Standardized phytocomplex of Perilla frutescens derived from in vitro cell cultures: maintenance of vaginal mucosa integrity and use in vaginal gel formulations
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Presented by: Giovanna Pressi
Introduction: Plant cell culture technology is a technique for growing of plant cells under strictly controlled environmental conditions that makes it possible to provide preparations with a standardized content of active substances and with a high safety profile for the consumer. Perilla frutescens L., also known as Shiso, is a specie of Perilla that belongs to the Lamiaceae family, commonly used as an aromatic and medical plant. Perilla frutescens phyocomplex, derived from in vitro plant cell cultures, has a high and standardized content of rosmarinic acid and anthocyanins and can be used as a new active ingredient for cosmetic and intimate products. The object of this study is to demonstrate the activity of this phytocomplex to maintain vaginal mucosa integrity acting with anti-inflammatory and hydrating activity and its application in intimate gel formulations.
Methods: Perilla frutescens phytocomplex was produced by using in vitro plant cell cultures technology. UPLC-ESI-MS and UPLC-DAD analysis were used for the identification and quantification of the secondary metabolites (rosmarinic acid and anthocyanins) of the phytocomplex. In vitro tests on HaCaT cells were used to evaluate expression of inflammatory cytochines with ELISA kit after an inflammatory stress. On HaCaT cells were studied the expression of tight junctions (Zonula occludens-1 and Occludin) and skin barrier proteins (Filaggrin and Loricrin) by using Western blot assay after inflammatory and oxidative insult with and without pretreatment with Perilla frutescens phytocomplex. We evaluated the moisturizing activity of this new active ingredient through the reduction of TEWL, after irritative damage with sodium lauryl sulfate, using in vivo test on 20 Caucasian subjects. The soothing capacity of Perilla frutescens phytocomplex was evaluated on in vitro vaginal mucosa after irritation induced by lactic acid. Perilla frutescens phytocomplex was used in intimate gels to evaluate the compatibility and the stability with different rheological modifiers classes.
Results: The Perilla frutescens phytocomplex prepared by suspension cell cultures, was analyzed by UPLC-ESI-MS which provided the presence of secondary metabolites such as rosmarinic acid,metyl ester of rosmarinic acid, salvionolic acid and anthocyanins (shisonin and methyl shisonin). The standardized content of polyphenols, expressed as rosmarinic acid, is 3,3% w/w and the content of anthocyanins, expressed as cyanidin-3-O-glucoside, is 0,16% w/w, and they were determined by UPLC-DAD analysis. The phytocomplex has shown a high anti-inflammatory activity significantly inhibiting the release of inflammatory cytochines TNF-α, IL-1β e IL-6. It was able to prevent the alteration induced by LPS + H2O2, bringing the Occludin and Zonula Occludens-1 levels back to those of the control on human keratinocytes pretreated for two hours. Pretreatment with the active at 100 µg/mL for 2 hours is able to countervert the action of LPS and H2O2 by reducing the levels of filaggrin and loricrin. In vivo it demonstrated moisturizing activity by decreasing TEWL after 24 and 48 hours of application of a product containing the active ingredient. Perilla frutescens phytocomplex was found to be compatible in vaginal gel formulations with both natural, semi-synthetic and synthetic polymers but precautions must be used for pH range and polymer percentage.
Discussion and Conclusion: Perilla frutescens phytocomplex produced by in vitro plant cell culture technology is a new cosmetic ingredient with sustainable and safety features that can be used in cosmetic product, particularly for intimate care. It demonstrated high anti-inflammatory and hydrating activity. Mucosa and skin barrier function were maintained by regulation of tight junctions and skin barrier proteins levels. The active ingredient is compatible with different classes of rheological modifiers, but precautions must be used in formulation for pH range and polymer percentage.
Methods: Perilla frutescens phytocomplex was produced by using in vitro plant cell cultures technology. UPLC-ESI-MS and UPLC-DAD analysis were used for the identification and quantification of the secondary metabolites (rosmarinic acid and anthocyanins) of the phytocomplex. In vitro tests on HaCaT cells were used to evaluate expression of inflammatory cytochines with ELISA kit after an inflammatory stress. On HaCaT cells were studied the expression of tight junctions (Zonula occludens-1 and Occludin) and skin barrier proteins (Filaggrin and Loricrin) by using Western blot assay after inflammatory and oxidative insult with and without pretreatment with Perilla frutescens phytocomplex. We evaluated the moisturizing activity of this new active ingredient through the reduction of TEWL, after irritative damage with sodium lauryl sulfate, using in vivo test on 20 Caucasian subjects. The soothing capacity of Perilla frutescens phytocomplex was evaluated on in vitro vaginal mucosa after irritation induced by lactic acid. Perilla frutescens phytocomplex was used in intimate gels to evaluate the compatibility and the stability with different rheological modifiers classes.
Results: The Perilla frutescens phytocomplex prepared by suspension cell cultures, was analyzed by UPLC-ESI-MS which provided the presence of secondary metabolites such as rosmarinic acid,metyl ester of rosmarinic acid, salvionolic acid and anthocyanins (shisonin and methyl shisonin). The standardized content of polyphenols, expressed as rosmarinic acid, is 3,3% w/w and the content of anthocyanins, expressed as cyanidin-3-O-glucoside, is 0,16% w/w, and they were determined by UPLC-DAD analysis. The phytocomplex has shown a high anti-inflammatory activity significantly inhibiting the release of inflammatory cytochines TNF-α, IL-1β e IL-6. It was able to prevent the alteration induced by LPS + H2O2, bringing the Occludin and Zonula Occludens-1 levels back to those of the control on human keratinocytes pretreated for two hours. Pretreatment with the active at 100 µg/mL for 2 hours is able to countervert the action of LPS and H2O2 by reducing the levels of filaggrin and loricrin. In vivo it demonstrated moisturizing activity by decreasing TEWL after 24 and 48 hours of application of a product containing the active ingredient. Perilla frutescens phytocomplex was found to be compatible in vaginal gel formulations with both natural, semi-synthetic and synthetic polymers but precautions must be used for pH range and polymer percentage.
Discussion and Conclusion: Perilla frutescens phytocomplex produced by in vitro plant cell culture technology is a new cosmetic ingredient with sustainable and safety features that can be used in cosmetic product, particularly for intimate care. It demonstrated high anti-inflammatory and hydrating activity. Mucosa and skin barrier function were maintained by regulation of tight junctions and skin barrier proteins levels. The active ingredient is compatible with different classes of rheological modifiers, but precautions must be used in formulation for pH range and polymer percentage.