Phytochemical Profile and Antimicrobial Potential of Propolis Samples from Kazakhstan

In the current paper, we present the results of Kazakh propolis investigations. Due to limited data about propolis from this country, research was focused mainly on phytochemical analysis and evaluation of propolis antimicrobial activity. uHPLC-DAD (ultra-high-pressure-liquid chromatography coupled with diode array detection, UV/VIS) and uHPLC-MS/MS (ultra-high-pressure-liquid chromatography coupled with tandem mass spectrometry) were used to phytochemical characteristics while antimicrobial activity was evaluated in the serial dilution method (MIC, minimal inhibitory concentration, and MBC/MFC, minimal bactericidal/fungicidal concentration measurements). In the study, Kazakh propolis exhibited a strong presence of markers characteristic of poplar-type propolis—flavonoid aglycones (pinocembrin, galangin, pinobanksin and pinobanskin-3-O-acetate) and hydroxycinnamic acid monoesters (mainly caffeic acid phenethyl ester and different isomers of caffeic acid prenyl ester). The second plant precursor of Kazakh propolis was aspen–poplar with 2-acetyl-1,3-di-p-coumaroyl glycerol as the main marker. Regarding antimicrobial activity, Kazakh propolis revealed stronger activity against reference Gram-positive strains (MIC from 31.3 to above 4000 mg/L) and yeasts (MIC from 62.5 to 1000 mg/L) than against reference Gram-negative strains (MIC ≥ 4000 mg/L). Moreover, Kazakh propolis showed good anti-Helicobacter pylori activity (MIC and MBC were from 31.3 to 62.5 mg/L). All propolis samples were also tested for H. pylori urease inhibitory activity (IC50, half-maximal inhibitory concentration, ranged from 440.73 to 11,177.24 µg/mL). In summary Kazakh propolis are potent antimicrobial agents and may be considered as a medicament in the future.


Introduction
Propolis, also called "bee glue" is a natural product of different bee species. Its viscous form is due to the fact that exudates collected from the buds and flowers of different plant species growing in the vicinity of the bee hive are used to produce propolis [1]. These exudates of botanical origin are chewed by the honeybees and mixed later with pollen and the bee's saliva (containing several enzymes, among them β-glucosidase) and, finally, after              Tables 1 and 2).
Molecules 2023, 28, x FOR PEER REVIEW 5 of 32 Apart from poplar, the presence of 2-acetyl-1,3-di-p-coumaroyl glycerol suggested aspen origin of some samples. This substance is specific marker of P. tremula (aspen) as well as some other Populus species (e.g., P. lasiocarpa [24]). Aspen is widely spread across whole Eurasia [23] while P. lasiocarpa is naturally present in China. Outside China, it is rather planted in botanical gardens and parks than easily spreading in the natural environment. For these reasons, presence of 2-acetyl-1,3-di-p-coumaroyl glycerol in propolis rather proves the presence of P. tremula exudates more than another species.
Previous research exhibited mixed aspen-poplar origin of Kazakh propolis sample [19]. In the current research, most samples exhibited a strong presence of poplars markers (Almaty 1, 4, 6, Bozovoe and Kogaly and Kegen) and lower (Almaty 2, 3, 5) or a strong of aspen ones (Almaty 7). However, there were some unidentified components, which may not be connected with Populus genus origin, especially in Almaty-1 (see Tables 2 and 3). Moreover, a high occurrence of cinnamic acid is also not usual for black poplar trees, but hydroxycinnamic acids are presented in its place [25,26]. According to the plant distribution map [22], P. nigra should be rather present in northern Kazakhstan. Therefore, the sample from Bozovoe may exhibit black poplar origin. Propolis from the southern part (Almaty, Kegen and Kogaly) should be rather originated from another balsamic Populus tree (e.g., P. laurifolia or P. euphratica). However, literature studies exhibited that the situation of Populus genus distribution in Kazakhstan is complex. During the Soviet Union time in the central Asia region, there were introduced many Populus species such as P. nigra, P. bolleana and P. deltoides and other cultivars [27]. Moreover, many Populus cultivars are known for the easy creation of crossbreed species. In a result in the same Almaty, many crossbreed species were observed (e.g., P. nigra × P. maximowiczii, P. nigra × deltoides or P. laurifolia × P. canadensis) [28]. Dependences between Populus bud exudate compositions and their genetic origin were not well investigated and, for this reason, accurate tracking of plant precursors of Kazakhstani poplar type propolis may be very difficult.
Apart from the Populus genus, it is worth to add that Kazakhstani propolis may have some other plant sources. In the previous research [19], we observed also the presence of flavonoid ermanin which may be connected with birch origin. However, in the present research, ermanin was only a trace component and the same Betula genus may be a marginal plant precursor. Moreover, there are possibly more minor or marginal plant precursors. Confirmation of birch presence requires further research with additional techniques such as GC-MS [25].
The possibility of non-Populus plant precursors suggested also caffeoylmalic acid (phaseolic acid) isomer and some unidentified components, especially described in Table 2. They were strongly presented in the mainly two samples (Kogaly and Almaty-1) which may suggest additional unknown plant precursors.
In the end, it is worth adding that not all components presented in propolis have natural origin. Sometimes, there may be traces of different beekeeping techniques (e.g., treatment of honeybees to American and the European foulbrood [29]) or even pollutants occurring in the environment [30]. For this reason, the presence of an uncommon component in propolis should be analyzed in detail.

Comparative Analysis of Chemical Composition of Extracts for Kazakh Propolis Samples
The results of comparative analysis of chemical composition are presented in Figure 2. An investigation based on the uHPLC-DAD matrix (Table 3) with the spectral properties of polyphenols allowed to group of the samples into three main clusters.
The first cluster was composed of six propolis samples with a high presence of different prenyl (methylbutenyl) esters of caffeic acid (mainly 2-methyl-2-butenyl ester, 3-methyl-2butenyl and 3-methyl-3-butenyl) and pinobanksin-3-O-acetate. In this cluster Bozovoe was less similar to the other samples due to a higher concentration of flavonoid aglycones and lower prenyl esters of caffeic acid. Samples in this cluster exhibited a strong presence of P. nigra and similar poplars resins. There were possible other plant precursors; however, they are rather minor or even marginal in most cases.

Comparative Analysis of Chemical Composition of Extracts for Kazakh Propolis Sam
The results of comparative analysis of chemical composition are presented in An investigation based on the uHPLC-DAD matrix (Table 3) with the spectral pr of polyphenols allowed to group of the samples into three main clusters. The first cluster was composed of six propolis samples with a high presenc ferent prenyl (methylbutenyl) esters of caffeic acid (mainly 2-methyl-2-butenyl methyl-2-butenyl and 3-methyl-3-butenyl) and pinobanksin-3-O-acetate. In thi Bozovoe was less similar to the other samples due to a higher concentration of fl aglycones and lower prenyl esters of caffeic acid. Samples in this cluster exhibited presence of P. nigra and similar poplars resins. There were possible other plant pre however, they are rather minor or even marginal in most cases.
The second cluster contained three samples with strong presence of p-coum cinnamic acids and a lower amount of prenyl esters of caffeic acid than cluster 1 ver, in this cluster, there were also some of hydroxycinnamic acid glycerides prese erally, this cluster represented mixed, aspen-poplar type of propolis.
The last cluster (3) had only one sample (Almaty-7). Its main components we maric, 2-acetyl-1,3-di-p-coumaroyl glycerol and p-coumaric acid benzyl ester. The sition of Almaty-7 suggested a strong aspen origin with lower amount of poplar m In summary clusters presented in dendrogram reflected presence of poplar pen markers described in section 2.1. Generally, presence of unidentified compo some samples (Kogaly and Almaty 1, 2 and 6) exhibited rather low impact on their grouping-all these samples were presented in two main clusters. For this reason, suspect that Populus genus was main plant precursor of these samples.

Antimicrobial Activity of Propolis Samples from Kazakhstan
The results of the antimicrobial assays are presented in Table 4. The main go study was a general screening of antimicrobial properties of propolis from Kazak our study, we evaluated activity of 70EEP against the following reference mic isms: six strains of Gram-positive and six strains of Gram-negative bacteria as wel strains of yeasts. The second cluster contained three samples with strong presence of p-coumaric and cinnamic acids and a lower amount of prenyl esters of caffeic acid than cluster 1. Moreover, in this cluster, there were also some of hydroxycinnamic acid glycerides present. Generally, this cluster represented mixed, aspen-poplar type of propolis.
The last cluster (3) had only one sample (Almaty-7). Its main components were pcoumaric, 2-acetyl-1,3-di-p-coumaroyl glycerol and p-coumaric acid benzyl ester. The composition of Almaty-7 suggested a strong aspen origin with lower amount of poplar markers.
In summary clusters presented in dendrogram reflected presence of poplar and aspen markers described in Section 2.1. Generally, presence of unidentified components in some samples (Kogaly and Almaty 1, 2 and 6) exhibited rather low impact on their clusters grouping-all these samples were presented in two main clusters. For this reason, we may suspect that Populus genus was main plant precursor of these samples.

Antimicrobial Activity of Propolis Samples from Kazakhstan
The results of the antimicrobial assays are presented in Table 4. The main goal of our study was a general screening of antimicrobial properties of propolis from Kazakhstan. In our study, we evaluated activity of 70EEP against the following reference microorganisms: six strains of Gram-positive and six strains of Gram-negative bacteria as well as three strains of yeasts.
Among Gram-positive bacteria, Kazakh propolis samples were more active against Micrococcus luteus with MIC values of 31.3 µg/mL; however, some samples also showed weak activity (>4000 µg/mL). According to criterium of bioactivity presented by O'Donnell [31], it is defined as good activity. Against two strains of Staphylococcus aureus tested, 70EEPs exerted activity 62.5 -> 4000 µg/mL. It is worth mentioning that most of propolis samples presented good bioactivity (62.5-125 µg/mL), and only samples from Kogaly were inactive. Distinguished from the rest of the samples, propolis from Kogaly had its own specific markers in uHPLC-DAD (Table 3) analysis which suggested the presence of an additional unknown plant precursor. It is possible, that its presence may cause strongly lower activity of this sample. Antibacterial activity of Kazakh propolis against Staphylococcus epidermidis and Bacillus cereus can be described as good as 31.3-125 µg/mL and 62.5-125 µg/mL, respectively, except the samples obtained from Kogaly, which were inactive (MIC > 4000 µg/mL). Against Enterococcus faecalis, propolis from Kazakhstan expressed good or moderate antibacterial activity (62.5-250 µg/mL), which deserves attention. Gram-negative bacteria were insensitive to propolis collected in Kazakhstan (MIC = 4000 µg/mL or more). This may be related to the structure of the bacterial cells and the double cell membrane, which, when exposed to a fraction of surface-active compounds, can stiffen and remodel, increasing its resistance [32]. The presence of the periplasmic space may also cause compounds that have already penetrated the cell to be cut by hydrolytic enzymes, losing their activity [33]. H. pylori was the only exception of the Gram-negative bacteria which was inhibited by all propolis samples showing good antibacterial activity against this pathogen (MIC = 31.3-62.5 µg/mL). This high activity can often be associated with impaired urease activity and may affect stick adhesion and cell viability [34]. Moreover, MIC values in all 70EEPs were equal to MBCs. The results presented by the other authors [18,19,35], as well as our own research, clearly indicate that Gram-positive bacteria are susceptible to lower propolis concentrations than Gram-negative ones. S. aureus and B. cereus are well-known because of their involvement in the gastrointestinal and respiratory tract diseases [15]. Since propolis is usually administered orally, its antimicrobial activity against these pathogens is of great practical importance in its possible therapeutic use [15].
The results of this study showed higher activity of 70EEPs obtained from Kazakhstan in comparison to green and brown propolis ethanolic extracts from Brazil (MIC = 125 and 250 µg/mL, respectively) [15]. Interestingly, partitioning in dichloromethane has enhanced the extraction of antibacterial compounds from Brazilian propolis samples, as it can be inferred from the lower MIC values observed for green (MIC = 7.8 µg/mL) and brown propolis (MIC = 62.5 µg/mL), what was correlated with enhanced levels of phenolic compounds in the extracts [15]. Better activity against M. luteus was reported for propolis samples collected in Anatolia (Turkey). Four samples from a different locations, and characterized by the presence of flavonoid compounds (pinocembrin, pinostrobin, isalpinin, pinobanksin, quercetin, naringenin, chrysin and galangin) showed MIC values from 4 to 16 µg/mL [36]. The different species of staphylococci are the microorganisms most often used as models for antimicrobial activity of propolis. This is probably due to their high importance for human morbidity. Staphylococci colonize about 30% of humans (usually asymptomatically) and are responsible for a wide spectrum of difficultto-treat infections (eye inflammation, pneumonia, meningitis and others) [37]. The antistaphylococcal potential of tested Kazakh propolis samples against the three reference strains: S. aureus ATCC 25923, S. aureus ATCC 43300 and S. epidermidis ATCC 12228 were better (31.3-250 µg/mL) than results obtained by Grecka et al.  µg/mL) [37] and our studies concerning Georgian propolis samples (64-512 µg/mL) [20] and similar to our other result presented in paper about antimicrobial activity of poplar-type propolis (10-2500 µg/mL) [18]. It should be underlined that the MIC is equal to the MBC for most of the tested propolis samples from Kazakhstan. Similar antimicrobial properties (similar ranges of MIC values) of the propolis in question correlate with data on the chemical composition of propolis samples. All samples are characterized by a phytochemical profile (flavonoids and derivatives of phenolic acids) indicating different species of Populus as the plant precursor of propolis. The biological activity of propolis is related to time (plant source of exudate), harvest time and geographic origin. Due to these considerations, propolis from a particular geographic region should exhibit similar physicochemical characteristics and other properties, e.g., antimicrobial activity. Several independent studies have shown high susceptibility of S. aureus and S. epidermidis to different types of propolis originating from Brazil. For example, Reguiera et al. revealed the good bioactivity of Brazilian red propolis hydroalcoholic extracts against S. aureus ATCC 6538 and clinical isolates with MIC in the range of concentration 64 to 1024 µg/mL [3]. Another study confirmed the antimicrobial efficacy of ethanolic extracts of three different types of propolis from Brazil: brown, red and green against S. aureus ATCC 25923 and S. aureus ATCC 25923. The lowest MIC values characterized red type of propolis (25-50 µg/mL), higher in green ones (200-400 µg/mL) and highest (lowest antimicrobial activity) in brown propolis (200-800 µg/mL) [38]. In the same study, the authors compared two methods of raw propolis extraction methods: classical low-pressure extraction with ethanol and supercritical fluid extraction (SFE). Taking into consideration the antibacterial activity, the most potent were ethanolic extracts of propolis, characterized by the highest content of phenolic compounds and high values of flavonoids [38]. These findings confirmed the method we used for the extraction of propolis samples from Kazakhstan due to the increased amount of polar compounds in the extract (phenolic acids and their derivatives and flavonoids) that determine antimicrobial activity. Interestingly, anti-staphylococcal activity of Brazilian red propolis was presented by Regueira et al. [3]. Samples of propolis were collected in the rainy and the dry season. Hydroethanolic extracts showed MIC values for S. aureus ATCC 6538 and clinical isolate ≥1024 and 101.6 µg/mL as well 512 and 64 µg/mL for the rainy and the dry season samples, respectively [3]. Comparison of two extracts demonstrated two-times higher concentration of phenolic compounds in the dry season sample, which had the crucial influence on the antibacterial activity [3]. The study reported on the inhibitory and bactericidal properties of 39 South African and three propolis samples from Brazil and was conducted by Suleman et al. [1]. Some samples of African propolis displayed substantial antimicrobial activity with MIC and MBC values at a very low level of 6 µg/mL against S. aureus ATCC 25923 [1]; however, the remaining samples had weaker bioactivity (24 up to 1563 µg/mL). The main bioactive constituents of propolis were identified as chrysin, pinocembrin, galangin or 3-pinobankin-3-O-acetate, ingredients, that are also found in poplar type of propolis [1]. The activity of tested Kazakh propolis against the rest of Gram-positive bacteria was similar to results presented in the literature and our own studies [18,20,35,36].
Among all tested Gram-negative bacteria, only H. pylori was sensitive for 70EEPs form samples collected in Kazakhstan. To our best knowledge, it is the first communication on the anti-Helicobacter activity of Kazakh propolis (except two papers reported one propolis sample from Kazakhstan from different origin). Our group assessed ten samples of 70EEP obtained from different propolis samples derived from various parts of Kazakhstan. The highest bioactivity (31.3 µg/mL) against reference H. pylori strain was expressed by 70EEP from Kogaly, Bozove, and four samples from Almaty (Almaty-2, Almaty-3, Almaty-5 and Almaty-6). The rest of samples were characterized by MIC values 62.5 µg/mL. Generally, the antibacterial activity of Kazakh propolis against H. pylori and, according to O'Donell criterium, is regarded as good [31]. Moreover, the PE from the sample obtained in Kolgaly has one of the highest activities, despite its inactivity against all Gram-positive bacteria. Surprisingly, for all 70EEPs evaluated against H. pylori, MBC/MIC ratio was 1, which confirmed the bactericidal activity of tested propolis extracts [39]. In the frame of our studies, we tried to combine the results of the microbiological evaluation of the inhibition of H. pylori growth by propolis extracts from several locations in Kazakhstan with qualitative analysis of their composition by using chromatographic and spectral analysis (uHPLC-DAD and uHPLC-MS/MS). The antibacterial activity of Kazakh 70EEPs were similar to our previous studies focused on the activity propolis from Georgia (MIC = 31.3-125 µg/mL) [40] or different European propolis samples (MIC = 20-30 µg/mL) [18]. Similar results were obtained in the study performed by Santiago et al. [41] with hydroalcoholic extracts of Brazilian propolis against H. pylori ATCC 43526 (MIC = MBC = 50.0 µg/mL) and clinical isolate of H. pylori (MIC = MBC = 100.0 µg/mL) [41]. The weaker activity against H. pylori was presented by 19 propolis samples from Northern Spain (Basque Country) extracted with ethanol and propylene glycol (MIC from 6 to 14 mg/mL) [42]. Indonesian propolis produced by a stingless bee, belonging to Trigona spp. was tested against ten clinical isolates of H. pylori (from dyspeptic patients) [43]. The results of experiments indicate very weak activity of ethanolic extracts of propolis (MIC = 1024-8192); however, there were promising results of an additive effect against H. pylori when used together with clarithromycin and metronidazole [43]. The chemical composition of tested Kazakh propolis, showed a similar phytochemical profile, which is a good explanation of the activity of 70EEPs from Kazakhstan against H. pylori. However, results presented by Romero and coauthors showed that activity of complex natural mixtures as propolis is more than just simple sum properties of all constituents and interaction among them, which should be taken into consideration [44]. Finally, analyses by transmission electron microscopy at subinhibitory concentration showed vesicle formation and bacterial cell lysis after exposition to individual polyphenols and in the mixture, suggesting a potential bactericidal activity of propolis [44].
Antifungal activity of 70EEPs was tested against three Candida species. Propolis exhibited moderate antifungal activity (125-500 µg/mL) against C. glabrata and moderateto-mild bioactivity (125-1000 µg/mL) against C. albicans and C. parapsilosis. There are numerous experimental works on the antimicrobial activity of various kinds of propolis collected from different geographical locations [1, 15,36,45]. Depending on the content of the samples, propolis may inhibit the process of filamentation and yeast adhesion and increase intracellular oxidative stress [46]. The bioactivity of tested samples against pathogenic yeasts (62.5-1000 µg/mL) is weaker than our research concerning propolis for samples obtained from Georgia and central Europe, but similar to the activity of propolis samples from South Africa (MIC between 98-1563 µg/mL] [1] and Cretan propolis (370-1560 µg/mL) [45]. Better activity against C. albicans was exerted by Anatolian propolis (MIC range 4-32 µg/mL) [36]. Propolis owes its antimicrobial activity mainly to the presence of polyphenolic compounds (phenolic acids and flavonoids).
The mechanism underlying the antimicrobial activity of propolis involves the flavonoid and phenolic acids present in propolis. The literature data, among them some reviews confirmed and summarized these mechanisms of activity [3]. Polyphenols are responsible for the inhibition of nucleic acid synthesis (DNA and RNA) and the inhibitory mechanism on DNA gyrase (procaryotic enzyme plays an important role in processes of replication, transcription and recombination) [47][48][49]. Galangin (flavonol) and derivatives of caffeic acids derivatives (polyphenolic acids) have the ability to uncouple the energy-transducing cytoplasmic membrane and inhibit bacterial motility. Moreover, these effects on the bioenergetic status of the membrane may contribute to the antimicrobial action of propolis and its observed synergism with selected antibiotics [50]. Flavonoids, among other phenolic compounds, interfere with the energy metabolism of the bacterial cell due to the damage to the cytoplasmatic membranes, their permeability alteration and the perturbance in the exchange of nutrients and metabolites [35,[47][48][49]. Additionally, flavonoids from propolis inhibit adhesion and biofilm formation [3,35,[47][48][49].
In summary observed differences of the antimicrobial activity of Kazakh propolis may bresultesult of different factors. The basic one may be differences between Kazakh propolis plant precursors. Usually, propolis with a stronger presence of poplar markers is expected to be stronger than propolis with aspen-poplar origin [18,40]. However, P. treumula exudates sometimes exhibited stronger activity than P. nigra resins [51][52][53]. Moreover, the same propolis may exhibit lower or stronger activity than its plant precursor [51][52][53]. Propolis as well as Populus genus bud exudates are a complex matrix and their antimicrobial activity is an effect of interaction between many components. The same exudates of this same Populus species may be observed in different chemotypes [26,51,52] that should also exhibit an impact on propolis antimicrobial activity. In results, total effect may also be connected with presence of minor and even marginal plant precursors as well as specific chemical composition of plant precursor.

Urease Inhibitory Activity and Anti-Helicobacter Activity of Tested Kazakh Propolis Extracts
The results for the assessment of selected 70EEPs from Kazakh propolis are listed in Table 5. The presented study is the first attempt to evaluate the effects of 70EEPs obtained from propolis sample collected in Kazakhstan, a natural bee product used in the treatment of gastric diseases, on H. pylori growth in vitro as well as the activity of its enzyme urease which is crucial for the ability of the pathogen to colonize the stomach. Results of bioassay shows IC 50 values for 70EEPs ranging from 440.73 to 11,177.24 µg/mL and IC 50 = 92.7 µg/mL for thiourea (reference inhibitor) ( Table 5 and Figure 3). Obtained results suggested the same plant origin of Kazakhstan propolis. Presented results suggested that inhibition of urease is not directly connected with bactericidal activity against H. pylori. Moreover, variability of activity inside clusters also suggested that the same type of propolis may not be directly connected with urease inhibition activity. It is more possible that the obtained effect is a result of interaction between components in complex natural matrix of propolis. For this reason, some samples may exhibit better activity inside this same cluster.
Molecules 2023, 28, x FOR PEER REVIEW 26 which is crucial for the ability of the pathogen to colonize the stomach. Results of bioa shows IC50 values for 70EEPs ranging from 440.73 to 11,177.24 µg/mL and IC50 = 92.7 µg for thiourea (reference inhibitor) ( Table 5 and Figure 3). Obtained results suggested same plant origin of Kazakhstan propolis. Presented results suggested that inhibitio urease is not directly connected with bactericidal activity against H. pylori. Moreover iability of activity inside clusters also suggested that the same type of propolis may n directly connected with urease inhibition activity. It is more possible that the obta effect is a result of interaction between components in complex natural matrix of prop For this reason, some samples may exhibit better activity inside this same cluster.  Results showed in described experiments are similar to the other research of 70EEPs inhibitory activity and indicate that searching for a novel, natural urease inhibitors among bee products is the proper direction. For example, Baltsas et al. [54] tested 15 PEs of Turkish propolis samples for urease inhibitory activity. The tested propolis samples exerted IC 50 in the range of 0.260 to 1.525 mg per mL, similar to the results exhibited in this study. Inhibition activity of 70EEPs from Kazakhstan is distinctly weaker than other natural products, e.g., essential oils. For example, essential oil from Origanum vulgare (MIC = 31.3 µg/mL) presented IC 50 against H. pylori urease equal to 208.3 µg/mL [55]. Moreover, the most active essential oil (cedarwood essential oil) has IC 50 = 5.3 µg/mL (MIC = 15.6 µg/mL) [55].
In research performed by Can [56], 11 propolis samples from the Marmara region of Turkey were tested. Their activity concerning urease inhibition was in the range from 1.110 to 5.870 mg/mL and authors suggested that it indicates good bioactivity of tested propolis extracts [56]. In another experiment done by Can [57], where enzyme inhibition of urease was examined by different bee products-honey, pollen and propolis. The IC 50 values were changed from 7.02 to 33.25 mg/mL, 5.00 to 8.78 and 0.16 to 1.98 mg/mL in the honey, pollen and propolis samples, respectively [57].
Urease is a crucial enzyme for H. pylori to survive in an acidic environment of the stomach. Propolis extracts, which contain numerous polyphenolic compounds that have the ability to inhibit urease, can be considered a useful component of H. pylori eradication therapy.

Determination of Antimicrobial Activity
The propolis extracts dissolved in dimethylo-sulfoxide (DMSO) were screened for antibacterial and antifungal activities by microdilution broth method according to both the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (www.eucast.org (accessed on 3 January 2023) using Mueller-Hinton broth or RPMI with MOPS for growth of fungi as we described elsewhere [58,59]. Minimal inhibitory concentration (MIC) of the tested extracts were evaluated for the wide panel of the reference microorganisms, including Gram-negative bacteria (Escherichia coli ATCC 25922, Salmonella typhimurium ATCC14028, Klebsiella pneumoniae ATCC 13883, Pseudomonas aeruginosa ATCC 9027), Grampositive bacteria (Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 43300, Staphylococcus epidermidis ATCC 12228, Micrococcus luteus ATCC 10240, Enterococcus faecalis ATCC 29212, Bacillus cereus ATCC 10876) and fungi (Candida albicans ATCC 10231, Candida parapsilosis ATCC 22019, Candida glabrata ATCC 90030). The sterile 96-well polystyrene microtitrate plates (Nunc, Roskilde, Denmark) were prepared by dispensing 100 µL of appropriate dilution of the tested extracts in broth medium per well by serial two-fold dilutions in order to obtain final concentrations of the tested extracts ranged from 0.0195 to 10 mg/mL The inocula were prepared with fresh microbial cultures in sterile 0.85% NaCl to match the turbidity of 0.5 McFarland standard were added to wells to obtain final density of 5 × 10 5 CFU/mL for bacteria and 5 × 10 4 CFU/mL for yeasts (CFU, colony forming units). After incubation (35 • C for 24 h), the MICs were assessed visually as the lowest concentration of the extracts showing complete growth inhibition of the reference microbial strains. Appropriate DMSO control (at a final concentration of 10%), a positive control (containing inoculum without the tested derivatives) and negative control (containing the tested derivatives without inoculum) were included on each microplate.
The MIC for H. pylori ATCC 43504 was determined using a two-fold microdilution method in MH broth with 7% of lysed horse blood at extract concentration ranging from 1000 to 1.95 mg/L with bacterial inocula of 3 McFarland standard. After incubation at 35 • C for 72 h under microaerophilic conditions (5% O 2 , 15% CO 2, and 80% N 2 ) the growth of H. pylori was visualized with the addition 10 µL of 0.04% resazurin. The MIC endpoint was recorded after 4 h incubation as the lowest concentration of extract that completely inhibits growth [55].
Minimal bactericidal concentration (MBC) or minimal fungicidal concentration (MFC) was obtained by culture of 5 mL from each well that showed through growth inhibition, from the last positive one, and from the growth control onto recommended agar plates. The plates were incubated at 35 • for 24 h for all microorganisms but H. pylori which were incubated for 72 h in microaerophilic conditions. The MBC/MFC was defined as the lowest concentration of extract without the growth of microorganisms. The MBC/MIC ratios were calculated to determine the bactericidal or bacteriostatic effect of the assayed extract. Vancomycin, clarithromycin, ciprofloxacin and nystatin were used as the reference drugs appropriate for different group of microorganisms.
The experiments were repeated in triplicate. Representative data are presented.

Urease Inhibitory Assay
In short, H. pylori were incubated for 72 h in the MH broth with 7% of horse serum (Sigma-Merk, Saint Louis, Missouri, USA) in microaerophilic conditions. Bacterial biomass was collected by centrifugation at 4000× g at 4 • C for 10 min, then the cells were dissolved in ice-cold phosphate buffer (pH 7.3) with a protease inhibitor cocktail (Sigma). The urease enzyme was prepared by disturbing H. pylori cells by sonication, followed by centrifugation at 12,000× g at 4 • C for 10 min.
Initial urease inhibitory activity of all the obtained extracts were evaluated at the concentration of 2 mg/mL with the modified Berthelot spectrophotometric method with phenol-hypochlorite reaction at the absorbance of 570 nm. The enzyme reaction was activated in 96-well plates by mixing the appropriate volume of 2% urea, sodium phosphate buffer solution (100 µL), different concentrations (2000-3.9 µg/mL) of propolis extract, and the reaction mixture was incubated for 15 min at 37 • C, then the concentration of ammonia was determined using the Berthelot method. The amount of the ammonia is equivalent to the hydrolysis of urea using the urease enzyme. The experiments were performed in triplicate. Activity of uninhibited urease was chosen as the control activity of 100% [60]. Inhibition rate (%) was calculated following the formula: I% = (1 − average with inhibitors/average activity without inhibitors) × 100%. The IC 50 was expressed as the concentration of inhibitor that decreased urease activity by 50% and calculated by plotting the percent of inhibition using the internet IC 50 Calculator (AAT Bioquest).

Statistical Analysis
Statistical analysis was performed by Statistica 14.0.0.5 software (Tibco Sofware Inc., Palo Alto, CA, USA). Analysis included hierarchical fuzzy clustering trees (dendrogram) from the prepared matrix. It was composed of % of UV chromatograms (280 nm) relatively peak area. Substances about at least 1% of relatively area (in any sample) were used to construct matrix.

Conclusions
The phytochemical profile and activity against 15 microorganisms 70EEP from ten propolis samples collected in Kazakhstan were evaluated. This is the first wider study on Kazakh propolis extracts phytochemical composition and the antimicrobial potential. Tested extracts exhibited good activity against Gram-positive bacteria, fungal species (yeasts) and H. pylori (the only Gram-negative bacterium sensitive to the tested propolis). In addition, bioactivity tests were conducted for urease inhibition. Propolis from Kazakhstan seems to belong to the poplar type, but analysis of the chemical composition showed the presence of polyphenolic compounds from other plant sources (especially aspen) which requires further research. Dependences between their plant origin and activity was ambiguous. This may be caused be specific chemotype of Kazakstani Populus species or presence additional, unknown plant precursors. An attempt of the isolation the active components from tested propolis samples should be performed in the future, in order to study more the origin of propolis and its various biological activities.