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Modifect® EV

Modifect® EV Bioactive is composed of three different ingredients that were carefully chosen based on their chemistry and previously reported biological activity and biophysical properties.

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Properties

Features And Benefits
Ingredient Claims
Renewable
End Use Claims
Anti-aging
Toning
Firming
Antioxidant
Wrinkle reduction
Protection
Detoxifying
Stress reducing
Elasticity
Collagen synthesis enhancement
Smooth feel
Regulatory Status
China Compliant
Product Characteristics
Odor
Characteristic
Shelf Life
24 Months
Formulation Conditions
Use Level
1.0 – 3.0%
Soluble in
Water

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Technical product information

Product Background

A multifunctional cosmetic ingredient designed to impart a more youthful appearance. It helps to detoxify and fortify skin against oxidative damage, showing a reduction of age spots, a more even skin tone and a smoother skin texture.

Reactive oxygen species (ROS) are recognized for their ability to cause damage to essential skin proteins and lipids. Over time, these individual events accumulate and manifest themselves in the slowing down or disruption of essential skin functions. The breaking down of these skin functions, in turn, lead to perceivable and undesirable changes in the visual and tactile attributes of the skin, including wrinkling, loss of firmness and elasticity, dry skin, and hyperpigmentation. the majority of consumers are familiar with external environmental factors raising ROS production, but what most of them don’t know is that ROS also arises from healthy behavior.

Coffea Arabica (Coffee) Seed Extract

One of these ingredients is an aqueous extract of green coffee (Coffea arabica L.) beans press cake. The coffee beans press cake is residual biomass left after extraction of coffee bean oil from coffee beans. It has been reported to be rich in bioactive components, such as chlorogenic acids, caffeine, diterpenoid alcohols, and polyphenols which have profound antioxidant activity. While extracts of both green and roasted coffee have been shown to possess skin benefits, the roasting process creates Maillard products, and a study comparing the two extracts revealed that green coffee seed extract may provide additional benefits when compared to its roasted counterpart. Topical application of these bioactive components have been shown to reduce the visible signs of photoaging.

Trehalose

Trehalose is a non-reducing disaccharide comprising two glucose units. Studies have shown that trehalose solutions may protect and stabilize biomolecules exposed to environmental stress. Due to its non-reducing structure, trehalose is not susceptible to Maillard chemistry, and cannot participate in the formation of AGEs. On the contrary, it has been shown that trehalose may be an effective inhibitor of nonenzymatic glycation reactions.

Carnosine

Further anti-glycation activity may be provided by Lcarnosine, a dipeptide of beta-alanine and histidine. Additional reported biological activity includes the regulation of enzyme activity and the inhibition of various oxidative processes. Human fibroblasts cultured in the presence of L-carnosine were rejuvenated, and exhibited fewer characteristics of senescent cells compared to controls cultured without L-carnosine.

Balance of Reactive Oxygen Species (ROS) in Mitochondria

Cellular reactive oxygen species (ROS), such as superoxides; arising either from the exposure to extrinsic stress factors (UV and pollutants), or as a consequence of normal cell function (respiration), are more abundant with increasing chronological age. This is due to an imbalance between ROS creation and sufficient cellular antioxidant production in the mitochondria. In order to maintain tightly controlled ROS levels, mammalian cells utilize multiple defense mechanisms. Within the mitochondria, these processes have been hypothesized to be regulated by a member of the sirtuin family; Sirtuin 3 (SIRT3), which is localized in the organelle. SIRT3 has been proposed as the master regulator of mitochondrial activity in the respiratory chain.

Expression and activity of SIRT3 is modulated by exposure to oxidative stress, resulting in the upregulation of transcription factor FOXO3, triggering an increase in the levels of mitochondrial manganese superoxide dismutase (SOD2), as well as the glutathione and thioredoxin antioxidant systems.These cellular mechanisms are key to maintaining the needed cellular ROS levels, and prompt removal of toxic species

Superoxide content of mitochondria was measured in cultured keratinocytes, in order to elucidate the effect of Modifect® EV Bioactive at 0.1% on viable cells. Results showed that there was a 38% decrease in fluorescence due to the presence of superoxide in cells incubated in media containing 0.1% Modifect® EV Bioactive, compared to baseline levels (Fig. 2b). The decrease in red fluorescence is significant and clearly evident in the representative images shown, as the intensity of the fluorescence signal correlates with the oxidation of the dye by superoxide (Fig. 2a). These results indicate that Modifect® EV Bioactive exhibits antioxidant potential under the conditions tested, and may be used to help reduce mitochondrial superoxide concentration.

Figure 2: Mitochondrial superoxide levels in cultured keratinocytes. (a) Fluorescence staining for superoxide (red) at 400X magnification of keratinocytes incubated with control media, or media containing 0.1% Modifect® EV Bioactive for 48 hours. Detection was carried out using the MitoSOX™ RED Mitochondrial Superoxide Indicator (Thermo Fisher) and images were taken with a Leica DM1000 system. (b) Quantification of fluorescence intensity was performed using ImageJ software. Six images were quantified for each experimental condition. Significance was *p<0.01 for the 0.1% Modifect® EV Bioactive sample compared to baseline levels in media (ANOVA, Dunnett).

In addition to examining the superoxide levels directly, SIRT3 protein levels were also evaluated to further understand the effect of Modifect® EV Bioactive on the mitochondria. When 3% Modifect® EV Bioactive was applied topically to ex vivo skin samples, a significant upregulation (267%) in the fluorescence signal due to increased SIRT3 protein was observed, compared to levels observed in the presence of a control emulsion (Fig. 3b). Consistent with current insights into the role of SIRT3 in the regulation of mitochondrial function, its increased expression in the presence of Modifect® EV Bioactive is expected to contribute to improved cellular antioxidant function.

Figure 3: SIRT3 protein expression in ex vivo human skin. (a) Emulsions both with and without 3% Modifect® EV Bioactive were applied topically at a dose of 12 mg/cm2 to ex vivo human skin explants and incubated at 37°C for 72 hours. Explants that received no topical application of any kind served as a control. All samples were rinsed, fixed with 4% paraformaldehyde (PFA), sectioned, stained for sirtuin 3 (green) and nuclei were counterstained with DAPI (blue). Immunofluorescence of 10 µm sections was imaged at 400X magnification. (b) Quantification of SIRT3 fluorescence signal was performed using Leica Application Suite software. Six images were quantified for each experimental condition. Significance was *p<0.001 for the 3% Modifect® EV Bioactive sample compared to control emulsion (ANOVA, TukeyKramer)

Effect of Oxidative Stress on Nuclear DNA

Because of extensive cellular communication between the mitochondria and the cell nucleus, oxidative stress within the mitochondria is well known to have deleterious effects on nuclear DNA and the resulting gene expression.23 Therefore, the observed benefits of Modifect® EV Bioactive for mitochondrial function may also mitigate the visible effects of DNA damage from environmental stressors such as UVA. This hypothesis was tested by studying the effect of oxidative stress in the presence and absence of Modifect® EV Bioactive. Oxidative stress was modeled by exposing cultured keratinocytes to either UVA irradiation or to the pollutants found in cigarette smoke.

A UVA dose of 5 J/cm2 causes a substantial increase in DNA fragmentation compared to the baseline level observed in cultured keratinocytes (Fig. 4b) as measured in a Comet Assay. This method allows the detection of DNA damage at single cell resolution, when cells are exposed to oxidative stress, embedded in an agarose gel, lysed, the DNA separated using electrophoresis, and incorporating a DNA-specific fluorescent dye to allow visualization and quantitation. The appearance of comet-like “tails”, indicating damaged DNA, is clearly visible in the microscope images as small green points trailing behind the main circular comet head (intact DNA) (Fig. 4a, middle panel). In the presence of 0.1% Modifect® EV Bioactive, the amount of UVA-induced DNA fragmentation detected is nearly reduced to baseline levels, whereas there is an 18% increase in the COMET score in the absence of Modifect® EV Bioactive (Fig. 4b).

Figure 4: DNA fragmentation in cultured keratinocytes. (a) Relative DNA damage visualized using the COMET assay method at 1000X magnification with normal human epidermal keratinocytes incubated with or without 0.1% Modifect® EV Bioactive for 48 hours, in the presence or absence of 5 J/cm2 UVA. The COMET assay was carried out as described by Olive & Banáth, with minor modifications. 24 (b) Quantification of the amount of DNA damage observed in the assay. The data represent the mean of triplicate samples. Significance was *p<0.01 both for the 0.1% Modifect® EV Bioactive sample compared to the UVA-irradiated sample, and for the UVA-irradiated sample vs baseline (ANOVA, Dunnett).

To model the consequences of exposure to pollutants, normal cultured epidermal keratinocytes were exposed to cigarette smoke, and the DNA integrity determined in the presence & absence of 0.1% Modifect® EV Bioactive (Fig. 5). There was a significant increase in the COMET score of cells exposed to pollutants from cigarette smoke compared to baseline control. That increase was 18% lower in the presence of 0.1% Modifect® EV Bioactive (Fig. 5b).

The data generated shows that Modifect® EV Bioactive can help mitigate the visible effects of DNA damage under different types of oxidative stress.

Figure 5: DNA fragmentation in cultured keratinocytes. (a) Relative DNA damage visualized (green) using the Comet assay method at 1000X magnification with normal human epidermal keratinocytes incubated with or without 0.1% Modifect® EV Bioactive Bioactive for 48 hours, in the presence or absence of pollutants from cigarette smoke (EPS - Environmental pollutant stress). The COMET assay was carried out as described by Olive & Banáth, with minor modifications. 24 (b) The data represent the mean of triplicate samples. Significance was *p<0.01 both for the 0.1% Modifect® EV Bioactive Bioactive sample compared to the pollutant-exposed sample, and for the pollutant-exposed sample vs baseline (ANOVA, Dunnett).

Oxidative Stress on Skin in vivo

Effective control of cellular oxidative stress by mitochondria may also have a benefit on the integrity and function of proteins affected by nonenzymatic glycation (NEG). As the site of glucose metabolism, the mitochondria itself is sensitive to varying sugar levels. Although the detailed pathways through which mitochondrial proteins may affect the levels of AGEs and their presence on structural proteins, such as collagen, have not been fully elucidated, a connection has been proposed, possibly involving SIRT3.

The reaction between the sugar and the protein substrate may yield products that are fluorescent AGEs such as pentosidine, or non-fluorescent products such as Ncarboxymethyllysine (CML). These reactions may further lead to intermolecular or intramolecular cross-links which then alter the physical properties of the protein (i.e. collagen or keratin), such as fiber stiffness, enzyme resistance, and ultimately function. Given the potential link between mitochondrial function and glycation, an in vivo study was carried out to elucidate the effect of topically applied Modifect® EV Bioactive.

A blind monadic study was carried out with 20 subjects (19-55 years old) divided into two groups. Subjects received either a control gel (Gel A) or the control gel with 3% Modifect® EV Bioactive added (Gel B); to apply twice daily on designated sites of the forearms for 60 days. Raman spectroscopy was used to evaluate the presence of CML & collagen at Day 0, Day 30 and Day 60.

There was no significant change in CML level, as detected by Raman on the forearm after 30 days of applying either the control gel or the same gel with 3% Modifect® EV Bioactive (Fig. 6). However, at day 60 a 35% decrease in detectable CML was quantified with application of 3% Modifect® EV Bioactive, whereas the CML level remained unchanged with the control gel.

Figure 6: Quantification of changes in CML level detected by Raman spectroscopy in vivo. The effect of 3% Modifect® EV Bioactive (blue bars) and the control gel (grey bars) are shown. Significance was calculated using Anderson-Darling, followed by Student’s t-test.

Changes in collagen were also assessed by monitoring changes in collagen-specific spectra peaks over the duration of the study (Fig. 7). There were small increases in levels of collagen measured after 30 days of applying either the control gel, or the gel containing 3% Modifect® EV Bioactive, but these changes were not significantly different from each other or in comparison to measurements at T0. After 60 days of formulation application, a significant increase of 19% in collagen was observed at sites where gel containing 3% Modifect® EV Bioactive was applied (Fig. 7). There was no such increase with the control formulation. These measurements indicate that topical application of a formulation with 3% Modifect® EV Bioactive may help address some consequences of oxidative stress resulting in nonenzymatic glycation and a reduction in the signs of skin ageing.

Figure 7: Quantification of changes in collagen level in vivo, as measured by Raman spectroscopy. The effect of 3% Modifect® EV Bioactive (blue bars) and the control gel (grey bars) are shown. There is a significant difference between the formula with 3% ModifectEV® and the control gel at T60-T0 (p=0.038).

To confirm the effect of Modifect® EV Bioactive on AGE formation, we followed the formation of fluorescent glycation products in an in vitro system, where a protein or peptide substrate is incubated in the presence of a reducing sugar. The rate at which AGEs are formed depends on the identity of the sugar present in the reaction, with fructose generating glycation products more quickly than glucose, as exhibited by the larger change in fluorescent signal in our experiments (Fig. 8). After 14 days incubation, 3% Modifect® EV Bioactive was able to reduce the formation of fluorescent glycation products by 97% in the presence of glucose, and 99% in the presence of fructose (Fig. 8). There was also slight inhibition observed in the presence of either glucose or fructose, 17% and 11% respectively, with 0.1% Modifect® EV Bioactive.

Figure 8: Nonenzymatic glycation in vitro. GKpeptide was incubated with either 100 mM fructose or 500 mM glucose in 100 mM phosphate buffer (pH 7) at 37° C for up to 21 days. The data shown here represents the change in fluorescence after 14 days. All reactions were carried out in triplicate, and 0.8 mM aminoguanidine (AG) served as a positive control. Significance was *p<0.001, as calculated using the Student’s t-test.

The observed inhibition may be due to the presence of trehalose in the composition of Modifect® EV Bioactive, as this is a non-reducing sugar that may interfere with nonenzymatic glycation reactions.When BSA is used as the protein substrate, in place of GK peptide, in this in vitro system, similar inhibition is observed (data not shown). These data further support the in vivo glycation data described above, and show that Modifect® EV Bioactive reduces levels of a key glycation marker.

Given the multifunctional benefits identified in vitro, ex vivo and in vivo, it was of interest to further examine the effects of Modifect® EV Bioactive on multiple signs of aging: wrinkles, spots and skin tone. A double-blind monadic study was carried out with 59 subjects (45- 69 years old) presenting with periorbital lines and wrinkles and facial age spots.

The subjects were divided in two groups, and given either a control emulsion (Cream A) or control emulsion with 3% Modifect® EV Bioactive added (Cream B) to apply twice daily on the face for 56 days. All were asked to maintain a diary and answered a questionnaire on perceived efficacy of the product after 28 or 56 days of use.

In addition, facial images were taken using a Visia CR imaging system (Canfield Scientific, Inc.) at days T0, T28 and T56. Facial images and were analyzed for wrinkles, skin homogeneity and spots. The comparison between products was performed using Student t-test with unilateral hypothesis. The response variable was the difference with initial timepoint (T14-T0 and T28-T0). The confidence level used on the comparative analysis was 95%. The analysis software packages were MINITAB 14 and XLSTAT 2019.

Application of 3% Modifect® EV Bioactive significantly reduced the appearance of periorbital wrinkles, compared to the control emulsion (Fig. 9). Image analysis using two parameters, the coefficient of visibility and the occupancy rate, revealed a 13.9% and 12.1% reduction, respectively, after 56 days. Application of the control emulsion yielded an improvement of 4.3% in the occupancy rate (Fig. 10), and a 5.4% improvement in the coefficient of visibility (data not shown).

Another common manifestation of skin aging is the appearance of age spots. Analyses of the total surface of facial spots in a region of interest were performed using the images taken with the Visia CR system. When the images were quantified, a statistically significant decrease of 18% in the total surface of spots was observed (Fig. 12), which translates to reduced visibility of spots at T56, compared to T0 after application of 3% Modifect® EV Bioactive (as shown in Fig. 11). Application of the control formulation did not yield an improvement over the same time frame

The reduction in spot visibility was also reflected in an improvement in skin homogeneity. There were statistically significant improvements after both 28 and 56 days of 3% Modifect® EV Bioactive application, when compared to application of the control emulsion (Fig. 13). Therefore, application of the formulation containing 3% Modifect® EV Bioactive leads to the appearance of a more even skin tone.

Figure 13: Quantification of skin homogeneity, as measured by the Haralick parameter. There was a significant difference between the formula with 3% Modifect® EV Bioactive and the control emulsion at T28-T0 (p=0.038) and T56-T0 (p=0.048).

 The topical application of a formulation containing 3% Modifect® EV Bioactive resulted in the improvement of multiple visible aspects of skin aging. Our data shows that it helps to detoxify and fortify skin against oxidative damage, showing a reduction of age spots, a more even skin tone, and a smoother skin texture.

Key Attributes of Modifect® EV Bioactive

In vitro/ Ex vivo tests demonstrate that Modifect® EV Bioactive:

- Increases mitochondrial Sirtuin-3 protein level

- Decreases cellular ROS levels

- Mitigates the visible effects of DNA damage from environmental stressors such as UVA or pollutants such as cigarette smoke

- Decreases the formation of advanced glycation end products (AGEs)

 

Clinical tests demonstrate that Modifect® EV Bioactive:

- Reduces the appearance of fine lines, wrinkles and age spots

- Reduces Carboxymethyl Lysine (CML) and increases collagen levels present in the skin

- Contributes to a healthier skin, which is able to produce higher levels of collagen

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