Study of Plant Growth and Essential Oil of Nepeta cataria L. in Kazakhstan

: The genus Nepeta exhibits a wide spectrum of biological activity in medicine, agriculture, and the food industry. The purpose of the research is to develop the optimal agricultural technology for plantation cultivation of Nepeta cataria L . with the preservation of useful properties and to increase the content of essential oil in the conditions of Northern Kazakhstan. The objects of research are annual plants N. cataria L. growing on a plantation in the Akmola region. It has been reliably proven that the increase in the growth of N. cataria L., the biomass, and the yield is positively affected by the application of complex fertilizer N 100 P 80 K 120 . The essential oil yield was 0.52% in the experiment without fertilizers and 0.57% in the experiment with fertilizers. The main components of the essential oil of N. cataria L. grown in the experimental plots (plot #1 and plot #2) were 4aα7α7aα-Nepetalactone (76.5612 and 79.3200%), β-caryophyllene (4.0001 and 4.0351%) and caryophyllene oxide (1.9789 and 2.5969%). The remaining components were contained in the essential oil in small quantities. The composition of the essential oil of plants from Kazakhstan was similar to the composition of the essential oil of plants from central Iran, Egypt, and Korea, because the main substance was nepetalactone. The conducted studies prove the success of growing catnip in the conditions of Northern Kazakhstan.


Introduction
The purpose of the research is to develop the optimal agricultural technology for plantation cultivation of N. cataria L. with the preservation of useful properties and to increase the content of essential oil in the conditions of Northern Kazakhstan.
Currently, herbal treatment is of great importance worldwide and because of the profound research, there is a diverse collection of the centuries-old experience growing medicinal plants (Malik, 2014). The genus Nepeta exhibits a wide spectrum of biological activity in medicine, agriculture, and the food industry (Prescott et al., 2011;Tan et al., 2019). Nepeta cataria L. is used for making cheeses, sausages, alcoholic and non-alcoholic drinks as a condiment, it can be used as an ornamental plant (Aćimović et al., 2021). In the near future, flavonoids extracted from N. cataria L. can be used to treat human lung cancer (Fan et al., 2017). The essential oil of N. cataria L. consists of terpenoid hydrocarbons, which act as an antimicrobial, sedative, antipyretic, antiviral, and diuretic substance (Gilani et al., 2009). One of the useful properties of the essential oil of Nepeta species is its repellent and insecticidal activity (Reichert et al., 2019). It is assumed that the antibacterial activity of the essential oil of Nepeta species against a strain of Escherichia coli and Staphylococcus aureus depends on the presence of nepetalactone in the composition. The presence of β-caryophyllene and β-caryophyllene oxide gives the plant antitumor activity, affecting the growth of cancer cells (Fidyt et al., 2016).
N. cataria L. is the introduced species for the conditions of Northern Kazakhstan. Until now, its useful properties have not been used in the pharmaceutical industry of the Republic of Kazakhstan and for this reason, the plant is not grown on plantations of medicinal herbs. According to the literature data, it was revealed that there were no scientific studies of the growth, yield, phytomass, and composition of the essential oil of N. cataria L. in Kazakhstan, which indicates the relevance of our research.

Materials and Methods
The experiments were carried out on a site located in Northern Kazakhstan, Akmola region, Zerendi district. The climate of the region is sharply continental, with frequent droughts, dry winds, and dust storms. Winter is characterized by low temperatures, late spring frosts, and early autumn frosts, which are repeated annually, the average annual temperature is 0.8-2.6C.
The objects of research are the two-year-old plants of N. cataria L. Seeds were sown in containers in April 2021 and kept at room temperature. Young plants were planted in the open ground on May 14, 2021, and covered with material that protected the plants from late spring frosts. Seedlings were planted with a row spacing of 50 cm in length and in between rows of 60 cm in length. Planting of seedlings was carried out on two experimental plots (Plot #1 and Plot #2) with approximately the same chemical composition of the soil. Before planting seedlings, complex fertilizer N100P80K120 was applied to the soil of the experimental plot (Wieteska et al., 2018). Fertilizers were not applied to the soil of the control plot. On the experimental and control plots rectangular lots were developed in three repetitions, on which measurements were carried out. The width of each lot was 1.6 meters (3 rows with edges), the length 17.5 m, and the area of a squared lot = 28 м 2 . There were 100 plants in each measurement of biometric indicators, which was followed by a 3-fold repetition. Measurements of plant height and root length were carried out with a ruler with an accuracy of 0.1 cm. To assess the yield, the entire phytomass of medicinal plants during the flowering period was collected on all lots capturing the area of 1  1 (m), which was weighed on a laboratory balance with an accuracy of ±5 g.
The obtained data were processed by statistical methods using the Tibco Statistica program. Cluster analysis was carried out between the main components of the essential oil of plants from Kazakhstan and other countries to determine the similarity in composition. The significance of the differences between the experimental plants was determined using an analysis of variance.
Essential oil from plants' phytomass was obtained by hydro distillation process from air-dry raw materials for 3 h, using a Clevenger laboratory installation. To study the composition of the essential oil, we used the method of Gas Chromatography-Mass Spectrometry (GC-MS) using an Agilent Technologies 7890 A gas chromatograph with an MSD 5975 C quadrupole mass spectrometer as a detector. The capillary column HP-5MS had a size of 30 m  0.25 mm (0.25 µm film thickness). The temperature of the evaporator is 230C. The gas chromatography column was kept at a temperature of 40C for 10 min; with temperature programming up to 240C with a temperature change rate of 2C/min and then kept in isothermal mode for 20 min. Sample injection mode flow division 100:1. The sample volume is 0.2 µL. Conditions for recording mass spectra 70 eV, mass range m/z 10-360. Data processing was performed using MSD chem station software supplied by Agilent Technologies in combination with AMDIS 32 and NIST 2017.

Results
Soils used in these experiments are typical chernozem with an average content of humus (5.33% on experimental plots and 6.03% on control plots). Humus content in soils was 4.3-5.2% respectively), and the pH scale indicated soil alkalinity (рН respectively 8.07-8.48). There was an acute shortage of easily hydrolyzable nitrogen in the soil, which content was 39.60 mg/kg on experimental plots and 46.41 mg/kg on control plots. The maximum nitrogen content in the soil should be from 50-100 mg/kg. Weather conditions are shown in (Table 1).
In the year of sowing of the cottontail, the air temperature and precipitation were optimal for seedlings. The amount of precipitation at a sufficiently high air temperature had a favorable effect on the emergence of seedlings, survival, and growth of young plants. In the summer months (June, and July of 2021), precipitation fell within 30 mm, but the lack of moisture in plants was compensated by irrigation. The greatest amount of precipitation was observed in August of 2021. The average temperature of the first and second growing seasons was approximately the same (16.2 and 15.9C, respectively). The average amount of precipitation in the first year of plant life was 7.7% higher than in the second year. Consequently, the weather conditions of the two growing seasons were favorable enough for the emergence and growth of young plants.     Table 3A: Raw biomass of stem and root of N. сataria L.
Raw biomass of one plant (g)   Laboratory seed germination was high at 89.9%, along with field germination at 84.1%. It should be noted that the rate of germination of seeds was good, as well as the survival rate of plants in open ground, which amounted to more than 90%. A comparative analysis showed that when fertilizers were applied, the length of the aboveground (by 10.7%) and underground parts (by 15.2%) was greater in the experimental plants (Table 2A-B). The number of lateral shoots of experimental plants is less than that of the control plants, but this difference is insignificant. More abundant flowering was observed in experimental plants, in which the number of inflorescences was higher than in the control plants by 6.3%. The length of the stem is more variable compared to the length of the roots, having a coefficient of variation in the range of 33.9-34.6%, while the length of the roots varies according to the coefficient of variation from 16.5-18.5%. The grass yield index was the most uniform, which varied at a low level (10.3-12.2%).
Without fertilization, the growth yield of plants was 178.8 g/m 2 , with fertilization 201.3 g/m 2 . All quantitative indicators of plant growth and yield in the experiments are significantly different according to student's t-test: Fexs = 17.8> Ftab = 5.14, which confirms the effect of fertilizer application on increasing plant growth of N. cataria L.
Fertilizers had a greater effect on increasing the biomass of the stem; in the experimental plot, the mass of the stem exceeded that of the control plants by 8.8% in the raw form and 8.5% in the dry form (Table 3A-B). The weight of the roots of experimental plants in the dry state exceeded the same indicator of control plants by 12.2%, and in the raw state by 2.1%. The coefficient of variation of the crude and dry phytomass of the aboveground and underground parts varied at a high level (34.8-37.9%). This indicates the heterogeneity of the trait and the different phytomass of plants and that the use of fertilizer had a positive effect on the increase in the mass of medicinal raw materials.
The composition of N. cataria L. essential oil was determined. Light yellow essential oil with a pleasant smell of freshness with a yield of 0.57% in the experimental plot and 0.52% in the control plot. Еighty-nine essential oil components have been isolated and identified, of which 29.4% are monoterpenes and 27.1% are sesquiterpenes. Figure 1 and Table 4 show the components of N. cataria L. essential oil. In total, the presence of 93.2% of the components in the control and 96.9% in the experimental plants was determined. Five components were present in the essential oil in the amount of more than 1%.
The main components of the essential oil of N. cataria L. grown on the control plots and experimental plots were 4aα7α7aα-nepetalactone (76.5612 and 79.3200% respectively), β-caryophyllene (4.0001 and 4.0351%) and caryophyllene oxide (1.9789 and 2.5969%), in addition, more than 1% contained cis β-Ocimene, β-copaene-4α-ol. The percentage composition of the components in the experiment with the application of fertilizers was generally higher than in the experiments without fertilizers.     Fig. 2).  (Patel (Zomorodian (Safaei-(Chalchat (Saeidnia (Zenasni (Kim (Suschke, (Aćimović, (Aćimović, (Aćimović, (Aćimović, (Aćimović, Ahl, et al., et al., Ghomi and Lamy et al., et al., et al., et al., et al., et al., et al., et al., et al., Components Kazakhstan 2018  As a result of the cluster analysis of the components of N. cataria L. essential oil from different countries, it was revealed that all the studied samples were divided into 3 clusters. The essential oil from the plants of central Iran, Egypt, and Korea was closer in composition to the essential oil of plants from Kazakhstan. In plants from these countries, nepetalactone was the main component. The second cluster included plants from Germany (Nerol and 4a,7a,7a, nepetalactone (Z, E-nepetalactone), France (Nerol and Geraniol), Poland (Nerol and Geraniol), Iran (Tehran) (α-citral and Nerol), Italy (Geraniol), Latvia (Geranyl acetate) and Ukraine (Geraniol and Geranial). In this cluster, almost all countries, except Iran, are geographically located in Europe. In the essential oil of plants from these countries, the main components were Geraniol and Nerol. The third cluster includes plants growing in warmer climate countries Iran (Shiraz) (4aaa,7a,7aß nepetalactone), Pakistan (α-Pinene, 11-Dodecenol), USA (Oklahoma) (Geranyl acetate) and Morocco (4aaa,7a,7aß nepetalactone). Cluster analysis showed that the composition of the essential oil of plants from different countries is similar depending on the region of growth, most likely its composition is influenced by weather conditions.

Discussion
N. cataria L. prefers well-lit places and slightly drained alkaline soils (Manju et al., 2019). It was experimentally found that for the germination of Nepeta seeds in conditions of Iran, the best soils are the ones with the light soil texture such as the sandy soils and the rate of seed germination on a certain soil texture depends on the origin of the seeds (Hakimzadeh et al., 2016).
The increase in the yield of medicinal raw materials of Nepeta cataria L. was influenced by mulching crops with flax straw, an additional positive effect was the reduction of weeds on the plantation (Duppong et al., 2004). It was found that with an increase in the level of water stress, the yield of green mass decreased and the content of essential oil, on the contrary, increased. The negative effect of water stress was reduced by spraying plants with salicylic acid which affected the growth of plants (Said-Al Ahl et al., 2016). Although soil salinization mainly affected the yield of Nepeta cataria L., small concentrations of soil salts increased the content of polyphenols, and flavonoids and stimulated antioxidant activity (Lungoci et al., 2022). To increase the yield of a plant of the genus Nepeta, it is necessary to develop not only optimal agrotechnical techniques but also plant breeding methodology (Srivastava et al., 2021).
The composition and main components of Nepeta essential oil are largely influenced by the soil and climatic conditions of the growing region, and its еnvironmental and seasonal changes (Gomes et al., 2020). It is indicated that the low yield of essential oil may be due to the influence of changes in altitude and temperature conditions during the growth period of the plant (Srivastava et al., 2021). In Poland, to increase the essential oil yield, it is recommended to apply fertilizers of the NPK group: 100:80:120 kg/ha. Lack of nitrogen can negatively affect the metabolism of plants leading to a reduction in plant growth. It was found that under greenhouse conditions, the introduction of nitrogen at a concentration of 300 mg/L had an effect on the growth of N. cataria L., which increased the yield of essential oil (Wieteska et al., 2018).
An experiment with potassium humate as a soil conditioner was carried out on three species of Nepeta, including N. cataria L. All experimental plants were found to have an increase in their growth, yield, and essential oil and flavonoid content (Mohamed et al., 2018). This is consistent with our studies, in which N. cataria L. showed an increase in growth and productivity on slightly alkaline soils, where the height of two-yearold plants reached more than 60 cm. The use of complex NPK fertilizer increased almost all quantitative indicators of the growth and development of N. cataria L. The yield of experimental plants was 11.2% higher than that of control plants, which indicates the undoubted benefits of using fertilizers. According to our observations, N. cataria L. does not demand long-term agrotechnical care, it is easy to cultivate and it gives a large yield of its phytomass.
Nepetalactone and its derivatives are the most common essential oil components of most Nepeta species, including N. cataria L. (Zenasni et al., 2008;Safaei-Ghomi et al., 2009;Reichert et al., 2016;Ibrahim et al., 2017;Vukovic et al., 2016;Said-Al Ahl et al., 2016;. This result is the same as in our studies in Kazakhstan when the composition of the essential oil contained more than 70% nonpetalactone with antibacterial activity. If we compare the content of essential oil in the experimental and control plants, it can be seen that the content of 4aa7a7aa-nonpetalactone was 3.5% higher in the experimental samples, and karyophyllene oxide by 23.8%. The yield of essential oil of plants at the experimental site was 8.8% higher, which indicates an improvement in the composition and an increase in the yield of essential oil when growing plants with fertilizer.
Antibacterial activity of Nepeta cataria L. essential oil can be associated not only with the main monoterpinoid component nepetalactone, but also with other monoterpenes, since in the composition of the essential oil of plants from different countries, the main components can be 1,8-cineol, α -pinene, geraniol, and others.
The components that are contained in the essential oil of plants from Kazakhstan more than 1% are β-caryophyllene, Caryophyllene oxide, cis β-Ocimene, β-copaene-4α-ol, the remaining components occupy an insignificant part (less than 1%). The absence of nepetalactone in the essential oil of N. cataria L. was revealed; the main components were citronellol, nerol, geraniol, and geranial (Saeidnia et al., 2008;Gomes et al., 2020), which were not found in our studies. The main components of the essential oil in Nepeta plants were β-caryophyllene and caryophyllene oxide (Suschke et al., 2007;Senatore et al., 2005), which were present in plants from Kazakhstan in a fairly large amount. The literature mentions only one studied species of the genus Nepeta growing in Kazakhstan, that is Nepeta pannonica L. The composition of the essential oil showed that the main component was 1,8-cineol, it contained less amount of nepetalactone two-fold (Kobaisy et al., 2005). In our studies of N. cataria L., 1,8-cineole was not present at all and nepetalactone was the leading component.

Conclusion
It has been reliably proven that the increase in the growth of N. cataria L., the phytomass, and the yield is positively affected by the application of complex fertilizer N100P80K120. The growth yield on the fertilized plots increased by 12% compared to the control plot, and the raw and dry phytomass increased by 8.8 and 8.5%, respectively. Only the number of shoots decreased by 0.9% with the use of complex fertilizer, but this indicator does not play a major role in comparison to the increase in other biometric indicators of the plant that was observed in this study. The essential oil yield was 0.52% (control plots) and 0.57% (experimental plots). It was revealed that the main components of the essential oil of N. cataria L. grown on experimental plots and control plots were 4aα7α7aα-Nepetalactone (76.5612 and 79.3200%), β-caryophyllene (4.0001 and 4.0351%) and caryophyllene oxide (1.9789 and 2.5969%), respectively. The remaining components were contained in the essential oil in small quantities. In total, there were 5 components in the samples, which contained more than 1% of the essential oil. In the control, the content of essential oil components was less than in the experimental plot. The composition of the essential oil of plants from Kazakhstan was similar to the composition of the essential oil of plants from central Iran, Egypt, and Korea, because the main substance was nepetalactone. According to the literature data, catnip grows mainly in warmer temperate regions (Aćimović et al., 2021). But a sufficiently large yield of essential oil and the content of nepetalactone, compared to countries with a warmer climate, indicates the possibility of successful cultivation of catnip in the conditions of Northern Kazakhstan.