Antimicrobial Properties and Assessment of the Content of Bioactive Compounds Lavandula angustifolia Mill. Cultivated in Southern Poland

Lavender is a valued plant due to its cosmetic, perfumery, culinary, and health benefits. A wide range of applications is related to the composition of bioactive compounds, the quantity and quality of which is determined by various internal and external factors, i.e., variety, morphological part of the plant, and climatic and soil conditions during vegetation. In the presented work, the characterization of antimicrobial properties as well as the qualitative and quantitative assessment of bioactive compounds in the form of polyphenols in ethanol extracts from leaves and flowers of Lavandula angustifolia Mill. intended for border hedges, cultivated in the region of southern Poland, were determined. The composition of the fraction of volatile substances and antioxidant properties were also assessed. The conducted research shows that extracts from leaves and flowers significantly affected the viability of bacterial cells and the development of mold fungi. A clear decrease in the viability of bacteria and C. albicans cells was shown in the concentration of 0.32% of extracts. Leaf extracts were characterized by a much higher content of polyphenols and antioxidant properties than flower extracts. The composition of volatiles measured by GC-MS was significantly different between the extracts. Linalyl acetate and ocimene isomers mix dominated in flower extracts, whereas coumarin, γ-cadinene, and 7-methoxycoumarin were identified as dominant in leaf extracts.


Introduction
Lavender (Lavandula angustifolia Mill.) is one of the most valued plants used in cosmetology and aromatherapy. It has a deodorizing, relaxing, antiseptic, and anti-inflammatory effect. In the Mediterranean countries, it is used in the design of creative cuisine as an additive that improves the taste of meat, ice cream, and drinks and also as a food preservative. Lavender is also a highly valued plant in landscaping, as it is suitable for borders, hedges, The aim of the presented work was to determine the antibacterial and fungicidal properties of ethanol extracts from the flowers and leaves of L. angustifolia grown in southern Poland (Wyżyna Miechowska). At the same time, the chemical composition of volatile substances, polyphenol profile, and antioxidant properties were analyzed in order to confirm the relationship between the composition of the extracts and the obtained antimicrobial properties. Lavender is not a plant popular in cultivation in Poland on a large scale for use as a raw material by the cosmetic or pharmaceutical industry; it rather appears only in home architecture, in gardens. We established a large experimental crop in order to demonstrate that this plant obtained from Poland also has good antimicrobial and antioxidant properties, which could be successfully used by the pharmaceutical and domestic market of cosmetic products as an ingredient in the formulation of these products, giving them antimicrobial and antioxidant properties.

Antimicrobial Activity
Figures 1 and 2 characterize the effect of lavender flower and leaf extracts on the viability of Stapchylococcus aureus and Pseudomonas aeruginosa bacteria and Candida albicans yeast. In each of the analyzed cases, it was found that the extracts inhibited the viability of microorganisms. Similar results were obtained in both the XTT and Presto Blue tests, indicating that extracts in very low concentrations of 0.31% are sufficient to significantly reduce the viability of microorganisms. It was also shown that lavender extracts inhibit the growth activity of mold fungi. The Trichoderma viride fungus is one of the organisms characterized by dynamic growth, as can be observed in control tests, in which complete overgrowth of the microbial substrate was obtained on the third day of cultivation. The growth of the tested fungus was completely inhibited when the extract content in the medium was at the level of 1 mL/100 mL ( Table 1). The Chaetomium globosum fungus was more sensitive than T. viride to the biocidal effect of lavender extracts. No growth was observed on the microbial medium for any of the concentrations used (Table 2). In order to confirm or exclude the biocidal effect of 60% ethanol on the growth of fungi, control tests were performed. It was not found that the ethanol used in amounts from 0.5 to 5 mL had an inhibitory effect on the growth of the tested microorganisms. overgrowth of the microbial substrate was obtained on the third day of cultivation. The growth of the tested fungus was completely inhibited when the extract content in the medium was at the level of 1 mL/100 mL ( Table 1). The Chaetomium globosum fungus was more sensitive than T. viride to the biocidal effect of lavender extracts. No growth was observed on the microbial medium for any of the concentrations used (Table 2). In order to confirm or exclude the biocidal effect of 60% ethanol on the growth of fungi, control tests were performed. It was not found that the ethanol used in amounts from 0.5 to 5 mL had an inhibitory effect on the growth of the tested microorganisms.

Antioxidant Assay
The antioxidant activity of extracts from lavender leaves and flowers was assessed on the basis of the ability of the extracts to reduce ABTS •+ , DPPH • radicals, and TPTZ compound (FRAP). Total polyphenol content (TPC) was also measured. In all analyzed cases, a higher antioxidant activity and a higher content of polyphenols were found in lavender leaf extracts (ZO2) ( Table 3). The ability of lavender leaf extracts to reduce free radicals and iron was almost twice as high as that of extracts obtained from flowers. At the same time, a very high level of influence (99.9%) of the type of extract (ZO1 and ZO2) on their antioxidant activity was indicated ( Table 4). The error in this respect was only 0.1%, which proves the negligible influence of other factors, which were not taken into account in the conducted research. With regard to the content of polyphenols, a significant effect of the type of extract was also noted, amounting to 98.9%.

Chemical Composition
GC-MS analysis showed clear differences in the chemical composition of leaves and flowers. The predominant component of the flowers turned out to be linalyl acetate, whose percentage in the extract was estimated at 25.83% (Tables 5 and 6). A sizable content of ocimene isomers belonging to monterpenes was also identified. Linalool, which dominates the composition of essential oils, was identified at the level of 2.23% in the obtained ethanol extracts. Small amounts of β-pinene (2.39%), limonene (7.09%), and myrcene (4.79%) have also been identified in flower extracts. Eleven substances were identified in lavender leaf extracts, and only four of the identified substances coincided with the substances identified in flower extracts. The leaf extracts were dominated by coumarin, γ-cadinene, 7-methoxycoumarin, and α-santalene (Tables 7 and 8). The identification of p-cymene, m-cymene, limonene, and eucalyptol were within detection limits. The confirmation of correctly identified compounds is the calculated linear retention indexes (LRI). In addition to the calculated LRI, the index values given in the literature have also been added for direct comparison and to verify the accuracy of the identification. Also, it should be stated that in the evaluation of the content of phenolic compounds (HPLC), leaf extracts are characterized by a higher content of these components. Based on the conducted research, 22 substances belonging to phenolic compounds were characterized (Table 9). Rosmarinic acid and ferulic acid glucosie III were dominant in both leaf and flower extracts. In addition, very high amounts of undefined caffeic acid derivative were identified in lavender leaf extracts. In the case of flower extracts, only two of the identified substanceschicoric acid and apigenin-were present in higher amounts than in leaf extracts.

Graphical Interpretation of Results
The use of multivariate analysis (clusters) made it possible to capture the hidden relationships between the analyzed variables within leaf extracts and do so separately from lavender flower extracts (Figure 3a,b). The applied data agglomeration showed connections between antimicrobial properties (antibacterial, fungicide) and the content of volatile compounds, phenolic acids, and flavonoids. The cluster analysis of compounds extracted from lavender flowers (ZO1) (Figure 3a) formed three main clusters, of which phenolic acids (9P) were in the first one and volatile compounds represented by the variables 6VC, 7VC, 8VC, and 11AA in the second. The third cluster included the remaining analyzed data (1B, 2B, 3B, 13AA, 4MM, 5MM, 10F, 14AA, 12AA). The graphical interpretation of the data resulting from the cluster analysis in the case of lavender leaf extracts (ZO2) (Figure 3b) also showed three main data clusters characterized by common variability. Within the first cluster, as in the case of ZO1, there were phenolic acids, whereas the second cluster contained antioxidant activity expressed by the ABTS test (12AA), the DPPH test (13AA), and the content of flavonoid compounds (10F). The remaining data were included in cluster 3. The use of a heat map with the scaling function allowed for a graphical comparison of the qualitative characteristics of both types of extracts ( Figure 4). It is clearly visible that lavender leaf extracts (ZO2) have a higher antioxidant activity expressed by TCA (11AA), ABTS (12AA) and FRAP (14AA) tests, and a higher content of phenolic acids (9P), flavonoids (10F), and volatile compounds than lavender extracts from ZO1 lavender flowers. Lavender flower extracts, on the other hand, have a slightly higher antibacterial activity than lavender leaf extracts.

Discussion
In recent years, there have been there have been an increasing number of detailed scientific reports on the assessment of the properties of lavender raw material. This plant, due to its health care and culinary values, is more and more often cultivated in various regions of the world. In the studies of many authors, the quality of plant materials largely depends

Discussion
In recent years, there have been there have been an increasing number of detailed scien tific reports on the assessment of the properties of lavender raw material. This plant, due

Discussion
In recent years, there have been there have been an increasing number of detailed scientific reports on the assessment of the properties of lavender raw material. This plant, due to its health care and culinary values, is more and more often cultivated in various regions of the world. In the studies of many authors, the quality of plant materials largely depends on climatic and soil conditions [27,28]. In our own research, it was found that extracts from lavender leaves and flowers obtained from cultivation in a temperate climate on very good wheat complex soils are characterized by good antibacterial and antifungal properties. The effect on the viability of microorganisms obviously depends on the dose of the extract, however, in this study, it was proved that small concentrations of the extract, amounting to about 0.32%, were sufficient to significantly reduce the viability of bacteria and fungi of the genus Candida. High activity inhibiting the growth of bacterial cells was also demonstrated by other researchers [29][30][31]. In turn, Adaszyńska et al. [32] found that three out of five varieties of lavender grown in Poland do not inhibit the growth of P. aeruginosa. Literature data indicate that the essential oil is an effective fungicide. Cavanagh and Wilkinson [33] found that L. angustifolia's oil is effective against Aspergillus niger and Trichophyton mentagrophytes. Also, in this study, it was proven that other types of extracts effectively inhibit the development of mold fungi. Different sensitivity of fungi to extracts was shown. The Trichoderma viride fungus turned out to be more resistant to the growth-inhibiting effect of lavender leaf and flower extracts than the Chaetomium globosum fungus. Similarly, different sensitivity of fungi to the tested lavender oils was shown by Caprari et al. [34].
Antibacterial and antifungal properties are determined by the composition of chemical components contained in the extracts. Significantly higher amounts of phenolic compounds were identified in lavender leaf extracts than in flower extracts. At the same time, using a statistical cluster analysis diagram, it was shown that there is a relationship between the antimicrobial activities and the presence of specific phenolic compounds in the extracts. According to Tăbăraşu et al. [3], the quantity and quality of phenolic compounds in the plant material depends on both the ambient temperature, the season, the nutrients in the soil, and the anatomical part of the plant. The total phenol content in the leaf extracts was 31.64 mg GAE/g and was less than half that of the flowers. Obviously, the extraction methods also have a significant impact on the final quality of the extract. Costa et al. [35] showed that water and an ethanol-water mixture are the most suitable solvents for the extraction of phenolic compounds. In turn, Dobros et al. [36] confirmed that the type of extraction, temperature, and ratio of solvent to sample may affect the ability to extract polyphenolic compounds. The obtained lavender extracts were rich in phenolic compounds such as rosmarinic acid, ferulic acid glucosie III, or coumaric acid glucoside I. Similar results were obtained by other researchers [37]. The presented analyzes showed significant differences in the content of some phenolic compounds in individual types of extracts. The content of caftaric acid in leaf extracts was 10 times higher than in lavender flower extracts. In turn, the content of chicoric acid was almost three times higher in flower extracts. The richer the quantitative composition of phenolic compounds in leaf extracts, the higher the number of antioxidant properties observed. The antioxidant activity of flower extracts in the DPPH radical method and the FRAP method was 97.47 and 62.88 µmol Trolox/g, respectively, and was similar to the antioxidant activity of L. angustifolia flower extracts determined by Dobros et al. [36]. In the studies conducted by other authors, the antioxidant activities of ethanol extracts from lavender flowers also oscillated at a similar level [38].
In this study, differences in the qualitative and quantitative composition of volatile substances extracted from various morphological parts of plants were identified. Small amounts of linalool were identified in flower extracts, which, according to literature data, is the dominant component of essential oils from lavender flowers [39]. The difference identified in the linalool content between the essential oil and the HS volatile fraction of the flower extract can be attributed to the relatively lower volatility of terpene alcohols (such as linalool) when compared to hydrocarbons with an equivalent number of carbon atoms. According to literature data, this ingredient also has a biocidal effect against C. albicans fungi [40]. The dominant component in HS fraction of the flower extracts was linalyl acetate due to the higher vapor pressure of these compounds compare to linalool In addition, compounds belonging to sesquiterpenes and monoterpene hydrocarbons, such as pinene, myrcene, carene, and santalene, have also been identified. According to Pokajewicz et al. [25], the composition of essential oil substances, even within the same species, depends on many factors, such as the place of cultivation, weather conditions, or harvest time. However, it should be noted that the qualitative composition of ethanol extracts from flowers was similar to the composition of volatile substances identified in oils from flowers of the L. angustifolia cultivar [41,42]. The lavender leaf extracts were dominated by coumarin, cadinene, and methoxycoumarin. Hajhashemi et al. [43] also indicate that the dominant components in the above-ground parts of the plant are ursolic acid and coumarins.

Plant Material
The material for the study was one of the three ecotypes of narrow-leaved lavender (Lavandula angustifolia Mill.)-a dwarf form with short stems used for low border hedges (ZO) and cultivated in southern Poland. The field experiment was carried out in Poland, in Polanowice (50 • 19 N 20 • 07 E), a farm belonging to the Małopolska Plant Breeding. The research was carried out on chernozem (valuation class 1), with a calcareous substrate (pH 6.8). Seedlings obtained generatively (from seeds) were planted on 19 June 2019, in 9-point strips, 0.5 × 0.5 m apart. Mineral fertilization was applied in the spring, before planting. During the lavender growing season at the turn of March and April, spring pruning was performed (2020 and 2021), and faded inflorescences were removed. From three-year-old plants (7 July 2021), flowers and leaves, each weighing 100 g, were collected. Fresh flowers and leaves were vacuum packed and sent for analysis. The course of weather in the growing season in 2019-2021 was varied. The years 2019 and 2020 were similar in terms of precipitation (646.3 and 652.75 mm respectively), and in 2021, there was 200 mm more (845 mm). Their distribution was also different. In 2019, the most rain occurred in May and August, in 2020 in May and June, and in 2021 in July and August. In turn, the warmest year was 2019, and the highest average temperatures occurred in June, July, and August.

Preparation of Ethanol Extracts
The amount of 10 g of lavender (flowers and leaves separately) was flooded with 200 cm 3 of 60% ethanol (PPS Polmos SA, Warsaw, Poland) and shaken for 72 h on a laboratory shaker (IKA KS 3000 icontrol, IKA-Werke GmbH & Co.KG, Staufen, Germany). After this time, the prepared extract was separated from lavender and then sterilized by syringe filters with a pore diameter of 0.22 µm. Flower extracts were marked with the symbol ZO1 and leaf extracts with the symbol ZO2.

Evaluation of Fungicidal Properties against Mold Fungi on a Malt-Agar Medium
The effect of the extracts on the growth of mold fungi was carried out in Petri dishes on a maltose agar medium. Two species of fungi were used in the study: Trichoderma viride Pers., strain A-102 and Chaetomium globosum Kunze, strain A-141 (ATCC 6205), coming from the collection of pure cultures of the Department of Wood Science and Wood Preservation, Warsaw University of Life Sciences. Ethanol extracts of lavender in the amounts of 0.5, 1.0, 2.5, and 5 cm 3 were added to a sterile Petri dish, followed by an amount of maltose agar medium, so that the total volume of the mixture was 10 cm 3 . Fungi were inoculated centrally into Petri dishes 24 h after gelation of the medium. 60% ethanol was used as a control. The size of the inoculum was 5-6 mm. Cultivation was carried out in a Thermolyne Type 42,000 model thermal incubator (ThermoFisher Scientific, Waltham, MA, USA) under temperature and relative humidity conditions of 26 ± 2 • C and 65 ± 2%, respectively. The assessment of fungicide properties on the culture medium was carried out by measuring the diameter of mycelial growth in two perpendicular directions. Measurements were made at 48 h intervals. The tests were completed on the day of complete overgrowth of the Petri dish in the control samples. The analysis of variance using the Snedecor statistics was used to verify the statistical analysis. Statistical inference was carried out for the significance level α = 0.05. In case of rejection of the null hypothesis, Tukey's test was performed.

Antioxidants Properties
The total content of phenolic compounds was assessed by the method described by Gao et al. [44]. Distilled water, Folin-Ciocalteau reagent, and 20% sodium carbonate solution were added to the extracts. After 1 h, the absorbance at 765 nm was measured with a UV-VIS spectrometer (Type UV2900, Hitachi Ltd., Chiyoda, Tokyo, Japan). The results were expressed in mg GAE/g.
The scavenging activity of extracts on ABTS •+ radicals was determined according to the method of Re et al. [45]. The solution of ABTS •+ (diluted with distillated water to an absorbance of 0.7) was added to the extracts. After 6 min, the absorbance at 734 nm was measured with a spectrophotometer. Results are expressed as µmol Trolox equivalent (TE)/g.
The scavenging activity of flower extracts on DPPH˙radicals was determined according to the method of Blois [46]. Briefly, 2.0 mL of a methanolic DPPH solution (0.1 mM) was mixed with plant extracts and left for 10 min. After this time, the absorbance was measured at 517 nm. Results are expressed as µmol Trolox equivalent (TE)/g. The ferric ion reduction by FRAP assay was determined by the method described by Benzie and Strain [47]. Three mL of FRAP solution were added to 0.5 mL of the sample. The absorbance was recorded at a wavelength of 593 nm after 10 min of reaction. Results are expressed as µmol Trolox equivalent (TE)/g.

GC-MS Analysis
The prepared sample was analyzed by SPME solid-phase microextraction using 100 µm polydimethylsiloxane (PDMS) fiber (Supelco Ltd., Bellefonte, PA, USA). Prior to the analyses, the fiber was conditioned according to the manufacturer's instructions at 250 • C for 30 min in a gas chromatograph dispenser. The tested material was placed in a 100 mL conical flask secured with aluminum foil. The fiber exposure was carried out using the over-surface method for 30 min at 20 • C. Then, the fiber was transferred to the gas chromatograph injector (temp. 250 • C), where the analytes were thermally desorbed for 5 min. Using a gas chromatograph (GC-MS, Varian 450GC compressed with 240 MS), the composition of compounds desorbed from the SPME fiber was examined. Helium was the carrier gas used, the flow rate of which was 1 mL/min. The dispenser temperature was 250 • C. Separation of the analytes was carried out using a 30 m × 0.25 mm capillary column with a moderately polar HP-5 (polysiloxanmethylphenyl) stationary phase and a layer thickness of 0.25 µm. The column oven temperature program was as follows: start-50 • C for 5 min isotherm, then set to a temperature gradient of 10 • C/min to 300 • C (5 min isotherm). Based on NIST.08 and the Willey database, compounds found in the extracts were identified. GC-MS analysis was performed in duplicate.

Phenolic Compound Identification
Polyphenolic compounds were analyzed by reverse-phase, ultra-performance liquid chromatography (UPLC)-PDA-MS/MS Waters ACQUITY (Waters, Mil-ford, MA, USA) consisting of binary pump manager, sample manager, column manager, photodiode array detector (PDA), and tandem quadrupole mass spectrometer (TQD) with electrospray ionization (ESI). Separation was performed using a BEH C18 column (100 mm × 2.1 mm id, 1.7 µm, Waters USA) maintained at 50 • C. The following solvent system was used for the study of anthocyanins: mobile phase A (2% formic acid in water v/v) and mobile phase B (2% formic acid in 40% ACN in water v/v). For the remaining polyphenolic compounds, a lower concentration of formic acid (0.1% w/w) was used. The gradient program was set as follows: 0 min 5% B, 0 to 8 min linear to 100% B, and 8 to 9.5 min for rinsing and return to baseline conditions. The injection volume of the samples was 5 µL (partial loop with needle overflow), and the flow rate was 0.35 mL/min. The following parameters were used for the TQD: capillary voltage, 3.5 kV; con voltage, 30 V in positive and negative mode; the source was kept at 120 • C; the desolvation temperature was 350 • C; gas flow con, 100 l/h; and desolvation gas flow, 800 l/h. Argon was used as the collision gas at a flow rate of 0.3 mL/min. Detection and identification of polyphenolic compounds was based on specific PDA spectra, mass-to-charge ratios, and ion fragments obtained after collision-induced dissociation (CID). Quantification was achieved by injecting solutions of phenolic compounds with known concentrations ranging from 0.05 to 5 mg/mL as standards [28,48]. All assays were performed in triplicate and expressed as µg/g. Waters MassLynx v.4.1 software was used for data acquisition and processing.

Statistical Analysis
Statistical analysis of the results was carried out in Statistica version 13 (TIBCO Software Inc., Palo Alto, CA, USA). Analysis of variance (ANOVA) was used to test (α = 0.05) for significant differences between factors. A comparison of the means was performed by Tukey test, with α = 0.05. In order to describe the mutual relations between the examined variables, variable reduction techniques based on cluster analysis and scaled heat maps made in the R studio program were used. For easier interpretation of the results, the following codes were assigned to individual measurable features: phenolic acids (9P) and flavonoids (10F); volatile compounds: 6VC, 7VC, and 8VC; antioxidant properties: (11AA), ABTS (12AA), DPPH (13AA), and FRAP (14AA); bacteria: 1B and 2B; yeast-like fungi: 3B; mold fungi: 4MM and 5MM.

Conclusions
Extracts obtained from the flowers and leaves of Lavandula angustifolia grown in southern Poland were characterized by good antibacterial and fungicidal properties. Extracts from leaves and flowers showed different antioxidant properties, with the determined anti-free radical activity being higher for extracts from lavender leaves. The content of volatile substances, phenolic acids and flavonoids, as well as their activity, differs between the extracts. Based on the conducted research, it should be concluded that leaf extracts are characterized by a higher activity of biologically active compounds. The results confirm that the areas of southern Poland allow for field cultivation of Lavandula angustifolia with good antimicrobial activity and a rich composition of bioactive substances.
Based on the conducted research, it seems that lavender grown in southern Poland may be a useful plant for those industries that are looking for natural raw materials with effective antimicrobial and antioxidant properties. However, in order to confirm our initial assumptions, further analyses would be necessary to confirm how the climatic and soil conditions of Poland affect the variability of the analyzed features. Another important aspect is obtaining a high-quality extract. In our research, we obtained ethanol extracts, which economically seem to be less expensive than those obtained by traditional steam distillation, which is characterized by higher energy consumption. However, in future experiments, it would be necessary to assess the quality of the obtained raw material by isolating bioactive active substances from it and using modern extraction methods. In order for lavender grown in Poland to become a useful export raw material with good antimicrobial and antioxidant properties, further research is needed, with particular emphasis on the optimization of cultivation conditions and activity-controlled extraction.