Moringa oleifera L. Extracts as Bioactive Ingredients That Increase Safety of Body Wash Cosmetics

The work attempts to obtain a multifunctional plant extract derived from Moringa tree leaves. Obtained results indicate a strong antioxidant potential of the tested extracts. It was shown that Moringa oleifera leaf extract is a rich source of flavonoid and phenolic compounds. Furthermore, it shows a strong antioxidant activity by scavenging free radicals. In vitro toxicity studies showed that the tested extracts in concentrations up to 5% showed a positive effect on cell proliferation and metabolism and may contribute to the reduction of oxidative stress in cells. It was noted that the tested model formulation of cosmetic (1% SCS) with the addition of different types of extracts might contribute to the reduction of skin irritation and improve the safety of the product.


Introduction
In recent years, the cosmetic industry was one of the fastest growing industries in the world. Strong competition on the cosmetic market and high consumer expectations force manufacturers to look for innovative solutions in every aspect of the product life cycle. Until recently, cosmetic manufacturers obtained the innovative advantage of their products by incorporating new raw materials and ingredients that were less common and not used by the competition. Examples include substances such as hyaluronic acid, peptides, polysaccharides, exotic oils and plant extracts, and snail slime. With time, however, these raw materials became very popular in cosmetic manufacturing and the solutions became outdated. e innovativeness of cosmetics can also be generated through the form in which skincare and beauty products are offered. Increasingly, novel forms such as foams, jellies, creams, or essences are commercially available. However, after the launch of cosmetics in innovative forms, the market tends to be almost flooded by products of the same type offered by various manufacturers. Consequently, the innovative advantage of such solutions is shortlived [1][2][3][4][5].
In the last few years, there has been a new trend in the cosmetic market, involving the formulation of innovative products on the basis of multifunctional ingredients. ese types of substances are characterized by multidirectional activity, combining biologically active properties with moisturizing effects and the ability to give cosmetics an appropriate form or improve their safety to people and the environment. e last of the abovementioned properties are particularly sought after by present-day consumers. e strong trend for "naturalness" in cosmetics has contributed to an increase in consumer awareness with regard to substances used in cosmetic production. Consumers are looking for products which-in addition to delivering the desired usually multifaceted activity-are safe for people and the environment and are able to reduce adverse environmental impacts on the skin (antismog, antipollution cosmetics). Examples of such ingredients include plant extracts that, as has been shown in many studies, can be used as multifunctional cosmetic raw materials with moisturizing, soothing, antiwrinkle, and antioxidant properties and minimizing the adverse effects of other ingredients of the cosmetic product on the skin. e above characteristics result from the complex chemical composition of plant extracts that represent solutions of active substances derived from plants in a suitable solvent [6][7][8][9][10][11][12][13].
In the course of research in this area, much attention has been focused on the Moringa tree (Moringa oleifera L.), also called the tree of life, as a source of active ingredients valuable for the cosmetic industry. Due to the presence of a broad spectrum of bioactive compounds, the plant has powerful antioxidant, antibacterial, toning, astringent, and anti-inflammatory properties [14][15][16][17][18][19][20][21][22][23]. Leaves of the Moringa tree have been found to contain flavonoids including myricetin, quercetin, kaempferol, isorhamnetin, or rutin, as well as phenolic acids. Fresh leaves are a good source of carotenoids such as lutein, β-carotene, and zeaxanthin. In addition, the Moringa tree is characterized by a high content of vitamins C and A. e active substances contained in the plant have shown to have beneficial effects on human skin and successfully replace the synthetic ingredients [20][21][22][23][24][25][26][27]. e present study was an attempt to evaluate the effect of Moringa oleifera leaf extracts on the irritant potential of body wash gels. For the purpose of the study, a technology for obtaining extracts in the process of solvent extraction was developed. Water and mixtures of water with glycerin were used as natural extraction solvents. e extracts obtained this way were used for further analysis to determine their basic biochemical properties: the ability to neutralize free radicals and the content of polyphenols and flavonoids. Analyses of cytotoxicity as well as the intracellular level of reactive oxygen species were performed on in vitro model: keratinocytes (HaCaT) and fibroblasts (BJ) cell lines. In addition, the model cosmetics with applied extracts were also analyzed on cell lines.

Extract Derivation Method.
Plant material was obtained commercially from Dary Natury-a Polish producer and distributor of herbs. Moringa tree leaves (Moringa oleifera L.) were collected on controlled and ecological crops in India and dried with warm air at 40°C for two days. e plant material has been confirmed by the distributor and Dr. Zofia Nizioł Łukaszewska (specializing in pharmacognosy). A voucher specimen of Moringa oleifera L. is stored in the Department of Technology of Cosmetic and Pharmaceutical Products (University of Information Technology and Management in Rzeszow, Poland). Extraction of bioactive compounds from dried leaves was carried out using the ultrasound-assisted extraction method. 5 g of grounded plant material and 100 mL of water with glycerin in various proportions (50/50, 60/40, and 80/20) were used to obtain the extract. Extraction was carried out for 10 cycles of 10 min at room temperature. en, the obtained extracts were collected and filtered through Whatman filter paper No. 10. e material was stored in the dark at 4°C for further analysis.

Identification of Active Component. ESI-MS detection
was performed in a 4000 QTRAP Mass spectrometer (Sciex, USA) equipped with an electrospray ionization source (ESI) and a triple quadrupole-ion trap mass analyzer that was controlled by the Analyst 1.5 software. ESI worked in the negative ion mode at the following conditions: curtain gas at 20 psi, nebulizer gas at 10 psi, and negative ionization mode source voltage−4500 V. Nitrogen was used as curtain and collision gas. MS 2 fragmentation was used to confirm the structure of the detected compounds. e extracts of Moringa oleifera were diluted 100x with methanol and infused directly with a rate of 10 µl/min via a syringe pump to a mass spectrometer (ESI-MS) and scanned from m/z 50 to 1000 Da under negative ion mode. High-performance liquid chromatography with tandem mass spectrometry (HPLC-ESI-MS) was used for the study of Moringa oleifera extracts. HPLC separation was performed on Dionex UltiMate 3000 RS Chromatograph system with computer program Chromeleon version 6.80. e eluent was monitored by electrospray ion mass spectrometer (ESI-MS) under negative ion mode and scanned from m/z 50 to 1000. HPLC separation was performed on a Kinetex 3.5 µm XB-C18 100Å column provided by Phenomenex. e mobile phases were composed of eluents A and B in gradient, where A was 0.1% (v/v) formic acid in water and B was methanol, with a flow rate of 0.6 ml/min. e elution conditions applied were as follows: e eluent was monitored by electrospray ion mass spectrometer (ESI-MS) under negative ion mode and scanned from m/z 20 to 1000 Da. Data analysis was processed with Analyst 1.5.1 software. e identification of selected compounds was done by molecular mass and fragment of anion entries of each individual compound and confirmed by MS 2 fragmentation. e identities of 14 compounds were determined along with their chemical formula, deprotonated molecular ions, and the characteristic fragment ions for each individual peaks.

Total Phenolic Content Determination.
e total phenolic content of Moringa oleifera L. leaf extracts was determined spectrophotometrically by the Folin-Ciocalteu method according to the procedure reported by Singleton et al. with some modifications [28]. 300 μL of leaf extract solutions and 1500 μL of 1 : 10 Folin-Ciocalteu reagent were mixed, and after 6 minutes in the dark, 1200 μL of sodium carbonate (7.5%) was added. After 2 h of incubation in the dark at room temperature, the absorbance at 740 nm was measured spectrophotometrically by Aquamate Helion ( ermo Scientific). e total phenolic concentration was calculated from a gallic acid (GA) calibration curve (10-100 mg L −1 ). Data were expressed as gallic acid equivalents (GA)·g −1 of extract averaged from three measurements.

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Total Flavonoid Content Determination.
e total flavonoid content of plant extracts was evaluated using aluminium nitrate nonahydrate according to the procedure reported by Woisky and Salatino with modifications [29]. 600 μL of plant extract solutions and 2400 μL of mixture (80% C 2 H 5 OH, 10% Al (NO 3 ) 3 × 9 H 2 O, and 1 M C 2 H 3 KO 2 ) were mixed. After 40 min of incubation at room temperature, the absorbance at 415 nm was measured spectrophotometrically by Aquamate Helion ( ermo Scientific). e total flavonoid concentration in extracts was calculated from a quercetin hydrate (Qu) calibration curve (10-100 mg·mL −1 ) and expressed as quercetin equivalents (Qu)·g −1 of extract averaged from three independent measurements.

DPPH Radical Scavenging
Assay. Antioxidant activity of the plant extract was analyzed using DPPH free radical scavenging assay, according to the method described in [30]. 167 µL of 4 mM ethanol solution of DPPH was mixed with 33 µL analyzed samples in different concentrations (250 µg·ml −1 -5000 µg·ml −1 ). e absorbance was measured at λ � 516 nm every 5 minutes for 30 minutes using UV-Vis spectrophotometer FilterMax 5 ( ermo Scientific). DPPH solution mixed with an equal volume of distilled water was served as a control. e percentage of the DPPH radical scavenging was calculated using the following equation: (1)

Zein
Test. e irritant potential of the products (1% SCS with different concentrations of the extracts) was measured using the zein test [10][11][12]. 40 mL of the sample solution with 2 ± 0.05 g of zein from corn was shaken on a shaker with a water bath (60 min at 35°C). en, the solutions were filtered on Whatman No. 1 filters and centrifuged at 6720g for 10 min. e nitrogen content in the solutions was determined by the Kjeldahl method. 1 mL of the centrifuged filtrate was mineralized in sulfuric acid (98%) containing copper sulfate pentahydrate and potassium sulfate. e mineralized solution was transferred (with 50 mL of Milli-Q water) into the flask of the Wagner-Parnas apparatus. 20 mL of 25 wt% sodium hydroxide was added. e released ammonia was distilled with steam. Ammonia was bound by sulfuric acid (5 mL of 0.1 N H 2 SO 4 ) in the receiver of the Wagner-Parnas apparatus. e unbound sulfuric acid was titrated with 0.1 N sodium hydroxide. Tashiro solution was used as an indicator. e zein number (ZN) was calculated from the following equation: where V1 is the volume (cm 3 ) of sodium hydroxide used for titration of the sample. e final result was the arithmetic mean of five independent measurements.

Cell Viability
Assay. e resazurin sodium salt reduction assay was used to assess cell viability. e assay was performed according to Ivanov et al. with some modifications [33]. Cells were placed in 96-well plates at a density of 1 × 10 4 cells/well with fresh medium. After 24 h of preculture, the medium was aspirated and various concentrations (10,5,3, and 1%) of tested extracts were added into each well and cultured for another 24 h. e control group was nontreated cells. After the time of exposure, resazurin salt solution (Sigma, R7017) was transferred into the plates for a final volume of 250 µL/ well and a concentration of 60 µM in medium and incubated for 3 h at 37°C in darkness. e absorbance was measured at the wavelength λ � 570 nm using a microplate reader Filter-Max F5 ( ermo Fisher Scientific). e experiments were performed in triplicate for each extract concentration and presented as a percentage of control values.
To evaluate if model cosmetic formulation (1% SCS) containing various concentrations (10, 5, 3, and 1%) of Moringa oleifera leaf extract has an effect on cell viability, the resazurin sodium salt reduction assay was used. BJ fibroblasts and keratinocytes were seeded in transparent 96-well plates at a density of 1 × 10 4 cells/well with fresh medium. After 24 h of preincubation, cells were exposed to 30 min of model cosmetic with different concentrations of Moringa tree leaf extract. e control group was nontreated cells . After the time of exposure, resazurin salt solution (Sigma, R7017) was transferred into the wells for a final volume of 250 µL/well and a concentration of 60 µM in medium and incubated for 3 h at 37°C in darkness. e absorbance was measured at the wavelength λ � 570 nm using a microplate reader (FilterMax F5, Molecular Devices). e experiments were performed in triplicate for each tested substance concentration and presented as a percentage of control values.

Measurement of DCF Fluorescence.
To measure the intracellular level of reactive oxygen species in HaCaT and fibroblasts cells, the fluorogenic dye H 2 DCFDA was used.

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After passive diffusion into the cells, H 2 DCFDA was deacetylated by intracellular esterases into the nonfluorescent compound that upon oxidation by ROS is converted to the highly fluorescent 2′, 7′-dichlorofluorescein (DCF) [34]. e fluorescence was measured according to the protocol previously described in [35]. Cells were seeded in 96-well plates at a density of 1 × 10 4 cells per well and initially cultured before the experiment for 24 h. After this, the culture medium was changed on 10 µM H 2 DCFDA (Sigma) in serum-free medium (DMEM or MEM for HaCaT and fibroblasts, respectively). Cells were incubated with H 2 DCFDA for 45 min before treatment. After this time, intracellular oxidative stress as well as production of ROS was induced by the addition of hydrogen peroxide (H 2 O 2 ) to the cells at a final concentration of 1 mM in PBS for 1 h. en, the medium was changed and cells were exposed to different M. oleifera leaf extract concentrations (10, 5, 3, and 1%). e control group was unexposed cells and cells treated with 1 mM hydrogen peroxide (H 2 O 2 ) were used as a positive control. e DCF fluorescence was measured after 90 min of incubation, using a microplate reader FilterMax F5 ( ermo Fisher Scientific) at a maximum excitation of 485 nm and emission spectra of 530 nm.
To assay the capacity of model cosmetic formulation (1% SCS) containing various concentrations (5, 3, and 1%) of M. oleifera leaf extract to generate the intracellular level of reactive oxygen species, fluorogenic dye H2DCFDA was also used. HaCaT and BJ fibroblasts were seeded in 96-well plates at a density of 1 × 10 4 cells per well and initially cultured for 24 h. After this, the culture medium was changed on 10 µM H 2 DCFDA (Sigma) in serum-free medium (DMEM or MEM for HaCaT and fibroblasts, respectively). Cells were incubated with H 2 DCFDA for 45 min before treatment. After this, the medium was changed and cells were exposed to 1% SCS with different extract concentrations. e control group was untreated cells and cells treated with 1% of sodium coco sulfate (SCS) were used as a positive control. e DCF fluorescence was measured every 30 min for a total of 90 min using a microplate reader FilterMax F5 ( ermo Fisher Scientific) at a maximum excitation of 485 nm and emission spectra of 530 nm.

Statistical Analysis.
Each value is the mean of three or five independent measurements. e values of the analyzed parameters were expressed as the mean ± standard deviation (SD). e two-way analysis of variance (ANOVA) and Bonferroni posttest between groups were performed at a p value of <0.05 to evaluate the significance of differences between values. Statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego CA).

Total Phenolic and Total Flavonoid Content
Determination. To confirm the previous result that M. oleifera is a rich source of antioxidant compounds, in this work an attempt was made to determine total phenolic content (TPC) and total flavonoid content (TFC). e amounts of these compounds were assayed from the calibration curves of gallic acid (y � 0.0046x + 0.0452, R 2 � 0.9989) and quercetin (y � 0.0153x − 0.0053, R 2 � 0.9996), respectively. e analysis was performed for three different dilutions (50%, 25%, and 12.5%) of each extract. e obtained results show that the highest amount of phenols and flavonoids was characterized by 50 : 50 (vol/vol) aqueous/glycerin extract, while the lowest concentration of these compounds was characterized by 80 : 20 (vol/vol) aqueous/glycerin extract (Figure 1). e difference between the highest and the lowest TPC values for different types of 50% extract dilution was about 24%. Considering the TFC value, it was nearly 37%. It was also observed that the TPC and TFC were increasing in a dose-dependent manner for all tested types of Moringa oleifera leaf extracts.

Identification of Active Components.
e obtained results of HPLC-ESI-MS ( Figure 2) in the negative ion mode revealed the presence of polyphenols, of which flavonoids and phenolic acids were the principal compounds. e observed flavonoids were quercetin and kaempferol derivatives, while phenolic acids were gallic, caffeic, quinic, and chlorogenic. e structures of the detected polyphenols were confirmed by MS 2 fragmentation of selected m/z signals. Table 1 lists polyphenols detected using ESI-MS/MS (ESI-MS analysis spectra are included as Supplementary material (available here)).

DPPH Radical Scavenging Assay.
To determine the antioxidant properties of all tested extracts, three different concentrations 12.5%, 25%, and 50% were used. e measurements were performed every five minutes over 30 min time period. e obtained data have shown that each concentration of the extract has a different ability to reduce free radicals ( Figure 3). e highest ability to scavenge DPPH radical was shown by 50 : 50 (vol/vol) aqueous/glycerin extract at the highest tested concentration (50%). After 30 min of incubation, the level of reduced DPPH• was above 56%, while in the lowest concentration (12.5%), the level of scavenged radicals oscillated at 26%. Moringa tree 60 : 40 (vol/vol) aqueous/glycerin leaf extract was characterized by middling antioxidant ability in comparison with other extracts. e highest reducing power of this extract was observed for 50% of concentration and it was about 41% after 30 min of incubation. Considering the 80 : 20 (vol/vol) aqueous/glycerin extract, it has the lowest antioxidant potential. In the highest concentration, the ability to scavenge DPPH radical was only on 35% level. Furthermore, a relationship between used concentration and antioxidant potential of extracts was observed. When the concentration was increasing, the free radical reducing power was higher. e order of free radical scavenging capacity was as follows: 50 : 50 (vol/vol) > 60 : 40 (vol/vol) > 80 : 20 (vol/vol) aqueous/glycerin.

Cell Viability Assay.
e resazurin sodium salt reduction assay was used to assess cell viability. e obtained results indicate the cell-specific effect of the analyzed extracts on cell 4 Dermatology Research and Practice viability ( Figure 4). In HaCaT cells, all tested concentrations as well as types of extracts showed a stimulating effect on cell viability. e highest difference between control and treated cells was observed for all types of 1% concentration of extracts. e best ability on increasing cell viability was shown for 60 : 40 (vol/vol) aqueous/glycerin extracts. It also has been observed that the cell proliferation was increasing in a dose-dependent manner. In turn, in BJ fibroblast, extracts showed both inhibitory and stimulatory effects. e highest concentrations (10% and 5%) of all tested extracts showed antiproliferative effect. For 10% of extract concentration, the fibroblast viability was observed on 20%, 37%, and 45% levels for 80 : 20 (v/v), 60 : 40 (v/v), and 50 : 50 (v/v), respectively, in comparison with the control. As well as, in HaCaT, the 1% of extract concentration has shown the most positive effect on cell viability. e highest response was observed for 50 : 50 (v/v) aqueous/glycerin extract and it was about 157%, in regard to the untreated group. e less cytotoxic potential was noted for 80 : 20 (v/ v) M. oleifera leaf extract. It was also noticed that when the extract concentration was decreased, the cell viability increased.        Figure 6). To assay the capacity of model cosmetic formulation (1% SCS) containing various concentrations (5, 3, and 1%) of M. oleifera leaf extract to generate intracellular level of reactive oxygen species, fluorogenic dye H2DCFDA was also used. e reactive oxygen species production was measured by the use of H 2 DCFDA method. As the results showed, the 60 : 40 (v/v) and 80 : 20 (v/v) aqueous/glycerin Moringa tree leaf extract did not significantly generate oxidative stress in HaCaT cells. e highest ROS production was exhibited by 1% SCS. In addition, the most intracellular increase in ROS level was noticed for 1% SCS + 5% 50 : 50 (v/v) extract and it was twofold higher than that of the nontreated group. Values  for other types of extracts and concentrations oscillate about the control. It was also observed that when the extract concentration decreased, the amount of ROS in cells was also lower (Figure 7). Considering the results of BJ fibroblast, it has been shown that the highest intracellular level of reactive oxygen production was for 1% SCS + 5%

Zein Test.
To assess the irritant potential of the products, the zein test was used. e results showed that the M. oleifera leaf extracts might improve the safety of use of the model washing system in terms of its effect on the skin. e addition of the analyzed extracts to 1% SCS causes a decrease in the value of the zein number and thus reduces skin irritation potential. e value of the measured parameter in the base sample was at 451 mgN/100 mL. e test results are presented in Figure 9. e skin irritation potential of the samples with the addition of extracts was about 10-20% lower than the baseline. It was observed that the extract concentrations used did not significantly affect the value of

Discussion
e major group of phytochemicals contributing to the antioxidant capacity of plant material includes polyphenols, carotenoids, and vitamins C and E. ere are several previous studies, which show that M. oleifera leaves are rich source of bioactive compounds with antioxidant properties. It has been reported that Moringa tree leaves have sufficient amounts of quercetin, kaempferol, β-carotene, α-tocopherol, and vitamin A [36,37].
With regard to these findings, the present investigation was undertaken to evaluate the antioxidant capacity of aqueous/glycerin extracts of M. oleifera leaves. We were shown that leaf extract exhibited a high content of phenols and flavonoids. e obtained results show that the highest amount of phenols and flavonoids was found in 50 : 50 (vol/ vol) aqueous/glycerin extract, while the lowest concentration of these compounds was found in 80 : 20 (vol/vol) aqueous/ glycerin extract. As it was previously mentioned, the most important properties of phenolic compounds derived from plant material are their antioxidant properties. e phenols consist of a hydroxyl group and play an essential role in the antioxidant ability by donating hydrogen and forming stable radical intermediates [38]. e mechanism of action of phenols mainly relies on neutralization of free radicals, chelation of metal ions, and induction of dismutase enzymes, as well as peroxidases [39]. e phytochemical profiles of Moringa oleifera were studied by ESI-MS. Via the screening analysis, the presence of polyphenols in investigated Moringa oleifera extracts was identified of which phenolic acids and flavonoids were the principal compounds (Table 1)    Dermatology Research and Practice Phytochemical analysis with ESI-MS was conducted to correlate the antioxidant activities with chemical compositions of Moringa oleifera. e results indicated that leaves of Moringa oleifera are rich in phenolic compounds and thus have a potential to be used as source of components with antioxidant activities. e high amounts of phenolic compounds in Moringa oleifera as a potential source of phytochemicals were also pointed out byÖzcan et al. [20]. e next stage of this work was to evaluate the ability of M. oleifera leaf extracts to scavenge free radicals. For this, we used DPPH• reducing assay, where changes of color solution are directly linked with the decrease of absorption values and the number of formed DPPH radicals [40]. Results from DPPH• scavenging assay obtained in this paper correspond to the other research. Atawodi et al. [36] in their work indicated on strong antioxidant activity of ethanolic extracts from different parts M. oleifera ethanolic extracts. Using the 2-deoxyguanosine assay model, he has shown that the highest free radical scavenging activity was characterized by leaves. Remarkable antioxidant activity of drumstick leaves might be possessed due to the presence of quercetin and kaempferol as well as chlorogenic acids and their derivatives, which were detected in this part of the plant [36]. In addition, it was hypothesized that antioxidant activity of M. oleifera leaf extracts could be accomplished through donating protons as well as reductones, which exert activity by breaking the free radical chain [41]. e biological activity of leaves of Moringa oleifera was also investigated on cells as in vitro model. e cytotoxicity of tested extracts was assessed on HaCaT and BJ fibroblast cell lines using resazurin method. Both cell types were treated with various extract concentrations, ranging from 1% up to 10% in the cultured medium. e obtained results indicate the cell-specific and dose-dependent effect of the analyzed extracts on cell viability. Whereas in HaCaT cells all tested concentrations as well as types of extracts showed stimulating effect on cell viability, in fibroblasts extracts showed both inhibitory and stimulatory effects. e equal connection in cell viability in response to increasing concentration of M. oleifera leaf extracts was also observed by other researchers. e antiproliferative effect was shown in KB (tumor cells), HEK-293 (human lung carcinoma), and A549 cells (human embryonic kidney cells). ese papers indicated that antiproliferative effect was associated with induction of apoptosis, morphological changes, and DNA fragmentation [37,42,43]. e mechanism of cell death may be reflected in the above reports; however, to be confirmed, a more thorough analysis should be carried out. In the next stage of this experiment, it has been evaluated if M. oleifera leaf extracts could inhibit H 2 O 2 -induced ROS production on in vitro cell models. As a substrate to determine the intracellular formation of ROS generation, H 2 DCFDA assay was used. As is recommended, we examined whether plant extracts without cells affected the fluorescence of the H 2 DCFDA [44]. Additionally, the separated experiment showed that there were no interactions between plant extracts and H 2 DCFDA substrate in DMEM or MEM. is experiment has shown that M. oleifera leaf extract possess antioxidant properties. e tested extracts were able to significantly decrease the high number of intracellular ROS to the level oscillating to the control group, which were untreated cells. e high ability of extracts to reduce oxidative stress might be correlated with rich content of phenols and flavonoids. According to Vongsak et al., it was directly indicated that major bioactive substances present in leaf extracts that provide strong inhibition of H 2 O 2 -induced oxidative stress are cryptochlorogenic acid and isoquercetin [42]. Given the antioxidant nature of tested substances, based on the previous experience and available information, a model washing gel formula containing M. oleifera leaf extract was prepared. Resazurin reduction assay has been used to check if model cosmetic formulation (1% sodium coco sulfate-SCS) with various concentrations (5,3, and 1%) of tested extracts has an effect on cell viability. e obtained results indicated that the use of the extract in the cosmetic formula is not dangerous to human skin cells and additionally introduces the desired properties such as antioxidant activity. In order to further confirm the above reports, the ability of model cosmetic formulation containing M. oleifera leaf extract to generate intracellular ROS in keratinocytes and fibroblast was assayed. e reactive oxygen species production was measured using the H 2 DCFDA method. e above findings correlate with the cell viability assay, and therefore, it seems that all types of tested extracts are a desirable plant material with remarkable antioxidant activity. ese features can protect it from aging and other diseases, where the reactive oxygen plays major role. Moringa tree leaf extracts were also tested by evaluating its ability to reduce the irritant potential of sodium coco sulfate (SCS), the anionic surfactant used in the formulation of body wash cosmetics. For this purpose, four samples constituting model washing systems were prepared. Each sample contained 1 wt% of SCS combined with 1, 3, and 5 wt % of the extracts prepared with different ratios of water and glycerin as extractant.
e reference (baseline) sample contained 1 wt% solution of SCS without any extract. e pH of each sample was adjusted with 25% citric acid to a value of 5.5, the physiological pH value of the skin.
e irritant potential of model body wash systems was analyzed by zein value measurements. In the surfactants, solution zein protein is denatured and then is solubilized in the solution. is process simulates the behavior of surfactants in relation to the skin proteins. According to the literature [2-4, 7, 8, 10-13, 45, 46], the sample should be classified as highly irritating for the value of the zein number exceeding 400 mgN/100 mL. e skin irritation potential of the samples with the addition of extracts was about 10-20% lower than the baseline. It was observed that the extract concentrations used did not affect the value of zein number, and the differences noted between the samples were within the margin of error. e decrease in the zein number following the application of the extract to the model cosmetic formula might be the result of the presence of active substances in the extracts. In aqueous solutions and at low concentrations, surfactants occur in the form of individual particles referred as monomers [2,4,5,[10][11][12][13]. When specific concentration unique to a compound is reached, referred as the critical micelle concentration (CMC), the micellar aggregates start to appear in surfactant solutions.
Dermatology Research and Practice e irritation potential is significantly correlated with the type and concentration of the surface active agent. e highest skin irritation ability is attributed to anionic surfactants and surfactants present in the form of monomers. ey demonstrate the capacity to form strong and longlasting bonds with epidermal proteins. Following their ability to bind with proteins, surfactants may cause denaturation and elimination (elution) proteins from the skin, which results as a cutaneous irritation. Available data indicate that the irritant potential might be reduced by bindings of monomers with various types of substances including peptides, polymers, polysaccharides, and mineral salts, where all of them were found in plant extracts [2,4,5,[10][11][12][13][45][46][47][48][49][50][51][52]. Another factor which is potentially contributing to a reduction of irritant potential is the stabilization of micelles formed in solutions. Micelles are thermodynamically unstable aggregates, which constantly disintegrates and releases monomers into the volume phase of the solution. Stabilization of micelles in the presence of plant extracts may take place through the incorporation of such substances as proteins, polyphenols, and flavonoids, as well as solvents used in the extraction process (glycerin and glycols) into their structure [2,4,5,[10][11][12][13][46][47][48][49][50][51][52][53]. e effect of Moringa extracts on the irritation potential of surfactants has not been studied before. Our previous research indicates that plant extracts may reduce the irritation potential of cleansing cosmetics and surfactants. It has been shown that the addition of dogwood extracts [13], natural saponins [11], blackcurrant [8], or chicory [54] reduces the irritation potential of cleansing cosmetics and anionic surfactants.

Conclusions
In the present paper, an attempt was made to determine the properties and the applicability of extracts from Moringa oleifera leaves in model products.
e tested extracts were characterized by a high content of phenolic compounds, flavonoids, and high antioxidant potential. In vitro toxicity studies showed that the tested extracts in concentrations up to 5% showed a positive effect on cell proliferation and metabolism. It has also been shown that the extracts may contribute to the reduction of oxidative stress in cells. It was noted that the tested model formulation of cosmetic (1% SCS) with the addition of different types of extracts in various concentrations does not negatively affect cell metabolism. Analyses defining the ROS level showed that model cosmetic formulation (1% SCS) with the presence of tested extracts does not cause an increase in the formation of intracellular reactive forms of oxygen. To summarize, the results of the research conducted show that application of extracts from the Moringa oleifera leaves to the model cosmetic formulation might contribute to the reduction of skin irritation and improve the safety of the product.

Data Availability
Readers can access the data underlying the findings of this study by contacting the author through tbujak@wsiz.rzeszow.pl.

Conflicts of Interest
e authors declare no conflicts of interest.