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Safeguarding Squalene: Lemon Myrtle Antioxidant for Pollution Protection and Oleostasis

Contact Author Joan Attia-Vigneau Ph.D., Marty Shortt, Rachelle Seguin, Isabelle Lacasse and Estelle Loing, Ph.D., IFF/Lucas Meyer Cosmetics, Québec
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Sebum, the lipid film produced by sebaceous glands in the skin, has important functions including reducing water loss from the skin surface, serving as a vehicle for lipophilic antioxidants, protecting against harmful microorganisms and shielding against environmental aggressors.1 Human sebum is a complex mixture of lipids consisting of triglycerides, diacylglycerols and fatty acids (50–60% altogether); wax esters (20–30%); squalene (10–16%); and cholesterol esters (2–4%).2

Variations in sebum production and composition have been reported among different ethnic groups. For example, African-American skin produces more sebum than Asian skin, which in turn produces more than Caucasian skin. African-American skin also contains higher squalene content.3 The presence of squalene in sebum is of particular interest since it is unique to humans and has recently been identified as a reliable marker of environmental damage to the skin.4, 5

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Squalene is a triterpene comprising six non-conjugated double bonds, making it one of the most unsaturated lipids. However, this means that squalene is highly prone to oxidative damage. Indeed, squalene is particularly sensitive to singlet oxygen (1O2), which is generated in the skin upon exposure to UVB and UVA radiation. Atmospheric pollutants, ozone (O3) and cigarette smoke are also powerful oxidizing agents of squalene.5

When squalene is oxidized, peroxides are formed, creating important biological consequences. First, peroxidized by-products are potent inflammatory mediators associated with acne, hyperpigmentation, skin roughness and wrinkle formation.6 Moreover, in response to squalene deterioration, sebaceous glands are activated, resulting in excess sebum production to compensate for its poor quality. This creates an unbalanced sebum composition abnormally rich in peroxidation-prone squalene and glycerides that can only perpetuate oily skin problems.

With the purpose of finding a means to protect squalene, the present work focused on Backhousia citriodora, commonly known as lemon myrtle. This flowering tree is endemic to the subtropical rainforests of Australia. In traditional medicine, indigenous Australians applied a paste derived from the leaves to help heal wounds. Today, lemon myrtle is popular for its lemony flavor and is added to various foods and beverages.

The leaves of the plant are especially rich in flavonoids and polyphenols, with antioxidant activity eight times that of blueberries.7 Given this high antioxidant index, it was hypothesized that an extract of B. citriodora leaves could be a suitable candidate for maintaining cutaneous squalene quality, in turn supporting normal sebum balance (oleostasis). Thus, the current study aimed to test the efficacy of B. citriodora leaf to protect squalene against peroxidation and inflammatory reactions, as well as normalize sebum production in skin of various ethnic origins exposed to urban environmental antagonists.

In vitro Materials and Methods

Ingredient: The cosmetic activea characterized in this study is a polyphenol-enriched aqueous extract of B. citriodora leaf.

Antioxidation of squalene: Squalene was obtained from skin explants using liquid/liquid extraction and exposed in vitro to singlet oxygen in the presence/absence of 0.4% and 2% B. citriodora leaf extracts. The lipid oxidation of squalene was assessed by measuring malondialdehyde (MDA) formation via gas chromatography–mass spectrometry (GC/MS).

Inhibition of pro-inflammatory cytokines: Non-confluent normal human dermal fibroblasts (NHDF) were treated for 24 hr with interleukin-1 (IL-1, 0.003 ng/mL) to induce inerleukin-8 (IL-8) production in the presence or absence of 0.1%, 0.2% and 0.4% B. citriodora leaf extract, or 1 µM dexamethasone as the positive control. The release of IL-8 was quantified in cell culture media using the enzyme-linked immunosorbent assay (ELISA).

Inhibition of cell differentiation and lipid accumulation: Human sebocyte cells (SEB0662), were seeded in 96-well plates and cultured for 24 hr in sebocyte medium.8 The medium was then removed and replaced by assay medium containing (or not, as control) 0.1% B. citriodora leaf extract or 1 μM of the androgen inhibitor negative reference dutasteride; the cells were pre-incubated for 4 hr.

After pre-incubation, 1 nM testosterone was added or not (non-stimulated control) in the wells and the cells were incubated for seven days. Sebocyte differentiation was evaluated by fluorescent immunolabeling of the epithelial membrane antigen (EMA)b. In parallel, the synthesis and storage of lipids in sebocytes was followed with a fluorescent probec while cell nuclei were dyed using a DNA stain solutiond. Images were acquired by confocal microscopye and labeling was quantified by measuring fluorescence intensity.

Clinical Study Protocol

Thirty-one healthy male and female urban volunteers, aged 18–55 years and presenting combination or oily skin, were recruited for this study. The panel was multiethnic, including Caucasian, Asian and African-American volunteers. Participants were asked to apply a cream containing 2% of the test extract or a placebo for 28 days (see Formula 1), to a randomized side of their cleansed face, morning and evening. Application was followed by lightly massaging the skin.

Shine evaluation: High-resolution photographs of each half-face were acquired using a facial imaging systemf at D0 and D28. Gloss analysis was performed using dedicated software. Results were corrected for possible lighting variations. Statistical significance was calculated using the Wilcoxon test.

Sebum production: Sebum production was determined at D0 and D28 using a sebumeterg. This photometric device uses a special plastic strip that becomes increasingly transparent with increasing fat absorption. A photocell measures the transparency. The total mass of lipids excreted by the surface unit (in μg/cm2) was quantified with the help of a microprocessor. Statistical significance was calculated using the Wilcoxon test.

The antioxidative activity of the extract significantly protected purified squalane from peroxidation after exposure to
singlet oxygen.

Results: In vitro Tests

Antioxidation of squalene: The antioxidant potential of B. citriodora leaf extract was first investigated using the DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) assay, which is commonly used to assess the free radical-quenching efficacy of products. A complete inhibition of free radical release was observed within 30 min in the presence of 0.4% extract (data not shown). In a second experiment, the antioxidative activity of the extract significantly protected purified squalene from lipid peroxidation following exposure to singlet oxygen. Protection levels increased by 18% in the presence of 0.4% extract, vs. the control (see Figure 1).

Inhibition of pro-inflammatory cytokines: Since inflammation is a natural consequence of squalene oxidation, the ability of B. citriodora leaf extract to inhibit the release of inflammatory mediators in fibroblasts also was tested. These cells generate IL-8 production when challenged with IL-1α. In turn, IL-8 mediates inflammatory reactions in the skin through recruitment of neutrophils and other immune cells to invade injured or inflamed tissues.

As expected, the results showed the production of IL-8 following the introduction of IL-1α into the cell culture media. However, inflammatory cytokine release was dose-dependently inhibited in the presence of the extract, up to 96% when the extract was used at a concentration of 0.4% (see Figure 2). Furthermore, the extract proved to be even more potent than dexamethasone (positive control).

Inhibition of cell differentiation and lipid accumulation: Next, the effect of B. citriodora leaf extract on sebocyte activity was assessed using testosterone as an inducer of cell differentiation and lipid production. Following the expression of the epithelial membrane antigen (EMA) as a marker of sebocyte differentiation, 0.1% extract significantly inhibited testosterone-induced cell differentiation by 30% (see Figure 3, top). Also at a concentration of 0.1%, the extract significantly reduced the testosterone-induced lipid accumulation in sebocytes by 32% (see Figure 3, bottom).

Results: Clinical Study

As stated, the ability of B. citriodora leaf extract to rebalance sebum content under real-life conditions was tested in a panel of 31 urban volunteers of various ethnic origins, i.e. African-American, Asian and Caucasian, presenting oily skin. Participants applied a cream containing 2% of the test extract or a placebo for 28 days, morning and evening, randomly to one-half of their cleansed face.

For all ethnic groups, facial gloss analysis at D28 provided evidence of a significant amelioration in shiny skin appearance of the treated zone, compared with D0 and the control, with an average decrease of 11% for African-American skin, 3% for Asian skin and 1% for Caucasian skin. Maximum values for reductions in glossy skin reached 28% for African-American skin, 22% for Asian skin and 5% for Caucasian skins. Representative photographs are shown in Figure 4 (top).

Accordingly, significant reductions in skin sebum production were documented across all ethnic skin types. At D28, sebum production was reduced by an average of 9% for African-American skin, 9% for Asian skin and 6% for Caucasian skin—compared with D0 and the control—particularly in Millennials, i.e., volunteers < 35 years old. Maximum values for inhibiting skin sebum production reached 32% for African-American skin, 19% for Asian skin and 34% for Caucasian skin (see Figure 4, bottom).

One month of treatment with B. citriodora extract
significantly mattified skin.

Discussion

In consumers’ minds, oily skin is most commonly associated with hormonal imbalance, diet or a bad genetic make-up. However, in recent years, other factors have emerged as significant triggers of shiny skin. Exposure to UV radiation, pollutants, ozone and cigarette smoke can all affect the lipid composition and quality of sebum.5 As noted, a major consequence of such environmental aggressors is the oxidative alteration of squalene, an important lipid in human sebum composition, whose degradation triggers inflammatory reactions and sebum overproduction in exposed skin.6

The current paper demonstrates an extract of B. citriodora leaf acts as a potent antioxidant, capable of protecting the integrity of squalene. The extract also is shown to block the release of IL-1-induced IL-8 secretion in fibroblasts, and IL-1-driven IL-8 production represents a potent pathway for the amplification of inflammatory signals. Indeed, a recent study associated the release of both cytokines with squalene peroxidation in the skin.9 The extract additionally inhibits differentiation and lipid accumulation in sebocytes, and therefore could be used to treat acne—a condition that is easily exacerbated under adverse environmental conditions.10

Finally, the potential of B. citriodora leaf extract to protect the skin from environmentally induced oily skin was fully demonstrated in a split-face, placebo-controlled clinical study involving urban volunteers. In this study, the extract efficiently restored the balance of lipid production in skin of various ethnic types. One month of treatment with a cream containing 2% extract significantly mattified the skin, while placebo treatment did not (see Figure 5).

Conclusion

B. citriodora leaf extract is a potent antioxidant that protects squalene from peroxidation, inhibits the release of inflammatory cytokines in skin, controls proliferation and lipid accumulation in sebocytes, and efficiently reduces a shiny skin appearance under environmentally challenging conditions. B. citriodora leaf extract is therefore a suitable candidate to naturally restore and maintain oleostasis—i.e., lipid homeostasis in the skin.

In a world dominated by social media where selfies are constantly exchanged, appearance is of prime importance. This is especially true for millennials, who feel they must always look their best, while at the same time facing increased challenges from the environment. B. citriodora leaf extract may be the answer for this new generation of self-conscious citizens of the world.

References

  1. C Barrault et al, Androgens induce sebaceous differentiation in sebocyte cells expressing a stable functional androgen receptor, J Steroid Biochem Mol Biol 152 34-44 (2015)
  2. E Camera, M Ludovici, M Galante, JL Sinagra and M Picardo, Comprehensive analysis of the major lipid classes in sebum by rapid resolution high-performance liquid chromatography and electrospray mass spectrometry, J Lipid Res 51(11) 3377-88 (2010)
  3. CC Zouboulis, JM Baron, M Böhm, S Kippenberger, H Kurzen, J Reichrath and A Thielitz, Frontiers in sebaceous gland biology and pathology, Exp Dermatol 17(6) 542-51 (2008)
  4. A Pappas, J Fantasia and T Chen, Age and ethnic variations in sebaceous lipids, Dermatoendocrinol 5(2) 319-24 (2013)
  5. C De Luca and G Valacchi, Surface lipids as multifunctional mediators of skin responses to environmental stimuli, Mediators Inflamm Article ID 321494, 11 pages (2010)
  6. DM Pham, B Boussouira, D Moyal and QL Nguyen, Oxidization of squalene, a human skin lipid: A new and reliable marker of environmental pollution studies, Int J Cosmet Sci 37(4) 357-65 (2015)
  7. I Konczak, D Zabaras, M Dunstan, P Aguas, P Roulfe and A Pavan, Health benefits of Australian native foods—An evaluation of health-enhancing compounds, in Australian Government, Rural Industries Research and Development Corporation, RIRDC Pub. No 09/133, Canberra, Australia (2009) pp 1-52
  8. B Boussouira and DM Pham, Squalene and skin barrier function: From molecular target to biomarker of environmental exposure, in Skin Stress Response Pathways, G Wondrak, ed, Springer, Cham (2016) pp 29-48
  9. V Kostyuk, A Potapovich, A Stancato, C De Luca, D Lulli, S Pastore and L Korkina, Photo-oxidation products of skin surface squalene mediate metabolic and inflammatory responses to solar UV in human keratinocytes, PLoS One 7(8) e44472 (2012)
  10. J Krutmann, D Moyal, W Liu, S Kandahari, GS Lee, N Nopadon, LF Xiang and S Seité, Pollution and acne: Is there a link? Clin Cosmet Investig Dermatol 10 199-204 (2017)

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Figure 1. Squalene antioxidation

Figure 1. Squalene antioxidation

The antioxidative activity of the extract significantly protected purified squalene from lipid peroxidation following exposure to singlet oxygen. Protection levels increased by 18% in the presence of 0.4% extract, vs. the control.

Figure 2. Inhibition of proinflammatory cytokine release

Figure 2. Inhibition of proinflammatory cytokine release

Inflammatory cytokine release was dose-dependently inhibited in the presence of the extract, up to 96% when the extract was used at a concentration of 0.4%.

Figure 3. Inhibition of differentiation (a) and lipid accumulation (b)

Figure 3. Inhibition of differentiation (a) and lipid accumulation (b)

Following the expression of the epithelial membrane antigen (EMA) as a marker of sebocyte differentiation, 0.1% extract significantly inhibited testosterone-induced cell differentiation by 30%. Also at a concentration of 0.1%, the extract significantly reduced the testosterone-induced lipid accumulation in sebocytes by 32%

Figure 4. Reduction in shiny appearance and sebum production in select volunteers

Figure 4. Reduction in shiny appearance and sebum production in select volunteers

Maximum values for reductions in glossy skin reached 28% for African-American skin, 22% for Asian skin and 5% for Caucasian skin.

Figure 5. B. citriodora leaf extract supports skin oleostasis

Figure 5. <em>B. citriodora</em> leaf extract supports skin oleostasis

One month of treatment with a cream containing 2% extract significantly mattified the skin, while placebo treatment did not.

Footnotes [CT1805 Loing]

a OilessCity (INCI: Backhousia Citriodora Leaf Extract) is a product of IFF/Lucas Meyer Cosmetics

b GAM-Alexa 568

c Bodipy fluorescent dyes, Thermo-Fisher

d Hoechst 33258 solution, MedChemExpress

e LSM 510 NLO, Zeiss

f Visia-CR, Canfield Scientific Inc.

g SM 815, Courage & Khazaka GmbH

Formula 1. Clinical Study Test Cream

Formula 1. Clinical Study Test Cream

Participants were asked to apply cream containing 2% of the test extract or a placebo for 28 days, to a randomized side of their cleansed face, morning and evening. Application was followed by lightly massaging the skin.

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