W Vanuden

DETECTION AND IDENTIFICATION OF PODOPHYLLOTOXIN 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.

PRODUCTION OF THE NEW ANTIMALARIAL DRUG ARTEMISININ IN SHOOT CULTURES OF ARTEMISIA-ANNUA L

From aseptically grown Artemisia annua plantlets, shoot cultures were initiated. Using different concentrations of auxine, cytokinine and sucrose, a suitable culture medium was developed, with respect to the growth of the shoots and their artemisinin accumulation. Nitrate concentration and conductivity appeared to be suitable growth parameters. The artemisinin content was measured gas chromatographically. The shoot cultures were maintained in the developed standard medium, consisting of a half concentration of MS-salts with vitamins, 0.2 mg l-1 BAP, 0.05 mg l-1 NAA and 1% sucrose. The growth of the shoots and the artemisinin content remained stable for a longer period. They showed considerable photosynthetic activity and generally contained ca. 0.08% artemisinin on a dry weight basis. The highest artemisinin content found was 0.16% in the above mentioned standard medium, but also on the same medium with 0.5% sucrose. Attempts were made to further improve the artemisinin production by varying the medium composition through addition of gibberellic acid or casein hydroly-state; by omitting plant growth regulators; by precursor feeding, i.e. mevalonic acid; by influencing the biosynthesis routing through inhibition of the sterol synthesis by miconazole, naftifine or terbinafine; by changing gene expression with 5-azacytidine or colchicine; and by elicitation, using cellulase, chitosan, glutathione or nigeran. Enhanced artemisinin production was found with 10 mg l-1 gibberellic acid, 0.5 g l-1 casein hydrolysate, 10 mg l-1 or 20 mg l-1 naftifine. Relative increases of 154%, 169%, 140% and 120% were found, respectively. Other additions caused the growth to cease and the artemisinin contents to drop.

PROGRESS IN THE RESEARCH OF ARTEMISININ-RELATED ANTIMALARIALS - AN UPDATE

Artemisinin, a sesquiterpene lactone endoperoxide isolated fromArtemisia annua L., and a number of its semisynthetic derivatives have shown to possess antimalarial properties. They are all eflective againstPlasmodium parasites that are resistant to the newest and commonly used antimalarial drugs. This article gives a survey of the literature dealing with artemisinin-relaled antimalarial issues that have appeared from the end of 1989 up to the beginning of 1994. A broad range of medical and pharmaceutical disciplines is covered, including phytochemical aspects like the selection of high-producing plants, analytical procedures, and plant biotechnology. Furthermore, the organic synthesis of artemisinin derivatives is discussed, as well as their mechanism of action and antimalarial activity, metabolism and pharmacokinetics, clinical studies, sideeffects and toxicology, and biological activities other than antimalarial activity.

ON THE IMPROVEMENT OF THE PODOPHYLLOTOXIN PRODUCTION BY PHENYLPROPANOID PRECURSOR FEEDING TO CELL-CULTURES OF PODOPHYLLUM-HEXANDRUM ROYLE

In order to improve the production of the cytotoxic lignan podophyllotoxin, seven precursors from the phenylpropanoid-routing and one related compound were fed to cell suspension cultures derived from the rhizomes of Podophyllum hexandrum Royle. These cell cultures were able to convert only coniferin into podophyllotoxin, maximally a 12.8 fold increase in content was found. Permeabilization using isopropanol, in combination with coniferin as a substrate, did not result in an extra increase in podophyllotoxin accumulation. Concentrations of isopropanol exceeding 0.5% (v/v) were found to be rather toxic for suspension growth cells of P. hexandrum. When coniferin was fed in presence of such isopropanol concentrations, β-glucosidase activity was still present, resulting in the formation of the aglucon coniferyl alcohol. In addition, podophyllotoxin was released into the medium under these permeabilization conditions. Entrapment of P. hexandrum cells in calcium-alginate as such or in combination with the feeding of biosynthetic precursors, did not improve the podophyllotoxin production. Cell-free medium from suspension cultures at later growth stages incubated with coniferin, resulted in the synthesis of the lignan pinoresinol.

The accumulation of podophyllotoxin-β-d-glucoside by cell suspension cultures derived from the conifer Callitris drummondii

SummaryPodophyllotoxin was produced by cell cultures derived from needles of Callitris drummondii. The needles of this conifer contained 1.56% podophyllotoxin on a dry weight basis, 32% being present in the β-glucosidic form. Trace amounts of desoxypodophyllotoxin and matairesinol were also detected. In dark-grown cell cultures, ca. 0.02 % podophyllotoxin was accumulated, 85–90 % in the β-D-glucosidic form. Moreover, traces of the lignans matairesinol, hinokinin and asarinin were detected. Illumination stimulated the endogenous production of podophyllotoxin-β-D-glucoside; contents of up to 0.11 % could be measured.

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 β-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.

Structure-cytotoxicity relationships of a series of natural and semi-synthetic simple coumarins as assessed in two human tumour cell lines.

Abstract The cytotoxicity of 22 natural and semi-synthetic simple coumarins was evaluated in GLC4, a human small cell lung carcinoma cell line, and in COLO 320, a human colorectal cancer cell line, using the microculture tetrazolium (MTT) assay. With IC50 values > 100 μᴍ , following a continuous (96h) incubation, most coumarins exhibited only low cytotoxicity. Several compounds, however, displayed significant potencies. As far as the structure -cytotoxicity relationship is concerned, it is conspicuous that all the potentially active natural compounds possess at least two phenolic groups in either the 6,7-or 6,8-positions. In addition, the 5-formyl-6-hydroxy substituted semi-synthetic analogue was found to be potent, reflecting the importance of at least two polar functions for high cytotoxicity.

MUCUNA PRURIENS - IMPROVEMENT OF THE BIOTECHNOLOGICAL PRODUCTION OF THE ANTI-PARKINSON DRUG L-DOPA BY PLANT-CELL SELECTION

Routinely grown cell suspension cultures ofMucuna pruriens L. (Fabaceae) were able to endogenously accumulate the anti-Parkinson drug L-dihydroxyphenylalanine (L-dopa) in the range between 0.2 and 2% on a dry weight (DW) basis. The green colour that developed in light-exposed cultures, appeared to be a suitable marker to select cells with an increased L-dopa biosynthesis and/or phenoloxidase activity. For this purpose, saccharose concentrations from 0 to 4% (w/v), and light intensities of 1,000 and 2,000 lux, were involved in the selection procedure. After 6 months, photomixotrophic callus cultures with a rapid growth and a high L-dopa content of 0.9% (DW) were obtained on 2% saccharose and under 1,000 lux. The cell suspensions, derived from these calli, accumulated up to 6% (DW) L-dopa, which was the highest stable content ever measured in cultures ofM. pruriens. An L-dopa yield of approximately 1.2 g/l was calculated after 6 days of growth. In contrast, compared with the standard-grown parent cell line, the phenoloxidase activity, and consequently the bioconversion capacity as measured after entrapment in calcium alginate, of these high-producing cultures was approximately threefold lower.

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: β-cyclodextrin, γ-cyclodextrin, dimethyl-β-cyclodextrin and hydroxypropyl-β-cyclodextrin were used to improve the solubility of podophyllotoxin by complexation. Dimethyl-β-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-β-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-β-cyclodextrin complex at a final concentration of 1.35 mM.

Bioconversion potential of plant enzymes for the production of pharmaceuticals

Plant enzymes are able to catalyze regio- and stereospecific reactions. Freely suspended and immobilized plant cells as well as enzyme preparations can therefore be applied for the production of pharmaceuticals by bioconversion, as such or in combination with chemical syntheses. This review paper deals with bioconversions of added precursors from natural or synthetic origin by several biocatalytic systems.