1. Screening and selection of highly productive cell lines
1.1. Introduction
Plant cell culture is a genetically heterogeneous system. In addition, epigenetic changes cause genetic instability leading to product accumulation only in some population of cells. The overall production of secondary metabolites in a cell culture depends on the rate of accumulation within the productive cells. By cell culture system only low yields of desired secondary metabolites are obtained. The reason for low production in culture may be due to:
- Competition between primary and secondary pathways for common intermediates
- Low levels of expression of key enzymes at rate limiting steps in a pathway
Screening and selection are often used as exchangeable terms. Screening is a passive technique by which a great number of cells alone analyzed for a certain traits and those showing the desired features are cultivated and screened. Selection process is an active process, which deliberately favor only the survival of the desired variants while wild type cells are killed.
1.2. Screening and selection
Callus culture is an easiest system for screening and selection program. Callus which shows the desired coloration is picked up and subcultured until pure cell line is established. The heterogeneity in the biochemical activity of cells has been exploited to obtain highly productive cell lines. The selection with cell suspension cultures can also be performed with a fine, rapidly growing suspension of cells consisting of small aggregates of up to 50 cells. Selection procedure can be easily achieved if the product of interest is a pigment. For example, in cultures of Lithospermum erythrorhizon, screening of a number of clones resulted in 13–20-fold increase in shikonin production. Increased production of anthocyanin by clonal selection and visual screening has been reported in Euphorbia milii and Daucus carota. High performance liquid chromatography (HPLC), radioimmuno assays (RIA) and mutation strategies have also been employed in order to obtain overproducing cell lines. The use of selective agents can be employed as an alternative approach to select high yielding cell lines. In this method, a large population of cells is exposed to a toxic (or cytotoxic) inhibitor or environmental stress and only cells that are able to resist the selection procedures will grow. P-Fluorophenylalanine, an analogue of phenylalanine, was extensively used to select high-yielding cell lines with respect to phenolics. Other selective agents consists of 5-methyltryptophan; glyphosate and biotin have also been used to select high-yielding cell lines.
2. Procedure for extraction of high value industrial products
2.1. Introduction
Plant-derived substances are of great interest due to their versatile applications. These plant derived substance are richest bio-resource of drugs of traditional systems of medicine, modern medicines, folk medicines, pharmaceutical intermediates and chemical entities for synthetic drugs. Extraction is the separation of medicinally active compounds from plant tissues using selective solvents through standard protocol. The products so obtained from plants are complex mixtures of metabolites, in liquid or semisolid state or in dry powder form, and are intended for oral or external use. These include decoctions, infusions, fluid extracts, tinctures, pilular extracts or powdered extracts.
The purpose of standardized extraction protocol for crude drugs is to attain the therapeutically desired portions and to eliminate unwanted material by treatment with a selective solvent known as menstruum. The extract obtained, after standardization, may be used as medicinal agent as such in the form of tinctures, fluid extracts or further processed to be incorporated in any dosage form, such as tablets and capsules. These products consist of complex mixture of many medicinal plant metabolites, such as alkaloids, glycosides, terpenoids, flavonoids and lignans. In order to be used as a drug, an extract may be further processed through different techniques of fractionation to isolate individual chemical entities, such as vinblastine, hyoscyamine, hyoscine, pilocarpine, forskolin and codeine.
The quality of an extract is influenced by several parameters. These parameters include plant parts used as starting material, the solvent used for extraction, the manufacturing process used with the type of equipment employed, and the crude-drug: extract ratio. The use of appropriate extraction technology, plant material, manufacturing equipment and solvent and the adherence to good manufacturing practices certainly help to produce a good quality extract. All the conditions and parameters can be modeled from laboratory scale to pilot scale, using process simulation for successful industrial-scale production.
Effect of extracted plant phytochemicals depends on:
• the nature of the plant material
• its origin
• degree of processing
• moisture content
• particle size