Multidirectional Characterization of Phytochemical Profile and Health-Promoting Effects of Ziziphora bungeana Juz. Extracts

Ziziphora species (Lamiaceae) have been used in traditional medicine as sedatives, antiseptics, carminatives, or expectorants. Despite their common applications in phytotherapy, there is still lack of evidence about the composition of their extracts and its impact on biological properties of the plants. The aim of this study was to evaluate the content of Ziziphora bungeana, a less studied species growing in Kazakhstan, using HPLC-ESI-QTOF-MS/MS instrumentation and to determine its antimicrobial, antioxidant, and cytotoxic activity together with inhibitory properties against tyrosinase and toxicity in erythrocyte lysis assay. Extracts from Z. bungeana were found to be sources of flavonoids, phenolic acids, organic acids, and terpenes that determined their antiradical activity. The minimum inhibitory concentrations of extracts were lower for Gram-positive bacteria (1.25–10 mg/mL) than for Gram-negative bacteria and fungi (5–20 mg/mL). The EC50 value calculated for antiradical activity ranged between 15.00 ± 1.06 µg/mL and 13.21 ± 3.24 µg/mL for ABTS and DPPH assays, respectively. Z. bungeana extracts were found to decrease the activity of tyrosinase by 50% (at 200 µg/mL) similarly to kojic acid and were slightly cytotoxic for human melanoma A375 cell line (at 200 µg/mL) with no effect on HaCaT keratinocytes. In the end, Z. bungeana did not reveal toxic effects in hemolytic assay as compared to the positive control Triton X-100. The performed tests show potential application of the plant in the treatment of infectious diseases, disorders caused by free radicals, and skin problems.


Introduction
Plant biodiversity is certainly an invaluable wealth of our planet. Herbal remedies have been used in the treatment of various diseases for centuries as plants are capable of  Ursolic acid Z1, Z3 [31] study was designed to meet the diverse expectations towards plant extracts in the context of their potential use in the treatment of civilization diseases or in the skin care because of the harmful effects caused by the environment. The determination of the antioxidant, antimicrobial, and whitening properties of the extracts of different polarities will allow for the study on the potential use of Ziziphora bungeana in cosmetics. Moreover, the toxicity studies in relation to normal and cancer skin cells will bring evidence for a discussion about safety of its use. On the other hand, the qualitative analysis of different polarity extracts will develop a fingerprint responsible for the determined action of the plant.

Chemical Composition of Extracts by HPLC-ESI-QTOF-MS/MS
The applied chromatographic method was capable of separating metabolites present in the extracts from the aerial parts of Z. bungeana, whereas the applied mass spectrometer settings provided MS/MS spectra that helped in the identification of metabolites from different groups.
The HPLC-ESI-QTOF-MS/MS analysis confirmed the presence of phenolic acids, flavonoids, triterpenes, and monoterpene glucosides in the tested extracts. The list of 26 tentatively identified components is presented in the Table 1 and Figure 1, whereas the recorded mass chromatograms from positive and negative ionization modes are shown in the supplementary file ( Figures S1 and S2). It was proven that the components of Z. bungeana were previously described in other species from the same gender. The applied LC-MS technology enabled the identification of components, e.g., thymol, carvacrol, or ziziphorosides, that were previously determined in Ziziphora spp. by GC-MS technique. Milder fragmentation conditions (fragmentation voltage of 110 V, capillary voltage of 3000 V, gas temperature of 275 °C and collision energy of 10 V) increased the chance to observe terpene compounds in the chromatogram in the liquid chromatography-based system. Ziziphoroside isomers, possibly ziziphoroside A, B, and C, were present in the mass spectra in the form of adducts with sodium ions. The remaining compounds were traced in the form of molecular ions with or without a proton.  The applied LC-MS technology enabled the identification of components, e.g., thymol, carvacrol, or ziziphorosides, that were previously determined in Ziziphora spp. by GC-MS technique. Milder fragmentation conditions (fragmentation voltage of 110 V, capillary voltage of 3000 V, gas temperature of 275 • C and collision energy of 10 V) increased the chance to observe terpene compounds in the chromatogram in the liquid chromatographybased system. Ziziphoroside isomers, possibly ziziphoroside A, B, and C, were present in the mass spectra in the form of adducts with sodium ions. The remaining compounds were traced in the form of molecular ions with or without a proton.
Z. bungeana was proven to contain different types of metabolites whose presence was revealed in the HPLC-MS assessment of the extracts tested in this study. Among them flavonoids constituted the leading group of components, followed by phenolic acids and, interesting from the structural point of view, terpenes, e.g., ziziphorosides or schizonepetaside E. It is worthwhile to note that the scientific literature still lacks sufficient information about the composition of this plant species. For the moment, to the best of the authors' knowledge, there is only one original manuscript that discusses the composition of extracts based on the HPLC-MS results. The researchers confirmed the presence of twelve flavonoids in Z. bungeana, that included: kaempferol-7-O-rutinoside, kaempferol-3-Orutinoside; rutin; apigenin-7-O-rutinoside; 3 -hydroxyacacetin-7-O-rutinoside; acacetin-4 -O-rutinoside; pinocembrin-7-O-rutinoside; chrysin-7-O-rutinoside; linarin; 5,7,3 -trihydroxy-6,4 ,5 -trimethoxyflavone, 5,4 -dihydroxy-6-methoxy-7,8-methylenedioxyflavone, and 5,7dihydroxy-6-methoxyflavone. The above list of components presented in the Table 1 expands information on the composition of this species.
The analysis of previously published papers provided a more detailed list of components of Ziziphora genus that helped to enrich the list of the tentatively identified metabolites of Z. bungeana extracts. Previous investigation of the chemical profile of ethyl acetate, methanol, and water extracts from the aerial parts of Ziziphora taurica subsp. cleonioides showed that among the identified compounds, rosmarinic acid and chlorogenic acid were the most abundant components of the methanol extract with the calculated concentration of 3375.67 ± 38.02 and 3225.10 ± 16.44 µg/mL, respectively [32]. Both compounds were also determined in the studied species. Moreover, flavonoids constituted the major group of bioactive compounds present in Ziziphora clinopodioides Lam. [2] together with organic acids, alkaloids, and glycosides that were listed by other authors [33].
As mentioned above, Ziziphora species belong to the plants that synthesize secondary metabolites from different classes, which explains their various therapeutical applications.
In the previous studies Ziziphora spp. were found to be rich sources of phenolic compounds. Interestingly, both extracts and the EO were sources of polyphenols. For example, the measured total phenolic content in Z. tenuior was equal to 49.0 ± 1.4 mg mg gallic acid/100 g of EO [15].
The metabolites that are mentioned above are important from the pharmacological point of view. Phenolic acids and flavonoids are known scavengers of free radicals that are efficiently inhibiting the progression of different inflammatory conditions and civilization diseases progressing with an important role of radicals [34]. Their presence in the final extracts from edible plants is certainly related to the type of the plant, but also to the extraction conditions [35].
Based on this information, the authors found it crucial to study the biological potential of Z. bungeana and focus on its antimicrobial, antioxidant, and anti-tyrosynase properties, as well as to evaluate safety.

Antimicrobial Activity Assessment
The data presented in Tables 2 and 3 indicate that the extracts from Ziziphora bungeana showed some potential antimicrobial activity. They were more effective against reference Gram-positive bacteria than towards Gram-negative bacteria and yeasts. The lowest concentration of extracts which inhibited the growth of these microorganisms or killed them ranged from 1.25 mg/mL to 20 mg/mL and from 2.5 mg/mL to 20 mg/mL, respectively. The representative (modal) data are presented. The sensitivity of the fungi belonging to Candida spp. to the tested extracts Z1-Z3 was similar to that of Gram-negative bacteria (MIC = 5-20 mg/mL and MFC = 20 mg/mL). Candida parapsilosis ATCC 22,019 was the most susceptible to Z2 and Z1 at MIC = 5 mg/mL and 10 mg/mL, respectively. Z2 showed also activity towards other Candida spp. with MIC = 10 mg/mL, except Candida glabrata ATCC 90,030 (MIC = 20 mg/mL). Moreover, the minimal concentrations of these extracts, which inhibited growth or killed these microorganisms were 20 mg/mL ( Table 3).
As shown in Table 2, in the case of Gram-positive bacteria, the MIC values of the extracts were in the range of 1.25-10 mg/mL. Their activity was the same towards staphylococci, both Staphylococcus aureus ATCC 43,300 (MRSA-Methicillin Resistant S. aureus), S. aureus ATCC 29,213 (MSSA-Methicillin Susceptible S. aureus) and Staphylococcus epidermidis ATCC 12,228 with MIC = 2.5 mg/mL and MBC = 2.5-5 mg/mL (except S. aureus ATCC 29213; MIC = 5 mg/mL for Z2). In turn, Micrococcus luteus ATCC 10,240 was the most (MIC = 1.25 mg/mL and MBC = 5 mg/mL) and the least sensitive (MIC = 5 mg/mL and MBC = 10 mg/mL) to Z2 and Z3, respectively. The antibacterial effect against two reference Bacillus spp. strains was lower. MIC values were mainly 5 mg/mL. However, MIC = 2.5 mg/mL was shown for the Z3 against B. subtillis ATCC 6633 and MIC = 10 mg/mL for Z1 towards B. cereus ATCC 10876. MBCs of all extracts for bacilli were the same-10 mg/mL. The representative (modal) data are presented. As shown our results (Tables 2 and 3), the most common MIC value of 20 mg/mL was found for Z1 (7 (43.75%) strains) and Z3 extracts (10 (62.5%) strains). In the case of Z2 extract, the values of MIC = 20 mg/mL and MIC = 10 mg/mL, occurred with the same frequency (25% each) against reference strains of microorganisms. The same frequency of MIC = 10 mg/mL was shown for Z1. MIC values of 5 mg/mL were shown for 5 (31.25%), 2 (12.5%) and 1 (6.25%) strains in the case of Z2, Z3 and Z1 extracts, respectively. The Z1 and Z3 inhibited the growth of microorganisms at the minimum concentration of 2.5 mg/mL (4 strains (25%) each). Additionally, Z2 inhibited the growth of 1 (6.25%) and 2 (12.5%) strains with MIC = 1.25 mg/mL and 2.5 mg/mL, respectively.
The activity of extracts towards Gram-negative rods-shaped, was slightly weaker with MIC = 5-20 mg/mL and MBC = 10-20 mg/mL. Among them, Z2 showed the highest effect towards Bordetella bronchiseptica ATCC 4617 (MIC = 5 mg/mL and MBC = 20 mg/mL). For Z1, MIC = 10 mg/mL against B. bronchiseptica and Pseudomonas aeruginosa ATCC 9027 was shown. In the case of Z2, the same MIC value towards reference Klebsiella pneumoniae strain was indicated. The growth of remaining Gram-negative bacteria was inhibited by these extracts at a concentration of 20 mg/mL.
Taking into account the MBC/MIC and MFC/MIC ratios, as presented at Figure 2, it was shown that extracts from Z. bungeana had a beneficial bactericidal or fungicidal effect towards reference microorganisms. The values of MBC/MIC or MFC/MIC were in the range 1-4. MICs equal to MBC or MFC (MBC/MIC = 1 and MFC/MIC = 1) were shown for most, i.e., 12 (75%) and 11 (68.75%) strains in the case of Z1 and Z3, respectively. For Z2, these values were different. However, these ratios were mainly 1 (for six (37.5%) strains) and 2 (for seven (43.75%) strains). The value of 4 was found the least frequently (only for Z2 and Z3). The bacteriostatic effect (MBC/MIC > 4 or MFC/MIC > 4) of the tested extracts was not demonstrated.
In the next stage of this study, the total antimicrobial activity (TAA) was assessed. The total antibacterial activity or total antifungal activity of the studied extracts Z1-Z2 was shown in Table 4. The MIC and TAA, both total antibacterial activity and total antifungal activity values are important pharmacological tools. They are useful in determining the activity of extracts in mg/mL (potency) of plants extracts for isolating bioactive compounds and total activity on mL/g (efficacy) for the selection of appropriate plant species [36]. Generally, their TAA values were the highest against Gram-positive bacteria: Staphylococcus spp., Micrococcus luteus, followed by Bacillus spp. (3.35 ± 0.0-20.20 ± 0.0 mL/g) and the lowest against Gram-negative bacteria and fungi belonging to Candida spp. (1.09 ± 0.0-4.21 ± 1.46 mL/g). Z1 had higher TAA towards reference strains of S. aureus, S. epidermidis and M. luteus (13.39 ± 0.0 mL/g). TAA values of Z2 varied slightly and were in the range 4.21 ± 1.46 to 20.20 ± 0.0 mL/g against these bacteria. In turn, TAA values of Z3 were slightly lower (4.38 ± 0.0-8.75 ± 0.0 mL/g). towards reference microorganisms. The values of MBC/MIC or MFC/MIC were in the range 1-4. MICs equal to MBC or MFC (MBC/MIC = 1 and MFC/MIC = 1) were shown for most, i.e., 12 (75%) and 11 (68.75%) strains in the case of Z1 and Z3, respectively. For Z2, these values were different. However, these ratios were mainly 1 (for six (37.5%) strains) and 2 (for seven (43.75%) strains). The value of 4 was found the least frequently (only for Z2 and Z3). The bacteriostatic effect (MBC/MIC > 4 or MFC/MIC > 4) of the tested extracts was not demonstrated. In the next stage of this study, the total antimicrobial activity (TAA) was assessed. The total antibacterial activity or total antifungal activity of the studied extracts Z1-Z2 was shown in Table 4. The MIC and TAA, both total antibacterial activity and total antifungal activity values are important pharmacological tools. They are useful in determining the activity of extracts in mg/mL (potency) of plants extracts for isolating bioactive compounds and total activity on mL/g (efficacy) for the selection of appropriate plant species [36]. Generally, their TAA values were the highest against Gram-positive bacteria: Staphylococcus spp., Micrococcus luteus, followed by Bacillus spp. (3.35 ± 0.0-20.20 ± 0.0 mL/g) and the lowest against Gram-negative bacteria and fungi belonging to Candida spp. (1.09 ± 0.0-4.21 ± 1.46 mL/g). Z1 had higher TAA towards reference strains of S. aureus, S. epidermidis and M. luteus (13.39 ± 0.0 mL/g). TAA values of Z2 varied slightly and were in the range 4.21 ± 1.46 to 20.20 ± 0.0 mL/g against these bacteria. In turn, TAA values of Z3 were slightly lower (4.38 ± 0.0-8.75 ± 0.0 mL/g).   As shown in Figure 3, Z1, Z2, and Z3 extracts had the mean total antibacterial activities of 6.49, 6.01 and 4.41 mL/g, respectively. In turn, the mean total antifungal activity of extracts was lower in the range 1.24-2. mL/g. In conclusion, Z1 and Z2 had a similar and As shown in Figure 3, Z1, Z2, and Z3 extracts had the mean total antibacterial activities of 6.49, 6.01 and 4.41 mL/g, respectively. In turn, the mean total antifungal activity of extracts was lower in the range 1.24-2. mL/g. In conclusion, Z1 and Z2 had a similar and better efficacy against both bacteria and fungi compared to Z3. The higher the TAA value, the more efficacious the plant extract. The results indicated that Z1-Z3 extracts from Z. bungeana showed some antimicrobial activity with bactericidal or fungicidal effect. Among all studied reference microorganisms, Gram-positive bacteria were the most sensitive to them. The lowest concentrations of Z1-Z3 extracts which inhibited the growth of the tested microorganisms or killed them ranged from 1.25 mg/mL to 20 mg/mL and from 2.5 mg/mL to 20 mg/mL, respectively. Overall, the Gram-positive bacteria were more sensitive to the extracts than the Gram-negative bacteria and yeasts from Candida. The difference in the sensitivity between these microorganisms may be due to the variation in their cell wall structure. The Gram- The results indicated that Z1-Z3 extracts from Z. bungeana showed some antimicrobial activity with bactericidal or fungicidal effect. Among all studied reference microorganisms, Gram-positive bacteria were the most sensitive to them. The lowest concentrations of Z1-Z3 extracts which inhibited the growth of the tested microorganisms or killed them ranged from 1.25 mg/mL to 20 mg/mL and from 2.5 mg/mL to 20 mg/mL, respectively. Overall, the Gram-positive bacteria were more sensitive to the extracts than the Gramnegative bacteria and yeasts from Candida. The difference in the sensitivity between these microorganisms may be due to the variation in their cell wall structure. The Gram-positive bacterial cell wall consists of 70-100 layers of peptidoglycans. Peptidoglycan is comprised of two polysaccharides, N-acetyl-glucosamine and N-acetyl-muramic acid cross-linked by peptide side chains and cross bridges [37]. It is possible that active compounds from extracts can easier break important bonds in cell wall structure in these bacteria. On the other hand, the cell wall of Gram-negative bacteria is far more complex, and it is among other things the reason they are more resistant for biologically active compounds.
There is little information in the literature on the biological activity of Z. bungeana extracts. However, there are reports on other Ziziphora species. Some authors showed antimicrobial effect of different extracts, EO or selected compounds derived from Ziziphora gender. The results and findings described herein are in accordance with some other studies.
The antibacterial activity of EO and its two main components (pulegone and 1,8cineole) obtained from the aerial flowering parts of Ziziphora clinopodioides subsp. bungeana (Juz.) Rech. f. was analyzed by Sonboli A. et al. [38] against seven bacteria. It was found that the EO exhibited interesting activity against S. epidermidis, S. aureus, E. coli, and B. subtilis with MIC values of 3.75 mg/mL. These results were similar to ours for Gram-positive bacteria.
In turn, the inhibitory effect of methanol extract and EO from Ziziphora persica was tested against 98 strains belonging to 51 bacteria species by standard dilution methods. The results showed that both extract and EO had antibacterial activity against many tested bacteria. The lowest MIC values (7.81 µg/mL) of EO were obtained against Bacillus dipsauri, Corynebacterium cystitidis, and Corynebacterium flavescens [39].
Z. clinopodioides was studied by subsequent researchers. The LC-MS/MS results of Özkan E.E. et al. [3] indicated that quinic acid, malic acid and rhoifolin are the abundant compounds in aerial and root ethanol extracts of Z. clinopodioides. Both extracts exhibited moderate antifungal activity with MIC = 39.06 µg/mL against Candida tropicalis. Moreover, these extracts showed some better or the same antibacterial effect against reference S. aureus, S. epidermidis, and E. faecalis strains (MIC = 0.312-1.25 mg/mL) as our extracts of Z. bungeana.
In the case of Gram-negative bacteria (P. aeruginosa, E. coli, K. pneumoniae and P. mirabilis) and C. albicans, no activity was showed.
Moreover, the studies of Anzabi Y. et al. [40] showed that the Z. clinopodioides EOs was effective on many tested bacteria and can be used as natural antimicrobial drug against microorganisms causing urogenital tract infections in women. The aerial parts of Z. clinopodioides were also screened by other authors [41] for their possible antimicrobial activities. Methanol extract was found to have moderate antimicrobial activity against some microorganisms tested. Acinetobacter lwoffii and Candida krusei were the most sensitive for this extract. The antimicrobial properties were also found in Z. clinopodioides EOs collected from provinces in western Iran. The studied EO inhibited the growth of Listeria monocytogenes, S. typhimurium, E. coli O157:H7, B. subtilis, B. cereus, and S. aureus at MIC values between 0.03% and 0.04%. The Gram-positive bacteria were the most susceptible to it, while Gram-negative bacteria were resistant [1]. The interesting antibacterial activity against seven Gram-positive or Gram-negative bacteria exhibited also EO and methanol extract of Z. clinopodioides subsp. rigida (BOISS.) RECH. f. from Iran. The obtained results indicated that B. subtilis was the most sensitive microorganism to this EO, with the lowest MIC = 3.8 mg/mL. The growth inhibition of S. epidermidis and S. aureus was observed at similar MIC = 7.5 mg/mL. The inhibitory activity of EO against E. faecalis, K. pneumoniae, and E. coli was also determined with MIC values equal to or greater than 15 mg/mL. No activity was observed against P. aeruginosa [42].
The subsequent results of Hazrati et al. [2] showed 17 and 21 different compounds (comprising 99.7% of total EO) in Z. clinopodioides and Z. tenuior, respectively. The major identified compounds in EO analysis reported as pulegone and menthone for Z. clinopodioides, or pulegone and limonene for Z. tenuior. Both Ziziphora species were also rich in phenolic compounds. These authors investigated the antibacterial activity of EOs against important foodborne pathogenic bacteria and showed that they could be considered as good sources of natural antibacterial material as well as food preservative [2,15].
Additionally, Celiket al. Considering all above information, it can be concluded that Ziziphora plants may deliver extracts that are important from a pharmacological point of view, as they may help combat the occurring bacterial and fungal infections.

Antioxidant Activity Assessment
Antioxidant activity of Ziziphora bungeana extracts was compared using DPPH and ABTS radical scavenging assays ( Figure 4A) and determination of superoxide dismutase (SOD) activity ( Figure 4B). In respect of the radical scavenging potential, extract Z3 showed the most significant activity with EC 50 values of 15.00 ± 1.06 µg/mL and 13.21 ± 3.24 µg/mL for ABTS and DPPH assays, respectively. All tested extracts also showed significant SOD activity, dependent on the extract concentration. The most effective was extract Z1, showing > 90% SOD activity in all three tested concentrations. Extract Z2 was the least effective. At the concentration of 50 µg/mL, the mean SOD activity detected for this extract was 64.4 ± 0.55%.
The antioxidant activity of 50% (v/v) ethanolic extract from Z. bungeana was recently compared with other plants from Lamiaceae family by measuring its influence on the level of lipid peroxidation in the liver microsome and the membrane-stabilizing properties [44]. The antioxidant potential of the extracts was significant in both assays but moderate in comparison with other Lamiaceae plants.
(SOD) activity ( Figure 4B). In respect of the radical scavenging potential, extrac showed the most significant activity with EC50 values of 15.00 ± 1.06 µg/mL and 13. 3.24 µg/mL for ABTS and DPPH assays, respectively. All tested extracts also showed nificant SOD activity, dependent on the extract concentration. The most effective was tract Z1, showing > 90% SOD activity in all three tested concentrations. Extract Z2 was least effective. At the concentration of 50 µg/mL, the mean SOD activity detected for extract was 64.4 ± 0.55%. The antioxidant activity of 50% (v/v) ethanolic extract from Z. bungeana was rece compared with other plants from Lamiaceae family by measuring its influence on the l of lipid peroxidation in the liver microsome and the membrane-stabilizing properties The antioxidant potential of the extracts was significant in both assays but moderat comparison with other Lamiaceae plants.
However in the publication of Gursoy and co-investigators [41], the aerial parts o clinopodioides were found to be the strongest radical scavengers among the tested spe from Lamiaceae family, namely: Z. clinopodioides, Cyclotrichium niveum, and Mentha lo folia subsp. typhoides var. typhoides in the DPPH and beta-carotene/linoleic acid assays. calculated IC50 values for the tested extracts were 37.73 ± 1.18 µg/mg for DPPH and 8 ± 1.19% in the inhibition capacity of the linoleic acid. Moreover, the total phenolic con of its methanolic extract was the highest among the tested species and was equal to 12 +/− 2.26 µg/mg.
Recently, the antioxidant activity of the aqueous, ethyl acetate, and methanolic tracts from other Ziziphora species, Ziziphora taurica subsp. taurica, were compared u DPPH and ABTS scavenging assays. In both assays, the methanolic ectract from Z. tau was the most effective with IC50 values of 5.74 ± 0.08 mg/mL and 2.74 ± 0.10 mg/mL DPPH and ABTS scavenging, respectively. The EC50 values obtained in our study sug that the antioxidant potential of Z. bungeana extracts is higher than that of Z. taurica [4 However in the publication of Gursoy and co-investigators [41], the aerial parts of Z. clinopodioides were found to be the strongest radical scavengers among the tested species from Lamiaceae family, namely: Z. clinopodioides, Cyclotrichium niveum, and Mentha longifolia subsp. typhoides var. typhoides in the DPPH and beta-carotene/linoleic acid assays. The calculated IC 50 values for the tested extracts were 37.73 ± 1.18 µg/mg for DPPH and 83.56 ± 1.19% in the inhibition capacity of the linoleic acid. Moreover, the total phenolic content of its methanolic extract was the highest among the tested species and was equal to 129.55 +/− 2.26 µg/mg.
Recently, the antioxidant activity of the aqueous, ethyl acetate, and methanolic extracts from other Ziziphora species, Ziziphora taurica subsp. taurica, were compared using DPPH and ABTS scavenging assays. In both assays, the methanolic ectract from Z. taurica was the most effective with IC 50 values of 5.74 ± 0.08 mg/mL and 2.74 ± 0.10 mg/mL for DPPH and ABTS scavenging, respectively. The EC 50 values obtained in our study suggest that the antioxidant potential of Z. bungeana extracts is higher than that of Z. taurica [45].

Tyrosinase Activity Assay
Tyrosinase (EC 1.14.18.1) is a cooper containing metalloenzyme catalyzing the first two, rate-limiting steps of mammalian melanogenesis. Neither increased nor decreased activation of tyrosinase is desirable as it may lead to hyper-or hypopigmentation disorders, respectively. Natural extracts and compounds with tyrosinase inhibitory activity are particularly desired by the cosmetic industry as they serve as active ingredients in skin lightening cosmetics and rituals [46]. On the other hand, the compounds increasing the activity of tyrosinase might be considered as topical treatment for vitiligo [47].
Investigating the influence of novel extracts and compounds on tyrosinase activity is commonly performed using the assay utilizing commercially available mushroom tyrosinase, incubated with its substrate L-3,4-dihydroxyphenylalanine (L-DOPA), in the presence or absence of tested compound. Despite the low costs, simplicity, and high throughput of the procedure, the assay has several limitations, resulting from substantial differences between mushroom and mammalian tyrosinase [48]. Therefore, the influence of plantderived extracts and compounds on the activity of mushroom and mammalian tyrosinases may vary significantly [49,50].
As shown in Figure 5, none of the analyzed Ziziphora extracts significantly inhibited mushroom tyrosinase up to the concentration of 200 µg/mL ( Figure 5B). Extract Z2 slightly increased the activity of this enzyme at 25 and 50 µg/mL. In respect of the murine tyrosinase all Ziziphora extracts showed significant, dose-dependent inhibitory potential ( Figure 5A). The most potent inhibitor of murine tyrosinase was extracts Z2, decreasing the activity of tyrosinase by 50% at 200 µg/mL which was comparable with the inhibitory activity of kojic acid (KA), a tyrosinase inhibitor widely used in skin lightening cosmetics [49]. Extracts Z3 showed the lowest activity, significantly decreasing the activity of tyrosinase only at the highest analyzed concentration (200 µg/mL). To our knowledge, this is the first study investigating the effect of Ziziphora spp. extract on the activity of mammalian tyrosinase.
increased the activity of this enzyme at 25 and 50 µg/mL. In respect of the murine tyrosinase all Ziziphora extracts showed significant, dose-dependent inhibitory potential ( Figure  5A). The most potent inhibitor of murine tyrosinase was extracts Z2, decreasing the activity of tyrosinase by 50% at 200 µg/mL which was comparable with the inhibitory activity of kojic acid (KA), a tyrosinase inhibitor widely used in skin lightening cosmetics [49]. Extracts Z3 showed the lowest activity, significantly decreasing the activity of tyrosinase only at the highest analyzed concentration (200 µg/mL). To our knowledge, this is the first study investigating the effect of Ziziphora spp. extract on the activity of mammalian tyrosinase. Several phytochemicals identified in Ziziphora extracts were described in scientific literature as effective mushroom tyrosinase inhibitors, including acetophenone [51] identified in Z. tenuior [52] and cuminyl aldehyde (syn. cumaldehyde) [53] from Z. clinopodioides subsp. rigida [54]. The last compound was also shown to suppress melanin synthesis in B16F10 murine melanoma cells [55].
Z. clinopodioides extracts were previously found to exhibit weak tyrosinase inhibitory potential. The extracts from the overground parts of the plant exhibited weak inhibitory potential against the enzyme at the concentration of 200 µg/mL with 8.60 ± 0.87% inhibition compared to kojic acid (KA) for whom the inhibition percentage was calculated as 95.26 ± 0.23% [3]. Several phytochemicals identified in Ziziphora extracts were described in scientific literature as effective mushroom tyrosinase inhibitors, including acetophenone [51] identified in Z. tenuior [52] and cuminyl aldehyde (syn. cumaldehyde) [53] from Z. clinopodioides subsp. rigida [54]. The last compound was also shown to suppress melanin synthesis in B16F10 murine melanoma cells [55].
Z. clinopodioides extracts were previously found to exhibit weak tyrosinase inhibitory potential. The extracts from the overground parts of the plant exhibited weak inhibitory potential against the enzyme at the concentration of 200 µg/mL with 8.60 ± 0.87% inhibition compared to kojic acid (KA) for whom the inhibition percentage was calculated as 95.26 ± 0.23% [3].
Mushroom tyrosinase activity was also analyzed by Tomczyk and co-workers in respect of Z. taurica extracts [45]. The IC 50 values for aqueous, ethyl acetate and methanolic extracts were 2.29 ± 0.13, 1.37 ± 0.07 and 1.46 ± 0.06 mg/mL, respectively. These values suggest that Z. bungeana extracts Z1, Z2, and Z3 might be effective against mushroom tyrosinase, but at higher concentrations than tested in this study.
In this study, the authors focused on the assessment of Z. bungeana cytotoxic effect on human and murine melanoma cells ( Figure 6B-D) in comparison with a known chemotherapeutic agent, 5 fluorouracil (5 FU). Human keratinocytes HaCaT served as noncancerous control cells ( Figure 5A).
Extract Z1 at 200 µg/mL was slightly cytotoxic for human melanoma A375 cell line, reducing the number of viable cells by ca. 20%. It was not cytotoxic for HaCaT keratinocytes, B16F10, and SKMEL-3 melanoma cells. Extract Z2 at 200 µg/mL significantly reduced the number of viable A375 and SK-MEL3 melanoma cells by ca 28% and 23%. However, it showed comparable cytotoxicity towards HaCaT keratinocytes. Extract Z3 was cytotoxic only for B16F10 murine melanoma cells, reducing their viability by 15% at 200 µg/mL.
The cytotoxicity of Ziziphora spp. Extracts towards murine and human melanoma cells as well as human noncancerous skin cells has not been described in the scientific literature to date. Several compounds found in Ziziphora preparations, such as extracts and EO, including linalool and α-terpineol (Z. clinopodioides), carvacrol (Z. tenuior, Z. clinopodioides), thymol (Z. tenuior), and terpinen-4-ol (Z. clinopodioides), are known to induce apoptosis in melanoma cell lines [11]. In the study of Yousefbeyk and co-investigators [59] Z. clinopoides n-hexane extract that was found rich in pulegone, menthone and menthol exhibited strong cytotoxic activity against K-562 and T-47D cell lines with EC 50 values of 80 ± 2.56 µg/mL and 77.41 ± 12.89, respectively. Interestingly, more polar fractions did not show cytotoxic effects.
suggest that Z. bungeana extracts Z1, Z2, and Z3 might be effective against mushroom tyrosinase, but at higher concentrations than tested in this study.
In this study, the authors focused on the assessment of Z. bungeana cytotoxic effect on human and murine melanoma cells ( Figure 6B-D) in comparison with a known chemotherapeutic agent, 5′fluorouracil (5′FU). Human keratinocytes HaCaT served as noncancerous control cells ( Figure 5A).  Available scientific data on the in vitro cytotoxic effect of Ziziphora spp. preparations were obtained using EOs. Azimi and co-workers showed that the EO from Z. tenuior induces apoptosis in human colorectal cancer cells HT-29 in a concentration range of 50-200 µg/mL. The apoptotic effect was mediated by increased caspase 3 and 9 expression at mRNA and protein levels and decreased levels of Bcl-2 [56]. Ghavan et al. showed that Z. clinopodioides subsp. rigida EO is cytotoxic for human ovarian cancer cells (OVCAR-3) [60].

The Hemolytic Activity Assay (Toxicity towards Erythrocytes)
In the present studies, the toxicity of Z1-Z3 extracts from Z. bungeana towards red blood cells was calculated in vitro hemolytic assay. The erythrocyte model (erythrocyte lysis assay; ELA) was used to analyzed their effect on cell membrane [60]. The results revealed that studied extracts exhibit negligible toxicity as compared to the positive control Triton X-100 (100% erythrocyte lysis). As presented in Figure 7, hemolytic activity of each extract was related to their concentration. The highest concentrations of the studied extracts (20 mg/mL) showed some hemolytic activity in the range 6.1-30%. In turn, their concentrations that did not exert any hemolytic effect ranged from 1.25 to 2.5 mg/mL and the percentage of lysed red blood cells of 0-4.5 was within the permissible limit of 5% hemolysis [61]. The Z1 and Z3 extracts exhibited lower hemolytic activity (0-18.9%) than Z2 (2.5-30%) and did not affect the stability of the erythrocyte membrane. Data obtained using ELA confirm that antimicrobial effect, especially against Gram-positive bacteria (MIC = 1.25-5 mg/mL), was observed at non-cytotoxic concentrations of extracts from Z. bungeana. mRNA and protein levels and decreased levels of Bcl-2 [56]. Ghavan et al. showed that Z. clinopodioides subsp. rigida EO is cytotoxic for human ovarian cancer cells (OVCAR-3) [60].

The Hemolytic Activity Assay (Toxicity towards Erythrocytes)
In the present studies, the toxicity of Z1-Z3 extracts from Z. bungeana towards red blood cells was calculated in vitro hemolytic assay. The erythrocyte model (erythrocyte lysis assay; ELA) was used to analyzed their effect on cell membrane [60]. The results revealed that studied extracts exhibit negligible toxicity as compared to the positive control Triton X-100 (100% erythrocyte lysis). As presented in Figure 7, hemolytic activity of each extract was related to their concentration. The highest concentrations of the studied extracts (20 mg/mL) showed some hemolytic activity in the range 6.1-30%. In turn, their concentrations that did not exert any hemolytic effect ranged from 1.25 to 2.5 mg/mL and the percentage of lysed red blood cells of 0-4.5 was within the permissible limit of 5% hemolysis [61]. The Z1 and Z3 extracts exhibited lower hemolytic activity (0-18.9%) than Z2 (2.5-30%) and did not affect the stability of the erythrocyte membrane. Data obtained using ELA confirm that antimicrobial effect, especially against Gram-positive bacteria (MIC = 1.25-5 mg/mL), was observed at non-cytotoxic concentrations of extracts from Z. bungeana.  The erythrocyte model presents general indication of membrane toxicity. The red blood cell membrane shows similarity to other cell membranes. Hemolysis is due to erythrocyte cells destruction resulting from lysis of the membrane lipid bilayer [60,61]. The obtained data using ELA confirm antibacterial activity of Z1-Z3 extracts at their non-cytotoxic concentrations (MIC = 1.25-5 mg/mL) against staphylococci, micrococci, and some bacilli. Therefore, it seems practical to use these extracts in the future in the prevention and treatment of infections caused by selected microorganisms, mainly Gram-positive bacteria.

Chemometric Assessment
Principal component analysis (PCA) was conducted separately for the Z1-Z3 relative compositions (C rel ) and the Z1-Z3 relative activities (A rel ) (See Tables S2 and S3 in the Supplementary File), whereas the extracts were treated as vectors defined on C rel or A rel values. The resulting spaces were two-dimensional, since in both cases the first two principal components extracted almost 100% of the information (expressed as variance) of studied ensembles. In the case of the C rel system (Figure 8A), the relative composition of the Z3 and Z1 extracts were reversely correlated, constituting the first dimension of the studied dataset, whereas the Z2 extract exhibited rather unique proportions of the selected eleven analyzed compounds (named in the Table 1 as 1, 2, 4, 6, 9, 10, 11, 13, 15, Table S2 in the Supplementary File), defining the second dimension of the vector space. The obtained conclusions are logical, as dichloromethane is characterized by a much lower polarity than ethanol and water, and that is why the extracts obtained using dichloromethane can show a different fingerprint from alcoholic or water ones. In the case of A rel dataset ( Figure 8B), the relative biological activities of Z2 and Z3 extracts were reversely correlated (Dimension 1), while the Z1 extract exhibited different properties, possibly thanks to other metabolites, e.g., peptides, sugars, or proteins whose identity was not analyzed in this study, solely explaining the second dimension of the studied space. methane can show a different fingerprint from alcoholic or water ones. In the case of Arel dataset ( Figure 8B), the relative biological activities of Z2 and Z3 extracts were reversely correlated (Dimension 1), while the Z1 extract exhibited different properties, possibly thanks to other metabolites, e.g., peptides, sugars, or proteins whose identity was not analyzed in this study, solely explaining the second dimension of the studied space. The linear maps of the studied compounds ( Figure 9A) and the activity tests ( Figure  9B) were presented in the same spaces as the respective linear maps of the Z1-Z3 vectors in Figure 8. In the case of Crel ensemble, the relative amounts of the 1, 2, 6, 9, 11, and 13 compounds were very similar for all three extracts. Z3 clearly excelled in the relative amounts of 23 and 26, whereas it was very low on 10′s concentration. On the contrary, Z1 The linear maps of the studied compounds ( Figure 9A) and the activity tests ( Figure 9B) were presented in the same spaces as the respective linear maps of the Z1-Z3 vectors in Figure 8. In the case of C rel ensemble, the relative amounts of the 1, 2, 6, 9, 11, and 13 compounds were very similar for all three extracts. Z3 clearly excelled in the relative amounts of 23 and 26, whereas it was very low on 10 s concentration. On the contrary, Z1 contained impressive amounts of 10 while lacking 23 and 26. Z2 was quite low in 4 and 10 yet exhibited higher-than-average amounts of 15 and 26. While taking into account the relative values of activity tests, Z3 excelled with the B16F10 cell line (I and II), on the contrary to Z1. Moreover, Z2 was quite good at IV (A375 cell line) and V (SKMEL-3 cell line), yet toxic to HaCaT cells (III), whereas Z1 exhibited poor activity at VIII (SOD assay).

23, 26-see
While comparing the above with the distribution of the relative compositions of the eleven analyzed compounds (named in Table 1 as 1, 2, 4, 6, 9, 10, 11, 13, 15, 23, 26) within the Z1-Z3 extracts, one might conclude that eventual toxic effects towards HaCat cell line (III) at the highest dose, exhibited solely by Z2, could result from the presence of very high, relative amounts of the compounds 15 and 26. These findings may be due to the fact, that the analyzed concentration used in the calculations was high and exceeded safe doses for both diosmin and ursolic acid, respectively. In the meantime, Z2 utterly failed at the tests I and II (B16F10 cell line), similarly to Z1. Since Z3 succeeded at I and II, while it was also rich with the compounds 23 and 26, the good result at I and II could be directly associated with high relative amounts of 23 (acacetin). Finally, Z1 extract did relatively well at the tests IV (A375 cell line) and VII (mushroom tyrosinase assay), while it exhibited high amounts of a ziziphoroside isomer 2 (10). Possibly, its presence influences the total activity of the extract. Previously, other species of Ziziphora were proven to inhibit tyrosinase [10]. On the basis of the resulting images, no other compounds could be related to the biological activities of Z1-Z3 extracts in a straightforward manner.
contained impressive amounts of 10 while lacking 23 and 26. Z2 was quite low in 4 and 10 yet exhibited higher-than-average amounts of 15 and 26. While taking into account the relative values of activity tests, Z3 excelled with the B16F10 cell line (I and II), on the contrary to Z1. Moreover, Z2 was quite good at IV (A375 cell line) and V (SKMEL-3 cell line), yet toxic to HaCaT cells (III), whereas Z1 exhibited poor activity at VIII (SOD assay). While comparing the above with the distribution of the relative compositions of the eleven analyzed compounds (named in Table 1 as 1, 2, 4, 6, 9, 10, 11, 13, 15, 23, 26) within the Z1-Z3 extracts, one might conclude that eventual toxic effects towards HaCat cell line (III) at the highest dose, exhibited solely by Z2, could result from the presence of very high, relative amounts of the compounds 15 and 26. These findings may be due to the fact, that the analyzed concentration used in the calculations was high and exceeded safe doses for both diosmin and ursolic acid, respectively. In the meantime, Z2 utterly failed at the tests I and II (B16F10 cell line), similarly to Z1. Since Z3 succeeded at I and II, while it was also rich with the compounds 23 and 26, the good result at I and II could be directly associated with high relative amounts of 23 (acacetin). Finally, Z1 extract did relatively well at the tests IV (A375 cell line) and VII (mushroom tyrosinase assay), while it exhibited high amounts of a ziziphoroside isomer 2 (10). Possibly, its presence influences the total activity of the extract. Previously, other species of Ziziphora were proven to inhibit tyrosinase [10]. On the basis of the resulting images, no other compounds could be related to the biological activities of Z1-Z3 extracts in a straightforward manner.

Microorganisms
The reference strains of microorganisms from American Type Culture Collection (ATCC) (Manassas, VA, USA) were used in the study.

Extraction Procedure
First, the aerial parts of the plant were powdered using an electric mill (type WZ-1, ZBPP, Poland). Next, 10 g portions of the aerial parts of the plant were divided into three parts to provide three extracts using the following extracting solvents-water (Z1), dichloromethane (Z2) and 96% ethanol (Z3). After adding 50 mL of solvents, the extraction was performed three times, 30 min each, at room temperature using an ultrasonic bath with no heating. Then, the extracts were centrifuged at 3500 rpm for 10 min, filtered through a nylon syringe filter (pore diameter 0.22 µm), and evaporated in the weighted vials using the Eppendorf Concentrator Plus (Hamburg, Germany) at the temperature of 45 • C. Weighted samples were kept in the freezer before chromatographic studies and bioactivity evaluations.
The extracts' constituents were separated in gradient method composed of 0.1% formic acid (solvent A) and acetonitrile with the addition of 0.1% formic acid (solvent B) in the following program: 0 min: 10% B, 10 min: 20% B, 15 min: 40% B, 17-22 min: 95% B, 22.10 min: 10% B. The run lasted 30 min, the flow rate was set at 0.200 mL/min and the injection volume was set at 5 µL, and the concentration of the extracts was 10 mg/mL. Chromatographic separation was performed on the RP-18 chromatographic column (dimensions: 150 mm × 2.1 mm; dp = 3.5 µm) (Zorbax Eclipse Plus by Agilent Technologies, Santa Clara, CA, USA).
The detection on the mass spectrometer was achieved in the following settings, using both negative and positive ionization mode: m/z range of 100-1700 Da, capillary voltage of 3000 V, gas and sheath gas temperatures of 275 and 325 • C, gas flows of 12 L/min, respectively, fragmentation voltage of 110 V, skimmer voltage of 65 V, and collision energies of 10 and 20 V. In the used method, the MS/MS spectra were recorded for the two most intense peaks per scan The structure determination was based on the fragmentation spectra, literature data, retention times, and open databases (Metlin).

In Vitro Antimicrobial Activity Assay
The three extracts Z1-Z3 from Ziziphora bungeana were investigated in vitro for antibacterial and antifungal activities. In these studies, the broth microdilution was used.
The tests were performed in accordance with the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [14,62,63] and Clinical and Laboratory Standards [64,65]. The used microbial cultures were first subcultured and on nutrient agar Next, the bacterial or fungal suspensions were introduced into each well of the microplate to obtain final density of 1.5 × 10 6 CFU/mL for bacteria and 5 × 10 4 CFU/mL for yeasts. After 18-24 h incubation at 35 • C, the MIC value was assessed in the BioTek spectrophotometer (Biokom, Janki, Poland) as the minimal concentration of the samples that showed complete microbial growth inhibition. The inhibition of bacterial and fungal growth was assessed by comparison with control cultures in media without any sample tested. Standard drugs: ciprofloxacin (antibacterial chemotherapeutic) and nystatin (antifungal antibiotic) (Sigma-Aldrich Chemicals, St. Louis, MO, USA) were used as reference substances. Appropriate DMSO, sterile, and growth controls were prepared. The media with and without tested extracts/DMSO were used as controls [13,[62][63][64][65][66][67].
Subsequently, minimal bactericidal concentration (MBC) or minimal fungicidal concentration (MFC) values of extracts were determined by transferring the cultures from each MIC determination well to the appropriate solid medium. After incubation, the lowest concentrations of extracts with no visible bacterial or fungal growth were evaluated as MBC or MFC. All the experiments were repeated three times as independent assays, and representative data are presented [13,[62][63][64][65][66] The total antibacterial activity (TAA) is a function of the extraction yield in milligram per 1 g of plant material and the minimal inhibitory concentration (MIC), expressed in milliliter per gram (mL/g). TAA indicates the volume of water or solvent, when added to 1 g of the extract, that will still inhibit the growth of the pathogen [68][69][70].

Antioxidant Activity DPPH Scavenging Assay
The DPPH radical scavenging assay was performed as described by Matejic et al. [71]. Briefly, 100 µL of Z1, Z2 or Z3 diluted extracts (0.48-1000 µg/mL) was mixed with equal volume DPPH working solution (25 mM DPPH in 99.9% methanol; A540 ≈ 1). 100 µL of the solvent mixed with 100 µL DPPH served as a control sample. After 20 min incubation at RT in darkness, the absorbance of the samples was measured at λ = 540 nm using a where Abs(S) is the absorbance of the sample and Abs(C) is the absorbance of the control sample (DPPH + solvent). Obtained results were used to calculated EC 50 values defined as the concentration of dried extract/fraction that is required to scavenge 50% of the DPPH radical activity.

ABTS Scavenging Assay
ABTS radical scavenging assay was performed according to Re and co-workers [72] with some modifications. Briefly, 135 µL of ABTS working solution (7 mM ABTS in 2.45 mM K 2 S 2 O 8 diluted in distilled H 2 O up to A405 ≈ 1) was mixed with 15 µL of Z1, Z2 or Z3 diluted extract (0.48-1000 µg/mL) or solvent control. Following 15 min incubation at RT in darkness, the absorbance of the samples was measured at λ = 405 nm using a microplate reader (FilterMax F5 Molecular Devices, USA). The obtained values were corrected by the absorbance value of the sample without ABTS. The percentage of ABTS radical scavenging was calculated based on the equation: where Abs(S) is the absorbance of the extract and Abs(C) is the absorbance of the control sample (ABTS + solvent).
Obtained results were used to calculated EC 50 values defined as the concentration of dried extract/fraction that is required to scavenge 50% of the ABTS radical activity.

SOD Inhibitory Assay
The influence of Z1, Z2, and Z3 extracts on the activity of superoxide dismutase (SOD) was measured using SOD Determination Kit (cat. No. 19160, Sigma Aldrich, Merck, Darmstadt, Germany), according to manufacturer's instructions.
Mushroom tyrosinase activity assay was performed according to the protocol described by Uchida and co-workers [74] For this analysis, 120 µL phosphate buffer (100 mM, pH = 6.8) was mixed with 20 µL of diluted extracts (final concentrations 10-200 µg/mL) and 20 µL of mushroom tyrosinase (500 U/mL) and pre-incubated at room temperature for 10 min. Following the addition of 40 µL 4 mM L-DOPA, the samples were incubated for another 20 min at RT.
The activity of murine tyrosinase was assessed by Incubating the volume of B16F10 cell lysate containing 20 µg protein with 20 µL of diluted extracts (final concentrations 10-200 µg/mL), 40 µL 4 mM L-DOPA and 100 mM phosphate buffer pH 6.8 (up to 200 µL). The reaction was carried out for 4 h at 37 • C. Control samples (100% tyrosinase activity) for both assays contained an appropriate volume of the solvent instead of the extract. In both assays, the dopachrome formation was measured spectrophotometrically at λ = 450 nm using FilterMax F5 microplate reader (Molecular Devices, USA). The obtained values were corrected by the absorbance value of the extracts without mushroom or murine tyrosinase and L-DOPA. Each sample was analyzed in 3 independent repetitions. Kojic acid was used as a known tyrosinase inhibitor control.

In Vitro Cytotoxicity Assay
The cytotoxicity of Z1, Z2 and Z3 extracts was established by Neutral Red Uptake Test, as described by Repetto et al. [75] using human immortalized keratinocytes HaCaT (CLS Cell Lines Service GmbH, Eppelheim, Germany) [76], murine melanoma B16F10 (ATCC CRL-6475) and human melanoma A375 (ATCC CRL-1619) and SK-MEL3 (ATCC HTB-69) (LGC Standards, Łomianki, Poland). All cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM)/high glucose supplemented with 10% fetal bovine serum (FBS, Pan Biotech, Aidenbach, Germany) at 37 • C in a humidified atmosphere with 5% CO 2 . For the experiments 3000 cells were plated per well onto a 96-well plate and grown overnight. Then, the cells were treated with various concentrations of Z1, Z2, or Z3 extracts (12.5-200 µg/mL) or an equal volume of the solvent control. Following 48 h of culture, the cells were incubated for 3 h in DMEM containing 1% FBS and 33 µg/mL neutral red, following by washing in PBS and lysis using acidified ethanol solution (50% v/v ethanol, 1% v/v acetic acid). The absorbance of the released neutral red was measured using FilterMax F5 microplate reader (Molecular Devices, San Jose, CA, USA) at λ = 540 nm. The mean measurement value for the lysate from control cells was set as 100% cellular viability and used to calculate the percentage of viable cells following extracts treatment.

Toxicity to Erythrocyte Assay
The erythrocyte lysis assay (ELA) was performed to study the toxicity of the extracts Z1, Z2 and Z3 from Ziziphora bungeana on red blood cells. In the first, erythrocytes were harvested from 5.0 mL fresh sheep blood (BioMaxima S.A., Poland) by centrifugation for 10 min at 1000× g and washed with 0.85% NaCl. Subsequently, 2% erythrocyte suspension was prepared in sterile phosphate buffer saline and in a volume of 100 µL was added to each well of a 96-well microtiter plate. The serial dilutions of these extracts ranging from 0.01 to 20 mg/mL were performed. To estimate the relative hemolytic potential of Z1, Z2, and Z3, the appropriate controls, i.e., 100% erythrocyte lysis using 4% Triton X-100 (Pol-Aura, Różnowo, Poland) and 0% lysis in saline solution, were used. Plates with samples were incubated for 1 h at 37 • C, then centrifuged for 10 min at 1000× g to separate the unlysed erythrocytes, and subsequently, the supernatant was transferred to a new plate. The absorbance was measured spectrophotometrically at 450 nm. The ELA represents an advantageous bioassay, because the lytic response can be measured photometrically by the amount of released hemoglobin. The hemolysis percentage was calculated according to the equation: % hemolysis = [(A450 of tested extract treated sample-A450 of buffer treated sample)/(A450 of 4% Triton X-100 treated samples-A450 of buffer treated sample)] × 100 [60,[77][78][79].

Chemometric Analysis
All the chemometric analyses and visualizations were performed using R v4.2.0 [80] programming language in RStudio [81] software with pracma [82], factoextra [83], matlib [84], and corrplot [85] packages installed. After the standard, formal decomposition of the covariance matrices was calculated for the C rel (relative compositions) and A rel (relative activities) autoscaled datasets, and two principal components (PCs) were considered relevant in both cases. After the selection of the relevant PCs, their vectors were rotated in space in order to maximize the values of correlation coefficients between the original variables and the two orthogonal factors using the VARIMAX algorithm. In every case, compound/activity test scores in the space of the resulting varivectors (dimensions) were calculated by multiplying the matrix of the autoscaled C rel /A rel dataset by the matrix of the original variables' loadings in the space of the resulting varivectors.

Conclusions
The presented results show the significance of Ziziphora bungeana extracts in terms of their composition and bioactivity. Twenty-six secondary metabolites were identified in the prepared extracts from Z. bungeana in the HPLC-ESI-QTOF-MS/MS analysis, that belonged to flavonoids, phenolic acids, terpenes, and organic acids. The results of antimicrobial studies indicated that extracts Z1, Z2, and Z3 showed potential activity with bactericidal or fungicidal effects. Among reference microorganisms, Gram-positive bacteria strains Staphylococcus spp., Micrococcus luteus, followed by Bacillus spp. were the most susceptible to the tested extracts (3.347-20.202 mL/g) in comparison with Gram-negative bacteria and fungi. Spectrophotometric assays proved the strongest antiradical properties of Z3 (EC 50 values of 15.00 ± 1.06 µg/mL and 13.21 ± 3.24 µg/mL for ABTS and DPPH assays, respectively) and a marked SOD stimulatory action (>90% SOD activity) for Z1. In the murine tyrosinase assay all Ziziphora extracts showed significant, dose-dependent whitening properties. The most potent inhibitor of murine tyrosinase was extract Z2, decreasing the activity of tyrosinase by 50% at 200 µg/mL which was comparable with the inhibitory activity of kojic acid. All extracts were slightly cytotoxic for melanoma cells. However, Z2 at the concentration of 200 µg/mL showed a comparable cytotoxicity towards HaCaT keratinocytes. Moreover, our data suggest that the extracts Z1 and Z3 are not toxic for HaCaT cell lines or for erythrocyte membranes at the tested concentrations, which gives hope for its potential internal and external administration. The chemometric analysis performed to deliver the connections between the composition and biological properties of the extracts confirmed a different identity of all three extracts. According to the obtained results, the presence of the ziziphoroside isomer could induce anti-tyrosinase properties to the highest extent, whereas the presence of a higher quantity of acacetin could increase the anticancer potential of an extract.