PLANT SMALL RNA TECHNOLOGY 117 The small RNAs, a class of noncoding RNAs, play a role in the regulation of genomes and transcriptomes targeting chromatin or transcripts. They are considered epigenetic regulators. Epigenetics is the study of modifi cations that are not caused by changes in the DNA sequence it allows us to perceive the infl uence of the environment on the transcrip- tional potential of a cell through modifying epigenetic marks (6). For example, baobab seeds must contend with harsh environmental conditions, such as drought and heat. Their resistance to these conditions is possible, thanks to the small RNAs that will regu- late their gene expression. Small RNAs are also involved in effi cient defense systems that provide resistance to environmental stresses, and scavenge reactive oxygen species (ROS). Small RNAs include miRNAs, consisting of 22–24 ribonucleotides. miRNAs are involved in post-transcriptional regulation through interaction with messenger RNAs. In the cells of both humans and plants, they are involved in numerous processes such as development, metabolism, and reproduction, as well as in the response to biotic or abiotic stresses and diseases. Regulation by miRNAs accounts for more than a third of all gene expression (7). To act on miRNA targets, in plants, this process is performed by a single RNase III enzyme, DCL1 (Dicer-like 1), in the nucleus (8). In animals, miRNAs follow a matura- tion process divided into two major steps. The primary miRNA is processed by the RNase III endoribonuclease Drosha to generate a shorter precursor miRNA (pre-miRNA). After export in the cytoplasm by the nucleoplasmic transport protein exportin-5, the pre- miRNA is cleaved by Dicer. Thereby, the miRNA is matured and assembled into the RNA-induced silencing complexes (RISC) and is ready to inhibit its mRNA targets (9). On senescent WI-38 human diploid fi broblasts from the lung, the expression of these two key enzymes has been shown to decrease, involving the dysregulation of 20% of miRNAs (10). Among the miRNAs dysregulated during cellular aging, miRNA-19b is described as a biomarker of cellular aging. Indeed, Hackl et al. showed a downregulation of this miRNA with aging, in different replicative cell and organismal aging models (11). In addition, this miRNA is part of small noncoding RNA signature in centenarians. Indeed, miRNA-19b expression was shown to be similar in the cells of both young and centenar- ian people (12). On the basis of their functions, plant miRNAs could be considered as a new class of mi- cronutrients, along with vitamins. Thus, an exclusive and novel extraction process maxi- mizing the potential of PSRs was developed and named PSR technology. Tests on ex vivo skin and in vitro fi broblasts demonstrated the benefi ts of PSR plant presence in extract on maintaining skin homeostasis by stabilizing the maturation of the skin’s miRNA machinery. MATERIALS AND METHODS PLANT MATERIAL The seedcake of Adansonia digitata used in this study originated from Senegal. We ap- preciate the partnership of two complementary companies in obtaining the seedcake: Biomega, an Austrian society specializing in food technology and Baonane, a Senegalese company and leading producer of baobab products, particularly baobab fruit valorization. The baobab fruits are matured 5–6 mo after fl owering. During the harvest, they are

JOURNAL OF COSMETIC SCIENCE 118 hand-picked, and the seeds are removed from the pulp and red fi ber. Oil is obtained from the seeds by cold pressing. The coproduct of the oil extraction is the seedcake. The seed- cake is micronized and was used to perform the extraction. PREPARATION OF PLANT EXTRACT Baobab seedcake was fi rst ground with a blender to obtain a powder. Then, PSR technol- ogy patented process was applied to obtain the small RNA-enriched baobab extract and other phytochemicals. The extract was then diluted by 30% with either water or a cos- metic solvent such as glycerin. The extract was then transferred into sterile bottles and sterilized at low temperature. To obtain a plant extract placebo for the biological evalua- tion, another baobab extract that did not contain small RNAs was prepared. These two baobab extracts were comparatively studied for their effects on skin. PHYTOCHEMICAL ANALYSIS OF THE EXTRACT Phytochemical screening was performed on the baobab seedcake and on the fi nal extract to determine the dry weight and the quantifi cation of total protein and sugar. Polyphenol and amino acid content analysis was performed only on the fi nal extract. Dry weight of the raw material and of the fi nal extract was determined by placing 2 g of baobab seedcake powder in a metal cup in duplicate for 2 h at 105°C in a ventilated oven (Memmert, Schwabach, Germany). Protein content was determined by Lowry protein assay, which was used to quantify the total protein content of the extract (13). Protein measurement with the Folin phenol re- agent was used to quantify the total protein content of the extract. The Lowry method is based on the reaction of Cu+, produced by the oxidation of peptide bonds, with Folin– Ciocalteu reagent. The absorbance of the sample is read on the spectrophotometer at 550 nm. The protein content is determined using a BSA standard curve. Sugar content was determined colorimetrically via an adaptation of the assay described by Dubois et al. (14). This analysis consisted of concentrated sulfuric acid, which was then reacted with phenol to form a colored complex. The absorbance of the complex was read on the spectrophotometer at 490 nm. The sugar content was determined using a glucose standard curve. Polyphenolic compound quantity was determined using the Folin–Ciocalteu assay (15). Polyphenol compounds in the sample reacted with the Folin–Ciocalteu reagent, and the oxidation of the reagent turns the polyphenol compounds blue. The absorbance of the sample was read on the spectrophotometer at 760 nm. The content was expressed as gal- lic acid equivalents using a gallic acid standard curve. Amino acids were quantifi ed using a protocol published by Moore and Stein (16). The free amino acid content of the baobab extract was assessed by the formation of a colored complex, following the rupture of the amine and carboxylic functions by the reagent ninhydrin. The absorbance of the complex was read on the spectrophotometer at 570 nm. The total amino acids content was determined using a standard curve of amino acids pool. For the analysis of quality and integrity of the small RNAs present in our baobab extract, the Agilent 2100 Bioanalyzer system was used, with a small RNA Chip Kit according to the manufacturer’s instructions (Agilent, Santa Clara, CA).