Isolation, Purification, and Cytotoxicity of 5-Methoxypodophyllotoxin, a Lignan from a Root Culture of Linum flavum

Abstract

A method has been developed for the large scale isolation of 5-methoxypodophyllotoxin [1] from a high-producing root culture derived from Linum flavum. A closely related lignan, 5'-demethoxy-5-methoxypodophyllotoxin [2], was also present in the root culture and was the cause of the main isolation difficulties. Essential steps in the isolation procedure are CH2Cl2 and XAD-4 extraction and XAD-8 cc followed by Si gel chromatography, using two different mobile phases. The isolated 5-methoxypodophyllotoxin [1] was very pure (greater than 99%) and possessed the desired stereochemical configuration, namely (-)-5-methoxypodophyllotoxin [1]. The in vitro cytotoxicity of 5-methoxypodophyllotoxin [1] against EAT and HeLa cells was determined and compared with those of podophyllotoxin [3], etoposide (VP-16-213) [4], teniposide (VM-26) [5], and 5-methoxypodophyllotoxin-4-beta-D-glucoside [6]. It appeared that 5-methoxypodophyllotoxin [1] has about the same cytotoxic potency as podophyllotoxin [3].

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

“…Podophyllotoxin (Sigma P-4405), 5-methoxypodophyllotoxin (isolated from cell cultures of L. flavum, Van Uden et al 1992), 5-methoxypodophyllotoxin-9-D-gtucoside, ~-peltatin (both kindly provided by TNO-Zeist, The Netherlands) and desoxypodophyllotoxin (prepared at the Laboratory for Organic and Molecular Chemistry, University of Groningen, The Netherlands) were analysed and detected using the HPLC system as reported previously by Van Uden et al (1989), equipped with a reversed phase Lichrosorb RP-18 (Chrompack, Middelburg, The Netherlands) column (100 × 3 mm i.d.) and a guard column.…”

“…Mass spectrometry (El) was performed as described previously (Van Uden et al 1992). In addition, positive ion chemical ionisation (PICI) was performed with NH3 as the reactant gas.…”

“…In addition, the same experiments were performed with cell suspensions of Linum flavum (yellow flax; Linaceae), which normally accumulate 5-methoxypodophyllotoxin ( Fig. 1C) as the major lignan (Berlin et al 1986(Berlin et al , 1988Van Uden et al 19-90, 1991a, 1991b, 1992, 1994Wichers et al 1990Wichers et al , 1991.…”

Reduced Coniferin and Enhanced 6-Methoxypodophyllotoxin Production in Linum flavum Cell Cultures

“…Calibration curves were made using coniferyl alcohol (Sigma), coniferin and 6-MPT, which were isolated from L. flavum cell suspension cultures as published previously. [9,13] For the statistical evaluation of the data the student's t-test was used. A p-value <0.05 was considered as significant.…”

“…[4] Several investigations to enhance the production of podophyllotoxin-derived lignans by manipulation of cell and organ cultures have been carried out. [3,5,6,7,8] The production of podophyllotoxin, 6-MPT and its glucoside could be enhanced in cell cultures of Podophyllum hexandrum Royle, [7] Linum flavum L. [5,9,10] Callitris drummondii F. Mueller, [11] and Linum album Kotschy. [3] Based on the close chemical resemblance with podophyllotoxin (see Figure 1), 6-MPT is considered also as an interesting starting compound for the preparation of new semi-synthetic derivatives with antitumor properties.…”