The production of podophyllotoxin and its 5-methoxy derivative through bioconversion of cyclodextrin-complexed desoxypodophyllotoxin by plant cell cultures

The bioconversion of the lignan desoxypodophyllotoxin by cell suspensions of Linum flavum and of Podophyllum hexandrum was investigated. The apolar substrate could be easily dissolved in the culture medium at a concentration of 2 mM by complexation with dimethyl--cyclodextrin. Growth parameters of the cell suspensions were not affected by either the addition of cyclodextrin itself, or when cyclodextrin-complexed desoxypodophyllotoxin was present in the medium. The complexed lignan disappeared from the medium within 7 days for both cell cultures. Cellularly only small amounts of desoxypodophyllotoxin were found. After feeding of desoxypodophyllotoxin, the cell culture of L. flavum accumulated 5-methoxypodophyllotoxin and 5-methoxypodophyllotoxin--D-glucoside. After 7 days a total maximal content of 2.38% on a dry weight basis of 5-methoxypodophyllotoxin was formed, corresponding with 249 mg l^-1 suspension. The highest bioconversion percentage of 52.3% was found at day 14. The desoxypodophyllotoxin-fed culture of P. hexandrum accumulated podophyllotoxin and its -D-glucoside with a maximal content of 2.87% on a dry weight basis after 9 days, corresponding with 192 mg 1^-1 suspension. The highest bioconversion percentage of 33.2% was also found at day 9.     

Increased podophyllotoxin production in Podophyllum hexandrum cell suspension cultures after feeding coniferyl alcohol as a β-cyclodextrin complex

Summary Cell suspension cultures, derived from roots of Podophyllum hexandrum Royle (Berberidaceae), accumulate podophyllotoxin. In this study the use of -cyclodextrin in feeding the poorly water-soluble precursor coniferyl alcohol to these cultures is described. By complexation with -cyclodextrin, a solution of 3 mM coniferyl alcohol could be fed, resulting in enhanced podophyllotoxin accumulation. The same concentration of non-complexed suspended coniferyl alcohol had only little effect on the podophyllotoxin accumulation. -Cyclodextrin itself was proven to be non-toxic for the cells. It did not influence the podophyllotoxin content and it was not metabolized or used as a carbon source by the cells. For comparison, coniferin, the water-soluble -D-glucoside of coniferyl alcohol, was also fed in the same concentration. The effect of coniferin on the podophyllotoxin accumulation was stronger than that of coniferyl alcohol complexed with -cyclodextrin, but coniferin is not commercially available.Abbreviations -CD -cyclodextrin - NAA naphthaleneacetic acid     

Glucosylation of cyclodextrin-complexed podophyllotoxin by cell cultures of Linum flavum L.

The glucosylation of the cytotoxic lignan podophyllotoxin by cell cultures derived from Linum flavum was investigated. Four cyclodextrins: -cyclodextrin, -cyclodextrin, dimethyl--cyclodextrin and hydroxypropyl--cyclodextrin were used to improve the solubility of podophyllotoxin by complexation. Dimethyl--cyclodextrin met our needs the best and the solubility of podophyllotoxin could be enhanced from 0.15 to 1.92 mM, using a podophyllotoxin/cyclodextrin ratio of 1:1. Growth parameters of the cell suspensions were not affected neither by the addition of cyclodextrins alone, nor when complexed podophyllotoxin was dissolved in the medium.The complexed lignan disappeared rapidly from the culture medium, within 24h, under all experimental conditions. Almost simultaneously, between 73 and 100% of detectable podophyllotoxin was bioconverted into podophyllotoxin--d-glucoside. A maximal bioconversion rate of 0.51 mmol l^-1 suspension day^-1 was calculated for the L. flavum cells growing in a medium which included the podophyllotoxin/dimethyl--cyclodextrin complex at a final concentration of 1.35 mM.     

Production of tumour-inhibitory lignans in callus cultures of Podophyllum hexandrum

Callus cultures have been established from root explants of aseptically-grown Podophyllum hexandrum seedlings. A fully defined medium based on Gamborg's B5 salts supplemented with 2/4-dichlorophenoxyacetic acid, gibberellic acid and 6-benzylaminopurine was effective for both initiation and sustained growth of callus tissue. Cultures produced anticancer lignans podophyllotoxin, 4-demethylpodophyllotoxin and podophyllotoxin 4-O-glucoside at levels similar to those found in the expiant material as assayed by high performance liquid chromatography. The relative proportions of podophyllotoxin and 4-demethyl-podophyllotoxin were markedly influenced by the presence of plant growth regulators. Particularly high levels of podophyllotoxin were associated with growth regulator induced tissue differentiation.     

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.     

Development of suspension culture of Podophyllum hexandrum for production of podophyllotoxin

A suspension culture of Podophyllum hexandrum was established. As the cultures grew, reduction in cell viability, biomass and product yield were associated with browning of culture medium, clumping of cells and drop in medium pH. Supplementation of the medium with both polyvinylpyrrollidone (PVP) and pectinase eliminated these problems. PVP at 10 g l^–1 was optimum for both growth of and product formation in P. hexandrum suspension cultures.     

Plant cell factories as a source for anti-cancer lignans

The review places podophyllotoxin, a powerful anti-cancer material used in clinical treatment of small cell cancers, in focus. The economical synthesis of podophyllotoxin is not feasible and demand for this material outstrips supply. At present, Podophyllum hexandrum (Indian May apple) is the commercial source but it grows in an inhospitable region (the Himalayas) where it is collected from wild stands. Furthermore, the plant is now an endangered species. Alternative sources of podophyllotoxin are considered, e.g., the supply of podophyllotoxin and related lignans by establishing plant cell cultures that can be grown in fermentation vessels. Increase of product yields, by variation of medium and culture conditions or by varying the channelling of precursors into side-branches of the biosynthetic pathway by molecular approaches, are discussed.     

Linum persicum: Lignans and placement in Linaceae†

Aryltetralin lignans (podophyllotoxin type) are the main lignan constituents of species belonging to Linum section Syllinum (Linaceae). Linum persicum, a perennial plant native to Iran closely related to L. album, has not yet been studied. To evaluate the lignan profile, fresh plants of L. persicumwere collected and divided into different parts and analyzed by HPLC. The main aryltetralin lignans found inL. persicumplant parts, callus and cell cultures were podophyllotoxin (PTOX), 6-methoxypodophyllotoxin (MPTOX) and - and -peltatin. Furthermore, the systematic relationship between L. persicum and other Linum species are discussed in the light of morphological and phytochemical aspects. Abbreviations: MPTOX – 6-methoxypodophyllotoxin; PTOX – podophyllotoxin; DOP – deoxypodophyllotoxin.     

The accumulation and isolation of coniferin from a high-producing cell suspension of Linum flavum L.

Cell suspensions of Linum flavum L. contained large amounts (2 g·l^–1) of the glucoside coniferin which was accumulated endogenously up to 12.4% on a dryweight basis. Callus material contained 5.6%, while in leaves of in-vitro-grown plantlets, the origin of the callus and suspension cultures, no coniferin could be detected. Leaf, callus and suspension material were compared for metabolite accumulation and associated enzyme activities. High coniferin contents corresponded with low 5-methoxypodophyllotoxin levels. A reciprocal relationship between -glucosidase (E.C. 3.2.1.21) activity and coniferin accumulation was found. No relationship between peroxidase (E.C. 1.11.1.7) activity and coniferin accumulation or 5-methoxypodophyllotoxin could be demonstrated. Finally, a rapid and effective isolation procedure for coniferin was developed.Abbreviation HPLC high-performance liquid chromatography This study has been performed within PDI (Plant Disciplines Integrated), a cooperation between the Free University of Amsterdam, TNO-ITC, Zeist and the University of Groningen, The Netherlands. We would like to thank Dr. H.J. Wichers for taking care of mass spectrometric analysis.     

Aspects of cytotoxic lignan biosynthesis in suspension cultures of Linum nodiflorum

Plant cell suspension cultures of Linum flavum, Linum nodiflorum and Linum album have been used to characterize the growth and production of cytotoxic lignans as well as to study the biosynthesis of these lignans. A cell culture of Linum nodiflorum accumulated up to 1.7% of the cell dry weight as 6-methoxypodophyllotoxin in only nine days of cultivation. The biosynthesis of podophyllotoxin and 6-methoxypodophyllotoxin follows the formation of the first aryltetralin lignan deoxypodophyllotoxin. Hydroxylation in position 7 by deoxypodophyllotoxin 7-hydroxylase leads to podophyllotoxin. Hydroxylation in position 6 by the cytochrome P450 monooxygenase deoxypodophyllotoxin 6-hydroxylase yields -peltatin which is further methylated by S-adenosylmethionine:-peltatin 6-O-methyltransferase to -peltatin-A methylether and then hydroxylated to 6-methoxypodophyllotoxin. Both, podophyllotoxin as well as 6-methoxypodophyllotoxin are stored as glucosides in the vacuole. Certain enzymes of these transformations have been isolated and characterized from Linum cell cultures.     

Polyacetylenes accumulation in <Emphasis Type="Italic">Ambrosia maritima</Emphasis> hairy root and cell cultures after elicitation with methyl jasmonate

Three lines of hairy root culture of Ambrosia maritima induced by Agrobacterium rhizogenes ATCC15834 were established. Thiarubrine A, thiarubrine A epoxide, thiarubrine A diol and their precursor pentayneene were produced by the hairy roots after elicitation with methyl jasmonate, the common signal molecule in the plant defense and development. Thiarubrine A diol was the main form detected in the medium. Maximum yield was achieved when the 13-day-old hairy root cultures were exposed to 40 M methyl jasmonate for 72 h. Callus and cell suspension cultures were established and maintained on Murashige and Skoog medium supplied with -naphthylacetic acid (NAA) and kinetin. When the cell suspension cultures were elicited with methyl jasmonate, pentayneene was the only polyacetylene produced. The yield of pentayneene in hairy root cultures was much higher (9.6 times) than that of cell suspension cultures.     

Detection and identification ofPodophyllotoxin produced by cell cultures derived from podophyllum hexandrum royle

The phenylpropanoid derived lignan podophyllotoxin, occurring inPodophyllum species, is used as a starting compound for the chemical synthesis of the antitumour agents etoposide (VP-16-213) and teniposide (VM-26). At present, the availability of this lignan becomes increasingly limited. As an alternative source, cell cultures originating fromPodophyllum hexandrum Royle were initiated. Analysis of the cell extracts using different HPLC systems as well as TLC, indicated the presence of podophyllotoxin. After prepurification of the extracts by means of ITLC, the identity was confirmed by mass spectrometric analysis. Dark-grown cultures accumulated considerable higher amounts of podophyllotoxin in comparison with the light-grown cultures.     

Lignans in plant cell and organ cultures: An overview

Lignans are found in a wide variety of plant species. The lignan podophyllotoxin is of special interest, since its derivatives like e.g. etopophos® are used in anticancer therapy. As chemical synthesis of podophyllotoxin is not yet economic, it still has to be isolated from wild growing Podophyllum species, some of which are considered to be endangered species. Therefore plant in vitro cultures may serve as alternative sources for podophyllotoxin and for other types of lignans as well. This review describes the establishment of plant cell and tissue cultures for lignan production and the experiments to improve product yields by changing the cultivation parameters, addition of elicitors and feeding of precursors. It also summarizes the use of plant cell and organ cultures to study the biosynthesis of lignans on enzymological level. Abbreviations: DOP – deoxypodophyllotoxin; LARI – lariciresinol; MATAI – matairesinol; 6MPTOX – 6-methoxypodophyllotoxin; PINO – pinoresinol; PTOX – podophyllotoxin; SECO – secoisolariciresinol     

The effect of cultural conditions on the accumulation of formononetin by suspension cultures of Glycyrrhiza glabra

Secondary product accumulation by suspension cultures of Glycyrrhiza glabra has been studied. The cultures did not accumulate either glycyrrhizin of glycyrrhetic acid but did produce the isoflavonoid formononetin. The effect of initial pH, sucrose concentrations, medium composition, auxin and cytokinins on formononetin accumulation was studied. Initially formononetin accumulation was non-growth related but this changed to growth associated when the medium was modified.Abbreviations 2,4-d dichlorophenoxyacetic acid - NAA napthalenacetic acid - IAA indoleacetic acid - GA glycyrrhizic acid - -GA -glycyrrhetic acid     

Growth and respiration of rice (Oryza sativa L.) Cells in suspension culture

Studies on the growth and respiration of batch suspension cultures of rice (Oryza sativa L.) in a reference medium containing Murashige-Skoog salts, 2% (w/v) sucrose and yeast extract are reported. It was found that the yeast extract contributed 70% of the phosphate in this medium, and that the cells grew equally well in continued subculture in a defined medium which contained 6 mM phosphate and 3% (w/v) sucrose and the remaining Murashige-Skoog salts. Cell clumps (up to 1.5 mm diameter) were prevalent in the initial cultures in the reference medium. In such cultures the critical O₂ pressure of cell respiration was high (125 M), and ethanol accumulated. When cell clumps were routinely removed during several weekly subcultures on the defined medium cultures were obtained in which no clumps were present, the critical O₂ pressures was decreased to 40 M and no ethanol accumulated.This work was supported by grant PCM-84-03542 from the U.S. National Science Foundation.     

Induction of lignans and phenolic compounds in cell culture of Linum album by culture filtrate of Fusarium graminearum

Cell suspension cultures of Linum album Kotschy ex Boiss. have been reported to produce anticancer podophyllotoxin and its related lignans. In the present study, we investigated the effect of culture filtrate of Fusarium graminearumon growth, and lignan and phenolic compounds in L. album cell culture. After 7 days of pre-culture, the cells were treated with 1% (v/v) of the culture filtrate. Cell growth was reduced, while phodophyllotoxin and lariciresinol production was stimulated reaching a maximum 0.0187 mg/g fresh weight (FW) and 0.0136 mg/g FW 5 days after the treatment, respectively. Also, our results provide evidence that the culture filtrate of F. graminearum can be effective on phenylalanine ammonia-lyase activity and phenolic compounds.     

Production of podophyllotoxin from Podophyllum hexandrum: a potential natural product for clinically useful anticancer drugs

Podophyllum hexandrum Royle of family Berberidaceae is an endangered medicinal plant. Rhizome ofP.hexandrum contains several lignans which posses antitumor activity. Podphyllotoxin is the most active cytotoxic natural product. It is used as starting compound for the synthesis of anticancer drug etoposide and teniposide. Podophyllotoxin acts as an inhibitor of microtubule assembly. These drugs are used for lung cancer, testicular cancer, neuroblastoma, hepatoma and other tumors. Besides this, it also shows antiviral activities by interfering with some critical viral processes. Availabilityof podophyllotoxin from plants has its limitations because of its intense collection from nature and lack of organized cultivation. The chemical synthesis of podophyllotoxin is considered to be very complicated as yet. The use of biotechnological approaches for the production of podophyllotoxin using cell cultures, organ cultures, and biotransformation route or by manipulating biosynthetic pathway proves to be an attractive alternative for production of podophyllotoxin. The present paper discusses the current status of research, limitations and future prospects for theproduction of podophyllotoxinin vitro.     

Biosynthesis of podophyllum lignans—i. cinnamic acid precursors of podophyllotoxin in podophyllum hexandrum

Feeding experiments in Podophyllum hexandrum plants have established that phenylalanine, cinnamic acid and ferulic acid are good precursors of the two major aryltetralin lignans podophyllotoxin and 4′-demethylpodophyllotoxin. Sinapic and 3,4,5-trimethoxycinnamic acids were poorly utilized, showing that the substitution pattern of the pendent aryl ring is built up after coupling of the two phenylpropane units. Degradation studies on podophyllotoxin derived from [3-O¹⁴CH₃] ferulic acid show that the two halves of the lignan molecule are equally labelled supporting a biosynthetic sequence involving oxidative coupling of two similar phenylpropane precursors having the substitution pattern of ferulic acid. Although 3,4-methylenedioxycinnamic acid was readily incorporated, degradative studies prove that this compound is not incorporated intact, but via a metabolic sequence in which the methylenedioxy carbon atom enters the C₁-pool and then labels the methylenedioxy and methoxyl substituents of podophyllotoxin. The rest of the skeleton is incorporated via ferulic acid, presumably by way of caffeic acid.     

Production of higher levels of trigonelline by cell cultures of Trigonella foenum-graecum than by the differentiated plant

Callus cultures of Trigonella foenum-graecum contained 3 to 4 times more trigonelline than the seeds of this plant and 12 to 13 times more than the roots and shoots. Even higher levels of this alkaloid were produced by suspension cultures. This high productivity was maintained during successive subculturing of calli and cell suspensions for eight months. Thus, trigonelline is to be added to the group of the few metabolites whose synthesis in cell cultures exceeds its production in the differentiated plants. Media that had supported the growth of suspension cultures contained one third or more of the total alkaloid, whereas media of callus cultures contained about one tenth of this substance. Trigonelline accumulated in callus and suspension cultures with aging. Raising the level of nicotinic acid in the nutrient medium resulted in some increase of trigonelline production by the culture.Abbreviations 2.4 D 2.4-dichlorophenoxyacetic acid - IAA indoleacetic acid - IPA indolepropionic acid - NAA -naphthaleneacetic acid - GA Gibberellic acid - K kinetin