Volume 23 | Number 3 | November 2020 MAGAZINE...IFSCC Magazine archives starting in 2000 most...

MAGAZINEThe Scientific Publication of the International Federation of Societies of Cosmetic Chemists

Volume 23 | Number 3 | November 2020

AWARDS

Holistic Beauty: 3D Macroscopic Visualization of Vasculature in Skin and Its Physical Relevance in Skin Aging

How Can We Control Unpleasant Body Malodor Effectively? Development of Novel Fragrances Using the Olfactory Receptor Technology

SUSTAINABILITY

A New Sustainable Anti-Aging Product Using Aquaponics Technology

Impact of Anti-dandruff Piroctone-Olamine Designed Formulations on the Formation of Smart Deposit for Hair Surface

PROTECTION AGAINST ENVIRONMENT DAMAGES

A New Inositol Derivative to Mitigate the Effects of Air Pollution on Skin

A Global and Powerful Approach to Circumvent the Harmful Effects of Blue Light and IR-A Irradiation on the Skin

supported by

162 IFSCC Magazine 3 | 2020© 2020 International Federation of Societies of Cosmetic Chemists

SCIENTIFIC PAPERS Holistic Beauty: 3D Macroscopic Visualization of Vasculature in Skin and Its Physical Relevance in Skin Aging Kazuki Takagaki, Yuko Matsumoto, Mika Sawane, Yusuke Hara, Akiko Miyake, Kentaro Kajiya (Japan) �� 165

How can we control unpleasant body malodor effectively? Development of novel fragrances using the olfactory receptor technology Hisashi Mihara, Azusa Miki, Masato Murai, Ikuo Terada, Yukari Kuwahara, Tsuyoshi Kobayashi, Noboru Sano,

Kenya Ishida, Kunihide Hoshino, Naomi Hirano (Japan) �� 173

A New Sustainable Anti-Aging Product Using Aquaponics Technology You Ah Kim, Tae Byeong Jeon, Chae Eun Lim, Ki Yong Lee, Dong Hee, Kim, Ji Eun Woo, Byoung Jun Park, Hakhee Kang (Korea) �� 179

Impact of Anti-dandruff Piroctone-Olamine Designed Formulations on the Formation of Smart Deposit for Hair Surface Nawel Baghdadli, Frans Leemarkers, Gustavo S. Luengo, Christian Mazilier, Jean-Charles Cheneble,

Anne Potter, Fabien Leonforte (France, The Netherlands) �� 189

A New Inositol Derivative to Mitigate the Effects of Air Pollution on Skin Yuko Saeki, Shinji Yamaki, Ichiro Fujita, Naoko Motohashi, Eiko Kato (Japan) �� 199

A Global and Powerful Approach to Circumvent the Harmful Effects of Blue Light and IR-A Irradiation on the Skin Valérie Bicard-Benhamou, Marina Lefort, Lilia Heider, Jutta zur Lage, Silke Hornung, Heike Hanau,

Alexander Kielbassa, Christophe Carola, Joerg von Hagen (Germany) �� 207

Abstracts of Papers published in the International Journal of Cosmetic Science, Volume 42, No� 5, 2020 �� 219

AD INDEXKOSMET ��163 IFSCC Congress 2022 ��164

IFSCC Congress 2023 ��218 IFSCC Conference 2021 ��228

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IFSCC Magazine 3 | 2020 165© 2020 International Federation of Societies of Cosmetic Chemists

Holistic Beauty: 3D Macroscopic Visualization of Vasculature in Skin and Its Physical Relevance in Skin AgingKazuki Takagaki, Yuko Matsumoto, Mika Sawane, Yusuke Hara, Akiko Miyake, Kentaro KajiyaShiseido Global Innovation Center, 1-2-11, Takashima, Nishi-ku, Yokohama, Japan

Keywords: 3D visualization, capillary, elasticity, skin aging, holistic beauty

This publication was originally presented as a podium presentation at the 25th IFSCC Conference in Milan, Italy, September 30 - October 2, 2019.

We used various cutting-edge methods and models, including clearing techniques for human skin tissue, in vivo noninvasive visu-alization of vascular structures, in vitro gel model co-culturing and its measurement, and our ex vivo angiogenesis model in order to shed light on the key role of the blood vessels in maintaining the skin’s elasticity, as they seem to serve as the steering func-tion for positive circulation in the dermis. A better understanding of this topic could lead to innovative beauty solutions to serve the growing trend of “holistic beauty.”

INTRODUCTION

Holistic beauty, where beauty consists of not only one’s external appearance, but also the state of one’s entire body, is a trending idea. This idea of understanding the body as a whole is also described in the ancient Chinese medical book, Huang Di Nei Jing. It is included in the UNESCO Memory of the World register, showing it has existed for thousands of years. In line with this idea in western medicine, as Wil-liam Osler stated, “A man is as old as his arteries.” Gaining acceptance is the con-ception of skin not as a separate organ but as physically and functionally connected to the rest of the body through the vascular, immune, neuronal and endocrine systems. In our previous research we discovered that we can estimate vascular function as par-ticularly related to the aging process, which includes skin aging. We reported that the severity of photodamaged skin correlates with reduced numbers of blood vessels in the dermis [1]. However, the relevance of blood vessels, including their physical properties and molecular mechanisms, to the process of skin aging remains unclear.

Typically, the main symptoms of skin ag-ing – including photoaging – are sagging and wrinkling, and many people are very concerned about these perceived blemish-es because of the adverse effects on their appearance. Since it has been reported that these troubles are strongly related to the elasticity of the skin, including the collagen,

elastin and hyaluronic acid status, elasticity can be defined as one of the indicators of skin aging [2]. Although involvement of human fibroblasts in the elasticity of the dermis has been investigated, interactions between elasticity and other body compo-nents such as blood vessels have not been fully understood.

The purpose of this study was therefore to examine the relationship between blood vessels and physical properties, especially the elasticity of skin, both in vitro to in vivo.

Abstract

There seems to be ever increasing in-terest for “holistic beauty” in the cos-metic field, which refers to the idea that skin is not a separate organ but is physically and functionally connected to the body through its vascular, im-mune and endocrine systems. Vascu-lar function is particularly related to the aging process, which includes the skin. However, the relevance of blood vessels to the process of skin aging, including their physical properties, still remains to be elucidated.

In this study, we succeeded in making a 3D macroscopic visualization of cap-illaries in human skin. It revealed that capillaries are densely and uniformly networked in the upper dermis – par-

ticularly with a regular loop structure just below the epidermis – and were reduced with photoaging. This inno-vative visualization allowed examina-tion of the relationship between the capillaries and physical features of the skin, such as elasticity, with in vivo testing using optical coherence to-mography angiography and in vitro model. This work suggested that elas-ticity was strongly correlated with the density and size of capillaries. Further-more, our ex vivo skin culture model clearly demonstrated that type I col-lagen was found closely surrounding the blood vessels, suggesting a pos-sible mechanism of increased elastic-ity by vessel-associated collagen pro-duction. These results indicate that blood vessels play an important role in maintaining skin elasticity.

This paper received the

Best Podium Award 2019

at the IFSCC Conference in Milan


EXPERIMENTAL

Human skin samplesCheek skin samples from ILSBIO LLC (Ches-tertown, MD, USA) were collected accord-ing to US and International ethical guide-lines. All ILSBIO protocols were approved by a Health and Human Services-registered In-stitutional Review Board. Informed consent was obtained from each patient prior to collection. We also obtained skin samples including subcutaneous adipose tissues from the Gakugeidai-Nishiguchi Clinic (To-kyo, Japan). We confirmed that those sub-jects did not suffer from atopic dermatitis or rosacea. All procedures involving human subjects were approved by the Institutional Review Board of the Shiseido Global In-novation Center, and all subjects provided written informed consent.

Immunostaining and clearing for 3D visualizationWe selected the iDISCO as the best of several various methods of tissue clear-ing and performed all procedures in ac-cordance with our previously described report [3]. Polyclonal antibodies against CD31 (R&D Systems, Minneapolis, MN, USA) were used as the primary antibody and an anti-sheep antibody conjugated with Alexa Fluor 594 (Life Technologies, Carlsbad, CA, USA) was used as the sec-ondary antibody.

Optical coherence tomography angiography (OCTA) for in-face vascular imagingWe performed basic measurement proce-dures of optical coherence tomography angiography (OCTA) and its quantitative analysis as previously described [4]. The following two protocols for human studies were approved by the Ethics Committee of Shiseido Co., Ltd. Healthy volunteers were enrolled after obtaining their written informed consent. Prior to the studies vol-unteers were acclimatized for 15 minutes at 22 °C and 40 % relative humidity.

Human study 1:Investigation of the relationship between skin elasticity and vascular conditionFifty-four female Asian volunteers, vary-ing in age from their 20 s to 60 s, were enrolled in the study. First, the skin’s vas-

culature was visualized using our OCTA system. To identify the measurement posi-tion, facial photographs were taken using a COOLPIX4500 digital camera (NIKON Corp., Tokyo, Japan). Then, skin elasticity was measured using a Cutometer SEM575 Skin Elasticity Meter® (Cutometer, Cour-age & Khazaka Electronic GmbH, Cologne, Germany) whose suction hole is 2 mm in diameter with a suction pressure setting of 200 mbar. In the Cutometer measurement a constant load was applied for 2 seconds followed by a 2-second relaxation period. We defined Ur / Uf, the recovery ratio of retraction (Ur) to maximal skin deforma-tion at the end of the suction period, as the skin elasticity.

Human study 2: Evaluation of the effect of temperature-controlled thermal instrumentsFourteen female Asian volunteers, vary-ing in age from their 20 s to 40 s, were enrolled in the study. First, the skin’s vas-culature was visualized using our OCTA system. Afterwards our own originally-designed temperature-controlled thermal instrument, set to 40 °C, was applied to the skin for 5 minutes. Then the skin’s vasculature was visualized again.

CellsHuman umbilical endothelial cells (HU-VECs, PromoCell, Heidelberg, Germa-

ny) and human neonatal dermal fibro-blasts (HFs, Lonza, Basel, Switzerland) were used for these studies. HUVECs and HFs were cultured in Endothelial Cell Basal Medium-2 (EBM-2, Lonza) supplemented with growth factors in-cluding 0.5 % fetal bovine serum (FBS, Life Technologies, Carlsbad, CA, USA) and Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10 % FBS, respectively, in humidified 5 % CO2 / air at 37 °C.

In vitro gel model and measuring elasticityStirling 0.3 % collagen type I-A (Nitta- gelatin, Osaka, Japan), DMEM without FBS, HUVECs suspended in EBM-2 with 0.5 % FBS and HFs suspended in DMEM without FBS at each concentration, de-pending on the experiment, with 6 ml of this mixture pipetted per well onto 6-well plates. After 5 days of incubation in humidified 5 % CO2 / air at 37 °C, the elasticity was measured using a rheom-eter (MCR300, Paar Physica, Stuttgart, Germany). For immunostaining, poly-clonal antibodies against CD31 (R & D Systems, Minneapolis, MN, USA) were used as the primary antibody and an anti-sheep antibody conjugated with Alexa Fluor 488 (Life Technologies, Carlsbad, CA, USA) was used as the secondary antibody. We performed the

Figure 1 3D visualization of blood vessels in human cheek skin.

Immunofluorescence stains of whole cheek skin for the pan-endothelial marker CD31 (red) observed

using light-sheet microscopy. These representative images are snapshots of a 3D movie.

The left side is 22-year-old male tissue, the right side is 54-year-old male tissue. The lower side is an

enlarged view focusing on a point just below the epidermis. Capillaries are densely and uniformly

networked in the upper dermis and decrease with aging. Bar: 300 µm.

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image analysis using Imaris software (BITPLANE).

siRNA transfectionsiRNA transfection was performed using the Basic Nucleofactor Kit for primary mammalian endothelial cells (Amaxa Biosystems, Cologne, Germany). Briefly, cells were transfected by electroporation (Nucleofactor II, Amaxa Biosystems) using 3 silencer selective, pre-designed siRNAs for VE-Cadherin (s2780, s2781, s2782, Applied Biosystems Inc., Foster City, CA, USA), integrin-α3 (s7541, s7542, s7543), integrin-α5 (s7547, s7548, s7549), integrin-α6 (s7492, s7493, s7494), or the control siRNA (Silencer negative con-trol #1 siRNA, Ambion, Cambridgeshire, UK). The control siRNA, comprising a 19-bp-scrambled sequence with 3’dT overhangs, was used as the control as it has no significant sequence homology to any known gene sequence. Knockdown of target molecules was confirmed by a quantitative polymerase chain reaction (qPCR) using the following probes; VE-cadherin: Cat#_Hs00170986_m1,Itg-α3: Cat#_Hs01076879_m1, Itg-α5: Cat#_Hs01547673_m1, Itg-α6: Cat#_Hs01041011_m1 (Applied Biosystems Inc.).

Measuring contact force and the ex vivo gel model The contact forces of human cheek skin and collagen gels were measured as an elasticity index using a softness sensor system as previously reported [5]. Em-bedding the human skin tissues in the gels of collagen type I-A (Nitta-gelatin), it was cultured for 21 days in EBM-2 (Cambrex, Verviers, Belgium), including 50 ng / ml VEGFA. Immunostaining was performed using polyclonal antibodies against CD31 (R&D Systems) and type I collagen (Abcam, Cambridge, UK) as the primary antibody after fixation with 4 % paraformaldehyde. An anti-sheep antibody conjugated with Alexa Fluor 488 (Molecular Probes, Eugene, OR, US) and an anti-rabbit antibody conjugated with Alexa Fluor 594 (Molecular Probes) were used as the secondary antibod-ies. Images were taken using a confocal microscopy LSM880 (CarlZeiss, Jena, Germany).

Figure 2 Skin elasticity as a function of capillary density and diameter.

a) Representative OCTA images acquired by projecting maximum signal intensities from the top

of the dermis to 494 µm of young (20s) and old (60s) cheek. The field of view is 7.5 mm x 7.5 mm.

b), c) The density and diameter of the capillaries correlated significantly with skin elasticity measured

by a Cutometer. N = 54, 20 s to 60 s, p < 0.1, *p < 0.05, respectively.

Figure 3 Establishment of an in vitro gel model to assess the effects of capillaries on dermal elasticity.

a) To examine the effects of capillaries on dermal elasticity, an in vitro gel model was established

and its elasticity was measured by a rheometer.

b) Human umbilical vein endothelial cells (HUVECs: red) formed a luminal structure in the gel model

with human fibroblasts (HFs: green). The HFs were labeled in green using the PKH67-Green fluorescent

cell labeling kit (Sigma) and HUVECs were immunofluorescent stained with anti-CD31. Bar: 100 µm.

c) The models in which HUVECs and HFs were co-cultured had significantly greater elasticity than

the models with only HFs. **p < 0.01.

d) The diameters of the models including HUVECs were significantly smaller than those of the models

with only HFs. Both models used a total of 5 x105 cells, but for the co-culture model, 2.5 x105 HFs

and HUVECs were used. *p < 0.05.


RESULTS

3D visualization of human skin Although the skin is a complex 3D tissue including various organs such as sweat glands, sebaceous glands, nerves and vessels, histological studies of the skin pronominally use 2D sections. Therefore, to understand the structure of capillar-ies in the human skin in more detail, we used a tissue clearing technique and light-sheet microscopy to visualize the capillary structures in the human skin three-dimen-sionally. Previously, we reported iDISCO treatment using a solvent as the best of various clearing methods for human skin tissue [3]. Clearing the immunolabeled hu-man cheek skin tissue with the anti-CD31 antibody as a pan-endothelial marker us-ing the iDISCO treatment revealed that capillaries are densely and uniformly net-worked in the upper dermis, particularly with a regular loop structure just below the epidermis (Figure 1, upper). In ad-dition, a decrease in the density of the capillaries and an increase in irregular loop structure just below the epidermis were observed with aging in the cheek skin (Figure 1, lower). These data suggested that capillaries just below the epidermis seem to physically hold up the skin.

Capillary visualization using a noninvasive methodAs the capillary loop structure visualized by clearing techniques looked like it was lifting the skin, the 3D capillary structure allowed us to hypothesize that capillaries play a role in physical features like elasticity. To test this hypothesis, the elasticity of the cheek skin was measured using a Cutometer and the same position of vascular structure was visualized noninvasively using optical coher-ence tomography angiography (OCTA) [4]. Consistent with the 3D visualization of the capillaries, a decrease in blood vessel density with aging was also observed in our OCTA images (Figure 2a). Surprisingly, it was re-vealed that the cheek skin’s elasticity had a positive correlation with capillary density (Figure 2b, N = 54, p < 0.1) as well as di-ameter (Figure 2c, N = 54, p < 0.05). Based on the results above, we estimated that the presence of dense capillaries, as seen in young people, could control the physical properties, especially the elasticity of skin.

of the cells was the same. Consistent with this, the size considered to indicate the contraction force of this model was sig-nificantly smaller in the co-culture models (Figure 3d).To investigate the molecules involved, the expression of vascular endothelial cadherin (VE-Cadherin), an endothelial-specific ad-hesion molecule, was suppressed with siR-NA in HUVECs (Figure 4a). The elasticity decreased significantly in the model with VE-Cadherin-suppressed HUVECs com-pared with control models (Figure 4b). The HUVEC network structure was also

Figure 4 Role of VE-Cadherin in dermal elasticity and formation of the HUVECs network structure.

a) To investigate the molecules involved, the expression of vascular endothelial cadherin (VE-cadherin),

an endothelial-specific adhesion molecule, was suppressed with siRNA in HUVECs.

b) The elasticity decreased significantly in the model with VE-Cadherin-suppressed HUVECs compared

with the control model. c) The HUVECs network structure (red) was not formed densely in the gel model

when expression of VE-cadherin in HUVECs was downregulated compared with the control.

d-f) Image analysis showed a considerable decline in the length (d), the volume (e) and the

cross-sectional area (f) in VE-cadherin-suppressed HUVECs network structure compared with

the control model. *p < 0.05, **p < 0.01.

Effect of capillary network structureon skin elasticity in the newly established in vitro gel modelTo examine the structural effects of capil-laries on dermal elasticity, an in vitro gel model was established (Figure 3a), and its elasticity was measured by a rheom-eter. The model in which human umbili-cal vein endothelial cells (HUVECs) and human fibroblasts (HFs) were co-cultured had tube structures derived from HUVEC (Figure 3b) and significantly greater elasticity than the model with only HFs (Figure 3c), although the total number

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not formed densely in the gel model when the expression of VE-Cadherin in HUVECs was downregulated compared with the control (Figure 4c). Furthermore, image analysis showed a considerable decline in the length, volume and cross-sectional ar-ea in VE-Cadherin-suppressed HUVEC net-work structures was observed compared with the control model (Figure 4d-f). Similar experiments were performed by adding Angiopoietin-1 (Ang-1), which is known to enhance vascular stabilization through its upregulating expression of VE-cadherin [6, 7], to the above in vitro gel model. The result showed an increase in elasticity and volume of capillaries, while there was no considerable difference in the length or cross-sectional area (Figure 5).

Integrin (Itg) -α3, -α5 and -α6 molecules, which are expressed in vascular endotheli-al cells, are known to play an indispensable role when interacting with the extracellular matrix contributing to the skin’s elasticity. To examine the importance of adhesion between blood vessels and the extracel-lular matrix, we suppressed each molecule in the HUVECs using siRNA (Figure 6a). The elasticity of the gel model with Itg-α5 siRNA significantly decreased, while those with Itg-α3 and α6 siRNA were equivalent

to the control siRNA (Figure 6b). The HU-VECs network structure in the gel model with Itg-α5 siRNA looked thinner and longer than the control (Figure 6c), and the image analysis also showed that the network structure of HUVECs with Itg-α5 siRNA was significantly longer and thin-ner than the control, although there was no considerable difference in the volume (Figure 6d-f). Due to the selective binding of Itg-α5 to fibronectin, while Itg-α3 and α6 bind to collagen and laminin, respec-tively, it was suggested that the binding between the blood vessel and fibronectin is important for skin elasticity.

Moreover, to evaluate aging, senescent HUVECs were used as models and com-pared with young HUVECs. There was a significant decline in the elasticity of the model with senescent HUVECs compared with the young HUVECs, accompanied by a significant decrease in expression levels

Figure 5 Effect of Ang-1 on the HUVECs network structure and dermal elasticity structure.

a) Angiopoietin-1 (Ang-1) has a vascular protective effect by promoting expression of VE-cadherin. The HUVECs

network structure in a gel model was enhanced by addition of Ang-1 compared with the control.

b) The elasticity of the gel model significantly increased with Ang-1 compared with the control.

c-e) The volume of the HUVECs network structure was significantly larger in the gel model with Ang-1 than that

of the control (d), while there was no considerable difference in the length (c) or cross-sectional area (e) *p < 0.05.

Figure 6 Contribution of interaction between HUVECs and the extracellular matrix to dermal elasticity.

The influence of the interaction between HUVECs and the extracellular matrix (ECM) on elasticity and the

HUVECs network structure was examined. a) The main molecules in HUVECs related to cell-ECM adhesion,

Itg-α3, -α5 and -α6, were suppressed by siRNA, respectively. b) The elasticity of the gel model with Itg-α5 siRNA

was significantly decreased, while those with Itg-α3 and α6 siRNA were equivalent to those of the control siRNA.

c) The HUVECs network structure in the gel model with Itg-α5 siRNA was lankier than the control.

d-f). The image analysis showed that HUVECs, whose Itg-α5 expression was suppressed, formed a significantly

longer (d) and thinner (f) network structure than the control, although there was no considerable difference in

the volume (e) *p<0.05.


of VE-Cadherin and Itg-α5 in aged HU-VECs (Figure 7). However, Itg-α3 and -α6 expressions were comparable between the young and aged HUVECs. These data suggested that senescent HUVECs show a decline in elasticity due to a deficient HUVECs network structure with lower ex-pression levels of VE-cadherin and Itg-α5.

Effect of thermal stimulation on skinelasticity and the density and diameterof capillaries in vivoSince a dense network structure of sta-ble capillaries was shown to be impor-tant for skin elasticity, we investigated the effect of thermal stimulation on skin elasticity via changes in the skin’s capillaries in vivo. We confirmed that 5 minutes of moderate thermal stimula-tion (40 °C) on a human cheek using a temperature-controlled thermal instru-ment significantly increased the skin’s temperature (Figure 8a). Blood flow is generally known to increase with the rise of skin’s temperature, but an in-crease in the density and diameter of the capillaries, possibly due to an increase in blood flow, was observed with OCTA (Figure 8b-d). Furthermore, consistent with the above results, the elasticity of the skin also significantly increased, along with enhancement of the capillaries via ther-mal stimulation (Figure 8e). In addition, the expression levels of VE-cadherin and Itg-α5 in HUVECs cultured at a high tem-perature (37 °C) were shown to be higher than in those at normal skin temperature (33 °C) or lower (29 °C) (Figure 8f, g).

Although it is necessary to differentiate between the results of the in vivo experi-ments, which show a short-term response, and the in vitro experiments, continuous long-term application of thermal stimu-lation may maintain and/or increase skin elasticity through capillary enhancement.

Induction of collagen synthesis aroundcapillary structures in models with adequate levels of elasticityTo clarify the action mechanism of the presence of stable capillaries and how it contributes to the elasticity of the skin, we used an ex vivo angiogenesis culture model that included human skin tissue in collagen gels for neovascular observation.

Figure 7 Effect of HUVEC aging on elasticity and expression levels of VE-cadherin and integrins.

a) To evaluate aging, aged HUVECs (50s) were compared with young cells (newborn). There was

a significant decline in the elasticity of the model with aged HUVECs compared with the young.

b, c) The expression levels of VE-Cadherin (b) and Itg-α5 (c) in aged HUVECs decreased significantly,

although Itg-α3 and -α6 expressions were comparable between young and aged HUVECs. *p<0.05, p<0.1.

Figure 9 Neovessel-induced collagen synthesis.

a) Contact forces of gels were measured by the softness sensor system. 0.9 mg / ml collagen gels showed

a 17.5 mN contact force, which is comparable to that of human cheek skin. b) Staining image of neovessel

(green, CD31) and type I collagen (red) in the ex vivo model. Arrows indicate collagen scaffolds for the neovessel.

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To set an elasticity environment close to that of human skin, contact forces of gels with various collagen concentrations were measured by the softness sensor system [5]. We found that 0.9 mg / ml collagen gels have a 17.5 mN contact force, which is comparable to that of human cheek skin (Figure 9a). We decided this model had adequate elasticity to be used as our ex vivo model. Interestingly, in this model production of type I collagen was observed around the neovasculature like scaffolds (Figure 9b). This data suggested that like the phenomenon of seeds in soil, capillar-ies might where they extend induce fibro-blasts to synthesize collagen, leading to an increase in skin elasticity.

DISCUSSION

In this study we found a strong relation-ship between capillaries and skin elasticity using in vitro to in vivo experiments. With regard to skin elasticity directly linked to skin problems, such as sagging and wrin-kling, attention has been focused only on fibroblasts and their products. Contrary to this, the possibility derived from our exper-iments that the presence of capillaries or their stability could control skin elasticity suggests this could be helpful for new skin aging care proposals. In addition, as the circulatory system, including blood ves-sels, has been shown to be important for the inhibition of UV-induced skin damage

[8], vascular-targeted approaches have the potential to be an innovative solution to photoaging itself as well as for condi-tions such as sagging and wrinkling. On the other hand, a large number of blood vessels is not always good for the skin, as it has been report that the number of blood vessels in the upper layer of the dermis are abnormally increased at the pigmented sites of solar lentigo [9]. As shown in this study, “living“ blood vessels with stable structures are important and could be a solution to improve this condition, but more studies are needed.

CONCLUSION

Acknowlegments

This research was supported by the Japan Science and Technology Agency ImPACT program. References

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(1996) 1171-1180.

[7] Lee SW, Won JY, Lee HY, Lee HJ,

Youn SW, Lee JY, Cho CH, Cho HJ, Oh S,

Chae IH, and Kim HS, Angiopoietin-1 Pro-

tects Heart against Ischemia / Reperfusion

Injury through VE-Cadherin Dephosphor-

ylation and Myocardiac Integrin-β1/ ERK /

Caspase-9 Phosphorylation Cascade. Mol

Med. 17 (2011) 1095-1106.

[8] Sawane M, and Kajiya K, Ultraviolet light-

induced changes of lymphatic and blood

vasculature in skin and their molecular

mechanisms. Exp Dermatol. 21 (2012)

22-25.

[9] Hasegawa K, Fujiwara R, Sato K,

Park JY, Kim SJ, Kim M, and Kang HY,

Increased blood flow and vasculature

in solar lentigo. J Dermatol. 43 (2016)

1209-1213.

Corresponding AuthorKentaro Kajiya

Shiseido Global Innovation Center Yokohama

[email protected]

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How Can We Control Unpleasant Body Malodor Effectively? Development of Novel Fragrances Using the Olfactory Receptor TechnologyHisashi Mihara, Azusa Miki, Masato Murai, Ikuo Terada, Yukari Kuwahara, Tsuyoshi Kobayashi,Noboru Sano, Kenya Ishida, Kunihide Hoshino, Naomi HiranoCorporate Research & Development Division, Takasago International Corporation, 1-4-11 Nishiyawata, Hiratsuka City, Kanagawa, Japan

Keywords: Deodorant, Olfactory Receptor, Antagonist, Aging Odor, Axillary Odor

This publication received the best poster award at the 31st (virtual) IFSCC Congress in Yokohama, Japan, October 21-30, 2020.

[1]. In addition, Specific odor generated in the armpits of people after puberty is one of the strongest body malodors and annoys many people. It was reported that vinyl ketones such as 1-octen-3-one and 1,5-octadien-3-one were identified as key components of axillary odor, with their very low odor thresholds and strong metallic odor. They had a great effect on sweat odor [2]. Figure 1 shows the chemical structure of the key compo-nents of each malodor.

Many deodorizing methods have been developed for various malodor so far. Generally, deodorizing methods are clas-sified into biological methods, chemical methods, physical methods, and sen-sory methods. Sensory methods include »masking« which is most commonly used in various products, where malodors are covered by fragrance components that are stronger than the malodors. How-ever, excessive “masking” often makes consumers uncomfortable due to the overly strong odor.

The odor that we are sniffing is an ag-gregate of volatile organic compounds (odor molecules) with a molecular weight

INTRODUCTION

To improve the living environment, there is a serious demand for more effective deodorization of the unpleasant odors in daily life. Among them, awareness of body odor has been increasing recently.

Body odors are roughly classified into “odor of each parts of the body” such as bad breath, foot odor, axillary odor and scalp odor, and “a combined odor emit-ted from the trunk”. The latter is known

to change with age, and there is very high tendency to dislike the age-related odors observed in middle-aged and the elderly. This odor is called the aging odor (Karei-shu). It was reported that unsaturated al-dehydes such as 2-nonenal and 2-octenal are the causative substances of this odor

Abstract

Due to the effects of global warming and diversification of our lifestyles, vari-ous unpleasant odors such as body mal-odors are becoming a social problem. Accordingly, market needs for various effective deodorant products are rap-idly increasing. For their development, techniques for reducing malodor using chemical deodorizers and/or fragrances having masking effects have been com-monly used. In the field of a malodor masking technology, olfactory receptor (OR) assay technique has attracted huge attention as the latest technology that allows high-throughput screening for highly effective masking agents. In this study, we focused on suppressing both aging and axillary malodors as body mal-

odors based on this technology. OR2C1 was found to respond to both the aging and the axillary key odor components, such as (E)-2-nonenal, (E)-2-octenal, 1-octen-3-one and 1,5-octadien-3-one, and potential antagonists of OR2C1 were discovered with fragrance materi-als. The antagonist fragrance materials clearly suppressed odor strength of the malodor materials in the sensory evalu-ation. Furthermore, the effect was also clearly verified in experiments using fra-grances that contain the antagonists. In conclusion, utilizing the olfactory recep-tor technology, fragrances to control the body malodor were successfully devel-oped. This methodology is believed to be efficient and rapid for the develop-ment of high performance masking fra-grance compositions.

Figure 1 The chemical structure of the key components of each malodor.

This paper received the

Best Poster Award 2020

at the IFSCC Congress in Yokohama


of about 30 to 300. The olfactory recep-tor plays the role of sensor to detect each odor molecule. It had been reported that there are 396 types of OR functions in humans and 1,130 types in mice [3]. A lot of research has been conducted on the mechanism of odor molecules’ ac-ceptance by olfactory receptors and the recognition of scents. The latest technol-ogy allows high-throughput screening for highly effective masking agents. In recent years, some deodorant products based on the technology to modulate ol-factory receptor activity have been found in the market.

We focused on developing odor con-trol fragrances with the olfactory recep-tor technology. Aging odors and axillary odors, which are globally important repre-sentatives of body malodor, were selected as the target odors. Using OR assay tech-nology, we searched for ORs that respond to major components of the malodors and searched for fragrance materials that sup-press OR activity. We applied these ma-terials and succeeded to develop newly excellent deodorant fragrances.

MATERIALS AND METHODS

Cloning of human ORsA total of 398 human OR genes were ob-tained by cloning from Human Genomic DNA: Female (Promega Corporation, Madi-son, WI, USA) by PCR. A human OR gene expression vector was obtained by incor-porating the N-terminal 20 amino acid resi-dues of bovine rhodopsin into the pME18S vector and further incorporating the ob-tained OR gene downstream thereof.

Luciferase reporter gene AssayHEK293T cells were cultured at 37 °C in 5 % CO2 atmosphere.

The luciferase reporter gene assay was performed by modifying the previously re-ported method [4]. The firefly and Renilla luciferase activities were measured by us-ing Dual-Glo Luciferase Assay System (Pro-mega). The pGL4.29 [luc2P / CRE / Hygro] vector (Promega), firefly luciferase vector containing a cAMP responsive element promoter, was used to measure receptor activities. The pGL4.74 [hRluc / TK] vector

(Promega), Renilla luciferase vector con-taining a thymidine kinase promoter, was used as an internal control for cell viabil-ity and transfection efficiency. Receptor-transporting protein 1 short (RTP1S) gene incorporated into pME18S vector was used to enhance functional expression of the ORs. HEK293T cells were transfected with 50 ng of human OR gene expres-sion vector, 10 ng of RTP1S vector, 10 ng of pGL4.29, and 5 ng of pGL4.74 using Lipofectamine 3000 (Life Technologies, Carlsbad, CA, USA) and cultured in poly-D-lysine coated 96 well plates (BioCoat, Corning Inc., New York, NY, USA) for 24 hours. After removal of the culture medium, transfected cells were stimu-lated with 50 μL CD293 medium (Life Technologies) containing odorants and supplemented with 20 μM L-glutamine.

After 3 hours of stimulation, their lumi-nescence was measured with microplate luminometer Centro LB960 (Berthold Technologies GmbH, Vienna, Austria). In the screening for ORs that respond to

Figure 2 (A), A plastic bottle used for the

evaluation; (B), How to evaluate the smell.

Table I Model malodor formulations

malodor components and screening for inhibitors of OR2C1 responses, cells were cultured in poly-D-lysine coated 384 well plates (ViewPlate, PerkinElmer, Waltham, MA, USA), and the experimental scale was reduced to one third of the amount mentioned above. Their luminescence was measured with microplate luminom-eter TriStar2 LB942 (Berthold).

Ratio of firefly luminescence to Renilla lu-minescence was calculated for each well. The olfactory receptor response intensi-ties were represented as fold increase (FI) values calculated by dividing the ratio val-ues of cells stimulated with odorants by the ratio values without odorants. In the experiments to inhibit OR2C1 response to malodor component, their inhibition efficiencies were represented as normal-ized response values calculated by divid-ing the FI values of the mixture of mal-odor component and antagonists by the FI values without antagonists. Data were analyzed with GraphPad Prism [6].

Sensory evaluation

MethodSensory evaluation was performed in accordance with the 6-point scale odor intensity measurement method by 20 trained panels. The odor intensity was

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classified into the following categories according to the intensity. 0; no odor, 1; very weak, 2; weak, 3; distinct, 4; strong, 5; very strong. When only malodor was presented, the score was set to 4. Two cotton balls were placed in a plastic bot-tle, one with 10 μL of a malodor and the other with 1 μL of a test substance. The bottle was sniffed, and the intensity of the malodor was scored (Figure 2).

Malodor Components• Aging malodor; (E)-2-nonenal (Tokyo Chemical Industry Co., Ltd. Tokyo, Japan) 0.1 wt% triethyl citrate• Axillary malodor; 1-octen-3-one (Tokyo Chemical Industry Co., Ltd. Tokyo, Japan) 0.1 wt% triethyl citrate

Model MalodorA model aging malodor containing (E)-2-nonenal, (E)-2-octenal (Tokyo Chemi-cal Industry) and a model axillary odor containing 1-octen-3-one, 1,5-octadien-3-one (Santa Cruz Biotechnology, Inc.

Texas, USA) were prepared according to Table I.

The acids listed in Table I were manufac-tured by Tokyo Chemical Industry. Both model malodors became a high degree of correlation reminiscent of actual aging malodor and axillary malodor by adding the key components.

Model FragranceA floral balsamic fragrance was prepared as a model fragrance by Takasago In-ternational Corp. Tokyo, Japan. The constituent raw materials of this fra-grance were selected not to suppress the response of OR2C1. Respective an-tagonists were added 0.1 to 1 % to this model fragrance.

RESULTS

Screening of human ORs for aging and sweat malodor key componentsWe screened human ORs which can re-

spond to (E)-2-nonenal. HEK293T cells transiently expressing each of 398 human ORs were exposed to 60 μM (E)-2-none-nal, and their responses were measured with the luciferase reporter gene assay. OR2C1 responded strongly to (E)-2-non-enal, and OR1D2, 2J2, 4E2, 5P3, and 52N2 also responded weakly (Figure 3).

We confirmed the dose-response relation-ship of OR2C1 to (E)-2-nonenal. OR2C1 expressing cells or mock cells were stimu-lated with different concentrations of (E)-2-nonenal and their responses were measured by the luciferase assay. More than 10 μM of (E)-2-nonenal activated OR2C1 in a dose-dependent manner, and mock cells showed no response to any of the concentrations of (E)-2-nonenal (Figure 4A).

Further, we also tried applying OR2C1 expressing cells to other malodor com-ponents. As a result, the cells showed re-sponses to not only (E)-2-octenal, known

Figure 4 OR2C1 and mock cells response to aging and axillary malodor key components. Responses of HEK293T cells transiently expressing OR2C1

to malodor components of each concentration were measured with luciferase reporter gene assay. Malodor components were (A), (E)-2-nonenal;

(B), (E)-2-octenal; (C), 1-octen-3-one; (D), 1,5-octadien-3-one. Black bars indicate fold increase values of OR2C1, and white bars mean values of mock.

Each bar represents the mean ± SE (n = 3).

Figure 3 Screening of human ORs for (E)-2-nonenal. Responses of HEK293T cells transiently expressing each of human ORs to 60 μM (E)-2-nonenal

were measured with luciferase reporter gene assay. Black bars indicate the ORs that responded to (E)-2-nonenal stimulation with a fold increase value

of 2 or more. Numbers (1-14, 51, 52, and 56) with white bars on the x-axis indicate OR families and T means TAARs.


as an aging malodor key component, but also1-octen-3-one and 1,5-octadi-en-3-one, known as axillary malodor key components, in a dose-dependent man-ner (Figure 4B - D). In summary, OR2C1 responded to all the four malodor com-ponents.

Search for the potential antagonists of OR2C1To develop fragrances with masking ef-fect, we screened 800 fragrance materi-als using (E)-2-nonenal. We stimulated OR2C1-expressing cells with mixtures of 60 μM (E)-2-nonenal and fragrance materials, and measured their respons-es using a luciferase assay. As a result, dozens of materials suppressed the cel-lular responses to (E)-2-nonenal (data

to 60 μM (E)-2-nonenal with various con-centrations of compound 1. Compound 1 significantly reduced the OR2C1 response to (E)-2-nonenal at 30 and 100 μM (Figure 5A).

We confirmed whether antagonists of OR2C1 found using (E)-2-nonenal could also suppress the same receptor response to the other malodor components. We examined in the same manner as above except changing the ligand to (E)-2-oc-tenal, 1-octen-3-one, and 1,5-octadien-3-one. Compound 1 showed suppression effects for the OR2C1 response to all the three malodor components in a dose de-pendent manner (Figure 5B-D).

We further selected 5 materials; com-pound 2, 3, Eucalyptus EO, Patchouli EO, and Vetiver EO, from the antago-nists and evaluated their inhibitory ef-fects of OR2C1 responses on aging and axillary malodor components. We stimu-lated OR2C1 expressing cells with mix-tures of 60 μM (E)-2-nonenal or 30 μM 1-octen-3-one and each 5 materials at different concentrations and measured their response with luciferase assay. All five materials significantly suppressed the OR2C1 response to both aging (Figure 6A-E) and axillary components (Figure 7A-E).

Sensory evaluationSensory evaluation was conducted to verify whether the antagonist works as a body odor suppressor in the human

not shown). Among these potential antagonists, we chose six materials: 1,4-Dioxacycloheptadecane-5,17-dione (compound 1), 2-Methyl-4-[(1R)-2,2,3-trimethylcyclopent-3-en-1-yl]but-2-en-1-ol (compound 2), Cyclohexanepropa-nol, 2,2,6-trimethyl-alpha-propyl (compound 3), Eucalyptus Essential Oil (EO), Patchouli EO, Vetiver EO (Table II). Compound 1-3 were manufactured by Takasago International Corp. Essential Oils were manufactured by Hayashi Pure Chemical Ind., Ltd. Osaka, Japan. Their inhibitory activities were confirmed in detail as follow.

First, we evaluated dose-inhibition rela-tionship of compound 1. We measured the responses of OR2C1-expressing cells

Table II Characteristics of candidate of antagonists

Figure 5 Inhibitory activity of Compound 1 on the response of OR2C1 to aging and axillary malodor key components. Responses of HEK293T cells

transiently expressing OR2C1 to malodor key components with compound 1 of each concentration were measured with luciferase reporter gene assay.

Malodor components were (A), 60 μM (E)-2-nonenal; (B), 60 μM (E)-2-octenal; (C), 30 μM 1-octen-3-one; (D), 60 μM 1,5-octadien-3-one.

Bars indicate normalized response values of OR2C1 and expressed as mean ± SE (n = 3).

The significances for the differences between control (malodor components alone) and test values were determined using one-way ANOVA followed

by Dunnett’s test. *, p< < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

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sense of smell. First, the sensory inhibi-tory effect of a fragrance material on each malodor was examined. It was veri-fied that compounds 1, 2, 3, and Euca-lyptus EO suppressed the odor intensity of (E)-2-nonenal and 1-octen-3-one. On the other hand, Benzyl Salicylate which did not suppress OR2C1 response, did not reduce the intensity of each malodor (Figure 8A, B).

Next, more practical tests were con-ducted to verify the effectiveness when applied to production-level applications. We prepared fragrances that contain the antagonists, a model aging malodor and a model sweat malodor that consists mainly of their key components, respec-tively. Sensory evaluation results of the fragrances showed excellent odor con-trolling effect on the model malodors (Figure 8C, D). Model fragrance without antagonist slightly reduced the intensity of malodor, which was used as a control. These results clearly demonstrated the meaning from the practical application perspective.

We screened fragrance materials that suppress the response of OR2C1 to (E)-2-nonenal and found dozens of antago-nist candidates. Among them, compound 1 inhibited OR2C1 activity against all four malodor components (Figure 5A-D). Further, each of compound 2, 3, and three essential oils also reduced OR2C1 response to both (E)-2-nonenal and 1-oc-ten-3-one (Figures 6A-E and 7A-E). This indicates that one antagonist can mask multiple types of malodors.

In sensory evaluation, the antagonists also reduced the odor intensity of ag-ing and axillary malodor components (Figure 8A, B). Sensory evaluation re-sults showed actual suppression of mal-odor by the antagonists, supporting the effectiveness of this method. In addition, fragrances containing these antagonists showed excellent odor controlling ef-fects of model aging and sweat malodor (Figure 8C, D). It was very interesting that one antagonist could suppress the mal-odors of aging odor and axillary odor. On the other hand, there was no clear correla-

Discussion

To develop new fragrances that effective-ly deodorize unpleasant odors, we used OR assay technique to search for ORs that respond to malodor key components and fragrance materials that reduce the OR activities.

We selected (E)-2-nonenal and (E)-2-oc-tenal as aging malodor components, and 1-octne-3-one and 1,5-octadinen-3-one as axillary malodor components. We found some ORs responsive to (E)-2-nonenal (Figure 3), and among them OR2C1 responded to all 4 malodor com-ponents in a dose-dependent manner (Figure 4A-D). Considering that OR2C1 also responds to octanethiol and non-anethiol [5], OR2C1 may recognize linear molecules with a chain length of approxi-mately [8, 9]. These OR2C1 ligands do not show similar odors to each other. In human olfaction system, OR2C1 may not contribute to odor quality discrimination alone, unlike musk odor perception that is highly dependent on OR5AN1 [6, 7].

Figure 6 Inhibitory activity of antagonists on the response of OR2C1 to (E)-2-nonenal. Responses of HEK293T cells transiently expressing OR2C1 to

60 μM (E)-2-nonenal with antagonists of each concentration were measured with luciferase reporter gene assay. The antagonists were (A), compound 2;

(B), compound 3; (C), Eucalyptus EO; (D), Patchouli EO; (E), Vetiver EO. Bars indicate normalized response values of OR2C1 and expressed as mean

± SE (n = 3). The significances for the differences between control ((E)-2-nonenal alone) and test values were determined using one-way ANOVA

followed by Dunnett’s test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

Figure 7 Inhibitory activity of antagonists on the response of OR2C1 to 1-octen-3-one. Responses of HEK293T cells transiently expressing OR2C1 to

30 μM 1-octen-3-one with antagonists of each concentration were measured with luciferase reporter gene assay. The antagonists were (A), compound 2;

(B), compound 3; (C), Eucalyptus EO; (D), Patchouli EO; (E), Vetiver EO. Bars indicate normalized response values of OR2C1 and expressed as mean

± SE (n = 3). The significances for the differences between control (1-octen-3-one alone) and test values were determined using one-way ANOVA

followed by Dunnett’s test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.


[3] Niimura, Y., Matsui, A., and Touhara,

K., Extreme expansion of the olfactory

receptor gene repertoire in African el-

ephants and evolutionary dynamics of

orthologous gene groups in 13 placental

mammals, Genome Res., 24 (2014) 1485-

1496.

[4] Zhuang, H., and Matsunami, H., Evaluat-

ing cell-surface expression and measuring

activation of mammalian odorant recep-

tors in heterologous cells, Nat. Protoc.

3 (2008) 1402-1413.

[5] Saito, H., Chi, Q., Zhuang, H., Matsuna-

mi, H., and Mainland, J. D., Odor Coding

by a Mammalian Receptor Repertoire, Sci.

Signal., 2(60), (2009) ra9-ra9.

[6] Shirasu, M., Yoshikawa, K., Takai, Y.,

Nakashima, A., Takeuchi, H., Sakano, H.,

and Touhara, K., Olfactory receptor and

neural pathway responsible for highly se-

lective sensing of musk odors, Neuron, 81

(2014) 165-178.

[7] Sato-Akuhara, N., Horio, N., Kato-Nam-

ba, A., Yoshikawa, K., Niimura, Y., Ihara,

S., Shirasu, M., and Touhara, K., Ligand

Specificity and Evolution of Mammalian

Musk Odor Receptors: Effect of Single

Receptor Deletion on Odor Detection, J.

Neurosci., 36(16), (2016) 4482-4491.

Corresponding AuthorHisashi Mihara

Takasago International CorporationCorporate Research & Development Division

Hiratsuka CityKanagawa

[email protected]

G

tion between the efficacy of the OR2C1 inhibitory activity and the masking perfor-mance in sensory evaluation. One of the reasons for the difference is the influence of physical properties such as volatility in sensory evaluation. Also, interactions with ORs other than OR2C1 cannot be ignored. Although the ORs assay is not a flawless method, it is still very efficient for select-ing masking agent candidates, and this method can significantly reduce the devel-opment workload.

CONCLUSION

OR2C1 was found as a receptor com-mon to both the aging and the axillary key odor components. And potential an-tagonists of OR2C1 were discovered with fragrance materials.

It was demonstrated in the sensory evaluation that the antagonist fragrance materials significantly suppressed odor strength of the malodor materials. Fur-thermore, the effect was also clearly veri-fied in experiments using fragrances that contain the antagonists. These results suggest the significant importance from a practical application perspective.

In conclusion, utilizing the olfactory re-ceptor technology, fragrances to control body malodor were successfully devel-oped. This methodology is believed to be efficient and rapid for the development of high performance masking fragrance compositions. We utilize the products to solve the odor problems of people whose lifestyles have changed due to global warming and improve their qual-ity of life. We believe that this approach will lead to the realization of a sustain-able society.

References

[1] Haze, S., Gozu, Y., Nakamura, S., Kohno,

Y., Sawano, K., Ohta, H., and Yamazaki,

K., 2-Nonenal newly found in human

body odor tends to increase with aging,

J. Invest. Dermatol., 116(4), (2001) 520-

524.

[2] Iida, S., Ichinose, N., Gomi, T., Someya,

K., Hirano, K., Ogura, M., Yamazaki, S.,

and Sakurai, K., Mechanism and Regu-

lation of Body Malodor Generation (1)

– Effect of Iron on Axillary Malodor and

of Anti-Oxidant on Deodorant-, J. Soc.

Cosmet. Chem., 37(3), (2003) 195-201.

Figure 8 Sensory evaluation for the inhibitory effect of the antagonists on each malodor.

Sensory evaluation was performed in accordance with the 6-point scale odor intensity measurement

method by 20 trained panels. 0; no odor - 5; very strong.

When only malodor was presented, the score was set to 4. The combination of malodor and

test substance were (A), (E)-2-nonenal – fragrance materials; (B), 1-octen-3-one – fragrance materials;

(C), a model aging odor – model fragrances with / without antagonists; (D), a model axillary odor –

model fragrances w / wo antagonists. Each bar represents the mean ± SE (n = 20).

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A New Sustainable Anti-Aging Product Using Aquaponics TechnologyYou Ah Kim1, Tae Byeong Jeon2, Chae Eun Lim3, Ki Yong Lee4, Dong Hee Kim5, Ji Eun Woo1, Byoung Jun Park1, Hakhee Kang1

1 Skin & Natural Products Lab., Kolmar Korea Co., Ltd., 61, Heolleung-ro 8-gil Seocho-gu, Seoul, Korea2 Agricultural Corporation, MANNACEA Co., Ltd., 702-10, Jingwang-ro, Iwol-myeon, Jincheon-gun, Chungcheongbuk-do, Korea3 Plant Resource Division, National Institute of Biological Resources, 42, Hwangyeong-ro, Seo-gu, Incheon, Korea4 College of Pharmacy, Korea University, 2511, Sejong-ro, Jochiwon-eup, Sejong-si, Korea5 National Institute for Korean Medicine Development, 94, Hwarang-ro, Gyeongsan-si, Gyeongsangbuk-do, Korea

Keywords: Nymphoides, aquaponics, DNA barcoding, anti-aging, 3,7-di-O-methylquercetin-4ʹ-O-β-glucoside

This publication was originally presented as a podium presentation at the 25th IFSCC Conference, Milan, Italy, September 30 - October 2, 2019.

Ség. have historically been used as multi-medicinal herbs [2, 3]. Nymphoides (family Menyanthaceae) consists of 40 - 50 species worldwide [4 - 6], some of which are of minor economic importance as foodstuffs and medicines [5, 7, 8], while others are cultivated for ornamental uses in aquaria and water gardens [5, 9-11]. According to an ethnobotanical study of Nymphoides, these aquatic species are traditionally used for the treatment of injuries, snake bites, jaundice, dysentery, and convulsion [12].

INTRODUCTION

Recent global cosmetics trends are oriented towards the use of phytochemicals present in natural resources. However, when plants are grown in the open field, their qual-ity and productivity tend to vary with the climate. In addition, the world is increas-ingly faced with global problems, includ-ing unusual weather and environmental pollution. Accordingly, the stable supply of plant-derived products is under threat. These issues drive the need for new so-lutions to the production of plant-derived products. Aquaponics is an innovative and sustainable hydroponic plant production system using fish waste (ammonia) that can play an important role in the future of environmental and socio-economic sus-tainability by following principles [1]: • The waste products of one biologicalsystem serve as nutrients for a second biological system.• The integration of fish and plants resultsin a polyculture that increases diversity and yields multiple products.• Water is re-used through biological filtration and recirculation.• Local industrial plant production enhances the local economy.

Therefore, we began researching aquatic plants to find a suitable material for an aquaponic system.

Aquatic vascular plants are adapted to liv-ing in the aquatic environment and mainly occur within or on top of water bodies. These aquatic plants account for ~ 2 % of all plants worldwide; however, their industrial uses are limited compared with those of land plants. We assume that these aquatic plants might make secondary metabolites different from existing terrestrial plants to survive in the unique underwater environ-ment. In Asia, many indigenous aquatic plants belonging to the genus Nymphoides

Abstract

To develop sustainable new anti-aging ingredients from Korean native plants, we investigated the cultivation potential of three Korean Nymphoides using the aquaponics system, and tested the anti-aging effects. We also assessed the ef-fectiveness of DNA barcoding and com-ponent pattern analyses using LC-QTOF MS/MS of the Nymphoides. As a result, nitrate, potassium and water tempera-ture were the most important factors in the cultivation of Nymphoides and their optimum values were 80 μg / mL, 63.5 μg / mL and 25 °C, respectively. In the component study, a similar pat-tern was observed in N. indica and N. coreana, while N. peltata showed a dif-

ferent pattern. The main components of N. indica and N. coreana were 3,7-di-O-methylquercetin-4ʹ-O-β-glucoside (C1). In the skin activity study, the N. indica extract showed that inhibited expres-sion of UVB-induced ROS, MMP-1 and increased pro-collagen. And C1 isolat-ed from N. indica has a regulating ef-fect on skin moisture and aging-related signaling substances. Further, a clinical trial with a test cream containing 1% of N. indica extract demonstrated sig-nificant improvement in skin wrinkling, lifting and elasticity. Taken together, the results suggested that the standardized extract of N. indica cultivated in aqua-ponics has considerable potential as a new cosmetics ingredient with an anti-aging effect.


In Korea, three Nymphoides spp., name-ly N. peltata, N. coreana, and N. indica, are distributed mainly in ponds, reser-voirs, and margins of shallow rivers in the southern part of the Korean Penin-sula [13, 14]. A recent study reported the pharmacological value of N. indica leaves and their phytochemicals owing to their antimicrobial, antiprotozoal, antioxidant, and antidiabetic properties [3]. Another study demonstrated that the rhizomes of N. indica exhibit anticonvulsant activity [15] and may potentially serve as an al-ternative source of Tagara (N. macrosper-mum) [16]. Additionally, previous studies on the biological activities of N. indica have demonstrated the inhibitory activity of whole plant methanol extracts on melanin synthesis [17]. The major component of the N. indica leaves, 3,7-di-O-methylquercetin-4ʹ-O-β-glucoside, has been reported to possess moderate anti- glycation and α-glucosidase inhibitory activities [18]. In addition, there has been an attempt to cultivate N. indica extracts in vitro for industrial application [19].

However, scientific investigations on the pharmacological components of Nymphoides spp. are still insufficient, and there is also no commercial culti-vation technique available for them. Unfortunately, Nymphoides spp. are in-distinguishable using only vegetative or-gans, and they are often misidentified in most herbaria [20, 21]. In this case, DNA barcoding is a system to aid species recognition and identification through the characterization of a standard gene region across all organisms [22]. In particular, the use of barcoding readily facilitates the identification of small plant fragments or the determination of spe-cies used in herbal supplements [23, 24].

Therefore, the present study aimed to reli-ably distinguish between, and accurately identify, the Nymphoides spp. in Korea. We examined the morphological char-acters and DNA barcode regions of the three Nymphoides spp. In addition, the components of these three species were identified by LC-QTOF MS/MS analysis and their cultivation conditions were investi-gated for use as new anti-aging materials with industrial cosmetic value.

EXPERIMENTAL

Plant materials and identification of speciesIn the DNA barcoding aspect of this study, we used four samples from each of the three Nymphoides spp., among which one sample per species was cultivated and the remaining samples were collected during field studies. Examined voucher specimens of the species were deposited in the her-barium of the National Institute of Biologi-cal Resources (NIBR). Genomic DNA was extracted from silica gel-dried leaves using the DNeasy Plant Mini Kit (Qiagen, Ger-many) according to the manufacturer’s instructions. PCR primers and protocols to amplify the four DNA barcode regions—rbcL, matK, ITS, and psbA-trnH—were fol-lowed by [25 - 28], respectively. The PCR products were purified using PCRquick-spin™ PCR Product Purification Kit (Intron, Korea) and then sequenced in both direc-tions with the primers used for PCR ampli-fication on a 3730XL sequencer (Applied Biosystems, USA).

Sequence alignment and data analysisIn total, 36 accessions related to the 4 can-didate DNA barcoding sites (rbcL, matK, ITS, and psbA-trnH) from 12 samples be-longing to 3 Nymphoides spp. were ana-lyzed in this study. The sequences of three DNA regions were assembled and edited using Sequencher 5.0 (Gene Codes Co., USA), and the sequences were aligned us-ing Clustal X v. 1.81 [29] with final manual adjustment using Se-Al v. 2.0a11 [30]. All DNA sequences obtained in this study were compared with known sequences in the BLAST method (NCBI, BOLD system) and deposited in GenBank (Appendix S1, Supporting information) and WIGIS (Wild Life Genetic Information System, http://species.nibr.go.kr). We used the neighbor-joining (NJ) analysis to evaluate whether our sequence datasets were in various combinations for species-specific clusters. For NJ analysis, pairwise genetic distances for each individual sequence dataset and all possible combinations of the three se-quence datasets were determined using the Kimura 2-parameter (K2P) method [31] and PAUP* 4.0b10 [32]. A bootstrap analysis [33] of 1000 replicates was per-formed in PAUP* to evaluate the support

for clades. The inter- and intra-specific variations of the samples were calculated. Genetic distances and nucleotide diver-gences were computed using the same search parameters as in the NJ analysis above in PAUP (ver. 4.0b100 [32]. To es-timate the species resolution ability using the three barcode regions, we calculated the percentage of the total number of species correctly identified in the NJ tree divided by the total number of species ex-amined in this study.

LC-QTOF MS/MS analysisWe performed a liquid chromatography quadrupole time-of-flight mass spec-trometry (LC-QTOF MS/MS) analysis of the three Nymphoides spp. to identify the major components and to compare the distribution patterns of these compo-nents. Samples for analysis were prepared by drying raw materials at ~ 25 °C for ~ 2 days, weighing ~ 1 g samples, adding 100 g of 99.5 % MeOH, and keeping the mixture undisturbed for 3 days at ~ 25 °C to obtain an extract solution. The extrac-tion was performed in triplicate. Through filtration and concentration methods, a powder of the extract was obtained. The samples were analyzed in triplicate. High-performance liquid chromatography (HPLC) and MS conditions for the analysis are as follows:

The LC-QTOF MS/MS analysis was per-formed on an Agilent 1260 series system and an Agilent 6530 Q-TOF mass spec-trometer (Agilent, US). The LC system was equipped with a binary pump, an auto sampler, and a photodiode array detector, and used a Shiseido CapCell PAK C18 column (150.0 × 4.6 mm, 5 μm). The mobile phase consisted of water con-taining 0.1 % formic acid (solvent A) and acetonitrile containing 0.1 % formic acid (solvent B). The gradient conditions were as follows: 5 % B, 0 - 5 min and 5 - 95% B, 5 - 30 min. The flow rate of the mobile phase was 0.6 mL·min-1 and the injection volume was 10.0 μL. The UV spectrum was monitored at 254 nm and 330 nm. The effluent of LC was analyzed using a mass spectrometer equipped with an ESI interface and was ionized in nega-tive mode. Before analysis, negative ion mass calibration was performed using

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the tuning solution. The tuning solution was prepared by ESI-L Low Concentration Tuning Mix diluted with HPLC-grade wa-ter and acetonitrile. In addition, 0.1 mM HP-0321, which is included in the Bio-polymer Reference Mass Standard Kit, was mixed in the positive tuning solution. The mass parameters were as follows: nebulizer pressure, 40 psi; capillary volt-age, 4000 V; fragmentor voltage, 175 V; skimmer voltage, 65 V; drying gas tem-perature, 325 °C; and flow rate of drying gas, 12.0 L·min-1. The MS / MS chromato-gram of samples was obtained using colli-sion energy of 10, 20, and 30V. The mass scan range was set at 50 - 1700 m / z. Mass Hunter Workstation software LC / MS Data Acquisition for 6530 series Q-TOF (version B.05.00) was used to set all acquisition parameters for obtaining data. The principal component analysis (PCA) was performed using Mass Profiler Professional (MPP, Agilent Technologies, Santa Clara, CA, USA).

CultivationCultivation studies of N. indica (i.e., the representative plant among the three Nymphoides spp.) were conducted for the reforestation and standardization. Throughout the cultivation period, chang-es in the pH and in the concentration of various trace elements, such as nitrate and potassium, were measured and quantified in extracts. To standardize the cultivation methods, various physical cultivation con-ditions were adjusted and the results were observed. To determine how temperature and nutrient composition affected the growth of specimens, fresh weights were determined based on growth conditions (Table I). Fresh weights were measured

Table I Calculated parameters of the initial weight (g) while being supplied nutrient solution

under two temperature conditions

Figure 1 Practical deep-water cultivation example for N. indica.

(A) free-floating and (B) floating leaved method.

7, 14, 21, and 28 days after the formal implantation of specimens in the growth conditions, under different temperature conditions.

To create a sub-leaf environment using a deep water culture (DWC) technique, cul-tivation beds and a root growth environ-ment without water flow were prepared in an aquaponics system. To prevent infec-tion from outside pests and diseases in the soil composition and in the root-growing environment, DWC cultivation tests were conducted using a combination of soil with peat moss, masato, and gravel, and the nutrients and drainage were consid-ered in an independent space (Figure 1). In addition to the soil composition, loess balls were used for the plant growth envi-ronment. Through this, we demonstrated effective propagation methods of N. indica in artificial culture fluid.

Isolation and structure determinationof compoundsThe air-dried samples were cut into pieces and extracted for 3 days with 95 % methyl alcohol (MeOH) (18 L × 3) at room tem-perature. The combined crude extracts

were concentrated under reduced pres-sure to yield a MeOH extract and dissolved in distilled water (H2O). The suspended extract was partitioned using n-hexane, methylene chloride (MC), ethyl acetate (EtOAc), and n-butanol to yield layers. A portion of EtOAc and n-butanol frac-tions were subjected to silica gel column chromatography and preparative reverse-phase LC (YMC Actus Triart C18 column; 250 × 20 mm, S-5 μm, 12 mm; UV detec-tion at 254 nm) to afford compounds 1 - 3 (C1 - C3), sequentially.

Cell culture and UVB irradiationHuman skin dermal fibroblast (CCD-986sk) and immortalized human kerati-nocytes (HaCaT) were purchased from the American Type Culture collection (Manas-sas, VA, USA). The cells were cultured in high glucose DMEM containing 10 % FBS, 1 % streptomycin/penicillin at 37 °C in a 5 % CO2 humidified atmosphere. The cells were exposed to UVB radiation using a UV irradiation system (BIO-LINK Crosslinker, WA, Australlia) delivering the 280 - 320 nm wavelength range, with maximum emis-sion at 312 nm. Seeded cells were ringed PBS and then exposed to the following doses of UVB: 20 mJ / cm2.

Measurement of ROSCCD-986sk cells were cultured in a 96-well plate for fluorescence measure-ment at 5 × 104 cells / well for 24 hours. After treating the extracts and fractions of the sample for 24 hours, 100 μL of DCFH-DA (2’,7-dichlorodihydro-fluorescein di-acetate) prepared in 20 μM was added to HCSS (HEPES buffer control salt solution), and incubated in a CO2 incubator for 20 minutes, and then washed with HCSS. Af-ter adding 100 μL of HCSS containing each


extract, which had been pretreated for 48 hours, the fluorescence intensity of DCF initially acidified as reactive oxygen species (ROS) was measured with a fluo-rescent plate reader.

Determination of MMP-1 and Pro-collagen secretion with ELISACCD-986sk cells were inoculated into a 6-well plate at a concentration of 5 × 104 cells / well and stabilized for 24 hours. After 24 hours, UV-B (312 nm) was irra-diated, and TNF-α was added at a con-centration of 10 ng / mL to increase the activity of MMP-1, and extract and frac-tions were treated at concentrations of 5, 10, 25 μg / mL, and cultured for 48 hours. The culture medium of the cells was col-lected and used for the test, and the ab-sorbance was measured at 450 nm with a plate reader using Matrix Metallopro-teinase-1 assay kit (Abcam, Cambridge, UK) and pro-collagen assay kit (TAKARA, Berkeley, CA, USA), and the calculation formula was obtained through a standard curve, and then expression of MMP-1 and pro-collagen in the cell culture medium was quantified.

Western blot20 μg of protein was separated by 10 % SDS-PAGE and transferred to a PVDF membrane at 120 V for 2 hours. Then, after blocking for 1 hour in 5 % skim milk, it was treated overnight at 4 °C in MMP-1 (Abcam, Cambridge, UK) primary an-tibody. After washing with TBST again

Reverse transcription polymerasechain reaction (RT-PCR) HaCaT cells (1 × 104 cells / mL) were cul-tured with indicated concentration of QDG in 6-well plates for 24 hours. Af-ter 24 hours, TRIzol reagent (Invitrogen, Carlsbad, CA) was used for the extraction of total RNA. RNA was used for cDNA synthesis with oligo(dT) primer and re-verse transcriptase (Promega, Madison, WI) for 1 h at 42 °C (Table II). PCR was performed with 56 °C annealing temper-ature for 30 s, 72 °C, 1 min for extension with a total of 40 cycles. PCR products were analyzed using 1.5 % agarose gel electrophoresis, stained with ethidium bromide (EtBr), and examined using an LAS4000 image analyzer (Fujifilm Life Sci-ence, Tokyo, Japan).

Clinical trialA blinded clinical study of wrinkles, lifting and elasticity was conducted in a group of 20 healthy females aged 47.65 years on average.

Skin safety evaluation was performed by assessing skin irritation through an inter-view and by medical examination after 4 and 8 weeks of product use. The product was applied to the face twice daily for a duration of 8 weeks. Skin improvement rates were then measured by a PRIMOS High Resolution (Phaseshift Rapid in vivo Measurement Of Skin, GF Messtechnik GmbH, Germany), Moire & Image analysis program (Image-Pro Plus, USA), and Cu-tometer MPA 580 (Courage and Khazaka, Germany), respectively.

three times, the secondary antibody (San-ta Cruz, CA, USA) was reacted at room temperature for 1 hour, and then quan-tified using an LAS 4000 image analysis device.

Cell migrationHaCaT cells were dispensed into a 12-well plate at 5 × 104 cells/mL per well, incu-bated for 24 hours, and then the culture medium was removed and scratched with a 10 μL pipette tip. After observation un-der a microscope, and replacement of the culture medium, quercetin 3,7-dimethyl ether 4ʹ-glucoside (QDG), HACaT cells were treated at a concentration of 1, 5, 10 μg / mL. Twenty-four hours after the extract treatment, the degree of migra-tion was observed with a microscope and a picture was taken.

Table II Primer sequence of the gene

Table III Statistics for the four DNA barcode regions used in this study

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code regions from the three Nymphoides spp. examined in this study. Although the topology of the NJ tree based on the ITS data showed congruent interspecific rela-tionships with an NJ tree based on psbA-trnH data (data not shown), the ITS region showed the highest species resolution in this study. Therefore, we presented the more resolved NJ tree of the ITS data (Figure 2). In addition, our DNA barcode data are consistent with the morphologi-cal distinctions of the three Nymphoides spp. in Korea [13]. Among the three spe-cies, N. peltata is clearly distinguishable from the other two species in Korea by having yellow flowers and crenate leaf margins [13]. N. coreana is morphologi-cally similar to N. indica, but distinct from it in having violet gland dots on the ab-axial surface (vs. yellowish brown glands), leaves 2 - 6 cm in diameter (vs. leaves 7 - 20 cm in diameter), and ca. 8 mm flow-ers in diameter (vs. ca. 1.5 cm flowers in diameter) [13]. Based on our findings, and in conjunction with the morphologi-cal differences, DNA barcode data offer a viable technique for the identification of the three Korean Nymphoides spp.

Analysis of Nymphoides spp. using LC-QTOF MS / MSThe MS and UV chromatogram of 99.5 % methanol extracts of the three Nymphoi-des spp., are shown in Figure 3 and Figure 4. The three Nymphoides spp. Extracts contained secoiridoids, flavo-noids, and phenolic acids. The peaks a, b, i, and l (i.e., chlorogenic acid, sweroside, 3,5-di-O-caffeoylquinic acid, and 3,7-di-O-methylquercetin-4’-O-β-glucoside, respectively) were observed in all three Nymphoides spp. extracts. The chemi-

Figure 2 Neighbor-joining tree based on the nuclear ITS region of the three Nymphoides spp. in Korea.

Numbers above branches indicate bootstrap (≥ 50) values. Asterisks (*) indicate cultivated plant samples.

Liparophyllum capitatum was selected as the outgroup [43].

Statistical analysisAnalysis of variance was performed in SPSS (SPSS Inc., Chicago, IL, USA), and all data are expressed as mean ± SD. For the biological activity, statistically significant differences between experimental and control values were analyzed by one-way ANOVA followed by a t-test. For the clini-cal activity, according to the results of the normality test, the Friedman test was used to check the significance of the sample be-fore and after application in the 95 % con-fidence interval, and the Wilcoxon signed rank test was performed to confirm the time point when a significant difference appeared.

RESULTS AND DISCUSSION

Plant materials and identification of speciesIn total, 2,881 sequences of the four DNA barcode regions (rbcL, matK, psbA-trnH, and ITS) were obtained from 12 accessions representing the three Nymphoides spp. in Korea. The sequence characteristics of the four DNA barcode regions examined in this study are summarized in Table III.

The degree of species resolution for the individual barcode regions ranged from 33.3 % (rbcL, matK) to 100 % (ITS, psbA-trnH) (data not shown). Therefore, ITS or psbA-trnH, which comprises the smallest number of DNA regions, is proposed to be the best option for barcoding of the Korean Nymphoides spp. The nuclear ITS region, which demonstrated the highest species resolution in this study, has been proposed as the most promising single-locus barcode for land plants [34]. Using the NJ tree-based method, we evaluated the resolution abilities of four DNA bar-

Figure 3 ESI-MS chromatogram (negative ionization mode) of the extracts from N. coreana (A),

N. indica (B), and N. peltata (C). (The lowercase letters is refer to Table III)


two principal components was 93.27 % (52.58 % first dimension and 40.69 % second dimension). Therefore, the PCA score plot clearly identified the differences between the three species.

CultivationIn the studies for the cultivation and stan-dardization of Nymphoides spp., nitrate, potassium, and water temperature were revealed as the most influential factors for the growth of N. indica, and the opti-mal values for these three environmental factors were determined as 80 μg / mL, 63.5 μg / mL, and 25 °C, respectively. As a result of changing the cultivation envi-ronment (owing to growth abnormality in the floating environment), normality in growth was achieved through circulation of water and balanced nutrition supply in the floating environment of the DWC cultivation system.

Both the soil with peat moss, masato, and gravel combination and the soil with the loess balls (630g·pot-1) resulted in the for-mation of the side leaf form (Figure 6).

cal profiles of N. coreana and N. indica extracts were similar, while chemical profile of the N. peltata extract differed from those of the other two Nymphoi-des spp. The major components of the N. coreana and N. indica extracts were sweroside and 3,7-di-O-methylquercetin-4ʹ-O-β-glucoside (peaks b and l); however, their content was higher in the N. coreana extracts (Table IV). The main components of N. peltata extracts were chlorogenic acid and 3,5-di-O-caffeoylquinic acid (peaks a and i). Despite the extracts of

the three Nymphoides spp. exhibiting the same peaks, the differences in the content patterns of the extracts can be used as markers to distinguish between the three species or their extracts. Statistical analy-ses were performed to investigate the dif-ferences between 3 types of Nymphoides spp. The PCA results revealed all extracts showing differences between the groups. Figure 5 depicts the distribution of com-pounds from N. coreana, N. indica, and N. peltata independently in the negative ionization mode. The total variance in the

Figure 4 UV chromatogram (254 nm) of the extracts from N. coreana (A), N. indica (B),

and N. peltata (C). (The lowercase letters is refer to Table III)

Table IV LC-QTOF MS/MS analysis of constituents in extracts of Nymphoides spp. (refer to Figure 3)

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In the case of the former, the conditions were unsuitable as a breeding environ-ment for N. indica owing to the covered gravel preventing the floating of mate-rial; however, the latter conditions were conducive to breeding N. indica owing to the smooth circulation of the nutrient solution. In addition, the latter conditions were easier to manage and were efficient. In response to the propagation method of Nymphoides spp. in artificial culture, N. peltata showed crawling stem propa-gation and N. indica showed rhizome propagation after rooting from a leaf sec-tion (Figure 7). No significant differences were found in the changes in fresh weight in response to differing nutrient compo-sitions. The compositional pattern of N. indica was not significantly different

from that of previous studies; however, quercetin-based compounds were dom-inant in N. indica and N. coreana, and chlorogenic acid-based compounds were

dominant in N. peltata [18]. As a result of assessing the Menyanthaceae family clas-sification, N. indica and N. peltata were clearly distinguishable, and the difference in the components of the same genus suggests that the metabolite production process differs between the two species and warrants further investigation [35]. However, the differences in the major components between these two groups offer an effective standard to discriminate between species during commercial analy-sis in quality management processes.

Isolation and identificationAmong cultivated Nymphoides, the ex-tract of N. indica exhibited strong anti-oxidant, skin hydration, anti-wrinkle, an-ti-inflammatory, and anti-pollution activ-ity [36, 37]. Phytochemical investigations of the N. indica resulted in the isolation of three main compounds, i.e., 3,7-Di-O-methylquercetin-4ʹ-O-β-glucoside (C1, QDG), sweroside (C2) and isoquercitrin (C3) (Figure 8). Especially, C1, the main component of N. indica, was not found in N. peltata. Accordingly, it can be used as an index component between two spe-cies for the industrialization of N. indica [3, 38 - 40].

Biological and clinical activityThe anti-aging effects of the extracts and compounds of Nymphoides spp. were measured using CCD-986sk and HaCaT cells. As a result, the ethyl acetate frac-tion of N. indica extract most inhibited UVB-induced ROS expression, increased pro-collagen expression and suppressed MMP-1 expression (Figure 9). Based on these results, it was confirmed that C1

Figure 5 Score plot for 3 types of Nymphoides spp.; (A) N. coreana, (B) N. indica, (C) N. peltata

and Loading plot of peaks in 3 species of Nymphoides spp..

Figure 6 (A) Soil composition using peat moss, masato, and gravel, and

(B) the soil environment using lightweight expanded clay aggregate.


isolated from N. indica has various skin protecting effects on skin, such as re-generation of cell damage, regulation of skin moisture and aging-related signaling substances (Figures 10, 11). In addition, we made a test cream containing 1 % of N. indica extract for clinical evaluation of its skin safety and anti-aging effect. As a result, no adverse dermatological events

were observed in any subjects during the test period. In addition, the test cream im-proved wrinkle and moisturizing relative to the control cream (base cream without N. indica extract) (Figure 12). Therefore, the test group was assumed to reduce skin aging. These results confirmed that N. indica and its compounds could be used as natural anti-aging ingredients.

CONCLUSION

One of the major issues in the industrial cultivation of wild plants is clear classifica-tion of the consistently used plant species. These problems may cause confusion in the supply and demand of raw materials for mass production. Therefore, the es-tablishment of clear classification criteria through DNA barcoding of the three Nym-phoides spp. in Korea is of great indus-trial and academic significance because the identity of genetic resources can be clearly identified in industrial applications. Researchers have studied the composition and efficacy of many plant resources; how-ever, one of the main reasons for them not being industrialized is the lack of research on standardized cultivation resources and techniques. In plants, the growth rate, size, and amount of internal metabolites differ in response to numerous nutrients in the soil, as well as external environ-mental conditions, such as amount of sun-shine, temperature, and humidity. Thus, extensive and comprehensive research is limited. Therefore, the present standard-ized cultivation and skin biological study of N. indica has great significance. We showed that this species can be cultivated using the demonstrated system, which is expected to be used more actively in the fields of cosmetic, health and pharmaceu-tical products in the future [41]. Finally, N. peltata and N. coreana, which have not been studied as extensively as N. indica, may also have value as medicinal crops. Recently, a case study demonstrated an antitumor effect from the application of N. peltata; it is likely that other pharma-cological effects may be discovered if the components and efficacy of N. peltata are tested further [42]. In addition, the major components of N. coreana were higher than those of N. indica; therefore, if ef-fective cultivation techniques are assessed and developed for N. coreana, this species may be more suitable for industrialization than N. indica.

Acknowledgements

This study was supported by a grant from the Korean Health Technology R&D Proj-ect, Ministry of Health & Welfare, Republic of Korea.(Grant No.: HN15C0103)

Figure 7 Cutting position for breeding (A) N. indica and (B) N. peltata in artificial culture.

Figure 8 Chemical structures of compounds 1 - 3

(1, 3,7-Di-O-methylquercetin-4ʹ-O-β-glucoside, 2, sweroside, and 3, isoquercitrin)

Figure 9 Biological activity (A) MMP-1 inhibitory activity of N. indica extract (B) MMP-1 inhibitory

activity of N. indica fractions (C) ROS generation suppression activity of N. indica fractions (D) Type-1

pro-collagen production activity of N. indica fractions. Nor.: Normal group not stimulated by UVB

and not treated by QDG. Cont.: Control group stimulated by UVB and not treated by QDG.

EGCG = positive control. Data are means ± SD, n = 3, *= p < 0.001 and ** = p < 0.0001 compared

with the control group.

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[26] CBOL Plant Working Group, A DNA bar-code for land plants, Proc. Natl. Acad. Sci., USA., 106 (2009) 12794-12797.

[27] White, T.J., Bruns, T., Lee, S., and Taylor, J.W., Amplification and direct sequencing of fungal ribosomal RNA genes for phy-logenetics, In PCR Protocols: A Guide to Methods and Applications (1990) 315-322.

[28] Sang, T., Crawford, D.J., and Stuessy T.F., Chloroplast DNA phylogeny, reticulate evolution, and biogeography of Paeonia (Paeoniaceae), Am. J. Bot., 84 (1997) 1120-1136.

[29] Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., and Higgins, D.G., The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools, Nucleic Ac-ids Res. 25 (1997) 4876-4882.

[30] Rambaut, A., Sel-Al: sequences alignment editor v 2.0011. Edinburgh: Institute of Evo-lutionary Biology, University of Edinburgh (2002). http://tree.bio.ed.ac.uk/software/

[31] Kimura, M., A simple method for estimat-ing evolutionary rates of base substitution through comparative studies of nucleotide sequences, J. Mol. Evol., 16 (1980) 111-120.

[32] Swofford, D.L., PAUP*4.0b Phylogenetic analysis using parsimony (* and other methods), Version 4. Sinauer Associates: Sunderland, MA, USA (2002).

[33] Felsenstein, J., Confidence limits on phy-logenies: An approach using the boot-strap, Evolution, 39 (1985) 783-791.

[34] Kress, W.J., and Erickson, D.L., A two-locus global DNA barcode for land plants: The coding rbcL gene complements the non-coding trnH-psbA spacer region, PLoS ONE. 2 (2007) e508, https://doi.org/10.1371/journal.pone.0000508.

[35] Tippery, N.P., Les, D.H., Regalado, J.C., Av-eryanov Jr., L.V., Long, V.N., and Raven,

P.H., Transfer of Villarsia cambodiana to Nymphoides (Menyanthaceae), Systemat. Bot., 34 (2009) 818-823.

[36] Kim, Y.A., Woo, J.E., Park, B.J., Hong, I.K., Kim, D.H., and Son, J.H., Cosmetic com-position for skin improvement, containing extract of nymphoides sp. plant, Patent WO2017138692A1 (2017).

[37] Kim, B.Y., Jeong, H.J., Kim, Y.A., Park, B.J., and Kim, D.H., Antifouling composi-tion for skin, containing extract of nym-phoides sp. plant as active ingredient, WO2018135761A1 (2018).

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Corresponding AuthorYou Ah Kim

Skin & Natural Products Lab.Kolmar Korea Co., Ltd.

SeoulKorea

[email protected]

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Impact of Anti-dandruff Piroctone-Olamine Designed Formulations on the Formation of Smart Deposit for Hair SurfaceNawel Baghdadli1, Frans Leemarkers2, Gustavo S. Luengo1, Christian Mazilier1, Jean-Charles Cheneble1, Anne Potter1 and Fabien Leonforte1

1 L’Oréal Research & Innovation, Aulnay-sous-Bois, France2 Department of Physical Chemistry and Soft Matter, Wageningen University, The Netherlands

Keywords: Eco-conception, eco-toxicity, numerical simulation, piroctone olamine, hair, deposit

A part of this publication was originally presented as a podium presentation at the 25th IFSCC Conference, Milan, Italy, September 30 - October 2, 2019.

The search for active ingredients to address and manage scalp dysfunction thus remains one of the main objectives of shampoos.

In addition to guaranteeing sustainability, new shampoo formulations need to con-tain more and more biodegradable ingre-dients that have a low impact on the en-vironment and a modest water footprint. Among the active ingredients that can ad-dress dandruff, Piroctone Olamine (OCT), also known as Octopirox (Figure 1), is of particular interest, specifically because of

INTRODUCTION

Cosmetic context and challengeIn hair care, the formulations used in sham-poos and conditioners are intrinsically com-plex to address very different functions si-multaneously. Very complex formulations contain ~10 to ~30 ingredients to satisfy different consumers’ demands, in addition to the primary washing function. Among these ingredients are detergents (surfac-

tants), conditioners, and additives to impart extra effects (foam, etc.), or to preserve or stabilize the formulations. In addition, consumers demand personalized health and well-being. This explains the interest in addressing scalp issues, greasy hair and, in particular, dandruff-free care. More than 200 million consumers suffer from dan-druff, which is a recurring problem, often accompanied by itching, scalp irritation and sensitivity, which in turn causes discomfort.

Abstract

Piroctone Olamine is an effective an-tidandruff active ingredient used in formulations to treat dandruff, a mild form of seborrheic dermatitis. It is an interesting alternative to Zinc Pyrithione because it is significantly less ecotoxic. Adapted formulas using this active can develop high levels of anti-dandruff per-formance together with hair care ben-efits (cosmetic acceptance, kindness to the scalp, hair conditioning).

The objective of this study was to char-acterize the impact of different types of shampoo formulas based on Piroctone Olamine on the biophysical properties of different hair types, as well as the physicochemical mechanisms associat-ed with the interaction with the surface

deposits of shampoo formulations that contain this ingredient. Applying a molecularly detailed Self-Consistent Field Theory, we illustrate how the dif-ficulty of a liquid not spreading on it-self preventing its growth (autophobic wetting) can be overcome by wetting additives. More specifically, we use an emulsifier additive that keeps the in-terfacial tension between the wetting component and the majority solvent low, and a co-solvent that partitions inside the film, destroying the internal molecular order.

We present experimental results for such complex formulations and, us-ing complementary modeling of such systems, we reveal all signatures of an additive-induced wetting transition and special hair surface properties. Figure 1 Structural formula of

Piroctone Olamine or Octopirox (OCT).

its low eco-toxicity impact (biodegrad-ability and low water footprint). Formulas using this active ingredient have had sig-nificant success in treating dandruff, as well as excellent cosmetic effects (i.e., hair conditioning and fiber benefits).

The objective of an antidandruff shampoo formulation is to bring large deposits of an active ingredient to the hair surface, close to the root, and near the scalp, to enable it


to function properly. Typically, it will not be enough to simply rely on an “adsorption-from-solution” mechanism as a way to ac-cumulate the active compound, because coverage is thereby limited to a molecu-larly thin layer with sub-milligram per meter squared adsorption amounts. The loading capacity can, in principle, be substantially enhanced if an active ingredient is absorbed inside a ‘wetting’ film that is ‘produced’ on top of a surface of interest. When analyzing the Octopirox antidandruff formulation, we considered the wetting characteristics of glycerol distearate (GD), a compound with low water solubility on hydrophobic surfaces (the root of a hair is hydrophobic), which is a ‘carrying’ component in such an antidandruff formulation that can absorb large amounts of the active compound. If a thick GD layer develops on the hair sur-face, a large amount of the active ingredi-ent can automatically be deposited. That is why wetting scenarios for this system are of more than average interest. Before going into further details, it is instructive to review the basic concepts of wetting.

Physicochemical context and challengeWhen three phases coexist in a system – for example, a mixture of two liquids (a minority liquid A and a majority liquid B) with a solubility gap, in the presence of a solid S – three interfaces are possible: S-A, S-B and A-B. However, there may be just two interfaces, either S-A and A-B, or S-B and A-B. One can classify the situation of three interfaces as partially wet (there is a three-phase coexistence line at the surface on the edge of droplets that are bound to the surface), whereas the second situ-ation is classified as either completely wet or completely dry (there is a homogeneous film of the A-rich or B-rich phase at the surface) [1, 2].

In practice, wetting scenarios are compli-cated. This is because surface interactions are not necessarily short ranged, or be-cause the molecules have a non-trivial ar-chitecture. In this paper, we focus on auto-phobic wetting [3]. Autophobicity is, for ex-ample, observed with molecules that have an amphiphile architecture with polar and non-polar fragments, such as surfactants. In water, when the substrate is non-polar, the tails will be directed to the surface, and

a densely packed monolayer is formed. The head groups are at the periphery and are solvated by water. Inversely for hydrophilic substrates, the head groups point towards the substrate. As the tails avoid the water phase, a second monolayer with opposite orientation develops on top of the surface monolayer. The bilayer configuration again has the head groups at the periphery and is readily solvated. The hydrophilic periph-ery of such a film prevents the subsequent spread of the (hydrophobic) surfactant so-lution on top of it. Hence, even though the adsorbed monolayer or bilayer readily takes place on the surface, the growth of the film to macroscopic dimensions (multilayer for-mation) does not take place. More-recent literature discusses a phenomenon termed pseudo-partial wetting, which is charac-terized by similar features, namely, after a surface layer has developed, the growth of the wetting film is hindered by an energy barrier [4, 5]. This scenario has gained at-tention, for example, for polymer spread on top of its own brush [6]. Here and below we will use the classical term autophobicity for these wetting features that allow the coexistence of a molecular thin film (not a macroscopic film) and a finite contact angle (partial wetting) of the same liquid nature.

To understand the physicochemical mech-anisms behind these cosmetic effects, we have combined different experiments and a theoretical model to describe the inter-action of the main components of formu-lations containing this active.

Objective The objective of the project is to design a formula that, upon deposition on the hair surface, wets the surface, forms a stable film containing glycerol distearate (GD) for its sensory and cosmetic benefits and a conditioner for care (PQ). In principle, such a scenario is hard to achieve on account of autophobic dewetting of GD, and we know from numerical calculations, that adding PQ loses the GD layer (and therefore the potential sensory benefits). The target of this work is thus to show how one can introduce wetting additives to change the wetting scenario from an autophobic to a completely wet case. In the analysis that follows, using numerical calculations, we will present the wetting characteristics of

GD on hydrophobic surfaces and show the effect of an emulsifier additive that influ-ences the interfacial free energy of the in-terface between the wetting component and the majority solvent, in combination with a co-solvent additive that partitions inside the GD-film. We will show rigorously that this set of additives can change the wetting characteristic from an autophobic to a completely wet state. This first set of results will set the stage to underpin the experimental results for an antidandruff shampoo formulation. We anticipate that being in the neighborhood of such wetting transition guarantees a swift deposit and spread of GD emulsion droplets on or near the root of the hair fibers. This large deposit is in line with the experimental finding, and it implies a large deposit of the active ingre-dient at the right spot with the right sensory and cosmetic properties.


1. Hair Sample Preparation Virgin Caucasian and Chinese hair col-lected from different healthy volunteers was bundled into standardized swatches, cleaned with a regular shampoo and dried. Hair swatches were purchased from IHIP International Hair Importers & Products. IHIP. 8729 Myrtle Ave. Glendale, NY, 11385-7847. Formulations containing 0.5 % OCT where compared with a placebo to evaluate the effect of this ingredient on the formula and hair properties.

2. Chemical ingredientsWe evaluated the impact of shampoo formulas based with OCT- on the bio-physical properties of hair as well as the physicochemical mechanisms associated with the interaction with the surface de-posits of shampoo formulations contain-ing this ingredient. OCT is an effective antidandruff active ingredient used in formulations to treat dandruff disorders. Formulas using this ingredient have been studied for this work and in the literature, and it was shown that they can develop high levels of antidandruff performance. The Piroctone Olamine-OCT- (CAS 68890-66-4) used was purchased from raw mate-rials company CLARIANT Corp.

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For this purpose, two different shampoo formulas were used to treat Caucasian natural hair. The placebo formula served as a control and had the same formulation backbone as the OCT formula. The back-bone formula, among other ingredients, broadly contains wetting ingredients, wet-ting additives and emulsifiers, plus sham-poo conditioners.

3. Electronic microscopy methodsOptical Microscopy (OM) and Scanning Electron Microscopy (SEM JEOL JSM 6300 F at 3 kV voltage at a magnification of 1000 after metallization of samples with a platform) have been used extensively to evaluate the morphological state of the hair fiber. These techniques use light, elec-trons and matter (small tip) to probe the surface of the hair. [10 -17]

4. Atomic force microscopy (AFM)To complement these studies, beyond imaging surface morphology investiga-tion, we evaluated the frictional forces at the hair surface using atomic force microscopy (AFM). AFM autoprobe CP Research (Veeco, Sunnyvale, USA) was used for the evaluation. We used a 10 -100 mm2 scanner and Si3N4 tips. All tips were unsharpened pyramidal, with a normal spring constant of k 0.06 N m1. All AFM images were acquired in the con-tact mode. Topographic and friction images were obtained in forward and reverse da-tasets, whereas a measure of the frictional force for a given applied load was calculated following well-established protocols.

5. Deposit chemical analysisFor the GC-FID analytical technique, the Gas Chromatography (GC) analytical tech-nique, 100 mg of hairs cut in 0.5 cm length sections was desorbed in 2mL dichloro-methane over 3 hours of continuous agita-tion. The extracted liquid phase was then analyzed by GC using flame ionization detection (FID), and the quantification of three retention pics corresponding to GD was obtained by external calibration using nonanol as internal standard.

6. Surface angle measurementWe systematically evaluated the hydro-phobic / hydrophilic balance of the fiber measuring the hair wettability and ana-

lyzing the characteristics of the deposit that was observed after treatment of these formulations.

The hydrophobic / hydrophilic balance of the hair surface was evaluated by means of the Wilhelmy balance method, using a Kruss K100 microbalance from KRUSS. Hairs were vertically suspended from the root side, using a microbalance, and then the level of the surface of the test liquid (water in this case) was raised to come into contact with the free extreme of the fiber. The force was measured as the fiber was immersed in the liquid (advancing). This force varied depending on the affinity (wetting) of the surface of the fiber with the liquid. As the surface was moved out, the receding forces were also measured. Contact angles were then calculated.

7. Friction methodWe also evaluated the frictional forces at the hair surface. This tribological charac-terization provided access to the friction coefficient, an important parameter in un-derstanding the sensory (touch) consumer appreciation. However, friction is only a manifestation of the state of the hair sur-face and its chemical state.

We used a homemade microtribometer apparatus with engine Escap® 22V28 213E and K27 979 scrubber: stainless steel skate (7 grams) sensor: blades de 0.18 mm, with acquisition parameters fre-quency 10 Hz time 2 minutes and friction speed: 1 mm / min (2 volts). The steel made friction bearing moves according to the sliding axis (similar to the fiber axis) and was activated by the motor. The normal force (friction bearing) being stable, the transversal force was recorded to obtain the friction coefficient.

The measured parameters were: static, dynamic friction coefficient and stick/slip amplitude, which help to understand ad-herence to the surface of non-treated or treated hair. We performed friction ex-periments from hair tip-root and root-tip direction. The fiber was fixed on a micro-scope slide that was immobile. All the measurements were performed at room temperature (24 °C) and hygrometry (45 % RH relative humidity).

8. Numerical simulationThe Scheutjens-Fleer self-consistent field theory SCFT [7 - 9] is well suited to describe at the coarse-grained level polymer and surfactants of complex architecture with-out loss of molecular and relevant realistic details. Such a method has been recently used to study bulk and surface behaviors of complex binary cosmetic formulations [8, 9]. The use of this numerical method helped us to calculate and predict the mechanism behind the complex interaction of the different ingredients, the aggrega-tion phenomena occurring in the bulk and the adsorption of the complexes formed in solution onto solid charged surfaces.

The use of a SCF method is based on the minimization of a mean-field free-ener-gy functional, here using the procedure described by Banerjee et al. [8, 9]. Short-range interaction parameters (Flory-Hug-gins parameters χ) between segments of the molecules, dielectric permittivity (ε) and valence (ν) should be introduced as inputs to the model. The valence accounts for the electrostatic contributions, where-as the Flory-Huggins parameters play a central role in the formation and stability of the self-assembly colloids.

It should be noted that we have consid-ered bare and charged surfaces, on which are deposited mixed micelles formed in the mass, with fixed chemical potential.

Furthermore, calculations are performed over a range of varying surface hydro-philicity, where the tuning parameter is the increasing grafting density) of fatty chains on the bare surface, which in turn aim to mimic the extreme surface of the hair. In that context, hydrophilic surface that corresponds to a bleached hair sur-face has very low fatty chains grafted on, whereas increasing leads to a more and more hydrophobic surface that cor-responds to a less damaged hair surface. Details about the numerical method are given in [8, 9].

9. Consumer in vivo performance evaluationA quantitative test was performed on a population of 12 0 women per formu-la; subjects were 20 - 40 years old and


had severely damaged hair. They were very concerned about the care of their hair, still using anti-dandruff shampoo (100 % beauty anti-dandruff shampoo users). This consumer study was conduct-ed in Shanghai and Beijing in October / November 2014. They tested two types of formula, one optimized formula with OCT, and one reference anti-dandruff formula containing Zinc Pyrithione (CAS 13463-41-7) also known as ZnPt.


Hair fiber is a complex structure. Howev-er, natural hair is hydrophobic in charac-ter owing to the presence of lipids in the cuticle (the outermost layer). In addition to sebum, other noncovalent bonded lipids are a particular characteristic of hair as they form a layer of 18-methyl eicosanoic acid (18-MEA) linked by a thioester linkage to the protein below [18]. The presence of this particular lipid has been the target of many studies that confirm a protective, lubricating effect. Exposure to air and aggression remove this surface layer in an irregular manner. This predisposes the aged hair fiber to gradually become more hydrophilic, as the exposed proteinaceous layer carries a net negative charge. Thus, a simple model of natural hair is a negatively charged surface onto which a layer of fatty acids (FA) is grafted.

cosmeticity benefits) and a conditioner for care (PQ). In principle, such a scenario is hard to achieve on account of autophobic dewetting of GD, and we know from nu-merical calculations that adding PQ loses the GD layer (and therefore the potential sensory benefits). In the following section, we provide a brief explanation of the meth-odology we used to solve this problem. For more comprehensive details, see [20].

Autophobic / pseudo-partial wetting scenarioIn the calculations, GD is modeled as a string of segments consisting of two coupled stearate molecules attached in the polar region by a C2 spacer. Sodium laureth sulfate (SLES) is used as the sur-factant to keep the GD in solution. Here we will first disregard the surfactant and assume that the GD concentration can be gradually increased to the saturation value numerically (a complicated task in practice).

Figure 3 presents the adsorption iso-therms of GD for several values of the grafting density of the 18-MEA chains on the hair surface. None of these iso-therms corresponds to complete wetting because all isotherms merge the binodal value coming from the super-saturated region (see inset in Figure 3) It is clear that the GD isotherms deviated in several ways from the classical wetting isotherms.

A large number of techniques have been applied to standardize hair and find direct or indirect evidence for the effect of a for-mulation on the hair properties.

1. Simulation results Thanks to the newly developed modeling approaches, SCF used with lattice approxi-mations to study polymers – surfactants interactions first on bulk and then at in-terfaces, taken in acout in particular bio-inspired hair model surfaces, it is possible to explore the physicochemical mechanism behind the formation of shampoo deposits. The procedure starts with a definition of the hair surface that aims to capture its relevant physicochemical features, as de-scribed above. Figure 2 presents a sketch of the model surface.

The hair surface is modeled as a substrate (S) with a layer of fatty acids (FA) attached to it that mimic the 18-MEA present at the ex-treme cuticle hair surface. The length of the chains is 20 C-segments. On the substrate it-self, there are negative charges (the valence is set to 0.1, which roughly corresponds to a surface charge of 200 mC.m-2). The hy-drophobicity of the surface is controlled by varying the grafting density, s, of the FA.

The objective is to design a formula that, upon deposition on the hair surface, wets the surface, forms a stable film contain-ing glycerol distearate (GD) (sensory and

Figure 2 Schematic illustration of the model used for the FA decorated

negatively charged surface (natural hair). The volume fraction of salt is

indicated. The surface charge (valence of surface groups) is set to vS = 0.1,

which means that 1 / 10 of the (surface) sites has a negative charge.

The grafting density of the FA chains s = 1 / a with an area per molecule

is indicated. The red bars are the grafting units (X) that hold the C20 fatty

acid chains on the surface. In the system there are as many negative

as positive charges.

Figure 3 Family of adsorption isotherms, that is, θexcGD (log ϕb GD) of GD

on the hair surface. The grafting density of fatty acids is here mentioned

in terms of the amount of 18-MEA in equivalent monolayers: θ18-MEA.

The inset presents an enlarged view of the region near the binodal

(vertical dotted line).

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For bleached or damaged hair (low graft-ing densities of 18-MEA), that is, for 0 < θ18-MEA < 1, there is a huge van der Waals loop in the isotherm, indicating that an energy barrier must be overcome be-fore the GD layer is formed. Indeed, when the ‘bell-shaped’ isotherm curve on the supersaturated side has a larger area than the one in the sub-saturated side there is only a very, very dilute gas phase on the hair surface (black horizontal line). Around a grafting density θ18-MEA = 1, we observe a step in the isotherm before the bimodal is reached (pre-wetting). Now the isotherm labeled ‘1’ , will go after the ‘pre-wetting’ step for the second time to the super-saturated region (see inset). This occurs at high adsorbed amounts of θexc

GD ≈ 7. The system is characterized by interaction on at least two length scales. The interac-tion with the surface promotes wetting (thanks to the 18-MEA chains). However, on a larger length scale, the system ex-periences the van der Waals loop, which prevents wetting, and the film remains of the thickness of a monolayer. As previ-ously indicated, the inability of molecules to spread on top of a monolayer of the same molecules is known as autophobicity or partial wetting.

With increasing grafting density of 18-MEA, the large van de Waals loop barrier disappears, and GD molecules can anchor inside or on top of the 18-MEA layer. Yet the stopping force that prevents wetting

This maximum indicates that the GD mol-ecules are oriented with the tails inside the 18-MEA layer. In other words, the film is not isotropic. The anisotropy (at the ori-gin of the autophobic effect) produces an unfavorable interaction with an incoming GD molecule.

GD plus hydrophobized cationic polysaccharide (PQ)As mentioned previously, we aim to ad-dress, the sensory and cosmetic benefits observed in the formulas studied experi-mentally. The key ingredients included are glycerol distearate (GD) and a hydrophobi-zed cationic polysaccharide (PQ).

We show that, on top of the 18-MEA lay-er, it is possible to find a good coverage of GD, but unfortunately a film is rather dif-ficult to form, fragile and easily destroyed by, for example, inhomogeneities along the hair surface.

It is natural to strive for a thicker GD film, which is possible when the autophobic barrier is overcome. In the absence of such barrier, the thickness of the film is de-termined by the transport of the GD (as emulsion droplets): a significant GD film thickness may build up rapidly through spreading of the droplets.

The conditioning ingredient (PQ) in OCT-based shampoos is hydrophilic but can insert itself in the interfacial film because

(the autophobic effect) also increases. The result is that the equilibrium film thickness and the amount of GD decrease with in-creasing 18-MEA coverage (the insertion of the GD in the 18-MEA becomes pro-gressively harder as the density of this layer increases).

Nevertheless, the decrease of the amount of GD is significantly less than the increase of the 18-MEA coverage. Hence, the combination of GD+18-MEA film thick-ness increases with 18-MEA coverage (not shown). To search for the cause of the autophobic effect, we considered a typical density profile of the film thickness at the first crossing of the saturation value for a surface with an intermediate grafting den-sity of θ18-MEA = 4. In Figure 4, we see the expected configuration of the molecules at the interface. The 18-MEA film (FA) sits directly on top of the substrate (positioned at z < 1), the GD molecules form a con-densed film on top of the 18-MEA layer (blue line). The two films interdigitate significantly (the FA and GD curves overlap). This in-terdigitation is possible because both the 18-ME and the GD are composed of the same type of segments (C). There is an en-tropic driving force for this interdigitation. The mixing entropy is gained, and this mix-ing entropy is balanced by a stretching entropy loss of the 18-MEA chains. The oxygen molecules have a weak maximum on the water side of the composite film.

Figure 4 The volume fraction profile for the fatty acid

with coverage θ18-MEA = 4, that for GD at the bulk volume fraction

equal to the binodal (saturation value), and the distribution

of the oxygen groups of the GD.

Figure 5 Overall density profiles for 18-MEA (black), GD (blue) and PA (red)

and the profile for the positively charged N (green), for the case that GD

is at coexistence, the θ18-MEA = 4, the amount of PQ at the interface is fixed

to θexcPQ = 4. The amount of GD is strongly suppressed (θDG = 0.84).

Salt concentration ϕs = 0.01.


er, the ‘bell curve’ in the super-saturated domain decreased in size (and area), the partial wet state was rather persistent. The system remains partially wet even for amounts of PQ θ = 5, as shown in the inset, where the grand potential is plotted as a function of the GD concentration. The fact that, in this system, two lines do not cross proves that the system (just) did not reach the wet state. The isotherms in Figure 6c are for ϕb

OCT = 1 × 10-3.

Now, for a coverage of θPQ = 2, the iso-therm increases continuously and diverges at the bulk binodal, indicating the com-plete wet state of this system. Hence, a sufficiently high concentration of the OCT surfactant in combination with the PQ polymer leads to the disappearance of the autophobic wetting barrier. Appar-ently, the combination of OCT+PQ func-tions as a spreading aid.

Taking account of all isotherms in the limit of θPQ → 0 proves that the OCT surfactant alone is not sufficient to induce the wet-ting transition. Even when the OCT con-centration is increased to 10-3, the partial

some hydrophobic tails are present at the ends of some of the side groups attached to the polysaccharide backbone. More specifically, there is a cationic polysaccha-ride backbone (here modeled as units A, which are given repulsive interactions with C that is decorated by PEO sides that are terminated with a quaternary amine, half of which carry a hydrophobic tail of 12 car-bons). The positive charges contribute to the preference of the PQ to be in the water phase. The hydrophobic tails make these molecules surface active, and therefore it is of interest to find out how the PQ mol-ecules influence the film formation of GD.

In Figure 5, the volume fraction profiles are shown for the case that the PQ amount was fixed to θexc

PQ = 4, while the salt concentration was reduced to ϕs = 0.01. The amount of GD at bulk coexistence was strongly reduced by the presence of PQ in this case. Inspection of the profiles reveals the expected picture. The 18-MEA den-sity is high near the surface (surface is in z < 1), the remainder of the GD film is di-rectly positioned on the top of the GD lay-er. The PQ molecules are on the waterside of the composite film. A brush-like profile is found. The hydrophilic groups tend to give a shoulder around z = 15, character-istic for the parabolic shape of the pro-file. We also have plotted the distribution of the positive charges ′N′ (green). Even though their density is low, it is clear that there is a small maximum of the charges of the polymer in the region where GD is placed. This already suggests that there is a non-trivial electrostatic effect. Without the negative charges on the surface, the positive charges would have tried to be as far as possible from the surface in the distal parts of the PQ profile.

GD plus hydrophobized cationic polysaccharide (PQ), plus OCTWhen a positive charge on the polymer suppresses the formation of a GD film, a molecule with negative charges may do the opposite. This idea sets the stage to see how the PQ-induced suppression of the GD film is modified by the addition of the OCT surfactant.

The effect of OCT on the film formation of GD is more complicated because the

mixing of OCT with the GD cannot be neglected. Therefore, we focus on adsorp-tion isotherms of GD at a fixed salt concen-tration, fixed OCT concentration and for a variable amount of PQ in the system. The hair surface is modeled with a reasonable amount of 18-MEA θ18-MEA = 4. As we are looking for a wetting transition, we pres-ent the results for a reasonably high salt concentration ϕs = 0.04, and note again that choosing more hydrophobic coun-terions may mean that the same wetting scenarios can be achieved at lower con-centration.

Figure 6a-c presents a selection of ad-sorption isotherms, wherein the OCT con-centration was systematically increased. It is noted that the corresponding binodal concentration shifted to lower and lower concentrations of GD.

In Figure 6a, the OCT volume fraction is represented by ϕb

OCT = 2 × 10-4, and all isotherms correspond to partial wetting. In panel b, the OCT concentration was increased to ϕb

OCT = 5 × 10-4. Even though with increasing amounts of PQ in the lay-

Figure 6 a-c Adsorption isotherms and d typical density profile for the system with18-MEA, GD,

PQ, and OCT. a-d Adsorbed amount of GD as a function of the bulk concentration ϕbGD for

(a) ϕbOCT = 2 × 10-4, (b) ϕb

OCT = 5 × 10-4, (c) ϕbOCT = 1 × 10-3.

Values for the amount of PQ polymer at the surface are indicated near the isotherms. In panel b

the amounts of PQ: θPQ = 1, 2, 3, and 4. The inset shows the grand potential as a function of the log

of the volume fraction of GD for θPQ = 5. d Typical density profile for a wetting film of GD,

for parameters as in panel c, for θPQ = 2. The overall profiles for 18-MEA, GD, OCT and PQ

are given for the case that θGD = 20. Parameters: θ18-MEA = 4, salt concentration ϕs = 0.04.

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Hair treated by specific formulas contain-ing OCT are characterized by a significant lower friction coefficient, as compared to formulas without this ingredient, as control.

3. Hair topography by AFMIn Figure 8a, SEM and AFM representa-tive images of hair surface after OCT treat-ment show no particular damage. Scales and edges are well preserved after the hair treatment. In Figure 8b, quantitative hair surface roughness measurements confirm this observation. There are no significant surface roughness differences between placebo and OCT hair treatments, as shown in Figure 8b.

Thanks to silico, we succeeded in designing a formula that fulfills the prerequisites in terms of ingredients. Piroctone-olamine-containing formulas, when applied onto hair model surfaces, promote the deposi-tion of fatty compounds and, in particular, glycerol distearate (GD), while preserving contribution of hair care products PQ.

It is now necessary to evaluate such for-mula on hair swatches.

2. Friction measurementsA decrease of hair friction is statistically observed on hair treated with OCT (right) versus placebo (left) (Figure 7).

wet state remains the case when PQ levels are low. Inversely, the OCT surfactant itself suppresses the formation of a thick GD film. This is judged from the fact that the loops in Figure 6 have the tendency to oc-cur at lower values of θGD with increasing ϕOCT. Hence, the combination of the PQ polymer and the OCT surfactant is neces-sary for wetting to occur. In all cases, we see that autophobic (partial) wetting is the result when PQ levels are low, even when OCT levels are increased. Wetting is seen in the limit of a sufficient amount of OCT and a high enough amount of PQ in the formulation.

Finally, to show the structure of a typical wetting film, we present the density pro-files for the system with a thick GD film in Figure 6d. As can be seen, the 18-MEA chains remain near the surface, the GD film is intermixed with an almost equal amount of the OCT surfactant. At the pe-riphery of this film, the OCT anionic surfac-tant has a local maximum that coincides with the cationic PQ profile. As this profile is taken for a relatively low concentration of PQ, the expected brush-like profile has not yet developed. It is expected, however, that with increasing amounts of PQ, this brush does develop. Such a hydrophilic brush may help to prevent the erosion of the wetting film after the deposition of it. Interestingly the OCT surfactant accu-mulated in large amounts in the wetting film. This result should correlate with the biological activity of this surfactant in pre-venting the growth of fungi.

Figure 7 Friction coefficient measured from

fiber–fiber experiments on natural Caucasian

hair for the placebo (classical shampoo formula)

and OCT formulas. Each formula has the same

formulation backbone, only differing by the

addition of active OCT for OCT formula.

Figure 8a SEM and AFM representative images of the surface of hair after OCT treatment.

No particular damage is observed, scales are well preserved. The AFM image is on 3d representation

10 x 10 µm surface, the color code from blue (lowes)t to red (highest) z values.

Figure 8b SAFM representative images of the surface of hair after placebo and OCT treatments,

and the variation of the hair surface roughness Sa (nm) obtained after large numbers of hair

surface analyses.


The measurements of roughness do not, however, explain the low lubrication and friction coefficient after OCTtreatment.

4. Surface angle measurementIn Figure 9, the variation of contact angle shows that Octopirox OCT-based sham-poo presents a slightly higher advancing contact angle interpreted as a more hy-drophobic hair surface, though this is not significant.

The measurements of surface hydropho-bicity do not, however, explain this in-creased lubrication.

(cationic polymers, fatty acids, etc.) is a complex phenomenon.

Piroctone-olamine-containing formulas, when applied to hair, promote the deposi-tion of fatty compounds and, in particular, glycerol distearate (GD). For example, as revealed in Figure 10, in the OCT formu-lation, a deposition of GD of 0.2 mg per gram of hair was recorded, which was about four times as high as for a compa-rable formulation in the absence of OCT.

The extraordinarily high deposition of GD is noteworthy and appears to be a strong function of the presence of OCT. The lat-ter observation indicates that OCT may mediate the lubricating characteristics of the GD on the natural hair surface.

6. Consumer in vivo performanceThe results shown in Table I, comparing an optimized OCT-containing formula with a bench formula containing ZnPt, show that the antidandruff performance is equivalent for both formulas.

The hair benefits perceived on hair by the consumers are significantly better for the piroctone-olamine-containing formula. Surprisingly, the hair benefits are signifi-cantly superior on numerous measures: “texture liking”, “repairs damaged hair”, “prevents split ends”, with a feeling of “no residue on dry hair”.

7. Sustainability formula performanceAfter use, cosmetic products are often discharged into the sewers and the aquatic compartment. This discharge is considered as dispersive and continuous. Steady progress in reducing the envi-ronmental impact of cosmetic products can be achieved by focusing on three strategic indicators: biodegradability, grey water footprint adapted for eco-design (GWFE) and a global indicator, complementary to these two endpoints. Biodegradability is the key process in the removal of organic ingredients from the environment. GWFE is defined herein as the theoretical volume of natural fresh-water required to dilute a cosmetic for-mula, after use by the consumer, to a concentration without foreseeable toxic effects on aquatic species. Finally, the

complementary indicator highlights a possible alert on a formula ingredient owing to an unfavorable environmental profile based on hazard properties [21]. For example, a H410 classification by the United Nations Globally Harmonized System of classification and labelling of chemicals (GHS) or by the European Classification, Labelling and Packag-ing of substances and mixtures (CLP). Anti-dandruff shampoos are among the cosmetic products which end up in the aquatic environment after use.

However, it has a very high chronic aquatic ecotoxicity towards algae, daphnia and fish leading to an unfavourable GWFE in-dicator of 735. ZnPt also has an ongoing H400-H410 harmonised CLP classifica-tion [22] with a GHS09 pictogram and a chronic M-factor of 10, leading to a very unfavourable third indicator.

The anti-dandruff active ingredient OCT is not readily biodegradable, leading to an unfavourable biodegradability indica-tor. It is however rapidly degraded by direct photolysis in water with a half-life of 0.9 days and therefore not persistent according to its REACH registration dos-sier. It is an interesting alternative to Zinc Pyrithione because it is significantly less ecotoxic. With only two chronic aquatic ecotoxicity results, and thus a less fa-vourable safety factor, OCT still has an improved GWFE indicator of 390. In ad-dition, OCT is only classified H412 with-out the GHS09 pictogram in its REACH registration dossier [23].

Therefore, considering the 1 % ZnPt and 0.5% OCT concentrations in the tested formulas, the loss in biodegradability is relative. The gain in GWFE would be sig-nificant for an equivalent amount, and is even more so considering the lower con-centration of OCT.

In the end, substituting ZnPt by OCT leads to an overall improvement of the impact on the aquatic environment due to the significant improvement in ecotoxicity. OCT, although not readily biodegradable, is also not persistent. The use of OCT is therefore part of a continuous improve-ment process.

Figure 9 Measurement of hydrophobicity

through advancing contact angle experiment

of water droplets on hair after treatment.

5. Deposit chemical analysisIn general, the interaction of OCT with the different components of a formulation

Figure 10 Experimental results of the adsorbed

amount of glycerol distearate (GD) using

Gas Chromatography with a Flame Ionization

Detector (GC-FID).

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CONCLUSIONEvidence of the hair repair effects of Piroc-tone Olamine-OCT-based specific formu-lations are presented as an add-on of the well-known antidandruff effect.

The OCT formula contains ecofriendly products. Besides low molecular weight salts, key ingredients include glycerol dis-tearate (GD), a hydrophobized cationic polysaccharide (PQ), and OCT. It can be speculated that the mode of action gen-erates a significant deposition of GD (re-placing a classical silicone wetting film) on natural hair, and this layer, together with additives, provides the friction reduction between hair fibers, such that disruptive contacts are suppressed.

The SCF (Self Consistent Field) calcula-tions give evidence for a rather complex (wetting) scenario, in which additives that accumulate on top and into the GD layer serve as spreading aids (for the build-up of a GD film) that switches off autophobic wetting in favor of a complete wetting scenario. In the absence of PQ and OCT, the build-up of the GD film on top of a fatty-acid-functionalized hair surface is limited to a microscopically thin mono-layer, even when in the bulk the solution is saturated with GD. This latter effect is known as autophobicity and results from the fact that the monolayer of GD on the surface is not an amorphous film but consists of a layer of somewhat oriented molecules.

In water, the outer part of this layer is slightly hydrophilic, and this prevents the build-up of second and subsequent layers. The aforementioned additives have the ef-fect that the layer of GD ‘randomizes’ and loses most of its anisotropic orientation, and this triggers a wetting transition. Of key importance is a hydrophobic anionic surfactant (OCT) that inserts itself into the GD film in significant amounts and forms an ion-complex on the water-side of the GD/surfactant film with the PQ polymer. The polymer is ‘adsorbed/complexed’ on top of this film, and this polymer/surfac-tant complex arguably provides mechani-cal strength to it. The polymer brush mo-tive of this layer is anticipated when the loading of PQ on top of this GD/surfac-tant film is sufficiently high. The polymer brushes at the periphery of this complex layer may provide the mechanical strength as the brush motive is known to survive significant normal forces.

Formulas using this active ingredient have achieve significant success in the treat-ment of dandruff, as well as excellent cosmetic effects (i.e., hair conditioning and fiber benefits).

It was shown that they can develop high levels of anti-dandruff performance to-gether with hair care (which in turn may increase cosmeticity, scalp kindness and hair repair efficiency).

Acknowledgments

The authors wish to acknowledge the tech-nical assistance of Sophie VICIC for AFM measurements and Julien LUCCHINI for all hair treatments and characterizations.

References

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Corresponding AuthorFabien Leonforte

L’Oréal Research & InnovationAulnay-sous-Bois

[email protected]

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A New Inositol Derivative to Mitigate the Effects of Air Pollution on SkinYuko Saeki1, Shinji Yamaki1, Ichiro Fujita2, Naoko Motohashi3 and Eiko Kato1

1 Institute for Integrated Product Development, Showa Denko K.K.13-9, Shiba Daimon 1-Chome, Minato-ku, Tokyo 105-8518, Japan2 Development Group, Development Department, Kawasaki Plant, Showa Denko K.K. 2-3, Chidori-cho, Kawasaki-ku, Kawasaki City, Kanagawa, 210-0865, Japan3 Functional Chemical Division, Showa Denko K.K. 13-9, Shiba Daimon 1-Chome, Minato-ku, Tokyo 105-8518 Japan

Keywords: Anti-pollution, skin barrier, inositol derivative, proteoglycans, aryl hydrocarbon receptor

care products containing functional ingre-dients, such as antioxidants, to counteract pollution-induced skin damage [6, 7].

Inositol, or more precisely myo-inositol, is a carbocyclic sugar found abundantly in the brain and other tissues. It medi-ates cell signal transduction in response to a variety of hormones, neurotransmit-ters, and growth factors and participates in osmoregulation as an osmolite. In the cosmeceutical field, inositol has been widely applied because of its humectant ability and efficacy in the treatment of ef-ficacies cutaneous disorders [8, 9]. To de-velop more effective bioactive materials for moisturizing skin, we designed a new inositol derivative (InoO) that consists of an inositol body and oligosaccharide resi-dues (Figure 1), both of which are known to promote skin hydration and barrier for-mation [10]. Our experimental data reveal how InoO improves the skin barrier via an increase in proteoglycans (PGs) rather than the activation of epidermal differ-entiation factors. The influence of InoO on PGs did not change under exposure to urban particulate matter (UPM), which contains PAHs and dioxins. Moreover, InoO exhibited anti-inflammatory effects on UPM-exposed keratinocytes.

The aryl hydrocarbon receptor (AhR) is an important regulator of drug metabolism that is activated by a broad spectrum of low-molecular-weight compounds, in-

INTRODUCTION

With continued global industrialization, atmospheric pollutants have caused se-rious human health problems [1]. Expo-sure to pollutants has major effects on the human skin, such as oxidative stress, premature aging, inflammation, pigment spot formation, skin rashes, eczema, and disease [2 - 4]. Previous studies have dem-onstrated that airborne particulate matter containing polycyclic aromatic hydrocar-

bons (PAHs), such as benzo(a)pyrene (BaP) and dioxins, are correlated with premature skin aging and dryness [2, 5]. The skin is continuously exposed to particulate mat-ter that attaches to the surface or pen-etrates the epidermis, and it is the first pro-tective barrier against external stressors. Therefore, highly effective and protective bioactive materials are required to address skin problems related to particulate mat-ter. A cosmetic approach to address this problem is the topical application of skin-

Abstract

Exposure to pollutants has major effects on human skin. A cosmetic approach to address this problem is the topical appli-cation of skincare products containing functional ingredients, such as antioxi-dants, to counteract pollution-induced skin damage. In this study, we present a new inositol derivative (InoO) as an anti-pollution moisturizer that reduces skin dryness and inflammation induced by pollutants.

Treatment with InoO suppressed tran-sepidermal water loss (TEWL) and in-duced expression of proteoglycans (PGs), but not of differentiation markers. When the reconstructed skin was exposed to urban particle matters (UPM), TEWL was significantly elevated. Treating the skin

with InoO for 24h before UPM exposure recovered TEWL by inducing PG synthe-sis in the epidermis. Furthermore, InoO exerted an anti-inflammatory effect on keratinocytes under UPM exposure con-ditions by inhibiting inflammatory cyto-kines and reactive oxygen species (ROS) generation. We revealed that these effects occurred because of the block-age of aryl hydrocarbon receptor (AhR) translocation to the cell nucleus by InoO.

InoO inhibited ROS generation and inflammation mediated by AhR acti-vation. Moreover, InoO induced the expression of PGs, which improved the skin barrier under polluted condi-tions. Thus, InoO can regulate the AhR pathway and PG generation and has the potential to be used as a new anti-pollution material for skin.


cluding dioxins and PAHs [11, 12]. Upon ligand binding, the AhR shuttles into the nucleus and binds to so-called xenobiotic-responsive elements in the promoter re-gion of genes to stimulate their expres-sion. Prototype AhR target genes encode for cytochrome P450 (CYP) 1A1, CYP1A2, and CYP1B1, which enhance cyclooxygen-ase 2 (COX-2) expression in rodents [13], increase interleukin secretion [14 -16], and stimulate the production of reactive oxygen species (ROS), such as superoxide anion radicals [17]. Further investigation of the effects of InoO under UPM exposure conditions revealed that InoO specifically inhibits AhR translocation to the nucleus, which is required before ROS generation and inflammatory responses. The inhibi-tion of AhR nuclear translocation by InoO led to the reduced expression of CYP1A1 and CYPB1, as well as COX-2 gene and inflammatory cytokines, such as Prosta-glandin E2 (PGE2) and IL-1a. Interestingly, inositol and oligo, which are the compo-nent elements of InoO, had no effect on the AhR pathways and UPM-induced bar-rier disruption and inflammation.

First, we introduce InoO-inhibited ROS generation and inflammation mediated by AhR activation in keratinocytes. Moreover, we report that InoO induced the expres-sion of PGs, which resulted in skin barrier improvement under polluted conditions. Thus, InoO was used to elucidate the AhR pathway and PG generation mechanisms as a new anti-pollution material for skin.


MaterialsInoO was enzymatically synthesized from inositol and cyclodextrin by Showa Denko K.K. UPM was purchased from NIST (1648a, MD, USA) [5], myo-inositol was obtained from Kanto Chemical co., Inc., and oligosaccharides (oligo) were obtained from Senshu Scientific Co. Ltd. Sugars were dissolved in water at the con-centrations indicated in each figure. UPM was dissolved in dimethyl sulfoxide and was diluted with ultra-pure water before use.

Cell culture and UPM treatmentThe normal human epidermal kerati-nocytes (NHEKs) were maintained in a

HuMedia KG2 medium (Kurabo, Osaka, Japan) supplemented with 5 μg / ml in-sulin, 0.5 μM hydrocortisone, 200 pg / ml human recombinant epidermal growth factor, and 0.2 % bovine pituitary extract. After 24 h, cells were incubated with sug-ars for the periods indicated in each figure. Prior to UPM exposure, cells were pretreat-ed with 0.0001 % of sugars for 24 h and then exposed to 500 ng / mL UPM for 6 h. The treated cells were washed with a fresh medium and cultivated for the appropriate time period indicated in each figure. Cell viability was analyzed by MTT assay prior to each experiment.

TEWL measurementThe reconstructed epidermal skin model was purchased from Kurabo (Osaka, Ja-pan). The surface of the skin model was treated with 1 % of InoO, inositol, or oli-go dissolved in ultrapure water for 48 h. Before measurement, the surface of the skin was washed with phosphate-buff-ered saline (PBS) and air-dried. For UPM-treated experiments, the skin was topi-cally treated with 1 % of InoO, inositol, or oligo dissolved in ultrapure water for 24 h before UPM exposure. After wash-ing with PBS, the skin was exposed with 1 mg / cm2 UPM for 5 h. The skin was then cultivated for another 24 h after washing out all UPM residue and was air-dried. The transepidermal water loss (TEWL) of each skin sample was measured three times using VAPO SCAN (Asch Japan, Tokyo, Japan). A viability assay was then per-formed. For the data analysis, the values from the TEWL measurement were nor-malized by the values from the viability assay of each skin sample.

Immunohistochemistry (IHC)The skin tissues were fixed in ALTFiX (Falma, Tokyo, Japan) and embedded in paraffin wax. Paraffin-embedded tis-sues were cut at 5 μm and placed on microscope slides. Using xylene and a series of ethanol washes slides were de-paraffinized and rehydrated. They were blocked in Blocking One Histo solution provided by Nacalai Tesque (Kyoto, Ja-pan) for 0.5 h at room temperature. Then, the sections were immunohisto-chemically stained using anti-flaggrin, in-

volucrin, loricrin (purchased from Abcam, Cambridge, UK), perlecan (Cusabio Tech-nology Inc., TX, USA), syndecan-1, CD44, hyaluronic acid synthase (HAS) 3, and bi-glycan (Proteintech, IL, USA) antibodies at 4 °C overnight. In all cases, the first antibodies were used at concentrations that yielded no observable nonspecific staining. Then, the sections were applied with biotin-labeled anti-rabbit IgG sec-ondary antibody for 0.5 h at 25 °C, and developed with 3,3’-diaminobenzidine (FUJIFILM Wako Pure Chemical Corpora-tion, Osaka, Japan). Stained tissue sec-tions were photographed using a Nikon TS100 (Tokyo, Japan). The signal inten-sity of three independent sections was measured using Image J image analysis software (NIH, MD, USA).

ROS analysisROS activity was measured using an ROS assay kit (BioVision, CA, USA). Briefly, NHEKs were treated with 0.5 μg / mL UPM or 0.5 mM hydrogen peroxide in the buffer provided by the manufacturer and then in-cubated for 1 h. The immunofluorescence (ex / em: 495 / 529) was then measured us-ing TECAN infinite M200 microplate read-er (Tokyo, Japan). After measurement, cells were lysed with RIPA buffer, and the protein quantity was measured by BioRad ECL kit (CA, USA), using the normalization of the ROS assay results.

PGE2 measurement by enzyme-linked immunosorbent assay (ELISA)After 48 h of UPM exposure, culture medi-um was collected and used to measure the released PGE2 using a kit provided by Cay-man Chemicals (MI, USA). The appropriate optical density was then measured using TECAN infinite M200 microplate reader (Tokyo, Japan). Each value from the ELISA assay was normalized by the values from the cell viability assay.

ImmunofluorescencePrior to UPM exposure, NHEKs were pre-treated with sugars for 24 h and then ex-posed to 500 ng / mL UPM for 1 h. The cells were fixed with 4 % neutralized formalde-hyde and permeabilized with 0.1 % Tri-ton-X100 in PBS. After blocking with 10 % fetal bovine serum in PBS, the cells were incubated with primary anti-AhR antibody

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(purchased from Abcam, Cambridge, UK) at 4 °C overnight. DAPI staining was used to identify the cell nuclei. The cells were observed and photographed with a Nikon TS100 (Tokyo, Japan). The AhR signal in-tensity of three independent images was measured using Image J image analysis software (NIH, MD, USA).

Quantitative RT-PCRRNA was reverse-transcribed into comple-mentary DNA using the PrimeScript RT reagent Kit (TaKaRa, Japan). All PCR reac-tions were performed using either SYBR Premix Ex Taq (TaKaRa) or KAPA SYBR Fast kit (KAPA Biosystems, South Africa) in a Thermal Cycler Dice Real Time System TP500 (TaKaRa). qRT-PCR was performed in triplicate for each sample, according to the manufacturer’s instructions. The stan-dard curve method was used to determine the relative quantity of mRNA. All qRT-PCR data were normalized to glyceraldehyde 3-phosphate dehydrogenase expression.

StatisticsThe results are expressed as mean values with the standard deviation (SD). The ver-tical bars in the graphs indicate SD. The significance was tested by using the one-way analysis of variance followed by Dun-nett’s test.

Figure 3 IHC staining for epidermal differentiation markers in human reconstructed skin

after treatment of 1 % InoO, inositol, and oligo. Representative images of a) Filaggrin, b) Loricrin,

and c) Involucrin. Bar: 10 μm. d) Quantitative image analysis for the expression of each differentiation

markers. N = 3. Statistics: P** < 0.01 vs control.


InoO improves skin barrierWe developed a novel inositol derivative to moisturize skin and reduce the risk of skin damage from pollutants. The deriva-tive consists of an inositol body and oligo (Figure 1, InoO), both of which are known to improve skin hydration and barrier for-mation [8, 10].

To identify the properties and efficacy of InoO, we measured TEWL using a recon-structed skin model. Figure 2 shows that InoO clearly reduced TEWL after 48 h of treatment, while the inositol and oligo treatment had no effect on TEWL. This suggests the improvement of TEWL by InoO is not because of its mother com-pounds. To investigate how InoO affects the skin barrier, factors related to epider-mal differentiation and skin barrier forma-tion were assessed by an IHC analysis. Ino-sitol and oligo increased the expression of differentiation markers such as filaggrin, loricrin, and involucrin in the epidermis (Figure 3), while InoO promoted the ex-pression of loricrin and involucrin. In con-

trast, with regard to PGs and GAG-related proteins, InoO significantly increased the levels of perlecan, syndecan-1, biglycan, CD44 (a hyaluronic acid receptor), and HAS3 (Figure 4f), while inositol and oligo had no or weak effect on the promotion of PG expression. These results indicate that InoO improves the epidermal barrier by mainly promoting the expression of PGs and GAGs but not differentiation markers. The skin section analysis revealed that InoO specifically induced the formation of PGs but not differentiation markers such as filaggrin; conversely, inositol and oligo, each of which has been reported to have a moisturizing effect [8, 10], promoted only the expression of differentiation markers but not PGs. These results suggested that the moisturizing mechanism of InoO is dif-ferent from other moisturizing bioactive materials, which is characterized by the activation of PG synthesis (Figures 3, 4) in the epidermis. This also indicates that the effect of InoO is due to its own structure and not the inositol body or oligosaccha-ride residues in its structure. The expres-sion of PGs and GAG-related proteins was tested in an age-dependent manner

Figure 1 Chemical structure of inositol oligo

(InoO). N = 2 - 30.

Figure 2 Effect of 1% InoO, inositol, and oligo

on TEWL of reconstructed skin. N= 3.

Statistics: P** < 0.01 vs control.


[18-20]. The results suggested that InoO could increase PG expression, which de-creases with age, and improve age-related skin changes, such as dryness and fine lines.

InoO protects skin from the damages by air-pollutantsRecent studies have revealed that air-pollutants such as BaP and dioxins affect skin dryness and promote skin problems,

analysis revealed that UPM exposure re-duced the expression of differentiation markers such as filaggrin and involucrin (Figure 6d) in the epidermis. Only the expression of loricrin was increased after UPM exposure. Pretreatment with ino-sitol and oligo showed no effect on the expression of filaggrin and involucrin, but that of loricrin was significantly elevated. InoO treatment had no effect on filag-grin and loricrin but significantly induced involucrin expression. With regard to PG expression in the epidermis (Figure 7f), it was clearly increased in the InoO-treated skin, while inositol and oligo treatment had no or weak effect on it, suggesting that InoO has a specific protective effect on the UPM-disrupted skin barrier and PG expression, which is different from that of inositol and oligo.

Figure 5 and Figure 7 illustrate how InoO improved the skin barrier damaged by UPM exposure by elevating the expres-sion of PGs and GAG-related proteins. As shown in Figure 6 and Figure 7, inositol and oligo treatment could not recover UPM exposure-induced dysregulated ex-pression of differentiation markers such as filaggrin and involucrin and PGs. These results suggest that InoO can inhibit ad-verse effects of UPM exposure on the skin barrier and directly improve the expres-sion of PGs and GAG-related proteins.

The generation of free radicals, especially ROS, by BaP and dioxins is mediated by the activation of AhR, which resulted in inflammation and premature aging [4, 17]. The gene expression analysis re-

such as premature aging. Thus, we evalu-ated the effects of InoO on air pollution-induced skin dryness.

When the reconstructed skin was ex-posed to UPM, TEWL was significantly elevated (Figure 5). The 24 h treat-ment with InoO before UPM exposure significantly recovered the TEWL, while the treatment with inositol or oligo el-evated TEWL. The immunohistochemical

Figure 4 IHC staining for PGs in human reconstructed skin after treatment with 1 % InoO, inositol,

and oligo. Representative images of a) Perlecan, b) Syndecan-1, c) Biglycan, d) CD44, and e) HAS3.

Bar: 10 μm. f) Quantitative image analysis for the expression of each PGs. N = 3. Statistics: P* < 0.05,

and P** < 0.01 vs control.

Figure 5 Effect of 24 h pretreatment with

1 % InoO, inositol, and oligo on TEWL of

reconstructed skin after UPM exposure.

N = 3. Statistics: P† < 0.05 vs N.C. and

P** < 0.01 vs control. N.C.: negative control.

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vealed that under UPM exposure condi-tions, keratinocytes treated with InoO showed a downregulated expression of CYP1A1 and B1, interleukin (IL) 1a, IL-1b, IL-6, IL-8, and COX-2 (Table I), which is regulated by the AhR. Therefore, InoO reduced ROS generation induced by UPM exposure, although it had no effect on ROS generated by hydrogen peroxide (Figures 8a, 8b), suggesting that InoO itself has no anti-oxidative property as a compound, but it has an inhibitory ef-fect on AhR-mediated ROS induction. The treatment with inositol or oligo significantly promoted ROS produc-tion. Notably, InoO inhibited inflamma-tory cytokines, such as IL-1a and PGE2 (Figures 9a, 9b), which was induced by AhR pathway activation. Neither inosi-tol nor oligo showed any effect on ROS generation and inflammatory media-tors. When we investigated the effect of InoO on the AhR nuclear translocation

creases involucrin expression among the tested differentiation markers, although inositol and oligo can more effectively promote filaggrin and loricrin expression than InoO, which is possibly due to the inhibitory effect of InoO on AhR nuclear translocation, as described previously. Moreover, in our experiments, inosi-tol and oligo were positively correlated with TEWL elevation (Figure 5) and ROS generation (Figure 8), although both induced the expression of filaggrin and loricrin, which induces barrier formation in epidermis; this indicates that negative cellular responses such as ROS generation may be more substantial than the positive effects on the skin barrier, which are both induced by AhR translocation.

To mitigate the deleterious effects of pollutants on skin, endogenous defense mechanisms, including two fundamental and closely interconnected biochemical pathways (AhR pathways and nuclear factor erythroid 2-related factor 2 [Nrf2]), are activated. The Nrf2 pathway plays an important role in skin homeostasis and repair [22, 23]. When we investigated the effect of InoO on the Nrf2 pathway, InoO promoted neither the expression nor the nuclear translocation of Nrf2 (data not shown). This indicates that InoO plays a role only in the AhR pathway but not in the Nrf2 antioxidant responses activated by particulate matters. Neither inositol nor oligo shows inhibitory effects on UPM activated AhR and Nrf2 pathway. From these results, we can hypothesize that the molecular structure of InoO may inter-fere with the binding of oxidative ligands, such as PAHs and dioxins, to AhR; how-ever, this requires further investigation.

CONCLUSION

In this report, we present the effects of a newly developed inositol derivative, InoO, on skin barrier and hydration. InoO im-proves skin hydration by promoting the

(Figure 10b), InoO significantly blocked the AhR translocation to the cell nucleus, while inositol and oligo did not. This in-dicates the possibility that the molecular structure of InoO may interfere with the binding of oxidative ligands such as BaP and dioxins to AhR.

Our experimental data illustrate that InoO specifically inhibits the nuclear transloca-tion of AhR, which sequentially induces the generation of CYP1A1 / B1, inflam-matory mediators, and ROS (Figure 11). Pretreatment with InoO before UPM ex-posure clearly inhibited the expression of CYP1A1 / B1 and the generation of IL-1a, PGE2, and ROS. Hashimoto-Hachiya et al. reported that the expression of epidermal differentiation markers such as filaggrin and loricrin required the nuclear trans-location of AhR, but that of involucrin is independent of AhR [21]. Figure 3 and Figure 6 show that InoO clearly in-

Figure 6 IHC staining for epidermal differentiation markers in human reconstructed skin under

UPM exposure conditions. The skin was pretreated with 1% InoO, inositol, and oligo 24 h before

UPM exposure. UPM (-), negative control, UPM (+), vehicle treatment before UPM exposure.

N.C.: negative control. Representative images of a) Filaggrin, b) Loricrin, and c) Involucrin.

Bar: 10 μm. d) Quantitative image analysis for the expression of each differentiation markers.

N = 3. P† < 0.05, P†† < 0.01 vs N.C., and P*< 0.05, P** < 0.01 vs control.

Table I Inhibitory effect of InoO in the gene expression of inflammatory mediators on AhR pathway

Table I. Inhibitory effect of InoO in the gene expression of inflammatory mediators on AhR pathway.

Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D.UPM ( - ) 1.000 0.161 1.000 0.208 1.000 0.036 1.000 0.212 1.000 0.267 1.000 0.175 1.000 0.085UPM ( + ) 7.464†† 0.642 0.976 0.164 1.737† 0.191 5.734† 1.752 43.479†† 0.751 689.781†† 56.226 2.591†† 0.054

InoO 5.250* 0.278 0.610* 0.125 1.245* 0.118* 3.294 0.676 31.068** 1.286 413.955** 72.792 1.401** 0.115† P<0.05, †† P<0.01,vs UPM ( - )* P<0.05, ** P<0.01,vs UPM ( + )

IL-8 COX-2CYP1A1 CYP1B1 IL-1a IL-1b IL-6


expression of PGs and GAGs but not dif-ferentiation markers. The properties of InoO on GAGs and PGs were maintained even under UPM exposure conditions, suggesting that the expression of PGs and GAG-related proteins is independent from the AhR pathway, unlike that of dif-ferentiation markers such as filaggrin and loricrin. However, the mechanism underly-ing the activation of PG expression by InoO remains to be elucidated.

To the best of our knowledge, this is the first study to introduce InoO-inhibited ROS generation and inflammation mediated by AhR activation in keratinocytes. More-over, we report that InoO induced PG expression, which resulted in skin barrier improvement under UPM exposure condi-tions. However, the expression of PGs and GAG-related proteins seems to be inde-pendent of the AhR pathway, suggesting that InoO must affect both the regula-tion of PG expression and AhR-related in-flammatory responses. Thus, InoO can be used to elucidate the mechanism of AhR pathway and PGs generation pathways. It can also be used as a new anti-pollution cosmeceutical product. References

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Figure 10 Immunofluorescence staining for

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The cells were treated with 0.001% InoO,

inositol, and oligo 24 h before UPM exposure.

UPM (-), negative control, UPM (+), vehicle

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Representative images of AhR localization

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CYP1A1CYP1B1

AhR

AhR

ARNTAhR

ARNT

GenesARNT

AhRmRNA

ROS DNA damage

Inflammatory Cytokines

Nc

IL1-α,IL-1β, IL-6 IL8, PGE2 etc.

NRF2

Nqo1

Anti-oxidative PathwayAhR

BenzopyreneDioxins

Anti-oxidative Ligands

Oxidative Ligands

Flavonoids, Ketoconazole

InoO

Differentiation MarkersFilaggrin, Loricrin etc.

Cell membrane

Figure 11 Schematic of InoO inhibitory effects on AhR oxidative pathway.

Various external and internal ligands including dioxins, flavonoids, and metabolites bind to and

activate AhR on cell membrane. Upon ligand binding, ligand–AhR protein complex translocates

into the nucleus (Nc) and then binds to the genes and induces the transcription of responsive genes,

such as CYP1A1 and B1, and several differentiation markers.

Ligand metabolism process by CYP1A1 / B1, produces a large number of ROS.

This ROS generation is closely related to various cellular responses, such as cytokine production

and DNA damage. Meanwhile, anti-oxidative phytochemical ligands, such as flavonoids and

ketoconazole, bind to AhR and induce CYP1A1 / B1 upregulation.

However, they are also good activators of antioxidant master transcription factors, namely, Nrf2.

By inhibiting the translocation of ligand–AhR protein complex, InoO blocks the AhR induced oxidative

pathway and results in ROS generation. InoO possibly structurally interferes ligand binding to AhR.

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Corresponding AuthorYuko Saeki

Institute for Integrated Product Development

Showa Denko K.K.TokyoJapan

[email protected]

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A Global and Powerful Approach to Circumvent the Harmful Effects of Blue Light and IR-A Irradiation on the SkinValérie Bicard-Benhamou, Marina Lefort, Lilia Heider, Jutta zur Lage, Silke Hornung, Heike Hanau, Alexander Kielbassa, Christophe Carola, Joerg von HagenMERCK KGaA, Frankfurter Strasse 250, 64293 Darmstadt, Germany

Keywords: Blue light, HEVL, IR-A, human explant protection, biochemical markers, first-line defense, second-line defense

This publication was originally presented as a podium presentation at the 25th IFSCC conference in Milan, Italy, September 30 - October 2, 2019.

complementary approach mainly target-ing second defense line, meaning reduc-ing damage that nevertheless may occur in the skin considering an over-exposure to HEVL (blue light) and / or IR-A radiations.

Cosmetic ingredients from different ori-gins were selected: two ingredients known for their antioxidant properties: a synthe-tic one, Bis-ethylhexyl hydroxydimethoxy

INTRODUCTION

Appropriate and controlled doses of UV, HEVL and IR may be beneficial for solving different skin problems; however, protec-tion in all these ranges is needed. It often remains in mind that photoprotection is mainly a matter of UV protection. However, all solar radiation leads to the formation of radical oxygen species (ROS), and an ex-cess of free radicals in the skin contributes to premature skin aging and wrinkling. Visible light, and especially high-energy visible light (HEVL, 400 - 500 nm), notably triggers pro-inflammatory cytokines, the expression of different matrix metallopro-teinases, and / or oxidation of proteins. All these markers play a role in accelerating skin aging (e.g. sagging skin, inflammation, wrinkles, and pigmentation disorders). In comparison, infrared (IR) radiation has the lowest energy level, but its contribu-tion to the solar spectrum reaching the human skin is about 45 % [1]. Therefore, the biological impact on the skin may be strong [2]. IR light, and particularly IR-A (700 - 1400 nm), induces significant free radicals in the dermis and diminishes the skin’s antioxidant capacity [3]. Additionally, at high doses, harmful effects are reported [4]. IR radiation has been shown to alter the collagen content of the dermal extracellular matrix (ECM), not only by causing increased expression of the collagen-degrading en-zymes MMPs, but also by decreasing de novo collagen synthesis [4].

There are already powerful solutions to achieve light protection beyond UV, nota-bly in the HEVL and IR-A ranges, considering the first-line defense strategy that means preventing any radiation from penetrating the skin. To achieve this first-line defense protection appropriate combina-tions of specific titanium dioxide UV filters and mica-based functional fillers could be used [5, 6]. In this work, we present a

Abstract

Research ObjectiveThe effect of solar radiation in the blue light or high energy visible light range, as well as in the longer wavelengths such as infrared, is an increasingly important topic of research. Indeed, the potential damage to the skin is a real issue for the skin. There is therefore a need to iden-tify efficient cosmetic solutions enabling protection of the skin beyond UV range.

Experimental methodsWe present here facts showing the per-formance of different ingredients on hu-man explants after irradiation with blue light and IR-A in 2 different ex-vivo stu-dies. Ingredients of different origin were applied to human explants. An emulsion containing a titanium dioxide UV filter grade (first-line defense equivalent to a physical barrier) was tested, as well as

second-line defense ingredients: 2 anti-oxidants, the natural extract Phyllanthus Emblica fruit extract and the synthetic Bis-ethylhexyl hydroxydimethoxy ben-zylmalonate, and a natural cell protector Ectoin. These ingredients are described as second-line defense ingredients (equivalent to a biochemical protection barrier in the skin). Finally, the perfor-mance of the self-tanner dihydroxyace-tone was also investigated. To assess the level of efficacy of the ingredients, different and representative biochemical markers were immunostained.

Main observationsOur findings showed that all tested products had very good performance on several biochemical parameters and in both wavelengths ranges. Photoag-ing damage may therefore be slowed down and the onset of aging may be delayed.


benzylmalonate (HDBM) and a natural extract, Phyllanthus Emblica Fruit Extract. Furthermore, we selected a natural cell protection agent: Ectoin. Validating the link between a first-line defense strategy and the impact on biochemical markers after a stress with HEVL / IR-A was also in focus. Therefore, a fourth ingredient, in the form of an emulsion containing a ti-tanium dioxide UV filter grade (UV-TiO2 /SiO2), was tested. Finally, we included a compound that may act on both the first and second defense line: the well-known self-tanner dihydroxyacetone (DHA). We tested DHA knowing that dark-colored materials absorb in the visible range, and that a certain photo-protection ability of topically applied DHA had been pre-viously briefly reported in the UV-A and HEVL ranges [7 - 8]. We also investigated the longer wavelengths (infrared range). The assessment of the protective effect of the different ingredients against HEVL and IR-A radiation was carried out on living human skin explants. To obtain a comprehensive picture of the mechanism of action and performance of the differ-ent ingredients, multiple parameters were tested. Immunostaining of tropoelastin and MMP-1 was investigated for explants irradiated with IR-A and the expression of MMP-1, 8-OHdG, Opsin-3 and oxidized protein level were assessed for explants irradiated with HEVL.

EXPERIMENTAL

1. Products tested

Ectoin (1% in water)Ectoin was first discovered in Ectothio-rhodospira halochloris, a halophilic bacte-rium that survives and grows in extreme conditions and high temperatures in salt lakes, saline soils, sea water and saline deserts. Meanwhile, other halophilic bac-teria species, such as Halomonas elongata, have been found to generate Ectoin as a protective mechanism. Ectoin is a multi-talented natural ingredient [9 - 10] nota-bly showing immune system protection, cell protection, super moisturizing: for example, it notably induces moisture ac-cumulation in skin that is still detectable 7 days after application [35]. In addition, Ectoin protects against protein oxidation

induced by dehydration stress (27), anti-aging (e.g., roughness reduced by 53% and wrinkle volume reduced by 23% after 4 weeks treatment on the skin (36)) and has anti-pollution properties. Ectoin is an ingredient from Merck KGaA, Darmstadt, Germany.

Phyllanthus Emblica fruit extract (Emblica) (0.5% in water)Emblica is obtained from Phyllanthus Emblica officinalis, also known as Indian gooseberry, a sustainable resource that is well known in Ayurvedic medicine. It has long-lasting antioxidant properties: a DDPH test showed how Emblica retains full antioxidant activity after 12 months at 45 °C, whereas other ingredients (vi-tamin C, rosemary extract, pine antioxi-dant, vitamin E) failed [11]. Emblica also shows excellent singlet oxygen/superoxide anion/hydroxyl radical quenching abili-ties (against 1O2 : IC50(Emblica) 61 μg / ml, IC50(Trolox) 84 μg / ml; against

.O2- :

IC50(Emblica) 12 μg / ml, IC50(Vit C) 26 μg / ml, IC50(Trolox) 360 μg / ml), as well as strong metal-chelating properties in comparison with other chelators tested (for instance, vitamin C, pine antioxidant, green tea) [12, 26]. Emblica fruit extract is an ingredi-ent from Merck KGaA, Darmstadt, Germany.

Bis-ethylhexyl hydroxydimethoxy benzylmalonate (HDBM) (1% in ethanol)It is a pure, stable, and transparent cos-metic oil that can be directly added to the oil phase of an emulsion and remains stable at higher temperatures (up to 80 °C) and over a broad pH range [4 - 8].

HDBM is a powerful and long-lasting antioxidant. It is notably as stable as α-tocopherol acetate and as active as ascorbic acid simultaneously (DPPH test). It has been proven to combine high efficacy with lasting stability in cosmetic emul-sions. It can provide four equivalents of hydrogen for radical scavenging purposes (one phenolic, two benzylic, one malonic). The oxidation product of HDBM itself has antioxidant properties and its activity is therefore maintained over time [13]. As a result of oxidation, the single bond (HDBM) can be converted into a double

bond (HDBMox), which then possesses the ability to quench singlet oxygen, thus ideally completing the system [14]. HDBM is an ingredient from Merck KGaA, Darm-stadt, Germany.

Titanium dioxide UV filter grade (INCI: titanium dioxide, silica) (acronym for the rest of the text UV-TiO2 / SiO2) used in 3 % o/w emulsion. UV-TiO2 / SiO2 is a 100 % inorganic UV filter, transparent on the skin, with an efficient broad-spectrum efficacy in the UVA/B range. It also pro-vides first-line defensive efficacy against HEVL, visible and IR radiation, as shown in previous experiments by Rozman et al. [6]. UV-TiO2 / SiO2 is an ingredient from Merck KGaA, Darmstadt, Germany.

Dihydroxyacetone (DHA) (2% in water)DHA of high purity, is a self-tanning agent produced by a biotechnological process providing strong skin coloration efficacy. DHA reacts with the proteins and amino acids of the horny layer of the skin (Mail-lard reaction), in which resulting polymers have the well-known intense brown color. DHA is an ingredient from Merck KGaA, Darmstadt, Germany.

2. Experiments investigating HEVL impact on human skin explants

Explant preparationHuman skin explants were prepared in an abdominoplasty from a 45-year-old Caucasian woman (skin phototype II). The explants were kept in survival in a spe-cific culture medium (BIO-EC’s Explants Medium) at 37 °C in a humid, 5 % CO2 atmosphere. Three explants per treatment were used for the experiments. On day 0, day 3 and day 4 (before blue light irra-diation), the tested products were applied topically. The blank batch did not receive any treatment except the renewal of the culture medium. On day 4, the explants of the relevant batches were irradiated with the Solarbox® blue light using a dose of 84.96 J/cm² for 4 hours.

Immunostaining of Opsin-3Opsin-3 is expressed in several tissues, in-cluding skin. It serves as a sensor for blue light in melanocytes and therefore func-tions as a sensor for visible light pigmenta-

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tion. Opsin-3 appears to be a new potential target for regulating melanogenesis [15].

Opsin-3 immunostaining was performed on paraffin sections with a polyclonal anti-Opsin-3 antibody (Novus Biologicals, ref. NBP2-55726) diluted at 1:50 in PBS-BSA 0.3 %-Tween 20 at 0.05 % for 1 hour at room temperature, using a Vectastain Kit Vector amplifier system avidin/biotin, and revealed by VIP, a violet substrate of peroxidase (Vector, ref. SK-4600). The immunostaining was performed using an automated slide processing system (Au-tostainer, Dako) and assessed by micro-scopic observation. Three pictures were taken per explant, meaning 9 pictures were available for each treatment.

8-OHdG immunostaining8-hydroxydesoxyguanosine (8-OHdG) reflects the degree of oxidative damage to DNA and contributes to signs of pre-mature skin aging [16]. 8-OHdG immu-nostaining was performed on paraffin sections with a monoclonal anti-8-OHdG antibody (Gentaur, ref. 50-MOG, clone N45-1) diluted at 1:100 in PBS-BSA 0.3 % at 0.05 % overnight at room temperature, using a Vectastain Kit Vector amplifier sys-tem avidin/biotin, and revealed by VIP, a violet substrate of peroxidase (Vector, ref. SK-4600). The immunostaining was per-formed manually and assessed by micro-scopic observation, with semi-quantifica-tion by image analysis. Three pictures were taken per explant, meaning 9 pictures were available for each treatment.

Oxidized proteins immunostainingAccumulation of damaged proteins is one of the most fundamental features of ag-ing. Carbonylated protein content is the usual general biomarker of severe oxi-dative protein damage/inactivation. The oxidized proteins were stained on frozen sections after pre-incubation with DNPH (2,4-dinitrophenylhydrazine, Millipore, ref. 90448) and incubation with an anti-DNP antibody (Millipore, ref. 90451) di-luted at 1:250 in PBS, BSA 0.3 % for 1 h at 37 °C, with a biotin/streptavidin-ampli-fying system and revealed with VIP, a violet substrate of peroxidase (Vector, ref. SK-4600). The immunostaining was assessed by microscopic observation. Three pictures

were taken per explant, meaning 9 pic-tures were available for each treatment.

MMP-1 immunostainingMatrix metalloproteinases (MMPs) are peptidase enzymes responsible for the degradation of ECM components. Irradia-tion of human skin equivalents with HEVL induced production of MMP-1. HEVL ex-posure therefore contributes to signs of premature skin aging [17].

MMP-1 immunostaining was performed on paraffin sections with a polyclonal anti-MMP-1 antibody (Sigma, Ref M4696) di-luted at 1:100 in PBS-BSA 0.3 %-Tween 20 at 0.05 % for 1 h at room tempera-ture, using Vectastain Kit Vecot amplifier system avidin/biotin and revealed by VIP, a violet substrate of peroxidase (Vector Laboratories, ref SK-4600). The immuno-staining was performed using an automat-ed slide processing system (Autostainer, Dako) and assessed by microscopic ob-servation. Three pictures were taken per explant, meaning 9 pictures were available for each treatment.

Known impacts of blue light exposure on these markers, as well as their desired modulation on treatment with the differ-ent products, are summarized in Table I.

Image analysis methodImage analyses were performed on all images in each batch, according to the following method using Cell^D software. For each batch of explants, the percentage of the region of interest covered by the staining (stained surface percentage) was determined by image analysis (see raw va-lues in Table II-VII). We thus expressed all our results in percentage terms relative to control and/or irradiated control.

Statistical analysisStatistical analysis was performed using Student t-test.

3. Experiments investigating infraredA light (IR-A) on human skin explants

Explant preparationHuman skin explants were prepared on an abdominoplasty from a 61-year-old Caucasian woman (Fitzpatrick skin pho-

totype II-III). The explants were kept alive in a specific culture medium (BIO-EC’s Explants Medium) at 37 °C in a humid, 5 % CO2 atmosphere. On day 0, day 3, day 4 (3 hours before IR irradiation), day 5 and day 6, the tested products were applied topically, followed by 10 minutes of drying. The blank batch did not receive any treatment except the renewal of the culture medium. The culture medium was half-renewed (1 ml / well) on day 3 and day 5. Irradiation of the explants was done using an infrared lamp (Dr Fischer 1000W, 235V 2500 K; 760 - 3000 nm), for 5040 seconds (720J / cm2) with an intensity of 143 mW / cm2. According to Weiss et al. [32], the NIR irradiance of the solar spec-trum is 72 mW / cm2. So, the device is ap-proximately twice as powerful as the sun: it takes half the time of the sun to deliver the same dose of NIR. The dose 720 J / cm2

was determined according to Schroeder et al [19]. According to Barolet et al. [33], this dose of 720 J / cm2 can be described as medium. It was delivered in 1 hour and 24 minutes, which corresponds to a solar exposure of 2 hours and 48 minutes.

To avoid temperature increases, and there-fore heat damage known to occur in the IR-B and IR-C ranges [18], we focused on the IR-A range, and water filter and IR760 filter were also used to achieve the follow-ing IR range 700 - 1150 nm. The explants’ temperature was measured before and after IR irradiation. The explants’ tempera-ture was maintained at 37 °C (max 39 °C) by a specially adapted refreshing system.

MMP-1 immunostainingSee above in the HEVL section. It is known from literature that the irradiation of hu-man skin equivalents with infrared light induces production of MMP-1 [19].

TropoelastinElastic fibers are found in the ECM of con-nective tissue, providing elasticity and re-silience to tissues.

Due to extreme insolubility of elastin, we focused on its soluble precursor, tropo-elastin.

Under a single and medium IR-A irradia-tion, elastic fibers are degraded and expres-


sion of tropoelastin units is reduced [20]. Tropoelastin immunostaining was carried out on frozen sections with a monoclonal anti-tropoelastin antibody (Chemicon, ref MAB2503, clone 10B8) diluted at 1:200 in PBS-BSA 0,3 % overnight at 4 °C, using Vectastain Kit Vector amplifier system avi-din / biotin and revealed by AF488 (Lifetech-nologies, ref. A11001). The nuclei were counterstained by propidium iodide. The immunostaining was assessed by micro-scopic observation. Three pictures were taken per explant, meaning 9 pictures were available for each treatment.

Known impacts on IR-A exposure on these 2 markers, as well as their desired modula-tion on treatment with the different prod-ucts, are summarized in Table I.


1. Results of the ex vivo study investigating HEVL impact on human skin explants

All raw data have been summarized in Tables II-VII.

Immunostaining of oxidized proteinsOxidized proteins were stained in the pa-pillary dermis, and the results showed the following outcome:

tion containing UV-TiO2 / SiO2 (- 47 %**; p < 0.01), Emblica (- 44 %**; p < 0.01), Ectoin (-59 %**; p < 0.01) and DHA (- 61%**; p < 0.01) (see Figure 1 and raw data on Table V). In the case of HDBM (solubilized in ethanol), no comparison with irradiated ethanol control explant is available. However, the intensity of the oxidized protein immunostaining vs the ir-radiated control could be very significantly decreased (data not shown).

Immunostaining of 8-OHdGNuclear and mitochondrial DNA oxidation occur most readily in guanine residues owing to the high ionization potential of this base. 8-hydroxydesoxyguanosine (8-OHdG) is one of the predominant forms of free-radical-induced oxidative lesions in humans [16]. The interaction of hydroxyl radicals with the double bond at the C-8 position of the guanine base leads to the production of 8-OHdG. This stable oxida-tive modified DNA product has extensively been used to reflect the degree of oxida-tive damage to DNA [21].

After HEVL exposure, the formation of 8-OHdG is significantly increased by 37 %* (p < 0.05) in the epidermis com-pared with the control batch at day 5. Upon exposure of the human explants to HEVL and application of the products,

The blue light irradiation induced a visible and very significant increase of 95 %** (p < 0.01) of oxidized protein formation in the papillary dermis relative to the control batch at day 5. Upon exposure of the hu-man explants to HEVL, and application of the products, the intensity of the oxidized protein immunostaining was visibly and significantly reduced for all products vs the irradiated untreated control: the formula-

– –

Table I Impact of blue light and IR light on literature-known biomarkers

and envisioned modulation upon treatment with the tested products.

Figure 1 Immunostaining of oxidized proteins. Representative pictures at day 5.

(a) control; (b) control + HEVL; (c) Ectoin + HEVL: - 59 %** vs (b); (d) DHA + HEVL: - 61 %** vs (b);

(e) formulation cont. UV-TiO2 / SiO2 + HEVL: - 47 %** vs (b); (f) Emblica + HEVL: - 44 %** vs (b).

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it could be observed that the 2 follow-ing products were able to decrease the levels of 8-OHdG compared with the irradiated control at a similar level: the formulation containing UV-TiO2 / SiO2 (-13 %*, p < 0.05), Emblica (-15 %*, p < 0.05). Ectoin did not show any im-pact on 8-OHdG levels vs the irradi-ated control. (Figure 2 and Table IV). In the case of HDBM (solubilized in etha-nol), no comparison with irradiated etha-nol control explant was available. How-ever, the intensity of 8-OHdG immunos-taining vs the irradiated control could be significantly decreased (data not shown).

Immunostaining of Opsin-3Opsins are members of the guanine nucle-otide-binding protein (G protein)-coupled receptor superfamily. In addition to the visual opsins, mammals possess several photoreceptive non-visual opsins that are expressed in extra-ocular tissues. This gene, Opsin-3, is expressed in several tis-sues, including the skin. It has been re-cently shown that melanocytes sense blue light and regulate pigmentation through Opsin-3 [15]. HEVL exposure induced a significant increase of 72 %** (p < 0.01) in Opsin-3 expression in the epidermis vs control at day 5. The strongest decrease in Opsin-3 after irradiating the explants with

provided a decrease in immunostaining of 23 % # (p < 0.1) vs irradiated control and Emblica, a decrease of -23 %* (p < 0.01) vs irradiated control.

The synthetic antioxidant (HDBM) did not show any significant decrease vs irradiated control in this test. See also raw data in Table III.

Immunostaining of MMP-1Matrix metalloproteinases (MMPs) are a family of peptidase enzymes responsible for the degradation of extracellular matrix components, including collagen I, gelatin, fibronectin, laminin and proteoglycan. HEVL exposure notably induces MMP-1 expression, and therefore contributes to signs of premature skin aging [17]. HEVL irradiation induced a significant increase of 77 %* (p < 0.05) of MMP-1 expres-sion in the epidermis vs control at day 5. The intensity of MMP-1 immunostain-ing is visibly and significantly decreased for all products vs the irradiated control. Nonetheless, HDBM showed the strongest performance and was able to decrease by 31 %** (p < 0.01) MMP-1 expression in the epidermis in comparison with the ir-radiated ethanol control and correspond-ing to -36 %** vs irradiated control. The other products also decreased MMP-1 lev-els vs irradiated control: the formulation

HEVL and treating them with the products was observed for the emulsion containing 3 % UV-TiO2 / SiO2 (-37 %** vs irradiated control; p < 0.01). The reduction in immu-nostaining intensity is significantly visible as shown in Figure 3. With Ectoin treat-ment and HEVL irradiation, a decrease in the Opsin-3 level of 24 %** (p < 0.01) vs irradiated control was observed. DHA

Figure 2 Immunostaining of 8-OHdG. Representative pictures at day 5.

(a) control; (b) control + HEVL; (c) Ectoin + HEVL: n.s. reduction vs (b);

(d) DHA + HEVL: n.s. reduction vs (b); (e) formulation cont. UV-TiO2 / SiO2 + HEVL: -13 %* vs (b);

(f) Emblica + HEVL: -15 %* vs (b).

Figure 3 Immunostaining of Opsin-3. Representative pictures at day 5.

(a) control; (b) control + HEVL; (c) Ectoin + HEVL: - 24 %** vs (b); (d) DHA + HEVL: - 23 %# vs (b);

(e) formulation cont. UV-TiO2 / SiO2 + HEVL: -37%** vs (b); (f) Emblica + HEVL: -23 % vs (b).


containing 3 % UV-TiO2 / SiO2 (-65 %**; p < 0.01). The natural antioxidant also showed a good performance vs irradiat-ed control: Emblica (-48 %**; p < 0.01). Finally, Ectoin moderately decreased MMP-1 expression in IR-A irradiated explants (-22 %*, p < 0.05). Pictures are shown in Figure 5 and raw data in Table VI. In the case of HDBM (solubi-lized in ethanol), no comparison with irra-diated ethanol control explant was avail-able. However, the intensity of MMP-1 immunostaining vs the irradiated control could be very significantly decreased (da-ta not shown).

Immunostaining of TropoelastinElastin is the major component (90 %) of the elastic fibers [23 - 24]. It is a protein of the connective tissue responsible for the elastic properties of the skin. It is initially synthesized as its precursor, tro-poelastin. The impact of infrared radia-tion on tropoelastin expression at mRNA and protein levels has been investigated [18, 25].

The staining of tropoelastin in the papillary dermis showed the following results:An IR-A irradiation of the explants induced a significant decrease of 45 %** (p < 0.01) in tropoeleastin expression in the papillary dermis at day 7.

containing 3 % UV-TiO2 / SiO2 (-20 %**; p < 0.01), Emblica (-23 %**; p < 0.01), Ec-toin (-20 %**; p < 0.01) and DHA (-23 %*; p < 0.05). See pictures in Figure 4 and raw data in Table II.

2. Results of the study investigatingIR-A impact on human skin explants

All raw data have been summarized in Tables VI and VII.

Immunostaining of MMP-1Matrix metalloproteinase-1 (MMP-1) is an enzyme also known as interstitial col-lagenase and fibroblast collagenase. It was previously shown that IR-A radiation elicits a retrograde signaling response, which is initiated in the mitochondria through gen-eration of reactive oxygen species (ROS) that originate from the mitochondrial elec-tron transport chain. One major biological consequence of this retrograde signaling response is the increased expression of ma-trix metalloproteinase (MMP-1) [22].

An IR-A irradiation of the explants induced a clear increase in MMP-1 expression in

the epidermis (+208 %**, p < 0.01). Up-on IR-A exposure, two products in par-ticular showed good ability to decrease MMP-1 levels vs irradiated control: DHA (-67 %**; p < 0.01) and the formulation

– –

Table II Surface percentage positive to MMP-1 in the epidermis – HEVL Study

Figure 4 Immunostaining of MMP-1. Representative pictures at day 5.

(a) control; (b) control + HEVL; (c) Ectoin + HEVL: - 20%** vs (b); (d) DHA + HEVL: - 23 %* vs (b);

(e) formulation cont. UV-TiO2 / SiO2 + HEVL:-20 %** vs (b); (f) Emblica + HEVL: -23 %** vs (b);

(g) HDBM + HEVL: - 31 %** vs (h), (h) control + ethanol+ HEVL.

– –

–

–

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Table III Surface percentage positive to Opsin-3 in the epidermis – HEVL Study

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–

–

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Table IV Surface percentage positive to 8-OHdG in the epidermis – HEVL study

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transmission data measurements, how specific titanium dioxide UV filters prevent radition from penetrating the skin [6]. In the light of our new results, we were able to make a link between first-line defense (physical barrier) and second-line defense (protection of biochemical endpoints).

HDBMIn the case of the HEVL study, we show the results for the MMP-1 biomarker vs irradiated ethanol control: -31 %**. For the other oxidized proteins and 8-OHdG biomarkers, there is no compar-

notably help to reduce the risk of blue-light-induced pigmentation (see Opsin-3 results), and in the case of the explants exposed to IR-A light, we could show a complete protec-tion of tropoelastin (+116 % vs control + IR-A) and thus a preservation of elastic fibers in the ECM. Knowing that HEVL and IR-A radiation both induce ROS formation, the use of the formulation containing 3 % UV-TiO2 / SiO2 may minimize oxidative damage and there-fore prevent premature aging.

These results also confirm previous inves-tigations, which showed, by means of

The formulation containing 3 % UV-TiO2 / SiO2 induced the strongest, and very significant, tropoelastin increase of 116 %** (p < 0.01) relative to the irradiated control. Three other ingredients also showed very good ability to increase tropoelastin expres-sion: Emblica (+79 %**, p < 0.01), HDBM (+86 %** vs irradiated ethanol control, p < 0.01; + 63 %** vs irradiated control, p < 0.01), DHA (+57%**; p < 0.01), and fi-nally Ectoin (+ 36 %*, p < 0.05) in a more moderate manner. Pictures are shown in Figure 6 and raw data in Table VII.

3. Discussion of both studies

UV-TiO2 / SiO2

In both studies investigating the impact of HEVL and IR-A radiation, all the biomark-ers tested were strongly and significantly impacted in a positive manner relative to the non-treated irradiated control ex-plants (control + HEVL or control + IR-A).UV-TiO2 / SiO2 (INCI titanium dioxide, sili-ca) formulated at 3 % in an o/w emulsion is an inorganic material that prevents/min-imizes free radical formation by absorb-ing, reflecting, and scattering properties of solar radiation, thus acting as first-line defense, and de facto provides a physical barrier. Radiation penetrating the skin is highly reduced. The studied biomarkers are known to be modulated by exposure to IR-A and HEV light (Table I).

In fact, the very limited amount of radiation reaching the skin after topical application of the emulsion containing UV-TiO2 / SiO2 notably explains the positive modulation of the biomarkers compared with irradi-ated control explants. The base emulsion used as placebo has been investigated in previous transmission / reflection / absorp-tion measurements in both HEVL and IR-A ranges and showed no protective effect in both wavelength ranges compared with the same emulsion but containing UV-TiO2 / SiO2 [6]. We therefore made the as-sumption that this placebo emulsion would not provide any protective effect in the cur-rent ex vivo studies.

We can conclude that a strong explant pro-tection may be achieved at a multi-parameter level for both wavelength ranges. The for-mulation containing 3 % UV-TiO2 / SiO2 may

Figure 5 Immunostaining of MMP-1. Representative pictures at day 5.

(a) control; (b) control + IR-A; (c) Ectoin + IR-A: -22%* vs (b); (d) DHA + IR-A: -67%** vs (b);

(e) formulation cont. UV-TiO2 / SiO2 + IR-A: - 65 %** vs (b); (f) Emblica + IR-A: - 48 %** vs (b).

– –

–

–

–

Table V Surface percentage positive to oxidized proteins in the dermis – HEVL study

– –

–

–

–

Table VI Surface percentage positive to MMP-1 in the epidermis – IR-A study


ison available vs the irradiated ethanol control. Nonetheless the comparison vs irradiated control showed good results (data not shown).

In the IR-A study, we show the results for tropoelastin indicating positive changes from treatment with HDBM and after IR-A exposure vs control + ethanol + IR-A: + 86 %**. For MMP-1, there is no com-parison available vs the irradiated ethanol control. Nonetheless, the comparison vs irradiated control explant showed good results (data not shown).

As stated above, IR-A and HEVL irradia-tions are oxidative processes, and we can explain the performance of HDBM by its strong radical neutralization power, as de-picted in Figure 7 and as described in the experimental section. Oxidative damage after HEVL and IR-A irradiation is therefore minimized, and human explants may be protected against premature aging.

treatment with the extract Emblica and af-ter blue light irradiation vs irradiated control explant: the oxidized proteins (- 44 %**), MMP-1 (- 23 %**), 8-OHdG (-15%*) and Opsin-3 (-23%*).

Investigated biomarkers indicate positive changes from explant treatment with Em-blica and after IR-A exposure vs irradiated control explant: (- 48 %** for MMP-1 and +79 %** for tropoelastin).

Emblica was previously shown to decrease MMP-1 levels at an in vitro level, and there-fore to protect most of the components of the extracellular matrix (ECM) [26]. This observation is further supported by our current results showing that Emblica is able to decrease MMP-1 expression in the presence of IR-A irradiation stress.

Emblica is a naturally powerful antioxidant, and its performance in these studies could

Figure 6 Immunostaining of tropoelastin. Representative pictures at day 7.

(a) control; (b) control + HEVL; (c) Ectoin + HEVL: +36 %* vs (b); (d) DHA + HEVL: + 57 %** vs (b);

(e) formulation cont. UV-TiO2 / SiO2 + HEVL: +116 %** vs (b); (f) Emblica + HEVL: +79%** vs (b);

(g) HDBM + HEVL: + 86 %** vs (h), (h) control+ ethanol+ IR-A.

–

Table VII Surface percentage positive to tropoelastin in the papillary demis – IR-A study

Figure 7: Structure and mechanism of action of HDBM

HDBM

HDBMox

R—Ofree radical

R—OH

.

[intermediate]R—Ofree radical

R—OH

.

OHO

O

O O

O

O

OO

O

O O

O

O

OHO

O

O O

O

O

HDBM

HDBMox

R—Ofree radical

R—OH

.


R—OH

.

HDBM

HDBMox

R—Ofree radical

R—OH

.R—Ofree radical

R—OH

.


R—OH

.R—Ofree radical

R—OH

.

OHO

O

O O

O

O

OO

O

O O

O

O

OHO

O

O O

O

O

OHO

O

O O

O

O

OO

O

O O

O

O

OO

O

O O

O

O

OHO

O

O O

O

O

Figure 7 Structure and mechanism of action of HDBM.

EMBLICAIn the case of the HEVL study, four biomark-ers were positively impacted by explant

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be largely explained by the broad antioxi-dative cascade of Emblicanin A and Embli-canin B, the main constituents of Emblica extract (see structures in Figure 8). Given that formation of free radicals can be re-duced, DNA damage,as well as premature aging, may be minimized. Considering the results in the IR-A study, we conclude that Emblica can protect elastic fibers and pre-serve the integrity of the ECM.

It is interesting to note that, despite their structural differences, the two antioxidants (HDBM and Emblica) showed similar per-formance profiles and positively modulated the same biomarkers connected with oxida-tive processes, albeit to differing degrees.

ECTOINIn the case of the HEVL study, three biomarkers were positively impacted by treatment with Ectoin after blue light irradiation vs control + HEVL: the oxidized proteins (- 59 %**; p < 0.01), MMP-1 (-20 %**; p < 0.01) and Opsin-3 (- 24 %**; p < 0.01).

The results obtained in oxidized proteins nicely confirm earlier results: Ectoin was indeed shown to reduce the carbonyl score and carbonylated protein expression in HaCat cells and human skin explants after exposure to HEVL [27].

Concerning MMP-1, earlier research also showed the photoprotective effect of Ectoin in the shorter wavelength range. Indeed, this substance could decrease MMP-1 expression in keratinocytes after exposure to UV-B [28].

Finally, we consider Opsin-3 modulation. This marker has recently attracted grow-ing attention in cosmetic research. Indeed, Regazetti et al [15] showed that blue light stimulated melanogenesis by acting di-rectly on melanocytes and via the Opsin-3 receptor.

In our experiments, the intensity of Op-sin-3 immunostaining was significantly increased in the control explant exposed to blue light, which fits with the experi-mental observations of Regazetti et al [15]. The explant treated with Ectoin and exposed to HEVL showed a visible and significant reduction in Opsin-3 immu-nostaining intensity.

Taking the results of Regazetti et al linking Opsin-3 up-regulation and melanogenesis into account, Ectoin may help reduce the risk of blue light-induced pigmentation.

In the case of the IR-A study, both in-vestigated biomarkers were positively impacted by treatment with Ectoin and after IR-A exposure vs control + IR-A. (- 22 %*; p < 0.05) for MMP-1 and + 36 %** (p < 0.01) for tropoelastin. To our knowledge, we show here for the first time that Ectoin also possesses a cer-tain level of photoprotection in the longer wavelengths (IR-A).

A very interesting aspect of these results is the finding that Ectoin, which does not have any antioxidant properties, is able to circumvent skin explant damage associated with oxidative processes in the blue light and IR-A ranges. Further

research is needed and is currently under way to fully understand these results and the specific mode of action and role of Ectoin.

DIHYDROXYACETONE

Within the HEVL study, three biomarkers were positively impacted by explant treat-ment with DHA and blue light irradiation vs irradiated control explants: the oxidized proteins (- 61 %**), MMP-1 (- 23 %**; p < 0.01) and Opsin-3 (- 23 %#; p < 0.1).

DHA is a well-established self-tanner in the cosmetic industry [29]. DHA reacts with proteins and amino acids in the horny layer of the skin following the well-known Maillard reaction, in which the resulting polymers (melanoidins) give the skin a brownish hue via a reaction route which has not yet been fully clarified. This reaction is complete after approximately 4 - 6 hours. The tan achieved in this way cannot be washed off and is removed only with normal skin desquamation.

DHA also shows some moderate photo-protective properties. In the UV-B range, topically applied DHA offers a modest SPF of 2 - 3 in humans lasting from days to weeks (given that it is bound to the skin) [30]. The melanoidins (brown pigments) that are formed after topical application of DHA have been shown to exhibit ab-sorption properties similar to those of melanin and provide moderate sunlight protection. Moreover, topical application of DHA gives the skin a certain photo-protection in the UV-A range [8, 31], as well as in the HEVL range. Our results confirm the earlier observations made by Johnson [7].

Nonetheless, to the best of our knowledge no data on the infrared range have previ-ously been published, and this led us to investigate the impact of topically applied DHA in this range.

The biomarkers investigated indicate pos-itive changes from explant treatment with DHA and after IR-A exposure vs irradiated control explant: - 67 %** (p < 0.01) for MMP-1 and + 57 %** (p < 0.01) for tro-poelastin. This unexpected outcome led

Figure 8 Main constituents of Emblica extract.


us to measure the IR absorption spectrum of a blend of DHA (10-3 M) mixed with the amino acid lysine (10-3 M) in glycerol to better understand these results. This blend mimicked the Maillard reaction on the skin (skin liquid model [32]) and we could observe a clear absorption in the IR-A range (results not shown). It is none-theless important to mention that this absorption is very low in comparison with common UV filter absorptions. These re-sults offered the first hints to explain the ability of DHA to mitigate damage caused to human skin explants by IR-A irradia-tion. Further research is currently under way to fully understand the mechanism of action in these wavelengths.

Topical application of DHA works at a multi-parameter level for both HEVL and IR-A ranges and minimizes oxidative damage. DHA may help reduce the risk of blue-light-induced pigmentation and preserve components and integrity of the ECM, such as collagen and elastic fibers.

CONCLUSION

This paper summarizes results investigat-ing the effects of several different cos-metic ingredients on human skin explants after exposure to HEV and near infrared A (IR-A) light. Ingredients with different ori-gins and different mechanisms of action were selected: two strong antioxidants, one of synthetic origin (HDBM), the sec-ond of natural origin (Emblica). A natural substance with cell-protecting properties but without any antioxidant properties was evaluated (Ectoin). Understanding the link between a first-line defense strat-egy and second-line biochemical defense strategy after stress with HEVL/IR-A was also investigated. Therefore, an emulsion containing a specific titanium dioxide UV filter providing a physical barrier to IR-A and HEVL rays was tested. Finally, we investigated the well-known self-tanner dihydroxyacetone (DHA). Different bio-chemical markers known to be modu-lated after an exposure to HEVL and / or IR-A were investigated.

After IR-A and HEVL irradiation, the for-mulation containing the selected titanium dioxide UV filter showed excellent protec-

tion of the human explants. All evaluated biochemical markers were modulated in a positive manner compared with the ir-radiated control explants. Therefore, the link between first-line defense (shown in earlier investigations) and second-line de-fense, that is, the impact on biochemical markers, could be validated. The antioxi-dants HDBM and the natural extract Em-blica also showed excellent performance in relation to the tested biomarkers. It is interesting to note that Ectoin, which neither provides a physical barrier nor is an antioxidant, helps to mitigate damage to skin explants after different irradiation stresses. Finally, DHA showed very good results in both studies. Further research is continuing to get a full picture of the underlying mechanism of action in the case of the infrared study, which, to the best of our knowledge, showed for the first time the protective potential of DHA in the NIR range.

These results show how the tested prod-ucts work on a multiple biochemical parameter level and help to minimizie damage to human explants in a broad wavelength band of the solar spectrum.

Natural sunlight is the main source of HEVL and IR-A radiation and determines the probability and frequency of expo-sure. Natural sunlight is essential to life; however, human skin needs to be pro-tected. The tested compounds are com-plementary for protection in both IR and visible light ranges at the cellular level. In line with consumer needs, a large choice of ingredients is now available, used ei-ther alone or possibly in different combi-nations to minimize the negative effects of HEVL and IR-A radiation on the skin: for instance, a first-line defense product (formulation containing UV-TiO2 / SiO2) associated with second-line defense products (HDBM, Emblica, Ectoin, or even dihydroxacetone).

Acknowledgments The authors would like to warmly thank Dr. Andrew Salazar (Merck KGaA, Darm-stadt, Germany) for his great support with statistics, as well as Dr. Elian Lati and Dr. Laurent Peno-Mazzarino from

Laboratoires BioEC (France) for the conduct of the ex vivo studies and the very helpful scientific discussions around the results.

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Corresponding AuthorValérie Bicard-Benhamou

MERCK KGaADarmstadtGermany

[email protected]

G


Rethinking Beauty Science

See you in Barcelona!

www.ifscc2023.com

September 2023 will be the summer in which Barcelona awaits you to redesign the new paths that will mark the future of cosmetic science. Rethinking Beauty Science will be the theme that will serve as a central axle to deal with the most current, new and relevant scientific topics, while we will put the pillars of cosmetic research for the following years. Barcelona, a creative and entrepreneurial city, known for its science, art, culture and being a high-level Mediterranean gastronomy benchmark, will host the 33rd IFSCC Congress from September 4th to 7th, organized by the SEQC (Spanish Society of Cosmetic Chemists).

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Abstracts of papers published in the International Journal of Cosmetic Science, Volume 42, No. 5, 2020 (Sept. / Oct.)

Original Articles

pp. 421 - 428

TitleValidation of a new in vitro Sun Protection Factor method to include a wide range of sunscreen product emulsion types

AuthorsM. Pissavini* , C. Tricaud†, G. Wiener‡, A. Lauer§, M. Contier¶, L. Kolbe** , C. Trullàs Cabanas††, F. Boyer‡‡, E. Meredith§§, J de Lapuente¶¶, E. Dietrich*** and P.J. Matts††† * Coty-Lancaster SAM, 2, rue de la Lujerneta, Monaco 98000, Monaco † L’Oréal, 188 rue Paul Hochart, Chevilly Larue 94550, France‡ Edgewell Personal Care, Ormond Beach FL, 32174, USA§ Chanel PB, 135 avenue Charles de Gaulle, Neuilly sur Seine 92200, France ¶ LVMH Recherche Parfums et Cosmétiques, 185 avenue de Verdun, Saint Jean de Braye 45800, France

** Beiersdorf AG, Unnastrasse 48, Hamburg 20245, Germany†† ISDIN, Provencàls 33, Barcelona, Spain‡‡ Pierre Fabre Dermo Cosmetique, Hotel Dieu, 2 Rue Viguerie BP 3071, Toulouse cedex 3 31 025, France§§ CTPA, Sackville House, 40 Piccadilly, London W1J 0DR, U¶¶ AC MARCA, Avenida Carrilet 293-297, L’Hospitalet del Llobregat Barcelona 08907, Spain

*** Cosmetics Europe - The Personal Care Association, Avenue Herrmann-Debroux 40, Brussels B-1160, Belgium††† Procter & Gamble, Greater London Innovation Centre, Rusham Park, Whitehall Ln, Egham TW20 9NW, UK

Received 23 March 2020, Accepted 4 May 2020

Abstract

In 2017, Cosmetics Europe performed a double-blinded ring test of 24 emulsion-type sunscreen products, across 3 in vivo test labora-tories and 3 in vitro test laboratories, using a new candidate in vitro SPF test method. Based on the results of this work, an article was published showing how data derived from a new lead candidate method conform to new International Standards (ISO) acceptance criteria for alternative SPF test methods (Any alternative method should consider the matrix effect and if required, specify the matrix applicability of the method; Criterion 1a: Systematic differences between methods should be negligible: 95 % of all individual results of an alternative method are within the range of _29 reproducibility standard deviation of the in vivo method, that is overall bias must be below 0.59 reproducibility standard deviation of the in vivo method; Criterion 1b: Measurement uncertainty of an alternative method should be below the measurement uncertainty of the in vivo method. Candidate method predicted values must fall within the full ‘funnel’ (SPF 6-50+) limits proposed by Cosmetics Europe (derived from the same minimum test design, that is using the ISO24444 Method to measure at least 24 products across at least 3 laboratories using at least 5 test subjects/laboratory, in a blinded fashion).). Of the 24 sunscreen products tested, the majority of emulsions were of the oilin- water (O/W) type, whereas only one was water-in-oil (W/O) and there were no products with a mineral-only sun filter system. In order to confirm the scope of this method, therefore, a new study was conducted that included 73 W/O (12 mineral + organic, 44 mineral only and 17 organic only) and 3 O/W mineral- only, emulsion-type sunscreen products (a total of 76 new sunscreen products). When combined with the previous 24 products (tested in 3 different laboratories), this yielded a new data set comprising a total of 100 emulsion-type sunscreen products, with SPF values ranging from 6 to 50+ (with a total of 148 data points). These products were tested using the double-plate in vitro SPF test method and compared with the ISO TC217/WG7 acceptance criteria for alternative SPF test methods. Over 95% of paired in vitro: in vivo SPF values lay within the upper and lower limits of the ISO acceptance criteria funnel, with no bias. This new in vitro SPF test method, therefore, meets the minimum requirements for an alternative SPF test method to ISO24444:2010, for emulsion-type sunscreen products (which make up the majority of marketed sunscreen products).


pp. 429 - 435

TitleMicroneedle patch based on dissolving, detachable microneedletechnology for improved skin quality of the periorbital regionPart2: Clinical Evaluation

AuthorsV. Zvezdin* , T. Kasatkina†, I. Kasatkin†, M. Gavrilova‡ and O. Kazakova‡

* Microneedles Inc., 919 North Market Street, Wilmington, DE, USA† Microneedle Industrial LLC, 1130-42 Bolshoj bulvar, Moscow, Russia‡ Medlife LLC, Perm, Russia

Received 25 February 2020, Revised 26 May 2020, Accepted 26 May 2020

Abstract

ObjectiveMicroneedle patches based on dissolving, detachable microneedle technology (Russian patent No. 2652567; US patent EFS No. 32735812; WO/2019/231360) are novel dermatological products that allow safe, painless and effective reduction of epidermal wrinkles after six procedures. The purpose of this study was two-fold (a) to assess the safety and efficacy of microneedle patches comprising 650 microneedles containing hyaluronic and ferulic acids after 6 procedures of applying the applicator to the skin for 25 min; and (b) to correlate our previously reported ex vivo findings.

MethodsThe microneedle applicator contains 650 microneedles, which dissolve in 25 min of exposure. The effectiveness of microneedle applica-tors was confirmed by a randomized split-phase study involving 82 subjects. Applications of microneedle patches were performed at intervals of two times per week, and the effectiveness was assessed at 6 weeks after the start of the study.

ResultsThe results of the profilometric evaluation of skin smoothness demonstrated a significant reduction in the average roughness index by 65.32 _ 2.99 % and in the mean skin waviness by 66.84 _ 1.6 % compared with these indicators in the control group (P < 0.05). The therapeutic effect of the procedure was confirmed by an ultrasound examination that registered a 72.2 _ 5.4% and 25 _ 1.4 % increase in echo-density of the epidermis and dermis, respectively. An independent blinded skin evaluation by dermatologists revealed steady decrease in puffiness of the application area in 89.9 % of patients, increased elasticity in 78.3 % of the patients, and reduced severity of epidermal wrinkles in 89.9 % of the patients.

ConclusionsThe applicator with 650 soluble microneedles on its surface containing hyaluronic and ferulic acids is considered a safe, effective and convenient way to improve the skin quality in the periorbital region after six procedures. Nevertheless, additional studies of soluble microneedles are required to fully assess the amount and distribution area of the injected hyaluronic acid and other active components, as well as to detail the mechanism of action of soluble microneedles to improve skin quality

pp. 436 - 443

TitleThe impact of different hair-removal behaviours on the biophysical and biochemical characteristics of female axillary skin

AuthorsR. L. Evans , S. Bates, R. E. Marriott and D. S. ArnoldUnilever Research & Development, Port Sunlight Laboratory, Quarry Road East, Bebington, Wirral, Merseyside CH63 3JW, UK

Received 12 May 2020, Revised 24 June 2020, Accepted 2 July 2020

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Abstract

Objective The impact of hair removal on the biophysical and biochemical characteristics of human axillary skin is not fully understood. This study investigated the effect of different hair-removal techniques on biophysical parameters and the concentrations of key inflammatory biomarkers in the axillae of female Thai subjects. Axillary hair was removed by shaving, plucking or waxing.

MethodsFollowing a 2-week washout phase without hair removal, subjects underwent visual assessment for erythema and skin dryness in one (randomized) axilla, then hair was removed from the axilla by shaving, plucking or waxing according to each. subject’s established habit. Erythema and dryness were assessed again 30 min after hair removal, and buffer scrubs collected from depilated and non-depilated axillae and analysed for inflammatory cytokines; after a further 48 h, erythema, dryness and post-inflammatory hyperpigmentation (PIHP) were assessed in the depilated axilla. Biophysical assessments (skin hydration, barrier integrity, elasticity and roughness) were made in depilated and non-depilated axillae.

ResultsAll three hair-removal techniques induced an increase in axillary erythema and skin dryness. Shaving was associated with significantly less erythema (P < 0.01), but significantly greater skin dryness (P < 0.05) versus the other techniques 30 min after hair removal. There were no between-technique differences in PIHP or biophysical parameters. Interleukins IL-1a and IL-1RA concentrations increased, and IL-8 concentration decreased following hair removal by each technique.

Conclusion This is the first study to identify the principal cytokines associated with the inflammatory process triggered by axillary hair removal. A single hair-removal treatment did not appear to induce PIHP or further biophysical changes to the skin.

pp. 444 - 451

TitleA blackberry–dill extract combination synergistically increases skin elasticity

AuthorsD. Meza , W.-H. Li , I. Seo , R. Parsa , S. Kaur , M. Kizoulis and M.D. SouthallJohnson & Johnson Consumer Inc., Skillman, NJ, USA

Received 30 April 2020, Revised 12 June 2020, Accepted 18 June 2020

Abstract

BackgroundThe loss of structural elastin due to intrinsic and extrinsic ageing results in the skin’s inability to stretch and recoil (decrease in elasticity) and manifests as loss of skin firmness and sagging. While other extracellular matrix (ECM) components such as collagen and hyaluronic acid are continually synthesized and assembled through life, elastic fibres are not. Elastic fibre assembly and functionality require fibre cross-linking, induced by the lysyl oxidase-like (LOXL) enzymes, which sharply decrease during ageing. OBJECTIVE: To evaluate the enhanced elastogenic effect of a blackberry–dill extract combination, which was hypothesized to induce elastin fibre component synthesis, fibre cross-linking and reduce elastin fibre degradation.

MethodsThe blackberry and the dill extracts were tested separately and in combination to confirm single ingredient bioactivity and synergistic benefits. Human skin explants, dermal fibroblasts, elastase assays, ELISAs, quantitative real-time PCRs and spectrofluorometer mea-surements were used. Moreover, a double-blinded, placebo-controlled clinical study was carried out to assess skin elasticity using Cutometer and histologically from biopsies.

ResultsThe blackberry extract induced elastin gene expression, elastin promoter activity and inhibited elastic fibre degradation by matrix metalloproteinases (MMPs) 9 and 12. The dill extract induced elastin, collagen and LOXL1 gene expression, resulting in enhanced


fibre cross-linking in human skin explants. Clinically, the blackberry and dill combination treatment displayed synergistic pro-elasticity activity as compared to each ingredient alone and placebo. ConclusionTaken together, these results demonstrated the two multimodal plant-based extracts complemented each other in terms of bioactivity and resulted in a synergistic elastogenesis induction

pp. 452 - 461

TitleChanges in facial signs due to age and their respective weights on the perception of age, on a tired-look or a healthy glow among differently aged Chinese men

AuthorsF. Flament* , A. Abric†, D. Amar‡, C. Ye‡, J. Caron§ and C. Negre§

* L’Oréal Research and Innovation, Chevilly-Larue, France† Eurosyn, Villebon-sur-Yvette, France‡ L’Oréal Research and Innovation, Shanghai, China§ L’Oréal, Levallois-Perret, France

Received 15 May 2020, Revised 12 June 2020, Accepted 18 June 2020

Abstract

ObjectiveTo determine the respective weights of certain facial signs on the assessment of perceived age, tired-look and healthy glow on Chinese men of different ages.

Material and MethodsPhotographs were taken of the faces of 420 Chinese men of different ages, under standardized conditions. These photographs allowed to focus and define 15 facial signs, which were then graded by 15 experts and dermatologists, using standardized scales provided by a reference Skin Aging Atlas. The facial signs were dispatched into 5 clusters, namely wrinkles/texture, ptosis/sagging, pigmentation disorders, vascular disorders and cheek skin pores. A native panel, composed of 80 Chinese women, of similar age range were asked, when viewing full-face photographs, to: (i) attribute on a 0–10 scale their perception of both the tired-look and healthy glow aspects and (ii) estimate the age of the subject.

ResultsWith the exception of vascular disorders, the severity of all 4 clusters increased with age, although at different rates. The ptosis/sag-ging or pigmentation disorders showed a rather regular progression. Although perceived ages and real ages were found to be closely correlated, the vast majority of subjects were judged older by 2 - 10 years. The changes in facial signs (and their related clusters) were significantly correlated with perceived age, with the exceptions of skin spot density and cheek skin pores. Although the aspects of tired-look and healthy glow were logically found to be anti-correlated, tired-look was more statistically associated with perceived age for the five clusters. Signs of eye contour appear to be closely correlated with the perception of a tired-look.

ConclusionWithin facial clinical clusters, wrinkles/texture and ptosis / sagging are major factors in the assessment of perceived age in Chinese men. Tired- look appears to be strongly associated with perceived age.

pp. 462 - 470

TitleA minimally invasive clinical model to test sunscreen toxicity based on oxidative stress levels using microbiopsy and confocal microscopy – a proof of concept study

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AuthorsMiko Yamada*,a, Lynlee L. Lin†,a, Lydia Y.T. Hang†,a, Paul J. Belt‡, H. Peter Soyer†, Anthony P. Raphael* and Tarl W. Prow** Future Industries Institute, University of South Australia, Adelaide, Australia† The University of Queensland Diamantina Institute, School of Medicine, The University of Queensland, Dermatology Research Centre, Brisbane, Australia ‡ Department of Plastic and Reconstructive Surgery, Princess Alexandra Hospital, Brisbane, Australia

Received 23 March 2020, Revised 28 May 2020, Accepted 25 June 2020

Abstract

Objective This proof-of-concept study demonstrated that using minimally invasive skin microsampling could enable significantly higher through-put of cosmetic testing in volunteers than conventional biopsy. Nanoparticle sunscreen was used as a model to test toxicity based on oxidative stress using microbiopsy and confocal imaging.

MethodsSix volunteers were recruited for this study (3 males and 3 females). Zinc oxide nanoparticle containing topical formulation was prepared at 10% w/v. Each volunteer had 3 areas of 4 cm2 each mapped on each inner forearm for a total of 6 treatment areas (intact/ tape-stripped and with/without treatment). The topical zinc-nanoparticle formulation was applied directly to volunteer skin (2mg / cm2) for 2 hrs. Microbiopsied tissue from each treatment group was stained for reactive oxygen and nitrogen species in addition to mitochondrial superoxide. The stained samples were then imaged using confocal microscopy prior to image analysis.

ResultsSkin exposed to zinc oxide nanoparticles did not show any significant increases in oxidative stress. Zinc oxide nanoparticle tape-stripped skin resulted in signal significantly lower (P < 0.001) oxidative stress levels than t-butylated hydroxytoluene treated tapestripped skin for oxidative stress markers. Topically applied zinc oxide nanoparticles had no detectable effect on the oxidative status in volunteer skin. No adverse reactions or effects were observed after all treatments including microbiopsy.

Conclusion The data support the hypothesis that microbiopsy is a viable approach to study cosmeceutical- skin interactions in volunteers with capacity for molecular assays and high throughput with very low risk to the volunteer.

pp. 471-481

TitlePerceived age and perceived health among a Chinese cohort: Does it mean the same thing?

AuthorsC. Messaraa*, T.J.C. Richard†, M. Walsh*, L. Doyle*, C. O’Connor*, N. Robertson*, A. Mansfield*, S. Hurley*, A. Mavon†, A. Grenz‡

* Oriflame R&D, Bray Business Park, Kilruddery, Bray A98 Y6W0, Ireland† Oriflame Skin Research Institute, Oriflame Cosmetics AB, Master Samuelsgatan 56, Stockholm 11121, Sweden‡ Oriflame Global insight, Oriflame Cosmetics AB, Master Samuelsgatan 56, Stockholm 11121, Sweden

Received 17 January 2020, Revised 15 May 2020, Accepted 18 June 2020

Abstract

Background and AimsPrevious investigations have aimed atinvestigating parameters affecting age perception on several ethnicities.Perceived health has been a newer focus on Caucasian skin,yet little is known on the skin features used to estimate the healthstatus of Chinese women and we aimed to investigate whetherthese cues are the same as those used for age perception.

MethodsAge and health appearance of 276 Chinese female volunteerswere estimated from their photographs by 1025 femalenative Chinese graders 20 - 69 years old. Models were built to predictperceived age and health from topographic, colour and biophysicalmeasured vari-


ables, in two subsets of the studiedvolunteers: below and above 50 years. Machine learning-based predictivemodels for age and health perception were built on the collecteddata, and the interpretability of the models was establishedby measuring feature importance.

ResultsAge perception was mostly driven by topographic features,particularly eye bags and eyelid sagging in the group below50 years old. Wrinkles, notably from the lower part of the face andoval of the lower face, were found to be more relevant in the groupabove 50 years. Health appearance was primarily signalled by skinimperfections and global pigmentation in the subset below 50 years, whereas colour-related parameters and skin hydrationacted as health cues for the subset above 50 years.

ConclusionDistinct skin features were acting as cues for ageperception and/or health perception and varied per age subset.Their contribution should be borne in mind when designing productsfor ‘younger looking skin’ and ‘healthier looking skin’.

pp. 482 - 493

TitleStratum corneum occlusion induces water transformation towards lower bonding state: a molecular level in vivo study by confocal Raman microspectroscopy

AuthorsC. Choe*, J. Schleusener†, S. Choe*, J. Ri*, J. Lademann† and M. E. Darvin†

* Kim Il Sung University, Taesong District, Ryongnam-Dong, Pyongyang, DPR Korea† Department of Dermatology, Venerology and Allergology, Center of Experimental and Applied Cutaneous Physiology, Charité – Universitätsmedizin

Berlin, Corporate Member of Freie Universität Berlin,Humboldt-Universität zu Berlin, Berlin Institute of Health, Charit_eplatz 1, Berlin 10117, Germany

Received 9 June 2020, Revised 15 July 2020, Accepted 17 July 2020

Abstract

ObjectiveIt is conventionally understood that occlusive effectsare the retention of excessive water in the stratum corneum (SC),the increase of SC thickness (swelling) and a decrease of thetransepidermal water loss. However, the influence of occlusion onwater binding properties in the SC is unknown.

MethodsThe action of plant-derived jojoba and almond oils, aswell as mineral-derived paraffin oil and petrolatum topically appliedon human skin, is investigated in vivo using confocal Ramanmicrospectroscopy. To understand the oils’ influence on the SC onthe molecular level, the depth-dependent hydrogen bonding statesof water in the SC and their relationship to the conformation ofkeratin, concentration of natural moisturizing factor (NMF) moleculesand lipid organization were investigated.

ResultsA significant SC swelling was observed only in petrolatum-treated skin. The water concentration was increased in oiltreatedskin in the intermediate SC region (40 - 70 % SC depth).Meanwhile, the amount of free, weakly and tightly bound waterincreased, and strongly bound water decreased in the uppermostSC region (0 - 30 % SC depth). The NMF concentration of oil-treatedskin was significantly lower at 50 - 70 % SC depth. The lateral organizationof lipids in oil-treated skin was lower at 0 - 30 % SC depth. The secondary structure of keratin was changed towards anincrease of b-sheet content in mineral-derived oil-treated skin andchanged towards an increase of a-helix content in plant-derivedoil-treated skin.

ConclusionThe occlusive properties can be summarized as theincrease of free water and the transformation of water from a morestrongly to a more weakly hydrogen bonding state in the uppermostSC, although some oils cause insignificant changes of the SCthickness. The accompanied changes in the keratin conformationat the intermediate swelling region of the SC also emphasize therole of keratin in the SC’s water-transporting system, that is thewater in the SC transports intercellularly and intracellularly in the intermediate swelling region and only intercellularly in the upper most non-swelling region. Bearing this in mind, almond, jojobaand paraffin oils, which are not occlusive from the conventionalviewpoint, have an occlusion effect similar to petrolatum on the SC.

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pp. 494 - 500

TitleInfluence of vegetable oils in the rheology, texture profile and sensory properties of cosmetic formulations based on organogel

AuthorsFrancine C.S. César and Patrícia M.B.G. Maia CamposFaculty of Pharmaceutical Sciences, University of Sao Paulo, Ribeirao Preto 14040-903, Brazil

Received 19 December 2019, Revised 11 July 2020, Accepted 17 July 2020

Abstract

ObjectiveThe knowledge about how ingredients in formulationcan influence the texture profile is an important factor on thedevelopment of a cosmetic product. In this context, the aim of thiswork was to evaluate the effect of vegetable oils in the texture profile,rheological and sensorial properties of cosmetic formulationsbased on organogel.

MethodsFour organogel-based emulsions were developed andsupplemented or not with sunflower, macadamia or olive oils.Analyses of rheo-logical behaviour, texture profile and sensory propertieswere performed.

ResultsThe vegetable oils added to formulation did not alterthe pseudoplastic rheological behaviour, but increased the area ofhysteresis and reduced the work of shear of the formulations. Inaddition, the sunflower seed oil increased the consistency indexand all texture parameters while the macadamia oil reduced firmnessand consistency. The cosmetic formulation based on organogelcontaining the sunflower seed oil showed the highest score on sensoryevaluation.

ConclusionThe vegetable oils affected the rheology behaviour,texture profile and sensory properties of the formulations understudy. However, the influence of sunflower oil in organogel-basedcosmetic formulation was more pronounced considering textureprofile and the response perceived by subjects in the sensorial analysis.

pp. 501 - 511

TitleNiacinamide mitigates SASP-related inflammation induced by environmental stressors in human epidermal keratinocytes and skin

AuthorsJohn C. Bierman*, Timothy Laughlin*, Makio Tamura*, Ben C. Hulette*, Catherine E. Mack*, Joseph D. Sherrill*, Christina Y.R. Tan†, Malgorzata Morenc†, Sophie Bellanger† and John E. Oblong** The Procter & Gamble Company, Mason OH, 45040, USA † Skin Research Institute of Singapore, A*STAR, 8A Biomedical Grove, #06-06 Immunos, Singapore 138648, Singapore

Received 1 June 2020, Revised 8 July 2020, Accepted 9 July 2020

Abstract

ObjectiveTo evaluate whether niacinamide (Nam) can mitigateproduction of inflammatory and senescence-related biomarkersinduced by envi-ronmental stressors.

MethodsHuman epidermal keratinocytes were exposed to UVB,urban dust, diesel exhaust and cigarette smoke extract and treatedwith Nam or vehicle control. Full thickness 3-D skin organotypicmodels were exposed to a combination of UVB and PM2.5 and treatedwith Nam


or vehicle control. Quantitation of the SASP-relatedinflammatory mediators PGE2, IL-6 and IL-8 was performed on culturedmedia. UVB-exposed keratinocytes treated with and withoutNam were immunostained for the senescence biomarker Lamin B1 (LmnB1). Transcriptomics profiling of cigarette smoke extracteffects on keratinocytes was performed. A double-blind, placebocontrolledclinical was conducted on 40 female panellists that werepretreated on back sites for two weeks with 5% Nam or vehicleand then exposed to 1.5 minimal erythemal dose (MED) solar-simulatedradiation (SSR). Treated sites were compared with non-treatedexposed sites for erythema and the skin surface IL-1aRA/IL-1ainflammatory biomarkers.

ResultsUltraviolet B induced synthesis of PGE2, IL-8 and IL-6and reduced LmnB1 levels in keratinocytes. Urban dust and dieselexhaust only stimulated synthesis of IL-8 whereas cigarette smokeextract only stimulated levels of PGE2. In all exposures, treatmentwith Nam signifi-cantly mitigated synthesis of the inflammatorymediators and restored levels of UVB-reduced LmnB1. In the 3Dskin equivalent model, Nam reduced IL-8 levels stimulated by acombination of topical PM2.5 and UV exposure. In a UV challengeclinical, pretreatment with 5% Nam reduced erythema and skinsurface IL-1aRA/IL-1a inflammatory biomarkers that were inducedby SSR.

ConclusionSince it is known that Nam has anti-inflammatoryproperties, we tested whether Nam can inhibit environmentalstress-induced in-flammation and senescence-associated secretoryphenotype (SASP) biomarkers. We show Nam can reduce PGE2, IL-6 and IL-8 levels induced by environmental stressors. Additionally, in vivo pretreatment with Nam can reduce UV-induced erythemaand skin surface inflammatory biomarkers. These findings add tothe body of evidence that Nam can mitigate the skin’s inflammatory response elicited by environmental stressors. Thissupports Nam can potentially inhibit senescence and prematureageing and thereby maintain skin’s functionality and appearance.

pp. 512 - 519

TitleHomogeneous distribution of fatty ester-based active cosmetic ingredients in hydrophilic thin films by means of nanodispersion

AuthorsEmilie Munnier* , Almar Al Assaad*, Stephanie David*, Frédéric Mahut†, Marylène Vayer†, Louise Van Gheluwe*, Florent Yvergnaux‡, Christophe Sinturel†, Martin Soucé*, Igor Chourpa* and Franck Bonnier** EA 6295 Nanomédicaments et Nanosondes (NMNS), Faculté de Pharmacie, Université de Tours, 31 avenue Monge 37200, Tours, France† Interfaces, Confinement, Matériaux et Nanostructures (ICMN), CNRS-Université d’Orléans, UMR 7374, 1b, Rue de la Férollerie,

C.S. 40059, Orléans Cedex 2 45071, France ‡ Bioeurope (Solabia group), Route d’Oulins, Anet 28 260, France

Received 15 May 2020, Revised 30 June 2020, Accepted 15 July 2020

Abstract

ObjectiveCosmetic films and patches are interesting forms topromote skin penetration of active ingredients as they ensure theirlong stay on the treated zone of the skin. Nevertheless, currentlydeveloped films and patches are most of all hydrophilic and are notadapted to the hydrophobic molecules. The aim of this study wasto establish whether nanodispersion of fatty acid-based active cosmeticingredients (ACI) could be a manner to introduce high concentrationsof those ACI in hydrophilic films.

MethodsPunica granatum seed oil hydroxyphenethyl esters(PHE) constitute a commercialized lipolytic cosmetic ingredientobtained by enzymatic conjugation of tyrosol to long-chain fatty acidsand to enhance its skin diffusion. Nanodispersions of PHE were preparedby a green emulsion-solvent evaporation process and dispersedin polyvinyl alcohol films. Raman imaging coupled to multivariateanalysis was used to study the distribution of PHE in the films.

ResultsNanodispersions of PHE combined with antioxidant vitaminE and stabilized by Pluronic_ F127 were successfully prepared.The nanodis-persions show a spherical shape and ahydrodynamic diameter close to 100 nm. Raman images analysiswith multivariate approaches showed a very homogeneous distributionof PHE nanodispersions in the films compared to free PHEintroduced as an ethanol solution.

AB

STRACTS


ConclusionNanodispersions of hydrophobic fatty acid-basedingredients seem to be relevant method to introduce this type ofingredient in hydro-philic film matrix. The co-suspension with vitaminE limits their degradation in time.

Research Communication

pp. 512 - 519

TitlePotential anti-ageing effect of chondroitin sulphate through skin regeneration

AuthorsD. Min*,†, S. Park*, H. Kim‡, S. H. Lee*, Y. Ahn§, W. Jung§, H.-J. Kim* and Y. W. Cho†

* Basic Research & Innovation Division, AMOREPACIFIC R&D Unit, Yongin, Republic of Korea† Department of Chemical Engineering, HanyangUniversity, Ansan, Republic of Korea ‡ Amorepacific R&D Unit, Yongin, Republic of Korea§ Department of Biomedical Engineering, UlsanNational Institute of Science and Technology (UNIST), Ulsan, Republic of Korea

Received 21 January 2020, Revised 3 May 2020, Accepted 16 June 2020

Abstract

ObjectiveSkin ageing is inevitably exposed through its typicalfeatures such as wrinkles and sagging. Therefore, skin anti-ageingis a major issue in cosmetic research to prevent and improve ageingsymptoms using effective ingredients. Chondroitin sulphate(CS), a type of glycos-aminoglycan, is an important structural componentof the extracellular matrix (ECM) and is involved in variousbiological processes, such as cell proliferation, differentiation andmigration. Here, we aimed to investigate the effects of CS on skinregeneration and examine its efficacy as a potential safe and effectiveskin anti-ageing ingredient.

MethodsWe investigated the effects of CS on cell proliferationin normal human keratinocytes and fibroblasts. Then, cell migration,ECM synthesis and related signalling pathways were examinedin fibroblasts through gene and protein expression analysis. Finally,the effect on skin wound healing and regeneration was validatedusing a full-thickness skin wound model and an aged skin model.

ResultsChondroitin sulphate treatment increased the proliferationof keratinocytes and fibroblasts. It also stimulated the migrationand synthe-sis of ECM components of fibroblasts. Furtheranalysis revealed that CS induced the expression of type I procollagenby activating the extracellular signal-regulated kinase pathway.Using a full-thickness skin wound model and an aged skinmodel, we confirmed that CS treatment promoted skin wound healingand regeneration.

Conclusion Together, our results indicated that CS has thepotential to facilitate skin regeneration, implying that CS could beclinically applied to improve skin ageing.

IFSCC GOLDEN BOOK | 2017 © 2017 International Federation of Societies of Cosmetic Chemists

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Volume 23 | Number 3 | November 2020 MAGAZINE...IFSCC Magazine archives starting in 2000 most...

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