Today, despite continuous improvements in plant cultivation and product manufacturing procedures, the required safety, availability and purity standards of plant ingredients cannot be entirely ensured because of the nature of the traditional production process itself. On the other hand, a biotechnological approach using cultures of plant cells could open new avenues in the production of plant ingredients. While plant cell culture biotechnology is well known in the scientific community, it has not yet been fully developed on an industrial scale despite the numerous advantages over conventional methods. As well as being a totally environmental friendly technique, it is able to ensure a high degree of safety, reproducibility and standardisation in the composition of the final product.
Products of botanical origin: issues
Safety
Botanicals are plagued by numerous problems, mainly concerning their safety and quality. In fact, environmental, chemical and microbiological contaminations have been widely documented, together with reported adulterations with synthetic pharmaceuticals and errors in the taxonomic identification of the plant itself.1 However, the intensification of organic cultivation, the application of rigorous protocols for the harvesting, storage and handling of the raw materials, and an increase in quality control testing in the various phases of the manufacturing process have determined a marked improvement in terms of safety of the finished products but are yet unable to guarantee the total absence of polluting or toxic agents.
Quality and purity
Secondary metabolites are substances belonging to different chemical classes (penylpropanoids, alkaloids and terpens) which are synthesised by plants in response to various biotic and abiotic factors and have a defensive function, namely to protect the plant against environmental mechanical, physical (e.g. exposure to UV rays), or biological (e.g. microorganism attack) threats.2,3 The profile and concentration of the secondary metabolites of the plant are therefore strongly dependent on the environmental conditions under which the plant is grown, which are numerous and intrinsically variable. The composition of the soil, including the microflora, climatic conditions, seasonal variations and chemical treatment can all affect the secondary metabolite content and the poor uniformity of the genetic material of the plants themselves.4,5 Consequently, according to their physiological role in plants, secondary metabolites are often tightly correlated to the claimed biological activity of the derived ingredient. Despite the application of strict, and often costly, cultivation and extraction protocols, control of all the environmental and seasonal variables becomes almost impossible with conventional cultivation techniques, thus limiting the chances to obtain plant products with a defined and standardised composition, and above all, a reproducible biological activity.
Availability
Numerous poorly controllable factors have hindered the full use of the beneficial properties of active substances of plant origin, rendering it difficult to properly fulfil the requests of an evergrowing market. Firstly, the low content of some active ingredients in plants force the collection of large amounts of plant tissue and use of time-consuming, costly and ecologically unsustainable purification procedures, i.e. the widespread use of organic solvents with great environmental hazard. Furthermore, many of these active ingredients are found in rare, protected or endangered plants, or in plants growing in remote, barely accessible areas, thereby making their harvesting problematic.6 In addition, the availability of these species for large-scale commercial purposes is hampered by factors intrinsic to the plants, such as their biological cycle and balsamic period.
Plant cell and tissue culture technology
The growth of living organisms in culture, or fermentation is widely used on an industrial scale in the production of raw materials of high value, also for cosmetic application, such as ascorbic, lactic and hyaluronic acid, all obtained by bacterial fermentation.
Theoretical basis
Plant cell culture technology comprises a group of means and methods employed for the growth of plant cells, tissues or organs in a nutritious medium in the absence of microorganisms. However its application has hitherto been limited to laboratory environments due to the prohibitive costs involved in research, development and industrial scale-up.7,8 In fact, the industrial development of this technology has thus far been limited to complex, difficult to synthesise molecules such as taxol (Paclitaxel), an anti-cancer drug initially obtained by extraction from the arils, needles and bark of Taxus spp., and nowadays derived mainly by biotechnological production. This technique represents an optimal solution that has negated problems such as the slow growth cycle of the plant source.9 Plant cell culture allows for the synthesis of the bioactive substances present in plants, and is often the only available source, unhampered by quantitative limitations, of active ingredients which are poorly available or difficult to manufacture by chemical synthesis. These aspects have also been highlighted by the FAO (Food and Agriculture Organization of the United Nations) which, as far back as 1994, proposed plant cell and tissue culture as a suitable biotechnological process for the production of substances and metabolites like food additives.10
Biological basis
The biological basis for the technique lies in the presence in all higher plants of a totipotent stem cell reservoir in specialised tissues (meristems) located mainly at the growth tips of the plant, namely roots and buds. These meristematic cells, while small and undifferentiated, provide the main source for renewal and generation of differentiated cells. Other cells, located in more differentiated plant tissues, maintain “stem cell like” properties (secondary meristems), may reactivate full “totipotency” under specific conditions such as those occurring following tissue damage.11,12 In the generation of plant cell lines, the capacity of adult plant cells to “reawaken” is harnessed and their staminal potential artificially released by making incisions in the plant tissue. Inflicting mechanical damage to the plant induces the rapid proliferation of a tissue repair response, the so-called “callus”, which is made up of aggregates of undifferentiated meristematic or stem cells. In the presence of adequate nutritional support, the callus can grow indefinitely in culture and, using suitable hormonal stimuli, can be induced to regenerate mature plants (a technique known as micropropagation).
Methodology
The first step for the generation of a new plant cell line is sterilisation of the tissue. This fundamental phase is extremely critical as all microorganisms (bacteria, fungi and moulds) which could impede or inhibit the development of the culture need to be removed without causing irreversible damage to the meristem cells required for the generation of a new cell line. Subsequently, the sterilised plant tissue is reduced to minute fragments (termed “explants”) and placed in Petri dishes containing a solid antibiotic-free nutrient medium (Figs. 1, 2). The callus tissue generated by this procedure is then regularly transferred to appropriate culture, containing all the substrates necessary for cellular metabolism medium, ensuring its growth and maintenance. The most important of these is a source of organic carbon and energy, usually sucrose, since cells cultured in the dark lose their photosynthetic capacity and thus behave as heterotrophic organisms. Plant hormones (auxins and cytokinins), and several vitamins, and inorganic macro- and microelements, must also be supplied.13 Variation of the composition of the culture medium allows the cell lines with the most advantageous biochemical and metabolic characteristics to be selected over time: repeated transfers allow selection of only the most productive cells, up to the point of acquiring cell lines with stable and homogeneous characteristics (Fig. 3). As no genetic engineering is carried out in this procedure, a non-GM plant cell line results, whose selection is based entirely on its morphological, biochemical and growth characteristics. An essential step for industrial scale up of the manufacturing process is the adaptation of the selected cell lines for growth in liquid media, as this substantially increases the biomass volume. Cultures in suspension need to be gradually adapted to growth in ever-higher capacity bioreactors (scale up) and must be continuously mixed to allow the degree of gaseous exchange required for cellular metabolism. The advantages and results obtained with this process have been termed a “High Tech Nature” (HTN) approach, thus underlying a new positive relation among generally conflicting “technology” and “nature”.
Sourcing ingredients from plant cell culture technology
At the end of the fermentation cycle, the biomass expanded in the liquid culture is processed differently in order to obtain two distinct types of cosmetic ingredients.
Stems G
The first type of products is made of a stable glycerine dispersion of whole plant stem cells. It therefore contains the secondary metabolites produced by the plant cell together with the complex of polysaccharides, phytosterols and amino acids with hydrating and nutrient properties. An example, particularly important to highlight the usefulness of the plant cell culture technology, is its application to Leontopodium alpinum, more generally known as edelweiss (Fig. 4). This is a rare plant growing in the Alps and it is a protected species because it is considered close to extinction. L. alpinum lives in a very harsh environment, particularly exposed to high levels of UV light, cold temperatures and other abiotic stresses. Its survival depends therefore on the ability of the plant to synthesise a number of defensive molecules that can protect it from the various types of environmental stresses. Some of these molecules have been recently identified and most belong to the class phenylpropanoids.14,15 Particularly interesting are the leontopodic acids (Fig. 5) which provide an antioxidant capacity at least three times that of trolox taken as a benchmark (Fig. 6).16 However, edelweiss plants are strictly controlled for commercial use and are available in limited amounts. Thus one of the few possibilities, if not the only chance, to provide such important substances is by plant cell cultures. Leontopodic acids, together with other interesting antioxidant molecules, such as dicaffeoyl quinic acids, have been found in L. alpinum cell cultures and are made fully available as cosmetic ingredients by a stem cell preparation together with polysaccharides and lipids of the same cells (trade name: Leontopodium alpinum stems G).
HTN actives
The second type of products is characterised by a higher content of secondary metabolites. It is obtained through the homogenisation of the plant biomass, to allow the release of the intracellular secondary metabolites, the filtration and the extraction so as to concentrate the compounds of interest. The final product is a powder with a defined composition and titrated in the active substance typical of the cell line. An example of this ingredient type is Syringa vulgaris (lilac) cell culture extract titrated in verbascoside (trade name: Dermasyr) (Fig. 7). Verbascoside, also known as acteoside, is a phenylpropanoid glycoside (Fig. 8) containing caffeic acid and hydroxytyrosol and it is a secondary metabolite widely present in many plant species but found in low amounts (0.01% of dry weight). Instead in plant cell cultures, the amount of verbascoside can be highly increased (up to 10% of dry weight) by the continuous selection of the highest producing cell strains (Fig. 8). Several studies within the last few years have shown that verbascoside has many important biological functions, some of which are extremely interesting for skincare applications. Indeed, further to its well known scavenger activity for oxygen and nitrogen free radicals, it also chelates transition metals and inhibits many pro-inflammatory processes. These include the activity of the COX-2 enzyme in macrophagic cells, the inducible nitric oxide synthase gene expression, and the nitric oxide production in endothelial cell lines; it suppresses the release of arachidonic acid and eicosanoid synthesis and inhibits NF-kB activation.16,17,18,19,20,21 A recent result has also shown that verbascoside can also reduce the enzymatic activity of 5-alpha-reductase, the key enzyme involved in the biotransformation of testosterone into dihydrotestosterone (DHT). DHT has been recognised as the major cause of skin alterations such as juvenile acne, and hormonal dependent hair loss (androgenic alopecia).22 All of these findings provide insights into many new skin and personal care applications for this cell culture derived active substances.
Advantages
Plant cells selected and cultured on an industrial scale in sterile bioreactors are effectively nurseries of plant substances, thus offering numerous advantages with respect to cultivation of plants in the open field. First and foremost, toxic substances such as herbicides, pesticides, heavy metals and other environmental culture is grown in sterile antibiotic-free conditions. Moreover, the strict control of the culture conditions and the continuous selection activity considerably reduce the appearance of spontaneous variation, and guarantee a reproducible profile of secondary metabolites, thereby overcoming the issue of variability linked to climatic and geographical conditions. Furthermore, this technology obviates obstacles such as the natural biological cycle of the plant and the seasonality of the secondary metabolites, thereby guaranteeing full availability of the constituents at all times. In addition, the degradation of active ingredients that usually occurs during storage of the plant material is greatly reduced as extraction is performed immediately following completion of the fermentation cycle, with minimal loss of the substances responsible for the beneficial properties of the extracts. The final result is thus the production of plant ingredients by rapid and flexible means, with no limitations on quantity, and with a greatly improved safety profile and a highly standardised composition. Moreover, this biotechnological process has a lower environmental impact than other conventional extraction methodologies, as neither massive harvesting of plant matter nor intensive exploitation of the land, especially critical in the case of slow-growing or protected species, are necessary. Finally, as the plant biomass obtained via fermentation has a very simple and homogeneous composition with a high concentration of the desired metabolites, a relatively simple and efficient extraction procedure is required, entailing a greatly reduced use of organic solvents with a major environmental impact.
Conclusion
Biotechnology applied to the production of natural compounds from plant cell cultures offers higher safety, availability and standardisation levels over more conventional processes using open field cultivation. Furthermore, this technology is fully eco-friendly and non-GM, and allows the availability of active substances even from protected or endangered plants, without affecting the delicate natural ecosystem balance and biodiversity. Despite being a very challenging technology, plant cell culture is potentially a huge source of new cosmetic ingredients able to answer the main needs of the cosmetics market: innovation, safety and eco-sustainability.
References
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