Biotechnological Production of Plant Secondary Metabolites

Plants are an important source of secondary metabolites that have been used throughout history as drugs, pesticides, pigments, flavors and fragrances. However, one of the main constraints to the use of cultivated plants as a source of these metabolites is the ability to ensure the constant and efficient supply of the compounds, since the yields are usually affected by the genetic background, as well as by the geographic location, edaphic and climatic conditions at the site of cultivation, combined with the potential effect of harvest and transport methods. The use of plant tissue culture has been proposed as an alternative to conventional agriculture for the production of secondary metabolites due to the possibility of controlling the quality and quantity of the compound of interest by controlling the factors affecting its synthesis and/or accumulation. Recent advances in the field of plant biotechnology show the potential of using plant cell and tissue cultures as a source for the large-scale production of valuable secondary metabolites instead of using whole plants and subsequent extensive land exploitation. Moreover, the employment of molecular biology techniques has allowed for obtaining novel products from genetically engineered plants.

The dependence of mankind upon the plant kingdom goes far beyond the production of food crops. A great number of plant species produce secondary metabolites that possess valuable properties, many of which have been studied and applied mainly to the pharmaceutical and food industries. Secondary metabolites such as terpenes and phenolic acid compounds have become very important due to their chemical and biological properties and play a major role in plant and human health. Terpenes are the primary constituents of the essential oils of many types of plants and flowers, and have been extensively used as natural flavor additives for food, as fragrances in perfumery, and in traditional and alternative medicines. On the other hand, phenolic acid compounds are plant metabolites widely distributed throughout the plant kingdom, which have a potential use as natural antioxidants in processed foods. The aim of this chapter is to provide an overview of the current knowledge on these metabolites regarding their biosynthesis, main sources and methods for their obtaining and use.

Plants are useful sources of molecules for the development of new pharmaceutical products. Coumarins are one of the most important secondary metabolites of plants and known as naturally occurring benzo-α-pyrone derivatives from the metabolism of phenylalanine. Many kinds of coumarins such as furocoumarins and pyranocoumarins arise from the biosynthetic pathway as being the substitution of the coumarin ring following by some steps such as prenylation, cyclization or glycosylation. The coumarins particularly exist in Apiaceae, Rutaceae, Fabaceae, Asteraceae and Rosaceae families, which have considerable pharmacological properties and usages. To date, more than 1000 different types of coumarins have been isolated from natural sources. Biotechnology is the most recent application in developing useful products used in medicine or industry. The coumarin production was determined in the presence of some precursors in suspensions. Thus, the biosynthesis of some coumarins has been carried out in cell cultures by different types of applications. Moreover, dipyranocoumarins as cancer chemoprevention agents and valuable dihydrocoumarin in flavor industry have been produced in callus cultures. Although the major problem of these productions is very low efficiency; most of the coumarins especially important ones in treatment are promising candidates for the production in the biotechnological process. In this chapter, these procedures will be elucidated, and the coumarin production will be debated in the optimized systems in relation to biotechnology under the light of recent articles. In addition, some beneficial information concerning coumarins will be presented.

Natural product chemistry is playing a key role in providing structural diversity and this feature makes them an important source of lead drug candidates to the drug discovery program. Nearly 50% of the prescribed drugs available on the market are of natural product origin and 25% of these commercially available drugs are of plant origin. Enzymes are responsible for performing several biochemical processes including metabolisms, catabolism, signal transductions, cell development and their growth. Over expression and hyper-activation of enzymes cause several human diseases. With the development of modern techniques in the field of molecular biology and enzymology, over expression and hyper-activation of enzymes in the body can easily be diagnosed that led to understand human diseases at the molecular level. An understanding of diseases at the molecular level resulted in the successful applications of enzyme inhibitors in clinics to treat these diseases. This chapter describes the discovery of novel glutathione S-transferase, acetylcholinesterase and α-glucosidase inhibitors from medicinally important plants and their biomedical applications. Additionally, biotechnological method to produce potent bioactive compounds on a large scale using biosynthetic information has also been proposed.

Plant cell and tissue culture strategies provide a valuable tool for the production of plant chemicals and have been extended to commercial use for biosynthesis of various high-value metabolites of importance to pharmaceutical, food and chemical industries. In this chapter, different systems for the production of anthocyanins under in vitro conditions are discussed. Anthocyanins are used to color food as a substitute of synthetic red dyes and recently, great attention has been focused on their multifaceted pharmacological potential. In vitro production of these pigments has been obtained from several plant species. Most systems are based on the use of callus and cell suspension cultures, although organ cultures have also been studied. Several studies on the regulation of anthocyanins biosynthesis under in vitro conditions have been reported, although additional research is still necessary in order to allow commercial production. In general, these studies have shown that anthocyanins biosynthesis is strongly influenced not only by physical conditions as light and temperature, but also by other parameters such as osmotic pressure, hormones, basal medium composition and nutrient stress. Strategies such as elicitation and use of conditioned medium have also been reported. In addition, the use of in vitro technologies has allowed the production of anthocyanins that usually are not found in field-grown plants. Large scale production of these pigments in standardized conditions remains as one of the great challenges for researchers in plant biotechnology.

Castilleja tenuiflora Benth. (Scrophulariaceae, “cancer herb”) is a wild plant widely recommended in Mexican folk medicine to treat tumors. Root and shoot cultures of this species were established for the production of secondary metabolites with cytotoxic and antioxidant activities. Root cultures were initiated from root tips induced in leaf explants from wild-grown plants using MS medium and 10 μM α-naphthalene acetic acid. Shoots spontaneously formed in 30-35 days and were excised. They presented continuous multiplication and elongation during subsequent subculture in free-hormone liquid medium. Antioxidant activity was measured using three in vitro models [scavenging of free radicals with 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), and the transition metal reduction in the phosphomolybdenum assay], and the strongest activity (p<0.05) was found in methanol extracts from shoot cultures. The highest contents of the phenolic compounds, flavonoid and iridoids, were also found in shoot cultures (p<0.05). Total phenolic compound levels correlated significantly with the antioxidant activity (p<0.05). Bioautography within TLC using DPPH as a detection reagent indicated that quercetin 3-β-_D-glucoside is one of the predominant contributors to the free radical scavenging of C. tenuiflora organ cultures. Aucubin, an iridoid with cytotoxic activity, was also found in the in vitro cultures. C. tenuiflora organ cultures are alternative sources for iridoids and natural antioxidants such as flavonoids and other phenolic compounds.

This chapter deals with the production of terpenoids, including pharmaceuticals and food additives through plant cell cultures, shoot cultures and root cultures obtained through biotechnological means. Plant cell and hairy root cultures are promising potential alternative sources for the production of terpenoids of industrial importance. Several strategies have been adopted for the enhancement of metabolites. There has been tremendous success in the production of terpenoids such as capsaicin from cell cultures of Salvia officinalis and Capsicum annuum. Procedures for the commercial production of paclitaxel (Taxol®) by this technique are in advanced stages of development and may soon be employed for the manufacture of these important drugs. The levels of sugar, nitrate, phosphate and growth regulators have been shown to affect the productivity of secondary metabolite-accumulating cultures. Precursor feeding has also been an obvious and popular approach to increase metabolite production in plant cell cultures. Culture environmental conditions such as light, temperature, medium pH and oxygen have been examined for their effect upon terpenoids accumulation in many types of cultures. Organ cultures are relatively more stable. There are a number of medicinal plants whose shoot cultures have been studied for terpenoids. Similarly, root cultures are valuable sources of medicinal compounds. Until now, there is no commercial process as an alternative for root-derived compounds, except in case of utilizing hairy root culture systems. The ability of Agrobacterium rhizogenes to induce hairy roots in a range of host plants has lead to studies on it as a source of root-derived pharmaceuticals and several hairy roots have been put to scale–up studies in bioreactors. Most remarkable developments of scale-up in large vessels have been in the cultivation of Panax ginseng hairy root biomass.

The Amaranthaceae family comprises of many species, which are used in nutrition and in traditional folk medicine for the treatment of several diseases such as infections, inflammation and fever. Gomphrena, Pfaffia and Alternanthera species are used in the extraction of natural pigments such as betaxanthin and betalains for application as food colorants and antioxidants. Pfaffia paniculata (Brazilian ginseng or Suma) has been indicated as a tonic, as well as having aphrodisiac, analgesic, and antidiabetic properties and may act against cancer. Gomphreneae is the major tribe of Amaranthaceae and previous chemical analyses have demonstrated the occurrence of anthraquinones, aurone, betacyanins, betaxanthins, betalains, chromoalkaloids, ecdysteroids, flavonoids, protoalkaloids, saponins, steroids and triterpenes. Biotechnological investigation with Amaranthaceae plants from the Gomphreneae tribe, demonstrated their potential for bioprospection of bioactive natural compounds such as flavonoids, steroids, terpenoides and saponins. Plant cell cultures, nowadays, are an important strategy for bioprospection of natural products. The in vitro large-scale production of bioactive compounds or extracts used as phytotherapics, pharmaceutical products, food additives and cosmetics should be encouraged because of their scientific, economical or ecological importance. Therefore, the present chapter reviews the literature data of the bioactive chemical constituents and biotechnological production of secondary metabolites in Amaranthaceae plants (Gomphreneae tribe), species that have many pharmacological properties and other applications.

Jojoba (Simmondsia chinensis (Link) Schneider) is a desert shrub which tolerates saline and alkaline soils and minimal water requirements. Jojoba seed storage lipids are liquid waxes which are esters of long chain monounsaturated fatty acids and alcohols. The oil content of the seed constitutes approximately 40-50% of the seed weight. The liquid wax is of economic importance in industry (machine lubricant) and medicine (e.g. cosmetics and anticancer compounds). Biotechnology approaches can be utilized for jojoba propagation and cloning of genes coding for economically important traits. Micropropagation of jojoba by in vitro seedling culture was achieved. This chapter will discuss the following aspects: 1) Jojoba production and economics, 2) The medicinal metabolites and their health related values, 3) Conventional and non-conventional, biotechnological, approaches to improve and utilize jojoba.

The controlled and uncontrolled use of pesticides along with unquestionable benefits can also affect other living organisms, including human beings and plant metabolism by causing abiotic stress in plants. Because possible effects on plants secondary metabolites are largely unknown, most researches focus on the influence of pesticides, their metabolism products, and residues on human health and environment. Other currently available data is related to effects of herbicides, fungicides, and plant growth regulators on phenylpropanoid and derived flavonoid metabolites as well as terpenoid metabolism.

Pesticides may influence levels of secondary metabolites like flavonoids, hydroxycinnamic acids, anthocyanins, tropane alkaloids, and volatile terpenoids by non-specific mechanisms or interfering the key biosynthesis steps. The quality of volatile oils can be altered by changing their chemical composition, especially the toxic or useful constituents. Moreover, pesticide residues can be solubilised in volatile oils. Also, pesticides are able to modulate plant metabolism affecting assimilation rate of micronutrients.

The complete assesment of plants exposed to pesticides requires knowledge of the biochemical and physiological responses of vegetal organisms to these substances in order to understand the magnitude of the agrochemicals action, but also for the rational engineering of plant secondary metabolites.

Leaves of Digitalis plants are still the major source for the isolation of cardenolides, especially digitoxin and digoxin that are used to treat cardiac insufficiency in humans. Cardenolides are characterized by a steroid nucleus with its four rings connected cis– trans–cis, having a 14β-hydroxy group and an unsaturated five-membered lactone ring at C-17β. Typically, sugar side chains of variable length are attached at position C-3 of the cardenolide genins. The use of tissue cultures for the production of cardenolides was examined quite extensively, using suspension cultures; morphogenic or embryogenic cell cultures as well as shoot or root cultures. It is important to found that cell cultures without clear tissue or organ differentiation (undifferentiated cultures) were found to be unable to produce considerable amounts of cardenolides, whereas cardenolide biosynthesis is restored as soon as morphogenesis is induced. In addition, most workers have reported that undifferentiated cultured cells either did not produce cardenolides or contained only trace amounts of it. However organredifferentiating cultures have reported to accumulate considerable amounts of cardenolides. The influence of light, phytohormones and nutrients on the production of cardenolides in Digitalis cultures was examined extensively in different studies. Expression of the cardenolide-biosynthesis system connected with differentiation of cells, different enzymes involved in biosynthesis, condition of plant tissue cultures and effect of several plant growth substances on cardenolide formation will be discussed together with the current expression systems used for the industrial production.

During the past four decades plant cell biotechnology has evolved as a promising new area within the field of biotechnology, focusing on production of secondary metabolites and in vitro propagation of plants. Boraginaceae is one of the family that biotechnological tools were applied extensively because of their economically, ornamentally and medicinally valuable seconder metabolites as well as their endangered species. The Boraginaceae family is known as Borage or Forget-me-not, contains more than 156 genera and about 2000 species including annual, perrenial herbs, shrubs and trees. Members of the family were distributed mostly in sandy -drier regions of the world. The most well-known members of the family are Forget me not (Myosotis sp.), Borage (Borago sp.), Comfreys (Symphytum sp.), and Heliotrope (Heliotropium sp.). A good number of the family members are used as a source of dye in cosmetics, food, textile and also in medical field. Selected species of the family utilized for obtaining secondary metabolites including naphtaquinone derivatives, rosmarinic acid and pyrrolizidine alkaloids. Due to their medicinal, economical and ecological importance, biotechnological tools such as plant tissue culture, metabolic engineering and in vitro micropropagation have been applied to produce biologically active compounds, pigments and to increase the population of the endangered species.

The purpose of this chapter to review studies performed utilizing biotechnological methods in diverse members of this family. Botanical aspects, traditional usage, chemical constituents and production of secondary metabolites in cultures and via metabolic engineering in some of the family members were also reviewed in brief.

Higher plants produce a wide spectrum of secondary metabolites that have been used as sources of a large number of industrial products (e.g. agricultural chemicals and pharmaceuticals). Although some of the natural products have been replaced by synthetic substitutes because of cost considerations, a number of medicinally important high value chemicals are still being extracted from plants. These products are used as intermediate/model compounds for chemical synthesis of many potent analogues/pharmaceuticals. Various natural products are used as antitumour agents or for the synthesis of antitumour agents. Of note, the readily available baccatin III have been exploited for the synthesis of paclitaxel by coupling baccatin III and the N-benzoyl-b-phenylisoserine side chain. However, novel biotechnological approaches (e.g. cell based bioprocess engineering) appears to be the best strategy since the extraction of natural products from plant sources may result in extinction of medicinal plant species (e.g. Taxus species). In fact, this approach confers cost-effective technology for the large-scale production of clinically/commercially important secondary metabolites such as paclitaxel. Since the emergence of the tissue culture technology in early 1950s, it has been increasingly advanced towards industrial production of secondary metabolites to overcome many problems associated with such approach. Given the fact that the plant cells/tissue culture systems not only provide means for biosynthesis of natural products but also serve as 'factories' for bioconversion of low value compounds into high value products, in the current chapter, we will focus on impacts of this robust technology regarding production of anticancer secondary metabolites.