Suppressive effect of platycodin D on bladder cancer through microRNA-129-5p-mediated PABPC1/PI3K/AKT axis inactivation

Platycodin D (PD) is a major constituent of Platycodon grandiflorum and has multiple functions in disease control. This study focused on the function of PD in bladder cancer cell behaviors and the molecules involved. First, we administered PD to the bladder cancer cell lines T24 and 5637 and the human uroepithelial cell line SV-HUC-1. Cell viability and growth were evaluated using MTT, EdU, and colony formation assays, and cell apoptosis was determined using Hoechst 33342 staining and flow cytometry. The microRNAs (miRNAs) showing differential expression in cells before and after PD treatment were screened. Moreover, we altered the expression of miR-129-5p and PABPC1 to identify their functions in bladder cancer progression. We found that PD specifically inhibited the proliferation and promoted the apoptosis of bladder cancer cells; miR-129-5p was found to be partially responsible for the cancer-inhibiting properties of PD. PABPC1, a direct target of miR-129-5p, was abundantly expressed in T24 and 5637 cell lines and promoted cell proliferation and suppressed cell apoptosis. In addition, PABPC1 promoted the phosphorylation of PI3K and AKT in bladder cancer cells. Altogether, PD had a concentration-dependent suppressive effect on bladder cancer cell growth and was involved in the upregulation of miR-129-5p and the subsequent inhibition of PABPC1 and inactivation of PI3K/AKT signaling.

Guiding mechanism of platycodin D in treatment of mouse lung cancer with doxorubicin / 中国中药杂志

This study is to observe whether platycodin D has the guiding role in treatment of mouse lung cancer with doxorubicin and explore its guiding mechanism. In vitro, platycodin D and doxorubicin(alone or in combination) were added into Lewis lung cancer(LLC) cells to detect the cell proliferation and doxorubicin uptake. Cell morphological changes were analyzed by cell holographic analysis system; cell gap junctional intercellular communication(GJIC) was tested by fluorescent yellow tracer; lyso-tracker red was used to examine lysosomal function; LC-3 B(Light chain 3 beta)and P62(heat shock 90-like protein)staining were used to test auto-phagy and autophagic degradation respectively; and P-glycoprotein(P-gp) expression was examined by Western blot. In vivo, lung solid tumor was formed in mouse LLC cells via intravenous injection. Platycodin D and doxorubicin(alone or in combination) were used to treat tumor-bearing mice for four weeks, and then the tumor size was examined, mouse survival time was recorded, doxorubicin uptake in lung tissues was tested, and lung tissues were stained for observation by HE(hematoxylin-eosin) and immunohistochemistry. The results showed that platycodin D at the experimental concentration had no effect on LLC cell proliferation but decreased LLC cell volume, promoted the cells to uptake doxorubicin and enhanced the inhibitory action of doxorubicin on cell proliferation. Platycodin D could promote GJIC and lysosomal function, increase autophagy and autophagic degradation and suppress P-gp expression. Platycodin D at the experimental dose in this study had no effect on LLC lung solid tumors in mice, increased doxorubicin uptake in lung tissues and enhanced the therapeutic efficacy of doxorubicin on lung solid tumors. Platycodin D could improve the extracellular matrix deposition in lung solid tumors, decreased the lung mucin 5 AC secretion and pulmonary vessel permeability. In summary, platycodin D had the guiding role in treating mouse lung cancer with doxorubicin, and its guiding mechanism may be associated with the promotion of cell communication, lysosomal function, and improvement of extracellular environment.

Study on Quality Standard of Compound Platycodon grandiflorum Antitussive Tablets / 中国药房

OBJECTIVE： To establish the quality standard of Compound Platycodon grandiflorum antitussive tablets. METHODS： TLC was used to identify the P. grandiflorum， Polygala tenuifolia and Glycyrrhiza uralensis qualitatively in Compound P. grandiflorum antitussive tablets. HPLC-ELSD method was used to measure the content of platycodin D in Compound P. grandiflorum antitussive tablets. The determination was performed on Agilent C18 column with mobile phase consisted of acetonitrile-water （26 ∶ 74， V/V） at the flow rate of 1.0 mL/min. ELSD was used with drift tube temperature of 105 ℃， gas flow rate of 3.0 L/min and column temperature at 35 ℃. RESULTS： TLC chromatograms of P. grandiflorum， P. tenuifolia and G. uralensis had clear spots with good separation and no same spot from negative samples. The linear range of platycodin D was 0.421 9- 5.062 8 μg （r＝0.999 9）. The quantitative limit and detection limit were 0.364， 0.109 μg， respectively. RSDs of precision， stability， reproducibility and durability tests were all lower than 3.0％. The recovery rates were 87.32％-91.96％ （RSD＝1.73％，n＝6）. The platycodin D contents of 178 samples ranged from 0.004 to 0.73 mg/tablet. The content of platycodin D in 55 batches （30.9％） of samples was lower than the content limit （0.10 mg/tablet） proposed in this study. CONCLUSIONS： Established method is accurate and reliable， and can be used for the quality control of Compound P. grandiflorum antitussive tablets.

Inhibitory effect and mechanism of platycodin D combined with imatinib on K562/R / 中国中药杂志

Platycodin D(PD) has a significantly inhibitory effect on multiple malignant tumors, and can inhibit the proliferation of leukemia cells K562 and induce apoptosis. However, its effect in improving the sensitivity of drug-resistant cells to imatinib and their molecular mechanism remained unclear. To investigate the effect and mechanism of PD alone or combined with imatinib (IM) in inhibiting CML imatinib resistant cell line K562/R, the cell proliferation was examined by CCK8 assay to reveal the effect of PD on the inhibitory function of imatinib. Cell apoptosis was detected by Annexin V-FITC/PI double staining. Protein expressions of cleaved caspase-3, cleaved caspase-9, PARP, cleaved PARP, Bcr/abl, p-AKT and p-mTOR were detected by Western blot. The results showed that the inhibitory effect of PD combined with imatinib on the proliferation and apoptosis of K562/R cells was significantly higher than that of the control group and the single drug group. Protein expressions of cleaved caspase-3, cleaved caspase-9 and cleaved PARP were significantly up-regulated in the combination group, and protein expressions of PARP, Bcr/abl, p-AKT and p-mTOR were down-regulated. The results indicated that PD increased the sensitivity of drug-resistant cells to imatinib, and the inhibitory effect of PD combined with imatinib was significantly better than the single drug on cell proliferation, induction of apoptosis, inhibition of Bcr/abl protein and PI3K/AKT/mTOR signaling pathway.

Platycodin D May Improve Acne and Prevent Scarring by Downregulating SREBP-1 Expression Via Inhibition of IGF-1R/PI3K/Akt Pathway and Modulating Inflammation with an Increase in Collagen

BACKGROUND: Although many therapeutic agents have been developed, only a few drugs are known to target multiple pathogenic factors in the treatment of acne. OBJECTIVE: The purpose of this study was to identify a new drug candidate, platycodin D, which is a substance extracted from the root of Platycodon grandiflorum. METHODS: Using western blotting and Cell Counting Kit-8 assay, we studied the effects of platycodin D on SEB-1 sebocytes, fibroblasts, and keratinocytes. We investigated its effects in view of lipogenesis, collagen production, anti-inflammatory activity, and dyskeratinization. RESULTS: In SEB-1 sebocytes, platycodin D showed a sebosuppressive effect by downregulating ERK and insulin- like growth factor-1R/PI3K/Akt/sterol-regulatory element binding protein-1 signaling pathways. In addition, adiponectin, one of the adipokines responsible for sebum production, was decreased in platycodin D-treated SEB-1 sebocytes. In fibroblasts, platycodin D increased collagen production and reduced inflammation by inhibiting nuclear factor kappa B and matrix metalloproteinases. Platycodin D also showed anti-inflammatory effects on keratinocytes. It also suppressed keratin 16 expression induced by lipopolysaccharide. Furthermore, platycodin D showed no cytotoxicity on both SEB-1 sebocytes and fibroblasts. CONCLUSION: Our data demonstrate the clinical feasibility of platycodin D for acne treatment and the prevention of acne scarring by sebosuppressive and anti-inflammatory effects, as well as through an increase in collagen levels.

Platycodin D induces necroptosis of prostate cancer PC-3 cells through FOXO3a pathway / 肿瘤

Objective: To investigate the effect of platycodin D on necroptosis of prostate cancer PC-3 cells, and to explored its mechanism related to forkhead transcription factor O3a (FOXO3a). Methods: PC-3 cells were treated with caspase inhibitor Z-VAD-FMK or caspase-3-specific inhibitor AC-DEVD-CHO, and then were exposed to different concentrations of platycodin D for 72 h. The survival rate of PC-3 cells was detected by MTT method. The cell morphology and membrane integrity of PC-3 cells treated with platycodin D were detected by typan blue staining and lactic dehydrogenase (LDH) release test, respectively. The expression levels of key necroptosis factors including mixed lineage kinase domain-like (MLKL), phospho-MLKL (p-MLKL) and its tetramer, as well as FOXO3a and its downstream molecules including factor-associated suicide ligand (FasL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) proteins in PC-3 cells treated with different concentrations of platycodin D were detected by Western blotting. The expression level of FOXO3a protein in the nucleus of PC-3 cells was detected by immunofluorescence. After PC-3 cells were transfected with FOXO3a-siRNA or the negative control (NC)-siRNA by liposome, and the expression levels of FOXO3a, FasL and TRAIL proteins were detected by Western blotting. The cell viability and cell membrane integrity of PC-3 cells after FOXO3a gene silencing and treatment with platycodin D were detected by MTT method, typan blue staining and LDH release test, respectively. Results: The survival rate of PC-3 cells treated with platycodin D was decreased in a concentration-dependent manner. There was no significant difference in the survival rate of PC-3 cells treated with DMSO, Z-VAD-FMK and AC-DEVD-CHO combined with the same concentration of platycodin D (all P > 0.05). After the treatment with platycodin D, the trypan blue staining rate and LDH release rate of PC-3 cells were significantly increased (both P < 0.05). The expression levels of MLKL, p-MLKL and its tetramer in PC-3 cells treated with platycodin D were up-regulated (all P < 0.05), and the effect on p-MLKL tetramer was the most significant. Platycodin D also promoted the transposition of FOXO3a into the nucleus, and up-regulated the expressions of FOXO3a and its downstream molecules FasL and TRAIL (all P < 0.05). After transfection with FOXO3a-siRNA, the expression level of FOXO3a protein in PC-3 cells was down-regulated (P < 0.05). Compared with NC-siRNA + platycodin D group, the survival rate of PC-3 cells in FOXO3a-siRNA + platycodin D group was increased (P < 0.05), and the positive rate of typan blue staining and LDH release rate were decreased (both P < 0.05). Conclusion: Platycodin D can induce caspase-independent necroptosis of prostate cancer PC-3 cells, which is involved in FOXO3a pathway and promoting the phosphorylation of MLKL.

Comparison of the Content of Total Saponins and Platycodin D in Platycodon grandiflorum from Sichuan of Different Cultivated Years / 中国药房

OBJECTIVE：To establish a method for content determination of total saponins and platycodin D in Platycodon grandiflorum from Sichuan and investigate the difference of 2 indexes in P. grandiflorum from Sichuan of different cultivated years. METHODS：The content of total saponins in P. grandiflorum from Sichuan was determined by UV spectrophotometry. The content of platycodin D was determined by HPLC. The contents of total saponins and platycodin D were compared among each 10 samples of annual，biennial and triennial P. grandiflorum from Sichuan. RESULTS：Average contents of total saponins in annual，biennial and triennial P. grandiflorum from Sichuan were 2.47％ ，3.01％ ，2.47％ ，respectively；average contents of platycodin D were 0.23％，0.27％，0.33％，respectively. The contents of 2 indexes in annual P. grandiflorum from Sichuan were in relative low level， while the content total saponins in biennial P. grandiflorum from Sichuan was the highest；the content of platycodin D in triennial P. grandiflorum was the highest. CONCLUSIONS：The contents of indexes are different among P. grandiflorum from Sichuan of different cultivated years. But there is no correlation between them. It is suggested to select biennial and triennial P. grandiflorum from Sichuan.

Platycodin D protects oral epithelial cells against infection of Candida albi-cans / 中国病理生理杂志

AIM: To investigate the effect of platycodin D on Candida albicans infection in oral epithelial cells.METHODS:The viability of the oral squamous carcinoma KB cells was detected by MTT assay after treated with different concentrations of platycodin D .The KB cells were infected with Candida albicans, and then were incubated with platycodin D at different concentrations .Adherent numbers of the Candida albicans were counted by Gram staining , and the bacterial activity and conversion were measured by Trypan blue staining .Furthermore , the protein levels of IL-18 and hu-man β-defensin 2 ( HBD-2) were analyzed by ELISA , and the expression of HBD-2 at mRNA and protein levels was deter-mined by RT-qPCR and Western blot , respectively .RESULTS:The viability of the KB cells was not affected by platycod-in D at the concentrations used .The adherent numbers , bacterial activity and conversion were decreased by treatment with platycodin D in a dose-dependent manner .In addition, the protein level of IL-18 in the culture supernatant and the mRNA expression of HBD-2 in the KB cells were also reduced after platycodin D treatment .CONCLUSION:Platycodin D has a bacteriostasis effect and prevents oral epithelial cells from Candida albicans infection.

Effect of platycodin D on radiosensitivity of human hepatoma cell line and related mechanisms of action / 中华肝脏病杂志

Objective@#To investigate the effect of platycodin D on the radiosensitivity of human hepatoma cell lines HepG2 and SMMC-7721 and related mechanisms of action.@*Methods@#MTT assay was used to analyze the effect of different concentrations of platycodin D with different treatment times on cell viability. The cells were pretreated with 5 μg/ml platycodin D for 24 hours followed by X-ray irradiation at different radiation doses. Colony-forming assay was used to measure the radiosensitizing effect of platycodin D on cells. The quasi-threshold dose (Dq), mean lethal dose (Do), extrapolation number (N), sensitizer enhancement ratio (SER), and survival fraction (SF) at different radiation doses were calculated, and the multi-target single-hit model was used to fit the cell survival curve according to the formula SF = l-(l-e-D/D0)N. Flow cytometry was used to investigate the distribution of cell cycle, and Western blotting was used to measure the changes in the protein expression of phosphorylated phosphatidylinositol 3’-kinase (pPI3K), phosphorylated protein kinase (pAkt), nuclear factor-κB (NF-κB), and phosphorylated nuclear factor inhibiting protein (pIκBα). A one-way analysis of variance, the t-test, or the least significant difference test was used for statistical analysis based on the type of data.@*Results@#Platycodin D reduced the viability of HepG2 and SMMC-7721 cells in a dose-dependent manner; the IC50 value for HepG2 cells was 24.2 ± 0.61 μg/ml at 24 hours and 7.68 ± 0.46 μg/ml at 48 hours, and that for SMMC-7721 cells was 23.8 ± 0.57 μg/ml at 24 hours and 8.63 ± 0.86 μg/mL at 48 hours. After the combined treatment with platycodin D and irradiation, there were significant reductions in Dq (P = 0.002), Do (P = 0.002), and N value (P = 0.003), the survival curve markedly shifted to the left, and SER was 1.347 ± 0.04 in HepG2 cells and 1.418 ± 0.05 in SMMC-7721 cells. In addition, platycodin D significantly inhibited the increase in the proportion of cells in G2/M phase, the increases in the protein expression of pPI3k (P = 0.002), pAkt (P = 0.003), and NF-κB (P = 0.002), and the reduction in the protein expression of pIκBα (P = 0.003).@*Conclusion@#Platycodin D can increase the radiosensitivity of HepG2 or SMMC-7721 cells, possibly by enhancing the growth inhibition effect of irradiation and inhibiting the activation of the PI3k/Akt and NF-κB pathways.

Content Determination of 3 Components in Sangju Ganmao Pills by HPLC-MS/MS / 中国药房

OBJECTIVE:To establish the method for simultaneous determinations of rutin,forsythin and platycodin D in Sangju ganmao pills.METHODS:HPLC-MS method was adopted.The determination was performed on Waters Atlantis C18 column with mobile phase consisted of acetonitrile-0.1％ formic acid (gradient elution) at the flow rate of 0.2 mL/min.The column temperature was set 35 ℃,and sample size was 10 μL.The ionization mode was electrospray ion,and the reaction mode was multi-reaction monitoring.By positive ion detection mode,the drying gas and nebuliser gas were all high purity nitrogen.The drying gas temperature was 270 ℃.The drying gas flow rate was 25 L/min.The sheath gas flow rate was 10 L/min.The capillary voltage was 4 500 V.The nozzle voltage was 2 000 V and the scanning time was 0.1 s.RESULTS:The linear range of rutin,forsythin and platycodin D were 0.010 82-2.164 μg/mL (r=0.999 7),0.010 18-2.036 μg/mL (r=0.999 4),0.010 27-2.054 μg/mL (r=0.999 7),respectively The limits of quantification were 1.250,0.260,2.720 ng/mL,and the limits of detection were 0.380,0.078,0.820 ng/mL.RSDs of precision,stability and reproducibility tests were all no more than 3.0％.The recoveries were 97.88％-99.88％ (RSD=0.72％,n=6),98.48％-103.13％ (RSD=1.91％,n=6),98.79％-101.41％ (RSD=1.05％,n=6).CONCLUSIONS:This method is simple,precise,stable and reproducible,and can be used for simultaneous determination of rutin,forsythin and platycodin D in Sangju ganmao pills.

Proteomic analysis of hepatocellular carcinoma HepG2 cells treated with platycodin D / 中国天然药物

Platycodin D (PD), a triterpenoid saponin isolated from Platycodonis Radix, is a famous Chinese herbal medicine that has been shown to have anti-proliferative effects in several cancer cell lines. The aim of this study was to determine the changes in cellular proteins after the treatment of hepatocellular carcinoma HepG2 cells with PD using proteomics approaches. The cell viability was determined using the MTT assay. The proteome was analyzed by two-dimensional difference gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Western blot analysis was used to confirm the expression of changed proteins. Our results showed that PD inhibited the proliferation of HepG2 cells in concentration- and time-dependent manners. Sixteen proteins were identified to be up-regulated in PD-treated HepG2 cells, including ATP5H, OXCT1, KRT9, CCDC40, ERP29, RCN1, ZNF175, HNRNPH1, HSP27, PA2G4, PHB, BANF1, TPM3, ECH1, LGALS1, and MYL6. Three proteins (i.e., RPS12, EMG1, and KRT1) decreased in HepG2 cells after treatment with PD. The changes in HSP27 and PHB were further confirmed by Western blotting. In conclusion, our results shed new lights on the mechanisms of action for the anti-cancer activity of PD.

Determination of platycodin in Platycodi Radix from different habitats / 中草药

Objective: To establish a method of the determination of total platycodin and platycodin D; And to determine the contents of total platycodin and platycodin D in Platycodi RadiX from different habitats. Methods: The content of total platycodin was determined by 1ultrasonic extraction and weight method; And the content of platycodin D was determined by HPLC. The chromatographic conditions were Waters Symmetry-C18 column, mobile phrase of CH3CN-H2O (22:78), flow rate of 1.0 mL/min, detection wavelength at 210 nm and column temperature 30℃. Results: There was a greater difference between the contents of total platycodin and platycodin D in Platycodi Radix from different habitats. In measuring Platycodi Radix, the content of total platycodin in Platycodi Radix from Zhejiang Province was the highest, which was 12.03%; the contents in Platycodi Radix from Heilongjiang, Liaoning, Anhui, Shandong, and Jiangxi Provinces were more than 6%; However, the contents in Platycodi Radix from the other provinces were lower than 6%, and it was the lowest in Platycodi Radix from Henan province, which was 1.57%. The contents of platycodin D in Platycodi Radix from Yunnan province was the highest, and it was the lowest in Platycodi Radix from Hebei province. Conclusion: The methods could be used for the determination of total platycodin and platycodin D in Platycodi Radix because of theirs simple operations with accurate and reliable results. The contents of total platycodin and platycodin D are different obviously in Platycodi Radix from different habitats, and the contents of total platycodin and platycodin D in the same drug with the level of inconsistency don't show correlation.

Determination of eight active components in Xuanmai Ganjie Granules by UPLC-MS/MS / 中草药

Objective: To develop a UPLC-MS/MS method for simultaneously determining harpagide, liquiritin, harpagoside, platycodin D, ammonium glycyrrhetate, ophiopogonin D, methylophiopogonanone A, and methylophiopogonanone B in Xuanmai Ganjie Granules (composed with Scrophulariae Radix, Ophiopogonis Radix, Glycyrrhizae Radix et Rhizoma, and Platycodonis Radix) from different pharmaceutical companies. Methods: The chromatographic separation was achieved on Phenomenex Kenetix C18 column (50 mm × 2.1 mm, 5 μm) by using a mobile phase consisted of acetonitrile and 0.1% formic acid water at the flow rate of 0.3 mL/min for gradient elution. Simultaneous monitoring of positive and negative ions and multiple reaction monitoring (MRM) scan mode were applied to the quantification of the components in Xuanmai Ganjie Granules; Sample volume was 5 μL. Results: There was good linearity between the absorption peak area and the concentration for harpagide, liquiritin, harpagoside, platycodin D, ammonium glycyrrhetate, ophiopogonin D, methylophiopogonanone A, and methylophiopogonanone B in the ranges of 9-2250, 8-2000, 3.4-850, 96-24000, 12.4-3100, 3.6-1900, 1.7-425, and 1.5-375 ng/mL, respectively. The average recoveries were ranged from 97.2% to 102.8% (RSD ≤ 2.7%). The contents of harpagide, liquiritin, harpagoside, platycodin D, ammonium glycyrrhetate, ophiopogonin D, methylophiopogonanone A, and methylophiopogonanone B in eight batches of samples were in the ranges of 32.8-107.6, 54.8-178.0, 14.6-70.7, 31.2-280.0, 106.4-287.9, 0.1-0.6, 0.01-0.07, and 0.03-0.17 μg/g, respectively. Conclusion: The developed method is simple, effective, and credible for determining the eight components in Xuanmai Ganjie Granules. It provides more helpful information for the comprehensive quality evaluation of Xuanmai Ganjie Granules.

Research on mechanisms of PD-induced inhibition of adhesion, invasion and migration in non-small cell lung cancer H460 and A549 cells / 中国药理学通报

Aim To explore the effects of the inhibition of cell adhesion , invasion and migration in non-small cell lung cancer ( NSCLC ) H460 and A549 cells in-duced by platycodin-D ( PD ) and its mechanism. Methods Cell adhesion assay, wound-healing assay and Transwell chamber migration assay were used to detect the ability of cell adhesion, migration and inva-sion. Regulation of MMP-2 and MMP-9 mRNA was de-termined by RT-PCR. Meanwhile, Western blot was performed to determine the expression levels of MMP-2/9 , its upstream related proteins of ERK signaling pathway and p-Akt. Results PD effectively inhibited the ability of cell adhesion, invasion and migration in H460 and A549 cells in a dose-dependent manner ( P<0. 05 ) . PD reduced the expression of MMP-2 and MMP-9 mRNA in H460 and A549 cells ( P<0. 01 );meanwhile, PD down-regulated the expression levels of MMP-2/9 , and inhibited the expression of its upstream proteins Ras, p-c-Raf, p-ERK 1/2 and p-Akt in a time- and dose-dependent manner. Conclusions PD inhibits cell adhesion, invasion and migration in NSCLC cells, and these effects are related to the down-regulation of the expression of MMP-2 and MMP-9 mR-NA and protein, and inhibition of its upstream expres-sion of ERK signaling pathway and p-Akt.

Research Progress in Anti-tumor Mechanisms of Platycodin-D / 中国中医药信息杂志

Platycodin-D (PD) is the major monomer of triterpene saponins in the root of Platycodon grandiflorum. Recent studies have demonstrated that PD has a wide range of anti-tumor effect and its efficacy is satisfying. This article reviewed anti-tumor mechanisms of PD from the aspects of proliferation, apoptosis, invasion and metastasis, immune and anti-inflammation, etc., with a purpose to provide a theoretical basis and reference for the further development and better utilization of PD and taking its anti-tumor advantage.

An HPLC-MS/MS method for the quantitative determination of platycodin D in rat plasma and its application to the pharmacokinetics of Platycodi Radix extract / 中国天然药物

AIMS@#To develop an HPLC-MS/MS method for the quantification of platycodin D (PD) in rat plasma, and to acquire the main pharmacokinetic parameters of PD after oral administration of pure PD or of Platycodi Radix extract (PRE) containing PD.@*METHOD@#Plasma samples were pretreated with solid-phase extraction using an Oasis® HLB SPE cartridge. Madecassoside was used as the internal standard (IS). Chromatographic separation was achieved on an ODS column (100 mm × 2.1 mm i.d., 3.5 μm) with a mobile phase consisting of acetonitrile/water (30 : 70, V/V) containing 0.1 mmol·L(-1) ammonium acetate at a flow rate of 0.25 mL·min(-1). The detection was performed on a triple quadruple tandem mass spectrometer using an electrospray ionization (ESI) source with a chromatographic run time of 3.0 min. The detection was operated by multiple reaction monitoring (MRM) of the transitions of m/z 1 223.6→469.2 for PD and of m/z 973.6→469.2 for madecassoside (IS), respectively.@*RESULTS@#The calibration curve was linear from 5 to 2 000 ng·mL(-1) (r(2) >0.99) with a lower limit of quantification (LLOQ) of 5 ng·mL(-1). The intra- and inter-day precision (relative standard deviation, RSD) values were below 15% and the accuracy (relative error, RE) was from -15% to +15% at three quality control (QC) levels. Plasma concentrations of PD were determined for 24 h after i.v. administration of PD, and oral administration of PD and PRE, respectively. The absolute oral bioavailability of PD in rats was found to be (0.48 ± 0.19)% when administered PD, and to be (1.81 ± 0.89)% when administered PRE.@*CONCLUSION@#The developed HPLC-MS/MS method was successfully applied to assess the pharmacokinetic parameters and oral bioavailability of PD in rats after administration of PD and Platycodi Radix extract.

Inhibitory effect of platycodin D on human colonic cancer SW620 cell proliferation / 中国药学杂志

OBJECTIVE: To explore the inhibitory effect and its mechanism of platycodin D (PD) on human colonic cancer SW620 cell proliferation. METHODS: The inhibitory effect of PD on SW620 cell proliferation was analyzed by MTT assay, while the effect of PD on cell cycle distribution and apoptosis were evaluated by flow cytometry. The effect of PD on expression of cyclin and ap-optosis associated proteins were detected by Western blot. RESULTS: The cell proliferation of human colonic cancer SW620 cell was inhibited by PD in a dose-dependent manner. After cells were treated with PD(15, 20 μmol · L-1) for 24 h, the proportions of cells in G0/G1 phase were (72.83±5.26)% and (76.82±5.83)% respectively, while the control group cells was (56.78±4.92)%, which suggested PD could block SW620 in the G, phase compared with the control group cells. Furthermore, the expressions of cyclinD1, c-myc and GDK6 were reduced obviously. Compared with the control group cells, the apoptotic rates were increased [(19.5±5.1)%, (35.8±5.3)% and (43.8±4.0)% respectively] after cells were treated by PD (10, 15, 20 μmol · L-1) for 48 h. The expression of procaspase 3 and PARP with proenzyme form were reduced. CONCLUSION: PD could inhibit the growth of colon cancer cell by blocking SW620 in the G, phase through regulating the expression of cyclinD1, c-myc and CDK6 and thus inducing the apoptosis by cell cycle arrest.

Comparison of Vasodilator Effects of Platycodin D and D3 in Rats

The aim of the present study was to examine the effects of platycodin D and D3, which are active components derived from the roots of Platycodon grandiflorum A. DC., on the contractile force of the i3olated rat aorta and blood pressure of the anesthetized rat, and also to elucidate its mechanism of action. Both phenylephrine (an adrenergic alpha1-receptor agonist) and high potassium (a membrane- depolarizing agent) caused great contractile responses in the isolated aortic strips. Platycodin D at high concentration (24microgram/ml) inhibited contractile responses induced by phenylephrine (10 (-5) M) and high potassium (5.6x10 (-2) M), while low concentrations of platycodin D (4~8microgram/ml) did not affect those responses. However, platycodin D3 (8~32microgram/ml) did not alter the contractile responses evoked by phenylephrine and high K+. Interestingly, the infusion of platycodin D3 (1.0 mg/kg/30 min) significantly reduced the pressor responses induced by intravenous norepinephrine. However, platycodin D3 (1.0 mg/kg/30 min) did not affect them. Taken together, these results show that intravenously administered platycodin D depresses norepinephrine-induced pressor responses in the anesthetized rat, at least partly through the blockade of adrenergic alpha1-receptors. Platycodin D also caused vascular relaxation in the isolated aortic strips of the rat via the blockade of adrenergic alpha1-receptors, in addition to an unknown direct mechanism. However, platycodin D3 did not affect both norepinephrine-induced pressor responses and the isolated rat aortic contractile responses evoked by phenylephrine and high potassium. Based on these results, there seems to be much difference in the mode of action between platycodin D and platycodin D3.

Preparation of Platycodin-D for Reference Substance / 中药新药与临床药理

Objective To establish the preparation techniques for preparing the reference substance of platycodin-D.Methods Raw material of platycodi radix was extracted with 70 %MeOH.The concentrated extract passed through D101 macroporous resin column,then the 40 %alcohol eluate was separated by silica gel column chromatography with chloroform-methanol gradient elution.The fractions contained platycodin-D were collected and purified by HPLC.Results HPLC analysis and normalization of peak areas showed the purity of the platycodin-D was 98.8 %.Conclusions The preparation techniques are simple and high yield,can be used for preparing the reference substance of platycodin-D.

HPLC fingerprints of Platycodon grandiflorum from different habitats and the determination of platycodin-D / 中成药

AIM: To establish HPLC-fingerprints and quantitatively determine platycodin-D from Platycodon grandiflorum.METHODS: HPLC analysis was carried out on Hypersil C18 column(250 mm ? 4.6 mm,5 ?m),with a mobile phase of acetonitrile-0.05 mol/L phosphoric acid system,gradient elution,with a flow at 0.5 mL/ min,an ultraviolet detection wavelength was at 210 nm for fingerprint and at 206 nm for platycodin-D,column tem-perature at 30 ℃.RESULTS: Twelve common peaks were identified in chromatograms with reference to platycod-in-D peak from the 18 batches of the samples.CONCLUSION: The method of the HPLC-fingerprint and quantita-tive analysis is rapid,simple and accurate with a good reproducibility and can be used for the quality control of Platycodon grandiflorum.