CPP can induce apoptosis in human lung cancer cell lines A549, and the probable mechanism is related to the down-regulation of survivin mRNA and protein.

Inhibitory Effects of Periplocin from Cortex Periplocae on Human Lung Cancer Cell Line QG56 / 天津医药

Objective To investigate the inhibitory effects of periplocin from cortex periplocae (CPP) on human lung cancer cell line QG56 and to discuss its mechanism. Methods QG56 cells were cultured in vitro. The final concentrations of CPP in control group were 1.25, 2.50, 5.00, 10.00 and 20.00μg/L. QG56 cells were treated with ascending concentration of CPP for 24 h, 48 h and 72 h. The cell proliferation was measured using MTT method. The morphological changes of QG56 cells were observed under inverted microscope. Flow cytometry (FCM) was used to detect the effects of CPP on cell cycle and cell apoptosis. The expression of apoptosis associated gene bax mRNA in QG56 cells was detected by RT-PCR. The expres-sion of bax protein before and after treatment of CPP was examined by SP immunocytochemistry. Results The inhibitory ef-fect of CPP on the proliferation of QG56 cells was increased with the increasing concentrations of CPP and the prolonged du-ration of treatment. The morphological changes were displayed in QG56 exposed to CPP. The results of FCM showed that CPP caused cell cycle arrest at G0/G1 phase. The apoptotic rate of QG56 cells was significantly increased after CPP treatment for 48 h (P<0.05). The expression of bax mRNA was increased in QG56 exposed to CPP. The result of immunocytochemis-try indicated that CPP up-regulated the expression of bax protein. Conclusion CPP showed significant inhibitory effect on human lung cancer cell lines QG56 through inducing cell cycle arrest and apoptosis.

Ethyl acetate extract from Cortex periplocae induced apoptosis of human esophageal carcinoma cell line TE-13 / 肿瘤

Objective:To investigate the effect of ethyl acetate extract from Cortex periplocae (CPEAE) on apoptosis of human esophageal carcinoma cell line TE-13 and to elucidate its mechanism. Methods:Inhibitory effect of CPEAE on TE-13 proliferation was tested by MTT assay. The morphological changes of cell apoptosis were observed by Giemsa staining and transmission electron microscopy. Cell cycle and apoptotic ratio were tested by flow cytometry (FCM). The protein expression of CDK4 was observed by Western blotting.Results:CPEAE inhibited proliferation of TE-13 cells in a concentration-dependent and time-dependent manner, and its IC_(50) value was (2.443±0.005) μg/mL at 48 h (P<0.05). The characteristic morphological changes of apoptosis were observed in TE-13 cells after treatment with CPEAE under transmission microscope. A typical subdiploid peak was detected by flow cytometry. CPEAE decreased the expression of gene CDK4 in TE-13 cells. Conclusion:CPEAE can induce apoptosis of TE-13 cells. The effect is related with down-regulation of CDK4 expression.

Immunoregulatory effects of Periplocin from Cortex Periplocae in tumor-bearing mice / 细胞与分子免疫学杂志

AIM: To investigate the immune mechanisms for Periplocin from Cortex Periplocae (CPP) in tumor-bearing mice. METHODS: H_(22) tumor-bearing model BALB/c mice were applied to evaluated in vivo immunoregulatory effect of CPP. The influence of different dose CPP (0.25, 0.50 and 1.00 mg/kg) on immune organs in tumorbearing mice were observed. T cell subsets of mice spleen were detected by flow cytometry. MTT assay was used to determine the influence of CPP on lymphocyte proliferation of mice spleen stimulated by ConA. The levels of TNF-α, IL-2 and IL-12 in serum from mice were detected by means of ELISA. RESULTS: Thymus index and spleen index of H_(22) tumor-bearing model control mice became less than that of normal mice (P < 0.05). Compared to both model and normal control groups, thymus index and spleen index of H_(22) tumor-bearing mice treated with CPP increased obviously (P < 0.05). CPP had no influence on the number of CD8~+ T cells, but up-regulated markedly the number of CD3~+, CD4~+ T cells and the ratio of CD4~+/CD8~+ in tumorbearing mice. In CPP-treated mice, the percentage of CD3~+, CD4~+ T cells were not different from normal mice (P<0.05), the ratio of CD4~+/CD8~+ was higher than that of normal mice (P < 0.05). CPP enhanced obviously lymphocyte proliferation of mice spleen induced by ConA, the SI scores were even higher than that of nornal mice. The levels of TNF-α, IL-2 and IL-12 in serum from CPP-treated mice, increased significantly compared to model control group (P<0.05) in a dose-dependent manner, were similar to or higher than that of normal mice. CONCLUSION: CPP protected immune organs of tumor-bearing mice, increased obviously the percentage of CD4~+ and CD4~+/ CD8~+ among the T cell line, and enhanced lymphocyte proliferation of mice spleen significantly, stimulated the production of TNF-α, IL-2 and IL-12. The results suggested that CPP possessed potent immunoregulatory effect.

Inductive effect of Cortex Periplocae extract on apoptosis of human gastric cancer cells BGC-823 / 中草药

Objective To study the inductive effect of Cortex Periplocae extract(CPE) on apoptosis of human gastric cancer cells BGC-823 and its mechanism.Methods The cell morphology and super-microstructural changes of apoptosis were analysed by Giemsa staining and electric microscope,respectively.The BGC-823 apoptosis ratio,cell cycles,and changes of apoptosis in DNA level were studied by Flow Cytometry and agarose gel electrophoresis.The genes mRNA and protein expression of apoptosis-related genes bcl-2,bax,and survivin were studied by RT-PCR and immunology cell chemistry method.Results After treatment with CPE,BGC-823 cells showed some typical morphologic features and super-microstructural changes of apoptosis.DNA agarose gel electrophoresis showed characteristic "DNA ladder" pattern.Most BGC-823 cells were arrested at G_2/M phase.Some typical subdiploid peaks before G_0/G_1 phase were observed.The apoptotic rate of BGC-823 was 18.9% after 250 ?g/mL CPE treated for 48 h.The gene mRNA and protein expression of bcl-2 and survivin were inhibited by CPE,whereas that of bax was up-regulated.CPE could enhance the life span of S_(180) bearing mice in a dose-dependent manner.Conclusion(CPE can) inhibit the tumor growth by arresting the BGC-823 cell cycle at G_2/M phase and inducing BGC-823 apoptosis.Its mechanism is related to the inhibition on gene mRNA and protein expression of bcl-2 and survivin,and enhancement of those of bax.

Periplocin of cortex periplocae inhibits Stat3 signaling and induces apoptosis in human hepatocellular carcinoma cell line SMMC-7721 / 第三军医大学学报

Objective To investigate the effect of periplocin of cortex periplocae (CPP) on Stat3 signaling and its probable molecular mechanism of inducing apoptosis and anti-tumor activity. Methods Cell proliferation was detected by MTT. Cell apoptosis and cell cycle were investigated by flow cytometry. Expression of Stat3 protein in SMMC-7721 cells was analyzed by Western blot. Mcl-1, Survivin and XIAP mRNA expressions were measured by RT-PCR. Results CPP inhibited the proliferation of SMMC-7721 cells significantly, induced their apoptosis and arrested their cell cycle at G2/M phase. Decreased expression of Stat3 protein in the cell nucleus was observed after CPP treatment, but no significant changes were found in cytoplasma. Mcl-1, Survivin and XIAP mRNA expression levels were decreased in a time-dependent manner. Conclusion CPP inhibits cell proliferation and induces apoptosis by inhibiting Stat3 signal transduction in human hepatocellular carcinoma cell line SMMC-7721.