Flower Extracts As Multifunctional Dyes in The Cosmetics Industry Part 2

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Many natural products are used in traditional medical systems to treat the relief of symptoms from pain and inflammation, [61] therefore, the effect of the extracts analyzed on the inhibition of lipoxygenase and proteinase activity was investigated. In the range of concentrations analyzed (100-500 ug/mL), CTE water extract showed the strongest ability to inhibit proteinase (Figure 4)(at 57% for a concentration of 500 ug/mL). This activity was compared to the well-known proteinase inhibitor diclofenac, used as a control (about 89%inhibition at the highest concentration tested). However, similar results were obtained for GGE and KTE extract (about 56% and 53%, respectively, for the highest concentrations). Lower proteinase inhibition was observed for PRE and PGE extracts. In a further test measuring the ability to inhibit lipoxygenase, the aqueous extracts from KTE and CTE show the highest values(about 67% and 64% inhibition, respectively, for a concentration of 500 μg/mL）. Diclofenac was also used as a control. GGE， PGE， and PRE extracts also feature significantly high values(60%,57%, and 54%, respectively). It was also noted that the ability to inhibit LOX and proteinase enzymes depends on the extract concentration (Figure 5).

Previous studies indicated that many polyphenolic compounds significantly contributed to the anti-inflammatory activities of many plant extracts[62]. Studies have shown the involvement of ROS in the inflammatory process, and phenolic compounds such as gallic and quinic acid may block arachidonic acid metabolism by inhibiting the activity of lipoxygenase activity, or they may serve as scavenging reactive free radicals, which are produced during arachidonic acid [63]. The results obtained by BenSaad et al. indicate that ellagic acid, gallic acid, and punicalagin A&B isolated from P. granatum inhibited the production of nitric oxide(NO), prostaglandin E2(PGE2), and interleukin 6 (IL-6)in lipopolysaccharide (LPS)-induced RAW 267.4 macrophages. Whether these compounds work as sole agents or have a synergistic effect still remains a question [64]. Since many flavonoids show anti-inflammatory properties, due to their intrinsic antioxidant behavior, they have been implicated in various inflammatory disorders. flavonoid extraction method pdf, In particular, quercetin is the most interesting molecule because it interferes with specific biological pathways. Moreover, studies suggest that it can reduce the inflammatory process involved in several models through different mechanisms[65]. In particular, the AMP-activated protein kinase and the histone/protein deacetylase(AMPK/SIRT1) pathway results in more interesting inflammation management. Thus, AMPK activators can reduce macrophage inflammation. Quercetin and other flavonoids, as activators of AMPK and SIRT1, may reduce inflammation by interfering with this pathway [66]. It has been also shown that quercetin and quercetin monoglucosides exert a higher LOX inhibition potential [67]Nair et al. have shown anti-inflammatory properties of KTE extract by evaluating the presence of flavonols with the quercetin moiety such as manghaslin Qu 3-[2G] rhamnosylrutinoside, Qu 3-O-dirhamnoside, and rutin. These molecules have shown strong inhibition of COX-2 activity and partial ROS suppression. In general, polyphenols present in CTE showed anti-inflammatory properties in LPS-induced inflammation in RAW 264.7 macrophage cells[68].

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2.5.Cytotoxicity Assessment

In creating new cosmetic raw materials, one of the most important properties is their safety of use. Substances dedicated for use in cosmetics must be non-toxic, especially in relation to skin cells, such as keratinocytes and fibroblasts. Two types of tests have been used to determine the toxicity of the analyzed extracts on HaCaT and BJ cells. flavonoids The first study, using the neutral red uptake assay, enables us to assess of the viability of the cells treated with the analyzed extracts. This dye enters the lysosomes of a living cell and is released into the cytoplasm of dead cells. It was observed (Figure 6) that the CTE extract has the highest ability to increase the proliferation of both the HaCaT and the BJ cells. In comparison with the control, this extract achieved about 20% and 40% higher values than the tested parameter at the concentration of 250 μL/mL （HaCaT and B】）and 500 μL/mL（BJ cells）， respectively. The GGE extract at concentrations of 100 and 250 μL/mL and the KTE extract at the concentration of 500 μL/mL were characterized by a little toxic effect on BJ cells. Other extracts had a positive influence on the viability of these cells. The PRE and PGE extracts at a concentration of 100 μL/mL did not differ significantly from the control， and they increased the proliferation of BJ cells by about 10-15% compared to the control at the concentration of 250 and 500 μL/mL. In the case of keratinocytes， no decrease in cell viability was observed. For PGE, PRE, and CTE extracts, an increase in proliferation with increasing concentration was noted, while a decrease in cell viability was demonstrated with increasing concentration of the KTE and GGE extracts.

The second test carried out to determine the cytotoxicity of the tested extracts was the resazurin test(Alamar Blue). It has been shown (Figure 7)that the viability of cells depends on the concentration of the extract with which the cells were incubated. In the case of fibroblasts, the PRE, PGE, and CTE extracts cause higher cell proliferation with an increase in their concentration. At the highest analyzed concentration （500 μL/mL）， about 20% higher proliferation was observed compared with the control. hesperidin uses In the case of KTE and GGE extracts, a decrease in cell viability was observed with an increase in the concentration of extracts. These extracts showed a little toxic effect on BJ at concentrations of 250 and 500 μL/mL. In the case of keratinocytes， a similar effect of the analyzed extracts on the skin cells was observed, but their ability to proliferate was not as strong as in the case of fibroblasts. The highest ability to increase keratinocyte proliferation was observed for the CTE extract in the entire range of analyzed concentrations. Similar values were obtained for the PRE extract at a concentration of 250 μL/mL and for the PGE extract at a concentration of 500 μL/mL. The KTE extract showed a significantly higher toxic effect on HaCa cells than the GGE extract.

The analyzed extracts have not been extensively tested for their toxicity to skin cells before. There are only a few cytotoxicity studies on extracts or their main active ingredients, especially towards cancer cells. The authors of previous studies indicated that these extracts generally do not have a toxic effect on skin cells, and their ability to increase cell proliferation is most often attributed to the high content of polyphenols, anthocyanins, and flavonoids [45,69-73]. Some authors also indicated that the individual components contained in the extracts may exhibit a toxic effect on skin cells, while the extract as a whole does not. Ali Hijazi et al. [69] showed that alkaloids extracted from Punica granatum are toxic to normal and cancer cell lines, while the whole extract is less toxic. The study of Nasiri et al. [70] indicates that Punica granatum flower extract may be useful in accelerating the wound healing process due to its ability to increase the proliferation of skin cells. In our previous research [45], it was shown that the water-ethanolic extracts obtained from the analyzed plants were characterized by a higher ability to increase the proliferation of BJ cells, and the KTE and GGE extracts showed a lower toxic effect on these cells. lost empire cistanche The effect of water-ethanol extracts on HaCaT cells was similar to that of pure aqueous extracts, but in the case of extracts obtained with ethanol, slightly more favorable proliferation properties were observed than in the case of aqueous extracts. The differences between the composition of both types of analyzed extracts may cause differences in their toxicity. As shown, water extracts are not as rich in bioactive ingredients as water-ethanol extracts. The greatest differences are observed in the content of rutin and isoquercitrin.

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2.6.Determination of Sun Protection Factor

The unfavorable effects of UV radiation on the skin may be revealed both shortly after exposure as well as even years later. Solar radiation has an immunosuppressive effect which accelerates the aging process of the skin with all the consequences associated with it, including increased carcinogenesis[74]. The currently observed trends indicate a growing need to develop products characterized not only by a very high level of safety in use but also multifunctionality in the sense that products will feature the skin's anti-radiation protection with a broader scope of action than used so far. Some plant substances play an invaluable role in this aspect, which is able not only to provide sun protection but also to neutralize the already existing negative effects of solar radiation on the skin [75-77]. The conducted research showed that the analyzed plant extracts PRE, PGE, KTE, CTE, and GGE are characterized by high SPF coefficients.

The analysis of the coefficients (SPF) was carried out for the obtained water extracts from the above-mentioned plants at concentrations of 10 and 50 mg/mL. For each investigated plant higher concentration of the extract resulted in a significantly higher value of SPF. micronized purified flavonoid fraction 1000 mg uses Comparison between extracts shows that the highest values of the SPF were observed for the KTE extract, which holds for both investigated concentrations. Another interesting observation is that even in the lower of investigated concentrations the KTE extract still exhibited high SPF, while the values for other extracts decreased noticeably. This is clear when we analyze how much the result for KTE was higher than other extracts. For the concentration of 50 mg/mL, the SPF was higher by the factor of 1.2(when compared to PGE, CTE) to the factor of almost 1.9 (when compared to PRE, GE). The same calculation for the concentration of 10 mg/mL will give factors from 1.4(when compared to PGE) to factors of the range 2-3 (when compared to CTE, GGE), even up to factors like 10 when compared to PRE. When focusing on the KTE extract, one can notice that a decreasing concentration of the extract by factor 5 (from a value of 50 mg/mL to the value of 10 mL/mL)results in a decrease of the SPF value by factor3.4, which means that SPF decreases slower than the concentration. The above shows that the KTE extract can be efficient even when applied in low concentrations (Figure 8).

2.7.Transepidermal Water Loss(TEWL)and Skin Hydration Measurements

Due to the wide range of biological and pharmacological activity of plant raw materials, plant substances contained in extracts have a significant impact on the condition of the skin. In particular, we are talking here about the influence of secondary metabolites on the condition of our skin [78,79].

In the next stage of the research, the analyses of hydration and TEWL were carried out. The effect of the tested extracts on the skin was assessed. Measurements were made at two-time intervals of 60 and 360 min for the extract concentration of 10mg/mL. For the TEWL measurement, the greatest percentage decrease was shown for PGE extract, where the control value of 13.9 fell to 8.71, resulting in a percentage decrease of 37%. It is also worth analyzing the KTE extract from that perspective, as this was the one exhibiting the highest SPF values. For the KTE extract, the TEWL control value decreased to the value of 10.22, which means a decrease of 26% (Figure 9A).

However, in the case of the second instrument measurement, it was shown that the analyzed extracts cause an increase in moisturization in relation to the control sample, both after 60 as well as after 360 min.

As a result of the analyses, it was found that the analyzed PRE, PGE, KTE, CTE, and GGE extracts increase skin hydration (Figure 9B). An increase in moisturizing properties was observed along with an increase in the concentration of the extract in the preparations. The strongest moisturizing properties have been observed for PRE and PGE extracts equaling 32% and 29% after 60 min and equaling 21% and 22% after 360 min. The lowest level of skin hydration, though, has been found for KTE extract equaling 18% after 60 min and close to 3% after 360 min.

2.8. Application Analysis

2.8.1. Determination of the Color Parameters of Extracts

Due to their natural color, the active ingredients of plant flowers can be used as natural dyes in many commercial products, such as cosmetics and food. Color analysis was performed for the obtained extracts (Table 5).

PRE, KTE, and CTE extracts were found to have the highest potential as natural cosmetic pigments. However, the highest chroma values (C*)was observed for CTE. Based on the value of the h°parameter, it was found that it is the yellow color of this extract that is observed and visible to the naked eye. In the case of KTE and PRE extracts, despite the low C* value (2.8), the color of these extracts can be clearly seen with the naked eye and was specified as red-purple for PRE and blue-violet for KTE extract. The water extracts of PGE and GGE were reddish and slightly orange in color, and the obtained chroma values were at the level of 1.3. For this color, these values were not significantly discernible to the naked eye.

2.8.2. Determination of the Color Parameters of Cosmetics Based on the Extracts

In recent years, there has been a strong need to develop new dyes of natural origin, especially in the food and cosmetic industries. Compared to dyes obtained synthetically, they may have a lower negative impact on human health and the environment [80]. The obtained extracts were used in the formulation of a model micellar make-up remover liquid. In each formulation, they were used in a concentration of 1%. The results of the color parameters of model cosmetics are presented in Table 6.

It was observed that the addition of 1% water extracts from PRE, PGE, CTE, and GGE significantly influenced the color of make-up remover. Each sample was clearly visible to the naked eye in color. The PRE extract changed the color of the cosmetic to orange, and the PGE, CTE, and GGE extract changed it to yellow.

The possibility of using the analyzed extracts as potential dyes is confirmed by the relatively high values of △Make-up remover with extract/base make-up remover, which indicates a significant change in the color of model cosmetics with the addition of the extract compared to the base sample (without adding of the extracts). Literature data [73] show that if AE values are higher than 5, the color is perceived by the naked eve and perceived as a color effect. For make-up removers containing PRE, KTE, and CTE extracts, the AE values of 8.83,9.17 and 8.14, respectively, were obtained. In the case of products with the extracts of PGE and GGE, no significant influence in the extract on the color of the preparation was observed. The values of AE are in the range of 2.51-2.82. This means that the difference in color is only discernible to an experienced observer [80,81].

3. Materials and Methods

3.1. Plant Material and Extraction Procedure

The plant material used in the research was dry flowers of P. rhoeas L., P.granatum L., C.ternatea L., C.tinctorius L., and G.globosa L., which were obtained from the local herbal store. The extraction process was performed in an ultrasonic bath (Digital Mgtrasonic Cleaner, Berlin, Germany), which was carried out using the method described by Yang et al.[82]. 10 grams of dry flowers and 100 g of water were used to prepare water extracts of the tested plants. The process was carried out for 20 min at room temperature. The obtained extracts were then collected and filtered three times through Whatman No. 1 filter paper. After filtration, the extracts were evaporated under reduced pressure at 40 °C. A stock solution at the concentration of 100 mg/mL was prepared from the dried extracts and was stored in the dark at 4°C until further analysis. The following abbreviations are used: PRE—Papaver rhoeas extract, PGE—Punica granatum extract, GGE—Gomphrena globosa extract, CTE—Carthamus tinctorius extract, KTE—Clitoria ternatea extract.

3.2. Determination of Bioactive Compounds by HPLC-UV-ESI-MS

The obtained extracts were analyzed to determine their main bioactive compounds using an HPLC(DionexUltiMate 3000 RS Thermo Fisher Scientific, Sunnyvale, CA, USA), coupled with a mass spectrometer(4000 QTRAP, AB Sciex, Concord, ON, Canada), equipped with an electrospray ionization source(ESI) and a triple quadrupole-ion trap mass analyzer. Chromatographic separation was achieved with a gradient reverse-phase system. Furthermore，100×4.6 mm chromatographic column Kinetex 3.5 μm XB-C18 100 A with iso-butyl side chains and with TMS end-capping stationary phase used with a similar composition guard column was purchased from Phenomenex and maintained at 30°C. A binary solvent system comprising 0.1% (o/v) aqueous formic acid as solvent A and methanol as solvent B was used under gradient mode during 19.1 min of the run time. The elution conditions applied were as follow:0.0-15.0 min 25-100% B,15.0-17.0 min 100%B,17.0-17.1 min 100-25% B,17.1-19.1 min 25% B.The flow rate of the mobile phase was 0.6 mL/min and the injection volume was 10 μL. The eluent was monitored by electrospray ion mass spectrometer (ESI-MS) under negative ion mode and scanned from m/z 20 to 1000 Da. For quantification analysis, the triple quadrupole MS detector was working in multiple reaction monitoring (MRM) scan mode. Optimal mass analyzer conditions and the selection of product ions for individual compounds were determined experimentally. For this purpose, standard solutions of investigated compounds (1 ng/mL) in mobile phase composition were introduced using an infusion pump operating in constant sample delivery. After ensuring that the correct precursor ion was selected, declustering potential (DP), entrance potential(EP), collision cell exit potential (CXP), and collision energy(CE)were optimized for each MRM transition (Table S1). Two MRM transitions were monitored, one for quantification and one for confirmation. The MS parameters were set as follows: capillary temperature of 600 C, curtain gas at 35 psi, nebulizer gas at 60 psi, and drying gas at 50 psi. Negative ionization mode source voltage -4500 V was applied for the determination of bioactive compounds. Nitrogen was used as curtain and collision gas. Data analysis was processed with Analyst 1.5.1 software. The identification of selected compounds was done by molecular mass and fragment of anion entries of each individual compound and confirmed by MS2 fragmentation. The identities of nine compounds were determined along with their chemical formula, deprotonated molecular ions, and the characteristic fragment ions for each individual peak. Six compounds were quantified based on the calibration curve generated using peak areas of the most intense MRM transitions of analytical standards. The linearity of the detector response for quantified compounds was demonstrated by injection of calibration standards at eight concentration levels ranging from 0.01 μg/mL to 2μg/mL. Calibration curves were linear with the coefficients of correlation (R) greater than 0.99. In case the samples did not fall in the linear range of the CMS detector, the samples were diluted.

Analytical standards of quinic acid, gallic acid, caffeic acid, caffeoylquinic acids(CQA, two isomers:3- and 5-CQA), and quercetin were purchased from Sigma-Aldrich, St. Louis, MO, USA). All standards used were of analytical grade (2≥99% purity).

Standard stock solutions were prepared by accurately weighing and dissolving 20 mg of each standard in 10 mL LC-MS grade methanol to give a concentration of 2 mg/mL. Serial dilutions of 2.0 μg/mL， 1.5 ug/mL，1.0 μg/mL， 0.5ug/mL，0.1 μg/mL， 0.05 μg/mL，0.02 μg/mL， and 0.01 μg/mL were then made using LC-MS grade methanol solution. The limit of quantitation (LOQ) was defined as 0.01 ug/ml.

LC-MS/MS assay was performed in triplicate. Obtained data were presented as means ± standard deviations.

3.3. Determination of Antioxidant Properties

3.3.1.ABTS·+ Scavenging Assay

First, the ABTS solution was prepared by mixing 19.5 mg ABTS and 3.3 mg potassium persulfate with 7mL phosphate buffer (pH=7.4) and dissolved for 16 h in darkness. Then, the solution was diluted to the absorbance at a level of about 1.0. Absorbances were measured at wavelength 入=734 nm. Next， 20 mL of KTE， PGE， PRE， CTE， and GGE extracts(10,100,250,500 ug/mL)was mixed with 980 mL of diluted ABTSe+ solution and then incubated for 10 min in darkness. In the next step, the absorbance of the prepared samples was measured at 入=734 nm using a UV/VIS spectrophotometer Aquamate Helion (Thermo Fisher Scientific, Waltham, MA, USA). Distilled water was used as a blank. The ABTS+ scavenging was calculated from Equation (1):

where: As—absorbance of the sample; Ac—absorbance of the control sample. Measurements were carried out in triplicate for each extracted sample. The procedure was described by Gawel-Beben et al. [83].

3.3.2.DPPH Radical Scavenging Assay

The ability of the extracts to scavenge free radicals was carried out using the method described by Brand-Williams et al. [84]. It is based on the use of the 1,1-diphenyl-2-picrylhydrazyl （DPPH） radical. First， 33 μL of aqueous solutions of extracts at concentrations of 100ug/mL were mixed with 167 μL methanol solution of DPPH（4 mM） and transferred to a 96-well plate, then mixed by shaking. Afterward, the absorbance of the samples was measured at a wavelength of 517 nm. Measurements were made every5 min for 30 min on a UV-VIS Filter Max入=5 spectrophotometer（Thermo Fisher Scientific， Waltham, MA, USA). Three independent replicates were performed for each extract. Water with a DPPH solution was used as a control. The antioxidant capacity was expressed as a percentage of DPPH inhibition using Equation (2):

where: As—absorbance of the sample; Ac—absorbance of the control sample. Measurements were carried out in triplicate for each extracted sample.

3.3.3.Detection of Intracellular Levels of Reactive Oxygen Species (ROS)

To determine the ability of the analyzed extracts to generate the intracellular production of reactive oxygen species in HaCaT and BJ cells, a fluorogenic H, DCFDA dye was used. This compound has the ability to enter cells by passive diffusion, where it is deacetylated by intracellular esterases to a non-fluorescent compound. If reactive oxygen species are present in the cell, this compound is transformed into highly fluorescent DCF. To determine the intracellular level of ROS in HaCaTs and BJ, cells were seeded in 96-well plates. Then, cells were cultured in an incubator for 24 h. DMEM medium was removed and replaced with 10 μM H2DCFDA（Sigma Aldrich， St.Louis， MO， USA） dissolved in serum-free DMEM medium. HaCaT and BJ cells were incubated in H, DCFDA for 45 min and then incubated with the extracts in the concentrations∶ of 100， 250， and 500 μg/mL. Cells treated with1 mM hydrogen peroxide (H2O2)were used as positive controls. The control samples were cells untreated with the tested extracts. DCF fluorescence was measured every 90 min using a FilterMax F5 microplate reader (Thermo Fisher Scientific) at a maximum excitation of 485 nm and emission spectra of 530 nm [85].

3.4. Assessment of Matrix Metallopeptidases Inhibition

3.4.1.Determination of Anti-Elastase Activity

To determine the possibility of inhibiting matrix metalloproteinase, neutrophil elastase (NE), a fluorometric kit(Abcam, ab118971)was applied. The test was carried out in accordance with the instructions attached to the kit and with the procedure described by Niziol-Lukaszewska et al. [86]. Analyses were performed in a standard 96-well plate with a clear flat bottom. For the analysis, plant extracts in a concentration of 100 and 250 ug/mL were used. Initially, NE enzyme solutions, an NE substrate, and an inhibitor control (SPCK)were prepared according to the instructions. Diluted NE solution was added to all wells and then, test samples, the inhibitor control, and the enzyme control (Assay Buffer) were added to subsequent wells. Afterward, samples were mixed and incubated at 37 °C for 5 min. In the meantime, a reaction mixture was prepared by mixing the Assay Buffer and NE substrate. The mixture was added to each well and mixed thoroughly. Fluorescence was measured immediately at excitation wavelength 入=400 nm and emission 入=505 nm using a microplate reader(FilterMax F5, Thermo Fisher Scientific, Waltham, MA, USA)The ability to inhibit NE activity of the analyzed samples was calculated from Equation (3):

The final result was the arithmetic mean of three independent measurements.

3.4.2. Determination of Anti-Collagenase Activity

To assess the ability of the obtained extracts to inhibit collagenase activity, a fluoro-metric kit(Abcam, Cambridge, UK, ab211108) was applied. The test was carried out in accordance with the instructions attached to the kit and with the procedure described by Niziol-Lukaszewska et al. [86]. Analyses were performed in a standard 96-well plate with a clear flat bottom. For the analysis, plant extracts in a concentration of 100 and 250 μg/mL were used. First， collagenase（COL） was dissolved in a collagenase analysis buffer(CAB). Then, analyzed samples were added to COL and CAB. Inhibitor control samples were prepared by mixing the collagenase inhibitor(1,10-phenanthroline(80 mM)with collagenase and CAB buffer. Enzyme control wells were prepared by mixing diluted COL with CAB. The CAB buffer was used as a background control. Then, samples were incubated at room temperature for 15 min. A reaction mixture was prepared by mixing the collagenase substrate with CAB. The reaction mixture prepared in this way was added to all analyzed samples and mixed thoroughly. Afterward, fluorescence was measured at an excitation wavelength of 490 nm and emission of 520 nm. The measurement was performed in kinetic mode for 60min at 37 C. The ability to inhibit COL activity of obtained extracts was calculated by Equation (4):

3.5. Determination of Anti-Inflammatory Properties

3.5.1. Inhibition of Protein Denaturation

Proteinase inhibitory activity of PRE, PGE, KTE, CTE, and GGEextracts was performed according to the method of Sakat et al.[87], which was modified by Gunathilake et al.[88]. Briefly, the reaction solution (2 mL)consisted of 1 mL of 1% trypsin in 20 mM Tris-HCl buffer(pH7.4) and 1 mL of test sample (0.02 mL extract 0.980 mL water). The solution was incubated (37 °C for5 min), and then 1 mL of 0.8% (w/v) casein was added and the mixture was further incubated for 20 min. At the end of the incubation,2 mL of 70% perchloric acid was added to complete the reaction. The mixture was centrifuged, and the absorbance of the supernatant was measured at 210 nm against the buffer as a blank. The phosphate buffer solution was used as control. The percentage inhibition of protein denaturation was calculated using the following formula:

where A1= absorption of the control sample, and A2= absorption of the test sample.

3.5.2. Inhibition of Lipoxygenase Activity

The ability of obtained extracts to inhibit lipoxygenase activity was determined using the method described by Sarvesvaran et al. 【89】. First，10 μL of plant extracts in different concentrations（100， 250, and 500 ug/mL） were mixed in a 96-well plate with 160 μL of 100 mM PBS and 20 μL of soybean lipoxygenase solution（167 U/mL）. Samples were incubated at 25°C for 10min and after this time 10 μL of sodium linoleic acid was added to initiate the reaction. Then, the absorbance of samples was measured at 234 nm over a period of 3 min in every minute using a FilterMax F5microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). Diclofenac was used as a positive control. The percent of lipoxygenase activity inhibition was calculated from Equation (6):

where: As is the absorbance of the tested sample, Ac is the absorbance of the negative control.

The final result was the arithmetic mean of three independent measurements.

3.6. Cytotoxicity Analysis

3.6.1. Cell Culture

In this study, two skin cell lines were used: normal human keratinocytes(HaCaT)and fibroblasts(BJ).HaCaTs were obtained from CLSCell Lines Service(CLS Cell Lines Service GmbH, Eppelheim, Germany) and BJs from the American Type Culture Collection (Manassas, VA, USA). Cells were grown in Dulbecco's Modification of Eagle's Medium (DMEM, Biological Industries, Cromwell, CO, USA)with sodium pyruvate, L-glutamine, and high glucose content (4.5g/L). The medium was also enriched with 10% fetal bovine serum (Gibco, Waltham, MA, USA) and 1% with antibiotics(100 U/mL penicillin and 1000 ug/mL streptomycin, Gibco) to prevent microbial contamination. Cells were grown in an incubator at 37°C in a humidified atmosphere of 95% air and 5%carbon dioxide.

3.6.2.Alamar Blue Assay

After the cultured cells(HaCaT and BJ) had reached the desired confluence, the DMEM medium was aspirated in the culture flasks. The bottom-attached cells were washed twice with sterile phosphate-buffered saline. The cell layer was detached with trypsin and then the cells were placed in a fresh DMEM medium. Cells were plated in 96-well flat-bottom plates (VWR, Radnor, PE, USA) and after attaching to the bottom of the plates, cells were treated with extracts（100， 250, and 500 μg/mL）. Cells were incubated for 24 h.

The cytotoxicity test was performed with the Alamar Blue assay (Sigma, R7017, Life Technologies, Bleiswijk, The Netherlands). After incubation, a resazurin solution at a concentration of 60 uM was added to the wells, then plates were placed in an incubator at 37°C for 2 h. After this time， fluorescence has been measured（入=570nm）. Each extract concentration was performed in three replications.

3.6.3. Neutral Red Uptake Assay

Neutral Red Uptake Assay is the second test used to determine the cytotoxicity of PRE, PGE, KTE, CTE, and GGE. First,96-well flat-bottom plates were prepared as described in the previous section. After 24 h of exposure of the cells to the extracts, they were aspirated and replaced with neutral red dye(40 ug/mL)and incubated for 2 h. After this time, cells were washed with phosphate-buffered saline. In the next step, decolorizing buffer（150 μL）was added to the wells. Then， the uptake of neutral red dve was determined by measuring the optical density (OD) at 540 nm. Each extract concentration was performed in three replications.

3.7. Determination of Sun Protection Factor (In Vitro)

The sun protection factor(SPF) was determined by measuring the absorbance of an aqueous solution of extracts in concentrations of 10 ug/mL and 50 μg/mL， within the wavelength range from 290 to 320 nm at 5-nm intervals. From the obtained results, the SPF was calculated from the Mansur Equation [90]: where∶ EE（λ）—erythemal effect spectrum， I（入）—solar intensity spectrum， ABS（入）—absorbance of the sunscreen product， CF—correction factor（=10）， E（N）×I（λ）—values determined by Sayre were used [91].

3.8. Transepidermal Water Loss (TEWL) and Skin Hydration Measurements

TEWL and skin hydration measurements were conducted using a TEWAmeter TM 300 probe and Corneometer CM 825 probe connected to an MPA adapter (Courage+ Khazaka Electronic， Köln， Germany）. Five volunteers participated in the study. On their forearm skin， six areas (2 ×2 cm in size) were marked. An amount of 0.2 mL of the tested plant extracts was applied in five places, the sixth place was the control (not treated with any samples). After 60 and 360 min, the hydration level and TEWL measurements were taken. The final result was the arithmetic mean(from each volunteer)of five independent measurements (skin hydration) and 20 measurements (TEWL).

3.9.Preparation of Model Cosmetics (Make-Up Remover)Containing Extracts

A model cosmetic (make-up remover)was prepared. All the components used were in line with EcoCert and COSMOS requirements. The formulation is shown in Table 7.

The product was produced by mixing the ingredients(from item 1 to item 6) at room temperature until a homogeneous liquid was obtained. In the last step, the pH of the formulation was adjusted. The make-up remover was divided into portions. An amount of 1%of the stock solutions of the extract was added to each portion and mixed thoroughly.

3.10.Determination of the Color Parameters of Extracts and Cosmetics(Make-Up Removers)Containing Extracts

Samples of extracts and cosmetics with extracts were tested at room temperature, 48 h after their preparation. A CHROMA METER CR-400(Konica Minolta, Sensing Inc., Tokyo, Japan) was used to evaluate the color parameters(CIELAB coordinates). The CIELAB system was defined by the International Commission on illumination in 1978. It is based on three color attributes:L*, a*,b, where L* is a brightness variable proportional to the value in the Munsell system, and a* and b* are chromatic coordinates. The a* and b* coordinates indicate positions on the red/green and yellow/blue axes, respectively (+a = red, -a = green; + b = yellow, -b =blue).

Based on the data obtained: L*, a*, and b*, the following color parameters were calculated: chroma (C*) and hue (h). The following equations were used:

4. Conclusions

Based on the obtained results, it can be concluded that the tested plant extracts show several positive features, thanks to which they can be used in the production of cosmetics as their safe and bioactive ingredient. It has been shown that these plants are a rich source of polyphenols, which gives them antioxidant properties. PRE showed the best ability to scavenge free radicals, which is probably because it contains the most polyphenols compared to other plants. PGE and PRE showed the best ability to reduce ROS production in cells. Moreover, plants do not show any cytotoxic activity. All tested extracts showed an inhibitory effect on the elastase and collagenase enzymes, with P. granatum and GGE having the greatest inhibitory effect. This may indicate that these plants can be used in cosmetics as substances that slow down the aging processes. Moreover, the obtained results indicate that these plants have anti-inflammatory properties. In this case, CTE and KTE appeared to be the best. KTE and PGE showed a protective effect against UV radiation, even at low concentrations. At higher concentrations, all plants had a UV protective effect. Furthermore, the plants had a positive effect on skin hydration and reduce transepidermal water loss. PRE, KTE, and CTE extracts may be used as effective colorants in cosmetics products. The model liquid for make-up removal containing the above-mentioned extracts was characterized by an intense and stable color over time. The color of the cosmetics with PGE and GGE extracts can be noticed only by experienced observers, and they are not significantly differing from the blank cosmetic sample, without the addition of the extract. Considering all the obtained results, it can be concluded that Papaver rhoeas, Clitoria ternatea, and Carthamus tinctorius can be successfully used as sources of yellow, orange, blue, and purple dyes in the production of cosmetics that will be safe to use, and, what is more, will have a positive effect on the skin.

This article is extracted from Molecules 2022, 27, 922. https://doi.org/10.3390/molecules27030922 https://www.mdpi.com/journal/molecules