Flower Extracts As Multifunctional Dyes in The Cosmetics Industry

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Abstract: Flowers are a natural source of bioactive compounds that not only have antioxidant, anti-inflammatory, and anti-aging properties but can also be used as natural dyes. For this reason, nowadays plants are widely used to produce natural cosmetics and foods. In these studies, the properties of the water extracts of Papaver rhoeas L., Punica granatum L., Clitoria ternatea L., Carthamus tinctorius L., and Gomphrena globosa L., as bioactive, natural dyes, were investigated. Plant flower extracts were tested for their antioxidant(ABTS and DPPH radical methods) and anti-inflammatory effects by determining the ability to inhibit the activity of lipoxygenase and proteinase. The extracts were tested for their cytotoxic effect on skin cells, using Alamar Blue and Neutral Red tests. The ability to inhibit the activity of enzymes responsible for the destruction of elastin and collagen was also studied. Research has shown that extracts have no toxic effect on skin cells, are a rich source of antioxidants, and show the ability to inhibit the activity of elastase and collagenase enzymes. P. rhoeas extract showed the strongest antioxidant properties with IC50 values of 24.8± 0.42 ug/mL and 47.5±1.01 ug/mL in ABTS and DPPH tests, respectively. The tested plants are also characterized by an anti-inflammatory property, for which the ability to inhibit lipoxygenase at a level above 80% and proteinase at a level of about 55% was noted. Extracts from P. Thomas, C. ternatea, and C. tinctorius show the strongest coloring ability and can permanently dye cosmetic products, without significant color changes during the storage of the product.

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Keywords: plant extracts; natural dyes; cosmetics; biologically active dyes; anti-inflammatory properties; antioxidants

1. Introduction

In recent years, there has been a growing interest in natural and ecological products. It is especially noticeable in the cosmetics, food, and other fast-moving consumer goods (FMCG) industries. Based on the observation of the current market trends, more and more consumers are looking for natural products, which, in their opinion, are safer in use and more effective. For that reason, producers are forced to look for natural replacements for synthetically derived substances in order to prepare products that meet the requirements of the consumers 1]. cistanche UK A lot of substances like emulsifiers, rheology modifiers, or surfactants were substituted by their natural equivalents, but many raw materials are still a big problem for producers [1]. One of the most problematic ingredients is dyes, for which there are not too many effective solutions in nature. Most of the dyes used by the food or cosmetics industry are continuously produced by chemical synthesis due to their lower cost and higher stability compared to the natural coloring agents.

Cistanche can anti-aging

Unfortunately, synthetic dyes have several disadvantages, the most important of which is their irritating and sensitizing potential as well as their negative impact on the environment [2-5]. Naturally, sourced ones are safer for human health and the environment, but they are not stable and may change color during product storage. They are also sensitive to changes in pH, UV radiation, and changes in temperature [6-8]. Looking for new dyes of natural origin, attention was paid to extracts obtained from plant flowers with a strong color. The plant does in the form of extracts may be more stable and more resistant to color changes due to the presence in their composition, in addition to coloring substances, of ingredients that are able to prevent their oxidation under the influence of external factors, such as UV radiation or the action of free radicals. These are naturally occurring substances, mainly from the group of antioxidants, which can prevent changes in plant color and maintain an intense color even when exposed to strong UV radiation |5,7,9,10]. In the case of isolating individual color substances from plants (like, for example, in the case of betalains extracted from beetroot), the resulting dye is devoid of these components, and in a lot of cases, it is necessary to add synthetic antioxidants to the final product to prevent color changes [11].

Antioxidants are a group of chemical compounds that play an important role in defending against oxidative stress. Their main function is the neutralization of oxygen free radicals, called reactive oxygen species (ROS), which are highly reactive side products of metabolism.

A large proportion of antioxidants are plant-derived substances, which include, for example, phenolic acids or flavonoids. Some plant dyes also show an antioxidant effect, which, due to their properties, could replace synthetic pigments currently used in cosmetics. An example of such compounds can be anthocyanins, which belong to flavonoids. They are present in the leaves, fruits, and flowers of many plants, for example, berries (choke-berries, blackcurrants, blueberries, and others), grapes, red chicory, etc., and give them blue, red, and purple colors [12-14]. Moreover, anthocyanins exhibit anti-inflammatory, antioxidant, and hepatoprotective properties and support the proper functioning of the cardiovascular system [12-14]. The presence of anthocyanins has also been demonstrated in Punica granatum L., Clitoria ternatea L., and Papaver rhoeas L. [15-17]. Betacyanins, which are responsible for the red-violet color, is found in leaves, flowers, roots, plant fruits, and in mushroom caps. These dyes show anti-cancer, antioxidant, and anti-inflammatory properties. Betacyanins include gomphrena I, gomphrena II, and gomphrena III, which are found in Gomphrena globosa L. [18,19]. Another coloring compound that has an antioxidant effect is carthamin. cistanche wirkung It gives the plant organs a red color and is present, among others, in Carthamus tinctorius L. [20,21].

The aim of these studies was to investigate the properties of water extracts from plants that are the source of plant pigments. In preliminary studies, extracts from colorful flowers of 20 different plants were obtained, the use of which in cosmetic products is not prohibited. Among them, five extracts characterized by the strongest color and stability during exposure to UV radiation, changes in the pH of the aqueous solution, and the action of oxidizing agents (hydrogen peroxide) were selected. The extracts with the most preferable properties selected for further research were extracts of Papaver rhoeas L.(PRE), Punica granatum L.(PGE), and Clitoria ternatea L.(KTE), Carthamus tinctorius L.(CTE), and Gomphrena globosa L.(GGE). Bioactive compounds were determined for the listed plant extracts, as well as their antioxidant and anti-inflammatory properties. The extracts were tested for cytotoxic activity on fibroblasts and keratinocytes. The ability to reduce transepidermal water loss(TEWL) and the ability to inhibit the activity of enzymes responsible for the destruction of elastin and collagen were also studied. Obtained extracts were applied in the model make-up remover in micellar liquid form as the bioactive and multifunctional dyes.

2. Results and Discussion

2.1.Determination of Bioactive Compounds by HPLC-ESI-MS/MS

A chromatographic method was developed to deepen the chemical structures of the active compounds. The main phenolic compounds were determined in the negative-ion mode on the basis of the mass-to-charge ratio (m/z)of the detected precursor ions and confirmed by the resulting product ions from MS2 fragmentation using HPLC-ESI-MS/MS. The compounds were identified based on the resulting product ions. The MS data, MS/MS fragmentation profiles, and molecular formula were compared with authentic standards or literature data [22,23. citrus bioflavonoids Table 1 lists the active compounds identified in the water extracts using HPLC-ESI-MS/MS.

The extracted ion chromatograms obtained in the negative-ion mode for investigated extracts in water are presented in a supplementary file. The obtained results of HPLC-ESI MS/MS revealed the presence of polyphenols, of which phenolic acids and flavonoids were a well-represented group. The characterized flavonoids were quercetin and kaempferol derivatives, while phenolic acids were caffeic, quinic, gallic, and caffeoylquinic acids(CQA)with two isomers:3-and 5-CQA. Several other flavonoid glycosides including kaempferol-3-O-rutinoside and kaempferol-3-O-glucoside were also identified in the sample extracts.

Quinic acid, gallic acid, caffeic acid, 3-CQA,5-CQA, and quercetin were quantified based on the calibration curve generated using peak areas of analytical standards in multiple reaction monitoring (MRM) modes. The obtained results are presented in Table 2. Based on the sum of the determined compounds (Table 2), it was found that the aqueous extract of PGE was the most abundant in determined bioactive compounds. Quinic acid was determined in the highest amount in the aqueous extract of CTE, while the aqueous extract of PGE was characterized by the highest content of gallic acid. Caffeic acid was the most abundant compound determined in the KTE extract.

2.2. Determination of Antioxidant Properties

Analysis of the composition of the extracts showed the presence of flavonoids and phenolic compounds, such as quinic acid, gallic acid, quercetin, rutin, caffeic acid, and others. These substances are known for their antioxidant properties, which have been demonstrated in many studies. The antioxidant activity of the extracts was examined in the next part of this research.

The first study was carried out using the ABTSe+ radical. cynomorium benefits From the obtained results, the IC50 point was determined for each of the plant extracts, as shown in Table 3. The lowest IC50 value was shown for PGE extract （24.8 μg/mL）， and it was about 5.4 times slower than the value obtained for GGE, which was the highest. Therefore, PGE showed the best antioxidant capacity. Moreover, PRE and KTE achieved low IC50 values(65.5 and 63.3 ug/mL respectively), which contributes to their good antioxidant effect.

In the next part of the research, the ability of extracts to reduce the production of reactive oxygen species in cells was examined. When the levels of reactive oxygen species in cells exceed the number of antioxidants, it leads to oxidative stress. ROS can damage DNA, proteins, and lipids, which may contribute to the development of diseases and increase the aging process. In these studies, the effects on intracellular ROS production were investigated on fibroblasts and keratinocytes, using fluorogenic H2DCFDA dye. By analyzing the results shown on the graphs (Figure 1A, B), it can be concluded that all tested extracts reduce the amount of ROSin cells. All extracts showed the highest potential to minimize oxidative stress at the concentration of 500ug/mL. In BJ cells, the strongest ability to reduce ROS was shown for PGE and PRE extracts. The fluorescence values for these plant extracts， with a concentration of 500 μg/mL， were around 60% lower than for the cells not treated with extracts(control). In HaCaT cells, the strongest ability to reduce ROS was also shown for PGE and PRE, and the fluorescence was 25-30%lower compared to the control （concentration of 500 μg/mL）. The ability to reduce intracellular oxidative stress in HaCaT cells by KTE， CTE， and GGE at the concentration of 100 μg/mL was similar to the control.

Based on the described results, it can be confirmed that the tested plant extracts have an antioxidant capacity. This is due to the presence of various substances that are capable of neutralizing free radicals. The best antioxidative activity was shown by extracts of PGE and PRE. Water extract of P. rhoeas contained caffeic acid, quinic acid, gallic acid, rutin, and quercetin, which are known for their antioxidant properties [24-29]. Moreover, vitamin C has been shown to be present in the petals of this plant [30]. Vitamin C is an electron donor and by that, it prevents the oxidation of other compounds. As a result, it oxidizes itself, forming a relatively stable free radical. Due to these actions, it reduces oxidative damage [31,32]PRE also contains pigments from the group of anthocyanins [33], which have the ability to scavenge free radicals[34]. The presence of the above-mentioned compounds in PRE gives this plant good antioxidant properties, which were demonstrated in these studies and by other researchers [35,36]. Analysis by HPLC-ESI-MS showed that the PGE water extract contained caffeic acid, quinic acid, quercetin, and kaempferol-O-glucoside. In addition, the flowers of these plants are rich in ellagic acid, ursolic acid, maslinic acid, and Asiatic acid. These substances are known for their antioxidant capacity as well as anti-inflammatory properties [24-2937,38]. It is their presence that makes the extract of this plant show its positive effect on reducing oxidative stress 39,40].CTE contains caffeic acid, quinic acid, gallic acid, caffeoylquinic acids, isoquercetin, quercetin, rutin, and kaempferol-O-glucoside, also anthocyanins that are responsible for its antioxidant properties. Kamkaen and Wilkinson also proved the antioxidant activity of CTE using the DPPH method, obtaining the result for the water extract IC50=1 mg/mL[41]. GGE in addition to phenolic compounds and flavonoids determined using the HPLC-ESI-MS method, also contains betacyanins, which are pigments with antioxidant properties [18,42. Susilaningrum and Wijayanti have shown that ethanol extract of GGE has very strong antioxidant activity (IC50=49.9ug/mL)[43]. CTE contains caffeic acid, quinic acid, gallic acid, caffeoylquinic acids, isoquercetin, quercetin, and kaempferol-O-glucoside, which make this plant exhibit antioxidant capacity.

2.3.Assessment of Matrix Metallopeptidase Inhibition

In order to assess the possibility of using plant extracts in formulations intended to combat the signs of skin aging, an important element is to assess their ability to inhibit the activity of enzymes closely involved in the skin aging processes. The main enzymes whose increased activity leads to the degradation of collagen and elastin fibers, which accelerates skin aging, are collagenase and elastase [44]. As part of this work, the influence of the analyzed extracts from five studied plants on the possibility of statistically significant inhibition of the activity of these metalloproteinases was investigated. As part of the conducted experiments, measurements were made for two concentrations of each of the extracts∶100 and 250 μg/mL and the results are presented in Figures 2 and 3. It was observed that all of the analyzed extracts are able to a greater or lesser degree influence the activity of these enzymes under in vitro conditions. It was noted that at the higher of the tested concentrations, the anti-aging activity was greater. During the measurements of elastase activity, the greatest inhibition was observed for the PGE extract (44.97%), followed by GGE (39.11%), PRE (30.99%), CTE (30.33%), and KTE (27.7%), respectively. In the case of the second enzyme, collagenase, the PGE extract (41.30%) also showed the greatest inhibition, followed by GGE (40.61%), CTE (39.09%), KTE (26.68%), and PRE (21.83%). As part of the analysis, measurements were also made for commonly known inhibitors of these enzymes, SPCK for elastase and 1,10-phenanthroline for collagenase, for which inhibition of 57.88%and 51.84%was observed, respectively. Thus, the inhibition obtained for the analyzed extracts, especially PGE and GGE, indicates that they exhibit only slightly lower activity than the commonly known inhibitors of these metalloproteinases, which may indicate their use in cosmetic and pharmaceutical preparations used against skin aging.

We have already demonstrated the anti-collagenase and anti-elastase activity of the studied plants in previous studies for a different type of extract (water-ethanol)[45]. The activity confirmed in this study also for aqueous extracts indicates that various types of extracts obtained from these plants can be a source of biologically active compounds with anti-aging activity. Chromatographic analyzes of the tested extracts showed the presence of numerous compounds with proven anti-aging properties, such as caffeic acid, quinic acid, gallic acid, quercetin, or rutin. The ability to inhibit skin aging is related to the wide spectrum of action of these compounds, which has been shown in numerous scientific papers. Chiang et al. in their study indicated that caffeic acid can inhibit skin photoaging as a result of UVB radiation by inhibiting metalloproteinases and increasing the production of I-type procollagen [46]. Moreover, Staniforth et al.showed that this phenolic acid can inhibit UVB-induced IL-10 mRNA expression and decrease mitogen-activated protein kinases activation [47]. desert hyacinth The possibility of a slight inhibition of elastase activity by quinic acid was shown in the studies by Shoko et al. [48]. Chaika et al. in their work demonstrated anti-aging properties of gallic acid manifested by inhibition of melanin formation by suppressing the activity of tyrosinase and tyrosinase-related protein-2, high antioxidant properties, and the possibility of inhibiting matrix metalloproteinase-2 [49]. Moreover, Hwang et al. found that this acid reduces skin dryness and limits the formation of wrinkles. This is the result of inhibition of the secretion of matrix metalloproteinase-1 and an increase in the level of elastin, type I procollagen, and transforming growth factor-β1 [50]Other authors have shown that quercetin inhibits the activity of elastase and reduces lipid peroxidation[51,52]. The bioflavonoid rutin is also characterized by a very strong anti-aging effect. As shown by Seong et al., it can increase the expression of type I collagen mRNA and decrease the expression of matrix metallopeptidase 1 mRNA in human dermal fibroblasts. Moreover, rutin can positively affect skin elasticity and significantly reduce the number and length of wrinkles [53]. Thus, the possibility of the interaction of compounds present in the extracts tested in this study on many cellular processes results in the anti-aging properties of these plants. The ability of test plants to inhibit collagenase and elastase activity may involve several mechanisms. This may be related to the interaction of the polyphenolic compounds present in the extracts, mainly their hydroxyl groups, with the enzyme skeleton or side chains, or conformational changes that lead to the inactivation of the enzyme [54,55]The inhibition may also be related to the ability of polyphenolic compounds and flavonoids to chelate metal ions that are found in the active site of metalloproteinases such as elastase and collagenase [56,57].

2.4. Determination of Anti-Inflammatory Properties

Over the past few decades, inflammation has been recognized as a major risk factor for various human diseases. Chronic inflammatory responses are predisposed to a pathological progression of chronic illnesses characterized by infiltration of inflammatory cells, excessive production of cytokines, dysregulation of cellular signaling, and loss of barrier function. Targeting the reduction of chronic inflammation is a beneficial strategy to combat several human diseases. Proteinases and lipoxygenases are the enzymes that take part in various types of inflammation. Proteinases have been linked to arthritic reactions. Neutrophils, in their lysosomal granules, carry many serine proteinases. Leukocyte proteinases play a significant role in the development of tissue damage during inflammatory processes [58]. Lipoxygenases are key enzymes in the biosynthesis of leukotrienes, which in turn are crucial mediators in many inflammatory diseases. The mechanism of anti-inflammatory action involves a number of issues in which the metabolism of arachidonic and linoleic acids plays an important role [59,60].

Figure 2. The effect of plant extracts on the activity of the elastase enzyme. Data are the mean of three independent experiments in which each sample was tested in triplicate. Different letters on the charts indicate significant differences between the individual results (p<0.05).

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