Juniper berry extract containing Anthricin and Yatein suppresses lipofuscin accumulation in human epidermal keratinocytes through proteasome activation, increases brightness and decreases spots in human skin.

OBJECTIVE: Skin brightness and spot have a significant impact on youthful and beautiful appearance. One important factor influencing skin brightness is the amount of internal reflected light from the skin. Observers recognize the total surface-reflected light and internal reflected light as skin brightness. The more internal reflected light from the skin, the more attractive and brighter the skin appears. This study aims to identify a new natural cosmetic ingredient that increases the skin's internal reflected light, decreases spot and provides a youthful and beautiful skin appearance. METHODS: Lipofuscin in epidermal keratinocytes, the aggregating complex of denatured proteins and peroxidized lipids, is one factor that decreases skin brightness and causes of spot. Aggregates block light transmission, and peroxidized lipids lead to skin yellowness, dullness and age spot. Lipofuscin is known to accumulate intracellularly with ageing. Rapid removal of intracellular denatured proteins prevents lipofuscin formation and accumulation in cells. We focused a proteasome system that efficiently removes intracellular denatured proteins. To identify natural ingredients that increase proteasome activity, we screened 380 extracts derived from natural products. The extract with the desired activity was fractionated and purified to identify active compounds that lead to proteasome activation. Finally, the efficacy of the proteasome-activating extract was evaluated in a human clinical study. RESULTS: We discovered that Juniperus communis fruits (Juniper berry) extract (JBE) increases proteasome activity and suppresses lipofuscin accumulation in human epidermal keratinocytes. We found Anthricin and Yatein, which belong to the lignan family, to be major active compounds responsible for the proteasome-activating effect of JBE. In a human clinical study, an emulsion containing 1% JBE was applied to half of the face twice daily for 4 weeks, resulting in increased internal reflected light, brightness improvement (L-value) and reduction in yellowness (b-value) and spot in the cheek area. CONCLUSION: This is the first report demonstrating that JBE containing Anthricin and Yatein decreases lipofuscin accumulation in human epidermal keratinocytes through proteasome activation, increases brightness and decreases surface spots in human skin. JBE would be an ideal natural cosmetic ingredient for creating a more youthful and beautiful skin appearance with greater brightness and less spot.

OBJECTIF: La luminosité et les taches de peau ont un impact significatif sur la jeunesse et la beauté de l'apparence. L'un des facteurs importants influençant la luminosité de la peau est la quantité de lumière interne réfléchie par la peau. Pour les observateurs, la luminosité de la peau correspond à la somme de la lumière réfléchie par la surface et de la lumière réfléchie par l'intérieur de la peau. Plus la quantité de lumière interne réfléchie par la peau est importante, plus la peau semble attrayante et lumineuse. Cette étude vise à identifier un nouvel ingrédient cosmétique naturel qui augmente la lumière interne réfléchie par la peau, diminue les taches et donne à la peau une apparence jeune et belle. MÉTHODES: La lipofuscine dans les kératinocytes de l'épiderme, le complexe agrégé de protéines dénaturées et de lipides peroxydés, est un facteur qui diminue l'éclat de la peau et qui est à l'origine des taches. Les agrégats bloquent la transmission de la lumière et les lipides peroxydés entraînent une coloration jaune de la peau, un aspect terne et des taches de vieillesse. On sait que la lipofuscine s'accumule au niveau intracellulaire avec le vieillissement. L'élimination rapide des protéines dénaturées intracellulaires empêche la formation et l'accumulation de lipofuscine dans les cellules. Nous avons mis l'accent sur un système de protéasome qui élimine efficacement les protéines dénaturées intracellulaires. Pour identifier les ingrédients naturels qui augmentent l'activité du protéasome, nous avons passé au crible 380 extraits dérivés de produits naturels. L'extrait présentant l'activité souhaitée a été fractionné et purifié afin d'identifier les composés actifs qui conduisent à l'activation du protéasome. Enfin, l'efficacité de l'extrait activant le protéasome a été évaluée dans une étude clinique humaine. RÉSULTATS: Nous avons découvert que l'extrait de Juniperus communis fruits (baie de genièvre) augmente l'activité du protéasome et supprime l'accumulation de lipofuscine dans les kératinocytes épidermiques humains. Nous avons découvert que l'anthricine et la yateine, qui appartiennent à la famille des lignanes, sont les principaux composés actifs responsables de l'effet activateur du protéasome de l'extrait de baies de genévrier. Dans une étude clinique humaine, une émulsion contenant 1 % de JBE a été appliquée sur la moitié du visage deux fois par jour pendant 4 semaines, ce qui a entraîné une augmentation de la lumière interne réfléchie, une amélioration de la luminosité (valeur L) et une réduction de la jaunisse (valeur b) et des taches dans la zone des joues. CONCLUSION: Il s'agit du premier rapport démontrant que l'EBJ contenant de l'anthricine et de la yateine diminue l'accumulation de lipofuscine dans les kératinocytes épidermiques humains par l'activation du protéasome, augmente la luminosité et diminue les taches superficielles de la peau humaine. Le JBE serait un ingrédient cosmétique naturel idéal pour créer une peau plus jeune et plus belle, plus lumineuse et moins tachetée.

Arnica montana L. extract containing 6-O-methacryloylhelenalin and 6-O-isobutyrylhelenalin accelerates growth and differentiation of human subcutaneous preadipocytes and leads volumizing of skin.

OBJECTIVE: An important factor in the aging of the face is a reduction in the volume of adipose tissue. This reduction in adipose tissue contributes to decreased skin elasticity, which is also part of the aging process. Overall, these lead to wrinkle formation. Fat injection is a common means of addressing this issue and is used to reduce the effects of aging on the face and to increase the fullness of the lips and breasts. However, fat injection is an invasive surgical procedure. This study aimed to discover novel cosmetic ingredients that increase the volume of subcutaneous (pre)adipocytes to create the appearance of more youthful skin. METHODS: We focused on the number of subcutaneous preadipocytes and the accumulation of lipid droplets. To discover natural ingredients that increase both of these, extracts of 380 natural products were prepared and screened for their effects on both growth and differentiation (i.e., lipid droplet accumulation) of human subcutaneous preadipocytes. One extract was found to have the desired effects, and this was further studied to determine the active compounds. We then evaluated its efficacy in a human clinical study. RESULTS: We found that Arnica montana L. flower extract (AFE) accelerates both the growth and the differentiation of human subcutaneous preadipocytes. AFE was found to significantly increase the volume of adipocyte spheroids. The active compounds 6-O-methacryloylhelenalin and 6-O-isobutyrylhelenalin were found to be responsible for the effects of AFE on preadipocytes. In a human clinical study, gels containing 1% AFE successfully enhanced the volume of the lips and face with reduction of wrinkles with no adverse reactions. CONCLUSION: This is the first report to demonstrate that AFE and the included compounds, 6-O-methacryloylhelenalin and 6-O-isobutyrylhelenalin, act on preadipocytes. AFE would be ideal for use in products that plump the face to reduce wrinkles and create a more youthful appearance.

OBJECTIF: Un facteur important du vieillissement du visage réside dans la réduction du volume du tissu adipeux. Cette réduction du tissu adipeux contribue à une diminution de l'élasticité de la peau qui fait également partie du processus de vieillissement. Globalement, ces facteurs induisent la formation des rides. L'injection de graisse est un moyen courant pour remédier à ce problème et sert à réduire les effets du vieillissement sur le visage et à augmenter la plénitude des lèvres et des seins. Cependant, l'injection de graisse est une intervention chirurgicale invasive. Cette étude visait à découvrir des ingrédients cosmétiques innovants qui augmentent le volume des (pré)adipocytes sous-cutanés pour créer l'apparence d'une peau plus jeune. MÉTHODES: Nous avons mis l'accent sur le nombre de préadipocytes sous-cutanés et sur l'accumulation de gouttelettes lipidiques. Pour découvrir des ingrédients naturels qui augmentent ces deux facteurs, des extraits de 380 produits naturels ont été préparés et analysés en vue de la détermination de leurs effets sur la croissance et la différenciation (c'est-à-dire l'accumulation de gouttelettes lipidiques) des préadipocytes humains sous-cutanés. Un extrait s'est avéré avoir les effets escomptés et il a fait l'objet d'études approfondies visant à déterminer les composés actifs. Nous avons ensuite évalué son efficacité dans une étude clinique chez l'homme. RÉSULTATS: Nous avons découvert que l'extrait de fleur de l'Arnica montana L. (AFE) accélère à la fois la croissance et la différenciation des préadipocytes humains sous-cutanés. L'AFE s'est avéré augmenter considérablement le volume des sphéroïdes des adipocytes. Les composés actifs 6-Ométhacryloyl-hélénaline et 6-O-isobutyryl-hélénaline se sont avérés responsables des effets de l'AFE sur les préadipocytes. Dans une étude clinique chez l'homme, des gels contenant 1 % d'AFE ont permis d'améliorer le volume des lèvres et du visage avec une réduction des rides sans effets indésirables. CONCLUSION: Il s'agit du premier rapport démontrant que l'AFE et les composés inclus, 6-O-méthacryloyl-hélénaline et 6-O-isobutyryl-hélénaline, agissent sur les préadipocytes. L'AFE serait idéal pour les produits qui repulpent le visage afin de réduire les rides et de donner un aspect rajeuni.

Corrigendum: Identification of an alternative glycyrrhizin metabolite causing liquorice-induced pseudohyperaldosteronism and the development of ELISA system to detect the predictive biomarker.

[This corrects the article DOI: 10.3389/fphar.2021.688508.].

Identification of an Alternative Glycyrrhizin Metabolite Causing Liquorice-Induced Pseudohyperaldosteronism and the Development of ELISA System to Detect the Predictive Biomarker.

Liquorice is usually used as crude drug in traditional Japanese Kampo medicine and traditional Chinese medicine. Liquorice-containing glycyrrhizin (GL) can cause pseudohyperaldosteronism as a side effect. Previously, we identified 18ß-glycyrrhetyl-3-O-sulfate (3) as a GL metabolite in Eisai hyperbilirubinuria rats (EHBRs) with the dysfunction of multidrug resistance-related protein (Mrp2). We speculated that 3 was associated with the onset of liquorice-induced pseudohyperaldosteronism, because it was mainly detected in serum of patients with suspected to have this condition. However, it is predicted that other metabolites might exist in the urine of EHBRs orally treated with glycyrrhetinic acid (GA). We explored other metabolites in the urine of EHBRs, and investigated the pharmacokinetic profiles of the new metabolite in EHBRs and normal Sprague-Dawley rats. We further analyzed the serum concentrations of the new metabolite in the patients of pseudohyperaldosteronism. Finally, we developed the analyzing method of these metabolites as a preventive biomarker for the onset of pseudohyperaldosteronism using an enzyme-linked immunosorbent assay (ELISA). We isolated a new GL metabolite, 18ß-glycyrrhetyl-3-O-sulfate-30-O-glucuronide (4). Compound 4 significantly inhibited rat type-2 11ß-hydroxysteroid dehydrogenase (11ß-HSD2) and was a substrate of both organic anion transporter (OAT) 1 and OAT3. Compound 4 was also detected in the serum of patients with suspected pseudohyperaldosteronism at an approximately 10-fold lower concentrations than 3, and these concentrations were positively correlated. Compound 4 showed a lower serum concentration and weaker inhibitory titer on 11ß-HSD2 than 3. We developed an enzyme-linked immunosorbent assay system using an anti-18ß-glycyrrhetyl-3-O-glucuronide (3MGA) monoclonal antibody to measure the serum concentration of 3 to facilitate the measurement of biomarkers to predict the onset of pseudohyperaldosteronism. Although we found 4 as the secondary candidate causative agent, 3 could be the main potent preventive biomarker of liquorice-induced pseudohyperaldosteronism. Compound 3 was detected in serum at a higher concentration than GA and 4, implying that 3 may be a pharmacologically active ingredient mediating not only the development of pseudohyperaldosteronism but anti-inflammatory effects in humans administered GL or other liquorice-containing preparations.

18ß-glycyrrhetyl-3-O-sulfate would be a causative agent of licorice-induced pseudoaldosteronism.

Licorice-induced pseudoaldosteronism is a common adverse effect in traditional Japanese Kampo medicine, and 3-monoglucuronyl glycyrrhetinic acid (3MGA) was considered as a causative agent of it. Previously, we found 22α-hydroxy-18ß-glycyrrhetyl-3-O-sulfate-30-glucuronide (1), one of the metabolites of glycyrrhizin (GL) in the urine of Eisai hyperbilirubinuria rats (EHBRs) treated with glycyrrhetinic acid (GA), and suggested that it is also a possible causative agent of pseudoaldosteronism. The discovery of 1 also suggested that there might be other metabolites of GA as causal candidates. In this study, we found 22α-hydroxy-18ß-glycyrrhetyl-3-O-sulfate (2) and 18ß-glycyrrhetyl-3-O-sulfate (3) in EHBRs' urine. 2 and 3 more strongly inhibited rat type 2 11ß-hydroxysteroid dehydrogenase than 1 did in vitro. When EHBRs were orally treated with GA, GA and 1-3 in plasma and 1-3 in urine were detected; the levels of 3MGA were quite low. 2 and 3 were shown to be the substrates of organic anion transporter (OAT) 1 and OAT3. In the plasma of a patient suffering from pseudoaldosteronism with rhabdomyolysis due to licorice, we found 8.6 µM of 3, 1.3 µM of GA, and 87 nM of 2, but 1, GL, and 3MGA were not detected. These findings suggest that 18ß-glycyrrhetyl-3-O-sulfate (3) is an alternative causative agent of pseudoaldosteronism, rather than 3MGA and 1.

Isolation of a novel glycyrrhizin metabolite as a causal candidate compound for pseudoaldosteronism.

Pseudoaldosteronism is a common adverse effect associated with traditional Japanese Kampo medicines. The pathogenesis is mainly caused by 3-monoglucuronyl glycyrrhetinic acid (3MGA), one of the metabolites of glycyrrhizin (GL) contained in licorice. We developed an anti-3MGA monoclonal antibody (MAb) and an ELISA system to easily detect 3MGA in the plasma and urine of the patients. However, we found that some metabolites of GL cross-reacted with this MAb. Mrp2-deficient Eisai Hyperbilirubinemia rats (EHBRs) were administered glycyrrhetinic acid (GA), and we isolated 22α-hydroxy-18ß-glycyrrhetyl-3-O-sulfate-30-glucuronide (1) from the pooled urine with the guidance of positive immunostaining of eastern blot as the new metabolite of GL. The IC50 of 1 for type 2 11ß-hydroxysteroid dehydrogenase (11ß-HSD2) was 2.0 µM. Similar plasma concentrations of 1 and GA were observed 12 h after oral administration of GA to EHBR. Compound 1 was eliminated via urine, whereas GA was not. In Sprague-Dawley (SD) rats orally treated with GA, compound 1 was absent from both the plasma and the urine. Compound 1 was actively transported into cells via OAT1 and OAT3, whereas GA was not. Compound 1, when produced in Mrp2-deficiency, represents a potential causative agent of pseudoaldosteronism, and might be used as a biomarker to prevent the adverse effect.