Plant Cell Culture As Emerging Technology For Production Of Active Cosmetic Ingredients

Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com

Vasil Georgiev1,2 Anton Slavov2 Ivelina Vasileva2 Atanas Pavlov1,3

Plants have always been the main source of active cosmetic ingredients, having proven health beneficial effects on humans, such as anti-aging, antioxidant, anti-inflammatory, UV-protective, anti-cancer, anti-wrinkle, skin-soothing, whitening, moisturizing, etc. Extracts from herbal, aromatic, and/or medicinal plants have been widely used as effective active ingredients in cosmeceuticals or nutricosmetics, especially in products for topical application and skin-care formulations. However, over the past decade, there has been an increasing interest to plant cell culture-derived active cosmetic ingredients. These are “new generation” of high-quality natural products, produced by the modern plan biotechnology methods, which usually showed stronger activities than the plant extracts obtained by the classical methods. In this review, the advantages and the current progress in plant cell culture technology for the production of active cosmetic ingredients have been summarized and discussed in detail within a presented case study for calendula stem cell product development.

Keywords: Calendula officinalis L. / Cosmeceuticals / Nutricosmetics / Plant cells / Plant stem cells / Polysaccharides / Tissues and organ cultures

cistanche——natural nourishing plants

1 Introduction

Plants are the oldest source of natural compounds with medicinal and cosmetic properties explored by mankind. Even nowadays, between 70 and 80% of people worldwide rely on traditional herbal medicine to cover their basic healthcare needs [1]. In fact, 11% of the existed essential drugs used in modern human medicine are still of plants origin [2]. The percentage of plant-derived natural products, applied as active ingredients in cosmetics, is much higher. In accordance with the continuous increase of the world population, the global demand for natural plant ingredients is expected to continue its exponential growth [1]. As result, we are already witnesses of mass overexploitation of natural habitats of some herbal and aromatic plants, placing them under threat or even leading to their extinction [3]. This negative tendency is especially dangerous for medicinal plants, which in most cases are rare and endemic species. These species often grew under extreme climate, unique soil composition, or specific latitude. Their populations usually have been characterized by low levels of genetic diversity, which makes these species extremely vulnerable to genetic erosion and decreases their chance to survive in case of environmental pollution [4]. Moreover, plant-derived active ingredients are usually a very complex mix of bioactive molecules, which cannot be easily replaced by chemically synthesized analogs. To solve the above problems, plant biotechnology has provided the tool, which can secure a more eco-friendly and sustainable supply of valuable phytochemicals with healthbeneficial properties, to cover the growing demands of the cosmetic industry. Over the last decade, the advantages of plant cells and tissues culture technologies have been widely explored in the development of highly-efficient platforms for more rapid production of pharmaceutically important molecules of plant origin or for heterologous expression of therapeutic proteins [5]. Nowadays, there are many cosmetic products, including both cosmeceuticals and nutri-cosmetics, which have active ingredients, derived from plant cell culture technology. The aim of this paper is to review the recent progress in plant cell technology for cosmetic application and to provide a short overview of commercialized plant cell culture-derived active cosmetic ingredients available on the market. The information in this review was retrieved by an exploratory electronic survey of plant tissue culture-derived cosmetic ingredients and their technical specification datasheets, conducted by search in the technical websites of the major suppliers such as Prospector-R search engine, SpecialChem, Cosmetic Design Europe, the academic search engines Scopus, ScienceDirect, Google Scholar, and PubMed, and the popular search engine Google. In addition, the main steps in the development and characterization of plant cell culture-derived active ingredients are closely discussed in the presented case study for the development of the InnovaStemCell Calendula product.

whole cistanche roots

2 Plant extracts as active ingredients in cosmetics

Plants are a rich source of countless metabolites with potential application in cosmetic products. Since time immemorial, mankind has used different plants and their extracts to create cosmetics with the aim of establishing a state of eternal youth [6]. Nowadays, plant extracts are becoming the most popular active ingredients of cosmetics due to the ever-increasing demand for natural compounds which in addition to esthetic looks can provide additional health benefits. The development of such products, known as “cosmeceuticals”, reflects a most recent trend in the modern cosmetics and personal care industries. Indeed, plants are rich in endogenous bioactive metabolites with potential cosmetic and pharmaceutical applications [7–9]. Most of these phytochemicals, such as polyphenols, phenolic acids, triterpenes, flavonoids, stilbenes, steroids, carotenoids, steroidal saponins, sterols, fatty acids, polysaccharides, sugars, peptides, etc., could be extracted with appropriate solvents and used as active ingredients present in cosmetic formulations [10]. Because of this, huge numbers of plant sources have been explored by the cosmetics industry in search of innovative active ingredients which combine some specific pharmacological properties, such as antioxidant, antimicrobial, antiviral, anti-cancer, antifungal, anti-inflammatory, anti-allergy, etc., and also showing strong moisturizing, anti-aging, anti-wrinkle, and UV protective effects [11]. However, the quality and phytochemical profiles of plant extracts varied in a wide range, depending on climate, soil, latitude, seasonal factors, time of harvest, and the field management practice, which could be a challenge to standardize their activities [12]. The search for novel natural phytochemicals has led to the gathering of bioactive extracts not only by plants, but also from mushrooms, algae, and also, by utilization of by-products of plant origins [13–17]. Nowadays, with the growth in consumers’ interest in effective and safe natural products, a “new generation” of high-quality bioactive phytochemicals, produced by the plant cell culture technology, have been introduced in the past decade, and now, their presence in the cosmeceuticals market has been steadily on the rise.

cistanche roots & cistanche extract

3 Plant cell culture technology: principles for the production of active cosmetic ingredients

Plant cell culture technology is a technique for the growth of plant cells under strictly controlled environmental conditions. Because plant cells are considered totipotent, they have the potential to express the full genetic machinery coded in the nucleus, and thus, they are able to produce the full spectrum of characteristic secondary metabolites, found in mother plants. Plant cells are amenable to good manufacturing practice procedures and can be easily propagated by using large volume bioreactors independently of climate or soil or field management practices [5, 18]. Moreover, in vitro cultured plant cells are characterized by fast growth, and the ability to accumulate a large amount of uniform biomass for a short period of time [19, 20]. This is a very important advantage especially for the production of rare bioactive compounds, such as resveratrol, paclitaxel, or terpenoids, which are usually found in low concentrations in plants and their isolation and purification requires the processing of large amounts of plant biomass [21–25]. Additionally, plant cell culture technology offers a reliable and powerful production platform for a continuous supply of contamination-free, phytochemically uniform biomass from herbal, aromatic, medicinal, and even from rare and threatened plant species [26]. The perspective to obtain natural phytochemicals by using an environmentally sustainable biosynthetic platform made the plant cell culture technique exceptionally attractive for the production of active ingredients for high added values “green” cosmetic formulations [27–30]. It should be noted, that active cosmetic ingredient, obtained by plant cell culture technology are popularized among the customers under the name “plant stem cells”. Here it is important to understand that the term “stem cells” used in this phrase is not always referred to real plant stem cells. Most of the existed plant cell culture production platforms are in fact developed on the basis of the use of dedifferentiated plant cells rather than on the culturing of the true plant stem cells. Dedifferentiated plant cells are obtained by dedifferentiation of already differentiated mature plant cells from different specialized tissues, whereas the true plant stem cells should be never differentiated in their life cycle. Because raised from the dedifferentiation of differentiated cells, the dedifferentiated plant cell cultures could inherit some epigenetic modifications, characteristic for the type of the tissues they have obtained from, and thus, they could be very heterogeneous in their biosynthetic and growth properties. This fact made it possible to generate almost unlimited numbers of plant cell lines with unique phytochemical profiles and growth characteristics even from the same plant, used for their initiation. For this reason, the terms “leaf stem cells”, “meristems stem cells”, “root stem cells” “rhizome stem cells”, “flower stem cells”, “fruit stem cells”, etc. could be often seen in INCI names of active cosmetic ingredients, but in fact, all of these refers to dedifferentiated plant cell cultures. On the other hand, there are plant cell technologies existed, which are developed on the basis of the cultivation of true plant stem cells – the cambial meristematic cells (CMC). These cells are isolated by the cambial layer and consist only of true meristem stem cells [31, 32]. Cambial meristematic cells are characterized by fast and uniform growth, lack of epigenetic modifications, and ability to produce predictable yields of secondary metabolites when treated with stimulating factors such as elicitors [33]. This technology has been used by the Korean company “Unhwa Corp.” to produce several active cosmetic ingredients, by using the patent-protected expression platform DdobyulR, developed on the basis of propagation of cambial meristematic cells (Table 1). Another important fact, concerning the plant cells used for cosmetics, is that the term “plant stem cells” is often equally used for active ingredients, produced by either callus cultures, cell suspensions, or hairy roots. It is important to understand, that the callus cultures are plant cells, cultured on a solid medium, whereas the cell suspension cultures are single plant cells or small cell aggregates cultivated under submerged conditions in a liquid medium. Both callus cultures and cell suspensions could consist of dedifferentiated or true stem cells. On the opposite, the hairy roots are organ cultures, obtained by genetic transformation of plant cells [23, 34]. Some of the plant cell culture technology–derived active cosmetic ingredients, currently available on the market are reviewed in Table 1.

3.1 Plant stem cells

Plant cells can be propagated and used for a continuous supply of fresh plant biomass for cosmetic formulations. However, it is of great importance to understand that we cannot introduce the entire plant cells in cosmetic products and keep them alive as active ingredients there [30,35]. Plant cells are extremely sensitive to environmental factors, nutrient medium composition, osmotic and mechanical (shear) stress, gas exchange and oxygen supply, temperature, light, ionic strength and water content, and thus, they can survive neither during cosmetic products preparation nor during storage or application of cosmetics on skin. Even a delivery system, able to maintain and supply live stem cells could be developed, the size and the specific structure of plant cells will not allow them to attach or penetrate the skin surface. Because of all discussed concerns, the plant stem cells are rather used as a raw material for preparing different types of extracts, which then could be included in cosmetic formulations as active ingredients. However, there are various products, available on the market, which are based on whole dried plant cells and standardized on the cells count per gram of active ingredient (Table 1). Such examples are the anti-aging active ingredient CeltosomeTM Eryngium Maritimum ST (based on sea holly cell culture with 100 million cells/g of active ingredient), and the tyrosinase inhibitory skin lightening active ingredient CeltosomeTM Crithmum Maritimum ST (based on rock samphire cell culture with more than 1 billion cells/g of active ingredient) (Table 1).

3.2 Plant stem cells extracts

Most of the available active cosmetic ingredients, obtained by plant cell culture technology are marketed in the form of different extracts (Table 1). In contrast to the plant-derived extracts, the extracts obtained from plant cell cultures can be easily standardized and perfectly comply with the strict safety requirements that the high-end cosmetic market constantly demands [35]. Plant cell extracts are free of pathogens, agrochemical, toxic substances, allergens, and pollutants because they are produced under controlled conditions, complying with the procedures of the good manufacturing practice. Depending on the type of used solvent, the plant cell extracts used in cosmetics could be contingently divided into liposoluble (extracted with oils) and hydrosoluble (extracted with glycerin) extracts, dried extracts (conditioned with maltodextrin), plant cell wall extracts (rich in peptides and sugars), nanoemulsions or suspension extracts [35]. However, most of the existed extracts have been developed on the basis of the extraction of a target compound or group of closely related bioactive compounds, and thus, not the entire health beneficial potential of the extracted plant cells could be utilized. Some exceptions could be found, where the entire plant cells were freeze-dried and powdered for direct application in cosmetic formulations (see some products offered by “Vytrus Biotech” and “Naolys”, Table 1), or the entire cell suspensions have been, emulsified or encapsulated in liposomal complex (see some products offered by “Innova BM” and “Mibelle AG Biochemistry”, Table 1) by using high-pressure homogenizers.

3.3 Molecular farming for production of recombinant proteins

Plants are excellent production matrixes for the expression of recombinant proteins with important pharmaceutical properties [36]. This powerful recombinant protein expression technique, known as “molecular farming” has been widely used for the production of vaccines [37–39], cytokines [40], and even for the production of therapeutic protein for human use [5, 41, 42]. The classical molecular farming technology is based on the genetic modification of plants for recombinant protein expression. This could be realized by the permanent integration of foreign genes into the host DNA to generate stable transgenic lines, or by the transient transformation of intact plant leaves [43]. However, both methods require the growing of whole plants under strictly controlled environmental conditions into contained greenhouses, complying with the strict regulatory standards [36]. By using such technology, The Iceland Company “BIOEFFECT” has applied for the first time a plant-based transgenic platform for large-scale production of Epidermal Growth Factors (EGF) for cosmetic use, expressed in genetically engineered barley seeds. The company offers a wide range of skin-care products containing this cellular activator, which contribute to healthier and younger-looking skin. However, the recent interests in molecular farming have been focused on the adaptation of plant cell culture technology for the production of recombinant proteins. This technique offers sustainable and continuous heterologous proteins production by plant cells growing under precisely controlled micro-environmental in vitro conditions in bioreactors. Cultivation of genetically engineered plant cell cultures has been recognized as a much more powerful expression platform, when compared to the classical molecular farming techniques, relying on agricultural-scale production by growing genetically transformed plants [18, 41]. Recently, the Korean company “Natural Bio-Materials (NBM)”, has launched a series of active cosmetic ingredients containing growth factors (Epidermal Growth Factor, Basic Fibroblast Growth Factor, Insulin-like Growth Factor-1, Keratinocyte Growth Factor, Fibroblast Growth Factor-7, and Vascular Endothelial Growth Factor), expressed by recombinant rice (Oryza sativa L.) cell cultures (Table 1). However, even though the human recombinant proteins, produced by this technology have undeniable advantages and are characterized by a high level of purity, animal-free, virus-free, bacterial-free, and exotoxin-free production, there are still some consumers which have concerns to use such products because they are expressed by genetically modified organisms.

4 Case Study: Initiation, growth, phytochemical profile and physicochemical characteristics of exopolysaccharides in INNOVA StemCell Calendula

Calendula (Calendula officinalis L.), known as “marigold”, has been widely used in traditional herbal medicine and skincare cosmeceuticals for topical application [11]. The pant was shown to be rich in phenolic acids, flavonoids, triterpenes, carotenoids, aroma compounds, and a unique mix of polyunsaturated fatty acids [44–46]. Because of its high therapeutic value and the proven cosmetic effects, the Bulgarian company “Innova BM” has developed and released on the market two high-quality active cosmetic ingredients, based on Calendula dedifferentiated cell culture (Table 1). The development of these products is schematically presented in Fig. 1. The technological steps include a screening of calendula plants with superior phytochemical profiles, selection, sterilization, and cultivation of plant explants on callus induction medium, selection of friable cell lines with appropriate phytochemical profiles, initiation of liquid cell suspension culture, and optimization of cultivation conditions and nutrient medium composition. The optimization step is critical in our technology since a significant increase in biosynthetic potential and accumulated biomass of the selected cell line can be achieved (Table 2, Fig. 2). After optimization, the selected line was scaledup to large-scale cultivation in stirred tank bioreactor. The produced cell suspension (cells and culture liquid) was then processed by a high-pressure homogenizer to produce glycerin extract (50 % wt.) or calendula emulsion (75 % wt. cell suspension) [47]. The produced active ingredients have been found to have superior moisturizing, anti-wrinkle, hydrating and regenerative effects when applied to the skin. These effects are due to the high content of secreted exopolysaccharides, during the cultivation of calendula cells (Fig. 2D). The exopolysaccharides have been identified to belong to the pectin type. The crude exopolysaccharides fraction contains 879 μg/mg neutral sugars and 50 μg/mg proteins. The unique combination of polysaccharides and peptides made this exopolysaccharide fraction almost perfect for application as an emulsifier in cosmetic products for topical application. The potential synergistic interactions, when applied with other popular emulsifiers, as well as their emulsion stabilization properties are presented in Table 3. The polysaccharides have a molecular weight of 6.7 × 104 Da and contain 413 μg/mg of uronic acids. The full monosaccharide composition of exopolysaccharide fraction was determined as: glucuronic acid (13.6 μg/mg), galacturonic acid (399.7μg/mg), glucose (185.5μg/mg), galactose (179.9μg/mg), rhamnose (178.9 μg/mg), arabinose (166.7 μg/mg), fucose (0.6 μg/mg) and mannose (4.7 μg/mg). The presence of this exopolysaccharides fraction incorporated into Innova StemCell Calendula products, in combination with bioactive compounds from the released cell content (Table 2) made these products unique and one of the kind in the market of active cosmetic ingredients.

5 Concluding remarks

Following the ever-growing demand for high-quality natural products of plant origin for application as active ingredients in cosmeceutical formulations, the plant cell culture technology has developed powerful production platforms which can effectively supply the customers’ needs. We are witnesses of an exponentially growing number of commercialized plant cell-derived ingredients, offered on the cosmetic industry market and the diversity of utilized plant species, used for their production has continued to increase every year. In fact, the observed interest in the production of plant cell-derived ingredients for cosmetic needs could be correlated with the recent advance in the development and commercialization of plant cell culture technology in technologically advanced countries. Moreover, the newly developed techniques of gene editing, metabolite engineering, and synthetic biology could have a significant impact on improving the yields and development of tailor-made cosmetic products with desired activities. The advance in molecular farming has already led to the commercial production of rare human activator peptides, cytokines, and growth factors, which are the first step in the development of cosmetic products with the potential to extend skin life by using the body's self-repair mechanisms. However, till now there are many open questions, concerning regulatory standards and documentation, unification of health benefit claims, and the methodology for evaluation of pharmaceutical effects, which should be answered in order to help the consumers to make the right choice of their cosmeceutical product.

Cistanche extract benefits skin