Phenolic Composition and Skin-related Properties of the Aerial Parts Extract of Different Hemerocallis Cultivars

Hemerocallis plants are important vegetables with nutritional and health value, especially in eastern Asia, where they have been used as medicines to cure disease such as depression and inflammation for thousands of years. The present study concerns the determination of flavonoids and phenolic acids, as well as antioxidant, anti-collagenase, anti-elastase, anti-tyrosinase and antimicrobial properties of taxa cultivated in Poland. For chemical composition estimation, LC-ESI-MS/MS analysis and spectrophotometric assays were performed. The results show the presence of sixteen compounds in all analyzed species. Among the investigated cultivars, it was found that H. “Chicago Apache” and H. fulva var. kwanso have the highest total phenolic acid and flavonoid content. The most abundant compounds in all analyzed extracts were chlorogenic acid (209.8 to 1010.0 µg/g of DE) and quercetin-3-O-rutinoside (114.7 to 1049.7 µg/g of DE). The studied extracts exhibited moderate to high skin-related activities. These properties were correlated with a high concentration of polyphenols. The present study demonstrated that Hemerocallis cultivars contain significant amounts of phenolic compounds with good skin-related activities and could be interesting as novel sources of bioactive agents for the pharmaceutical, food and cosmetic industries.


Introduction
Plant extracts are a valued source of biologically active compounds. Numerous scientific studies confirm the antibacterial, antiviral, antifungal and anti-inflammatory effects of plant raw extracts [1]. Cosmetic products containing them have been gaining popularity in recent years. Their use in cosmetic preparations yield much better results than the application of single biologically active compounds. Using the health-promoting properties of plant substances, one primarily looks for ingredients with anti-inflammatory and antioxidant effects (that eliminate adverse changes in the body), as well as with a positive impact on the functioning of the skin. An example of such case is materials rich in polyphenols, such as phenolic acids and flavonoids, as natural antioxidants efficient in preventing free radical production. Phenolic acids, which have a protective role in many illnesses such as cancer, inflammation, and cardiovascular illnesses, particularly recognized due to their potent antioxidant

Total Flavonoid, Phenolic and Phenolic Acids Content
Total flavonoid (TFC) and total phenolic content (TPC) were established using the colorimetric assays as described previously [39]. The absorbance was measured at 430 and 680 nm, respectively, using Pro 200F Elisa Reader (Tecan Group Ltd., Männedorf, Switzerland). Total phenolic concentration was estimated from the calibration curve (R 2 = 0.9811), using gallic acid in (0.002-0.1 mg/mL) as a standard. The results were expressed as mg of gallic acid equivalent (GAE) per 1 g of dry extract (DE). Total flavonoid content was estimated from the calibrated curve (R 2 = 0.9999), using quercetin (0.004-0.11 mg/mL) as a standard. The results were expressed as mg of quercetin equivalent (QE) per 1 g of DE. Total phenolic acids (TPAC) content was assayed using Arnov's reagent as described in Polish Pharmacopoeia IX (an official translation of PhEur 7.0) [40]. The absorbance was measured at 490 nm. TPAC was estimated from the calibration curve (R 2 = 0.9963), using caffeic acid in concentration 3.36-23.52 µg/mL as standard. The results were expressed as mg of caffeic acid equivalent (CAE) per 1 g of DE.

LC-ESI-MS/MS Analysis
Agilent 1200 Series HPLC system (Agilent Technologies, Palo Alto, CA, USA) coupled to 3200 QTRAP mass spectrometer (AB Sciex, Redwood City, CA, USA) was used for qualitative and quantitative analysis of flavonoids and phenolic acids in Hemerocallis extracts. The separation of analyzed compounds, injected in a 3-µL amount, was performed on a Zorbax SB-C 18 analytical column (2.1 × 100 mm, 1.8 µm, Agilent Technologies, Palo Alto, CA, USA) at 25 • C. Elution was carried out using solvent A (0.1% HCOOH in water) and solvent B (0.1% HCOOH in acetonitrile). The following gradient elution program was used: 0-2 min-20% B, 3-4 min-25% B, 5-6 min-35% B, 5-6 min-35% B, 8-12 min-65% B, 14-16 min-80% B, 20-28 min-20% B. The flow rate was 300 µL/min. The mass spectra of analyzed compounds were acquired in the negative ESI mode, and the optimum values of the source parameters were as follows: capillary temperature 450 • C, nebulizer gas 50 psi, curtain gas 30 psi, source voltage −4500 V for phenolic acids and flavonoid glycosides, and capillary temperature 550 • C, nebulizer gas 30 psi, curtain gas 20 psi, and source voltage −4500 V for flavonoid aglycones analysis. Details of LC-ESI-MS/MS analysis are presented in Table 1 and were described in our previous research [41]. The Analyst 1.5 software (AB Sciex, Redwood City, CA, USA) was used for analysis and data acquisition. 2.6.1. DPPH Assay 2,2-diphenyl-1-picryl-hydrazyl (DPPH) free radical scavenging activity of Hemerocallis extracts and the ascorbic acid (positive control) was studied using a modified method, described previously [39]. Decreasing of DPPH absorbance, caused by the extracts, was controlled at 517 nm after incubation at 28 • C during 30 min. The results were expressed as IC 50 .

Metal Chelating Activity (CHEL)
The metal chelating activity was established using method described by Guo et al., (2001) [3], modified in our previous study [39,42]. The absorbance was measured at 562 nm. As a positive control, Na 2 EDTA*2H 2 O was used.
Results were expressed as the IC 50 values of the Hemerocallis extracts based on concentrationinhibition curves.

Enzyme Inhibitory Activity
All tests were made using 96-well plates (Nunclon, Nunc, Roskilde, Denmark) and were estimated in an Infinite Pro 200F Elisa Reader (Tecan Group Ltd., Männedorf, Switzerland). All experiments were performed in triplicate.

Anti-Tyrosinase Activity (TYR)
Anti-tyrosinase activity was studied with the method described earlier by Zengin and co-authors [43]. Mushroom tyrosinase (40 µL, 200 U/mL) and Hemerocallis samples (25 µL) in different concentrations were incubated in sodium phosphate buffer (100 µL, pH 6.8) for 10 min at 29 • C. To start the reaction, L-DOPA (40 µL, 0.5 mM) was added. A blank sample was without a tyrosinase solution. The change in absorbance after 10 min incubation was measured at 492 nm at 29 • C. Kojic acid (6.25-100 µg/mL) was used as positive control.
All results were expressed as the IC 50 values of the Hemerocallis extracts based on concentrationinhibition curves.

Antibacterial Activity
Zones of bacterial growth inhibition, caused by obtained extracts, were estimated for the reference microorganisms from the American Type Culture Collection (ATCC): Gram-positive bacteria (Staphylococcus aureus ATCC 25923) and Gram-negative bacteria (Escherichia coli ATCC 25992). Clinical strains (Escherichia coli, Staphylococcus aureus) isolated from infected wounds were obtained and stored since August 2016 in the strain bank at the Department of Biochemistry and Biotechnology, Medical University of Lublin, Lublin, Poland. Stock cultures were held at −70 • C. Tested bacterial strain, before the experiments, was passaged onto fresh Mueller-Hinton agar (M-H) (Oxoid, Basingstoke, UK) at 37 • C for 24 h. Next, inocula were made with fresh microbial cultures in sterile 0.9% NaCl to 0.5 McFarland turbidity standard. The antibacterial properties of extracts against bacteria was estimated by disk diffusion method, measuring the zones of inhibition. (Kirby-Bauer Disk Diffusion Susceptibility Test Protocol) on Petri plates with solid medium (M-H agar). Appropriate strain cultures were separately spread over the agar surface using a cotton swab. Next, tested liquid samples (100 µg) were placed using sterile disc (discs dispenser BioMaxima S.A., Lublin, Poland). After 18 h of incubation at 37 • C, zones of microbial growth produced around the studied extracts were estimated and recorded as the diameters of inhibition [mm]. All analyses were carried out in triplicate. The results are expressed as mean ± RSD.

Statistical Analysis
All results were stated as means ± standard deviation (SD) of three independent tests. One-way ANOVA with Tukey's post hoc test was used for statistical analysis of significance of differences between means. p values below 0.05 were accepted as statistically significant. Statistics were carried out in Statistica 10.0, whereas Principal Component Analysis was carried out in R version 3.6.3 (64-bit, Windows 10), using built-in "prcomp" function.

Phytochemical Analysis
Total phenolic content (TPC) was determined using Folin-Ciocalteu reagent and the results were estimated as gallic acid equivalents (GAE) per g of dry extract (DE) ( Table 2). Among eight Hemerocallis cultivars, H. "Chicago Apache" (H5) and H. fulva var. kwanso (H1) had the highest phenolic content (99.8 ± 1.1 mg GAE/g DE and 78.8 ± 0.4 mg GAE/g DE, respectively), followed by H. The values obtained in our study are slightly higher than those of Lin and co-authors (2011) for various extracts of the flowers of H. fulva (from 25.3 ± 2.7 to 34.6 ± 2.7 mg GAE/g of extract) and lower than those of H. fulva leaves (25 ± 0.001 to 749 ± 0.004 mg chlorogenic acid equivalent/g) [46]. Mao et al. [31] found high amounts of phenolic compounds in leaves of H. fulva, ranging from 41.25 to 160.42 mg/g of dry extract. Fu and Mao [47] recorded a range from 44.68 to 59.22 mg of polyphenols per kg of dry weight for five Hemerocallis cultivars from China, and these values were lower than those obtained in our study. Moreover, Stefaniak and Grzeszczuk [48] determined total phenolic content for Hemerocallis × hybrida flowers on the level equal to 2.06 ± 0.02 mg GAE/g per fresh weight.  78 The total flavonoid content of the flowering aerial parts of Hemerocallis cultivars was estimated by previously described colorimetric method [39] and was expressed as quercetin equivalents (QE) per g of dry extracts. The results presented in Table 1 show that among studied species, a significantly higher content of total flavonoids was observed for H. "Chicago Apache" (H5) and H. fulva var. kwanso (H1) (25.4 ± 0.0 and 25.0 ± 0.1 mg QE/g DE, respectively). The high content was also noted for H. "Jaskółka" (H7) (13.8 ± 0.2 mg QE/g DE) and H. "Danuta" (H6) (12.8 ± 0.1 mg QE/g DE). The data for the flowering aerial parts of Hemerocallis cultivars were higher than those obtained for H. fulva flowers (10.4 ± 2.3 to 19.5 ± 1.3 mg catechin equivalent/g of extract) [32].
The total phenolic acids content (TPAC) in the studied extracts are presented in Table 2. The amounts were in the range from 8.3 ± 0.1 to 18.9 ± 0.2 mg CAE/g DE. The highest TPAC content was observed in H2 (18.9 ± 0.2 mg CAE/g DE), followed by H5 (17.2 ± 0.1 mg CAE/g DE) and H1 (16.2 ± 0.1 mg CAE/g DE). The smallest content was found in H4 (8.3 ± 0.1 mg CAE/g DE).
In the next step of our study, phenolic acid and flavonoid composition of the extracts obtained from Hemerocallis cultivars was investigated using the LC-MS/MS method. The analysis was carried out using a previously validated and described method [41]. The results of the qualitative and quantitative analysis are presented in Table 3. The sample LC-ESI-MS/MS chromatogram is displayed in Figure 1.
Among the investigated cultivars, it was found that H. "Chicago Apache" (H5) and H. fulva var. kwanso (H1) have the highest total phenolic acids and flavonoids content. Chlorogenic acid was the most abundant phenolic acid in all Hemerocallis extracts (209.8 ± 1.0 to 1010.0 ± 10.0 µg per g of DE). The protocatechuic, p-coumaric and caffeic acids were also observed in all Hemerocallis cultivars. The protocatechuic and caffeic acid contents of H. "Rebel Cause" (H8) extract (577.5 ± 12.5 and 98.0 ± 1.0 µg/g DE) were found to be much higher than those of the other extracts (45.9 ± 0.6 to 116.8 ± 0.5 µg/g DE for protocatechuic acid and from 10.0 ± 0.1 to 72.4 ± 1.9 µg/g DE for caffeic acid). As for p-coumaric acid, its content in the flowering aerial parts of H. "Aten" (H2) (53.1 ± 0.9 µg/g DE) was determined to be richer comparing to those of the other cultivars (11.8 ± 0.0 to 65.0 ± 0.8 µg/g DE). The 4-hydroxybenzoic acid was detected in a quantifiable amount only in H2-H6 extracts (5.1 ± 0.1 to 373.8 ± 1.3 µg/g DE) and rosmarinic acid only in H2 (11.9 ± 0.2 µg/g DE).
According to the literature reports, Hemerocallis species were found as the plants with high contents of phenolic acids and flavonoids. The chlorogenic acids in a methanolic extract of Hemerocallis were qualitatively profiled using LC-MS 3 . Three caffeoylquinic acids, three p-coumaroylquinic acids and two feruloylquinic acids were identified [49]. In studies of aqueous-methanol and methanol extracts of Hemerocallis cv. Stella de Oro flowers, isorhamnetin 3-O-glycosides, kaempferol and quercetin were isolated [7]. Moreover, fifteen phenolic compounds including, among others, Therefore, our results are in agreement with previous investigations and it can be seen that extracts, especially aqueous-ethanolic, of Hemerocallis species are rich in terms of phenolics.

Skin-Related Activities
Skin aging is a natural, unavoidable, complex process, progressing over the years. Basic life functions disappear gradually, creating an incorrect response to external factors. Various intrinsic and extrinsic factors are responsible for this process, including hormonal, genetic, metabolic changes, as well as exposure to environmental stress [50]. As several enzymes and biomarkers are involved in the skin aging process, plant extracts, as inhibitors of specific enzymes involved in the aging, such as elastase or collagenase, are desirable. Thus, apart from qualitative and quantitative analysis of flavonoids and phenolic acids, this work focused on skin-related properties of Hemerocallis cultivars. In the study, we examined in vitro antioxidant, anti-collagenase, anti-elastase, anti-tyrosinase and antibacterial properties of eight Hemerocallis cultivars collected in Poland.

DPPH Radical Scavenging Activity
The antioxidant activity was studied on the microplate scale in cell-free systems. All samples were studied in a concentration range from 1.25 to 40 mg/mL. The extracts of the flowering parts of Hemerocallis cultivars exhibited moderate to high scavenging capacity in a concentration-dependent manner. For comparison, the radical scavenging activity of ascorbic acid was tested in the same conditions. The higher DPPH scavenging activity was showed for the extract of H. "Chicago Apache" (H5) (IC 50 = 0.9 ± 0.2 mg/mL) followed by H. fulva var. kwanso (H1) (IC 50 = 1.1 ± 0.1 mg/mL). However, the IC 50 value for Hemerocallis extracts was from two-to even forty-times higher (H. "Catherine Woodbuery"-IC 50 = 19.4 ± 0.1 mg/mL) than the for ascorbic acid (IC 50 = 0.5 ± 0.0 mg/mL) ( Table 4). Table 4. The IC 50 values determined in antioxidant tests. Data are expressed as mean ± SD, n = 3. AA-ascorbic acid; Na 2 EDTA*2H 2 O-ethylenediaminetetraacetic acid, disodium dihydrate; nt-not tested; H1-H. fulva (L.) L. var. kwanso, H2-H. "Aten", H3-H. "Bożena", H4-H. "Catherine Woodbuery", H5-H. "Chicago Apache", H6-H. "Danuta", H7-H. "Jaskółka", H8-H. "Rebel Cause"; CHEL-metal chelating activity. * Statistically significant differences compared to AA; # statistically significant differences compared to Na 2 EDTA*2H 2 O, p < 0.05. The results of other published reports can be tough to compare due to the other conditions of experiments used. Nevertheless, several phenolic compounds isolated from Hemerocallis cv. Stella de Oro flowers were tested for their antioxidant activity. The flavonol 3-O-glycoside from this species demonstrated low antioxidant activities at 10 µM [7]. The scavenging effects of extracts of the flowers of six daylily cultivars from China have been tested on the DPPH radical [47]. The authors found that extracts of daylily cultivars in the concentration of 150 µg/mL possess the scavenging effect from 92.26 to 72.32%. These values are comparable to our findings.

Metal Chelating Activity (CHEL)
It is well known that polyphenols can reduce oxidative stress by several mechanisms that depend on their chemical structure. One of these mechanisms is the chelation of metal ions, such as iron, which plays a key role in the production of harmful oxygen species [51]. Under normal conditions, iron is stored and transported by ferritin or transferrin, preventing the reaction of free iron ions with reactive oxygen species. The iron ions, by taking part in Fenton reaction, generate OH• radicals, which can react with lipids causing their peroxidation [52]. Therefore, it is important to search for new natural compounds with the potential ability to chelate metal ions.
High levels of chlorogenic acid and rutin in the extracts of Hemerocallis cultivars may be responsible for their strong antioxidant effects [32], which was also confirmed in our study.

Sample
In many studies, it has been proved that polyphenols are mostly compounds that are responsible for collagenase inhibition [54,55]. Moreover, chlorogenic acid and quercetin derivatives, which were found in large quantities in all Hemerocallis extracts, have demonstrated a high collagenase inhibitory effect which was shown by different reports [54,56].

Anti-Elastase Activity (ELA)
In normal adult skin, the elastin dominates, representing over 90% of the total content of a developed elastic fiber, and it is a protein responsible for the elasticity [53]. Because there are many reports showing that skin aging is directly related to the breakdown of elastin by the enzyme elastase [54], the elastase inhibitory activity was also determined for the daylilies extracts.
The analysis was achieved with using a N-Succinyl-Ala-Ala-Ala-p-nitroanilide (substrate molecule) and elastase from porcine pancreas (enzyme). The extracts were tested in various concentrations (6.25-100 µg/mL). EGCG was used as the positive control and it showed IC 50 = 62.4 ± 0.1 µg/mL. As in the case of collagenase inhibition, the extract from H. "Chicago Apache" (H5) shows the highest anti-elastase activity with the IC 50 value of 45.5 ± 0.1 µg/mL. The inhibition of elastase activity of H. fulva var. kwanso (H1) and H. "Jaskółka" (H7) was also high (IC 50 = 51.0 ± 0.1 µg/mL and 56.7 ± 0.2 µg/mL, respectively). All the results of anti-elastase activity are presented in Table 5.
As with anti-collagenase activity, it has also been demonstrated that polyphenols are significant elastase inhibitors [57].

Anti-Tyrosinase Activity (TYR)
Tyrosinase is a copper-containing enzyme widespread in nature, that is, a key enzyme in the biosynthesis of melanin, biopolymer responsible for the color of skin and hair, and also protect skin from ultraviolet [58]. Though melanin is a very important molecule, its overproduction in epidermal layers can cause various dermatological disorders, such as hyperpigmentation due to skin aging [54,58]. The hyperpigmentation in human skin, such as age spots, is not desirable, therefore it seems relevant to search for natural substances that inhibit the melanogenesis, especially tyrosinase inhibitors.
In our study, the extracts of flowering aerial parts of Hemerocallis species and kojic acid used as positive control were tested in various concentrations (6.25-100 µg/mL). The extract from H. "Chicago Apache" (H5) was the most effective amongst all samples with IC 50 = 16.6 ± 0.1 µg/mL. Other extracts showed slightly weaker activity with IC 50 values from 20.6 ± 0.1 (H1) to 79.3 ± 0.2 µg/mL (Table 5).
Much research has been performed to identify the inhibitors of tyrosinase from natural sources [58,59] and it was found that flavonoids show high anti-tyrosinase activity [59]. It was noted that some flavonols, such as quercetin and kaempferol, can competitively inhibit tyrosinase activity by their ability to chelate metal, which leads to the irreversible inactivation of enzyme [58,59].

Antibacterial Activity
The antibacterial activity of the Hemerocallis cultivars' extracts against Gram-negative (E. coli) and Gram-positive (S. aureus, S. epidermidis) bacterial species was examined using a plate antimicrobial test. The obtained effects are presented in Table 6. All samples showed some activity against tested reference and clinical Gram-positive strains (zones in range of 4.4 ± 0.3 mm-27.3 ± 3.1 mm). The aerial parts of Hemerocallis "Chicago Apache" (H5) and H. "Jaskółka"(H7) showed the highest activity among the eight tested samples. The zones of reference S. aureus and S. epidermidis growth inhibition caused by Hemerocallis "Chicago Apache" (H5) were respectively 27.3 ± 3.1 mm and 19.5 ± 2.3 mm. Importantly, the growth of the tested clinical strains of S. aureus and S. epidermidis was also inhibited in highest amount by H5, creating zones of 19.5 ± 2.3 mm and 16.5 ± 2.1 mm, respectively. Inhibiting the growth of clinical strains is important when samples with potential therapeutic properties are tested. However, the tested samples showed no inhibition zones against Gram-negative E. coli strains. Therefore, Hemerocallis samples demonstrated narrow spectrum, limited only to Gram-positive bacteria, which may be beneficial in the prevention and fight against classic infections caused by Gram-positive strains.

Multivariate Analysis of the Results
To perform a holistic view of the results, we used a chemometric multivariate approach. It allows us to examine some independent trends in changes among the investigated properties. During the introductory analysis of the correlations between investigated properties (Figure 2), one can conclude that DPPH, CHEL, TYR, ELA and COL are highly positively intercorrelated. On the contrary, they are negatively correlated with all other properties, mostly with TPC and TFC. TPCA is the last property, correlated positively with TPC and TFC and negatively with all other properties, however, these correlations are substantially lower. It should be underlined that negative correlations should be perceived and interpreted as positive in these cases, where the property is measured as the concentration with a particular effect (such as IC 50 ). Then, the lower value denotes the higher activity. Therefore, we also performed scaled Principal Component Analysis of the whole data matrix, containing 24 rows (three samples for each eight species) and eight columns (all investigated properties). This approach allows us to examine intercorrelations between the properties, graph them in one two-dimensional graph and place the investigated samples in this reduced space. From a mathematical point of view, this is equivalent to the projection of multivariate space to the bivariate plane, where this plane is located in the best possible way (to visualize as much information, as possible). Of course, the visualized information depends on the data structure, so if the dataset is not intercorrelated (spherical), this method would not help us to understand the internal structure, as there is no optimal position on this plane.
The dataset was found to be easy to compress and 81.8% of the overall variance was located in the first PC. Of the variance, 11.6% was located in the second one, so the PC1-PC2 plot explains 93.3% of the overall variability inside this dataset. Therefore, almost all information can be presented in a two-dimensional projection and only several percentages of information are lost inside the orthogonal complement of this plane. It was also found that interesting information was located inside the PC3, explaining 5.1% of variance. No explainable trends were found in the further PCs.
The first main trend in this dataset, placed in PC1 ( Figure 3) is connected (for high values of this PC) with simultaneous increase of DPPH, TYR, CHEL, COL and ELA, together with simultaneous decrease of TFC and TPC. Low PC1 values exhibit the opposite change. The highest PC1 was obtained for H. "Catherine Woodbuery" (H4) and H. "Rebel Cause" (H8), whereas the lowest for H. fulva var. kwanso (H1) and H. "Chicago Apache" (H5). It should be emphasized that species are spread uniformly according to the PC1 values, so this trend should be perceived as continuous and fluent. It can be concluded that flavonoids and phenols are responsible for all investigated biological activities, as the activities are expressed as IC 50 , and thus are negatively correlated with the content of particular compounds. that DPPH, CHEL, TYR, ELA and COL are highly positively intercorrelated. On the contrary, they are negatively correlated with all other properties, mostly with TPC and TFC. TPCA is the last property, correlated positively with TPC and TFC and negatively with all other properties, however, these correlations are substantially lower. It should be underlined that negative correlations should be perceived and interpreted as positive in these cases, where the property is measured as the concentration with a particular effect (such as IC50). Then, the lower value denotes the higher activity. Therefore, we also performed scaled Principal Component Analysis of the whole data matrix, containing 24 rows (three samples for each eight species) and eight columns (all investigated properties). This approach allows us to examine intercorrelations between the properties, graph them in one two-dimensional graph and place the investigated samples in this reduced space. From a mathematical point of view, this is equivalent to the projection of multivariate space to the bivariate plane, where this plane is located in the best possible way (to visualize as much information, as possible). Of course, the visualized information depends on the data structure, so if the dataset is not intercorrelated (spherical), this method would not help us to understand the internal structure, as there is no optimal position on this plane. The dataset was found to be easy to compress and 81.8% of the overall variance was located in the first PC. Of the variance, 11.6% was located in the second one, so the PC1-PC2 plot explains 93.3% of the overall variability inside this dataset. Therefore, almost all information can be presented in a two-dimensional projection and only several percentages of information are lost inside the orthogonal complement of this plane. It was also found that interesting information was located inside the PC3, explaining 5.1% of variance. No explainable trends were found in the further PCs.
The first main trend in this dataset, placed in PC1 ( Figure 3) is connected (for high values of this PC) with simultaneous increase of DPPH, TYR, CHEL, COL and ELA, together with simultaneous decrease of TFC and TPC. Low PC1 values exhibit the opposite change. The highest PC1 was obtained for H. "Catherine Woodbuery" (H4) and H. "Rebel Cause" (H8), whereas the lowest for H. fulva var. kwanso (H1) and H. "Chicago Apache" (H5). It should be emphasized that species are spread uniformly according to the PC1 values, so this trend should be perceived as continuous and fluent. It can be concluded that flavonoids and phenols are responsible for all investigated biological activities, as the activities are expressed as IC50, and thus are negatively correlated with the content of particular compounds.
The third PC (Figure 4) presents the difference between H1, H4, H5 and H8 (high PC3) and the other species (low PC3). Species with high PC3 increased COL, TPC, TFC and (slightly) DPPH together (simultaneously) compared to the other species.

Conclusions
Due to the fact that natural ingredients in cosmetics are no longer just a trend, but a standard in creating new care products, there is a constant search for new plant raw materials. Plant extracts could contain specific secondary metabolites that provide a very wide range of possibilities in use.
The results of this study suggest that the Hemerocallis cultivars may have a value in the production of cosmetics. We found that aqueous-ethanol extracts of the flowering aerial parts of eight daylilies were rich in phenolics. Among the investigated cultivars, it was found that H. "Chicago Apache" and H. fulva var. kwanso have the highest total phenolic acids and flavonoids content. The most abundant compounds in all analyzed extracts were chlorogenic acid and quercetin-3-O-rutinoside. The studied extracts exhibited moderate to high skin-related activities. These properties were correlated with a high concentration of polyphenols. The present study demonstrated that Hemerocallis cultivars contain significant amounts of phenolic compounds with good skin-related activities, and could be interesting as novel sources of bioactive agents for the pharmaceutical, food and cosmetic industries.