Production Of Podophyllotoxin Research Articles

Enhanced production of podophyllotoxins by co-culture of transformed Linum album cells with plant growth-promoting fungi

To investigate the plant growth-promoting effect and stimulation of lignan biosynthesis, the effect of culture filtrates/live co-culture of two arbuscular mycorrhizae-like fungi, Piriformospora indica and Sebacina vermifera, on growth of Linum album cells and on production of podophyllotoxin (PT) and 6-methoxypodophyllotoxin (6-MPT) was studied. For elicitation studies, different volumes of culture filtrates (autoclaved and membrane-filtered) of P. indica/S. vermifera were added to suspension cultures of L. album. The culture filtrates of both the fungi exhibited a positive effect on product formation. For co-culture experiments, both fungi were individually co-cultivated at different concentrations with L. album in suspension cultures for different time periods. This resulted in significant enhancement of PT and 6-MPT content in the plant cells. The activity of phenylalanine ammonia lyase (PAL) was observed to be related to the lignan accumulation, indicating its role as the key enzyme of the phenylpropanoid pathway. The study resulted in total lignan (PT and 6-MPT) production of 745.6 mg/l with a very high PT productivity of 52.4 mg/(l.d).

Lignan and Nutrient Concentrations in American Mayapple (Podophyllum peltatum L.) in the Eastern United States

Podophyllotoxin is an anticancer compound found in Indian mayapple (Podophyllum emodii Wall.), American mayapple (Podophyllum peltatum L.), and other species. Podophyllotoxin and its derivatives are used in several commercially available pharmaceutical products such as the anticancer drugs etoposide, teniposide, and etopophos. Currently, the commercial production of podophyllotoxin is based on Indian mayapple. The objective of this study was to estimate podophyllotoxin concentration in American mayapple across its natural habitats in the eastern United States and to identify high podophyllotoxin types that could be used for further selection and cultivar development. Analyses of American mayapple leaves collected from 37 mayapple colonies across 18 states indicated a significant variation in podophyllotoxin, α-peltatin, and β-peltatin content and the presence of chemotypes. Overall, the concentrations of podophyllotoxin, α-peltatin, and β-peltatin in the collected accessions ranged from below detectable levels to 45.1, 47.3, and 7.0 mg·g−1 dry weight, respectively. We classified American mayapple accessions into seven groups: 1) with very high concentration of podophyllotoxin (greater than 20 mg·g−1) and no α- or β-peltatin; 2) high podophyllotoxin (greater than 10 mg·g−1) and no α-peltatin but trace amounts of β−peltatin; 3) medium podophyllotoxin (1 to 10 mg·g−1) and no α- or β-peltatin; 4) low podophyllotoxin (0.05 to 1 mg·g−1) and high α-peltatin; 5) trace amounts of podophyllotoxin and high concentration of α-peltatin and α-peltatin; 6) high α-peltatin and trace amounts of podophyllotoxin or β−peltatin; and 7) high α−peltatin and no podophyllotoxin or β-peltatin. American mayapple was found to grow on various soil types with a range of pH (4.6 to 7.6) and dissimilar concentrations of phytoavailable soil nutrients. Tissue zinc concentration was positively correlated to podophyllotoxin, whereas soil and tissue phosphorus was positively correlated to the concentration of α-peltatin. The results from this study may contribute toward the development of high podophyllotoxin-containing varieties of American mayapple and the development of a new cash crop for American farmers.

Improved podophyllotoxin production by transformed cultures of Linum album

Various cell and hairy root cultures of L. album were developed and analyzed for podophyllotoxin content. Transformed callus and hairy root cultures developed from infection of stem portions of in vitro-germinated L. album plant with Agrobacterium rhizogenes NCIM 5140 strain were selected on the basis of high podophyllotoxin content and growth. Based on the integration of Ri T(L)-DNA and T(R)-DNA, integration of only the ags and not the rol gene in transformed cell culture indicated fragmented integration pattern. The effect of different cultivation media and carbon source on growth and podophyllotoxin production were studied in shake-flask suspension cultures. Detailed batch growth and production kinetics with sugar consumption profile were also established. Maximum volumetric productivity of 4.40 and 2.75 mg/L per day was obtained in cell suspension and hairy root cultures, respectively.

Podophyllotoxin production via cell and adventitious root cultures of Podophyllum peltatum

Podophyllum peltatum is an important medicinal plant that produces podophyllotoxin (PTOX) with anti- cancer properties. We established the embryogenic cell and adventitious root culture systems in P. peltatum and analyzed PTOX production. For the growth of embryogenic cell clumps in shake flask culture, the most efficient concentration of 2,4-dichloroacetic acid (2,4-D) was 6.78 μM, and the growth of embryogenic cell clumps was 15.9-fold increased in Murashige and Skoog MS liquid medium with 6.78 μM 2,4-D after 3 wk of culture. To induce adventitious roots, half-strength MS medium showed the best results for adventitious root induction compared to full strength MS medium and MS medium lacking NH4NO3. Optimal indole-3-butyric acid concentra- tion for adventitious root formation was 14.78 μM. In liquid medium, the frequency of adventitious root forma- tion from root segments was 87.7% and the number of laterally formed adventitious roots was 22.3 per segment. PTOX production in embryogenic cells and adventitious roots was confirmed by liquid chromatography and electro- spray ionization-tandem mass spectrometry analysis. High- performance liquid chromatography analysis revealed that adventitious roots contained higher PTOX than embryo- genic cell clumps. Elicitor treatment (20 μM methyl jasmonate) strongly enhanced the production of PTOX in both embryogenic cell clumps and adventitious roots. This observation suggests that both embryogenic cell and adventitious root culture can be adopted to produce PTOX.

Bioconversion of deoxypodophyllotoxin into epipodophyllotoxin in E. coli using human cytochrome P450 3A4

Biotransformation of deoxypodophyllotoxin to epipodophyllotoxin (ePTOX) by three major human hepatic enzymes, CYP1A2, CYP2C9 and CYP3A4, heterologously expressed in E. coli DH5α, was investigated. It was shown that CYP3A4 catalysed the hydroxylation of deoxypodophyllotoxin into epipodo-phyllotoxin in yields up to 90%. The structure of the metabolite was determined using HPLC-MS and HPLC-SPE-NMR techniques [1]. There was no detectable production of epipodophyllotoxin or podophyllotoxin by CYP1A2 and CYP2C9 enzymes. The CYP3A4 enzyme shows a distinctly different reactivity to deoxypodophyllotoxin compared to the semisynthetic derivatives etoposide and teniposide, which are degraded by 3-O-demethylation. These findings demonstrate a novel system for the production of 2,7'-cyclolignans, starting from the easily accessible deoxypodophyllotoxin. Further kinetic analysis revealed that the Michaelis–Menten Km and Vmax for hydroxylation of deoxypodophyllotoxin by CYP3A4at C-7 position were 1.93µM and 1.48 nmol/min/nmol, respectively. Deoxypodophyllotoxin was subjected to automated docking analysis in order to get better knowledge of the interaction between the CYP 3A4 enzyme and the substrate, using the PatchDock algorithm with distance constraints. Automated docking showed that the β-hydrogen atom at C-7 position is in the most appropriate binding orientation at the site of oxidation. The docking results are consistent with the experimental data for the bioconversion of deoxypodophyllotoxin into epipodophyllotoxin by CYP 3A4 [2]. In addition, the effects of five lignans, deoxypodophyllotoxin, epipodophyllotoxin, podophyllotoxin, demethylenedeoxyodophyllotoxin, and demethylenepodo-phyllotoxin, on CYP3A4 were compared in order to investigate the influence of the methylenedioxy group on the biotransformation process. The docking method hypothesized inhibition by epipodophyllotoxin when the methylenedioxy moiety is involved and the substrate is oriented towards the iron atom of the heme group of CYP 3A4.

Effect of Aeration on Production of Anticancer Lignans by Cell Suspension Cultures of Linum album

The effects of aeration within the range of 0.2-0.5 vvm on transformed and high yielding cell cultures of Linum album were investigated in a 5-L stirred tank bioreactor equipped with low shear Setric impeller. The kinetics of cell growth, substrate utilization, and production of lignans, namely, podophyllotoxin and 6-methoxypodophyllotoxin, were established. Maximum biomass of 23.2 g/L and lignan accumulation levels of 176.3 mg/L podophyllotoxin and 10.86 mg/L 6-methoxypodophyllotoxin were obtained with initial air flow rate of 0.3 vvm. Specified oxygen demand of cells was estimated to be 1.35 g O(2)/g biomass. The optimum oxygen transfer coefficient was found to be 16.7 h(-1) (,) which corresponded to aeration rate of 0.3 vvm. The effect of minimum dissolved oxygen (DO) concentration was investigated with respect to biomass and lignan production by comparing identically aerated and agitated bioreactor cultivations at dissolved oxygen concentrations of 10%, 30%, and 50%. Cell growth and podophyllotoxin accumulation were not affected significantly at these DO levels, but 6-methoxypodophyllotoxin production was enhanced when cells were cultivated at 30% DO level. The maximum volumetric productivities of 18.2 mg/L day and 3.2 mg/L day for podophyllotoxin and 6-methoxypodophyllotoxin, respectively, were obtained. These results establish the key role of oxygen on mass scale production of anticancer lignans by cell cultures of L. album. It may serve as a suitable parameter for scale-up.

Characterization of genetic structure of Podophyllum hexandrum populations, an endangered medicinal herb of Northwestern Himalaya, using ISSR-PCR markers and its relatedness with podophyllotoxin content

To obtain accurate estimates of genetic structure for purpose of conservation planning for wild Indian May apple (Podophyllum hexandrum) in the Northwestern region of Himalayas, Himachal Pradesh, genetic diversity among and within 28 populations were analyzed. Eleven microsatellite DNA markers were isolated and used to quantify genetic structure. Out of 68 ISSR loci tested, 88.23% were polymorphic. The genetic diversity was high (percentage of polymorphic bands, PBB = 83.82%; Shannon’s information index, I = 0.4413) at the population level, but low within individual study populations (PBB = 34.22%; Shannon’s information index I = 0.1879). The mean coefficient of gene differentiation (Gst) was 0.7484, indicating that 29.44% of the genetic diversity resided within the population. Analysis of molecular variance (AMOVA) indicated that 48% of the genetic diversity among the study populations was attributed to geographical location while 29% was attributed to differences in their habitats. An overall value of mean estimated number of gene flow (Nm = 0.1618) indicated that there was limited gene flow among the sampled populations. We found a clear tendency for higher Gst values and podophyllotoxin levels between the populations with increasing geographical altitude. However, the existing variation in podophyllotoxin content among the populations was proved to be coupled with geographical altitude (r = 0.922) but not with genetic variations (r = - 0.273). Hence we recommend that any further cultivation of this species requires optimization of environmental factors in order to increase the rate of production of podophyllotoxin from any collected population.

Production of lignans in callus culture of <i>Podophyllum hexandrum</i>

Purpose: Podophyllum hexandrum Royle, a source of highly valued podophyllotoxin has been subjected to heavy collection from the wild. The ever-increasing demand of podophyllum is mainly due to two semi synthetic derivatives of podophyllotoxin that is etoposide and teniposide, which are used in the treatment of various types of cancer. The anti cancer lignan derivative podophyllotoxin in Podophyllum hexandrum is biosynthesized at very low quantities in intact plant, so the biotechnological production of podophyllotoxin has been considered essential. Method: The aseptically germinated embryos of Podophyllum hexandrum were developed on solid nutrient agar slab. For the growth of callus culture, Murashigae and Skoog media (MS media)) with various concentrations of BAP, NAA and GA3 adjusted to pH 5.8 was used. Podophyllotoxin content in the alcoholic extract of calli and plant root was analysed by HPLC and HPTLC and was also compared with cultivated Podophllum hexandrum root extracts. Result: A fully defined MS medium supplemented with Naphthalene acetic acid and 6benzylaminopurine (BAP) were effective for both initiation and sustained growth of callus tissue. The relative proportion of callus was markedly influenced by presence of plant growth regulators. The amount of Podophyllotoxin obtained from callus was 0.78 and 0.79 percent as characterized by HPLC and HPTLC respectively. Conclusion: The study revealed that callus culture may be a fruitful tool for the production of Podophyllotoxin resin, an anticancer entity.

Isolation and identification of an endophytic strain of Fusarium oxysporum producing podophyllotoxin from Juniperus recurva

Podophyllotoxin, a well-known naturally occurring aryltetralin lignan occurs in few plant species that is used as a precursor for the chemical synthesis of the anticancer drugs like etoposide, teniposide and etopophose phosphate. The availiability of this lignan is becoming increasingly limited because of the scarce occurance of its natural sources and also because synthetic approaches for its production are still commercially unacceptable. This paper reports first time the production of podophyllotoxin by an endophytic fungus Fusarium oxysporum isolated from the medicinal plant Juniperus recurva. Further confirmation and quantification of podophyllotoxin was performed by HPLC, LC-MS, and LC-MS/MS.

Lignans from Dysosma versipellis with Inhibitory Effects on Prostate Cancer Cell Lines

A new monoepoxylignan, dysosmarol (1), along with eight known compounds, podophyllotoxin (2), 4'-demethylpodophyllotoxin (3), deoxypodophyllotoxin (4), 4'-demethyldeoxypodophyllotoxin (5), diphyllin (6), kaempferol, quercetin, and beta-sitosterol, were isolated from the roots of Dysosma versipellis. The structure of 1 was elucidated by spectroscopic methods. Aryltetralin lignans 2-4 showed the most potent inhibitory activities against the growth of androgen-sensitive (LNCaP) and androgen-independent (PC-3) human prostate cancer cell lines, with IC50 values in the ranges 0.030-0.056 and 0.032-0.082 microM, respectively. A quantitative HPLC analysis showed that compound 2 occurred at the highest concentration in the plant (37.21 mg/g) followed by compound 4 (5.01 mg/g) and compound 3 (2.75 mg/g). Furthermore, D. versipellis roots contain a similar content of compound 2 as compared with the rhizomes and roots of Podophyllum hexandrum, a commercial source of the lignan. Thus, cultivation of D. versipellis in suitable locations may serve as an alternative source for podophyllotoxin (2) production.

Bioconversion of deoxypodophyllotoxin into epipodophyllotoxin in E. coli using human cytochrome P450 3A4

Biotransformation of deoxypodophyllotoxin to epipodophyllotoxin by three major human hepatic enzymes, CYP1A2, CYP2C9 and CYP3A4, heterologously expressed in E. coli DH5alpha, was investigated. It was shown that CYP3A4 catalysed the hydroxylation of deoxypodophyllotoxin into epipodophyllotoxin in yields up to 90%. The structure of the metabolite was determined using HPLC-MS and HPLC-SPE-NMR techniques. There was no detectable production of epipodophyllotoxin or podophyllotoxin by CYP1A2 and CYP2C9 enzymes. The CYP3A4 enzyme shows a distinctly different reactivity to deoxypodophyllotoxin compared to the semi-synthetic derivatives etoposide and teniposide, which are degraded by 3-O-demethylation. These findings demonstrate a novel system for the production of 2,7'-cyclolignans, starting from the easily accessible deoxypodophyllotoxin.

Post-harvest and scale-up extraction of American mayapple leaves for podophyllotoxin production

Leaves of American mayapple ( Podophyllum peltatum L. ) are of interest to the pharmaceutical industry as an alternative source of podophyllotoxin, an aryltetralin lignan that is the precursor of widely used anticancer drugs etoposide, teniposide, and etopophos. In this study, the effects of post-harvest handling were evaluated by inflicting physical damage to leaves of mayapple to simulate rough handling. The effects of storage conditions before and after being dried were also evaluated. In addition, techniques for conducting large-scale extractions of podophyllotoxin from bulk samples were investigated. Crushing injury, damaging the leaves in more than 70% of its area, has improved podophyllotoxin content of leaves when dried at 40 °C within 24 h of harvest. In contrast, podophyllotoxin content was greatly reduced when the leaves were dried at room temperature at 15% relative humidity and 24 °C. Podophyllotoxin was stable with no significant changes over time when the leaves were dried, ground, and stored under different conditions for up to 60 days. Based on these findings, mayapple leaves do not require careful handling at harvest. In fact, leaves can be handled in a manner consistent with mechanical injury as long as leaves are dried at 40 °C within 24 h. Leaves can then be stored for up to 60 days, and probably much longer, when dry. If leaves cannot be dried in a timely manner, they can be stored at 4 °C for up to 4 weeks without significant loss of podophyllotoxin.

In Vitro.–Enhanced Production of Podophyllotoxin in Phytohormonal-Induced and Regenerated Roots of Podophyllum hexandrum.

ABSTRACTSixty-day-old in vitro–induced roots and roots regenerated from totipotent calli of Podophyllum hexandrum Royle produced an enhanced quantity of podophyllotoxin. Induced root cultures originated from aseptically germinated seedlings, and regenerated roots were established on both Gamborg's B5 and Murashige and Skoog media. The podophyllotoxin content was compared with normal plant root/rhizomes by HPLC. The highest values, both in growth/proliferation and in podophyllotoxin production, were obtained using Gamborg's B5 medium.

Biotechnological aspects of the production of the anticancer drug podophyllotoxin

The natural lignan podophyllotoxin, a dimerized product of two phenylpropanoid moieties which occurs in a few plant species, is a pharmacologically important compound for its anticancer activities. It is used as a precursor for the chemical synthesis of the anticancer drugs etoposide, teniposide and etopophose. The availability of this lignan is becoming increasingly limited because of the scarce occurrence of its natural sources and also because synthetic approaches for its production are still commercially unacceptable. Biotechnological production using cell culture may be considered as an alternative source. Selection of the best performing cell line, its maintenance and stabilization are necessary prerequisites for its production in bioreactors and subsequent scale-up of the cultivation process to the industrial level. Scale-up of growth and product yield depends on a multitude of factors, such as growth medium, physicochemical conditions, seed inoculum, type of reactor and processing conditions. The composition of the growth medium, elicitors and precursors, etc. can markedly influence the production. Optimum levels of parameters that facilitate high growth and product response in cell suspensions of Podophyllum hexandrum have already been determined by statistical design. P. hexandrum cells have successfully been cultivated in a 3-l stirred-tank bioreactor under low shear conditions in batch and fed-batch modes of operation. The batch kinetic data were used to identify the mathematical model which was then used to develop nutrient-feeding strategies for fed-batch cultivation to prolong the productive log phase of cultivation. An improvement in the production of podophyllotoxin to 48.8 mg l(-1) in a cell culture of P. hexandrum was achieved, with a corresponding volumetric productivity of 0.80 mg l(-1) day(-1), when the reactor was operated in continuous cell-retention mode. Efforts are being made to further enhance its production levels by the development of hairy root culture or by varying the channeling of precursors towards the desired biosynthetic pathway by molecular approaches.

Production of Podophyllotoxin by Immobilized Cell Cultures of Juniperus chinensis

Production of Podophyllotoxin by Immobilized Cell Cultures of Juniperus chinensis

Research progress on production of podophyllotoxin from Sinopodophyllum hexandrum

Podophyllotoxin is isolated mainly from the rhizomes of Podophyllum plants, and serves as the main precursor for synthesis of anticancer drugs, such as VP-16 and VM-26. VP-16 and VM-26 are used for curing lung cancer, testicular cancer, neuroblastoma, hepatoma and other tumors. However, these plants are all near-extinction species due to over-collection and their own biological characteristics. The chemical synthesis of podophyllotoxin is so complicated that its price is unbelievably high. This paper discusses the current status of the biosynthetic pathway of podophyllotoxin and that of the podophyllotoxin production using several biotechnological approaches such as plant organ cultures, plant cell cultures with both flasks and bioreactors, hairy root cultures, bioconversions and metabolic regulations.

Enhanced Production of Podophyllotoxin by Fed‐batch Cultivation of Podophyllum hexandrum

AbstractPodophyllum hexandrum (Indian May Apple) was successfully cultivated in a 3 L stirred tank bioreactor under low shear conditions in batch and fed‐batch modes of operation. A statistically optimized culture medium was used for the batch cultivation of Podophyllum hexandrum. Under optimum culture conditions of P. hexandrum, the batch culture showed a growth‐associated product formation with a maximum biomass of 21.4 g/L dry cell weight (DCW) basis and a podophyllotoxin production of 13.8 mg/L in 26 d. A mathematical model was developed to design the nutrient feeding strategies for a fed‐batch cultivation to prolong the productive log phase of cultivation. The fed‐batch cultivation was able to enhance the biomass and podophyllotoxin accumulation to 48 g/L (DCW basis) and 43.2 mg/L, respectively, in 60 d. The volumetric productivity of podophyllotoxin in fed‐batch cultivation was found to be 0.72 mg/(L. d) as opposed to 0.53 mg/(L. d) in batch cultivation under optimized culture conditions.

Effect of major nutrients on podophyllotoxin production in Podophyllum hexandrum suspension cultures.

The effect of major medium ingredients (sugar, nitrogen source and phosphate) in Podophyllum hexandrum suspension cultures was investigated in order to increase the production of podophyllotoxin, the raw material in the synthesis of anticancer drugs. Amongst B5, Eriksson, MS, Nitsch, Street and White's medium, MS medium resulted in high growth and podophyllotoxin accumulation. The optimum level of nitrogen was found to be 60 mM, with a combination of ammonium salts and nitrate in the ratio of 1:2. The highest level of podophyllotoxin was obtained at 60 g glucose/l and at 1.25 mM phosphate after 30 days. Statistical design was adopted to determine the optimum levels of the parameters for cell growth and podophyllotoxin production.

Enhancement of Podophyllotoxin Production by Biogenetic Precursors and Elicitors in Cell Suspension Cultures of Juniperus chinensis

Enhancement of Podophyllotoxin Production by Biogenetic Precursors and Elicitors in Cell Suspension Cultures of Juniperus chinensis

Stimulation of the Production of Podophyllotoxin by Biogenetic Precursors and an Elicitor in Juniperus chinensis Stem-derived Callus Cultures

Stimulation of the Production of Podophyllotoxin by Biogenetic Precursors and an Elicitor in Juniperus chinensis Stem-derived Callus Cultures