Cinchona alkaloids cell cultures

R. Wijnsma and R. Verpoorte, Quinoline alkaloids in cell and tissue cultures of Cinchona species. Cell cultures and somatic genetics of plants. Vol. 5. 

To remove the feedback regulation mechanism and to avoid product degradation various adsorbents have been used for the in situ separation of plant cell cultures as shown in Table 1. In situ removal with polymeric adsorbents stimulated anthraquinone production more than the adsorbent-free control in Cinchona ledgeriana cells [35]. It was found that nonionic polymeric resins such as Amberlite XAD-2 and XAD-4 without specific functional groups are suitable for the adsorption of plant metabolite [36]. The use of the natural polymeric resin XAD-4 for the recovery of indole alkaloids showed that this resin could concentrate the alkaloids ajmalicine by two orders of magnitude over solvent extraction [37] but the adsorption by this resin proved to be relatively nonspecific. A more specific selectivity would be beneficial because plant cells produce a large number of biosynthetically related products and the purification of a several chemically similar solutes mixture is difficult [16]. 

Cinchona alkaloids are a group of closely related compounds, isolated from the bark of tropical Cinchona sp. trees. They are mainly of medical importance, but have also found application as chiral inductors in chemistry. Newer methods for their production include isolation from transformed root cell cultures of Cinchona ledgeriana1. Most alkaloids are commercially available, so there is no need for their synthesis in the laboratory. However, total syntheses do exist and useful tips for the preparation of analogs are available2-5. 

Secondary metabolism is a form of differentiation, but cells grown in vitro are rapidly dividing, undifferentiated cells. Only at the end of the growth phase of batch-cultured cells may some form of differentiation occur, connected with the production of secondary metabolites. A plant produces a wide variety of secondary metabolites, all with different, mostly unknown functions. In in vitro cultured cells those compounds which defend the plant against microorganisms, namely, phytoalexins, are often easily formed. For example. Cinchona cell cultures produce large amounts of anthraquinones, but the alkaloids of interest, the quinolines, are produced in trace amounts only. Similarly Papaver cell cultures produce sanguinarine and closely related alkaloids, but no morphinane alkaloids. 

Of the various pharmaceuticals derived from plants, the Cinchona alkaloids are probably, by volume the largest market, with an estimated production of 300-500 metric tons a year of pure quinine (32) and quinidine (33). These alkaloids are extracted from the bark of Cinchona trees, which require about 10 years to mature before harvesting. Furthermore most of the plantations are in areas not easily accessible, often threatened by infections with Phytophthora cinnamomi. This leads to many uncertainties in planning of the production, and as a result alternative sources for the alkaloids are of interest. Various synthetic aproaches have been used (552) but are not of industrial interest. Therefore, interest in biotechnological approaches is large. Patents related to the production of quinoline alkaloids by means of plant cell cultures are summarized in Table XXVIII. 

Akad. Wissenschaft DDR. DD-205-184-A. 26-04-1982-DD-239293 (21-12-1983). Cinchona alkaloid production by cell or callus culture giving production free of plant material. 

Fig. 10. Relationship between degree of differentiation and alkaloid production for Cinchona plant cell and tissue cultures.

Although very low levels of alkaloids are produced by the Cinchona cell cultures, they are capable of producing considerable amounts of anth-raquinones, particuleu-ly after elicitation with fungal elicitors. The anth-raquinones are thought to act as phytoalexins in this plant genus (575). Anthraquinone production could be stimulated by adding polymeric adsorbents like Amberlite XAD-7 to the medium. A production rate of 20 mg/liter/day could be obtained in this way (576). 

Feeding of tryptophan to Cinchona cell suspension cultures has been reported to result in considerable production of alkaloids 572,577,578). However, other authors reported that tryptophan inhibits the growth of the cell cultures and is converted, probably nonenzymatically, to norhar-mane and related compounds, and no increase in alkaloid production could be observed 586,587). 

Cinchona can be considered to be recalcitrant with regard to cell and tissue culture. Although it has been possible to obtain cell and tissue cultures of some Cinchona species, they often require special treatments also, growth is usually slow, and viability of cells is rapidly lost. Moreover, the cell cultures are poor producers of alkaloids. In order to arrive at... 

A number of terpenoid indole alkaloids have pharmaceutical interest. These alkaloids are isolated from plants belonging to the families Apocy-naceae, Loganiaceae, and Rubiaceae. For the production of alkaloids by means of plant cell cultures, plants of the latter two families have proved to be rather recalcitrant (e.g., see Cinchona alkaloids). On the other hand, it has been reported by Pawelka and Stockigt that all apocynaceous cell suspensions they studied did produce terpenoid indole alkaloids 588). Here we confine ourselves to alkaloids which have direct commercial interest the production of new, potentially interesting, compounds is not reviewed here. For this we refer the reader to reviews by Balsevich (589), van der Heijden et al. (tribe Tabernaemontaneae) (590), and Omar (Rhazya stricta) (591). 

In the subsequent paragraphs some of the work in our laboratories in this field will be reviewed. It particularly involves the secondary metabolism in cell cultures of plants capable of producing terpenoid-indole and related alkaloids, i.e. Cinchona and Tabernaemontana. 

The alkaloid biosynthesis is thus not expressed in the cell suspension cultures. However, another secondary metabolite pathway is operative. The yellow-orange coloured products formed by the cell cultures were identified as anthraquinones, at that time not yet known as product from Cinchona trees. Further studies proved that these compounds probably acts as phytoalexins in Cinchona (37). They have antimicrobial activity, their production can be induced by microrganisms (e.g. fungi) and preparations thereof. Also in infected Cinchona trees we have been able to proof the presence of anthraquinones, whereas healthy ones are free of these compounds (38). 

P.A.A. Harkes, L. Krijbolder, K.R. Libbenga, R. Wijnsma and R. Verpoorte, Influence of various media constituents on the growth of Cinchona ledgeriana tissue cultures and the production of alkaloids and anthraquinones therein. Plant Cell, Tissue and Organ Culture, 4 (1985) 199. 

Because of the value and the widespread use of Cinchona alkaloids, many attempts to produce alkaloids in plant tissue cultures and cell cultures from this group of plants have been made (Verpoorte et al., 1991). Trees require about 10 years before they can be harvested. Cell cultures of Cinchona ledg-eriana and C. succirubra produce largely cinchonine (67) and cinchonidine (68), although small quantities of quinine and quinidine were produced. Only small amounts of total alkaloids were produced (Ellis, 1988). 

Interesting correlation between distribution of the strictosidine synthase activity and alkaloid content in the 6-month C. ledgeriana plants was found by Aerts et al. The enzyme was active in the top of stems, in young leaflets, and in the roots, and these parts also contained the highest concentration of the alkaloids. Quinoline alkaloids were accumulated in the roots, whereas cinchophyllines in aerial part of plants [218]. Similarly high activity of strictosidine synthase in the germinating Cinchona seeds and cell cultures has been reported [219]. Four isoforms of strictosidine synthase have been isolated and purified from the suspension culture of C. robusta [220]. 

Bioconversion of Cinchona alkaloids is relatively less explored as compared to other natural products. The reason for this is the fact that none of the Cinchona alkaloid precursors are available at lower prices than the final alkaloids. The only exception is tryptophan however, it has no straightforward influence on the increasing production of alkaloids in cell cultures [267, 268]. 

As part of our studies on pharmaceutically important alkaloids in C. roseus and Cinchona spp. several aspects of their bios5mthesis were chciracterized and a number of enzymes were purified [4]. In the accumulation of alkaloids in suspension cultured C. roseus cells, it was found that the supply of isoprenoid precursors was a limiting factor. This prompted us to fiarther studies, thereby focusing on the early steps of the isoprenoid biosynthesis (fig. 2). Little was known of these enzymes from plants and except for HMG-CoA reductase, none of the enzymes was characterized [5,6]. Furthermore, there exists much controversy on the subcellular localization of this pathway [7,8]. 

The enzyme strictosidine synthase (EC 4.3.3.2) is responsible for the stereospecific coupling of tryptamine and secologanin, yielding strictosidine (Fig. 12). This glucoalkaloid is the precursor for all terpenoid indole and related alkaloids, including among others the Cinchona quinoline alkaloids. Hampp and Zenk (707) isolated and purified this enzyme to homogeneity from a cell suspension culture of R. serpentina. The enzyme could successfully be immobilized on CNBr-activated Sepharose 4B, as was reported for this enzyme isolated from Catharanthus roseus (102,708). It proved to be more stable than the C. roseus enzyme the half-life of the immobilized enzyme was 100 days at a temperature of 37°C. 

Hamill JD, Robins RJ, Rhodes MJC (1989) Alkaloid production by transformed root cultures of Cinchona ledgeriana. Planta Med 55 354-357 Hamill JD, Robins RJ, Parr AJ, Evans DM, Furze JM, Rhodes MJC (1990) Overexpressing a yeast ornithine decarboxylase gene in transgenic roots of Nicotiana rustica can lead tq enhanced nicotine accumulation. Plant Mol Biol 15 27-38 Hamill JD, Rounsley S, Spencer A, Todd G, Rhodes MJC (1991) The use of the polymerase chain reaction in plant transformation studies. Plant Cell Rep 10 221-224 Hartmann T, Toppel G (1987) Senecionine N-oxide, the primary product of pyrrolizidine alkaloid biosynthesis in root cultures of Senecio vulgaris. Phytochemistry 26 1639-1643... 

Among others in our laboratories cell and tissue cultures of Cinchona species have been initiated. For a review of this work is referred to Wijnsma and Verpoorte (29). Summarizing the results cell suspension cultures of Cinchona are very poor producers of alkaloids. With an increase of organisation of the culture an increase of the alkaloid production can be observed (Fig. 2). 

Why the cells are capable of expressing the anthraquinone biosynthesis in in-vitro cell suspension cultures and not the alkaloid biosynthesis has still to be answered. Further studies on the genetic regulation of the secondary metabolism in Cinchona are needed to answer this question and to eventually open the way for a biotechnological production method. 

R. Wijnsma, T.B. van Vliet, P.A.A. Harkes, H.J. van Groningen, R. van der Heijden, R. Verpoorte and A. Baerheim Svendsen, An improved method for the extraction of alkaloids from cell and tissue cultures of Cinchona species. Plants Medics, 53 (1987) 80-84. 

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