ANEXO G: PROCEDIMIENTO RETIRO DE PRODUCTO NO CONFORME 120

In the same way that it is possible to culture undifferentiated plant cells, so aseptic suspension cultures of leaves and roots can be main- tained. The organs can be obtained in the culture either by differenti- HO 7-Hydroxycoumarin derivatives HO OH O R1 R1 R2 O R2 R1 R2 CH3O R1 O O R1 O O

Marmesin derivatives Psoralen derivatives

Herniarin derivatives Series A (R1 = R2 = H) Series B (R1 = H; R2 = CH3) Series C (R1 = CH3; R2 = H) C O C O O C O O C O C Fig. 13.2

Biochemical conversions involving Ruta graveolens cell cultures.

PRINCIPLES RELATED TO THE COMMERCIAL PRODUCTION, qUALITy AND STANDARDIzATION Of NATURAL PRODUCTS

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ation from callus tissue cultures by suitable hormonal manipulation, or by the use of sterilized roots or growing points from whole plants or seedlings. Often, these cultured organs will synthesize secondary metabolites which may be either non-existent or in poor yield in the normal cell culture. Thus, cardenolide production in Digitalis lanata and D. purpurea cultures increases as tissue differentiation proceeds. Enhanced production of alkaloids occurs when roots develop from the callus cultures of the tropane alkaloid-producing Solanaceae. In contrast to the latter, in which leaf differentiation alone produces little alkaloid, leaf organ cultures of Catharanthus roseus and Rauwolfia

serpentina synthesize a variety of alkaloids. Dimeric alkaloids have been detected in organ cultures of C. roseus, suggesting the possibil- ity of an efficient production system for these valuable alkaloids. The dimers occurred only in those cultures which also contained vindoline and catharanthine. Whereas cell suspension cultures of Papaver brac-

teatum were found to synthesize orientalidine and sanguinarine, the root and shoot cultures produced thebaine. For ginsenoside production, ginseng root cultures have been grown in 20 000-litre bioreactors.

As with cell culture, elicitation can give increased yields of second- ary metabolites. Thus both normal and hairy (q.v. below) root cultures of Hyoscyamus muticus when treated with jasmonic acid and its methyl ester at concentrations of 0.001 to 10 μM produced large increases in the levels of methyl putrescine and conjugated polyamines. However the increase of tropane alkaloid production was not remarkable (S.-Bionde et al., Plant Cell Rep., 2000, 19, 691).

Transformed root culture—‘hairy root’ culture

Certain soil bacteria of the genus Agrobacterium cause a transfor- mation of plant cells by introducing into their genome t-DNA from a bacterial plasmid. Such transformed roots, produced by inoculat-

ing the host plant, when grown in a hormone-free medium give rise to copious roots referred to as ‘transformed roots’ or ‘hairy roots’ (see Chapter 14). On removal of the Agrobacterium the roots continue to develop profusely and for some plants which normally produce secondary metabolites the hairy roots accumulate these metabolites in quantities comparable to those found in the normal intact plant.

Agrobacterium rhizogenes and A. tumefaciens are the bacterial species most commonly used to effect transformation.

Compared with the few examples given in the 13th edition of this book there is now a considerable bibliography on transformed root cultures of medicinal plants. Some examples are given in Table 13.2.

As with ordinary cell cultures and root cultures it is possible to utilize transformed roots to carry out biological conversions not normally associated with the intact plant. The rapid growth rate of hairy roots offers the possibility of rapid conversions. Ginseng hairy root cultures have been shown to convert digitoxigenin (the agly- cone of a number of Digitalis cardiac glycosides, q.v.) into new com- pounds by esterification at C-3 with stearate, palmitate and myristate, and by the formation of gentiobiosides and sophorosides. Parr et al. (Phytochemistry, 1991, 30, 2607) in studies on the biosynthesis of tro- pane alkaloids fed the S-analogue of tropinone (8-thiabicyclo[3.2.1] octan-3-one) to transformed root cultures of Datura stramonium and obtained the S-analogue of tropine, together with the 3-O-acetyl ester.

Cell cultures derived from A. rhizogenes-transformed Papaver

somniferum tissue have been studied for alkaloid production (R. D. Williams and B. E. Ellis, Phytochemistry, 1993, 32, 719). As with nor- mal cultures, no morphinan alkaloids were produced but large amounts of sanguinarine were.

Table 13.2 Metabolites of some transformed (hairy) roots. Alkaloid-containing plants

Atropa belladonna Increase in growth rate compared with untreated roots

Catharanthus roseus Optimization of selected lines may give source of catharanthine and vindoline Cinchona ledgeriana quinoline alkaloid production comparable with that of intact plants

Datura stramonium Alkaloid content comparable with normal roots of intact plants; increased by use of various elicitors, e.g. methyl jasmonate

Hyoscyamus albus New piperidone alkaloid obtained together with tropane alkaloids

H. muticus Culture treated with chitosan accumulated hyoscyamine 2.5 to 3-fold compared with untreated hairy roots Narcissus confusus Improved production of galanthamine in shake cultures by use of methyl jasmonate as elicitor

Solanum laciniatum Solasodine yield increased about four-fold flavonoids

Glycyrrhiza glabra Known and new flavonoids produced; also a new prenylated biaurone Iridoids

Valeriana officinalis yield of valepotriates four times that found in normal 9-month-old roots Lignans

Linum flavum 5-Methoxypodophyllotoxin production 2–5 times that of untransformed roots; 5–12 times higher than cell suspension culture; comparable with natural roots

Polyacetylenes

Lobelia inflata Polyacetylenes produced, some having a gentiobiose moiety. These compounds have anticancer activity quinones

Sesamum indicum yield of antimicrobial naphthoquinone increased by more than 50 times that found in intact plant Steroids

Panax ginseng Japanese patent for production of ginsenosides; diol and triol type ginsenosides produced. Production more effective than with ordinary root cultures

Trigonella foenum-graecum By optimization of the cultural conditions, up to three times the production of diosgenin compared with non-elicited roots

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CLONAL PROPAGATION

As noted earlier (Fig. 13.1), by adjustment of the plant growth regula- tors in the cell culture medium it is possible to promote differentiation of organs from callus tissues and to carry these forward to produce entire plants. As all cells of the callus are derived from a single meristem, all regenerated plants should be genetically identical. This fact has obvious commercial implications for the production, in a short period of time, of uniform crops derived from a small number of desirable plants (or even a single individual).

In 1980, Levy reported from Ecuador on the first large-scale com- mercial application of in vitro culture for the mass propagation of clones of pyrethrum plants. The scale of such projects is evident from the original objective, which was to produce, from 57 superior clones isolated over a 10-year research period up to 1976, 12 million plants per year to reach a plantation target of the number required for 1000 ha of land over 4 years. Further details of the process are given in the 15th edition of this book. Similar techniques are now practised for other crops of pharmaceutical interest, benefits being the ability to introduce selected high-yielding strains on a commercial scale in a short time, the creation of improved growth characteristics and a greater ease of plantation planning.

Clonal propagation is a potentially valuable method for producing high-yielding crops of species which tend to be variable when grown from seed. Fennel (Foeniculum vulgare), for instance, is genetically

heterozygous and produces wide variations in oil yield and compo- sition. In 1987, Miura et al. (Planta Med., 1987, 53, 92) recorded a 2-year trial involving the production of uniform clonal plants derived from somatic embryoids of suitable parent plants. In the normal sexu- ally propagated plants the anethol content of fruits varied from 0 to 12.9% (mean 2.82%), whereas in the clonal plants it showed a narrower distribution of 0.7–3.0% (mean 1.92%).

Genetic homogeneity

In practice, genetic changes can occur in cells during their arti- ficial culture, and careful regulation of the medium is necessary. However, it has been shown, at least for Datura innoxia, that on dif- ferentiation it is the normal 2n plants, as distinct from the abnormal chromosomal types, that are favoured. Clonally propagated spe- cies for which the homogeneity of the secondary metabolites has been shown include Aconitum carmichaelii, Angelica acutiloba,

Bupleurum falcatum, Gentiana scabra, Rehmannia glutinosa and

Stevia rehaubiana.

Further reading

Verpoorte R, Heijden R van der, Schripsema J 1993 Plant cell biotechnology for the production of alkaloids: present status and prospects (review with

130 refs). Journal of Natural Products 56: 186

Zafar R, Aeri V, Datta A 1992 Application of plant tissue and cell culture for production of secondary metabolites (review with 80 refs). Fitoterapia 63: 33

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