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Influence of ecological factors on pyrrolizidine alkaloids accumulation

The article gives results of estimating alkaloids accumulation in the aboveground and underground plant components of family Boraginaceae (Symphytum asperum Lepech., S. caucasicum M. Bieb., Echium vulgare L., Pulmonaria mollis Wulfen ex Hornem.) depending on the ecological conditions of their habitat. Alkaloids content in the aboveground phytomass decreases in the row S. asperum > S. caucasicum > E. vulgare, and in the underground one – S. caucasicum > S. asperum > E. vulgare > P. mollis. At the end of vegetation there is an increase in alkaloids accumulation in roots and decrease in stems and leaves. Rise in air temperature and decrease in precipitation increases alkaloids accumulation in roots and scions. The level of correlation between alkaloids accumulation and microelements content in soil varies from high (Cu, Zn, Mo) to mean (Mn, Pb). The revealed regularities are recommended to discover cenopopulations of S. caucasicum, S. asperum, E. vulgare and P. mollis with maximal accumulation of pyrrolizidine alkaloids or the forecast of their accumulation in certain sections.


Introduction
Alkaloids accumulation in plant parts depends on different ecological factors (sunlight intensity, water deficit, high air temperature and relative low moisture, increase of nitrogen and calcium in soil, soil salinification, soil contamination with heavy metals) [1][2][3]. The greatest alkaloidness is in aboveground parts of the herbaceous plants, it occurs during the blooming period and in rootstocksduring the decline of the aboveground part [4]. When there is a sharp deficit or overplus of mineral elements alkaloid accumulation decreases, which is the base for the universal M-shaped dependence of alkaloids formation and accumulation in plants on their provision with mineral elements [5].
By chemical composition alkaloids of Boraginaceae family refer to the pyrrolizidine group. Toxicity of pyrrolizidine alkaloids is based on their ability to alkylate DNA, cause mutations and oncological diseases [6]. The most toxic pyrrolizidine alkaloids are lasiocarpine, ehimidin and symphytin. The content of pyrrolizidine alkaloids in the plants of Boraginaceae family varies from 0.04 to 0.6 % [7].
Wide spread occurrence of borage family in Russia, including the North Caucasus, application of some parts in medicine necessitates alkaloids content control in different parts of plants. As there is little information on the influence of ecological factors on the pyrrolizidine alkaloids accumulation and their content in some species of Boraginaceae family the purpose of the study was estimation of the level of alkaloid accumulation in aboveground and underground parts of prickly comfrey

Methods and materials
The study was conducted in the Kabardino-Balkarian Republic during 2017-2019. Plant samples (10-12 pieces in every section) were picked up during the blooming and fruit-bearing periods. The collected material was broken up into the rootstocks with roots and stems with leaves. The material was rinsed and dried at the temperature 25-30 °С and crushed.
Alkaloids content was determined by the weight method based on the alkaloids quantitative precipitation with silicotungstic acid solution with the formation of complex salts. The extract was condensed in vacuum at temperature not higher than 30-40 °С, in order not to destroy alkaloids. The analytic repeatability is three-stage.
Soil samples were selected in the area of species habitat from the upper layers of the soil (0-20 сm). The content of heavy metals in active forms (Mn, Cu, Zn, Mo, Pb) in the soil samples has been determined with the atomic absorption method with electro-thermal atomization.
The statistical treatment of the research findings included determination of correlation coefficients (r) and variation coefficients (V).

Results
Most habitats of the plants are in steppes (150-450 m below the sea level) and submontane (500-900 m below the sea level) in Kabardino-Balkaria zones. Some parts are limited to the mountain zone -Gill-Su area 2380 m below the sea level), Tashly-Tala village (1120 m below the sea level), lower terrace of the tailing pit of Tyrnyauz tungsten-molybdenum industrial plant (1128 m below the sea level).
Soil conditions of the plants habitats also differ. The top soil of the flat part in Kabardino-Balkaria contains liver-colored, meadow, meadow-chernozemic (humusness is 3-4 %), chernozemic (humusness is 5-10 %) and alluvium soils. The main soil types in submontane and mountain parts are grey and reddish forest soils (humusness is 2.8-3.9 %). Section no. 14 is of special importance among the studied ecotopes. It is a neo landscape of the career-stoker type, a tailing pit terrace of Tyrnyauz tungsten-molybdenum industrial plant. Throughout the period of the enterprise work 80 mln m 3 of cleaning rejects wastes have been accumulated containing tungsten, molybdenum and copper-bismuthic concentrates, which are high toxic and have extended solubility in ground waters.
Active forms content of Cu, Mn, Zn, Mo and Pb in sections 1-4 (P. mollis) was 0.   Alkaloids content in roots of S. caucasicum, S. asperum and P. mollis exceeds this figure in the aboveground part both during the blooming and fruit-bearing periods. At the end of vegetation there is an increase in the total alkaloids content in roots (by 21.0-29.2 %) and decrease in stems with leaves (by 7.2-12.8 %). In Е. vulgare plants there is more intensive alkaloid accumulation in the aboveground part. During the fruit-bearing period the alkaloid content in stems with leaves decreases by 11.6 %, and in roots increases by 20.0 %. Alkaloid content in the aboveground phytomass in S. asperum, Е. vulgare and P. mollis has mild, and in S. caucasicum -a noticeable variability. Alkaloids content in roots varies little.
Between alcaloids accumulation in spcions, temperature and total precipitation there is average (r=0.53…0.55) and high negative (r=-0.81…-0.83) correlations correspondingly. Correlation between the alkaloid content in the underground phytomass and temperature is weak (r=0.39…0.41), and percipitation is mean negative (r=-0.53…-0.55). On the whole, high air temperature and decrease in percipitation results in alkaloid accumulation increase in roots and scions, that proves the importance of alkaloids in adaptation of plants to climate stress-factors. Temperature and air humidity have more significant influence on alkaloids content in the aboveground parts of plants, than in underground ones, which has been proved by some researches. [1,4].
Altitude above the sea level correlates weakly with the alkaloids content in scinos and roots (r=0.16), which is explained by the position of the most studied ecotopes below the area level with the predominant content of the short-wave radiation in the sunlight [8].
The correlation between alkaloids accumulation in the underground phytomass and metal active forms content in soil decreases in the row Cu˃Zn˃Mn˃Mo˃Pb ( fig. 3), and in the aboveground -Cu˃Zn˃Mo=Pb˃Mn ( fig. 4).  Revealed regularities fit data on the genetic determinacy of bounds between alkaloids and microelements [11], and prove the role of pyrrolizidine alkaloids in plants adaptation to negative ecological factors [4,12].
Most pyrrolizidine alkaloids are highly bioactive substances, but also they have high hepatotoxicity, cancerogenic and mutation effect. There are pyrrolizidine alkaloids, used in medicine. Most important of them are platyfyllin and sarratsyn, which have cholinolytic and relaxing effect and are widely used during spasticity of unstriated muscles of abdominal cavity organs, bronchial asthma, arterial hypertension.
Alkaloids cinoglossin, consolidin and lasiocarpine can cause paralytic of the central nervous system, as they determine partial block of ganglies and disturb impulse coduction to cross-striated muscles. Alkaloid lasiocarpine intoxicates the organism, causes necrosis, fat dystrophy and liver pulp cirrhosis. Symphytine induces liver cancer development. Lasiocarpine and cynoglosin are mutagenic poisons and cause mutagenic malformations. Heliotrin, viridiflorin, heliosupine and laserocarpine are hepatotoxic poisons, and though they have high selective anticancer activity in the experiment they are not used for medical purposes due to high hepatotoxicity and cancinoginecity [17]. In most countries plants which contain pyrrolizidine alkaloids, are forbiden or limited to use. Taking into account that pyrrolizidine alkaloids are a natural component of a range of plants used in medicine and can be used as a part of a food chain, Committee on herbal remedy (CHR, 2014) recommended a maximum permissable daily consumption of pyrrolizidine alkaloids not more than 0.007 mkg/kg of body weight (on average 0.35 mkg/daily for grown-ups and 0.14 mkg/kg for children).

Conclusion
The studied plant species of Boraginaceae family accumulate alkaloids weakly (˂1%). Alkaloids content in the aboveground phytomass decreases in the row S. asperum ˃ S. caucasicum ˃ Е. vulgare, and in underground -S. caucasicum ˃ S. asperum ˃ Е. vulgare ˃ P. mollis. The level of alkaloids accumulation in rootstocks and roots of S. caucasicum and S. asperum exceeds the one in scions; at the end of vegetation there is an increase of total alkaloids content in roots and decrease in stems and leaves. In Е. vulgare plants the alkaloids content is higher in the aboveground part; during the fruitbearing period alkaloid accumulation decreases in stems with leaves, and increases in roots. Alkaloids content in the aboveground phytomass of S. asperum, Е. vulgare is of mean variability and in S. caucasicum is of high variability. In the underground phytomass of the studied species the level of alkaloid accumulation varies little. The relation of alkaloid accumulation in scions and climatic factors is mean or high, and in roots it is weak or mean. The altitude above the sea level has no significant effect on the alkaloids accumulation in the phytomass of the studied plant species. The degree of relation between alkaloids accumulation and microelements content in soil varies from high (Cu, Zn, Mo) to mean (Mn, Pb). The received data evidence the necessity to take into account ecological factors (air temperature, amount of precipitations, soil contamination with heavy metals) when picking plants for medical purposes. The revealed regularities can be recommended for determining cenopopulations of S. caucasicum, S. asperum, Е. vulgare and P. mollis with maximum content of pyrrolizidine alkaloids and the forecast of their content in certain sections.