Chemical composition and biological activity of essential oil of Nepeta pannonica

Search for new sources of biologically active substances from plants of local flora is a promising area of modern phytochemical science. The article examines the composition of essential oil samples obtained from Nepeta pannonica, growing in the Karaganda region with the use of gas-chromatography-mass spectrometry method. The differences in the chemical composition of the oil depending by the plant organs have been identified. The main component of essential oil is nepetalactone. For the analysis, a unified method for determining the component composition of essential oils, as well as an Agilent Technologies 7890A chromatograph system with a 5975C inert MSD mass spectrometric detector were used. According to the data, the following substances were identified in the essential oils of the plant — 1,8-cineole, nepetalactone, germacrene D, screening of essential oil of Nepeta pannonica for antimicrobial and analgesic activity.

Introduction

Genus Nepeta L. (family Lamiaceae) comprises of 279 species; among them in Kazakhstan there are 16 species [1]. Many species belonging to the genus Nepeta have traditionally been used as biologically active agents for the treatment of many diseases. Pharmacological effects of plants include antimicrobial, antioxidant, anti-inflammatory, sedative, cholesterol-lowering, anti-asthmatic, diuretic, sweating, antipyretic, glycogenic, and other properties [2–9].

All of these effects are related to a certain chemical composition. Plants of the genus Nepeta contain unsaturated lactones called non-tetolactones. Depending on the content of this compound, genus Nepeta species can be divided into two groups, one containing nepetolactone (and isomers thereof) with a relatively high content and a second group with low nepetalactone content and major components such as 1,8-cyneol, β-caryophyllene, caryophyllene oxide, β-farnesene, α-citral, β-citronellol [10–11].

Essential oil from Nepeta species is characterized by presence of sesquiterpene lactones and their deri- vates, such as germacrenes, caryophyllenes, caryophyllene oxide [2, 10–11]. Many substances contained in the essential oil of Nepeta have pronounced biological activity [12–14].

The prospects for the use of Nepeta set us the task of studying the chemical composition of the essential oil of Nepeta pannonica L., growing in the Central Kazakhstan, and determining its biological activity.

Experimental

N. pannonica raw material was collected in June, 2020; phase flowering, Spassky hills, Abay district of the Karaganda region (N 49°32′19″; E 73°16′33″). Raw material was dried according to plant drying rules.

Essential oils of aboveground parts (leaves, shoots and inflorescences) were extracted by hydrodistillation method for 3 hours using a Clevenger apparatus.

To study the composition of the essential oil, a chromatograph-mass spectrometry method was used with the help of an Agilent Technologies 7890A Gas Chromatograph with a quadrupole mass spectrometer MSD 5975 C as a detector. The capillary column HP-5MS had a size of 30 m x 0.25 mm (film thickness 0.25 μm). Evaporator temperature was 230 ºC. The gas chromatographic column was kept at 40 °C for 10 minutes; with temperature programming up to 240 °C at a temperature change rate of 2 °C/min, and then held in isothermal mode for 20 minutes. Sample injection mode was flow division 100:1. Sample volume was 0.2 μl. The conditions for recording mass spectra are 70 eV, the mass range is m/z 10–360. For data processing, the MSD ChemStation software, supplied by Agilent Technologies, was used in combination with AMDIS 32 and NIST 2017.

The analgesic activity of the essential oil of N. pannonica was studied on an experimental model of chemical irritation of the peritoneum, induced by the introduction of acetic acid in white barren mice. The crusts were induced by intra peritoneal administration of 0.75 % aqueous acetic acid solution at a dose of 1 ml per 100 g of animal body weight. The essential oil was administered by intra-gastric for 30 minutes prior to administration of acetic acid. The number of crusts was calculated in 20 minutes after intra-peritoneal administration of acetic acid for 30 minutes. An essential oil in the form of starch mucus was administered by intra-gastric at a dose of 25 mg/kg by means of a special metal probe for 30 minutes prior to administration of acetic acid. A decrease in the amount of crusts in animals compared to the control group served as an indicator of the analgesic activity of the test substances. As a comparative preparation, sodium diclophenac was used in its effective dose of 8 mg/kg (YeD50 = 8 mg/kg). Control animals received an equivalent amount of starch mucus. Analgesic activity was expressed as a percentage of the reduction in the number of acetic crusts in experimental rats compared to controls [15].

Antimicrobial activity of a sample of essential oil of N. pannonica was studied on reference test microorganisms: Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and to yeast fungus Сandida albicans ATCC 10231 method agar diffusion (holes). Comparative preparations are benzyl penicillin sodium salt, gentamicine for bacteria and nystatin for Candida albicans. For the study, pure cultures of test strains were taken, which were previously grown on a liquid medium pH 7.3 ± 0.2 at a temperature from 30 to 37 ºC; for 24–48 hours on a beveled meat-peptone agar.

A standard bacterial suspension was prepared by diluting a 1:1000 culture in sterile 0.9 % sodium chloride isotonic solution. The corresponding bacterial suspension was added 1.0 ml each to dishes with appropriate elective, nutritional media for the test strains under study and seeded by a “continuous lawn” method. After drying, 6.0 mm wells were formed on the surface of the agar, into which 20 μl of the test sample was added. In the control, water for injection was used to dilute samples in equivalent amounts. The crops were incubated at 370 ºC for 24 hours for the bacterium and at 300 ºC for 48 hours for the yeast fungus Candida albicans [15, 16].

The antimicrobial activity of the sample was estimated by the diameter of the growth delay zones of test strains (mm) around the hole: i) the absence of a growth delay zone — the test culture is not sensitive to this concentration of the sample; ii) diameter of growth delay zones is less than 10 mm and continuous growth in the dish was evaluated as an absence of antibacterial activity; iii) 10–15 mm — weak activity; iv) 15– 20 mm — moderately expressed activity; v) over 20 mm — pronounced activity. The essential oil of N. pan- nonica was tested in three parallel runs.

Statistical processing of the results was carried out using the software package Statistica 8.0. Differences at the achieved significance level p < 0.05 were considered reliable.

Results and Discussion

Earlier, in the composition of the essential oil of N. pannonica, 36 components were identified, more of them from light fractions of monoterpenes and their oxidized forms. The major components in the essential are 1,8-cineole and γ-cadinene. The amount of eucalyptol was 28.9 %, nepetalactone was 14.3 % [14].

Other authors investigated the above-ground part of N. pannonica, which was collected in the Aksai gorge of the Transili Alatau (Almaty region) in 2000. The yield of essential oil was 0.2 %. The essential oil composition was examined by chromatograph-mass spectrometry. It was identified 92 components; among them nepetalactone — 41.5 %, 1,8-cineole — 12.2 %, germacrene D — 6.3 %, caryophyllene oxide — 5.2 %, pulegon — 2.9 %, α-terpineol — 1.6 %, and β-terpineol — 1.0 % [15].

The essential oil that we have isolated from N. pannonica is a light yellow moving liquid with a pleasant smell. The yield of essential oil is 0.35 % (Tab. 1).

In the essential oil isolated from the aboveground part of N. pannonica, 50 components were identified by the GC-MS method, the main ones being 1,8-cineole (11.77 %) and (4aR,7S,7aS)-nepetalactone (18.75 %). The main sesquiterpenoids are represented by caryophyllene (3.53 %) and germacrene D (5.21 %). We have determined the component composition of essential oils depending on the plant organs (Tab. 2).

Table 1

Chemical compositions of essential oil of Nepeta pannonica

Table 2

According to GC-MS, the following substances are determined in essential oils of plant flowers — (4aR,7S,7aS)-nepetalactone (46.55 %), germacrene D (10.69 %). The essential oils of plant leaves contained (4aR,7S,7aS)-nepetalactone (18.0), 1.8-cyneol (13 %), caryophyllene (5.23 %) and its oxide (11 %), ger- macrene D (6.95 %).

The main components of essential oil of Nepeta pannonica depending on the plant organs

The results of the analysis of the antimicrobial activity of N. pannonica essential oil sample by diffusion into agar are shown in Tables 3, 4.

Antimicrobial activity of Nepeta pannonica essential oil

The screening of Nepeta pannonica essential oil for analgesic activity

Table 3

Note: validity of differences p < 0.05 compared to comparison group.

Table 4

Note: validity of differences p < 0.05 compared to comparison group.

An antimicrobial study found that an essential oil sample exhibited moderate antimicrobial activity against the gram-positive test strains Staphylococcus aureus ATCC 6538 and Bacillus subtilis ATCC 6633, and also illustrated poor antibacterial activity against the gram-negative test strain Escherichia coli ATCC 25922. N. pannonica essential oil shows antifungal activity against yeast fungus Сandida albicans АТСС 10231.

As a result of the study, it was determined that a sample of essential oil of N. pannonica at a dose of 25 mg/g demonstrated analgesic activity in a model of chemical irritation of the peritoneum, illustrating a significant decrease in the development of experimental bark in rats by 53.3 %, respectively, in comparison with the control.

Conclusions

The work presented the result of study of N. pannonica essential oil, extracted by hydro-distillation method. The essential oil is a light yellow moving liquid with a pleasant odor; the yield was 0.35 %. For the first time, the GC-MS method determined the component composition of N. pannonica growing in the Karaganda region (the Central Kazakhstan). 1,8-cyneol (11.77 %), (4aR,7S,7aS)-nepetalactone (18.75 %), caryophyllene (3.53 %), germacrene D (5.21 %) are characterized as the main components.

Screening for antimicrobial and analgesic activity of N. pannonica essential oil showed the indicated antimicrobial activity against gram-positive test strains of Staphylococcus aureus ATCC 6538 and Bacillus subtilis ATCC 6633, and yeast fungus Сandida albicans АТСС 10231, and also demonstrated poor antibacterial activity against the gram-negative test strain Escherichia coli ATCC 25922.

The analgesic activity of N. pannonica essential oil is comparable to the diclofenac sodium drug.

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