Synthesis and anti-tumor activity of Z-Gly-Pro-OH-podophyllotoxin derivatives / 中国药科大学学报

@#Ten novel podophyllotoxin derivatives (IIIa-IIIi and IV) were synthesized by three-step reactions using podophyllotoxin and N-benzyloxycarbonyl glycine-L-proline as raw material. The structures of the target compounds were confirmed by 1H NMR, 13C NMR and MS. MTT method was used to test anti-tumor activity of the target compounds on HepG2, THP-1, HeLa and MCF-7 cells. The results showed that all the target compounds had inhibitory activity against HepG2, THP-1, HeLa and MCF-7 cells, and the inhibitory activity of IIIa on HepG2 cells was the most prominent with an IC50 value of 0.58 nmol/L. The binding mode of compound IIIa and FAPα was studied by molecular docking. Compound IIIa could bind to multiple sites of FAPα enzyme.

Research progress in biosynthesis of podophyllotoxin and its derivatives / 生物工程学报

Podophyllotoxin (PTOX) is an aryl-tetralin lignan of plant origin found in some species of Podophyllum such as Dysosma versipellis, Diphylleia sinensis, and Sinopodophyllum hexandrum. Etoposide and teniposide are produced semisynthetically from PTOX and used clinically to treat several forms of cancer. As a typical representative of new drug discovery from natural products, the production of PTOX solely depends on extraction from plants, resulting in severe contradiction between supply and demand. With the advantages of unconstrained resources and eco-friendly reaction conditions, biosynthesis method has become a trend in the production of PTOX and its derivatives. In this review, we summarize the research progress of PTOX biosynthesis in plants and expound the functions of the key enzymes as well as their subcellular location. The synthetic biology for production of PTOX intermediates in a tobacco chassis is also introduced. Finally, the heterologous expression and biotransformation of PTOX in microorganisms is summarized, which sets the foundation for the efficient microbial production of PTOX using cell factories.

Synthesis and cytotoxic activities of podophyllotoxin derivatives / 中草药

Objective: To modify the structure of podophyllotoxin derivatives and evaluate the antitumor activities of the derivatives. Methods: The target compounds were synthesized by multi-step reaction with podophyllotoxin and aldehyde compound as starting material.. MTT assay was used to test antitumor activity of all the target compounds on Hela cells, K562 cells, and K562/A02 cell. Results: Eleven novel derivatives were synthesized which had not been reported in any literature and the structures were characterized by 1H-NMR, 13C-NMR, HR-ESI-MS and melting point determination analysis. The antitumor activity screening results showed that all the target compounds had different degrees of cytotoxic activity in vitro. Most of the compounds had significant anti-MDR activity in vitro. Conclusion: Through structural modification of podophyllotoxin derivatives, the antineoplastic activities are enhanced.

Synthesis and antitumor activity of novel indole podophyllotoxin derivatives / 中国中药杂志

According to drug design flattening principle,a series of novel indole podophyllotoxin derivatives which were introduced different indole substituents in C-4 position on the basis of podophyllotoxin nucleus were synthesized with the starting material podophyllotoxin and 1 H-indole-5-carboxylic acid. Its anti-tumor activity in vitro was tested in order to screen for high-efficiency and low-toxic compounds. Six target compounds were synthesized,and were confirmed by~1 H-NMR,~(13)C-NMR,HR-ESI-MS and melting point determination analysis. All these target compounds were not reported by previous literature. Using etoposide as positive control drug,all the target compounds were screened for cytotoxicity against He La cells,K562 cells and K562/A02 cell in vitro by MTT method. The antitumor activity screening results showed that compounds 4 b,4 e,4 f exhibited higher inhibitory rate against He La cells and K562 cells than those of control drug VP-16. This route has the advantages on simple operation and reasonable design,provides some practical reference value for the further development on the structure modification of podophyllotoxin and study on anti-tumor activity.

Effects of exogenous IBA and fungal elicitor on growth of in vitro roots culture of Dysosma versipellis and production of podophyllotoxin / 中国中药杂志

Using the White as basic medium, the effects of the exogenous IBA and endophytic fungal elicitor on the growth of in vitro roots cultures of Dysosma versipellis and production of podophyllotoxin were investigated in this study. The results showed that the IBA and the endophytic fungus Zasmidium syzygii elicitor could increase the content of podophyllotoxin of in vitro roots of D. versipellis after 3 weeks. The White medium added with 3 mg·L~(-1) IBA induced the highest increase of podophyllotoxin(1 830.86 μg·g~(-1)), which was 2.07 folds greater than the control, and followed by 1.5 mg·L~(-1) IBA, fungal elicitor, 1 mg·L~(-1) IBA, 0.5 mg·L~(-1) IBA and 4.5 mg·L~(-1) IBA, which was 1.82, 1.71, 1.63, 1.43 and 1.1 folds greater than the control, respectively. The results also showed that the growth of roots was certain positively correlated with the change of IBA concentration. Therefore, 3 mg·L~(-1) IBA was the most suitable for the production of podophyllotoxin in the in vitro roots of D. versipellis, and the stimulating effect of Z. syzygii fungal elicitor was between 1.5 mg·L~(-1) and 1 mg·L~(-1) IBA, which was a potential natural elicitor to induce the accumulation of podophyllotoxin in future production.

Synthesis and Antitumor Activity of Indole Podophyllotoxin Derivatives / 中国药学杂志

OBJECTIVE: To synthesize 5-substituted indole-3-deoxypodophyllotoxin derivatives and study their antitumor activity. METHODS: The target compounds were synthesized through a series of reactions and their anti-tumor activity in vitro were evaluated against Hela, K562 and K562/A02 cell lines by MTT as assay. RESULTS: Ten target compounds were synthesized and confirmed by 1H-NMR, 13C-NMR, and HR-ESI-MS. All the target compounds had different degrees of cytotoxic activity in vitro. Most of the compounds had significant anti-MDR activity in vitro. CONCLUSION: 5-Substituted indole-3-deoxypodophyllotoxin derivatives have good antitumor activity and worth of further study.

Progress in structural modification of representative lignan compounds / 中草药

Lignans which are polymerized by two or more phenylpropanoids derivatives were mostly found in the xylem and resin of plants. Modern pharmacological studies have shown that lignans have extensive biological activities, such as anti-tumor, anti-HIV, antidiabetics, anti-oxidant, cardiovascular and liver protections and so on. However, owing to the poor solubility of structures, its clinical application is still limited. Therefore, chemical modification methods are usually used by domestic and overseas researchers in order to improve their solubility. In this paper, a series of typical lignans such as arctigenin, podophyllotoxin, schisandrin, magnolol, and honokiol were taken as examples to summarize their structural modification methods and different biological activities, aiming to provide scientific basis for further exploitation and studies of lignans.

Acute and chronic toxicity of 0.5% podophyllotoxin-loaded nanostructured lipid carriers to vaginal mucosa in rabbits and rats / 南方医科大学学报

OBJECTIVE@#To test the acute and chronic toxicity of topical application of 0.5% podophyllotoxin-loaded nanostructured lipid carriers (POD-NLC) to the vaginal mucosa.@*METHODS@#Twelve New Zealand rabbits were randomized into 3 groups and subjected to daily topical applications of normal saline (control group), 0.5% podophyllotoxin tincture (POD-T) or 0.5% POD-NLC on the vaginal mucosa for 10 consecutive days, and the pathological changes in the mucosa were graded using the Eckstein scoring system.The acute toxicity of POD-NLC was tested in 20 SD female rats, which received intravaginal administration of POD-NLC or vehicle for 3 times within 24 h; After 14 days of continuous observation, the rats were dissected for calculating the viscera coefficient.For testing the chronic toxicity of POD-NLC, 80 SD female rats were randomized into 4 groups and subjected to daily intravaginal administration of the vehicle or POD-NLC at low, moderate or high doses for 13 consecutive weeks.The rats were weighed once a week and at the end of the experiment, 2/3 of the rats from each group were sacrificed to collect blood samples, calculate the viscera coefficient, and examine the pathological changes in the liver.The remaining 1/3 rats were observed for another 2 weeks without further drug treatment and the same examinations were performed.@*RESULTS@#In the rabbits, 0.5% POD-NLC elicited only mild irritation while POD-T caused moderate irritation of the vaginal mucosa.In the acute toxicity test, the organ coefficients were comparable between the rats treated with the vehicle and POD-NLC (>0.05).Long-term intravaginal administration of POD-NLC did not produce significant changes in the behavior, activity, body weight, blood biochemical profiles or organ coefficient as compared with the vehicle control group (>0.05).@*CONCLUSIONS@#Intravaginal administration of 0.5% POD-NLC causes very mild irritation without obvious acute or chronic toxicity to the vaginal mucosa in rabbits and rats.

Preparation of Carboxylated Multi-Walled Carbon Nanotubes Loaded with Podophyllotoxin and Their Transdermal Penetration / 中国药学杂志

OBJECTIVE: To study the preparation of carboxylated multi-walled carbon nanotubes loaded with podophyllotoxin (PPT-CNTs-COOH) as well as the characteristics of the in vitro transdermal penetration. METHODS: PPT-CNTs-COOH was prepared by freezing milling method; IR, UV, XRD, and TGA were used to characterize the PPT-CNTs-COOH; HPLC method was used for determination of the content of podophyllotoxin loaded in the carboxylated multi-walled carbon nanotubes; franze diffusion cells method was used to determine the drug transdermal penetration rate. RESULTS: The IR spectrum of PPT-CNTs-COOH showed the main absorption peaks of PPT and CNTs-COOH and the peaks changed obviously. Compared with free PPT, the UV absorption peaks of PPT-CNTs-COOH changed obviously. The PPT content in the CNTs-COOH gel was 58.0 μg·mg-1; the transdermal penetration rate of PPT gel was 7.08 μg·cm-2·h-1 and that of the PPT-CNTs-COOH gel was 3.03 μg·cm-2·h-1; the skin retention of PPT-CNTs-COOH gel was 3.04 μg·cm-2, far less than the 1.52 μg·cm-2 of PPT gel. Mild irritation developed within 24 h following removal of the PPT-CNTs-COOH gel, and disappears after 72 h. CONCLUSION: Podophyllotoxin can successfully be loaded into the carboxylated multi-wall carbon nanotubes by using the frozen ball milling method. The product has remarkable sustained release effect in vitro and high retention in skin, which is beneficial to transdermal delivery.

Inhibition on proliferation of HeLa cells in vitro of nano sllicon equipped with 4'-demethylepi-podophyllotoxin / 药物评价研究

Objective To study the inhibitory effect on proliferation of Hela cells of podophyllotoxin,4'-demethylepi-podophyllotoxin and drug combination of different proportion of nano-sillca (SiO2) and 4'-demethylepi-podophyllotoxin in vitro,and discuss the mechanism.Methods Used ethyl silicate hydrolysis method to prepare 25 nm SiO2 sample,next carried 4'-demethylepi-podophyllotoxin after the surface modification,and measure cell campatibility by MTT method and Hoechst 33342.The inhibitory effect of podophyllotoxin,4'-demethylepi-podophyllotoxin and drug combination on proliferation of Hela cells was measured by MTT assay.Hoechst 33342 staining method was used to detect cell apoptosis.The effect ofdmg combination treatment on cell morphology was observed by inverted microscope.Western blotting technique was used to detected effect of 4'-demethylepi-podophyllotoxin and drug combination on expression of apoptosis related protein.Results Inhibitory effect onproliferation of Hela cells of 4'-demethylepi-podophyllotoxin is superior to podophyllotoxin,inhibitory effect of drug combination is superior to the single 4'-demethylepi-podophyllotoxin,the inhibition of drug combination with 0.125 μg/mL nano SiO2 and 6.25 μg/mL 4'-demethylepi-podophyllotoxin is the most obvious.MTT and Hoechst 33342 experimental results showed that the 25 nm SiO2 have good cell compatibility.Podophyllotoxin,4'-demethylepi-podophyllotoxin and drug combination can induce apoptosis.Western blotting results showed that 4'-demethylepi-podophyllotoxin and drug combination can up-regulate the ratio of Bax/Bcl-2 and the expression level of Caspase-3、P53 and P38.Conclusion In vitro experimental performance of drug combination is superior to single 4'-demethylepi-podophyllotoxin,it is may by effecting the expression of Bcl-2,Bax,Caspase-3,P53,and P38 and others apoptosis related protein to induce Hela cell apoptosis.

Study on podophyllotoxin induced rat epididymalepithelial cell apoptosis and its mechanism in vitro / 中国药理学通报

Aim To investigate the proliferative effect and the apoptosis of rat epididymal epithelial cells induced by podophyllotoxin and its underlying mechanisms.Methods Primary epididymal epithelial cells were cultured in vitro.CCK-8 assay was used to analyze proliferation of epididymal epithelial cells induced by podophyllotoxin on 24, 48 and 72 h.The ultra structural changes of the epididymal epithelial cells were observed by transmission electron microscope.AnnexinV-FITC/PI staining was used to quantify the percentages of apoptosis in the total cell population.The TdTmediated dUTP nick end labeling(TUNEL) technique was applied to observe the morphological changes of apoptotic cells.The expression of tumor necrosis factor alpha(TNF-α) mRNA was investigated by real-time RT-PCR.The level of TNF-α in cell culture supernatant was measured by enzyme-linked immunosorbent assay(ELISA) technology.Western blot was per-formed to determine the protein expression of cytochrome C, caspase-3, caspase-8, caspase-9.Results Podophyllotoxin significantly inhibited the activity of proliferation and induced apoptosis of epididymal epithelial cells in a dose-and time-dependent manner(P<0.05), with a 50％ inhibitory concentration(IC50) value and corresponding 95％ confidence intervals(CI) of 59.36(15.50～227.41), 0.37(0.080～1.70), 0.077(0.017～0.35) μmol·L-1 at 24, 48 and 72 h, respectively.Podophyllotoxin induced cell volume turned round and cell nuclear fragmentation and mitochondrial vacuolation.RT-PCR and ELISA results showed that podophyllotoxin improved the expression of TNF-α mRNA and protein.Western blot results demonstrated that podophyllotoxin activated the protein expression of cytochrome C, caspase-3, caspase-8 and caspase-9.Conclusion Podophyllotoxin can induce rat epididymal epithelial cell apoptosis through both the mitochondria-regulated intrinsic pathway and the TNF receptor-mediated extrinsic pathway.

Podophyllotoxin derivative LN-13 induced multidrug resistant cell K562/A02 apoptosis and its molecular mechanism / 中国药理学通报

Aim To study the mechanism of action of the new derivative of podophyllotoxin(LN-13)in indu-cing the apoptosis of K562/A02 cells.Methods The MTT method was taken to detect the inhibition of LN-13 and VP-16 on K562/A02 proliferation and inhibi-tion rate and IC50 values were obtained 48 hours later. The K562/A02 cell morphological change induced by LN-13 were observed through Hochest33342 and PI staining after 48 hours later.Flow cytometry was taken to detect the apoptosis of K562/A02 cells induced by LN-13.The reverse transcription-polymerase chain re-action was taken to detect the Bcl-2,Bax,caspase-3 and mdr-1 mRNA expression.The expression of P-gp was detected by Western blot.Results The growth of K562 /A02 cells was obviously inhibited by LN-13 when IC50 value was 3.32 μmol · L-1 .LN-13 could obviously induced cell apoptosis observed by Ho-chest33342 and PI staining.Flow cytometry detection showed that LN-13(2,4,8 μmol·L-1 )could induce cell apoptosis and apoptosis ratio reached 15.0%, 48.0%,68.96%,respectively.The reverse transcrip-tion-polymerase chain reaction showed that LN-13 in-creased the Bax and Caspase-3 mRNA expression,and meanwhile the expression of mdr-1 mRNA decreased. Western blot showed that P-gp expression was de-creased as the LN-13 dose increased.The data were significantly different from those of control group.Con-clusion Podophyllotoxin derivative LN-13 can induce the apoptosis of K562 /A02 cells,which may be close-ly-related to regulating P-gp expression and apoptosis related gene mRNA expression.

Cloning,expression and functional identification of secoisolariciresinol dehydrogenase gene from Dysosma versipellis callus / 中国中药杂志

Secoisolariciresinol dehydrogenase (SDH) is a key enzyme involved in the biosynthetic pathway of podophyllotoxin.In this study, two SDH candidate genes,SO282 and SO1223, were cloned from callus of Dysosma versipellis by homology-based PCR and rapid amplification of cDNA end (RACE).The SDH candidate genes were expressed in Escherichia coli and the subsequent enzyme assay in vitro showed that recombinant SO282 had the SDH activity. These results pave the way to the follow-up investigation of the biosynthetic of podophyllotoxin.

Study on Effects of Podophyllotoxin Derivative QW-83 on Human Cervical Cancer HeLa Cell Apoptosis and Its Mechanism / 中国药房

OBJECTIVE:To study the effects of podophyllotoxin derivative QW-83 on human cervical cancer HeLa cell apopto-sis and its mechanism. METHODS:After treated with 0(negative control),0.01,0.1,1 and 10 μmol/L QW-83 and positive drug etoposide(VP-16)for 48 h,proliferation inhibition rate and IC50 of HeLa cell were determined by MTT assay. The morphological changes of HeLa cell were observed by Hochest 33342 staining after treated with QW-83 [0(negative control),2.5,5,10μmol/L] for 48 h;flow cytometry was used to detect apoptosis rate;semi quantitative RT-PCR was adopted to detect the expression of apop-tosis related gene P53,Bax,Casepase-3,Casepase-8,Casepase-9 and Bcl-2 mRNA. RESULTS:Compared with negative control, 1,10 μmol/L VP-16 and QW-83 had obvious proliferation inhibition effect on HeLa cells (P<0.05 or P<0.01),and IC50 were (5.11±0.43)μmol/L and(4.96±0.54)μmol/L. Hochest 33342 staining results showed QW-83 could obviously induce cells apopto-sis and nuclear pyknosis. Flow cytometry showed QW-83 could increase apoptosis rate in concentration-dependent manner,being 16.89%-62.56%. RT-PCR showed mRNA expression of P53,Bax,Caspase-3,Casepase-8 and Casepase-9,Bcl-2/Bax increased, while mRNA expression of Bcl-2 decreased after treated with QW-83(P<0.05). CONCLUSIONS:Podophyllotoxin derivative QW-83 can induce HeLa cell apoptosis,and its mechanism may be associated with regulate mRNA expression of apoptosis related gene.

Research on mechanisms of deoxypodophyllotoxin-induced inhibition of cell proliferation and migration in human lung cancer NCI-H358 cells / 天津医药

Objective To investigate the effects and mechanism of deoxypodophyllotoxin on cell proliferation and mi?gration of human lung cancer NCI-H358 cells in vitro. Methods CCK-8 assay, flow cytometry assay, wound healing assay and DCFH-DA assay were used to detect the effects of deoxypodophyllotoxin on the proliferation, cells cycle, apoptosis, mi?gration and reactive oxygen species (ROS). The protein expressions of Cyclin B1, Cdc25c, CDK1, Caspase-3, p53, Bcl-2, MMP9, ERK1/2, p38MAPK and JNK were measured by Western blot assay, respectively. Results Deoxypodophyllotoxin inhibited cell proliferation and reduced migration in human lung cancer NCI-H358 cells. Flow cytometry analysis showed that treatment with deoxypodophyllotoxin resulted in cell cycle G2/M and S phase arrest, cell apoptosis and ROS production. The result of Western blot assay showed that protein expressions of Cyclin B1, Cdc25c, CDK1, Bcl-2 and MMP9 were down-regulated while Caspase-3 and p53 were up-regulated. Moreover, Deoxypodophyllotoxin treatment decreased the phosphory?lated levels of ERK1/2, p38MAPK and JNK obviously. Conclusion Deoxypodophyllotoxin could suppress the proliferation and migration of human lung cancer NCI-H358 cells in vitro, which is a potential anti-tumor drug.

Lignans constituents from callus culture of Dysosma versipellis / 中国药学杂志

OBJECTIVE: To study the lignans constituents from callus culture of Dysosma versipellis (Hance) M. cheng ex Ying.

Preparation and Application of Podophyllotoxin Sensor Based on Molecularly Imprinting Technique / 分析化学

A novel electrochemical sensor for the determination of podophyllotoxin (PPT) was prepared by modifying a PPT-imprinted membrane, which was synthesized by in situ polymerization with PPT as template, methacrylic acid ( MAA) as functional monomer and ethylene glycol dimethacrylate ( EGDMA) as cross linking agent at the molar ratio of PPT/ MAA/ EGDMA as 1 : 2 : 100, on glassy carbon electrode ( GCE) followed by eluting PPT from the modified GCE with methanol/ acetic acid (1: 1, V/ V). The properties of the electrochemical sensor modified with PPT-imprinted membrane were characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS). Under the optimum conditions, it was found that the response of peak currents was linear to the concentration of PPT in range of 1. 0 - 120 μmol/ L with the detection limit of 0. 47 μmol/ L. The sensor not only showed high selectivity, but also exhibited good stability and reproducibility. The sensor was satisfactorily applied to the determination of PPT in podophyllum hexandrum and human serum samples with the relative standard deviation (RSD) below 3. 9% and recovery ranging from 95. 0% to 103. 3% .

Synthesis of podo-and epipodo-11C-podophyllotoxin and their biodistribution in mouse bearing EMT6 tumor / 中华核医学与分子影像杂志

Objective To synthesize podo-and epipodo-podophyllotoxin labeled with 11C and investigate their biodistribution in mice bearing EMT6 tumor by microPET.Methods 11 C-podo-or epipodopodophyllotoxin was synthesized by 11 C-CH3-Triflate mixed with 4'-methyl-demethmyl-podophyllotoxin (podo-and epipodo-) and purified using HPLC.The radiochemical purity (RCP) was analyzed by HPLC.Thirty mice bearing EMT6 tumor were divided into 2 groups and injected with 3.7 MBq 11C-podo-or epipodo-podophyllotoxin.The biodistribution of 11C-podo-or epipodo-podophyllotoxin was evaluated with microPET.The ID％/g was calculated.Paired t test was used to analyze the data.Results The yields of 11C-podo-and epipodo-podophyllotoxin were both ＞90％.The RCP was ＞99％.The biodistribution of 11C-podo-and epipodo-podophyllotoxin was similar with slow blood clearance and high uptake in abdomen.The tumor uptake of 11C-epipodophyllotoxin and 11C-podophyllotoxin at 30 min was (3.63±0.98) ％ID/g and (3.16±0.27) ％ID/g,respectively.The uptake ratio of tumor to blood and to muscle was 0.68 vs 0.62 and 1.52 vs 1.22,respectively.There was no significant difference between the tumor uptake of the two agents (t=0.47,P＞0.05).The result of microPET imaging was consistent with that of mice experiment.Conclusion 11C-podophyllotoxin has limited clinical value for tumor imaging.

Determination of quercetin, podophyllotoxin and kaempferol in Diphylleia sinensis Li. by HPLC / 中国药学杂志

OBJECTIVE: To establish a method to determine quercetin, podophyllotoxin and kaempferol in Diphylleia sinensis Li. METHODS: Diamonsil C18 column was used. Methanol-0.4% glacial acetic acid solution was used as the mobile phaseat a flow rate of 1 mL · min-1. The column temperature was 30°C. The detection wavelength was 290 nm. RESULTS: The liner ranges of quercetin, podophyllotoxin and kaempferol were 0.023-0.287 μg (r=0.999 9), 0.208-2.6 μg (r=0.999 9), and 0.036 1-0.451 μg (r=0.999 9), respectively. The average recoveries were 99.64% (RSD=0.83%), 100.04% (RSD=0.61%), and 99.88% (RSD=1.19%), respectively. CONCLUSION: The method is sensitive, accurate and reproducible. It can be used for the determination of quercetin, podophyllotoxin and kaempferol in Diphylleia sinensis Li.

Percutaneous permeation characteristic of podophyllotoxin ethosomes in vitro / 中草药

Objective To study the percutaneous permeability characteristic of podophyllotoxin (POD) ethosomes in vitro. Methods Excised SD-rat abdomen skin was used as penetration barrier. The steady penetration rate and the skin residual amount were calculated to evaluate the percutaneous permeability of POD from ethosomes, tinctures, liposome, 30% hydro-ethanolic suspension, mixture of drug and blank ethosomes, respectively. Results The skin residual amount of POD in the ethosomes group was 8.17 ug/cm2 in 12 h, higher than those in the other groups. In addition, the steady penetration rate of POD in the ethosomes group decreased significantly compared with those in the liposome group with POD loading being 0.5%, and the hydro-ethanolic suspension group (P < 0.05), while no obvious difference was seen among the ethosomes group and the other groups. Conclusion Higher skin residual amount and lower steady penetration rate are observed in the ethosomes group.