Plant cell tissue culture for the production of active compounds

For plant compounds one can consider several possibilities of biotechnological production: plant cell tissue cultures, transgenic plants or plant cells, transgenic microorganisms and isolated enzymes. For transgenic systems it is necessary to know the pathway of production involved and have the genes available. Isolated enzymes can be used only for bioconversions, that is going from a precursor to the next product. One application of PCTC is to provide an alternative method for producing food ingredients and medi-cinals that have traditionally been extracted from field-grown plants. As more is known about the biochemical and genetic regulation of plant secondary metabolism, and more advances are made in the development of yield improvement strategies and design of large-scale bioreactors, commercial application of PCTC-derived molecules is expected to increase. Therefore plant cell tissue culture seems the most interesting alternative to plant metabolite industrial production, provided that

• the technology is feasible,

• the process is economically competitive with existing production systems.

At present, many cell culture systems have demonstrated this feasibility, even if as reported by Verpoorte and colleagues (1999) the optimum production (0.3 g L-1/14 days) of ajmalicine in Catharantus roseus cell cultures and that the 10-fold higher reported for berberine in Coptis japonica cells is still far below the productivity for antibiotics such as penicillin in culture of microorganisms (Verpoorte et al, 1999). Berberine, an isoquinoline alkaloid used as a remedy for intestinal disorders in the Orient is now produced by Mitsui Petrochemical group on a large scale (1.4 g L-1 in two weeks) (DiCosmo and Misawa, 1995).

Types of cultures and their applications

Several types of cell-organ culture are used to produce food ingredients and drugs of plant origin, including cell suspensions, organized tissues and transformed shoot and root cultures.

Shoot Cultures are employed as a source of axenic plant material and as a commercially viable method of plant micropropagation. Well-developed techniques are currently available to meet the demands of the pharmaceutical industry (Rout et al, 2000) and they are to be extended to dietary supplement production. These protocols of in vitro culture are designed to provide plant material with optimal levels of carbohydrates, organic compounds (vitamins), mineral nutrients, environmental factors (e.g. light, temperature and humidity) and growth regulators required to obtain high regeneration rates of many plant species. Moreover micropropagation enables cloning of the resulting plants, allows us to propagate plants that cannot form seeds because of adverse climatic conditions and assures the production of virus-free plants (Rout et al, 2000).

Cell Suspensions are plant cells freely suspended in a nutrient medium. They are the preferred type of culture for large-scale production because they are similar to micro-bial cultures and have rapid growth cycles. The existing microbial fermentation technology has been adapted to plant cell production, though some discrepancies in cell growth and yield between shake flask and fermenter cultures have been documented, as for antraquinones production in Morinda elliptica cells (Abdullah et al, 2000). Plant cells are totipotent; that is, cells in culture can produce the same metabolites as the whole plant. However, the product profiles of callus (undifferentiated cell mass) or cell suspension cultures often differ from those of parent plants. Effects of culture age and those following the application of conditioning factors are of great importance in stimulating metabolite production. Cell suspensions of strawberry, Fragaria ananassa L. cv. Shikinari, and those of Ajuga pyramidalis have been used to produce anthocyanins, which are useful as commercial dyes (Mori et al, 1994; Madhavi et al, 1996). The content of peonidin-3-glucoside, a major anthocyanin, increased as the number of days of cell growth proceeded, while that of cyanidin-3-glucoside decreased. The contents of these two major anthocyanins were also changed by adding a preculture broth as a conditioning factor (Mori et al, 1994).

Since the use of many synthetic food colourants has been restricted due to their potential risk for consumers' health, natural pigments and their biotechnological production have become of great interest in the food industry (Jimenez-Aparicio and Gutierrez-Lopez, 1999). This is the case for red-violet betacyanins and yellow betaxan-thins; betacyanins particularly have been extracted as food colourant only from beetroot (Beta vulgaris L.). Valuable betacyanin production by cell cultures of this species has been obtained by Akita and colleagues (2000). Here, as well in many other cell systems, the envisaged advantages coming from in vitro culture production are:

• no seasonal fluctuation of the product;

• reduced contamination by microorganisms;

• easy extraction of the product;

• sustainable production and conservation of natural resources in developing countries.

A recent article by Ekiert (2000) reviews the results of biotechnology applied to the family Apiaceae. These species are well-known sources of many important herbal products and drugs. The number of active compounds naturally contained by these plants and that can now be obtained in vitro is impressive: furanochromones, coumarin compounds (especially furanocoumarins), saponins, pigments (flavonoids, anthocyanins, carotenoids), phytosterols, phenolic acids and essential oils (Ekiert, 2000).

Furanocoumarins, and especially psoralen derivatives exhibiting antiproliferative and photosensitizing activity (psoralen drugs in oral and topical forms are routinely used in medicine for the treatment of vitiligo and psoriasis) (Turegun et al., 1999), have been synthesized in vitro from Ammi majus L., Heracleum sphondylium L., Pastinaca sativa L. and other plants (Ekiert, 2000). Many of these active substances advocated as natural remedies or dietary supplements (e.g. methoxy-psoralen) have been extensively studied because their consumption or topical contact (e.g. psoralens) has long been associated with adverse effects on humans (Clifford, 2000).

Carotenoids, a totally different class of plant pigments, have recently received considerable interest because of their potential in delaying or preventing degenerative conditions such as heart disease, cancer and ageing (Giugliano, 2000). Their role in the plant is to act as accessory pigments for light harvesting and in the prevention of photo-oxidative damage, as well as acting as attractants for pollinators. Their function as antioxidants in the plant shows interesting parallels with their potential role as antioxidants in foods and dietary supplements (van den Berg et al., 2000). ^-Carotene, lycopene and 7-carotene among others have been produced in cell suspension cultures of Daucus carota L. with a yield range of 3—1,000 fxg/g of cell dry weight (Ekiert, 2000).

A very important group of herbal products is the essential oils, useful in medicine and flavourings. Most articles on the biosynthesis of oil constituents have reported its failure in undifferentiated callus or suspension cultures and many authors have noticed that a high degree of cell differentiation is needed to obtain positive results. However, several papers reported the biosynthesis of essential oil components (e.g. terpenes) in callus or suspension cultures of Apium graveolens L., Coriandrum sativum L., Foeniculum vulgare Miller and many other Apiaceae (Hsu and Yang, 1996; Ekiert, 2000), in Citrus spp. among Rutaceae (Reil and Berger, 1996), in Melissa officinalis (Bolade and Lockwood, 1992) and in Artemisia dracunculus L. (Cotton et al., 1991).

Most of the economically important natural products are produced by cell cultures only at very low levels or not at all (e.g. quinine, morphine, vinblastine and vincristine). This lack of production is, first, due to the failure to find conditions in the culture to stimulate their production and, second, owing to the frequent instability of production, once it has been established (Walton et al., 1999). PCTC-derived products that have reached the market are shown in Table 6.1. Shikonin, a red naphthoquinone

Table 6.1 Economic processes for the production of secondary compounds by plant cell cultures (from Walton et al, 1999).

Product

Plant species

Company

References

Shikonin

Lithospermum erythrorhizon

Mitsui Petrochemical Ind. Ltd

Fujita et al. ,1982

Ginsenosides

Panax ginseng

Nitto Denko Corp.

Ushiyama, 1991

Purpurin

Rubia akane

Mitsui Petrochemical Ind. Ltd

Walton, et al, 1999

dye produced in Japan by plant cells of Lithospermum erythrorhizon and used to promote healing, can be mentioned as one of the few pure chemicals so far produced on an industrial scale (Verpoorte et al., 1999). This species can provide accumulation of the compound exceeding 20 per cent of the dry matter of the cell culture (Walton et al., 1999). Ginseng is the third best selling herbal supplement in the United States, both as a disease-healing drug and as a general tonic, and it is now also being used as a flavouring agent in foods. The predominant pharmacologically active constituents of Panax are ginsenosides, at least 25 of which have been identified and are present in variable amounts and ratios to one another; depending on the particular species, variety and conditions of growth (Carabin et al., 2000). Plant cell and tissue culture methods have been explored as potentially more efficient alternatives for the mass production of ginseng and its bioactive components. Research into ginseng cell and tissue cultures started in the early 1960s and commercial applications have developed since the late 1980s (Ushiyama, 1991; Wu and Zhong, 1999; Carabin et al, 2000).

Other PCTC-derived products are polysaccharide mixtures (Verpoorte et al., 1999), rosmarinic acid and sanguinarine and there has been recent success with the anti-tumoural taxol, though whether this expensive procedure will become commercially available is not clear yet.

Plant cell cultures play an additional important role during the industrial development of new plant-derived compounds as drugs, when agricultural production is not feasible or not yet available (Verpoorte et al., 1999), or when these valuable compounds have limited occurrence in plants. Such is the case of the furanochromones of Ammi visnaga (L.) Lam. which have interesting vasodilatation properties on coronary and renal vessels (Ekiert, 2000).

Another important application is the production of novel natural pharmaceutical and food products that cannot otherwise be found in nature, like paniculide from Andrographis paniculata Ness. (Allison et al, 1968; Mandal et al, 2001), pericine from Picralina nitida and podoverine from Podophyllum versipelle (Walton et al., 1999), two new substances active on the central nervous system. As reported by Walton and colleagues (1999), these new biologically active compounds are highly attractive to several companies since they can be patented. For such applications, an understanding of the manufacturing process will help to determine the intended or unintended modifications made to the product and thus help the safety evaluation of these novel products (Fu, 1998).

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