Accumulation of podophyllotoxin and related lignans in cell suspension cultures of linum album (2023)

Table of Contents
Phytochemistry Abstract Introduction Section snippets Results and discussion Experimental Acknowledgements Phytochemistry J. Pharm. Sci. Progr. Drug. Res. Nature Can. J. Bot. Naturwissenschaften Plant Cell Rep. Plant Cell Rep. Plant Cell J. Nat. Prod. Effect of in vitro morphogenesis on the production of podophyllotoxin derivatives in callus cultures of Linum album Kinetics of glucosylated and non-glucosylated aryltetralin lignans in Linum Hairy root cultures Reduced coniferin and enhanced 6-methoxypodophyllotoxin production in Linum flavum cell cultures Structural analysis of coniferyl alcohol 9-O-methyltransferase from Linum nodiflorum reveals a novel active-site environment Effect of ploidy level on podophyllotoxin content and expression of genes related to its biosynthesis in callus cultures of Linum album Production of bioactive plant secondary metabolites through in vitro technologies—status and outlook Protein release from electrospun nonwovens: Improving the release characteristics through rational combination of polyester blend matrices with polidocanol HR-MAS NMR reveals a pH-dependent LPS alteration by de-O-acetylation at abequose in the O-antigen of Salmonella enterica serovar Typhimurium Effects of cutting orientation in poplar wood biomass size reduction on enzymatic hydrolysis sugar yield Triterpenoid saponins from the roots of Clematis argentilucida Binding of the plant alkaloid aristololactam-β-d-glucoside and antitumor antibiotic daunomycin to single stranded polyribonucleotides Reply to “Requirements for accurate 1H NMR quantification of mineral oil hydrocarbons (paraffins) for pharmaceutical or cosmetic use”

Phytochemistry

Volume 48, Issue 6,

24 July 1998

, Pages 975-979

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https://doi.org/10.1016/S0031-9422(97)00957-6Get rights and content

Abstract

Cell suspension cultures of Linum album were established, which were able to synthesize and accumulate lignans. Podophyllotoxin and 5-methoxypodophyllotoxin were the main products and were present as glycosides, together with small amounts of deoxypodophyllotoxin, 5′-demethoxy-5-methoxypodophyllotoxin, lariciresinol, pinoresinol, matairesinol, α- and β-peltatin, as well as the monolignol glucoside, coniferin. In dark and light grown cultures, maximal product yields of lignans of up to 0.2% and 0.5% of the dry weight, respectively, were achieved, mainly consisting of podophyllotoxin.

Introduction

Lignans, long known as natural products, are distributed widely in the plant kingdom. Several hundreds of compounds in this general class have been identified, which are derived from two phenylpropanoid units. From a medicinal point of view, the most important compounds today are etoposide and teniposide, semisynthetic derivatives of podophyllotoxin (Fig. 1), which are used in cancer chemotherapy. Total chemical synthesis of both compounds is possible, but is not an economic proposition. Therefore, podophyllotoxin (Ptox) still serves as the starting material for semisynthesis of both compounds [1]. It is extracted from rhizomes of Podophyllum hexandrum and P. peltatum, which contain ca 4 and 0.2% of Ptox, respectively, calculated on a dry basis [2]. There is an increasing demand for podophyllotoxin due to its medicinal importance. However, a sufficient supply of Podophyllum plants is rather limited, since the occurrence of these plant species is scarce and the plants need a growth period of 5 to 7 years, before harvesting of the rhizomes is convenient. Podophyllum peltatum, moreover, has poor reproduction capacities [3]. Podophyllum hexandrum, which is the species in most demand, originates from Himalaya. There it even became a threatened species due to the intensive collection of this plant. Nowadays, additional sources for podophyllotoxin and related lignans in other plant species (for a review, see [4]) are identified and in vitro propagation methods for Podophyllum species 5, 6, 24, 25are being established. Attempts to produce podophyllotoxin by cell cultures of P. hexandrum and Callitris drummondii have been reported 7, 8, 9, 10, but the yields obtained were low and the cultures grew only slowly. Cell and root cultures of Linum flavum grow much better, but these cultures accumulate only 5-methoxypodophyllotoxin (5-Mptox) in substantial amounts 11, 12, 13, 14, 15. In 1975, Weiss et al. [16]reported that plants of L. album contained podophyllotoxin besides some other lignans.

Recently, we initiated callus and cell suspension cultures of this species, which accumulate podophyllotoxin besides small amounts of 5-Mptox, as well as traces of some other lignans [17]. Herein we describe more detailed experiments about the accumulation of Ptox, 5-Mptox and a possible biosynthetic precursor, coniferin, during the culture cycle of cell suspension cultures of Linum album grown in the dark and light.

Section snippets

Results and discussion

Shoot cultures were established from seeds, which sometimes needed several weeks to germinate. From single seedlings we established calli and, subsequently, suspension cultures (lines 2–1, 2–3 and 2–8). About two years after establishment of the cultures in a preliminary screening, the medium [11]was modified using as single growth hormone 0.4 mg l−1 and 0.8 mg l−1 NAA, respectively, and the cells investigated for the presence of lignans and monolignols after a subculture time of 1–3 weeks. The

Experimental

Seeds of L. album Kotschy were collected in 1988 near Teheran and germinated under sterile conditions on hormone-free MS-medium [23]at 25° in continuous light (50 μE m−2 s−1 photosynthetic active radiation, Philips 18 W fluorescent tubes). Shoot cultures from single seedlings were established on the same medium. These were used to initiate calli and, subsequently, cell suspension cultures in MS-medium with various amounts of auxins. MS-medium with 0.4 mg l−1 NAA proved to be best for good

Acknowledgements

Financial support by the Fonds der Chemischen Industrie (Frankfurt) is gratefully acknowledged. We thank Drs Buitelaar (Zeist), Heller (Munich), Lewis (Pullman), McChesney (Boulder) and van Uden for kind gifts of authentic lignans.

  • Uden W. van et al.

    Plant Cell

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    J. Nat. Prod.

    (1986)

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

      2018, Journal of Plant Physiology

      Citation Excerpt :

      Federolf et al. (2007) established a L. album cell line that mainly produced MPTOX and another, obtained from the same plant, whose main lignan was PTOX at the peak of production (8–16 days of culture). Smollny et al. (1998) found several lignans, predominantly PTOX, in L. album cell cultures. Another example of cells, calli or root cultures forming different lignans has been reported by Wawrosch et al. (2014), who found the pharmacologically active lignans leoligin and 5-methoxy-leoligin in Edelweiss hairy roots, mainly after elicitation.

      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.

    • Kinetics of glucosylated and non-glucosylated aryltetralin lignans in Linum Hairy root cultures

      2015, Phytochemistry

      Citation Excerpt :

      Berlin et al. (1986) studied the production of ATL in isolated roots of L. flavum. Smollny et al. (1998) described the accumulation of PTOX in L. album cell suspensions and showed that they could produce 0.2–0.5% of ATLs. Similarly, Petersen and Alfermann (2001) described the kinetics of PTOX in cell cultures of L. album and Chashmi et al. (2013) detailed the kinetics of PTOX and MPTOX in HRCs of L. album.

      Due to their pronounced cytotoxic activity, a number of aryltetralin lignans (ATLs), such as podophyllotoxin (PTOX), are used as antitumor compounds. The production of such molecules from entire plants or plant cell-tissue-organ cultures is thus of interest to the pharmaceutical industry. Hairy root cultures constitute a good tool not only for phytochemical production but also for investigating plant secondary metabolism. This work reports on the growth and ATL biosynthesis in two hairy root cultures of Linum album Kotschy ex Boiss. and Linum flavum. The kinetics of accumulation of the intermediates of MPTOX biosynthesis and of their glucosylated forms are described over a 21-day period of growth. An accumulation of non-glucosylated forms of the ATLs during the exponential phase of the cultures is followed by an accumulation of the glucosylated forms during the stationary phase. Our results show a strong coordination of the biosynthetic paths derived from deoxypodophyllotoxin via deoxypodophyllotoxin 6-hydroxylase and deoxypodophyllotoxin 7-hydroxylase, and a coordinated glucosylation of podophyllotoxin, methoxypodophyllotoxin, and 5′-demethoxymethoxypodophyllotoxin. Furthermore, our results suggest an important role of β-peltatin-6-glucoside formation in the control of ATL accumulation in Linum hairy root cultures.

    • Reduced coniferin and enhanced 6-methoxypodophyllotoxin production in Linum flavum cell cultures

      2010, Pharmacognosy Journal

      Treatment of cell suspension cultures of Linum flavum L. with Na2EDTA reduced the coniferin and enhanced the 6-methoxypodophyllotoxin (6-MPT) production in a concentration-dependent way, in a range of 0.1–5 mM. On day 14 after treatment with Na2EDTA , an inhibition of the coniferin production up to 88% was found. The maximum enhancement of the 6-MPT production was 400% on day 7 after treatment with 5 mM Na2EDTA . The reduction in coniferin accumulation in the suspension cultures correlated with and inhibition of coniferyl alcohol glucosyltransferase (CAGT ) activity as determined in cell homogenates. On day 14 after treatment with 2 and 5 mM Na2EDTA , the CAGT activity was inhibited up to > 89 %. The inhibitory effect of Na2EDTA on CAGT was also shown in a partially purified enzyme preparation. Several metal ions and the elicitors nigeran and salicylic acid had no significant effect on the production of coniferin and 6-MPT.

    • Structural analysis of coniferyl alcohol 9-O-methyltransferase from Linum nodiflorum reveals a novel active-site environment

      2013, Acta Crystallographica Section D: Biological Crystallography

    • Effect of ploidy level on podophyllotoxin content and expression of genes related to its biosynthesis in callus cultures of Linum album

      2022, Biologia

    • Production of bioactive plant secondary metabolites through in vitro technologies—status and outlook

      2021, Applied Microbiology and Biotechnology

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      Protein release from electrospun nonwovens: Improving the release characteristics through rational combination of polyester blend matrices with polidocanol

      International Journal of Pharmaceutics, Volume 477, Issues 1–2, 2014, pp. 273-281

      Nonwoven scaffolds consisting of poly-ε-caprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA) and polidocanol (PD), and loaded with lysozyme crystals were prepared by electrospinning. The composition of the matrix was varied and the effect of PD content in binary mixtures, and of PD and PLGA content in ternary mixtures regarding processability, fiber morphology, water sorption, swelling and drug release was investigated. Binary PCL/PD blend nonwovens showed a PD-dependent increase in swelling of up to 30% and of lysozyme burst release of up to 45% associated with changes of the fiber morphology. Furthermore, addition of free PD to the release medium resulted in a significant increase of lysozyme burst release from pure PCL nonwovens from approximately 2–35%. Using ternary PCL/PD/PLGA blends, matrix degradation could be significantly improved over PCL/PD blends, resulting in a biphasic release of lysozyme with constant release over 9 weeks, followed by constant release with a reduced rate over additional 4 weeks. Based on these results, protein release from PCL scaffolds is improved by blending with PD due to improved lysozyme desorption from the polymer surface and PD-dependent matrix swelling.

    • Research article

      HR-MAS NMR reveals a pH-dependent LPS alteration by de-O-acetylation at abequose in the O-antigen of Salmonella enterica serovar Typhimurium

      Carbohydrate Research, Volume 382, 2013, pp. 58-64

      NMR spectroscopy can detect biomolecules like lipopolysaccharide directly on the surface of the cell, thus avoiding isolation and purification, and providing a more realistic description than the one derived from in vitro studies. Here we present a high-resolution magic-angle spinning NMR study of the O-antigen of Salmonella enterica serovar Typhimurium (S. Typhimurium) performed directly on the cells showing the alteration of its acetylation state over time. The O-antigen region of S. Typhimurium consists of the repeating unit [→2)-α-d-Manp-(1→4)-α-l-Rhap-(1→3)-α-d-Galp-(1→] where Man stands for mannose, Rha for rhamnose, and Gal for galactose. Man is substituted with abequose (Abe) O-acetylated at carbon 2. Our studies revealed that the appearance of de-O-acetylated O-antigen in the stationary growth phase is due to the de-O-acetylation of already synthesized O-acetylated O-antigen and that this reaction is caused by the metabolism-induced basic pH of the growth medium. The labile O-acetylation of the O-antigen we observed in S. Typhimurium generates non-stoichiometric O-acetylation states and therefore changes the nature of an immunogenic epitope.

    • Research article

      Effects of cutting orientation in poplar wood biomass size reduction on enzymatic hydrolysis sugar yield

      Bioresource Technology, Volume 194, 2015, pp. 407-410

      The aim of this study was to understand how cutting orientation in poplar wood biomass size reduction affects enzymatic hydrolysis sugar yield of wood particles. A metal cutting (milling) machine was used to produce poplar wood particles from three cutting orientations. Results showed that cutting orientation significantly affected enzymatic hydrolysis sugar yield of wood particles. In this study, size reduction from the optimum cutting orientation produced 50% more sugars than the other two cutting orientations. Particles from the cutting orientation with the highest sugar yield had a large enzyme accessible area (125mg orange dye/g biomass, as evaluated by Simons’ stain procedure) and low crystallinity (50% crystallinity index, as calculated by the Segal method). Furthermore, small particle size did not necessarily lead to improvement in enzymatic digestibility.

    • Research article

      Triterpenoid saponins from the roots of Clematis argentilucida

      Fitoterapia, Volume 97, 2014, pp. 234-240

      Reinvestigation of the n-BuOH extract of the roots of Clematis argentilucida led to the isolation of a new ursane-type triterpenoid saponin 1 and a new taraxerane-type saponin 2, four known saponins 36 first isolated from the species, together with seven saponins 713 reported in the previous papers. The structures of saponins 16 were elucidated by extensive spectroscopic analysis and chemical evidences. The ursane-type and taraxerane-type triterpenoid saponins were obtained from genus Clematis for the first time, and the aglycone of saponin 1, 3β,28-dihydroxy-18αH-ursan-20-en was first encountered. The cytotoxicity of all the saponins was evaluated against human glioblastoma U251MG cell lines. The monodesmosidic saponins 1, 2 and 48 exhibited cytotoxic activity against the cells with IC50 values ranging from 6.95 to 38.51μM.

    • Research article

      Binding of the plant alkaloid aristololactam-β-d-glucoside and antitumor antibiotic daunomycin to single stranded polyribonucleotides

      Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1830, Issue 10, 2013, pp. 4708-4718

      Interaction of the plant alkaloid aristololactam-β-d-glucoside and the antitumor drug daunomycin with single stranded RNAs poly(G), poly(I), poly(C) and poly(U) has been investigated.

      Biophysical techniques of absorption, fluorescence, competition dialysis, circular dichroism, and microcalorimetry have been used.

      Absorption and fluorescence studies have revealed noncooperative binding of ADG and DAN to the single stranded RNAs. The binding affinity of ADG varied as poly(G) > poly(I) > > poly(C) > poly(U). The affinity of DAN was one order higher than that of ADG and varied as poly(G) > poly(I) > poly(U) > poly(C). This binding preference was further confirmed by competition dialysis assay. The thermodynamics of the binding was characterised to be favourable entropy and enthalpic terms but their contributions were different for different systems. The major non-polyelectrolytic contribution to the binding revealed from salt dependent data appears to be arising mostly from stacking of DAN and ADG molecules with the bases leading to partial intercalation to single stranded RNA structures. Small negative heat capacity values have been observed in all the four cases.

      This study presents the comparative structural and thermodynamic profiles of the binding of aristololactam-β-d-glucoside and daunomycin to single stranded polyribonucleotides.

      These results suggest strong, specific but differential binding of these drug molecules to the single stranded RNAs and highlight the role of their structural differences in the interaction profile.

    • Research article

      Reply to “Requirements for accurate 1H NMR quantification of mineral oil hydrocarbons (paraffins) for pharmaceutical or cosmetic use”

      Journal of Pharmaceutical and Biomedical Analysis, Volume 171, 2019, pp. 235-237

    fn2

    Present address: Philipps-Universität Marburg, Institut für Pharmezentische Biologie, Deutschhausstr. 17 1\2, D-35037, Marburg, Germany.

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