As a name, ZAG may sound more appropriate for a pop star rather than a protein but it reflects two properties of the molecule: the protein precipitates in the presence of zinc and moves like α2-microglobulins within an electrophoretic field. Hence, it is named zinc-α2-glycoprotein (ZAG).1 The soluble glycoprotein has a molecular weight of 42 kDa. It was first identified in human plasma in association with cachectic states.2 The ZAG structure is similar to that of class I major histocompatibility complex (MHC) molecules, with a groove for binding fatty acids.3
ZAG is expressed and secreted by various cells throughout the body, including keratinocytes and adipocytes.4, 5 In adipose tissue, its expression is inversely related to body fat mass and influenced by diet, suggesting a link with obesity.6 ZAG expression in adipocytes is regulated through the PPAR-γ receptor, and modulated downward by TNF-α and upward by glucocorticoids.4, 7, 8 ZAG has recently emerged as an adipokine involved in lipid mobilization.1, 9 It directly modulates lipid metabolism via β-3- and β-2(or β-1)-adrenergic receptors in rodents and humans, respectively, leading to activation of a cAMP-dependant pathway.1, 10 Increasing the level of cAMP activates hormone sensitive lipase (HSL) that catalyzes the hydrolysis of triglycerides into fatty acids and glycerol (see Figure 1).11
ZAG may also indirectly modulate lipid metabolism via the stimulation of adiponectin and inhibition of leptin secretion by adipocytes.9 In addition, it is increasingly being viewed as a potential target for lipid homeostasis.12 Therefore, the aim of the present study was to identify and test an extract to modulate ZAG protein expression in vitro, and to document the clinical effects of a complex containing that extract on the appearance of cellulite in humans.
Shea Butter Extract Preparation
In searching for a modulator of the ZAG protein, the authors evaluated the related potential of a new original shea butter (SB) extract obtained in a way that bypasses the traditional roasting step. Briefly, shea nuts are mechanically ground and hydrated to obtain a paste that is deeply churned to separate the water from the oil composing the butter. By avoiding high temperatures, this approach is a more sustainable manufacturing process and it supports better working conditions for the women of Burkina Faso in western Africa, who harvest the nuts to produce the extract. It also protects natural wood reserves that are otherwise burned in the process.
Interestingly, this process also was found to preserve viminalol content in the nuts. Viminalol is a triterpene that is normally degraded by heat during the roasting step. The presence of viminalol esters has previously been linked to anti-inflammatory and antioxidant activities.13 Thus, the viminalol esters were concentrated, and their quantity was standardized for this extract. Here, the authors assess the additional role of the esters in modulating ZAG protein expression in vitro. Further, to complete and reinforce the action of the unroasted SB extract, a complex was created including avocado seed extract and bentonite. Also harvested in Burkina Faso, the avocado seed extract is rich in unsaponifiable matter that is capable of supporting healthy connective tissue metabolism and providing anti-inflammatory action.14 Bentonite is a clay mineral valued for its regenerative and wound-healing properties,15 and its highly organized structure makes it an ideal carrier for other ingredients.16 The avocado seed extract and the SB extract were incorporated and entrapped in the bentonite to form a multilayer powder. This complexa was used in the clinical studies described.
Materials and Methods
Stimulation of ZAG protein expression: Normal human keratinocytes were obtained from a 45-year-old Caucasian donor. Cells were cultured to confluence at 37°C in the presence of fetal calf serum (FCS), in a humidified incubator under 5% CO2/95% air atmosphere. Confluent cells were then incubated for 48 hr in the presence or absence of 0.005% or 0.01% unroasted SB extract. At the end of the incubation period, released ZAG protein was quantified in the culture media using a highly sensitive and specific enzyme-link immunoassay (ELISA) kit.
In parallel, cells were lysed by sonication and the total amount of proteins in the lysates determined according to the Bradford method. The quantity of ZAG protein found in the media was expressed as a percentage of total cell protein content and compared to control values. Experiments were performed in triplicate and statistically analyzed using the student’s t-test.
In vitro stimulation of lipolysis: Normal human keratinocytes and adipocytes were derived from an abdominal biopsy and cultured to confluence as previously described. Keratinocytes were then incubated for 48 hr in the presence or absence of 0.001% unroasted SB extract or 0.002% caffeine as a positive control. At the end of the incubation period, supernatants containing secreted ZAG proteins were removed from the keratinocyte cultures and transferred to confluent adipocyte cultures for additional incubation. To allow for the best release, the non-esterified fatty acids (NEFA) were quantified two hours later within the adipocyte cell culture media by spectrophotometric analysis, as a measure of lipolysis status. Experiments were performed in triplicate and statistically analyzed using the student’s t-test.
In vivo anti-cellulite efficacy: The anti-cellulite potential of the shea butter complex was evaluated in a double-blind, placebo-controlled clinical study involving 22 healthy volunteers, ages 28 to 71 years (mean age = 57). All volunteers presented slight to moderate excess weight, with a Body Mass Index (BMI) ranging from 23 to 30, and had signs of cellulite on their thighs. Each volunteer applied a 1% active test gel containing the complex on one thigh and a placebo gel on the other thigh, using enough to cover the area as they would a typical slimming product.
Product application was performed twice daily, morning and night, for 28 days. Results were evaluated at the beginning (D0) and the end (D28) of the study using a scannerb to analyze the cutaneous relief of fat nodes on the thighs. This technology measures the average roughness and maximal amplitude of a surface in μm, as well as the average volume per unit of surface in mm3/mm2. Any reduction in value of one of these parameters was considered an anti-cellulite effect.17 Before-and-after photographs of the thighs were taken for each volunteer.
In vivo firming efficacy: The firming efficacy of the complex was evaluated at D0 and D28, on the same panel of volunteers, using echographyc. With this type of apparatus, ultrasounds are emitted by a probe, in this case a 20–MHz probe, to be partially reflected when hitting the skin. The reflected echoes can be visualized in a two-dimensional color image that provides information on the structures reflecting the ultrasounds. Connective tissue structures appear in green, red and yellow shapes while fat nodules, which do not reflect ultrasounds, are seen as black areas. A decrease in the number of black zones therefore indicates a reduction in fat inclusions with a parallel increase in skin density and firmness.18
In vitro ZAG protein expression: Keratinocytes incubated in the presence of unroasted SB extract for 48 hr showed a dose-dependent stimulation of ZAG protein expression and secretion in cell culture media. Compared to untreated cells, significant (p < 0.005) increases of 15% and 56% were obtained in the presence of 0.005% and 0.01% unroasted SB extract, respectively (see Figure 2a).
In vitro stimulation of lipolysis: Adding supernatants from SB-stimulated keratinocyte cultures to adipocytes and incubating them for 2 hr resulted in the dose-dependent stimulation of NEFA release by fat cells. Compared to untreated cells, a significant (p < 0.005) increase of 60% was obtained in the presence of 0.001% unroasted SB extract. In the same conditions, treatment with 0.002% caffeine, used as a positive control, increased NEFA release by 67% (see Figure 2b).
Anti-cellulite efficacy: Application of the 1% active test complex on the thighs of human volunteers, twice daily for 28 days, improved the appearance of cellulite as documented using the three-dimensional (3D) scanner technology. Compared to the placebo, skin roughness was significantly reduced by 7% on average and up to 19% at p < 0.05; maximum amplitude was reduced by 13% and up to 24% at p < 0.01; and volume was reduced by 13% up to 38%, at p < 0.01.
A reduction in skin roughness was seen in 68% of the participants, whereas 73% saw a reduction in maximum amplitude and 73% reported a reduction in average volume per surface unit (see Figure 3). Globally, a positive anti-cellulite response was documented for 82% of the volunteers. One photograph, representative of the anti-cellulite results obtained with the complex, is presented in Figure 4.
Firming efficacy: Application of the 1% active test complex on the thighs of human volunteers, twice daily for 28 days, also improved skin firmness and density as documented using the frequency echography device. Compared to D0, the ratio of echogenic surface/total surface (dermis density) was significantly increased, on average by 9% (p < 0.005) for the treated areas (see Figure 5). Globally, a positive firming effect was documented for 82% of the volunteers. Placebo treatments resulted in an insignificant reduction of 3%.
As described, shea butter extract was obtained through a non-roasting process, allowing for more sustainable harvesting of shea tree nuts and better preservation of its viminalol ester content. This extract was tested in the search for potential effectors of ZAG expression—a protein recently identified in lipid mobilization.1, 9 In vitro, the extract significantly stimulated ZAG expression and secretion by keratinocytes. In turn, ZAG-enriched supernatants could stimulate NEFA release by adipocytes with efficacy comparable to caffeine, a recognized lipolysis stimulator.19 In vitro results therefore suggested potential benefits for this extract in anti-cellulite applications.
To test this hypothesis, unroasted SB extract was incorporated in a complex also containing an extract of avocado seed, with documented anti-elastase firming action, and bentonite (from clay) acting as a carrier and having regenerative properties.15, 16 The anti-cellulite potential of the complex was clinically tested against a placebo on a panel of 22 volunteers with signs of cellulite on their thighs. Using 3D scanner and echography technologies, positive results were observed within 28 days of twice daily application for 82% of the participants. Benefits included a significant reduction in the volume of fat lobules and a significant increase in dermis density, which translated as a visual improvement in the appearance of cellulite in the treated areas.
The present paper identifies the ZAG protein as a new target to improve the appearance of cellulite and presents a shea butter extract that modulates it. Increasing ZAG protein expression and secretion by keratinocytes has the potential to stimulate the release of free fatty acids from adipocytes, reducing the volume of fat lobules. Also illustrated here is how new and existing cosmetic ingredients can be simply screened for their effects on ZAG using cell culture techniques and protein immunodetection.
Further, ingredients acting on ZAG can be formulated with actives targeting other aspects of cellulite, for optimal results. For instance, ZAG inhibitors would be natural complements to the action of caffeine on adipocytes. Both boost cAMP levels via synergistic pathways; ZAG inhibitors act through β-adrenergic stimulation of cAMP formation, while caffeine blocks adenosine receptors to prevent degradation of the molecule, thus extending its active life.
Lastly, the current work illustrates that strong leads for the cosmetic industry may emerge from basic medical science. Once a pathway of interest for cosmetic applications has been identified, it becomes possible to screen existing and new extracts for potential effectors.
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