Skin aging is a multifactorial process regulated by various biological mechanisms. It is often accompanied by the appearance of skin dryness, wrinkles, sagging, uneven skin tone, mottled pigmentation, etc.;1 and research has shown that each of the biological mechanisms responsible for a particular skin aging attribute is regulated by genes. Thousands of genes are expressed in the epidermis2 and their collective expression is known as the gene expression profile.3 Groups of genes coding for proteins having similar or complementary functions may be responsible for regulating specific skin aging attributes. These genes may be categorized into groups of “functional clusters”; herein, gene clusters associated with anti-aging activities are referred to as youth gene clusters.4
Recent research also has shown that topical as well as nutritional materials may influence the gene expression profile of the skin to reflect a more youthful profile. The present article describes in vitro analyses of a blend of two extracts, namely Narcissus tazetta bulb and Schizandra chinensis fruit, for such effects. Narcissus tazetta bulb extract, a bulb extract from the daffodil plant family, has previously been shown to delay cellular proliferation when applied topically.5 In addition, Schizandra chinensis fruit extract, a red berry fruit extract belonging to the magnolia plant family, has been used for decades in Chinese medicine to promote general well-being and vitality when taken orally.6 This has led to the hypothesis that when combined, the two extracts may change the activity of genes in the epidermis to reflect a more youthful gene expression profile, which is assessed here. Note that the individual actives were not tested; future research is necessary to determine whether they have similar effects alone.
Materials and Methods
A combination of 0.02% Narcissus tazetta extract and 0.01% Schizandra chinensis fruit extract were tested in vitro using human full-thickness 3D epidermal skin equivalentsa. These amounts were chosen based on results from in-house clinical testing, where this specific combination was used in finished formulations (data not shown).
FTEE cultures: A 100-μL sample of the test article was applied to each culture and incubated for 24 hr. Following incubation, the cultures were thoroughly washed with sterile phosphate buffered saline (PBS) to remove test materials and placed in RNAlater solution for gene expression analysis. The tissues were incubated for 2 hr at room temperature then stored at 4°C until processing.
qPCR analysis: Custom Taqman Low Density Arrays (TLDAs) were created using validated gene expression assaysb. Each TLDA card contained 379 skin-relevant target genes, selected based on published literature. In addition, five common endogenous control genes were included. One gram of total RNA from each tissue sample was converted into cDNA using a high capacity cDNA reverse transcription kitc. A fast real-time polymerase chain reaction (PCR) instrumentd was used for amplification and fluorescence detection.
Statistics: Data analysis for quantitative PCR (qPCR) was carried out according to the relative quantitation (RQ) analysis method using softwaree, f. Expression levels were determined based on relative quantification analysis and using a t-test with Benjamini and Hochberg false discovery rate correction (p value < 0.05) with a cycle threshold of less than 35.
qPCR data for cultures incubated with the extract blend for 24 hr showed the regulation of 242 out of 384 targeted genes,133 being downregulated and 109 being up-regulated, with a cycle threshold of < 35 (p ≤ 0.05), compared with the untreated control; see Table 1, Table 2, Table 3 and Table 4 for examples of groups or clusters of genes regulated by the blend of Narcissus tazetta bulb and Schizandra chinensis fruit extracts.
The results from qPCR analysis indicate this ingredient blend influenced the regulation of youth gene clusters.4 Table 1 shows the gene expression from different biological processes responsible for skin structure. Elastin and collagen are key proteins responsible for skin structure and integrity,7 and the gene that codes for collagen COL4A3 was upregulated. LOX, a gene known to increase the synthesis of elastin, also was upregulated.8 TIMP1, whose function is to degrade metalloproteinases, i.e., proteins that degrade collagen, was upregulated.9 Further, PKP2, a gene involved in desmosome assembly, was increased.10 In addition to genes associated with skin structure, this combination of extracts regulated the expression of genes related to skin hydration and barrier integrity (see Table 2); HAS1, a key gene involved in the synthesis of hyaluronic acid (HA), was upregulated.11 HA is one of the most abundant components of the extracellular matrix of the skin and is involved in maintaining moisture as well as protecting skin barrier. Further, both GBA and UGCG, involved in ceramide synthesis, were upregulated.12, 13
Along with hydration and skin structure, genes related to cellular proliferation were also influenced by the blend (see Table 3). The expression of KRT14 and KRT5, genes that are downregulated during keratinocyte proliferation, was decreased, indicating the occurrence of cellular proliferation.14 This proliferation is also supported by the upregulation of OCLN and TMPRSS11E, which are both expressed during proliferation.15, 16
Table 4 shows examples of genes associated with UV-induced skin pigmentation that were regulated by the Narcissus tazetta bulb and Schizandra chinensis fruit extract. SOD2, known to reduce UV-induced pigmentation, was upregulated along with its positive regulator FOXO3.17, 18 In addition to SOD2, MT2A, known to inhibit tyrosinase via nitric oxide scavenging, was also upregulated,19 and both GADD45A and SIRT1, again known to protect UV-induced pigmentation, were upregulated. 20, 21
In addition to the described in vitro findings, unpublished clinical testing with this combination of ingredients in finished product formulations showed their positive effects on the specific skin aging attributes studied here (data not shown).
Both Narcissus tazetta bulb extract and Schizandra chinensis fruit extract are water-soluble. They can be added directly to the aqueous phase of an emulsion or in a water and/or glycerin premix. However, if the temperature if the aqueous phase is expected to surpass 70°C, these ingredients are recommended to be added postemulsification at temperatures under 70°C. Ingredient levels other than those mentioned in this study have not been tested for clinical efficacy or safety.
Based on the findings from this in vitro study, the combination of Narcissus tazetta bulb extract and Schizandra chinensis fruit extract, when applied topically to human-equivalent skin, regulates functional youth gene clusters to reflect the genetic profile and activities of more youthful skin, and decreases gene expression activities associated with skin aging. Notably, the clusters or groups of genes identified in this research are related specifically to skin structure, hydration, cellular proliferation and pigmentation.
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- C Gebhardt et al, Dermal hyaluronan is rapidly reduced by topical treatment with glucocorticoids, J Invest Dermatol 130(1) 141–149 (2010)
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