Enhanced podophyllotoxin production of endophyte Fusarium proliferatum TQN5T by host extract and phenylalanine.

Fermentation technology using endophytes is considered a potential alternative approach for producing pharmaceutical compounds like podophyllotoxin (PTOX). In this study, fungus TQN5T (VCCM 44284) was selected from endophytic fungi isolated from Dysosma versipellis in Vietnam for PTOX production through TLC. The presence of PTOX in TQN5T was further confirmed by HPLC. Molecular identification indicated TQN5T as Fusarium proliferatum with 99.43% identity. This result was asserted by morphological characteristics such as white cottony, filamentous colony, layer and branched mycelium, and clear hyphae septa. Cytotoxic assay indicated both biomass extract and culture filtrate of TQN5T presented strong cytotoxicity on LU-1 and HepG2 with IC50 of 0.11, 0.20, 0.041, and 0071, respectively, implying anti-cancer compounds were accumulated in the mycelium and secreted into the medium. Further, the production of PTOX in TQN5T was investigated in the fermentation condition supplemented with 10 µg/ml of host plant extract or phenylalanine as elicitors. The results revealed a significantly higher amount of PTOX in the PDB + PE and PDB + PA at all studied time points in comparison with PDB (control). Especially, after 168 h of culture, PTOX content in the PDB with plant extract reached the peak with 314 µg/g DW which is 10% higher than the best yield of PTOX in previous studies, denoting F. proliferatum TQN5T as a promising PTOX producer. This is the first study on enhancing the PTOX production in endophytic fungi by supplementing phenylalanine-a precursor for PTOX biosynthesis in plants into fermented media, suggesting a common PTOX biosynthetic pathway between host plant and endophytes. KEY POINTS: • Fusarium proliferatum TQN5T was proven for PTOX production. • Both mycelia extract and spent broth extract of Fusarium proliferatum TQN5T presented strong cytotoxicity on cancer cell lines LU-1 and HepG2. • The supplementation of 10 µg/ml host plant extract and phenylalanine into fermentation media of F. proliferatum TQN5T improved the yield of PTOX.

Methyl jasmonate redirects the dynamics of carbohydrates and amino acids toward the lignans accumulation in Linum album cells.

Linum album accumulates lignans e.g., podophyllotoxin (PTOX) and 6-methoxy podophyllotoxin (6MPTOX). This study was aimed to figure out how different concentrations of MeJA (0, 50, 100, 150, and 200 µM) by affecting on free sugars and amino acids contents induce lignans accumulation in L. album cells. Results revealed that hydrogen peroxide (H2O2) content increased at 50µM, while it decreased at the high levels of MeJA (150 and 200 µM). Also, increasing trend of nitric oxide (NO) and lipid peroxidation levels peaked at 200 µM MeJA. An increased antioxidant enzymes activity was also observed in the treated cells. Moreover, an increase in rhamnose/xylose, glucose, and mannose was detected at 150 and 200 µM MeJA compared to the control. These compounds provide energy source and carbon skeleton for amino acids biosynthesis. Our results emphasized variations in amino acids levels in the presence of MeJA, where Phe level shifts along with synthesizing phenolics. Likewise, MeJA treatment switch on phenyl-ammonia lyase (PAL) and tyrosine-ammonia lyase (TAL) activities that regenerate phenolic compounds. Changes in phenolic acids (cinnamic, coumaric, caffeic, ferulic, and salicylic acid) and flavonoids (catechin, vitexin, myricetin, and kaempferol) were observed under MeJA treatment. Eventually, MeJA induced lignans production except for lariciresinol (LARI), so that the highest amounts of PTOX and 6MPTOX were analyzed at 50 µM, which were 4 and 5 time of control, respectively. Conclusively, it can be suggested that MeJA-induced oxidative status change redirects free sugars and amino acids toward the production of phenolic compounds especially lignans in L. album cells.

Discovery of Epipodophyllotoxin-Derived B2 as Promising XooFtsZ Inhibitor for Controlling Bacterial Cell Division: Structure-Based Virtual Screening, Synthesis, and SAR Study.

The emergence of phytopathogenic bacteria resistant to antibacterial agents has rendered previously manageable plant diseases intractable, highlighting the need for safe and environmentally responsible agrochemicals. Inhibition of bacterial cell division by targeting bacterial cell division protein FtsZ has been proposed as a promising strategy for developing novel antibacterial agents. We previously identified 4'-demethylepipodophyllotoxin (DMEP), a naturally occurring substance isolated from the barberry species Dysosma versipellis, as a novel chemical scaffold for the development of inhibitors of FtsZ from the rice blight pathogen Xanthomonas oryzae pv. oryzae (Xoo). Therefore, constructing structure-activity relationship (SAR) studies of DMEP is indispensable for new agrochemical discovery. In this study, we performed a structure-activity relationship (SAR) study of DMEP derivatives as potential XooFtsZ inhibitors through introducing the structure-based virtual screening (SBVS) approach and various biochemical methods. Notably, prepared compound B2, a 4'-acyloxy DMEP analog, had a 50% inhibitory concentration of 159.4 µM for inhibition of recombinant XooFtsZ GTPase, which was lower than that of the parent DMEP (278.0 µM). Compound B2 potently inhibited Xoo growth in vitro (minimum inhibitory concentration 153 mg L-1) and had 54.9% and 48.4% curative and protective control efficiencies against rice blight in vivo. Moreover, compound B2 also showed low toxicity for non-target organisms, including rice plant and mammalian cell. Given these interesting results, we provide a novel strategy to discover and optimize promising bactericidal compounds for the management of plant bacterial diseases.

Assembly of Plant Enzymes in E. coli for the Production of the Valuable (-)-Podophyllotoxin Precursor (-)-Pluviatolide.

Lignans are plant secondary metabolites with a wide range of reported health-promoting bioactivities. Traditional routes toward these natural products involve, among others, the extraction from plant sources and chemical synthesis. However, the availability of the sources and the complex chemical structures of lignans often limit the feasibility of these approaches. In this work, we introduce a newly assembled biosynthetic route in E. coli for the efficient conversion of the common higher-lignan precursor (+)-pinoresinol to the noncommercially available (-)-pluviatolide via three intermediates. (-)-Pluviatolide is considered a crossroad compound in lignan biosynthesis, because the methylenedioxy bridge in its structure, resulting from the oxidation of (-)-matairesinol, channels the biosynthetic pathway toward the microtubule depolymerizer (-)-podophyllotoxin. This oxidation reaction is catalyzed with high regio- and enantioselectivity by a cytochrome P450 monooxygenase from Sinopodophyllum hexandrum (CYP719A23), which was expressed and optimized regarding redox partners in E. coli. Pinoresinol-lariciresinol reductase from Forsythia intermedia (FiPLR), secoisolariciresinol dehydrogenase from Podophyllum pleianthum (PpSDH), and CYP719A23 were coexpressed together with a suitable NADPH-dependent reductase to ensure P450 activity, allowing for four sequential biotransformations without intermediate isolation. By using an E. coli strain coexpressing the enzymes originating from four plants, (+)-pinoresinol was efficiently converted, allowing the isolation of enantiopure (-)-pluviatolide at a concentration of 137 mg/L (ee ≥99% with 76% isolated yield).

Picropodophyllotoxin, an Epimer of Podophyllotoxin, Causes Apoptosis of Human Esophageal Squamous Cell Carcinoma Cells Through ROS-Mediated JNK/P38 MAPK Pathways.

Esophageal squamous cell carcinoma (ESCC), a major histologic type of esophageal cancer, is one of the frequent causes of cancer-related death worldwide. Picropodophyllotoxin (PPT) is the main component of Podophyllum hexandrum root with antitumor activity via apoptosis-mediated mechanisms in several cancer cells. However, the underlying mechanism of the PPT effects in apoptosis induction in cancer remains ambiguous. Hence, in this study, we evaluate the anti-cancer effects of PPT in apoptotic signaling pathway-related mechanisms in ESCC cells. First, to verify the effect of PPT on ESCC cell viability, we employed an MTT assay. PPT inhibited the viability of ESCC cells in time- and dose-dependent manners. PPT induced G2/M phase cell cycle arrest and annexin V-stained cell apoptosis through the activation of the c-Jun N-terminal kinase (JNK)/p38 pathways. Furthermore, the treatment of KYSE 30 and KYSE 450 ESCC cells with PPT induced apoptosis involving the regulation of endoplasmic reticulum stress- and apoptosis-related proteins by reactive oxygen species (ROS) generation, the loss of mitochondrial membrane potential, and multi-caspase activation. In conclusion, our results indicate that the apoptotic effect of PPT on ESCC cells has the potential to become a new anti-cancer drug by increasing ROS levels and inducing the JNK/p38 signaling pathways.

Enzymatic O-Glycosylation of Etoposide Aglycone by Exploration of the Substrate Promiscuity for Glycosyltransferases.

The 4-O-ß-d-glucopyranoside of DMEP ((-)-4'-desmethylepipodophyllotoxin) (GDMEP), a natural product from Podophyllum hexandrum, is the direct precursor to the topoisomerase inhibitor etoposide, used in dozens of chemotherapy regimens for various malignancies. The biosynthesis pathway for DMEP has been completed, while the enzyme for biosynthesizing GDMEP is still unclear. Here, we report the enzymatic O-glycosylation of DMEP with 53% conversion by exploring the substrate promiscuity and entrances of glycosyltransferases. Notably, we found 6 essential amino acid residues surrounding the putative substrate entrances exposed to the protein surface in UGT78D2, CsUGT78D2, and CsUGT78D2-like, and these residues may determine substrate specificity and high O-glycosylation activity toward DMEP. Our results provide an effective route for one-step synthesis of GDMEP. Identification of the key residues and entrances of glycosyltransferases will promote precise identification of glycosyltransferase biocatalysts for novel substrates and provide a rational basis for glycosyltransferase engineering.

Transferrin receptor-targeted redox/pH-sensitive podophyllotoxin prodrug micelles for multidrug-resistant breast cancer therapy.

Podophyllotoxin (PPT), a toxic polyphenol extracted from the roots of Podophyllum species, showed remarkable activity against P-glycoprotein (P-gp) mediated multidrug resistant (MDR) cancer cells. Many PPT-prodrugs based on nano-technology have been developed for increasing aqueous solubility and reducing the side effects of PPT; however, the sensitive linkers in almost all PPT-prodrugs were ester bonds, resulting in slow and incomplete drug release. We developed a redox/pH double-sensitive and tumor active targeted drug delivery system for PPT delivery, in which PPT was covalently coupled to T7-peptide (Pep) modified polyethylene glycol (PEG) or methoxy-polyethylene glycol (mPEG) through a disulfide bond to obtain the final polymer (Pep-PEG-SS-PPT or PEG-SS-PPT). The mixed micelles (Pep-SS-NPs) were made by mixing Pep-PEG-SS-PPT with PEG-SS-PPT, and the mixed micelles showed good size uniformity and high stability in serum solution. The in vitro release experiment showed that about (81.7 ± 2.8)% PPT was released from Pep-SS-NPs in 10 mM glutathione (GSH) at pH 7.4, and also about (64.6 ± 1.7)% PPT was released from Pep-SS-NPs at pH 5.0. In vitro cytotoxicity analysis suggested that Pep-SS-NPs exhibited 57- to 270-fold lower resistance index (RI) values for different drug-resistant cancer cell lines than paclitaxel (PTX) or docetaxel (DTX). The cell uptake assay indicated that the Pep-SS-NPs could significantly enhance the intracellular level of coumarin-6 compared to that of the control group. The maximum tolerated dose (MTD) of Pep-SS-NPs was increased greatly compared to that of free PPT (5.3-fold). In vivo research showed that Pep-SS-NPs significantly enhanced antitumor efficacy against MCF-7/ADR xenograft tumors compared to the control groups. These findings suggest that mixed micelles could be a potentially successful nanomedicine for MDR breast cancer therapy.

Improving the production of podophyllotoxin in hairy roots of Hyptis suaveolens induced from regenerated plantlets.

Ten Hyptis suaveolens hairy root lines were established by infecting nodal explants with K599+pGus-GFP+ and ATCC15834+pTDT strains from Agrobacterium rhizogenes. Genetic transformation was confirmed by epifluorescence and plagiotropic hairy root growth in absence of growth regulators. Cytotoxicity was determined using the sulforhodamine B method, and the production of podophyllotoxin (PTOX) was measured by high performance thin layer chromatography scanning. Through these methodologies, HsTD10 was identified as the hairy root line with the highest cytotoxicity and PTOX production, which was corroborated by liquid chromatography-mass spectrometry and micrOTOF-Q II. A suspension culture of HsTD10 was established in which PTOX and carbohydrate consumption during growth kinetics were quantified by high-performance liquid chromatography. Procedures to increase the production and retrieval of PTOX in the HsTD10 line included selection of culture medium, addition of thiamine, and modification of the PTOX extraction method. The best combination of these variables was MS medium at 75% of its components with the addition of 2 mg L-1 of thiamine, extraction with methanol-dichloromethane, and sonication at 40 ± 5°C. During kinetics, growth-associated PTOX accumulation was observed. The specific growth rate (µ) was 0.11 d-1. The highest concentration of PTOX obtained with HsTD10 (5.6 mg g-1 DW) was 100 times higher than that reported for roots of wild plants and 56 times higher than that for in vitro nontransformed roots of H. suaveolens.

H2O2-responsive nano-prodrug for podophyllotoxin delivery.

The tumor microenvironment is different from that of normal tissue; therefore, the development of a prodrug that retains its efficacy in the tumor microenvironment can be useful in enhancing the anticancer properties of podophyllotoxin. An innovative podophyllotoxin prodrug (POD-PEG) was designed by linking podophyllotoxin to poly(ethylene glycol)(n) monomethacrylate with a H2O2-responsive oxalate ester bond. POD-PEG can self-assemble into stable nanoparticles (POD-PEG NPs). In vitro experiments demonstrated that the POD-PEG NPs can be activated by hydrogen peroxide resulting in podophyllotoxin release and are highly toxic against colon carcinoma CT26 cells. In vivo biodistribution studies demonstrate that PEGylated POD-PEG NPs are capable of prolonging blood circulation. Intravenous injection of POD-PEG NPs into CT26 tumor-bearing Balb/c mice resulted in a significantly enhanced therapeutic efficacy against tumors, with no significant systemic toxicity. Therefore, this H2O2-responsive prodrug delivery system exhibits good biosafety and provides a novel strategy for the development of drug delivery systems.

Click chemistry-based synthesis and cytotoxic activity evaluation of 4α-triazole acetate podophyllotoxin derivatives.

A series of novel 4α-triazole acetate podophyllotoxin derivatives were synthesized via click chemistry. In vitro cytotoxic activity evaluation showed that most of the derivatives exhibited potent inhibitory activities against the tested cancer cell lines with low nanomolar IC50 values. Further studies demonstrated that compound 31 exhibited broad-spectrum cytotoxic activities, effectively overcame drug-resistance, and showed relatively weak cytotoxicity on non-cancer cells. Preliminary mechanistic studies indicated that 31 might have action on microtubule, cause cell cycle arrest at G2 /M phase, and induce apoptosis in human PC-3 cancer cells.

DNA damage and apoptosis induced by a potent orally podophyllotoxin derivative in breast cancer.

BACKGROUND: Targeting TopoisomeraseII (TopoII) and generate enzyme mediated DNA damage is an effective strategy for treatment of breast cancer. TopoII is known as a validated target for drug discovery and cancer chemotherapy. METHODS: XWL-1-48, a new orally podophyllotoxin derivative, was designed and synthesized. The effect of XWL-1-48 on TopoII binding and activity was determined by molecular docking software and kDNA-decatenation assay, respectively. In vitro and in vivo breast cancer models were used to document the antitumor activity of XWL-1-48. Cellular apoptosis, cell cycle and ROS were analyzed by flow cytometry. Alteration of XWL-1-48-mediated downstream pathways was determined by western blot analysis. RESULTS: The cytotoxicity of XWL-1-48 is more potent than that of its congener GL331. Molecular docking demonstrated that XWL-1-48 could bind to TopoII through forming two strong hydrogen bonds and potential pi-pi interactions. Noticeably, XWL-1-48 exerts potent antitumor activity in in vitro and in vivo breast cancer model. Treatment with XWL-1-48 caused ROS generation and triggered DNA damage through induction of γ-H2AX and activation of ATM/p53/p21 pathway. Further studies showed that XWL-1-48 led to S-phase arrest and mitochondrial apoptosis. Meanwhile, XWL-1-48 significantly blocked PI3K/Akt/Mdm2 pathway and enhanced Mdm2 degradation. CONCLUSION: XWL-1-48 may be a promising orally topoII inhibitor, its mechanisms are associated with suppression of TopoII, induction of DNA damage and apoptosis, blockage of PI3K/AKT/Mdm2 pathway.

Effect of in vitro morphogenesis on the production of podophyllotoxin derivatives in callus cultures of Linum album.

The anticancer compound podophyllotoxin and other related lignans can be produced in Linum album in vitro cultures, although their biosynthesis varies according to the degree of differentiation of the plant material. In general, L. album cell cultures do not form the same lignans as roots or other culture systems. Our aim was to explore how the lignan-producing capacity of organogenic cell masses is affected by the conditions that promote their formation and growth. Thus, L. album biomass obtained from plantlets was cultured in darkness or light, with or without the addition of plant growth regulators, and the levels of podophyllotoxin, methoxypodophyllotoxin and other related lignans were determined in each of these conditions. The organogenic capacity of the cell biomass grown in the different conditions was studied directly and also with light and scanning electronic microscopy, leading to the observation of.several somatic embryos and well-formed shoots. The main lignan produced was methoxypodophyllotoxin, whose production was clearly linked to the organogenic capacity of the cell biomass, which to a lesser extent was also the case for podophyllotoxin.

P-3F, a microtubule polymerization inhibitor enhances P53 stability through the change in localization of RPS27a.

Previously, we demonstrated that P-3F, a podophyllum derivative, exhibits a 297-fold enhancement in antitumor activity than VP-16, used as anticancer agent in clinical. The purpose of our present study was to investigate the precise antitumor mechanism action of P-3F. It showed that P-3F inhibited microtubule polymerization in a concentration-dependent manner. The results were in overall agreement with modeling and docking studies performed on P-3F and tubulin. In addition, P-3F increased the levels of P53, this in turn prolonged P53 half-life. Note as well that levels of P21 protein were increased along with P53 in a concentration dependent change. It suggested that enhancement in stabilization of P53 induced by P-3F may be critical for P53/P21 signaling pathway, resulting in cell cycle arrest at G2/M. Furthermore, release of RPS27a from the nucleolus into the nucleoplasm led to decrease phosphorylation of Mdm2 at serine residue 166 and inhibit Mdm2-mediated ubiquitination of P53 in (P-3F)-treated HeLa cells. Together, these data suggest that P-3F, a microtubule polymerization inhibitor, causes P53 accumulation via P53 stability enhancement, due to blockage of the P53-Mdm2 network through the change in localization of RPS27a.

Synthesis and evaluation of biological properties of ferrocenyl-podophyllotoxin conjugates.

Three types, esters, amides and 1,2,3-triazoles, of ferrocenyl-podophyllotoxin conjugates were synthesised, and their anticancer activity was evaluated. We observed that the most potent ferrocenyl derivatives were esters. Esters 15, 16 and 17 acted in a similar way to podophyllotoxin, i.e. reduced the number of G1 phase cells and induced G2/M blockage, while esters 14 and 18 and amide 19 blocked cells in S phase in a similar manner to etoposide.

In vitro polyploidy induction: changes in morphology, podophyllotoxin biosynthesis, and expression of the related genes in Linum album (Linaceae).

MAIN CONCLUSION: Induction of tetraploidy was performed and podophyllotoxin production increased by upregulating the expression level and enzyme activity of genes related to its biosynthesis in tetraploid compared to diploid Linum album. Linum album is a valuable medicinal plant that produces antiviral and anticancer compounds including podophyllotoxin (PTOX). To achieve homogeneous materials, in vitro diploid clones were established, and their nodal segments were exposed to different concentrations and durations of colchicine. This resulted in successful tetraploidy induction, confirmed by flow cytometry, and is being reported for the first time. The highest efficiency of tetraploid induction (22%) was achieved after 72 h exposure to 2.5-mM colchicine treatment. The stable tetraploids were produced after being subcultured three times, and their ploidy stability was confirmed after each subculture. The effects of autopolyploidy were measured on the morphological and phytochemical characteristics, as well as enzyme activity and the expression levels of some key genes involved in the PTOX biosynthetic pathway, including phenylalanine ammonia-lyase (PAL), cinnamoyl-Coa reductase (CCR), cinnamyl-alcohol dehydrogenase (CAD), and pinoresinol-lariciresinol reductase (PLR). The tetraploid plants had larger leaves and stomata (length and width) and lower density stomata. Increasing the ploidy level from diploid to tetraploid resulted in 1.39- and 1.23-fold enhancement of PTOX production, respectively, in the leaves and stem. The increase in PTOX content was associated with upregulated activities of some enzymes studied related to its biosynthetic pathway and the expression of the corresponding genes. The expression of the PAL gene and PLR enzymatic activity had the most positive correlation with the ploidy level in both leaf and stem tissues. Our results verified that autotetraploid induction is a useful breeding method, remarkably increasing the PTOX content in the leaves and stem of L. album.

Manipulation of pH-Sensitive interactions between podophyllotoxin-chitosan for enhanced controlled drug release.

Podophyllotoxin (PPT) offers a broad-spectrum of anticancer activities, but little has been reported for its controlled release. This work shows that by manipulating molecular interactions between PPT and Chitosan, efficient nanoscale capsulation of PPT can be realized. The drug encapsulation efficiency is as high as 52%, with a final particle drug loading in the order of 10% (wt/wt). It further demonstrates that changes in pH can also significantly affect the rate of drug release from the Chitosan nanoparticles. Upon contact with cancer cells, chitosan nanoparticles enable efficient internalization and drug release. In vitro evaluations with HepG-2 and MCF-7 cells indicate that the chitosan nanoparticle carriers can improve drug efficacy in comparison to free PPT, most likely by regulating the intrinsic apoptotic signaling pathway to induce apoptosis. Overall, PPT chitosan nanoparticles promise a safe and efficient drug delivery system for PPT.

Podophyllotoxin-Loaded Nanostructured Lipid Carriers for Skin Targeting: In Vitro and In Vivo Studies.

Nanostructured lipid carriers (NLC) exhibit high skin targeting efficiency and good safety. They are promising vehicles for topical drug delivery. This study aims to increase the skin distribution of podophyllotoxin (POD) by incorporating it into NLCs. Two kinds of POD-loaded NLCs (POD-NLCs)-POD-NLCformulation 1 and POD-NLCformulation 2-were prepared and characterized. Their skin targeting efficiencies were compared by conducting in vitro and in vivo experiments. Obviously smaller mean particle size was observed for POD-NLCformulation 1 (106 nm) than POD-NLCformulation 2 (219 nm), whereas relatively low POD loadings (less than 0.5%) were observed for both POD-NLCformulation 1 (0.33%) and POD-NLCformulation 2 (0.49%). Significantly higher in vitro and in vivo rat skin deposit amounts of POD (p ˂ 0.01) were detected after the topical application of POD-NLCformulation 1 compared to POD-NLCformulation 2. To visualize the skin distribution behavior of hydrophobic active pharmaceutical ingredients (APIs) when NLCs were used as carriers, POD was replaced with Nile red (NR-a hydrophobic fluorescent probe), and the distribution behavior of NR-NLCformulation 1 and NR-NLCformulation 2 in rat skin in vivo was observed using confocal laser scanning microscopy (CLSM). Higher fluorescent intensity was observed in rat skin after the topical application of NR-NLCformulation 1 than NR-NLCformulation 2, suggesting that higher skin targeting efficiency might be obtained when NLCs with smaller mean particle size were used as carriers for hydrophobic APIs. This result was in accordance with those of skin distribution evaluation experiments of POD-NLCs. Skin irritation property of POD-NLCformulation 1 was investigated and no irritation was observed in intact or damaged rabbit skin, suggesting it is safe for topical use. Our results validated the safety of NLCs when applied topically. More importantly, mean particle size might be an important parameter for formulation optimization when NLCs are used as carriers for hydrophobic APIs for topical application, considering that their loading is relatively low.

Interspecies differences in metabolism of deoxypodophyllotoxin in hepatic microsomes from human, monkey, rat, mouse and dog.

Deoxypodophyllotoxin (DPT) is a natural lignan product which has drawn much attention due to its pharmacological properties including antitumor effect. The purpose of this study was to investigate interspecies differences in metabolism of DPT in hepatic microsomes from human (HLM), cynomolgus monkey (CyLM), rat (RLM), mouse (MLM) and dog (DLM). Incubation of DPT with hepatic microsomes from five species in the presence of NADPH resulted in formation of seven metabolites, five of which were compared with the synthetic standards. M2 was the most abundant metabolite in microsomes from all species. Rank order of intrinsic clearance for M2 formation was RLM > CyLM > MLM > HLM > DLM. In HLM, sulfaphenazole showed the strongest inhibition effect on M2 formation, but neither ticlopidine nor ketoconazole inhibited M2 formation in HLM. Results from cDNA-expressed human CYP450s experiments showed that clearance of M2 formation was much higher in CYP2C9 and CYP2C19 than that in CYP3A4. Contributions of the three CYP450 isoforms to M2 formation in HLM were estimated using relative activity factor (RAF) method or correction by amount of CYP450 isoforms in HLM. M2 formation in HLM was mainly attributed to CYP2C9, followed by CYP2C19. Involvement of CYP3A4 was minor.

Endophytic Fungus from Sinopodophyllum emodi (Wall.) Ying that Produces Podophyllotoxin.

The aryltetralin lactone podophyllotoxin, which exhibits pronounced antineoplastic activity, is used as the precursor of the following three clinical anticancer drugs: Etoposide™, Etopophos™ and Teniposide™. The natural occurrence of this arylnaphthalene lignan is scarce and unable to meet the ever-rising demand in the medical industry. Thus, developing alternative sources for the production of podophyllotoxin is extremely urgent. This is the first report of the production of podophyllotoxin from endophytic Alternaria tenuissima isolated from Sinopodophyllum emodi (Wall.) Ying. The identification of podophyllotoxin was performed using high-performance liquid chromatography and liquid chromatography-mass spectrometry (MS)-MS and confirmed by comparison with authentic standards.