Plant cell culture as emerging technology for production of active cosmetic ingredients (2024)

Abstract

Plants have always been the main source for active cosmetic ingredients, having proven health beneficial effects on human, 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 details within a presented case study for calendula stem cell product development.

1. Introduction

Plants are the oldest source of natural compounds with medicinal and cosmetic properties explored by the mankind. Even nowadays, between 70 and 80% of the people worldwide relay 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 with 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 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 with low levels of genetic diversity, which makes these species extremely vulnerable to genetic erosion and decrease their chance to survive in case of environmental pollution 4. Moreover, plant derived active ingredients are usually 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 more eco‐friendly and sustainable supply of valuable phytochemicals with health‐beneficial properties, to cover the growing demands of cosmetic industry. Over the last decade, the advantages of plant cells and tissues culture technologies have been widely explored in 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 nutricosmetics, which have active ingredients, derived by 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 short overview on 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® 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 development and characterization of plant cell culture – derived active ingredients are closely discussed in the presented case study for development of InnovaStemCell Calendula product.

2. Plant extracts as active ingredients in cosmetics

Plants are rich source of countless metabolites with potential application in cosmetic products. Since time immemorial, the mankind has used different plants and their extracts to create cosmetics with the aim to establishing a state of eternal youth 6. Nowadays, the 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. 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, 8, 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, anticancer, antifungal, anti‐inflammatory, anti‐allergy, etc., and also showing strong moisturizing, anti‐ageing, anti‐wrinkle and UV protective effects 11. However, the quality and phytochemical profiles of plant extracts varied in 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 bioactive extracts not only by plants, but also from mushrooms, algae, and also, by utilization of by‐products of plant origins 13, 14, 15, 16, 17. Nowadays, with the growth in consumers’ interest to 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 have been steadily on the rise.

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

Plant cell culture technology is a technique for growing 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 on climate or soil or field management practices 5, 18. Moreover, in vitro cultured plant cells are characterized with fast growth, and the ability to accumulate large amount of uniform biomass for a short period of time 19, 20. This is very important advantage especially for the production of rare bioactive compounds, 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 plant biomass 21, 22, 23, 24, 25. Additionally, plant cell culture technology offers a reliable and powerful production platform for 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 plant cell culture technique exceptionally attractive for the production of active ingredients for high added values “green” cosmetic formulations 27, 28, 29, 30. It should be noted, that active cosmetic ingredients, obtained by the 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. The 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 as “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 cambial layer and consist only of true meristem stem cells 31, 32. Cambial meristematic cells are characterized with 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 have been used by the Korean company “Unhwa Corp.” to produce several active cosmetic ingredients, by using the patent protected expression platform Ddobyul®, 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 solid medium, whereas the cell suspension cultures are single plant cells or small cell aggregates cultivated under submerged conditions in 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.

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 skin care cosmeceuticals for topical application 11. The pant was shown to be rich in phenolic acids, flavonoid, triterpenes, carotenoids, aroma compounds and unique mix of polyunsaturated fatty acids 44, 45, 46. Because of its high therapeutic value and the proved 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 on Fig. 1. The technological steps include 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 selected cell line can be achieved (Table 2, Fig. 2). After optimization, the selected line was scaled‐up to large scale cultivation in stirred tank bioreactor. The produced cell suspension (cells and culture liquid) was then processed by 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 on skin. These effects are due to the high content of secreted exopolysaccharides, during cultivation of calendula cells (Fig. 2D). The exopolysaccharides have been identified to belong to 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 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 molecular weight of 6.7 × 10⁴ Da and contain 413μg/mg uronic acids. The full monosaccharide composition of exopolysaccharide fraction was determined as: glucuronic acid (13.6μg/mg), galacturonic acid (399.7 μg/mg), glucose (185.5μg/mg), galactose (179.9μg/mg), rhamnose (178.9μg/mg), arabinose (166.7μg/mg), fucose (0.6μg/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.

Table 3.

Synergistic effect of exopolysaccharides, released by Calendula officinalis stem cell suspension in relation to emulsion stabilizing properties in mixtures with cellulose gum, xanthan gum, guar gum, and sodium‐alginate

Model emulsion systems Oil/Water (1:1 v/v) with:	Light microscopy	Turbidimetric method
		Emulsifying activity index (EAI), m2/g	Emulsion stability index (ESI), min
0.6 % Calendula Cell Suspension Exopolysaccharides		175.0	16.8
0.6 % Calendula Cell Suspension Exopolysaccharides And 0.3 % Cellulose Gum		38.4	13.9
0.6 % Calendula Cell Suspension Exopolysaccharides and 0.3 % Xanthan Gum		47.6	22.1
0.6 % Calendula Cell Suspension Exopolysaccharides and 0.3 % Guar Gum		322.4	15.0
0.6 % Calendula Cell Suspension Exopolysaccharides and 0.3 % Sodium Alginate		1307.5	60.7

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The emulsifying activity index (EAI) and emulsion stability index (ESI) were determined according to 48. Model emulsion systems were prepared by mixing polysaccharides, water and sunflower oil for 5 min at 10 000 rpm by homogenizer (Ultra Turrax IKA T18 Basic, Germany).

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 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 continue to increase every year. In fact, the observed interest to production of plant cell ‐ derived ingredients for cosmetic needs could be correlated with the recent advance in development and commercialization of plant cell culture technology in technologically advanced countries. Moreover, the new developed techniques of gene editing, metabolite engineering and synthetic biology could have a significant impact on improvement the yields and development of tailor made cosmetic products with desired activities. The advance in molecular farming has already leaded to commercial production of rare human activator peptides, cytokines and growth factors, which are the first step in development of cosmetic products with potential to extend skin life by using the body self‐repair mechanisms. However, till now there are many open questions, concerning regulatory standards and documentation, unification of health beneficial 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.

The authors have declared no conflict of interest.

Acknowledgments

We would like to thanks the Innova BM, Bulgaria, for the financial support, close collaboration and the shared data.

6 References