Аннотация

Artemisia jacutica Drob. is a valuable source of chamazulene, which has anti-inflammatory and antioxidant properties. We experimentally introduced this plant in the climatic conditions of Buryatia and compared the compositions of the essential oils produced from both cultivated and wild plants.
The reserves of A. jacutica and the laboratory/field germination of seeds were assessed by standard methods. Macro- and microscopic features were determined in line with general pharmacopoeia monographs. The composition of the essential oil obtained by hydrodistillation was analyzed by gas chromatography–mass spectrometry. The resulting data were processed by the principal component method. The antiradical activity was measured by the DPPH test.
The reserves of A. jacutica were determined in the Yeravninsky district of Buryatia. The laboratory germination of A. jacutica seeds was 75.00 ± 5.35%, while the field germination was only 11–23%. Planting with seedlings showed a good survival rate of 67–80%. In the first year of cultivation, A. jacutica plants had similar macro- and microscopic features to those of wild plants. The soils from the experimental plots were superior to the soils of A. jacutica’s natural habitat in terms of fertility. The essential oils from cultivated and wild plants contained 51 components. The content of chamazulene, the dominant component, was 59.22–66.60% in the cultivated plants and only 15.98–47.77% in the wild plants. The essential oil of A. jacutica exhibited high antiradical activity (IC₅₀ = 49.47 μL/mL).
The primary introduction of A. jacutica showed good prospects for its cultivation in Buryatia. The macro- and microscopic features and dominant components found in the essential oil of A. jacutica grown on the experimental plots were similar to those found in the wild plants. Two chemotypes of A. jacutica, Yakutian and Buryatian, were identified according to the oil composition, with the chemotypes preserved in the cultivated plants. The oil’s high antiradical activity and a high content of chamazulene make A. jacutica a valuable material for the cosmetic, pharmaceutical, and agricultural industries.

Ключевые слова

Artemisia jacutica Drob., cultivation, laboratory and field germination, essential oil, chamazulene, antiradical activity, chemotype

ВВЕДЕНИЕ

Traditional healing systems (Chinese, Tibetan, Japanese, etc.) have been in the focus of international science in recent years. These systems are based on the use of natural bioactive substances, primarily of plant origin. Scientific advances in this area highlight the need for using secondary metabolites to create effective drugs [1–4]. Russia’s Pharma-2030 Program aims to increase the market of domestic innovative pharmaceutical products, including those based on natural compounds. In addition, the National Technological Initiative’s Healthnet Roadmap intends to involve more regions in cultivating medicinal plants and creating a whole medicinal plant sector.

The Republic of Buryatia is part of the Baikal unique ecosystem with rare and endemic plant species. Among them are the medicinal plants of the Artemisia L. (Asteraceae family) genus used as ethnopharmacological poly-target agents. For example, the antimalarial drug Artemisinin, which is based on Artemisia annua L., exhibits antiparasitic, antitumorous, anti-inflammatory, antioxidant, antiangiogenic, and immunomodulatory effects. Another example is the anticancer drug Arglabin based on Artemisia glabella L. [5].

The aromatic and pharmacological properties of Artemisia plants are largely due to their essential oils based on mono- and sesquiterpenoids. Multiple components of plant essential oils determine their antioxidant and anti-inflammatory properties. For example, the essential oil of Matricaria chamomilla L. contains chamazulene with anti-inflammatory action. In addition, chamazulene or chamazulene-containing oils have an apoptotic effect on the A375 human malignant melanoma cells, as well as strong antioxidant properties and a photoprotective effect [6–8]. Other sources of chamazulene-containing essential oils are the plants that are systematically close to the pharmacopoeial species Artemisia absinthium L., including Artemisia sieversiana L., Artemisia jacutica Drob., Artemisia macrocephala L., and others. Among these species only A. sieversiana and A. jacutica grow within the territory of Buryatia, the latter having the highest content of chamazulene [9].

A. jacutica Drob. is an East Siberian endemic used in Yakut traditional medicine to treat gastrointestinal diseases. In addition, this species has selective antifungal activity and is used for helminthic invasions in cattle. Previously, the essential oil of A. jacutica was shown to exhibit a wound-healing effect on the napalm-caused burn. A. jacutica was first introduced in the Yakutsk Botanical Garden. From 1991 to 1998, scientists studied the agrotechnical methods of growing A. jacutica in the Siberian Botanical Garden [10]. Kucharova et al. were the first to obtain strains of A. jacutica callus cells with stable growth parameters in vitro [11].

We aimed to assess a possibility of introducing A. jacutica in the natural and climatic conditions of Buryatia and to compare the essential oil of cultivated plants with the oil of wild plants.

ОБЪЕКТЫ И МЕТОДЫ ИССЛЕДОВАНИЯ

We studied the aerial parts and seeds of Artemisia jacutica Drob. collected in the flowering and fruiting phases, respectively, in Yeravninsky district of Buryatia in 2018–2019. The voucher samples are kept in the herbarium of the Institute of General and Experimental Biology (A. jacutica – UUH019308).

The yield was determined on specific thickets by the quadrat method. Fifteen plots of 1 m² were evenly distributed over the thickets. The area of the thickets was calculated in m². The method’s error did not exceed 15% [12].

To determine the germination of A. jacutica seeds in the laboratory conditions, we used four samples of 100 seeds. The seeds were spread evenly on moistened filter paper in Petri dishes and germinated at 29–30°С. The filter paper was checked for moisture on a daily basis and, if necessary, wetted with water at room temperature to prevent overwetting. The lids of the Petri dishes were opened for several minutes every day for ventilation. The samples were protected from direct sunlight during seed germination. The germinated seeds were counted during 12 days. The seeds with primary leaves were classified as germinated, whereas hard seeds that had not swelled or changed in appearance were classified as non-germinated.

The macro- and microscopic features of raw materials were determined by standard methods in line with the General Pharmacopoeia Monographs (General Pharmacopoeia Monograph.1.5.3.0003.15 and General Pharmacopoeia Monograph.1.5.1.0002.15).

The essential oil was obtained from A. jacutica’s aerial parts by hydrodistillation according to the GPhA (General Pharmacopoeia Monograph.1.5.3.0010.15) using a modified Clevenger nozzle. The oil’s components were determined by gas chromatography–mass spectrometry (GC-MS) on an Agilent 6890 gas chromatograph (Agilent Technologies, USA) equipped withan HP 5973N mass selective detector (Hewlett-Packard, USA) and an HP-5MS capillary column (30 m×0.25 mm×0.2 μm; Hewlett-Packard) [13].

For visualization, the data on the oil’s composition were processed by the principal component method (PCM analysis, Sirius version 6.0, Pattern Recognition Systems, a/s, Norway).

The antiradical activity of the essential oil was determined by the DPPH test (using 2,2-diphenyl-1-picrylhydrazyl). For this, a solution of DPPH (0.006% in 95% ethanol) was added to A. jacutica oil (3.9–31.25 μL/mL in ethanol) and incubated for 30 min in the dark at room temperature. The antiradical activity (% inhibition) was measured spectrophotometrically on a ClarioStar Plus multimodal plate reader at 517 nm and calculated as:

                             

where А₀ is the absorbance in the control and А₁ is the absorbance of the samples [14, 15].

The IC₅₀ index was determined by regression analysis.

РЕЗУЛЬТАТЫ И ИХ ОБСУЖДЕНИЕ

Determination of Artemisia jacutica Drob. reserves. The reserves of A. jacutica were determined in the vicinity of Shiringa village (Yeravninsky district, Buryatia) in 2019. The plant’s recovery period is 2 years. A thicket of A. jacutica occupies a small area of only 500 m². According to standard estimations, the average mass of A. jacutica collected from one plot was 26.38 g, the dispersion of the result was 209.01 g, and the standard deviation was 3.77 g. Thus, the yield of A. jacutica was estimated as 26.38 ± 3.77 g/m², its biological reserve was 16.92 kg, the operational reserve was 9.46 kg, with a possible annual harvest of 3.15 kg.

Thus, we proposed to introduce A. jacutica into the culture taking into account its limited potential reserves in Buryatia and high medicinal value as a source of bioactive compounds.

Laboratory and field germination. At the first stage, we determined the germination of A. jacutica seeds in the laboratory. The seeds were obtained from wild intact plants collected in the fruiting phase in 2018–2019. The oblong, dark brown achenes were 1.0–1.2 mm long, 0.4–0.5 mm wide, and covered with a golden film. The seeds began to germinate in 2–4 days. The primary introduction of A. jacutica was carried out on two experimental plots: in Oreshkovo settlement, the Republican Ecological and Biological Center (plot No. 1) and in Sotnikovo settlement (plot No. 2), the Republic of Buryatia (Table 1).

As can be seen, the laboratory germination of A. jacutica seeds was 75.00 ± 5.35%, with a standard deviation of 8% [12]. Our results were consistent with the previous studies [16, 17], where this indicator amounted to 70–100% without primary dormancy. The authors classified A. jacutica seeds as slow germination seeds with a maximum number of germinated seeds at the beginning of germination.

Since we did not use fertilizers or artificial irrigation on the experimental plots, the differences between the cultivated and the natural plants were associated with soils. The plots had a chestnut soil type. Nothing was grown on them before the experiments and no chemicals (pesticides) were used. The plots were periodically plowed up to get rid of weeds. The main agrochemical indicators of soil fertility were analyzed by standard methods. The reaction of the soil solution was determined in an aqueous extract. The soil samples were analyzed for carbon and humus contents by the Tyurin spectrophotometric method (The Standard Operating Procedure for Soil Organic Carbon. The Tyurin spectrophotometric method), for total nitrogen by the Kjeldahl method (General Pharmacopoeia Monograph. 1.2.3.0011.15), for mobile phosphorus and potassium by the Chirikov method (State Standard 26204-91), and for exchangeable calcium and magnesium by the complexometric method (trilon method) (State Standard 26487-85). The results are presented in Table 2.

The acidity of the soils from Buryatia’s Yeravninsky district (natural habitat of A. jacutica) and from the experimental plots was close to neutral. However, the pH of the water extract of the experimental soil was in the alkaline region of 7.3–7.6, compared to 6.5 for the soil from the natural habitat. However, the soils from the experimental plots were superior to the soils from the natural habitat in the main indicators of fertility.

As we know, soil formation is largely determined by organic matter. The activity of plants, animals, and microorganisms leads to the accumulation of organic carbon in the form of humus. The processes of humus formation and accumulation are significantly affected by climatic conditions. Humic substances contribute to an optimal soil structure for plants and are an important reserve of ash elements. Thus, they determine a number of soil’s physical and chemical characteristics. Soil organic matter contains nitrogen. The accumulation of nitrogen, along with carbon, is part of soil formation determined by the cycle of matter. Organic nitrogen is taken as total nitrogen in the soil since mineral nitrogen is found in insignificant amounts. Our analysis showed that plot No. 1 had the most fertile soil rich in easily digestible nutrients. Plot No. 2 was only superior in the content of mobile phosphorus P₂O₅.

A. jacutica was planted with seeds and seedlings (Table 1). The seeds were sowed in early June in strips of 100 seeds. Shoots appeared in mid-June. The seedlings (208 plants) were planted in rows (10 plants per 1 m2) with a 50 cm distance between them.

The laboratory germination of A. jacutica seeds reached 75.00 ± 5.35%, while the field germination on both plots was very low, ranging from 11 to 23%. Further planting with seedlings showed a good survival rate: 80% on plot No. 1 and 67% on plot No. 2. The highest germination of seeds and the best survival of seedlings was observed in the plot with more fertile and less alkaline soil (pH 7.3). Thus, planting with seedlings is the best way to cultivate A. jacutica under the natural and climatic conditions of Buryatia.

Macro- and microscopic features of A. jacutica. Macro- and microscopic features were determined for both the wild and the cultivated plants of A. jacutica. We found that the morphological features of the plants did not change during cultivation, so the cultivated plants had most macro- and microscopic features similar to those of the wild plants. A. jacutica stems were under 25 cm long and had whole or leafy tops. Some plants had ribbed stems, simple or branched. Basal and middle stem leaves had long petioles, half the length of the leaf blade, with simple or pinnate ears at the base. The leaves had narrowly linear, or almost filiform, pointed terminal lobes. Dense hairs gave them a gray felt color. The stems were grayish-brown or greenish-gray, and leaves were grayish-green. The plants had a strong, peculiar smell. The water extract had a spicy and bitter taste (Fig. 1).

The microscopic analysis of the leaf revealed slightly sinuous cells of the upper epidermis and strongly sinuous cells of the lower epidermis. Both the upper and the lower epidermis had an anomocytic stomatal apparatus. The hairs were of two types: T-shaped and capitate. The leaves were densely covered with T-shaped thin-walled hairs, consisting of a multicellular stalk and a long transverse cell with narrowed ends. There were also some capitate hairs consisting of a unicellular stalk and a multicellular oblong head. Numerous essential oil glands consisted of 6–8 excretory cells arranged in 2 rows and 3–4 tiers. Above were glands with a septum, covered with a cuticle (Fig. 2).

A. jacutica essential oil composition. Essential oils were isolated from the aerial parts of the cultivated and wild plants of A. jacutica collected in late July of the same year. They were dark blue liquids with a characteristic odor. The oil yield was determined in terms of air-dry raw material. From the cultivated plants (aerial parts), the yield was 0.6 and 0.5% from plot No. 1 and plot No. 2, respectively. From the wild plants, the oil yield was 0.9, 1.4, and 0.7% in the budding, flowering, and fruiting phases, respectively. The composition of essential oils was studied by GC-MS (Table 3).

A total of 51 components were identified in the essential oil samples. A number of components (γ-terpinene, terpineol-4, α-terpineol, α-pinene, 1,8-cineol, β-farnesene, neryl-2-methylbutanoate, neryl-3-methylbutanoate, geranyl- 3-methylbutanoate, γ-curcumene, germacrene D, caryophyllene, chamazulene) were found in the oils from both the cultivated and wild plants at all, or almost all, development phases. Several compounds (β-ionone, α-bergamotene, Δ-amorphene, amorph-4-en-7-ol, and 3,6-dihydrochamazulene) were found only in the oil from the cultivated plants.

The oils from cultivated and wild plants were similar in composition by group. The composition of the oil from wild plants was most diverse in the flowering phase (up to 32 components). In this phase, the oil had the highest content of chamazulene (47.77%), the dominant component, compared to its lowest content in the fruiting phase (15.98%). The oil obtained from wild plants in the budding phase had higher contents of 1,8-cineol (4.97%) and neryl-3-methylbutanoate (13.12%), while in the oil obtained in the fruiting phase, neryl-2-methylbutanoate (10.45%), nerylpentanoate (14.30%), γ-eudesmol (31.66%), and germacren D (2.06%) prevailed. Our analysis showed a high content of geraniol and a wide variety of its derivatives, as well as its isomer nerol. However, neryl isobutanoate and geranyl-2-methylbutanoate were found only in the essential oil from wild plants (Table 3).

The highest content of chamazulene was found in the oil from cultivated plants (59.22 and 66.60% on plots No. 1 and No. 2, respectively), which was higher than its content in the oil from wild plants both in the budding (41.17%) or flowering phase (47.77%). The oil from plants cultivated on plot No. 1 was more diverse (33 components) that the one from plot No. 2 (25 components). Despite the higher survival rate of A. jacutica in the more fertile plot, the highest content of chamazulene was found in the oil from the less fertile plot with a higher content of mobile phosphorus P₂O₅. Apparently, phosphorus increases the activity of enzymes that control the biosynthesis of proazulene substances [18]. The total content of geraniol derivatives and nerol was also higher in the oil from the plants cultivated on plot No. 2 (11.01%), compared to plot No. 1 (9.91%). In the essential oils of wild plants, it varied from 15.11 to 25.68%, depending on the phase of plant development.

We compared the oils from A. jacutica cultivated in Buryatia and in the Siberian Botanical Garden (Tomsk) and found that chamazulene was their main component [10]. However, its content was lower in the plants grown in Tomsk (up to 43.37%), compared to the plants cultivated in Buryatia (up to 66.60%) or wildgrowing plants in Yakutia (up to 45.75%) and Buryatia (up to 47.77%). We analyzed our results and literature data by the principal component method and identified two chemotypes, “Yakutian” and “Buryatian”. The chemotypes were preserved in the cultivated plants (Fig. 3).

The components identified in A. jacutica essential oil can be divided into three groups: 1) Components found in the essential oils of both wild and cultivated plants, regardless of chemotype. This group includes dominant (chamazulene, γ-eudesmol, neryl-2-methylbutanoate, 1,8-cineol) and minor (β-myrcene, α-terpineol, geranyl-3-methylbutanoate, caryophyllene) components. The acyclic sesquiterpenoid nerylpentanoate was found in noticeable amounts only in the wild plants of the Yakutian (up to 14.59%) and Buryatian (up to 14.30%) chemotypes, and in small amounts in the cultivated plants of the Buryatian (0.65%) chemotype.
2) Components found in the essential oils of both wild and cultivated plants of either Yakutian or Buryatian chemotype. For example, the Yakutian plants had a noticeable content of α-bisabolol (6.07–24.75%), geranyl tiglat (0.69–3.05%), and intermediol (0.25–2.78%), while the Buryatian plants contained some compounds that were absent in the Yakutian chemotype, namely γ-terpinene, α-pinene, β-farnesene, γ-curcumene, lavandulyl-hexanoate, and others.
3) Components found in the essential oils from cultivated plants of the Yakutian chemotype and from both wild and cultivated plants of the Buryatian chemotype: terpineol-4, neryl-3-methylbutanoate, germacren D, and seline-4,11-diene. Sesquiterpene hydrocarbon β-selinene (0.21–0.41%) was identified only in the introduced plants of the Yakutian chemotype.

Antiradical activity of A. jacutica essential oil. The antiradical potential of A. jacutica essential oil was determined by the DPPH test (2,2-diphenyl-1-picrylhydrazyl free radical inhibition). We found that the oil exhibited high antiradical activity (IC₅₀ = 49.47 μL/mL) (Fig. 4).

The antiradical potential of A. jacutica oil was higher than that of A. annua – 50.63 μg/mL, A. gmelinii – 2400 μg/mL, or A. alba – 1.50 mg/mL [19–21]. It could be due to synergistic and antagonistic interactions between individual components of essential oil as a complex system. Azulenes, including chamazulene, are known to have significant antioxidant activity [22].

ВЫВОДЫ

The primary introduction of Artemisia jacutica Drob. showed good prospects for cultivating this plant in the natural and climatic conditions of Buryatia. The macro- and microscopic features and dominant components found in the essential oil of A. jacutica grown on the experimental plots were similar to those found in the wild plants. Two chemotypes of A. jacutica, Yakutian and Buryatian, were distinguished according to the oil composition. Notably, the chemotypes were preserved in the cultivated plants. The oil’s high antiradical activity and a high content of chamazulene make A. jacutica a valuable material for the cosmetic, pharmaceutical, and agricultural industries.

Вклад авторов

E.P. Dylenova developed the research concept and design, analyzed the data, and wrote the first draft. S.V. Zhigzhitzhapova collected and analyzed the data and edited the article. D.B. Goncharova collected and analyzed the data. Zh.A. Tykheev edited the article. D.G. Chimitov conducted field work and edited the article. L.D. Radnaeva edited the article.

КОНФЛИКТ ИНТЕРЕСОВ

The authors declare no conflict of interest.

БЛАГОДАРНОСТИ

We thank the Resource Ecological and Biological Center of Buryatia in Ulan-Ude and Svetlana A. Dondokova for proving us with experimental plots and assistance with the introduction.

ФИНАНСИРОВАНИЕ

Our study was conducted within the state assignment given to the Baikal Institute of Nature Management of the Siberian branch of the Russian Academy of Sciences (BINM SB RAS) and the Institute of General and Experimental Biology of the Siberian Branch of the Russian Academy of Sciences (IGEB SB RAS) in line with the work of the Baikal Interregional Scientific and Educational Center using the facilities of the Research Equipment Sharing Center of the BINM SB RAS.

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