Berberine (Thalictrum spp.)

Berberine-containing plants have been used for more than 2000 years in traditional folk medicine for therapeutic treatment. Berberine alkaloids are widely distributed in plants of the families Berberidaceae, Ranunclaceae, Menispermaceae and Rutaceae. Coptis species (Ranunclaceae) and Phellodendron amurense (Rutaceae), typical berberine-containing plants, have been frequently used as a folk antidysenteric in Japan and East Asia, effective by its berberine alkaloids. Another berberine-containing plant, Berberis aristata (Berberidaceae) was used for cholera and other bacterial diarrhoea. The protoberberine alkaloids and their derivatives show at least four types of biological activity: antimicrobial, uterine, anti-inflammatory, and antileukemic and antineoplastic.. Protoberberines and their derivatives as potential anticancer agents have been reviewed and their chemistry has also been reviewed. The cultivation of the rhizome of Coptis species is very slow, and takes 5-6 years to use as the raw material and as a source of berberine. The price of Coptis rhizome is at present very high (20000 yen/kg, 1983 in Japan), almost the same as that of Panax ginseng.

It is of pharmaceutical significance to investigate callus culture of these plants for berberine production. For the first time we succeeded in culturing the callus of Coptis japonica and isolated berberine (1), palmatine (2), coptisine (3), jatrorrhizine (4) together with two other unknown protoberberine-type alkaloids and aporphine type alkaloid, magnoflorine (9) (). Coptis callus retains the synthesizing activity of the berberine and other alkaloids, undergoes differentiation to form whole plants from callus cultures, and the alkaloid content of the regenerated plants was similar to that in the rhizome of the original plant. These results suggested the production of berberine from Coptis callus tissue. Then we started with intact plants other than Coptis producing the berberine, including other protoberberine-type alkaloids, and have investigated the production of berberine or other protoberberine-type alkaloids in callus tissue of Thalictrum, a member of Ranunclaceae and also Berberis, Nandina (), and Mahonia () belonging to Berberidaceae. These original plants contain berberine, together with other protoberberine-type and isoquinoline-type alkaloids. Further, we have studied chemical constituents of the extract of these callus tissue from a chemotaxonomic point of view. The present review is primarily based on the work on Thalictrum species. Thalictrum, belonging to Ranunclaceae, is distributed in the Northern Hemisphere, North America, Europe, Asia, and Africa, and has about 80 species. In Europe these plants are popular weeds called meadow rue, its name has been found in old literature, and there are many varieties. These Thalictrum plants are used medicinally as folk medicines in Europe: Th. flavum L. for stomach diseases, as aperients, diuretic, antifebrile, etc., Th. angustifolium L. and Th. aquilegifolium L. in India and China. In Japan also, the plants grow wild in the fields or hilly districts as a perennial herbaceous, and 16 species are found in flora in Japan, some of which are medicinal. Thalictrum plants are rich in alkaloids. There seems to be considerable variation in the presence of individual alkaloids in the same species growing in different geographical areas. The alkaloids are reported in sufficient amounts in the roots, rhizome, and foliage, and in relatively less amounts in fruits and seeds. The alkaloids so far reported from various Thalictrum spp. number over 60.

Thalictrum minus L. var. hypoleucum {Thalictrum thunbergii DC, Japanese name Akikaramatsu) is a perennial herb and grows wild in field or hilly districts. The dried leaves and stems of Th. minus are used as folk medicine for stomach disorders (commercial crude drug “Takatogusa” in Japan). There are many reports on the investigation of alkaloids, berberine, takatonine, magnoflorine, and bisbenzyl types from this plant.

Berberine: Prospects

Only small amounts of berberine as the protoberberine-type alkaloids were found in Thalictrum minus original plant, but the callus cultures produced several protoberberine-type alkaloids, moreover, berberine is the main product. In plant tissue culture, it has been generally observed that production of useful secondary products is negligible or lower than that in the original plants. However, cultured cells of Thalictrum had a higher berberine content than that of the original plant, and its content of cultured cells even without the selection showed ca. 350 times higher as compared with that in the original plant. The cell suspension cultures of this callus produced a large amount of berberine (0.8 g/1 of medium), released large amounts of berberine into the liquid medium, and that considerable amounts of berberine crystallized as the nitrate or chloride depending on the kind of major anion present in the medium. Moreover, the production of berberine in the cultured cells was also greatly stimulated by the addition of BA. It is the first case that the alkaloids accumulate as crystals in the culture medium of plant cells.

In contrast with cell cultures of Thalictrum, Coptis and Berberis cells in culture accumulated most of the protoberberine alkaloids in the cell and released only a small amount of the alkaloids into the medium, when partial autolysis of senescent cells occurred. Crystallization of alkaloids in the medium was not observed in both cultures.

Although the total amount of berberine production was still higher in Coptis callus (1.3 g/1 medium) than in Thalictrum callus (0.8 g/1 of medium) in suspension culture because of the superior growth of the former, Thalictrum callus seems to present a possibility for the industrial production of berberine in the future because of the simple isolation and purification of berberine production. However, one still needs to investigate the culture medium.

Moreover, it is interesting that in the biosynthesis of the alkaloids the cell cultures produced (1-2, 4-9), but except for (7) and (9), other protoberberine alkaloids were not produced in the original plant, and the berberine content of the original plant differs slightly by its habitat. These results suggested that there are biochemical differences between the intact plant and the callus tissues derived from them.

A. Ikuta and H. Itokawa, Medicinal and Aromatic Plants I (1988)