Berberis cell cultures

Berberis cell cultures were also reported to produce bisbenzylisoquino-line alkaloids. Cassels et al. 412) screened 34 callus cell lines (33 species). In all cases the protoberberine alkaloid Jatrorrhizine was the major component in aU but 12 cell lines bisbenzylisoquinoline alkaloids were found. High levels of berbamine were found in B. angulosa (0.8% of dry weight) and B. henryana (0.48%). Berbamunine (16) and the new alkaloid 2-norberbamunine (17) were the major alkaloids in B. stolonifera cell lines... 

When the substrate l" C-labelled (S)-scoulerine was incubated with crude cell-free homogenates of various Berberis cell cultures in the presence of SAM, the formation... 

Bisbenzylisoquinoline alkaloids are dimeric benzyltetrahydroisoquinoline alkaloids that are known for their pharmacological activities. A well-described example is the muscle relaxant (+)-tubocurarine, which in crude form serves as an arrow poison for South American Indian tribes. In the biosynthesis of this broad class of dimeric alkaloids, it has been postulated that the mechanism of phenol coupling proceeds by generation of phenolate radicals followed by radical pairing to form either an inter- or intramolecular C - O or C - C bond. Enzyme studies on the formation of bisbenzylisoquinoline alkaloids indicated that a cytochrome P-450-dependent oxidase catalyzes C - O bound formation in the biosynthesis of berbamunine in Berberis cell suspension culture.15 This enzyme, berbamunine synthase (CYP80A1), is one of the few cytochromes P-450 that can be purified to... 

STEFFENS, P., NAGAKURA, N., ZENK, M.H., Purification and characterization of the berberine bridge enzyme from Berberis beaniana cell cultures, Phytochemistry, 1985, 24, 2577-2583. 

STADLER, R., ZENK, M.H., The purification and characterization of a unique cytochrome P-450 enzyme from Berberis stolonifera plant cell cultures, J. Biol. Chem., 1993,268, 823-831. 

Breuling, M., Alfermann, A. W. and Reinhard, E. 1985. Culivation of cell cultures of Berberis wilsoniae in 20-1 airlift bioreactors. Plant Cell Reports, 4 220-223. 

The formation of the methylenedioxy bridge in Berberis has been found to be caused by the demethylating activity of a peroxidase (POD) found within the vesicle. It was also found that the cytochrome P450-requiring enzyme (canadine synthase) from microsomes of Berberis, Thalictrum and Coptis species formed the methylene bridge in (S)-tetrahydrocolumbamine (Ikezawa et al, 2003), but not in the quaternary alkaloid columbamine (Galneder et al, 1988 Zenk, 1995). Because of the substrate specificity of canadine s)mthase, the berberine pathway is considered to be that presented in Fig. 2.5 (Rueffer and Zenk, 1994). Columbamine, once proposed as an alternative route to berberine, is however converted to palmatine by a specific methyltransferase first isolated from Berberis wilsoniae cell cultures (Rueffer and Zenk, 1985 Ikezawa et al, 2003). 

The biosynthesis of protoberberine alkaloids, including berberine, has been extensively studied, and all the enzymes of the biosynthetic pathway have been characterized (11,121,391,508). Interestingly the pathway leading to berberine in Berberis was found to be different from that in Coptis and Thalictrum (509). In the former species berberine is formed from columbamine, in the latter plant species from tetrahydroberberine (121). The production of isoquinoline alkaloids, including the protoberberine alkaloids, by plant cell cultures have been reviewed by Riiffer (390) and Ikuta (510). Table XXVI summarizes patents concerning the production of berberine by means of plant cell cultures. In Table XXVII a summary is given of the occurrence of berberine in some plant cell cultures. 

Nevertheless in 1985 an oxidase was reported from crude extracts of Coptis japonica cell cultures with different properties as compared to the enzyme from Berberis. This led to a systematic comparison of the two cell cultures concerning the last steps of the berberine biosynthesis. The purified oxidase from... 

The lack of strict specificity for the enzymes involved in the initial conversion to (5)-reticuline (20) suggests that stereochemical control does not occur at this stage of biosynthesis (Zenk, 1985). The use of plant cell cultures (both callus and suspension types) from different species of plants that produce benzylisoquinoline alkaloids has proven to be a powerful approach for the isolation of enzymes and intermediates in the pathways leading to these compounds (Zenk et al., 1985). For example, the use of cell-free extracts of Berberis cultures permitted Zenk and co-workers to isolate and characterize all eight enzymes involved in the conversion of dopamine (22) and 4-hydroxyphenylacetaldehyde (19) to (5)-reticuline and, subsequently, to berberine. 

Berberine (71) is produced by several plant cell cultures (Ellis, 1988). Suspension cultures of Coptis japonica produce large quantities (8% by dry weight) of berberine. Some cultures excrete berberine directly into the culture medium whereupon it crystallizes (Ellis, 1988). Cell cultures of some Berberis species synthesize jatrorrhizine (69) and colum-bamine (70) as the principal alkaloids, especially during the exponential growth phase and stationary phase (Verpoorte et al., 1991). Many of these same cultures produce berberine as the major alkaloid during lag-phase growth. 

Frenzel, T. and M. H. Zenk, Purification and characterization of three isoforms of S-adenosyl-L-methionine (/f,S)-Tetrahy-drobenzylisoquinoline-JV-methyl transferase from Berberis koe-tineana cell cultures. Phytochemistry, 29, 3491-3497 (1990a). 

Since Reinhard (1967) described the production of protoberberine alkaloids by callus cultures of Berberis vulgaris, many different structure types of benzyiisoquinoline alkaloids could be isolated from callus- and suspension cultures (Table l).The greatest variety in the substitution pattern of the basic structure is found in the group of protoberberine alkaloids. Ikuta and Itokawa (1982b) for example described the isolation of 11 different protoberberine alkaloids from one plant cell culture (Nandina domes-tica, Berberidaceae). 

Sometimes the ratio between the amounts of several alkaloids in the plant differs markedly from that of the cell cultures. In the culture of Berberis wilsoniae var. sub-... 

Fig. 2. Comparison between the alkaloid production of the cell culture, the seedling, and the intact root of Berberis wilsoniae var. subcaulialata (Berberidaceae)...

Looking for example at the use of so far described basal media the production of jatrorrhizine in Berberis stolonifera cell cultures (Hinz and Zenk 1981) was optimal in LS-medium (Linsmaier and Skoog 1965), the cell suspension cultures of Coptis japonica (Sato and Yamada 1984) gave the best results in White medium (White 1963), md Eschscholtzia califomica (Papaveraceae) (Berlin et al. 1983) was cultivated with the best results in B5 medium (Gamborg et al. 1968). 

Another notable example of the production of alkaloids in a cell-suspension culture was with Berberis species ( Berberis stolonifera) where the yield of berberine alkaloids (eg. jatrorrhizine (62) ) was as high as 10% of the dry weight of the cells °. Cell cultures of Peganum harmaZa produce harman alkaloids in minute amounts. ... 

H. Hinz and M.H. Zenk, Production of protoberberine alkaloids by cell suspension cultures of Berberis species, Nat.Wiss. 67 (1981), 620. 

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