Component Composition and Antimicrobial Activity of CO2 Extract of Portulaca oleracea, Growing in the Territory of Kazakhstan

In the medicine of many countries, the use of herbal healing agents included a significant contribution to improving human health and well-being. Many antibiotics have been widely used to treat infectious diseases caused by various pathogenic bacteria. However, increased multidrug resistance has led to increased severity of diseases caused by bacterial pathogens. Bacteria remain the main causative agents of diseases that cause human death, even in the present day. This cause prompted scientists to investigate alternative new molecules against bacterial strains. The significant interest for the study is Portulaca oleracea L. (family Portulacaceae), a widespread annual plant used in folk medicine. Thus, the production and study of CO2 extract of Portulaca oleracea is an actual problem. Methods. Raw materials were collected from Almaty and Zhambyl regions (Southeast and South Kazakhstan) in phase flowering. Portulaca oleracea herb's CO2 extract was obtained by subcritical carbon dioxide extraction (installation of carbon dioxide flow-through extraction- 5L). The Wiley 7th edition and NIST'02 library were used to identify the mass spectra obtained. The antimicrobial activity study was conducted by the micromethod of serial dilution and disco-diffuse method. Standard test strains of microorganisms were used: Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6538-P, Candida albicans ATCC 10231, and Escherichia coli ATCC 8739. Results. The use of carbon dioxide extraction (further CO2 extract) is a promising direction of obtaining total medicinal substances containing biologically active substances, from fractions of volatile esters of various composition and functional purpose until a fraction of fatty acids and fat-soluble vitamins. In the current study, we obtained CO2 extract at subcritical conditions from aboveground organs of Portulaca oleracea and investigated the component composition for the first time. From 41 to 66 components were identified in the composition of Portulaca oleracea‘s CO2 extract. Studies of antimicrobial activity showed that CO2 extract of Portulaca oleracea had the expressed effect against clinically significant microorganisms such as Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Candida albicans. Conclusions. This study showed that CO2 extract of Portulaca oleracea's raw material contained biological active compounds exhibiting a significant antimicrobial effect.

In the medicine of many countries, the use of herbal healing agents included a significant contribution to improving human health and well-being. Many antibiotics have been widely used to treat infectious diseases caused by various pathogenic bacteria. However, increased multidrug resistance has led to increased severity of diseases caused by bacterial pathogens. Bacteria remain the main causative agents of diseases that cause human death, even in the present day. is cause prompted scientists to investigate alternative new molecules against bacterial strains. e significant interest for the study is Portulaca oleracea L. (family Portulacaceae), a widespread annual plant used in folk medicine. us, the production and study of CO 2 extract of Portulaca oleracea is an actual problem. Methods. Raw materials were collected from Almaty and Zhambyl regions (Southeast and South Kazakhstan) in phase flowering. Portulaca oleracea herb's CO 2 extract was obtained by subcritical carbon dioxide extraction (installation of carbon dioxide flow-through extraction-5L). e Wiley 7 th edition and NIST'02 library were used to identify the mass spectra obtained. e antimicrobial activity study was conducted by the micromethod of serial dilution and disco-diffuse method. Standard test strains of microorganisms were used: Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6538-P, Candida albicans ATCC 10231, and Escherichia coli ATCC 8739. Results. e use of carbon dioxide extraction (further CO 2 extract) is a promising direction of obtaining total medicinal substances containing biologically active substances, from fractions of volatile esters of various composition and functional purpose until a fraction of fatty acids and fat-soluble vitamins. In the current study, we obtained CO 2 extract at subcritical conditions from aboveground organs of Portulaca oleracea and investigated the component composition for the first time. From 41 to 66 components were identified in the composition of Portulaca oleracea's CO 2 extract. Studies of antimicrobial activity showed that CO 2 extract of Portulaca oleracea had the expressed effect against clinically significant microorganisms such as Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Candida albicans. Conclusions. is study showed that CO 2 extract of Portulaca oleracea's raw material contained biological active compounds exhibiting a significant antimicrobial effect.

Introduction
Plants from ancient times are a natural source of biologically active substances [1]. In the medicine of many countries, the use of herbal healing agents made a significant contribution to improving human health and well-being [2]. e World Health Organization (WHO) made a comprehensive analysis of the role of folk medicine in the world and published the "WHO Strategy in the field of folk medicine for 2014-2023" for integrating folk medicine into national health systems [3]. Medical preparations of plant origin are characterized by relative safety and low toxicity and act comprehensively on the human body, which allows applying them for the prevention and long-term treatment of diseases. Currently, more than 80% of the world's human population depends on herbal preparations for treatment of various human health problems [4].
Many antibiotics have been widely used to treat infectious diseases caused by various pathogenic bacteria. However, increased multidrug resistance has led to increased severity of diseases caused by bacterial pathogens.
us, bacteria remain the main causative agents of diseases that cause human death even in the present day. e use of several antibacterial agents simultaneously (polypragmasy) in higher doses can cause toxicity for humans. is situation prompted scientists to investigate alternative new molecules against bacterial strains [5].
Conducting research on the introduction of plants with healing properties into official medicine is an actual problem; therefore, the use of local vegetative raw materials will increase production volumes and expand the range of medical preparations based on local plants. e significant interest for the study is widespread annual plant Portulaca oleracea L. (family Portulacaceae), used in folk medicine. It vegetates from April to October; blooms from June to August; seeds mature from August to September. is species grows in gardens, on the melon fields, on streets settlements, in weed places, along the sandy coasts of reservoirs, and on roadsides, as a weed plant [6]. In Kazakhstan and CIS countries, it is successfully cultivated as ornamental and food culture [7].
Minh et al. report that the biologically active compounds, namely, flavonoids, alkaloids, fatty acids, terpenoids, sterols, phenolic compounds, proteins, and minerals, are present in Portulaca oleracea herb ethanolic and aqueous extracts [8]. e value of Portulaca oleracea is that it is a source of polyunsaturated fatty acids and antioxidants, which are necessary for maintaining human life [9].
Alcohol and aqueous extracts of Portulaca oleracea's aerial part have a wide range of pharmacological properties, such as antioxidant, neuroprotective, anti-inflammatory, gastroprotective, hypoglycemic, hepatoprotective, antimicrobial, antipyretic, and antipyretic activities due to the content of various groups of biologically active compounds [10]. e authors of [11] studied polysaccharides from Portulaca oleracea, which have an antidiabetic activity, lowering blood glucose levels in alloxan-induced diabetic mice; in addition, the authors of [12] carried out studies where the polysaccharide component from this species exerts a pronounced antitumor effect on in vivo models. e authors of [13,14] present data on the biologically active compounds, namely, homoisoflavonoids portulaconones A-D and new alkaloid operaciamde C isolated from Portulaca oleracea's extract that exhibits cytotoxic activity against four lines of human cancer cells and stem cells derived from human adipose tissue.
Scientific studies carried out in different years confirm the antioxidant activity of Portulaca oleracea's methanol extract with the content of total phenols, flavonoids, carotenoids [15] and the phenolic compounds fraction from crude Portulaca oleracea's extract [16]. e use of different extractants can affect the final content of biologically active compounds; the amount and composition of fatty acids were determined in the petroleum ether extract [17]. e use of carbon dioxide extraction is a promising direction for the production of total medicinal substances containing biologically active compounds, starting from volatile esters, fractions of various compositions, and functional purposes, ending with the fatty acids and fatsoluble vitamins fraction [18]. In this regard, the production and investigation of Portulaca oleracea's CO 2 extract is an urgent problem.
In the current study, we obtained the CO 2 extract in the subcritical conditions from aboveground organs of Portulaca oleracea and studied the component composition and established the antimicrobial activity against pathogenic bacteria for the first time.

Sample Collection.
e raw materials of Portulaca oleracea are collected in the flowering phase in 2-3 decades of August 2018-2019 in the foothill zone of Trans Ili Alatau (Almaty region, Southeast Kazakhstan) and in the floodplain of Talas River (Zhambyl region, South Kazakhstan). e raw material was harvested in dry weather. e drying of raw materials was carried out in a well-ventilated room at a temperature of +25 ± 5°C. e moisture content of the raw material should not exceed 10-12%. Portulaca oleracea's raw material is stored at a temperature of +15°S-25°S and humidity of not more than 65%, in dry, well-ventilated rooms.
e plant samples were identified and transferred for storage to the herbarium fund of the Institute of Botany and 2 e Scientific World Journal Phyto-Introduction (Almaty city). e herbarium code of the sample of Portulaca oleracea is 2421/25, 2421/26.

Obtaining Carbon Dioxide Extract. Portulaca oleracea
herb's CO 2 extract was obtained from the aboveground part of the raw material in a laboratory facility for subcritical carbon dioxide extraction (installation of carbon dioxide flow-through extraction-5L). e optimal conditions for obtaining CO 2 extract were as follows: pressure 45-52, atmosphere, temperature +19-22°C, dynamic extraction time 540 minutes, and raw materials particle size 0.2-0.3 mm; the yield was 0.7%.

Component Composition Determination.
e composition was determined on a gas chromatograph with an Agilent 6890N/5973N mass spectrometric detector. Chromatography conditions were as follows: sample volume 0.2 μl and sample inlet temperature 240°C, without dividing the flow. e separation was carried out using a DB-35MS chromatography capillary column with a length of 30 m, an inner diameter of 0.25 mm, and a film thickness of 0.25 μm at a constant carrier gas (helium) velocity of 1 ml/min. e chromatographic temperature was programmed from 40°C (holding 2 min) to 200°C with a heating rate of 10°C/min (holding 5 min) and up to 300°C with a heating rate of 20°C/ min (holding 10 min). e detection was carried out in the SCANm/z 34-750 mode. Agilent MSD Chem Station software was used to control the gas chromatography system, recording, and processing the results and data.

Component Identification.
e Wiley 7 th edition and NIST'02 library were used to identify the mass spectra obtained. e percentage of components was calculated automatically based on the peak areas of the total ion chromatogram. e components were identified by mass spectra and retention times.  [19,20]. Mueller Hinton broth (for bacterial testing) and Sabouraud broth (for fungal testing) were introduced into the holes in an amount of 50 μl. e extract was added in pure form in a volume of 50 μl to the 1st and 2nd holes; starting from the 2nd hole, serial dilutions were prepared. e medium and test strain whole were used as a positive control to confirm growth for each test strain. A noninoculated hole containing nutrient broth without the test substance was used as a negative control for each test strain.

Antimicrobial Activity
To all holes with dilution and positive control, 10 μl of tested strain of the microorganism was introduced. e samples with bacteria were incubated at 36 ± 1°C for 24 hours. Samples with Candida albicans were incubated at 22 ± 1°C for 48 hours. e results were taken into account visually by the presence/absence of visible growth of test strains on the surface of the dense nutrient medium. e minimum bactericidal concentration (MBC) was considered the lowest concentration that suppressed microorganism growth.

Disco-Diffuse Method.
Suspension of microorganisms at a concentration of 1.5 × 10 8 CFU/ml was seeded with a continuous uniform lawn on the entire surface of the Mueller Hinton agar [21,22]. Candida albicans suspension at a concentration of 7.5 × 10 8 CFU/ml was seeded with a continuous uniform lawn over the entire surface of the Sabouraud agar. It was held for 15 minutes, after which commercial discs, impregnated with the studied concentrations of extract, were applied to the surface of the inoculated culture and dried agar. Samples with bacteria were incubated at 36 ± 1°C for 24 hours and with Candida albicans were incubated at 22 ± 1°C for 48 hours. e results were taken into account by measuring growth suppression zones around the disks.

e Carbon Dioxide Extracts Component Composition.
From 41 to 66 components were identified in the composition of Portulaca oleracea's CO 2 extract (Tables 1-3).

Results of Antimicrobial Activity
Antimicrobial activity was studied on CO 2 extract from the raw material of the Almaty region, Southeast Kazakhstan (2019), since the sum of terpenoids was 18.30% and fatty acids were 34.11%.
When determining the antimicrobial activity by the serial dilution method, the antibacterial and fungicidal activity of Portulaca oleracea's CO 2 extract was established in e Scientific World Journal    [25], which reported that flavonoid apigenin isolated from the ethanol extract of the aboveground part of Portulaca oleracea showed an antibacterial activity on five pathogenic bacterial strains (Pseudomonas aeruginosa, Salmonella typhimurium, Proteus mirabilis, Klebsiella pneumoniae, and Enterobacter aerogenes) in in vitro experiments. Lei et al. [26] provided data on significant antibacterial effects of portulacebroside B, C, and D and portula ceramide isolated from Portulaca oleracea's ethanol extract on enteropathogenic bacteria in in vitro experiments. In article of Syed et al. [27], an antifungal activity was detected in a sample of the plant Portulaca    After studying the antimicrobial effect of Portulaca oleracea's CO 2 extract by the disco-diffuse method, its activity was also established. During interpreting the data, it was conditionally accepted that the diameter of the growth zone delay was over 15 mm-high activity, 10-15 mm-medium activity, and less than 10 mm-low activity (  albicans. Portulaca oleracea's CO 2 extract component composition varied according to the raw materials origin, place, and collection timing, which is explained by the difference in soil, climatic, and weather conditions. e chromatographic analysis sum of the main groups of compounds by classes is presented in Figure 3. 8 e Scientific World Journal antimicrobial effect at MBS (minimum bactericidal concentrations) values below 500 μg/ml. us, according to the results of the study, it was found that Portulaca oleracea's CO 2 extract has a pronounced antimicrobial effect.

Conclusions
e results of the study of the component composition of Portulaca oleracea's CO 2 extract obtained from raw materials of different origins are presented. e obtained extract identified 66 components from raw materials collected in the Zhambyl region and 41 and 50 components from raw materials collected in the Almaty region. e difference between the component compositions is explained by the soil climatic conditions of the regions. e main components in the raw materials are terpenoids, sterols, fatty acids, and tocopherols.
Study of the antimicrobial activity by serial dilution and the disco-diffuse method showed that Portulaca oleracea's CO 2 extract had a significant effect on the following microorganisms: Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Candida albicans.
Data Availability e datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.  e Scientific World Journal 9