Chemical Composition, Antioxidant Potential, and Antimicrobial Activity of Novel Antiseptic Lotion from the Leaves of Betula pendula Roth

Abstract

Plants of the genus Betula pendula (Betulaceae) have been used as antiseptics in the treatment of skin diseases. While the bark of this plant has so far been used as a medicinal raw material, our research has revealed that the leaves are an extremely valuable source of health-promoting bioactive compounds, especially polyphenolic compounds. The antioxidant activity of a novel lotion from the Betula pendula leaf and its phenolic content was determined using the DPPH and FRAP assays and the Folin–Ciocalteu method, and a qualitative analysis was conducted using the GC-MS method. The antiseptic preparation was also subjected to HPLC analysis for an assessment of the phenolic acids content (gallic, 3,4-dihydroxybenzoic, 2,5-dihydroxybenzoic, chlorogenic, and 3-hydroxybenzoic acids). The shake flask method was used to determine the partition coefficient of the lotion to assess its lipophilicity. A new ethanol-reduced antiseptic lotion containing antimicrobial ingredients was evaluated using standard killing methods (EN 13697:2019 and EN 13697:2015) at 60 ± 10 s and 300 ± 10 s against six microorganisms. The aim of these tests was to determine whether the formulation exhibited performance like a traditional ethanol. In addition, hand disinfection tests were also carried out using the preparation obtained from B. pendula leaves in accordance with EN 1500:2013, using Escherichia coli strain K12 NCTC 10538. According to the results, high polyphenol contents (52 ± 2 mg GAE/g dry raw material) and other antioxidant and biologically active substances identified by GC-MS (phytol, alpha-tocospiro B, sitosterol, or dilauryl thiodipropionate) can be thought of as the parameters responsible for the effective activity of the preparation derived from birch leaves. Gallic acid and 3,4-dihydroxybenzoic acid had the greatest concentrations among the phenolic acids examined. The mean 60 s kill time was >99.9 for the new antiseptic against Staphylococcus aureus ATCC 6538 (at a concentration of 30 g/100 mL) and Pseudomonas aeruginosa ATCC 15442 (at a concentration of 40 g/100 mL), while ethanol showed the same reduction for these microorganisms at a concentration of 80 g/100 mL.

1. Introduction

The synthesis of antioxidants, a natural source of plant metabolism, serves as a means of defense against biotic and abiotic factors in the environment. Environmental stressors can increase levels of reactive oxygen species (ROS), leading to an imbalance in their production and scavenging, which causes oxidative stress. Reactive oxygen species are thought to be responsible for many cell disorders through their chemical attacks on biostructures. These species are constantly generated as a result of human metabolism due to oxidation. Furthermore, ROS can be generated because of various external factors. As a defense mechanism, plants activate enzymatic and non-enzymatic systems in their metabolism to prevent damage to proteins, membranes, and DNA, protecting them from oxidative stress and related diseases [1,2,3,4].

Many plant species, owing to their high amounts of essential oils, are of medicinal and economic importance. The plants show, e.g., anti-inflammatory, antispasmodic, antioxidant, antibacterial, antifungal, antidiarrheal, and antiviral activities. Most of the green plants, vegetables, and fruits are known as the main sources of natural antioxidants. Consuming these antioxidants in the human diet is a sensible method to minimize, among others, brain dysfunction, cardiovascular diseases, cataracts, or risk of cancer [5,6,7].

One of the groups of plants with confirmed pharmacological effects (including antioxidant effects) is Betula (birches), belonging to the Betulaceae family. There are over 140 species of trees within the genus Betula that are known in the world. The genus Betula is widespread in northern temperate and Arctic regions, from Afghanistan to the Himalayas, Japan, Kazakhstan, Korea, China, Kyrgyzstan, Russia, Nepal, Mongolia, Sikkim, Europe, and into both Americas. Various pharmacological properties of the Betula species have found application in ethnomedicine. Currently, there are many pharmacological research results that confirm a wide range of health-promoting properties. Researchers have also shown that these plants can heal skin and digestive system diseases (hepato- and gastroprotective effect) [8,9,10].

In Europe, three naturally occurring species are particularly important, including commercial ones: white birch (Betula alba), silver birch (Betula pendula Roth), and B. pubescens. Extracts and biologically active ingredients isolated from these species are used in cosmetics, dietary supplements, and biocides. Birch sap, extracted from the trunk, is used as an aid in the treatment of urinary tract diseases and dermatological diseases, as well as arthritis and rheumatism [11]. The antibacterial potential of extracts and essential oils from various Betula species, including B. pendula, is also often highlighted [12,13]. Duric et al. [12] analyzed the antibacterial potential of methanol extracts from various raw materials of B. pendula—external and internal flowers, bark, buds, and leaves. In the case of leaf decoctions and leaf methanolic extracts, the researchers observed the largest zones of growth inhibition against S. aureus. In the case of leaf and internal bark methanolic extracts, they observed a significant zone of growth inhibition for P. aeruginosa. Leaf extracts showed no activity against B. subtilis, while E. coli was not sensitive to any of the extracts tested (no visible inhibition zone) [12]. In addition to assessing the surface disinfection and hand disinfection of silver birch leaf preparation, the aim of our study was also to assess the antioxidant activity of an ethanolic preparation obtained from this raw material. Betula plants are known for their antioxidant potential. Researchers suggest that the polyphenolic compounds in the leaves of B. pendula are responsible for this activity. This raw material is a valuable source of antioxidants such as myricetin, catechin, p-coumaric acid, kaempferol, and quercetin [14]. Azman et al. [15] identified catechin, myricetin, quercetin, naringenin, and p-coumaric acid in the methanolic extract of leaves and confirmed the antioxidant potential in TEAC, FRAP, and ORAC assays [15]. Penkov et al. [16] showed the presence of flavonoids and antioxidant activity assessed using the DPPH and ABTS techniques of extracts from dried B. pendula leaves [16]. Bljajić et al. [17] compared the antioxidant potential and total phenols, phenolic acids, and flavonoids in aqueous and ethanolic extracts of B. pendula leaves. The extract prepared using ethanol (80%) was characterized by a higher content of flavonoids (including rutin and quercetin) and phenols, whereas the aqueous extract was a more valuable source of phenolic acids (including protocatechuic, ellagic, and chlorogenic acids). Antioxidant activity was assessed by the DPPH technique. There was no significant difference between the two extracts, but the ethanolic extract was a better scavenger of ABTS radicals. Aqueous extracts proved to be a better chelator of Fe³⁺ ions, while a higher potential as assessed by TAA and FRAP methods was found for the ethanolic extract [17]. Not only are B. pendula leaves a valuable source of antioxidants, including polyphenolic compounds, B. pendula raw materials are also rich in antioxidants in the outer bark [18], pollen [9], sap [10], buds, aglets, bines, and bark [15].

Despite the increasing use of alcoholic plant extracts as active ingredients in cosmetic preparations, there is still a lack of antiseptic preparations with antioxidant potential and reduced ethanol content. One of the key elements in preventing the development of infections is the use of disinfectants containing biocidal substances capable of removing microorganisms or inhibiting their growth on both skin and abiotic surfaces [19]. Despite the lifting of the epidemiological emergency, disinfectants are still used on a large scale. In promoting the EU’s environmental objectives, it seems important to look for modern solutions and alternatives to the disinfectants currently in use [20]. Extracts obtained from the leaves of plants (due to their content of phenolic compounds) are characterized by their high antioxidant potential [21]. The high polyphenol content is a key parameter responsible for the effective biological activity of these preparations [22].

The purpose of this study was to obtain a novel biocidal preparation based on birch leaves with high antioxidant potential and with a reduced ethanol content. The new antiseptic had a mean 60 s kill time of more than 99.9 against Staphylococcus aureus ATCC 6538 (at a concentration of 30 g/100 mL) and Pseudomonas aeruginosa ATCC 15442 (at a concentration of 40 g/100 mL), while ethanol reduced these microorganisms at a concentration of 80 g/100 mL [23].

2. Materials and Methods

2.1. Materials

The plant materials were collected between May and September 2019 from their natural state in the village of Siecino (West Pomeranian Voivodeship, Poland). The voucher specimens are deposited at the Pomeranian Medical University (Szczecin, Poland). Dr. Eng. of Agricultural Sciences Anna Nowak, prof. PUM, identified the leaves of Betula pendula material.

2.2. Chemicals

6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox, TE), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,4,6-tripyridyl-s-triazine (TPTZ) were purchased from Sigma Aldrich (Poznań, Poland). Potassium persulfate, sodium acetate anhydrous, ferric chloride, hydrochloric acid, potassium acetate anhydrous, ethanol, and n-octanol were from Chempur (Piekary Śląskie, Poland). Folin–Ciocalteu reagent was supplied by Merck (Darmstadt, Germany). All reagents were of analytical grade.

2.3. Obtaining of B. pendula Leaf Preparation

Obtaining the B. pendula leaf preparation was carried out according to a previously used procedure [22]. The raw material fraction obtained in the receiver (<0.25 mm) was used for extraction. Below is how the plant preparation derived from Betula pendula leaves was made: 10.0 g of dried Betula pendula leaf and 90 mL of ethanol (at a concentration of 70 g/100 mL) were put into a 250 mL conical flask and sonicated in a bath at a frequency of 40 kHz for 60 min. The extract was filtered using a pressure funnel and a Whatman paper filter (codified EEA03), yielding the antiseptic lotion from the leaves of B. pendula.

2.4. HPLC Analysis

The tested phenolic acids, i.e., gallic acid (GA), 3,4-dihydroxybenzoic acid (34DHBA), 2,5-dihydroxybenzoic acid (25DHBA), chlorogenic acid (ChA), and 3-hydroxybenzoic acids (3HBA), were separated on a 125 × 4 mm column Hypersil ODS using the HPLC system from Knauer (Berlin, Germany) along with the WellChrom UV K-2600 detector. The mobile phase included 1% acetic acid and methanol (80:20), and the flow rate was 1 mL/min. Twenty µL of the sample were injected into the column. The compounds were identified according to their retention time (RT) at a wavelength of 280 nm. The calibration curve exhibited correlation coefficients of 0.9989 for GA (y = 31740x − 3.7628), 0.9993 for 34DHBA (y = 21523x − 1.8144), 0.9997 for 25DHBA (y = 32115x − 0.9831), 0.9998 for ChA (y = 39852x − 2.2859), and 0.9991 for 3HBA (y = 15820x + 1.8199). Each sample was examined three times.

2.5. GC-MS Analysis of the Preparation from Betula pendula Leaves

GC-MS analysis was conducted on a Shimadzu GC-MS-QP2020 NX (Shimadzu, San Jose, CA, USA) apparatus using a Shimadzu SH-I-5MS column (30 m × 0.25 mm × 0.25 μm), the temperature of which was held at 40 °C for 2 min and programmed to 300 °C at a rate of 10 °C/min and kept constant at 300 °C for 2 min. The flow rate for the helium used as a carrier gas is 1 μL/min, with the MS obtained at 70 eV, and a split of 10. The analysis duration was 30 min, and the sample volume was 1 µL. We identified the compounds (in three repetitions) by comparing their mass spectra from the spectra library (NIST-2020).

2.6. Assessment of Lipophilicity of the Preparation from Betula pendula Leaves

The lipophilicity tests were carried out according to a previously used procedure [24] and Equation (1) and using the Thermo Scientific GENESYS 50 apparatus (Thermo Fisher Scientific, Norristown, PA, USA) [25]:

$$P={\displaystyle \frac{C_o}{C_w}}={\displaystyle \frac{C^0-C_w}{C_w}}={\displaystyle \frac{S^0-S}{S}}={\displaystyle \frac{{\int }_{{\mathsf{\Lambda }}_1}^{{\mathsf{\Lambda }}_2}{A}^{0}d\mathsf{\Lambda }-{\int }_{{\mathsf{\Lambda }}_1}^{{\mathsf{\Lambda }}_2}Ad\mathsf{\Lambda }}{{\int }_{{\mathsf{\Lambda }}_1}^{{\mathsf{\Lambda }}_2}Ad\mathsf{\Lambda }}}$$

(1)

where C is the concentration of the sum of the active substances in the n-octanol layer and in the aqueous layer, S is the area occupied by the active substances under the UV-Vis spectrum, A is the absorbance, 0 is the superscript in the birch preparation before extraction, Λ is the wavelength, and o and w are the subscripts for the octanol and water phases, respectively.

2.7. Antioxidant Activity and TPC of the Preparation from Betula pendula Leaves

Evaluations of the antioxidant activity of the preparation from the B. pendula were carried out using the DPPH method [24] and the FRAP method [26] to assess its iron-reducing power. The reference substance for these methods was Trolox. The antioxidant activity of the extract was measured in Trolox equivalents, which is expressed as the mg of Trolox equivalent per 1 g of dry raw material (mg TE/g of dry raw material).

We prepared the reagent for FRAP in the same manner as described in the previous publication [22] to assess the reducing power of iron. We assessed antioxidant activity using the DPPH method, as described in the previous publication [22]. Moreover, we used the Folin–Ciocalteau method [22] to determine the total polyphenol content, following the procedure in the previous publication [27]. The reference substance for this method was gallic acid (GA), and the total polyphenol content was measured in gallic acid equivalents, which is expressed as the mg of gallic acid equivalent per 1 g of dry raw material (mg GAE/g of dry raw material). The analysis was conducted on the Merck Spectroquant Pharo 300 apparatus at λ: 517 nm (DPPH method), 593 nm (FRAP method), and 750 nm (Folin–Ciocalteau method).

2.8. Evaluation of Hand Disinfection by Means of Rubbing by EN 1500:2013 [28]

Hand disinfection tests using preparation from B. pendula were carried out according to the procedure previously used [22], in accordance with EN 1500:2013. This study determined the reduction in the release of transient Escherichia coli K12 NCTC 10538 after the application of an antiseptic (in the amount of 6 mL) to the artificially contaminated hands of participants (a duration of 60 ± 5 s at a temperature of 20 ± 1 °C). To determine the number of microorganisms, the technique of depth culture on plates was used [29]. The procedure was validated using the solution neutralization method. The Wilcoxon test was used for statistical analysis of the test data, and the results obtained are shown in

Table 1. Conditions for assessing the preparation from B. pendula by EN 1500:2013.

Birch Preparation	Escherichia coli K12 NCTC 10538
N	8.6 ± 0.1
Nvb	84 × 104
B	81 ± 1
Nv	83 × 104
C	76 ± 1

Mean ± SD (n = 9); N—log from the amount of microorganisms/mL that was in the EN 1500:2013 test mixture; N_vb—the amount of microorganisms/mL that was used during the neutralizer toxicity test; B—the number of CFU/mL after the study test; N_v—the amount of microorganisms/mL that was used during the validation test; C—the number of CFU/mL after the validation test.

. The birch preparation will be considered effective when the results meet the acceptance criteria and the statistical analysis performed shows that it exhibits antimicrobial activity equivalent to or greater than the reference compound [22,30].

Table 1 provides the requirements for measuring the antibacterial activity of the preparation.

2.9. Evaluation of Surface Disinfection Assessments by EN 13697:2019 [31]

Surface disinfection tests of the new antiseptic lotion derived from B. pendula leaves were performed in accordance with EN 13697:2019. The efficacy of the novel lotion against the evaluated microorganisms was assessed at the active concentration (60 g/100 mL) and the inactive concentrations (0.6 g/100 mL) for durations of 60 ± 10 s and 300 ± 10 s at a temperature of 20 ± 1 °C, using bovine serum albumin as a loading material.

Disinfection tests were carried out according to a previously used procedure using sterile discs as in EN 13697:2019 [32]. A description of the method of evaluation of surface disinfection assessments is described in our paper [22].

The following formula was used to evaluate the log reduction (LR) of the number of microorganisms that were present during the test. The LR was shown as the difference between the log counts of viable cells before and after treatment (2) [33]:

$$LR=N-N_{ts}$$

(2)

where: N is the log of the number of cells alive applied to the test surface (N), and N_ts is the log of the number of cells alive remaining on the surface after the test.

Table 2. The criteria for establishing the surface disinfection evaluations of the preparation from B. pendula according to the standard EN 13697:2019.

Phase II, Stage II
Birch Preparation	Contact Time	Staphylococcus aureus ATCC 6538	Pseudomonas aeruginosa ATCC 15442	Escherichia coli ATCC 10536	Enterococcus hirae ATCC 10541	Candida albicans ATCC 10231	Aspergillus brasiliensis ATCC 16404
N		7.01	7.07	7.02	6.99	6.03	5.88
NT		6.96	6.98	6.92	6.87	5.93	5.79
NC		6.99	6.94	6.98	6.89	5.91	5.76
NW
ND CP = 60 g/100 mL	60 ± 10 s	<2.15	<2.15	<2.15	<2.15	<2.15	<2.15
ND CP = 0.6 g/100 mL		>5.52	>5.52	>5.52	>5.52	>5.52	>5.52
ND CP = 60 g/100 mL	300 ± 10 s	<2.15	<2.15	<2.15	<2.15	<2.15	<2.15

Mean ± SD (n = 9); NT—log of the amount of microorganisms/mL that was applied to the test glass surface during the neutralizer toxicity test; N—log of the amount of microorganisms/mL that was applied to the test glass surface during the EN 13697:2019 test; NC—log of the amount of microorganisms/mL that was applied to the test glass surface during the validation test; NW—log of the amount of microorganisms/mL that was applied to the test glass surface during the control tests with distilled water; ND—log of the amount of microorganisms/mL that was applied to the test glass surface during the antimicrobial testing; C_P—concentration of preparation from B. pendula.

delineates the criteria for assessing surface disinfection tests of preparation from the leaves of Betula pendula according to standard EN 13697:2019.

2.10. Evaluation of Surface Disinfection Assessments by EN 13697:2015 [34]

The surface disinfection tests of novel antiseptic lotion from the leaves of B. pendula were conducted according to EN 13697:2015 [32]. The activity of the preparation against the tested microorganisms (Staphylococcus aureus ATCC 6538 and Pseudomonas aeruginosa ATCC 15442) was checked at the concentrations of 14–70 g/100 mL for 60 ± 10 s and at 20 ± 1 °C, with the inclusion of a loading substance (bovine serum albumin). Disinfection tests were carried out according to a previous publication [22]. The bactericidal efficacy of undiluted ethanol (100 g/100 mL) and ethanol diluted with hard water (80 and 90 g/100 mL) was assessed against the reference strain, respectively, under identical conditions (

Table 3. The criteria for assessing the surface disinfection evaluations of the preparation according to the standard EN 13697:2015.

Phase II, Stage II
Preparation/Ethanol	Contact Time	Staphylococcus aureus ATCC 6538	Pseudomonas aeruginosa ATCC 15442
N		6.75	6.74
NT		7.12	7.06
NC		7.14	7.10
NW		7.14	7.13
ND CP = 20 g/100 mL	60 ± 10 s	4.20	4.31
ND CP = 30 g/100 mL		4.09	4.25
ND CP = 40 g/100 mL		3.03	3.15
ND CP = 50 g/100 mL		2.82	2.89
ND CP = 60 g/100 mL		<0.10	<0.10
ND CP = 70 g/100 mL		<0.10	<0.10
ND CP = 80 g/100 mL		<0.10	<0.10
ND CP = 90 g/100 mL		<0.10	<0.10
ND CP = 100 g/100 mL		<0.10	<0.10
ND CE = E 80 g/100 mL		3.12	3.08
ND CE = 90 g/100 mL		2.54	2.49
ND CE = 100 g/100 mL		<0.10	<0.10

Mean ± SD (n = 9); NT—log of the amount of microorganisms/mL that was applied to the test glass surface during the neutralizer toxicity test; N—log of the amount of microorganisms/mL that was applied to the test glass surface during the EN 13697:2015 tests; NC—log of the amount of microorganisms/mL that was applied to the test glass surface during the validation test; NW—log of the amount of microorganisms/mL that was applied to the test glass surface during the control tests with hard water; C_P—concentration of preparation from B. pendula; C_E—concentration of ethanol; ND—log of the amount of microorganisms/mL that was applied to the test glass surface during the antimicrobial testing.

).

Table 3 shows the criteria for evaluating the surface disinfection tests of the birch preparation and ethanol.

2.11. Statistical Analysis

We used a one-way analysis of variance (ANOVA) for the microbiological analysis. We conducted a cluster analysis to determine the properties of the novel preparation from birch and its impact on the examined microorganism. The importance of variations across different groups was assessed using Tukey’s test (p < 0.05). Moreover, the importance of disparities between groups in the surface disinfection and hand disinfection tests was assessed using the Wilcoxon test (p > 0.01).

3. Results

3.1. HPLC Analysis

The quantitative composition of the phenolic acids identified by HPLC is shown in Figure 1.

The highest concentration of the identified phenolic compounds by the HPLC method was found for gallic acid (194 ± 5 mg/L extract, which corresponds to 1.74 ± 0.05 mg/g dry raw material) and 3,4-dihydroxybenzoic acid (123 ± 7 mg/L extract, which corresponds to 1.11 ± 0.06 mg/g dry raw material). The lowest concentration of the analyzed phenolic acids was found for chlorogenic acid (20 ± 1 mg/L, which corresponds to 0.18 ± 0.01 mg/g dry raw material)—Figure 1 and Figure S1, Table S1.

3.2. GC-MS Analysis of the Preparation

Table 4. The components were identified in preparation from B. pendula leaves studied by the GC-MS method.

Chemical Compound	Chemical Class	RT (min)	Biological Activity
palmitic acid	saturated fatty acid	15.62	antifungal antibacterial activity [22]
ethyl palmitate	ester of saturated fatty acid	15.82	larvicidal and insecticidal agents [35]
phytol	unsaturated terpene ketone	16.86	antioxidant anti-inflammatory antimicrobial activity [36,37]
linolenic acid	unsaturated fatty acid	17.01	anti-cancer activity [38]
methyl linoleate	ester of unsaturated fatty acid	17.15	antibacterial antifungal activity [22,39]
ethyl linoleate	ester of unsaturated fatty acid	17.19	insecticidal action [40]
hexacosanol	fatty alcohol	19.01	anti-inflammatory activity [41]
alpha-tocospiro B	alpha-tocopheroids	20.97	antioxidant antidiabetic anti-inflammatory cardioprotective activity [42,43]
sitosterol	triterpenoid	24.28	anti-inflammatory antioxidant activity [44]
cabraleadiol	triterpenoid	26.65	anti-inflammatory activity [45]
dilauryl thiodipropionate	ester; thio compound	26.76	antioxidant activity [46]

displays the components of the preparation obtained from leaves of the Betula pendula along with their potential biological activities.

The GC-MS method determined 11 major components of the B. pendula, including saturated fatty acid (palmitic acid), ester of saturated fatty acid (ethyl palmitate), unsaturated terpene ketone (phytol), unsaturated fatty acid (linolenic acid), esters of unsaturated fatty acid (methyl and ethyl linoleate), fatty alcohol (hexacosanol), triterpenoids (sitosterol and cabraleadiol), thio compound (dilauryl thiodipropionate), and alpha-tocospiro B—Figure S2, Table 4.

3.3. Assessment of Lipophilicity

Assessment of lipophilicity has demonstrated that the novel antiseptic lotion from Betula pendula has a log P partition coefficient of −0.500 ± 0.001—Figure S3.

3.4. Antioxidant Activity and TPC

Table 5. AA and TPC of the birch preparation.

	AA		TPC
Birch preparation	DPPH	FRAP	F-C
	(mg TE/g dry raw material)		(mg GAE/g dry raw material)
	32 ± 6	640 ± 10	52 ± 2

Mean ± SD (n = 3).

displays the antioxidant activity (AA) and total polyphenol content (TPC) of the extracts derived from Betula pendula leaves.

The results shown in Table 5 indicate that AA and TPC for the plant preparation from B. pendula leaves were 32 ± 6 mg TE/g dry raw material (in the case of the DPPH method), 640 ± 10 mg TE/g dry raw material (in the case of the FRAP method), and 52 ± 2 mg GAE/g dry raw material (in the case of the Folin–Ciocalteau method).

3.5. Evaluation of Hand Disinfection by Means of Rubbing

Table 6. Statistical analysis of birch preparation (PB) and the reference preparation (PR): sorted results and calculation results for statistical tests.

RP-PB	0.31	0.26	0.08	0.03	−0.04	−0.14	−0.17	−0.18	−0.18	−0.29
0.31	0.31
0.26	0.29	0.26
0.08	0.19	0.17	0.08
0.03	0.17	0.15	0.05	0.03
−0.04	0.14	0.11	0.02	0.00	−0.04
−0.14	0.08	0.06	−0.03	−0.06	−0.09	−0.14
−0.17	0.07	0.05	−0.04	−0.07	−0.1	−0.15	−0.17
−0.18	0.06	0.04	−0.05	−0.08	−0.11	−0.16	−0.17	−0.18
−0.18	0.06	0.04	−0.05	−0.08	−0.11	−0.16	−0.17	−0.18	−0.18
−0.29	0.01	−0.01	−0.11	−0.13	−0.16	−0.22	−0.23	−0.24	−0.24	−0.29
−0.29	0.01	−0.01	−0.11	−0.13	−0.17	−0.22	−0.23	−0.24	−0.24	−0.29
−0.30	0.00	−0.02	−0.11	−0.14	−0.17	−0.22	−0.23	−0.24	−0.24
−0.31	0.00	−0.02	−0.12	−0.14	−0.17	−0.23	−0.24	−0.24	−0.25
−0.31	0.00	−0.02	−0.12	−0.14	−0.18	−0.23	−0.24	−0.25	−0.25
−0.49	−0.09	−0.11	−0.21	−0.23	−0.27
−0.57	−0.13	−0.15	−0.24	−0.27
−0.58	−0.14	−0.16	−0.25	−0.28
−0.61	−0.15	−0.17	−0.27	−0.29
−0.61	−0.15	−0.18	−0.27	−0.29
−0.68	−0.18	−0.21

The median value of PR-PB is −0.29, the mean values for each PR-PB pair were calculated, and values less than the median were discarded; the confidence level of the test was set at p = 0.025; PR—reference preparation (propan-2-ol at a concentration = 60 g/100 mL); PB—preparation obtained of the leaves of Betula pendula.

presents the organized values for the statistical test of hand disinfection by means of rubbing in preparation from B. pendula by EN 1500:2013.

Table 7. Statistical analysis of preparation from B. pendula (PB) and reference preparation (PR) utilizing the Escherichia coli K12 strain NCTC 10538 as a benchmark.

Number of Tests	LR		PR-PB
	PR	PB
1	3.39 ± 0.01	4.31 ± 0.01	−0.92
2	4.45 ± 0.09	4.76 ± 0.01	−0.30
3	4.41 ± 0.08	4.52 ± 0.03	−0.12
4	3.69 ± 0.02	3.83 ± 0.06	−0.13
5	3.17 ± 0.02	3.68 ± 0.09	−0.50
6	3.99 ± 0.08	4.07 ± 0.02	−0.08
7	4.91 ± 0.02	4.09 ± 0.08	0.82
8	3.14 ± 0.02	3.26 ± 0.03	−0.12
9	3.48 ± 0.02	4.06 ± 0.04	−0.58
10	3.78 ± 0.04	4.09 ± 0.05	−0.32
11	3.85 ± 0.09	4.22 ± 0.04	−0.37
12	3.93 ± 0.01	3.98 ± 0.03	−0.04
13	3.49 ± 0.02	3.99 ± 0.09	−0.50
14	3.56 ± 0.01	3.94 ± 0.03	−0.38
15	3.49 ± 0.02	3.91 ± 0.04	−0.43
16	3.37 ± 0.02	3.51 ± 0.08	−0.14
17	3.73 ± 0.02	4.74 ± 0.08	−1.01
18	3.69 ± 0.02	4.01 ± 0.01	−0.32
19	3.65 ± 0.01	4.07 ± 0.03	−0.43
20	3.70 ± 0.01	4.12 ± 0.05	−0.42

Mean ± SD (n = 9), The quotient control of the weighted averages of the successive dilutions used in the calculations is between 5.0 and 15.0; the number of probands is 20; the average number of microorganisms before the test is a minimum of 5.0 after logarithmic transformation; no more than 3 PR values are below 3.00; the average PR-PB value for probands 1–10 (test product before reference) does not differ by more than 2.00 from the average for probands 11–20 (test product after reference); the product is considered less active than the reference at an LR limit of 0.60; LR—the log reduction value was calculated from Equation (2); PR—reference preparation (propan-2-ol at a concentration of 60 g/100 mL); PB—preparation from the leaves of Betula pendula.

presents the statistical analysis of the birch preparation (PB) and the propan-2-ol (PR), in accordance with EN 1500:2013.

Evaluation of the new birch leaf antiseptic lotion (contact time 60 ± 5 s at 20 ± 1 °C) by the EN 1500:2013 method against Escherichia coli K12 NCTC 10538 showed that the obtained preparation did not show weaker activity than the reference product. The critical value of PB-RP for the Wilcoxon test (52) is less than the highest mean value (53) and does not increase above the LR value (0.60). The value of 53, the highest mean value for PR-PB, is −0.13, which means that the hypothesis of lower activity of the birch preparation compared to the reference preparation can be rejected (Table 6 and Table 7).

3.6. Tests for Surface Disinfection by EN 13697:2019

Table 8. The results of disinfection testing of preparation from B. pendula.

Phase II, Stage II
Birch Preparation	Contact time	Staphylococcus aureus ATCC 6538	Pseudomonas aeruginosa ATCC 15442	Escherichia coli ATCC 10536	Enterococcus hirae ATCC 10541	Candida albicans ATCC 10231	Aspergillus brasiliensis ATCC 16404
CP = 0.6 g/100 mL	60 ± 10 s	10−0: >330	10−0: >330	10−0: >330	10−0: >330	10−0: >330	10−0: >165
		10−1: >330	10−1: >330	10−1: >330	10−1: >330	10−1: >330	10−1: >165
		10−2: >330	10−2: >330	10−2: >330	10−2: >330	10−2: >330	10−2: >165
		Nts: >330	Nts: >330	Nts: >330	Nts: >330	Nts: >330	Nts: >165
		R: <1.47	R: <1.43	R: <1.46	R: <1.37	R: <0.42	R: <0.58
CP = 60 g/100 mL		10−0: <14	10−0: <14	10−0: <14	10−0: 67 ± 0.07	10−0: <14	10−0: <14
		10−1: <14	10−1: <14	10−1: <14	10−1: <14	10−1: <14	10−1: <14
		10−2: <14	10−2: <14	10−2: <14	10−2: <14	10−2: <14	10−2: <14
		Nts: >0	Nts: >0	Nts: >0	Nts: >0	Nts: >0	Nts: >0
		R: >4.84	R: >4.80	R: >4.83	R: >4.75	R: >3.79	R: >3.65
CP = 60 g/100 mL	300 ± 10 s	10−0: <14	10−0: <14	10−0: <14	10−0: <14	10−0: <14	10−0: <14
		10−1: <14	10−1: <14	10−1: <14	10−1: <14	10−1: <14	10−1: <14
		10−2: <14	10−2: <14	10−2: <14	10−2: <14	10−2: <14	10−2: <14
		Nts: >0	Nts: >0	Nts: >0	Nts: >0	Nts: >0	Nts: >0
		R: >4.84	R: >4.80	R: >4.83	R: >4.75	R: >3.79	R: >3.65

Mean ± SD (n = 9); Nts—the number of units remaining after the test is performed; R—reduction in the number of microorganisms during the test; C_P—concentration of preparation from B. pendula.

and

Table 9. The outcomes of surface disinfection evaluations of the preparation from B. pendula according to standard EN 13697:2019.

Phase II, Stage II
Birch Preparation	Contact Time	Staphylococcus aureus ATCC 6538	Pseudomonas aeruginosa ATCC 15442	Escherichia coli ATCC 10536	Enterococcus hirae ATCC 10541	Candida albicans ATCC 10231	Aspergillus brasiliensis ATCC 16404
LR/%R CP = 0.6 g/100 mL	60 ± 10 s	1.47/ 96.6	1.43/ 96.2	1.46/ 96.5	1.37/ 95.7	<0.41/ <61.1	<0.58/ <73.7
LR/%R CP = 60 g/100 mL	300 ± 10 s	>4.84/ >99.9	>4.80/ >99.9	>4.83/ >9.99	>4.75/ >99.9	>3.79/ >99.9	>3.65/ >99.9

Mean ± SD (n = 9); LR—the log reduction value was calculated from the Equation (2); R—reduction; C_P—concentration of preparation from B. pendula.

provide the results of disinfection testing conducted on the preparation derived from the leaves of Betula pendula against six microorganisms.

The preparation from the leaves of Betula pendula was evaluated using the surface disinfection assay. The criterion for passing the tests was >99.9% decrease in the viability of the test microorganisms (LR > 3). The surface disinfection test findings indicated that the preparation at a concentration of 60 g/100 mL exhibited biocidal activity against all tested strains, both after a contact period of 60 ± 10 s and after 300 ± 10 s. The microbiological reduction percentage was >99.9 (Table 8 and Table 9).

3.7. Tests for Surface Disinfection by EN 13697:2015

Table 10. The outcomes of the surface disinfection evaluations of the preparation from B. pendula according to the standard EN 13697:2015.

Phase II, Stage II
Birch Preparation/Ethanol	Contact Time	Staphylococcus aureus ATCC 6538	Pseudomonas aeruginosa ATCC 15442
LR/%R CP = 20 g/100 mL	60 ± 10 s	2.94/>99.8	2.82/>99.8
LR/%R CP = 30 g/100 mL		3.05/>99.9	2.88/>99.8
LR/%R CP = 40 g/100 mL		4.11/>99.9	3.98/>99.9
LR/%R CP = 50 g/100 mL		4.32/>99.9	4.24/>99.9
LR/%R CP = 60 g/100 mL		>7.04/>99.9	>7.03/>99.9
LR/%R CP = 70 g/100 mL		>7.04/>99.9	>7.03/>99.9
LR/%R CP = 80 g/100 mL		>7.04/>99.9	>7.03/>99.9
LR/%R CP = 90 g/100 mL		>7.04/>99.9	>7.03/>99.9
LR/%R CP = 100 g/100 mL		>7.04/>99.9	>7.03/>99.9
LR/%R CE = 80 g/100 mL		4.02/>99.9	4.05/>99.9
LR/%R CE = 90 g/100 mL		4.60/>99.9	4.64/>99.9
LR/%R CE = 100 g/100 mL		>7.04/>99.9	>7.03/>99.9

Mean ± SD (n = 9); LR—the log reduction—value was calculated from Equation (2); R—reduction; C_P—concentration of preparation from B. pendula; C_E—concentration of ethanol.

and

Table 11. The results of disinfection tests of the preparation from B. pendula by the standard EN 13697:2015.

Phase II, Stage II
Birch Preparation/Ethanol	Contact Time	Staphylococcus aureus ATCC 6538	Pseudomonas aeruginosa ATCC 15442
CP = 20 g/100 mL	60 ± 10 s	10−0: >330; >330	10−0: >330; >330
		10−1: 161; 153	10−1: 211; 193
		10−2: 10; 9	10−2: 11; 13
		Nd: 4.20	Nd: 4.31
		Nts: >100	Nts: >100
		R: 2.94	R: 2.82
CP = 30 g/100 mL		10−0: 245; 237	10−0: 254; 269
		10−1: 123; 122	10−1: 172; 181
		10−2: 84; 91	10−2: 94; 61
		Nd: 4.09	Nd: 4.25
		Nts: 100	Nts: 90
		R: 3.05	R: >2.88
CP = 40 g/100 mL		10−0: 115; 98	10−0: 138; 146
		10−1: 49; 58	10−1: 13; 11
		10−2: 5; 7	10−2: 0; 0
		Nd: 3.03	Nd: 3.15
		Nts: 90	Nts: 0
		R: 4.11	R: 3.98
CP = 50 g/100 mL		10−0: 74; 59	10−0: 72; 83
		10−1: 18; 23	10−1: 4; 6
		10−2: 0; 0	10−2: 0; 0
		Nd: 2.82	Nd: 2.89
		Nts: 0	Nts: 0
		R: 4.32	R: >4.24
CP = 60 g/100 mL		10−0: 8; 5	10−0: 1; 2
		10−1: 0; 0	10−1: 0; 0
		10−2: 0; 0	10−2: 0; 0
		Nd: <0.10	Nd: <0.10
		Nts: 0	Nts: 0
		R: >7.04	R: >7.03
CP = 70 g/100 mL		10−0: 0; 0	10−0: 0; 0
		10−1: 0; 0	10−1: 0; 0
		10−2: 0; 0	10−2: 0; 0
		Nd: <0.10	Nd: <0.10
		Nts: 0	Nts: 0
		R: >7.04	R: >7.03
CP = 80 g/100 mL		10−0: 0; 0	10−0: 0; 0
		10−1: 0; 0	10−1: 0; 0
		10−2: 0; 0	10−2: 0; 0
		Nd: <0.10	Nd: <0.10
		Nts: 0	Nts: 0
		R: >7.04	R: >7.03
CP = 90 g/100 mL		10−0: 0; 0	10−0: 0; 0
		10−1: 0; 0	10−1: 0; 0
		10−2: 0; 0	10−2: 0; 0
		Nd: <0.10	Nd: <0.10
		Nts: 0	Nts: 0
		R: >7.04	R: >7.03
CP = 100 g/100 mL		10−0: 0; 0	10−0: 0; 0
		10−1: 0; 0	10−1: 0; 0
		10−2: 0; 0	10−2: 0; 0
		Nd: <0.10	Nd: <0.10
		Nts: 0	Nts: 0
		R: >7.04	R: >7.03
CE = 80 g/100 mL		10−0: 129; 132	10−0: 107; 135
		10−1: 68; 83	10−1: 87; 95
		10−2: 29; 22	10−2: 8; 9
		Nc: 3.12	Nc: 3.08
		Nts: 20	Nts: 100
		R:4.02	R:4.05
CE = 90 g/100 mL		10−0: 33; 37	10−0: 28; 34
		10−1: 1; 7	10−1: 1; 1
		10−2: 0; 0	10−2: 0; 0
		Nc: 2.54	Nc: 2.49
		Nts: 20	Nts: 20
		R:4.60	R:4.64
CE = 100 g/100 mL		10−0: 0; 0	10−0: 0; 0
		10−1: 0; 0	10−1: 0; 0
		10−2: 0; 0	10−2: 0; 0
		Nd: <0.10	Nd: <0.10
		Nts: 0	Nts: 0
		R: >7.04	R: >7.03

Mean ± SD (n = 9); Nts—the number of units remaining after the test is performed; R—reduction in the number of microorganisms during the test; C_P—concentration of preparation from B. pendula; C_E—concentration of ethanol.

provides the outcomes of surface disinfection experiments conducted on the preparation from the leaves of Betula pendula, targeting Staphylococcus aureus ATCC 6538 and Pseudomonas aeruginosa ATCC 15442.

The birch preparation showed significant efficacy on surfaces against Staphylococcus aureus ATCC 6538 already at a concentration of 30 g/100 mL, while against the Pseudomonas aeruginosa strain ATCC 15442, efficacy >99.9% was observed at a concentration of 40 g/100 mL (Table 10 and Table 11).

4. Discussion

Several studies support the pharmacological effects of extracts from the Betula pendula plant, including its antibacterial and antioxidant potential [11,47,48]. Most published studies focus on extracts from the bark of this plant. The novelty of this study is the GC-MS analysis of the composition of the leaf preparation (Table 4) and the quantitative analysis of selected phenolic acids (gallic acid, 3,4-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid, chlorogenic acid, and 3-hydroxybenzoic acid) contained in the ethanolic leaf preparation (Table S1, Figure 1).

Analyses of the composition of the Betula pendula leaf preparation were not found in the literature, but analyses of oil and extracts from the bark of this plant are available [13,49,50]. The authors of this study [13] analyzed birch essential oil buds obtained by hydrodistillation using a Clevenger apparatus. Its qualitative and quantitative composition was determined by GC-MS, and the analysis identified 27 compounds, mainly sesquiterpene hydrocarbons (about 79%) and oxidized sesquiterpenes (about 15%), representing about 94% of all identified essential oil components. The most abundant compounds were germacrene D (about 22%) and δ-cadinene (17%) [13]. In this study [50], the chemical composition of bud extracts from two white birch species, i.e., Betula pubescens Ehrh. and Betula pendula Roth (which may be a promising source of compounds with cytotoxic activity against various cancers), was assessed by GC-MS. These extracts were obtained by three distinct procedures: supercritical CO₂ extraction, washing of the exudate covering the whole buds, and extraction of the ground buds with diethyl ether. The chemical composition of B. pubescens and B. pendula buds differed, with bud extracts of the former containing rather significant levels of sesquiterpenoids and flavonoids; triterpenoids were the predominant components of extracts from the latter [50]. The authors [49] of this study analyzed the main compounds contained in ethanolic extracts from the bark of Alnus glutinosa (L.) Gaertn, B. pendula Roth, and Platanus hybrida Brot., which were obtained by supercritical (scCO₂ + EtOH) and conventional extraction in a Soxhlet apparatus. The main constituents identified by GC-MS were betulin and lupeol, as well as β-sitosterol and betulinic acid, with supercritical extraction having a higher content of the main constituents in the supercritical extracts (scCO₂ + EtOH) than in the conventional Soxhlet extracts [49]. Table 4 shows the composition of the Betula pendula leaf preparation. Using the GC-MS method, we recognized the 11 main components of the ethanol extract from this plant. This preparation consisted mainly of saturated fatty acid, saturated fatty acid ester, unsaturated terpene ketone, unsaturated fatty acid, unsaturated fatty acid ester, fatty alcohol, triterpenoids, thio compound, and alpha-tocopherol B (Table 4, Figure S2).

We also assessed the n-octanol/water partition coefficient of the Betula pendula leaf preparation. The lipophilicity tests performed showed that the preparation is highly hydrophilic (log P = −0.500 ± 0.001), which is due, among other things, to the presence of phenolic acids (i.e., GA, 34DHBA, 25DHBA, ChA, and 3HBA)—Figure 1 and Figure S1. Muzykiewicz et al. [51] noted the high hydrophilicity of the optimized S. officinalis extract assessed by log P. The partition coefficient value for the optimized extract was −0.307 ± 0.001 [25]. The authors postulate that the high hydrophilicity of the extracts is most likely related to the high content of phenolic acids (mainly GA 224 ± 10 mg/L), whose total concentration was about 1.3 g/L. Our study showed that TPC = 52 ± 2 mg GAE/g of dry raw material, among which GA and 34DHBA had the greatest concentration (194 ± 5 mg/g dry raw material and 123 ± 7 mg/g dry raw material, respectively)—Figure 1. In our previous publication [22], we noted the high hydrophilicity of leaf extracts from plants of the genus Rubus assessed by log P. The partition coefficient values for the preparations were −0.18 and −0.19, respectively. The high hydrophilicity of these extracts is most likely related to the high level of polyphenols, which in turn affect the antioxidant potential of the samples tested [22].

The antioxidant activity measured by the DPPH test was 32 ± 6 mg TE/g of dry raw material (Table 5) and was much more than the activity reported by the authors of reference [52]. Tocai et al. [53] evaluated the antioxidant activity of ethanolic plant extracts derived from various plant parts, revealing that root extracts exhibited significantly greater antioxidant activity (measured by the FRAP method) compared to leaf extracts, indicating that roots are the most valuable source of compounds with antioxidant potential, including polyphenols [53]. Results obtained by other authors [52] showed that the iron ion-reducing capacity (assessed by the FRAP method) for ethanolic extracts of the plant’s roots, leaves, stems, and flowers was 0.3, 0.2, 0.13, and 0.09 mmol/g on a dry weight basis, respectively. The results of our study (Table 5) and those of other authors show that plant parts that grow above ground (like leaves) and below ground (like roots) can be beneficial sources of antioxidants like polyphenols. However, many variables, such as the pre-extraction treatment of the raw material, extraction parameters, and ethanol concentration, may dramatically impact the activity of extracts [25,51,52]. The antioxidant potential of a dry extract of Betula pendula Roth leaves was evaluated in this investigation [16]. This study’s findings indicated that these extracts exhibit relatively strong antioxidant potential and can therefore be used as a natural source of antioxidants, offering the possibility of preparing high-value products helpful in the prevention of various oxidative stress-related conditions. Studies using the Folin–Ciocalteu technique have shown that the TPC in dried birch (Betula pendula) leaf extract is 128.45 mg GAE/lg. The investigators of this study assert that the elevated total phenolic content in birch extract is essential for significant antioxidant activity [16].

The total polyphenol content of ethanolic extracts from leaves, buds, and bark of Cinnamomum cassia were 6.313–9.534 g GA/100 g dry weight, with bark extracts having the highest polyphenol content (9.534 g GA/100 g DW), followed by the leaf (8.854 g GA/100 g dry weight) and bud (6.313 g GA/100 g dry weight). In contrast, 95% ethanolic extracts from Cinnamomum cassia leaves had a TPC (8.854 g/100 g dry weight) about twice as high as the total flavonoid content (3.348 g/100 g dry weight) [54]. Our study showed that ethanolic preparations of Betula pendula leaves were characterized by more than five times higher polyphenol content (52 ± 2 mg GAE/g dry raw material) than Cinnamomum cassia leaf extracts (Table 5). In the case of 50% ethanolic extracts of Chinese Cinnamomum cassia leaves, the content of polyphenols (which are secondary metabolites that show free radical scavenging and peroxide degrading capacity) was 1558.7 μg GAE/g dry weight, with a total flavonoid content of 981.1 μg/g dry weight [49,55,56,57].

Recently, preparations with anti-ageing, antimicrobial, and antioxidant properties derived from selected plant raw materials have become increasingly popular. Plant extracts containing phenolic compounds have been shown to reverse antimicrobial resistance and have a synergistic impact when paired with routinely used chemotherapeutics [38]. The authors of this paper postulate [58] that phenolic acids contained in plant extracts are likely to interact with antibiotics [58]. By examining the antimicrobial activity of antibiotics (erythromycin, clindamycin, and cefoxitin) alone and in an antibiotic–phenolic combo (using caffeic acid as the phenolic compound) against strains of Staphylococcus aureus, it was shown that the interaction of caffeic acid with antibiotics increased the antimicrobial activity of the antibiotic–phenolic combination [57,59]. Other studies of the antimicrobial activity of caffeic acid, EDHB, and catechin hydrate (CH) showed that it was the polyphenolic compound that exhibited the strongest antimicrobial activity and the greatest synergistic effect with antibiotics, which may be due to the fact that caffeic acid contains a propene side chain that affects the change in polarity [57,58,59].

Polyphenols’ structures include phenolic hydroxyl groups, which means that they have a high affinity for binding to proteins and lipids in the cell membrane and thus can inhibit microbial enzymes while increasing their affinity for cytoplasmic membranes, thereby enhancing antimicrobial activity. The current research demonstrates that preparation is a promising source of effective, safe, and inexpensive compounds with antimicrobial potential, opening a wide range of possibilities for new antimicrobial therapies. The antimicrobial activity of the obtained preparation results from, among other things, the presence of phenolic acids (Figure 1), especially ChA and GA found in many plant species with proven antimicrobial activity. ChA is a component in cosmetics (dermatological) and pharmaceutical preparations, providing an alternative for strategies to combat microbial pathogenesis and chronic infections caused by microorganisms (such as bacteria, fungi, and viruses) [60]. GA is a bioactive phytochemical compound, and its derivatives are often present in cosmetic preparations and can be considered ‘safe’ and ‘natural’ in the context of cosmetic manufacturing [60].

5. Conclusions

Studies have demonstrated the biocidal activity and high antioxidant potential of the silver birch (Betula pendula Roth) leaf preparation with a reduced ethanol content. The Betula pendula preparation, due to the presence of phenolic acids and volatile biologically active compounds, exhibits antioxidant and antimicrobial activity against reference bacteria and fungi. The presence of phenolic acids makes the preparation highly hydrophilic, as evidenced by a log P = −0.500 ± 0.001.

Using the new antiseptic lotion to clean people’s hands according to PN-EN 1500:2013 showed that it was effective against Escherichia coli K12 NCTC 10538. The mean 60 s kill time was >99.9% for the new antiseptic against Staphylococcus aureus ATCC 6538 (at a concentration of 30 g/100 mL) and Pseudomonas aeruginosa ATCC 15442 (at a concentration of 40 g/100 mL). Polyphenols are considered highly effective against bacteria, but the thicker cell wall and larger cell structure may slow down the antimicrobial effect of the preparation against Gram-positive bacteria compared to Gram-negative bacteria.

Furthermore, the high content of polyphenols and other biologically active compounds may be one of the parameters responsible for the effective biological activity of the preparation, which supports the use of a balanced product in healthcare facilities, schools, or homes. Moreover, its high antioxidant potential allows its use in the cosmetic and pharmaceutical industries.