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Objective To study the chemical constituents from the whole plants of Pteris semipinnata. Methods The chemical constituents were isolated by silica gel, sephadex LH-20, ODS, macroporous resin, MCI Gel resin and semi-preparation HPLC. Their structures were determined on the basis of NMR and MS analysis. Results Twenty compounds were isolated and identified as apigenin (1), apigenin-7-O-β-D-glucopyranoside (2), apigenin-7-O-β-D-gentiobioside (3), apigenin-7-O-β-D-glucopyranosyl-4′-O-α- L-rhamnopyranoside (4), isoviolanthin (5), luteolin (6), luteoloside (7), luteolin-7-O-β-D-gentiobioside (8), quercetin-3-O-β-D- glucopyranoside (9), kaempferol-3-O-β-D-glucopyranoside (10), rutin (11), epigallocatechin (12), pinoresinol-4-O-β-D- glucopyranoside (13), phillyrin (14), bergeninum (15), protocatechuic acid (16), trans-caffeic acid (17), gallic acid (18), β-sitosterol (19), and β-daucosterol (20). Conclusion Compounds 3, 5, 12, 13, and 15—18 are isolated from the plants of Pteris, and 2—5, 8—13, and 15—20 are isolated from this plant for the first time.
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AIM To find a new method for purifying the active compound 5F isolated from Pteris semipinnata L.(PsL ) and observe its enhanced cytotoxicity when combined with other antitumor agents. METHODS Silica gel combined with AgNO 3 was made to purify 5F. Cytotoxicity was detected with trypan blue dye exclusion assay. RESULTS After purification, the concentration of compound 5F in purified products was higher than 99%. The inhibitory rates of 5F combined with 5 Fu or CDDP or VCR were higher than that of these drugs used alone. CONCLUSIONS Compound 5F could be purified effectively using silica gel combined with AgNO 3. 5F could enhance the cytotoxicity on HL 60 and K562 cells of the drugs mentioned above. The different effect of these agents on the various phases of cell cycle kinetics may explain the enhanced effect of 5F.
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AIM: To study the effects of a diterpenoid compound 5F (ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic acid) isolated from pteris semipinnata L on the the activity and expression of mitogen activated protein kinase of K562 cells. METHODS: K562 cells were treated with compound 5F for 24 hours, then MAPK activity was determined using MBP as substrate, Western blotting was used to detect the effect of compound 5F on the expression of MAPK. RESULTS: The activity and expression of MAPK increased after the treatment of 5F. CONCLUSION: One mechanism of the anti-tumor effect of compound 5F is through abnormal activation and abnormal expression of MAPK.
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[Objectivs] To investigate the principle of AgNO3-processed silica gel chromatography in isolation and purification of mixtures with carbon-carbon double bonds (CCDB) from herbal medicine. [Methods] AgN03-processed gel chromatography was used for the isolation of mixtures with carbon-carbon double bonds in the volatile oils and active components from Pteris semipinnata L. ; silver-ion complexion chromatography was applied to isolate arachidom'c acid from lipids. The principle of AgNO3-processed silica gel chromatography was analyzed by coordination theory. [Results] Stable AgNO3 coordinate complexes were obtained. [ Conclusion ] The principle of the formation of stable AgNO3 coordinate complexes may be: (1) the stability will increase with CCDB in free space; (2) exocyclic CCDB is more stable than that of intra-cyclic CCDB; (3) cis-form CCDB is more stable than that of trans-form CCDB; (4) terminal CCDB is more stable than that of cis-form CCDB; (5) aromatic hydrocarbon with multiple benzene rings is more stable than that with single ber zene ring. The possible principle will benefit to the isolation and purification of the mixtures with CCDB, which have similar structure and physiochemical property and are difficult to be isolated and purified by common chromatography and extriction method.
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AIM: To investigate the changes of cytosolic free calcium concentration ([Ca 2+ ] i) and expression of Bcl-2 in HL-60 cells treated by 6F isolated from Pteris semipinnata L. (PSL), and to discuss the relations between calcium ion and cytotoxicity and DNA fragment induction effects of 6F. METHODS: HL-60 cells were used as in vitro model. [Ca 2+ ] i was measured on fluorescent spectrophotometry using Fura-2/AM as Ca 2+ indicator. Bcl-2 expressing level was measured by flow cytometry. Tetrazolium salt (MTT) and diphenylamine staining methods were applied for cytotoxicity assay and DNA fragmentation detection, respectively. RESULTS: [Ca 2+ ] i increased obviously in a dose and time dependent manner after treated HL-60 cells with 6F. 6F decreased the expressing level of Bcl-2. Adding 2 mmol/L Ca 2+ to the medium, or 1 mmol/L EDTA to chelate Ca 2+ , or 4 ?mol/L calcium ionophore A 23187 to increase the concentration of cytosolic Ca 2+ , the DNA fragment induction by 6F was not affected, whereas the cytotoxicity of 6F was enhanced. 250 ?mol/L Zn 2+ attenuated the DNA fragment induction, and the cytotoxicity of 6F against HL-60 cells was enhanced significantly. CONCLUSION: It was speculated that the decreased expressing of Bcl-2 by compound 6F was related to increased [Ca 2+ ] i in HL-60 cells, and DNA fragment induction was possibly catalyzed by Ca 2+ - independent DNase.
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Objective To study the effect of a diterpenoid 5F (ent-11?-hydroxy-15-oxo-kaur-16-en-19-oic acid) isolated from Pteris semipinnata on the expressions of NF-?B (P65), Smad3 protein and NF-?B (P65), ETS-1 mRNA, and to investigate the antitumor mechanisms of 5F. Methods MTT assay was used to examine the effect of 5F on proliferation of HO-8910PM cells. The expressions levels of NF-?B (P65) and Smad3 proteins were assessed by Western blot analysis. RT-PCR assay was used to assess the expression levels of NF-?B (P65) and ETS-1 mRNA. Results The proliferation of HO-8910PM cells can be inhibited by 5F in a dose-dependent manner. The expression levels of NF-?B (P65) and Smad3 proteins decreased obviously in HO-8910PM cells after treatment with 25—100 ?mol/L 5F for 24 h, and the effects appeared in a dose-dependent manner. 5F down-regulated significantly the expression of NF-?B (P65) mRNA, down-regulated gently the expression of ETS-1 mRNA. Conclusion The antitumor activity of 5F is involved in the expression of NF-?B (P65), Smad3 protein and NF-?B (P65), ETS-1 mRNA.
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Object To establish a high efficient and reliable method for extraction and analysis of the diterpenoids in Pteris semipinnata L. Methods Supercritical CO 2 fluid modified with alcohol was used to extract the diterpenoids in P. semipinnata, the extracting conditions were optimized by orthogonal design method, and the modifying solvent was investigated through total ions chromatograghy normalization. A quadrupole mass spectrometer coupled with atmospheric pressure chemical ionization interface was employed as a detector for HPLC, the -1 ion was used as selective ion for the detecting of ent 11 ? hydroxy 15 oxo kaur 16 en 19 olic acid (5F) in selective ion monitoring (SIM) mode. The peak area of SIM and tolal ion chromatogram (TIC) were used for quantitative determination. As an example of its application, this method was used to determinate the content of 5F as antitumor diterpenoids in P. semipinnata. Results The optimized conditions for supercritical CO 2 fluid extraction are 25 MPa, 60 ℃, 15% methanol, flow rate 3 0 mL/min; analytical column was Diamonsil ODS (150 mm?4 6 mm, 5 ?m); the mobile phase of HPLC was CH 3CN 2 mmol/L NH 4AC (35∶65), flow rate 1 0 mL/min, injection volume 5 ?L; the standard curve showed good linearity over the range of 0 05-2 5 ?g; the limit of detection is 0 4 ng; the recovery is 97 8% (n=3). Conclusion This method is highly efficient, sensitive, and selective, which can be applied to study the antitumor drug of diterpenoids in P. semipinnata and to establish the drug standard.
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Aim To study the effect of 5F on tubulin polymerization.Methed 5F was added into the tubulin polymerization-depolymerization system and the changes of Absorption values at 350 nm, 37 ℃ were recorded. Results 5F decreased the Absorption values of the reaction system.and markedly inhibited not only tubulin polymerization but also tubulin depolymerization. Conclusion 5F inhibits tubulin polymerization and depolymerization, and leads to cell cycle arrested, and possibly induces apoptosis.