Plant cell culture technology has numerous advantages because they address issues such as carbon footprint and water requirements, as well as pesticide and herbicide requirements that may arise as a result of plant use. Plant cell culture technology allows for the preparation of safe and clean components with high consistency levels, independent of seasons or plant reproduction cycles, under controlled conditions, and without the use of agricultural land. Furthermore, when using plant cell culture methods for secondary metabolite production, the concentration of compounds, culturing conditions, and physical parameters can be changed, or the amount of the desired compound can be increased or optimized by adding an inducer compound to the culture medium (Barbulova et al., 2014; Eibl et al., 2018; Krasteva et al., 2021; Trehan et al., 2017). Secondary metabolites have the potential to be used in different industries, pharmaceuticals, agriculture, cosmetics, textiles, and food, making them economically significant (Smetanska, 2008).
Plant cells can also produce higher concentrations of secondary metabolites that are not obtained by methods such as chemical synthesis and extraction, or are obtained at low concentrations. Plant cells have also been used to develop and produce various types of cosmetic actives (Eibl et al., 2018). Several production processes have been established in the cosmetic industry using plant cell culture technology. Examples of commercially produced products are arbutin (a whitening ingredient) from Catharanthus roseus, shikonin (a cosmetic pigment) from Lithospermum erythrorhizon, and carthamin (a cosmetic pigment) from Carthamus tinctorius. (Schürch et al., 2008).
Comfrey (Symphytum officinale L.) is traditionally used in folk medicine to reduce inflammation, promote wound healing, and treat broken bones and skin conditions. The traditional use activities of comfrey preparations have been confirmed by experimental studies.
Comfrey has been used for centuries as a conventional medicinal herb for treatment (Staiger, 2013) and it has a wide range of therapeutic applications. Comfrey has therapeutic applications, including injuries, fractures, swollen bruises, varicose ulcers, burns, tissue repair, arthritis, rheumatic pains, and non-healing wounds (Mulkijanyan et al., 2009; Savic et al., 2015). Comfrey contains a high concentration of rosmarinic acid, which has antioxidant activity. Recent studies have shown that antioxidants help prevent inflammation and promote wound healing (Sowa et al., 2018). Allantoin is the most pharmacologically active component of comfrey root, with a ratio of 0.6–0.8% (Mulkijanyan et al., 2009; Savic et al., 2015). Phenolic acids and allantoin (Rosmarinic, caffeic, p-hydroxybenzoic, p-coumaric acids and chlorogenic, etc.) found in Comfrey root extract have a positive effect on human skin fibroblasts and also provide remarkable wound healing antioxidant effect (Mulkijanyan et al., 2009; Salehi et al., 2019; Savic et al., 2015; Shestopalov et al., 2006).
All layers of the dermis and epidermis are involved in the complex biological process of skin aging (Papakonstantinou et al., 2012; Weihermann et al., 2017). Aging of human skin occurs as a result of two independent processes, age-related aging, and photoaging. The aging process is characterized by the reduction of collagen, elastin, and hyaluronic acid synthesis in the skin and the deterioration of dermal collagen and elastin structure. Age-dependent aging is an unavoidable process in which genetic and metabolic factors that occur with age are effective. Photoaging is a process in which external factors such as ultraviolet (UV) light and air pollution are effective (Kang et al., 2016; Weihermann et al., 2017). In both age-dependent and premature aging aged skin, degraded collagen levels increase while collagen synthesis declines (Papakonstantinou et al., 2012). Skin aging is characterized by processes such as decreased fibroblast count, skin thinning, decreased collagen synthesis, deterioration of dermal elastin structure, decreased elastin synthesis, and loss of elasticity. All of these processes are effective at causing skin sagging and wrinkle formation (Duque et al., 2017; Weihermann et al., 2017).
The most abundant extracellular matrix (ECM) protein made by dermal fibroblasts is collagen. About 80% of the dry weight of the dermis is produced by type I collagen (Mei Xiong et al., 2017). Exposure to UV light can reason photoaging of the skin by downregulating collagen synthesis (Jung et al., 2014). Skin elasticity depends on elastin, one of the richest ECM proteins in the skin dermis. Elastin is synthesized by dermal fibroblasts and forms into higher-order structures with other ECM proteins. Elastin production remains relatively constant during physiological aging until the age of 30 to 40, after which it drops significantly (Mei Xiong et al., 2017). Hyaluronan, also known as hyaluronic acid (HA), is an ECM component that is used to fill areas where cells can migrate. Hyaluronic acid is responsible for moisture retention in the skin, and it forms a base for fibroblasts during wound healing and tissue repair (Lee et al., 2016).
Collagen type 1 is a heterotrimer, triple helix protein encoded by the COL1A1 and COL1A2 genes and occurs of two α1 chains and one α2 chain (Bornstein & Sage, 1989). Elastin is encoded by the single-copy ELN gene and it occurs as a soluble tropoelastin precursor that can be found as globules or expanded polypeptides (Kielty, 2006). Hyaluronic acid is an unbranched glycosaminoglycan and is synthesized by HA synthases (HAS1-3). Hyaluronan synthase 3, encoded by the HAS3 gene, is related to the synthesis of hyaluronic acid. When the HAS3 gene is compared with the proteins encoded by other members of the HA synthase family (HAS1 and HAS2), it was concluded that this protein is more a regulator of hyaluronan synthesis (Ota et al., 2022).
Plant cells have totipotent properties, they can produce specific secondary metabolites found in parent plants and comply with good manufacturing practices and procedures. Plant cells can be easily produced using high-volume bioreactors regardless of climate. This method provides a reliable contamination-free production for the continuous supply of medicinal, rare, and endangered plant species (Georgiev et al., 2018). In many studies to date, methods to slow down skin aging, and strengthen and protect skin cells have been investigated. Plant stem cells can induce fibroblasts to synthesize collagen, which stimulates skin regeneration, and may exhibit anti-aging properties (Miastkowska & Sikora, 2018). Although the metabolites of Comfrey are supported by different studies with medicinal effects on the skin, the anti-aging activity of comfrey cell suspension culture extract on aged CRL20-76 cells has yet to be determined. For this reason, it would be an appropriate approach to obtain cell cultures of the comfrey plant, which has the potential to be a cosmetic active ingredient, using plant cell culture technology and to investigate the efficiency of the obtained cells in human normal fibroblast cells. For this reason, the effect of comfrey cell suspension extract on the expression of the COL1A1, ELN, and HAS3 genes were analyzed to determine the anti-aging activity in aged CRL-2076 cells.