Investigation of CO2 Extract of Portulaca oleracea for Antioxidant Activity from Raw Material Cultivated in Kazakhstan

Medicinal plants remain as an important resource in the fight against many diseases, especially in developing countries. Antioxidants are substances capable of delaying, retarding, and preventing the oxidation of lipids or substances that delay or prevent free radical reactions during lipid oxidation. Natural antioxidants such as ascorbic acid, tocopherol, phenolic compounds, and flavonoids are a safe alternative to chemical antioxidants. In present work, results of antioxidant activity of raw materials from the cultivated plant Portulaca oleracea are presented. The extraction time was optimized to 780 minutes; the yield of extractive substances was 1.25% in the production of CO2 extract under subcritical conditions. For the first time, the antioxidant activity of Portulaca oleracea CO2 extract was determined by the amperometric method. Gas chromatography-mass spectrometry (GC-MS) chemical analysis of Portulaca oleracea CO2 extract dissolved in hexane revealed 37 components, including a complex mixture of aldehydes, alkanes, alkenes, esters, diterpenes, steroids, vitamin E, and carbohydrates. The investigation results showed that the Portulaca oleracea CO2 extract was promising for pharmaceutical, cosmetic, and food industries and had great potential for the prevention and treatment of diseases caused by oxidative stress.


Introduction
In the fight against diseases, medicinal plants still remain their importance and are a promising source of medicines, especially in developing countries [1]. About 3.4 billion of people use herbal medicines. Natural products were the integral parts of the ancient system of traditional medicine. According to the World Health Organization (WHO), medicinal plant is any plant which contains substances used for therapeutic purposes [2,3].
Natural antioxidants are the interest for medicine among different biological activities. Antioxidants are important substances produced in the organisms to suppress oxidative stress. ey can be divided into enzymatic and nonenzymatic ones. Oxidative stress is associated with a large number of lifestyle-related diseases, such as cardiovascular disease, cancer, diabetes, and aging. Excessive oxidative stress can lead to oxidation of biomolecules, which is accompanied by cell damage and intervenes in the pathogenesis of many human diseases [4].
Antioxidants are able to interact with free radicals to stop the chain reaction without damaging vital molecules [5].
ese are compounds capable of delaying, retarding, and preventing the oxidation of lipids or substances that delay or prevent free radical reactions during lipid oxidation. Natural antioxidants such as ascorbic acid, tocopherol, phenolic compounds, and flavonoids are a safe alternative to chemical antioxidants [6,7].
Currently, there are different methods for evaluating the antioxidant activity of compounds. So, electrochemical and spectrometric methods are widely used, as they are characterized by high sensitivity and the ability to analyze quickly. When using amperometric detection, compounds containing hydroxyl groups are well oxidized, and the detection level of polyphenols and flavonoids is 10 −9 -10 −12 grams. e amperometric method allows measuring the amount of all antioxidants in the sample, which makes this investigation method more accurate [8][9][10][11][12].
It is noted that the therapeutic effect of extractive preparations does not depend on one active substance, but on the complex of all biologically active substances contained in it, enhancing, slowing down, or changing the type of action of basic substances [13].
is indicates the potential for antioxidant activity. In our study, for the first time, the antioxidant activity of P. oleracea CO 2 extract was determined by the amperometric method.

Plant Material and Portulaca oleracea CO 2 Extract.
Raw material: the above-ground part of the cultivated plant P. oleracea was collected in 2-3 decades of August, 2020, in flowering phase. e place of collecting was N 42°52'07.8"; E 71°20'42.8" (Zhambyl region, Kazakhstan). e plant samples were identified by specialists of the Institute of Botany and Phytointroduction (Almaty city); typical specimen is stored in the herbarium fund of this institute.
Extraction: for the first time, CO 2 extract from the above-ground part of the wild plants of Portulaca oleracea was obtained under precritical conditions, namely, pressure was 45-52 atmospheres, temperature was 19-22°C, extraction time was 540 minutes, and yield was 0.7%, as well as its component composition [18]. e extraction parameters of the cultivated plant P. oleracea were compared, which made it possible to recommend a change in the extraction time. Crushed air-dried raw materials (stems, leaves, and flowers) were extracted under pressure of 45-52 atmospheres, temperature of 19-22°C, extraction time of 780 minutes, and yield of 1.25%. e component composition was determined for the new extract.

e Determination of CO 2 Extract Component Composition of Portulaca oleracea Cultivated Raw
Material. CO 2 extract of Portulaca oleracea and the different fractions from CO 2 extract of Portulaca oleracea were injected using the 10 μL Agilent syringe into the sample injection device of a Gas Chromatograph 7890A (Agilent, USA) coupled with a mass spectrometric detector 5975°C (Agilent, USA) in split mode-10 : 1, injected sample volume-1.0 μl and inlet temperature-250°C. Chromatography was performed using a DB-35MS capillary column with a length of 30 m, an inner diameter of 0.25 mm, and a film thickness of 0.25 μm (Agilent, USA). e carrier gas Helium (>99.995%, Orenburg-Tehgas, Russia) was supplied at a constant rate of 1.0 mL min − 1.
e temperature of the column thermostat was programmed from 50°S (holding time 1 min) to 270°S (holding time 15 min) with a heating rate of 5°S min − 1. e analysis time was 60 minutes. e MSD interface temperature was 320°C, the temperature of the quadrupole was 180°C, and the ion source temperature was 230°C. It is detected in the ionscanning mode in the range of mass numbers m/z 34-750 a.m.u.
Agilent MSD ChemStation software (version 1701EA) was used to control the gas chromatograph system and the system for recording and processing chromatographic data. e data processing included the determination of the retention times of the substance; peak areas as well as the processing of the spectral information obtained using the mass spectrometric detector. Mass spectra were identified applying the Wiley 11th edition and NIST'02 [18].

e Extraction of Biological Active Substances by Fractionation of Portulaca oleracea CO 2 Extract.
Fractions analysis of P. oleracea CO 2 extract was carried out on a chromatograph. Sample preparation: fractionation was carried out on a silica gel column. Name of samples: 1st fraction -hexane, 2nd fraction -dichloromethane, 3rd fraction-ethyl acetate, 4th fraction -methanol. Gas chromatography with mass spectrometric detection (Agilent 7890 A/5975°C) was used as a method of analysis.

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International Journal of Biomaterials CO 2 Extract. e investigation was carried out on the basis of the technique for measuring the total content of fatsoluble antioxidants in food [19], drinks and food products, dietary supplements, and extracts of medicinal plants [20] by the amperometric method using the "Tsvet Yauza 01-AA" developed by "Khimavtomatika," the scientific and production association (Moscow, Russia). Gallic acid was the standard for fat-soluble antioxidants; the range of determination was 0.00125-25 mg of gallic acid/g (%0). Acetone acidified with phosphoric acid was used as an eluent. Eluent preparation and calibration of gallic acid solutions with a mass concentration of 0.1, 0.2, 0.4, 1.0, and 2.0 mg/dm 3 were carried out according to the certified method.

Evaluation of Antioxidant Activity of Portulaca oleracea
Quercetin served as a standard for water-soluble antioxidants; the range of determination was 0.2-4000 mg of quercetin/dm 3 . 70% ethyl alcohol was used as an eluent. Preparation of eluent and calibration solutions of quercetin with a mass concentration of 0.2, 0.5, 1.0, 2.0, and 4.0 mg/ dm 3 were carried out according to the certified method.

Method for Determining Antioxidant Activity by the FRAP
Method. 0.25 ml of 0.2 M phosphate buffer (pH � 6.6) and 0.25 ml of 1% solution of potassium hexacyanoferrate (III) were added to 0.1 ml of the test substances in the concentration range of 0.25, 0.5, 0.75, and 1.0 mg/ml [21,22]. e reaction mixture was incubated for 20 minutes at 50°C; the reaction was stopped by adding 0.25 ml of 10% trichloroacetic acid solution. e mixture was centrifuged for 10 minutes (3000 rpm). e upper layer with a volume of 0.5 ml was mixed with 0.5 ml of distilled water and 0.1 ml of 0.1% FeCl 3 . e optical density was measured at 700 nm. e antioxidant activity (AOA) of the samples was compared with the AOA of ascorbic acid (AA).
Dilution was made at the rate of 1 mg of substance per 1 ml of solvent. Each sample was tested in three parallel runs. It was carried out at a temperature of 20 ± 2°C, natural light period. 2 Extract of Cultivated P. oleracea. Chemical analysis of P. oleracea CO 2 extract dissolved in hexane was carried out by the GC-MS method. irty-seven components were identified, including complex mixture of aldehydes, alkanes, alkenes, esters, diterpenes, steroids, vitamin E, and carbohydrates (Table 1).

Amperometric Method.
Determination of the antioxidants sum composition by the amperometric method is based on measuring the electric current caused by oxidation of the antioxidant molecule on the surface of the working electrode at a certain potential, which is converted into a digital signal. Output signals are displayed on the computer screen as peaks. e magnitude of the electric current depends on the nature and concentration of the test substance, the type and material of the working electrode, and the potential applied to the electrode. e content of antioxidants in the studied samples of the Portulaca plfracea CO 2 extract was calculated in units of the quercetin and gallic acid concentration. e content of water-soluble antioxidants is 35.5385 ± 0.1457 mg/g, and the content of fat-soluble antioxidants is 34.8361 ± 0.0488 mg/g. e reliability of the correlation coefficient was determined for water-soluble antioxidants r xy � + 0.998, p> 99.9% and for fat-soluble antioxidants r xy � + 0.994, p>99.9%. e relationship between concentration and peak area is direct, strong, and reliable, which indicates a high reliability of the approximation.

FRAP Method (Ferric Reducing Antioxidant Power Assay).
e FRAP method (ferric reducing antioxidant power assay) is based on the reduction of Fe 3+ ions to Fe 2+ by antioxidants. e reduction reaction of K 3 [Fe(CN) 6 ] with antioxidants is used, which is accompanied by the formation of a yellow-colored compound, namely, K 4 [Fe(CN) 6 ]. e measurements are based on the ability of antioxidants to suppress the oxidative effect of reaction particles generated in the reaction mixture. Ascorbic acid was used as a reference drug. Samples were tested at concentrations of 0.25, 0.5, 0.75, and 1 mg/ml (Table 6).
Based on the data analysis, it can be seen that the Portulaca oleracea CO 2 extract at concentrations from 0.25 to 1 mg/ml has a low antioxidant activity compared to the standard solution of ascorbic acid.
Earlier, 41-66 components were found, when studying the component composition of the Portulaca oleracea carbon dioxide extract from the raw material of a wild plant. Triterpenoids 6.62%-30.72%, tocopherols 1.46-3.41%, fatty acids 11.31-34.11%, and terpenoids 3.22%-7.07% made up the sum of the main compounds of chromatographic analysis by classes [18]. e component composition of the Portulaca oleracea carbon dioxide extract from the raw material of the cultivated plant included 37 components. e most known pharmacologically bioactive compounds of the therapeutic value are given in Tables 7and 8. Antioxidant properties of Portulaca oleracea are associated with biologically active substances such as gallotannin, omega-3 fatty acids, ascorbic acid, tocopherol, kaempferol, quercetin, and apigenin [29,30].
Using a simple, fast, and affordable single cell electrophoresis method to measure DNA fiber breakage, the results showed that an aqueous extract of Portulaca oleracea, in contrast to ethanol extract, had a high ability to reduce oxidative damage caused by high levels of fat by modulating the activity of antioxidant enzymes [14]. e antioxidant activity of the Portulaca oleracea medicinal plant material was determined at the plant maturation stages. It was determined by the reduction of 1,1diphenyl-2-picrylhydrazyl (DPPH), by reducing properties of iron extracts (FRAP) and by the amount of ascorbic acid. On the basis of the obtained results, it was concluded that the total phenol content and antioxidant activity in mature Portulaca oleracea plants were higher than in immature plant stages [31]. e antioxidant activity of methanol extracts of various parts of the Portulaca oleracea and Portulaca grandiflora plants was studied using DPPH. For the first time, it was found that the Portulaca oleracea root had effective antioxidant activity [32].
According to Naciye Erkan, using TBARS analysis, it was shown that the fraction of Portulaca oleracea extract with the highest total quantitative content of phenolic compounds International Journal of Biomaterials had antioxidant activity with the highest rate of lipid peroxidation suppression [33]. e antioxidant activity of aqueous and ethanol extracts of stems and leaves of the Tunisian species of Portulaca oleracea was determined using free radical discoloration ABTS, the reducing properties of Fe 3+ extracts, and phosphomolybdenum analysis [34].
ere were carried out the study of the antioxidant activity of methanol, ethanol, and aqueous extracts of Portulaca oleracea using such methods as FRAP [31,34], DPPH [31,32], phosphomolybdenum [34], TBARS [33], and single cell electrophoresis [14]. e study of the antioxidant activity of extracts from plant materials is of interest to scientists. e authors Syeda A.M. and Riazunnisa K. report on the determination by gas chromatography-mass spectrometry (GC-MS) of the component composition of aqueous and methanolic extracts of Madagascar periwinkle (Catharanthus roseus) and drumstick tree (Moringa oleifera) and found their antioxidant activity [35]. On the basis of plant extracts, a technology of multifunctional film has been developed [36,37], which has the prospect of application in the pharmaceutical and food industries.

Conclusions
For the first time, there has been revealed the antioxidant activity of Portulaca plfracea CO 2 extract from raw materials cultivated in Kazakhstan. e results of the study included determination of the composition of the sum of antioxidants in the Portulaca plfracea CO 2 extract by the amperometric method. e composition concentration of the combination of fat-soluble and water-soluble antioxidants was established. e investigation results of antioxidant activity by the FRAP method allow us to conclude that the Portulaca oleracea CO 2 extract in concentrations of 0.25-1 mg/ml has an antioxidant activity, which turned out to be lower than that of ascorbic acid, but promising for the pharmaceutical, cosmetic, and food industries.
Data Availability e datasets used and/or analyzed during the current study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.