Plant cell cultures: Chemical factories of secondary metabolites

The structure-activity relationship of flavonoids as inhibitors of xanthine oxidase and as scavengers of the superoxide radical, produced by the action of the enzyme xanthine oxidase, was investigated. The hydroxyl groups at C-5 and C-7 and the double bond between C-2 and C-3 were essential for a high inhibitory activity on xanthine oxidase. Flavones showed slightly higher inhibitory activity than flavonols. All flavonoid derivatives except isorhamnetin (30) were less active than the original compounds. For a high superoxide scavenging activity on the other hand, a hydroxyl group at C-3' in ring B and at C-3 were essential. According to their effect on xanthine oxidase and as superoxide scavengers, the flavonoids could be classified into six groups: superoxide scavengers without inhibitory activity on xanthine oxidase (category A), xanthine oxidase inhibitors without any additional superoxide scavenging activity (category B), xanthine oxidase inhibitors with an additional superoxide scavenging activity (category C), xanthine oxidase inhibitors with an additional pro-oxidant effect on the production of superoxide (category D), flavonoids with a marginal effect on xanthine oxidase but with a prooxidant effect on the production of superoxide (category E), and finally, flavonoids with no effect on xanthine oxidase or superoxide (category F).

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Structure−Activity Relationship and Classification of Flavonoids as Inhibitors of Xanthine Oxidase and Superoxide Scavengers

Plants are the tremendous source for the discovery of new products with medicinal importance in drug development. Today several distinct chemicals derived from plants are important drugs, which are currently used in one or more countries in the world. Secondary metabolites are economically important as drugs, flavor and fragrances, dye and pigments, pesticides, and food additives. Many of the drugs sold today are simple synthetic modifications or copies of the naturally obtained substances. The evolving commercial importance of secondary metabolites has in recent years resulted in a great interest in secondary metabolism, particularly in the possibility of altering the production of bioactive plant metabolites by means of tissue culture technology. Plant cell and tissue culture technologies can be established routinely under sterile conditions from explants, such as plant leaves, stems, roots, and meristems for both the ways for multiplication and extraction of secondary metabolites. In vitro production of secondary metabolite in plant cell suspension cultures has been reported from various medicinal plants, and bioreactors are the key step for their commercial production. Based on this lime light, the present review is aimed to cover phytotherapeutic application and recent advancement for the production of some important plant pharmaceuticals.

Current approaches toward production of secondary plant metabolites.

Strategies for the improvement of secondary metabolite production in plant cell cultures

Cardenolides are remarkable steroidal toxins that have become model systems, critical in the development of theories for chemical ecology and coevolution. Because cardenolides inhibit the ubiquitous and essential animal enzyme Na⁺/K⁺-ATPase, most insects that feed on cardenolide-containing plants are highly specialized. With a huge diversity of chemical forms, these secondary metabolites are sporadically distributed across 12 botanical families, but dominate the Apocynaceae where they are found in > 30 genera. Studies over the past decade have demonstrated patterns in the distribution of cardenolides among plant organs, including all tissue types, and across broad geographic gradients within and across species. Cardenolide production has a genetic basis and is subject to natural selection by herbivores. In addition, there is strong evidence for phenotypic plasticity, with the biotic and abiotic environment predictably impacting cardenolide production. Mounting evidence indicates a high degree of specificity in herbivore-induced cardenolides in Asclepias. While herbivores of cardenolide-containing plants often sequester the toxins, are aposematic, and possess several physiological adaptations (including target site insensitivity), there is strong evidence that these specialists are nonetheless negatively impacted by cardenolides. While reviewing both the mechanisms and evolutionary ecology of cardenolide-mediated interactions, we advance novel hypotheses and suggest directions for future work.

https://nph.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1469-8137.2011.04049.x

Toxic cardenolides: chemical ecology and coevolution of specialized plant–herbivore interactions

Suspension cultures of Berberis wilsonae produce 4 berberine-type alkaloids: berberine, palmatine, columbamine and jatrorrhizine. In particular the formation of the phenolic alkaloids columbamine and jatrorrhizine and of berberine proves to be dependent on the concentration of dissolved oxygen. With higher aeration rates, berberine and jatrorrhizine yields can be increased considerably. Thus we reached an alkaloid yield of more than 3 g × 1−1 with 50% dissolved oxygen tension in the medium. As far as we know this is one of the best results in fermenting of alkaloid-producing cell cultures.

Cultivation of cell cultures of Berberis wilsonae in 20-l airlift bioreactors.

Plant cell cultures are a potential source of pharmaceutically important plant metabolites. In the past few years a lot of success has been achieved in the field of the cultivation of plant cells on a large scale. Special bioreactor systems, such as airlift or drum-type fermentors have been devised for the mass cultivation of plant cells. Classical stirred-tank bioreactors (up to 75000 l volume) have also been used for the cultivation of plant cells and the production of important plant metabolites. Plant cells proliferate much slower than microbial cells. In consequence, the time taken to grow a plant cell suspension from the shake-flask level (300 ml) to the production scale (20,000 l) takes about 3 to 6 weeks. Nevertheless, the cost analyses available show that the production of valuable chemicals by a suitable plant cell culture process could be commercially viable. Plant cells have been grown in batch and repeated batch culture, and single- stage as well as two-stage processes have been developed for the production of secondary metabolites by fermentor-cultured plant cells. There are, however, only few cell culture processes that have attracted industrial attention as yet. Biological rather than technological problems are the main obstacles to a more common use of fermentor-cultured plant cells in industry.

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The production of secondary metabolites by plant cells cultivated in bioreactors1.

Suspension cultures from several cell lines of Digitalis lanata, as well as cultures from 6 other plant species were checked for their ability to form purpurea-glycoside A from digitoxin. An in-vitro assay for the UDP-glucose:digitoxin 16′-O-glucosyltransferase (DGT, EC 2.4.1.-) has been established based on an HPLC method. The enzyme is located in the soluble fraction. Its pH optimum is at 7.4. No enzyme activity was found in either purified vacuole preparations or lysed vacuoles. Ascorbate (10 mM) increased the transferase activity about 4-fold. Of the sugar nucleotides tested, only UDP-glucose served as a glucosyl donor. Digitoxin, digoxin, α -acetyldigitoxin, and α-acetyldigoxin are substrates for the glucosyltransferase. The role of the DGT during the biotransformation of cardenolides in Digitalis lanata cell suspension cultures is discussed.

UDP-glucose:digitoxin 16′-O-glucosyltransferase from suspension-cultured Digitalis lanata cells

Digitalis lanata cells were grown at dif-ferent dissolved-oxygen (DO) levels in 20-1 airlift reactors. A DO level of 30% saturation (using air for aeration) was found to be optimal for growth and the biotransformation ofβ-methyldigitoxin toβ-methyldigoxin. Product yield was further in-creased by using stirred tank reactors instead of the airlift reactor.

Ernst Reinhard

Papers

Cultivation of cell cultures of Berberis wilsonae in 20-l airlift bioreactors.

The production of secondary metabolites by plant cells cultivated in bioreactors1.

UDP-glucose:digitoxin 16′-O-glucosyltransferase from suspension-cultured Digitalis lanata cells

Biotransformation of β-methyldigitoxin by cell cultures of Digitalis lanata in airlift and stirred tank reactors

Selective Uptake and Vacuolar Storage of Primary Cardiac Glycosides by Suspension-cultured Digitalis lanata Cells