Determination of Chemical Composition and Antimicrobial Activity of the CO2 Extract of Eryngium planum L.

The article presents parameters for obtaining a carbon dioxide extract from the subterranean part of Eryngium planum that contains a valuable set of organic substances and has a certain antimicrobial effect. Methods. Raw materials were collected in the Almaty region (Republic of Kazakhstan). The CO2 extract of Eryngium planum herbs was obtained under subcritical conditions. A gas chromatograph with a mass spectrometric detector was used to determine the compositional breakdown of the extract. Antimicrobial activity was determined by two methods: the micromethod of serial dilutions and the disk-diffusion method. Three microbial test strains were used: Staphylococcus aureus ATCC 6538-P, Escherichia coli ATCC 8739, and Candida albicans ATCC 10231. Results. To extract biologically active substances from the subterranean part of Eryngium planum L., we have chosen carbon dioxide extraction technology, a technology for processing carbon dioxide (CO2) raw materials, which allows us to extract various substances in high concentrations. Carbon dioxide extraction technology is an effective and environmentally safe way to isolate various biologically active substances contained in medicinal plant raw materials. In the composition of the CO2 extract of Eryngium planum L. 43 components were identified, the main of which are α-linolenic acid, 8.30%; myristic acid, 6.40%; caryophyllene, 6.92%; spatulous, 6.62%; and other main identified compounds and their percentage. Conclusions. The study showed that the CO2 extract of Eryngium planum L. contains biologically active compounds that have a pronounced antimicrobial effect against clinically significant microorganisms, such as Escherichia coli, Staphylococcus aureus, and Candida albicans.


Introduction
A source of raw materials for the production of efective and safe medicines has been increasing as a late interest of scientifc medicine in medicinal plants. Preparations made of plant raw materials are currently widely used for the treatment and prevention of many diseases [1]. Teir assortment is expanding, and the number of phytopreparations is increasing annually [2]. Te advantages of phytopreparations over synthetic drugs are their mild action and low toxicity.
Te fora of Kazakhstan has great potential as a source of promising medicinal forms. It is known that many promising plant species for medicine are poorly studied from the standpoint of botanical resource studies and pharmacogenetic analysis [3]. Such plants also include species of Eryngium.
Te authors in [22] studied the composition of the essential oil of Eryngium planum. Te analysis of the composition of essential oils showed the oil from diferent parts of Eryngium planum in vivo, and also, in vitro sprouts can be a source of falcarinol and polyacetylenes, which are important compounds that promote health. Falcarinol is a biologically active compound in the group of falcarinol-type polyacetylenes. It shows pronounced cytotoxic activity against human tumor cells in vitro and also has antitumor activity in vivo.
Suciu and Pârvu evaluated the efect of liquid extract (ethanol 70%) of Eryngium planum to reduce acute infammation caused by turpentine oil in rats. Te tested extract showed an anti-infammatory efect due to a decrease in the total number of leukocytes and neutrophil distribution and activity [23].
Oxidative stress plays an important role in the development and progression of many diseases, including neurodegenerative diseases [24]. Te results of epidemiological studies indicate that higher intake of antioxidant compounds is associated with a lower risk of mortality from cancer and coronary heart disease [25].
Studies show the antioxidant potential of methanol and methanol-water extracts of two plant species Eryngium amethystine and Eryngium planum using beta-carotenelinolenic acid analysis. Te results show that the watermethanol extracts of amethystine and planum are more powerful in terms of antioxidant content than methanol extracts. It was also found that plant extracts exhibit a stronger antioxidant efect than similar extracts for E. amethystine [25].
Tus, extracts of the subterranean part of Eryngium planum L. have a wide range of pharmacological properties due to the content of various groups of biologically active compounds. Terefore, Eryngium planum L. is very promising for further phytochemical study of its composition and therapeutic efects.
Currently, the technology of extraction of vegetable raw materials with compressed and liquefed gases has been proposed and is actively developing. Liquefed CO 2 is used to isolate essential and fatty hydrophobic substances. Hydrophilic substances are well extracted by liquefed gas, having high dielectric conductivity. When extracted with liquefed gas, the extractant evaporates in a high-pressure stream and extractive substances remain in pure form [26,27]. Te tendency to use liquefed and compressed gases and, in particular, carbon dioxide for these purposes, the extraction process can be carried out under pre-and supercritical conditions [27].
Supercritical fuid extraction with carbon dioxide has some minor disadvantages. One of them is the extraction plant's high engineering requirements and investment costs due to high pressure requirements.
Advantages of CO 2 -extraction under subcritical conditions compared to supercritical are primarily due to the fact that this process is highly cost-efective, more technologically advanced, allows processing not only high-quality raw materials, but also production waste in order to extract the main components from them [28,29].
In this study, we obtained a thick CO 2 extract under subcritical conditions from the subterranean part of Eryngium planum and studied the compositional breakdown and its antimicrobial activity against pathogenic microorganisms for the frst time. Te collected vegetable raw materials were naturally dried in the shade and in a well-ventilated room at a temperature of +25 ± 5°. Te moisture content of vegetable raw materials did not exceed 10%. Te dried vegetable raw materials were crushed using a KDU-2 crusher. Te particle size of vegetable raw materials was 1-3 mm.

Materials and Methods
Te extraction mass of medicinal plant raw materials was 1800 g, of which 30 g of extract was obtained; the yield was 1.67%. In appearance, a thick CO 2 extract obtained under subcritical conditions from Eryngium planum has a brown color and a specifc smell.

Preparation of Carbon Dioxide Extract.
Te dried subterranean part of Eryngium planum was used as a medicinal plant raw material for the production of the carbon dioxide extract. Te extract was obtained at the production base of Zhanafarm Medicine Production LLP. Te extract was obtained under subcritical conditions on an extraction unit with a volume of 5 liters in accordance with the standard of the enterprise. Liquid carbon dioxide was used as an extractant. Optimal conditions for obtaining the CO 2 extract were maintained as follows: pressure was 40-51 atm, temperature was 18-21°C, and the extraction time was 11 hours.

Determination of the Compositional Breakdown of the
Extract. Te compositional breakdown of the extract for the content of organic substances was determined using a gas chromatograph with a mass spectrometric detector (Agilent 7890A/5975C).
Chromatography conditions were as follows: sample volume 0.2 μl and sample inlet temperature 240°C, without fow separation. Separation was carried out using a capillary chromatographic column DB-35MS (Agilent, USA), with a length of 30 m, an inner diameter of 0.25 mm, and a flm thickness of 0.25 microns at a constant velocity of carrier gas (helium) 1 ml/min. Te chromatography temperature was programmed from 50°C (1 min exposure) to 270°C at a heating rate of 10°C/min (15 min exposure). Detection was carried out in the SCAN mode m/z 34-750. Agilent MSD Chem Station software was used to control the gas chromatography system and record and process results and data. Te Wiley 7th edition and NIST′02 data libraries were used to identify the obtained mass spectra. Data processing included determination of retention times and peak areas, and verifcation of spectral information was obtained using a mass spectrometric detector.

Determination of Antimicrobial Activity.
Te antimicrobial activity of the carbon dioxide extract of Eryngium planum L. was determined by two methods: the serial dilution method and the disk-difusion method [30][31][32].
To study antimicrobial activity, standard test strains of microorganisms were used as follows: Staphylococcus aureus ATCC 6538-P obtained from the Republican Collection of Microorganisms (Nur-Sultan, Kazakhstan) and Candida albicans ATCC 10231 and Escherichia coli ATCC 8739 obtained from the American Collection of Type cultures (ATCC, USA).

Antimicrobial Activity Assay of the Extract by the Serial Dilution Method.
A 96-well plate was used to determine antimicrobial activity. Muller-Hinton nutrient broth (M391) was added to all wells for testing bacteria, and Sabouraud broth was used for testing mushrooms in the amount of 150 ml (from the 1st to the 24th well). Te extract was introduced into the 1st well at a base concentration of 150 μl, and serial dilutions were carried out, which were prepared using a mixture of Muller-Hinton broth/Saburo dextrose broth (150 μl) + test sample (150 ml) from the 1st well. a test tube in the amount of 150 ml into the 2nd test tube, already containing 150 ml of broth. 150 ml of the test sample in the broth was transferred from the 2nd tube to the 3rd tube, which also initially contained 150 ml of broth. Tis procedure was repeated until the required number of dilutions was reached. 150 ml of the mixture was taken from the last hole. Tus, the following dilutions were obtained: 1 : Te concentration of the extract used in in vitro experiments was 66.2 μg/ml.
After a series of dilutions, 20 μl of test strains of microorganisms was added to all test tubes at a concentration of 1.5 × 10 6 CFU/ml ( Figure 1). All samples were incubated at 37 ± 1°C for 18-24 hours. After the incubation time, seeding was carried out on Petri dishes with Muller-Hinton nutrient medium to determine living cells. Te presence of visible growth of microorganisms on the surface of a dense nutrient medium was taken into account in the results. Te minimum bactericidal concentration (MBC) was considered the lowest concentration in a test tube, suppressing the growth of microorganisms. Table 1 shows the labeling of Petri dishes according to dilutions.

Determination of the Antimicrobial Activity of the
Disk-Difusion Method. Te difusion method was carried out by the borehole method. To perform this method, holes with a diameter of 6 mm were made with a sterile cylinder at a distance of 15-20 mm from the edge of the cup and from each other. Te extract of Eryngium planum L. was added to the obtained wells, 80 ml each. Petri dishes were preinoculated with a suspension of test strains at a density of 1.5 × 10 8 CFU/ml. Sterile cotton swabs were used for sowing and were immersed in a suspension of the microorganism, and then, these swabs were lightly pressed against the walls of the tube and hatched in three directions by turning the cup by 60°. As a reference discs with antibiotics were used (Ampicillin and Fluconazole).
After sowing, the cups were placed in a thermostat for incubation for 18-24 hours at 37°C for bacteria.
Te results of the disk-difusion method were taken into account by calculating the diameter of growth retardation/ suppression zones with an accuracy of 1 mm.

Determination of the Compositional Breakdown of the CO 2
Extract. During the study, the phytochemical composition of the CO 2 extract of the subterranean part of Eryngium planum L. was determined under subcritical conditions. 43 components were identifed by chromatography-mass spectrometry, the main of which are α-linolenic acid, myristic acid, caryophyllene, spatulous, and other main identifed compounds, and their percentage is shown in Table 2 and Figure 2.
Te main components found in the CO 2 extract under subcritical conditions were terpenes, sesquiterpenes such as caryophyllene (6.92%), spatulous (6.62%), and phytol (4.01%), and other compounds such as α-linolenic acid (8.30%) and myristic acid (6.40%). Tey indicate signifcant specifc pharmacological activity, in particular antibacterial, anti-infammatory, and antioxidant. We have studied the pharmacological activity of the main components by analyzing the work of foreign scientists. Te nature of the chemical compound and its therapeutic activity are shown in Table 3.
It is worth noting that β-caryophyllene has antibacterial potential. In a study against T strains. reuse, S. aureus. and E. coli, with MIC values (minimum inhibitory concentrations), they were in the range from 3 to 14 microns [49].
Spatulous is one of the main compounds found in our study. It has antimicrobial, antiproliferative, and antiinfammatory efects. In the study of Eugenia calycina leaf essential oil, spatulous showed antimicrobial activity against anaerobic bacteria such as Prevelar nigricans and Porphyrins gingivitis with an MIC (minimum inhibitory concentration) of 100 mcg/ml [51].
A signifcant contribution of our research consists in the identifcation and quantifcation of chemical compounds for the study of plant extracts.

Results of Antimicrobial Activity.
When determining antimicrobial activity by the serial dilution method, antibacterial and fungicidal activity of the CO 2 extract was found against the analyzed strains of S. aureus, E. coli, and C. albicans microorganisms.
To determine antimicrobial activity, the medium and test strains were used as a positive control to confrm growth for each test strain. For each test strain, suitable nutrient broth (Mueller-Hinton broth for bacterial testing or Sabouraud broth for fungal testing) without the test material was used as a negative control.
Te results were detected visually by the presence/ absence of visible growth of microorganisms on the surface of a dense nutrient medium (Figure 3). Te minimum bactericidal concentration (MBC) was considered the lowest concentration, which suppressed growth of microorganisms. Te results of antibacterial activity of the carbon dioxide extract of Eryngium     Table 4.
It can be seen from the presented data that the test sample exhibits antimicrobial activity in relation to the test cultures under study. Te CO 2 Eryngium planum L. extract was active against all tested strains. It has been experimentally shown that the extract of Eryngium planum L. when tested by serial dilutions, it has a bactericidal efect at a concentration of 8.3 mcg/mcl in relation to Staphylococcus aureus ATCC 6538-P; 33.1 mcg/mcl against Escherichia coli ATCC 8739; against yeast fungi 16.55 mcg/ mclCandida albicans ATCC 10231.
Te test sample, carbon dioxide extract of Eryngium planum L. has antibacterial activity against Staphylococcus aureus ATCC 6538-P; against Escherichia coli ATCC 8739; and against yeast-like fungi Candida albicans ATCC 10231 by serial dilution in broth.
Te results of the study on the carbon dioxide extract of Eryngium planum L. by the disk-difusion method are shown in Figure 4 and Table 5.
Ampicillin is a broad-spectrum, beta-lactam antibiotic active against Gram-positive and Gram-negative bacteria, and fuconazole is an antifungal medicine highly active against yeast-like fungi of the genus Candida. Tey were used as reference drugs, ampicillin 10 μg/disc (HI media, India) and fuconazole 10 μg/disc (HI media, India).
Te test sample, a carbon dioxide extract of Eryngium planum, has antibacterial activity against Staphylococcus aureus ATCC 6538-P: its growth retardation zone is 18.67 ± 0.57 mm, which is 1.2 times more efective than control (the antibiotic ampicillin). Against the yeast-like fungusCandida albicans ATCC 10231, the growth retardation zone was 20.3 ± 0.57 mm, while the growth retardation zone of the antifungal drug fuconazole was 14.0 ± 0.0 mm.
Tus, the obtained results of the prototype indicate the efectiveness of the tested extract against Gram-positive bacteria and yeast-like fungi.
Antimicrobial activity of extracts has been described for some Eryngium species [6,[52][53][54]. Results of studies of antimicrobial activity of methanol extracts and fractions of Eryngium planum, E. campestral, and E. maritime by the microdilution method of broth showed that maritime and planum have the highest antimicrobial activity (MIC � 1-2 mg·ml −1 ) against aureus, followed by the saponin fraction of planum (MIC � 2.5 mg·ml −1 ). Te inhibitory efect of methanol extracts of the roots of all the tested species (MIC � 12.5 mg·ml −1 ) and the planum cell suspension culture (MIC � 7.8 mg·ml −1 ) was found against C. albicans [52].
It is worth noting that the chemical composition of the essential oil plant depends on the type, climatic conditions, soil type, harvesting seasons, geographical region, and the extraction process used. Seasonal fuctuations are one of the main factors that afect the composition of essential oils [55,56].
Te composition of essential oil and antimicrobial activity of three other species belonging to the genus Eryngium (creticum, campestris, and trifolium), infusions of which are obtained from the aboveground and root parts. Te antibacterial activity of essential oils was tested by the disk-difusion method against nine clinical strains of methicillin-resistantaureus (MRSA). It has been demonstrated that the essential oil obtained from trifolium, which causes an inhibition zone in the range from  Figure 2: Te analysis chromatogram of the Eryngium planum L. CO 2 extract.

Conclusion
To extract biologically active substances from the subterranean part of Eryngium planum, we have chosen carbon dioxide extraction technology, a technology for processing carbon dioxide (CO 2 ) raw materials, which allows us to extract various substances in high concentrations. Carbon dioxide extraction technology is an efective and environmentally friendly way to isolate various biologically active substances contained in medicinal plant raw materials. Te sterility of extracts, low energy consumption, and relative cheapness of hardware today determine the advantages of subcritical carbon dioxide extraction over traditional methods of extracting substances. Te parameters of the extraction process provide softer and gentler extraction of biologically active substances from plant raw materials, which makes it possible to obtain the substance in native form at the end of this process.
Te chemical composition of the carbon dioxide extract of Eryngium planum obtained under subcritical conditions was studied for the frst time. By chromatography-mass spectrometry, 43 components were identifed, the main of which are α-linolenic acid, 8.30%; myristic acid, 6.40%; caryophyllene, 6.92%; and spatulous, 6.62% Te results of screening for antimicrobial activity showed that the CO 2 extract of Eryngium planum exhibits antimicrobial activity against Gram-positive bacteria and yeast-like fungi, using the serial dilution method and the disk-difusion method.
It has been experimentally found that the carbon dioxide extract of Eryngium planum has a high content of biologically active substances, which shows the relevance of the pharmacological study of the extract in order to use it as a medicinal product.

Data Availability
Te data used to support the fndings of this study are included within the article.

Conflicts of Interest
Te authors declare that there are no conficts of interest.