Hairy root culture as source of novel plant-derived active compounds with applications in cosmetics

Abstract

Hyperpigmentation is the process by which an excess of melanin is produced by the skin. Typically, hyperpigmentation occurs as a result of stress, damage or prolonged inflammation of the skin. The most common cause is sun damage, though hyperpigmentation is often a consequence of inflammation following acne, eczema, psoriasis, dermatitis etc. Hyperpigmentation may also occur in the skin due to hormonal changes in the body typically associated with pregnancy or the taking of oral contraception. Beside this medical aspects, the global skin depigmenting product market has been forecast to reach a value of $19.8 billion by 2018, driven by the growing desire for light-coloured skin among both men and women primarily from the Asian, African and Middle East regions. Although products do exist that can actually bleach the skin, these products contain dangerous or toxic ingredients (such as hydroquinone and mercury) and are banned in most countries. Blocking or reducing the accumulation of melanin in the skin can be obtained either by switching off one or more components of the pathway that go from the receptor activation to the enzymatic inhibition of melanin formation catalyzed by the tyrosinase. For this purpose, several antimelanogenic reagents have been developed and discovered nowadays. However, only a few of these inhibitors have been introduced and used due to their problems in cytotoxicity (affecting the cell growth and survival), selectivity, solubility and stability. The present project was aimed at identifying new total plant extracts exerting beneficial effects in skin care, with special emphasis on the development of novel plant-derived actives with hypopigmenting effects. Experimental activities were carried out in collaboration with Arterra Bioscience S.r.l, in the frame of the programme “Dottorato di Ricerca in Azienda”, funded by European Commission and Regione Campania (POR Campania FSE 2007-2013). Arterra is an Italian research-based Biotech company mostly involved in developing new plant-derived extracts to be used as active ingredients with cosmetic application. Hairy root cultures of three different plant species (Cichorium intybus, Brassica rapa subsp. pekinensis and Helianthus annuus) were generated. Hairy roots of Brassica rapa subsp. pekinensis were selected for further studies on the base of a preliminary screening for anti-oxidant activity of a total crude ethanol extract and a sugar/peptides mixture derived from cell walls, coupled to an active growth. Crude ethanol extract and a sugar/peptides mixture derived from cell wall of Brassica rapa subsp pekinensis hairy roots were tested in murine melanoma cells (B16-F1) and human epidermal melanocytes isolated from lightly pigmented adult skin (HEMa-LP), by using a panel of in vitro and in vivo biological assays to assess their role in modulating melanogenesis. Both extracts at different concentrations demonstrated to inhibit the cellular tyrosinase, a key enzyme in melanin production, and to reduce melanin content in murine melanoma cells. In addition, the sugar/peptides mixture of Brassica rapa susp. pekinensis hairy roots significantly inhibited the levels of cyclic adenosine monophosphate (cAMP), an important second messenger within melanogenesis signalling pathway. Furthermore, the same extract significantly decreased the expression of microphtalmia-associated transcription factor (MITF) and its promoter activity of about 30%, analyzed by in vitro reporter (luc+)-assay. Altogether these data indicates that the sugar/peptides mixture isolated from cell wall of Brassica rapa subsp. pekinensis hairy roots might exert its inhibitory effect on melanogenesis through the downregulation of MITF transcription. Furthermore, Brassica rapa subsp. pekinensis ethanol extract was able to enhance the expression levels of important genes encoding for proteins involved into extracellular matrix (ECM) assembly. Finally, a competitive industrial production hairy-root based platform was developed by Brassica rapa subsp. pekinensis hairy root biomass scaling-up and improved extraction procedures. Overall, these results, under pending patent application, will contribute to introduce product and process innovations at Arterra Bioscience s.r.l, for the identification of new and safer plant-derived melanogenesis inhibitors. In general, the developed industrial production platform will be also extended to the screening of actives from other plant species and to the release of novel plant-derived products in different segments of the cosmetic market. [edited by author]