Toxic and genotoxic effects of aqueous extracts of Poligonum weyrichii Fr . Schmidt on the Allium test taken as an example

The plant Poligonum weyrichii Fr. Schmidt is a promising plant in Murmansk region because it is a valuable source of flavonoid compounds. The aim of the study is to investigate, using a sensitive and the well-established Allium test, toxic and genotoxic effects of aqueous extracts of inflorescences and leaves of the middle tier, which differ in concentration (20, 50, 80 and 100%). According to the observations, aqueous extracts of inflorescences and leaves of P. weyrichii of 50%, 80% and 100% concentration have a mitodepressive effect on the cells of the root meristem of Allium cepa L., and inhibit the root growth, causing chromosomal abnormalities. The further investigations are necessary on selection of aqueous extracts concentrations of P. weyrichii.


Introduction
The plants containing flavonoids are the promising sources for fitopreparations because of a wide spectrum of their medicinal effect. These valuable capillary strengthening, cholagogue, antiphlogistic, immunomodulatory, diuretic, antimicrobial, anticarcinogenic medications, like many others, are used in medicine. Of special interest is the antioxidant effect of flavonoids, their ability to prevent free radicals from causing much severe pathologies (Siasos et al. 2013). The oxidative stress caused by unfavourable exogenic factors, by activation of the endogenic active forms of oxygen, and by weakening of antioxidant protection of an organism, is currently considered to be an important pathogenetic link in appearance and development of many diseases (Vazhappilly et al., 2019). It can be explained by the key role of redox reactions in the cells of the organism in normal and general pathological processes. Oxidation induced by free radicals, which develops most readily in the membrane lipid phase enriched with unsaturated bonds, is the biochemical basis of the universal mechanism of the cell damage caused by different damaging factors. Genetic and environmental risk factors cause imbalance in the oxidants-antioxidants system, free radicals accumulation, oxidative stress development, and, as a consequence, imbalance in organ and tissues functioning. Flavonoids are a large group of polyphenols, which is synthesized by plants. A group of substances participates in many key processes of plants growth and development; however, flavonoids play the most significant role in the mechanism of non-specific plant adaptation to unfavorable environmental factors (Brunetti et al, 2013). It is possible due to their antioxidant activity.
As membrane mechanisms of damage and adaptation of plants and animals are uniform, plant ergogenic aids are successfully applied in medical practice. However, flavonoid compounds can act as prooxidants. This activity is expressed in much greater oxidation, in the formation of other radicals under redox transformations, which, in the long run, can cause a mutagenic effect. This action depends mainly on solubility, the oxidizing-to-reducing agent ratio in the environment, the presence of metals with variable valence, pH, and many other factors (Decker, 2009).
Flavonoid synthesis in different organs is a common adaptive plant reaction to the effect of damaging factors of the environment.
The climatic conditions of Murmansk region are characterized by a short summer period, a short vegetation period, high humidity and extreme lighting conditions; reverse flipping between polar day and polar night (Marshall et al., 2016).
As the region is located in high latitudes, the geomagnetic field is unstable and cosmic radiation activity is high. Taking into account the data on more intensive synthesis of flavonoids by plants experiencing the effect of the extreme factors of the different nature (Yang et al., 2018), we suppose that the Arctic regions may be an important source of valuable medicinal raw materials.
The increasing incidence of diseases and a complicated course of different pathologies, which are mainly caused by oxidative stress among the residents of Murmansk region in recent years (Statistical Yearbook…, 2019) indicate the urgent need in new sources of plant flavonoids capable of increasing the nonspecific protection of the human organism from physical, chemical and biological effects.
P. weyrichii was introduced in the Kola Peninsula in 1920. This plant was often attributed to Fam. Aconogonon (Meinh.) Rchb. as A. weyrichii (F. Schmidt) H. Hara (Tsvelev, 1987(Tsvelev, , 2012Hassannejad and Ghafarbi, 2017), and recently, based on the molecular-phylogenetic data, it was included into Fam. Koenigia L. (K. weyrichii (F.Schmidt) T. M. Schust. Et Reveal) (Schuster et al., 2015). As this plant is more widely known as P. weyrichii in the Resource Management and Phytochemistry literature, we also use P. weyrichii in this study.
The representatives of this species have successfully naturalized in the local conditions and are widely distributed over the industrial area of the region. P. weyrichii is a perennial herbaceous plant, frost-and cold-resistant, being distinguished for rapid growth and high productivity of the green mass. The leaves and inflorescences of P. weyrichii contain 3, 4% to 5, 6% of flavonoids by weight of dried tissue (Korovkina and Zhirov, 2019), so this plant can be used as a possible source of flavonoid compounds.
The utilization of any plant for medical purposes can lead to negative consequences, so it is necessary to study the ways of producing the flavonoids compounds, their concentrations and toxicity in order to have the reliable data on its safe and efficient application in medicine.
The aim of this study is, using the Allium test, to estimate genotoxicity and toxicity of the aqueous extracts of the inflorescences and leaves of P. weyrichii, of different concentrations.

Materials and methods Plant material
The plant material was the leaves of the middle tier and the inflorescences of naturalized P. weyrichii growing in the protected area of the N.A. Avrorin Polar-Alpine Botanical Garden-Institute of the Kola Science Centre, the Russian Academy of Sciences (PABGI, KSC RAS) located near the town of Kirovsk, Murmansk region (67°36', 33°40'), Russia.
The Kirovsk site (PABGI) is located at about 340m above sea level, near the Khibiny Mountains. The plant material was collected in the flowering season, on 20. 07. 2019, P. weyrichii was identified by experienced biologists working at PABSI, KSC RAS.

The extraction technique
The inflorescences and the leaves of the middle tier of the P. weyrichii were the plant material to be used in the experiment. According to the standards of drying and storage of medicinal plants (Waterhouse, 2001), the plant material was dried, ground into powder, 1mm mesh sieved, additionally dried for 3 h at 60°C to stabilize its mass. To produce the plant aqueous extracts, the ground plant raw materials composed of leaves of the middle tier and inflorescences, was placed into perforated infantry glasses and then into infudirkas heated before in a boiling water bath for 15 minutes, filled with distilled water of room temperature, which was used, taking into account the corresponding water saturation coefficient given in OFS "Determination of water absorption coefficient and consumption coefficient of medicinal plant raw materials", and was drawn on a boiling water bath for 15 minutes.
Then the material was cooled for 45 minutes at room temperature, strained, and distilled water was added to reach the required volume. The aqueous extracts were used to make the Allium test. To make the Allium test, it was necessary for the aqueous extracts of leaves of the middle tier and inflorescences to be watered with distilled water to reach the concentration of 20, 50, 80 and 100 μg ml. Hydrogen peroxide 1% (Akwu et al., 2019) was used as the positive control, distilled water was used as the negative control. To produce alcoholic extracts, the material was drawn in 70% ethanol for 24 h at room temperature; the plant material-to-solvent ratio was 1:10. The alcoholic extracts were used to quantify flavonoids.

Quantifying the flavonoids
The method based on the complexation reaction of flavonoids with aluminum chloride, was applied to determine the total flavonoid content (Belikov and Shraiber, 1970). The 0.05 ml extract was mixed with 0.1 ml of 2% solution of AlCl3 in 96% ethanol, and the volume was adjusted to 2.5 ml with 70% ethanol. Absorbance at 415 nm of the analyzed solutions was measured using a KFK-3-01 "ZOMZ" spectrophotometer. The calibration curve was obtained using the solutions of routine in 70% ethanol-water mixture (100 -1000 μg ml). The total flavonoid complex (TFC) is calculated by the formula W (flavonoids) = (k*A〖_415〗*V1*V2)/(M*V3*10〖^6〗)mq/g, where k -calibration coefficient, A415 -absorbance at 415 nm µg; V1 -extract volume, ml; V2dilution volume, ml; V3 -analyzed sample volume, ml; М -mass of dried plant material, g.

The Allium test
Onion (A. cepa L., 2n=16, fam. Amaryllidaceae), class Kupido, was purchased in the shop. Before the experiment, the bulbs were preserved in a dark cool place for 14 days and then were selected to be of similar diameter, were examined and pilled from old scales.
The experiment was made in accordance with Fiskesjo (Fiskesjo, 1997), with the bulbs being preliminary sprouted in distilled water for 24 h. Then 40 bulbs were selected, 5 bulbs per each concentration and per each control, with roots of 2-3 mm long, which were placed into test tubes filled with aqueous extracts of inflorescences and leaves of P. weyrichii of 20, 50, 80 and 100 % concentrations. The aqueous extracts and controls were changed for new ones once a day, with the roots being cut and placed into ethanol solution (96%) and glacial acetic acid (3:1) for 24 h. The roots were placed into sealed test tubes, in 80% alcohol. In total, it took 96 h to make the experiment in darkness at room temperature, in an encrypted form.
The cytotoxic and genotoxic effects of the plant extracts were analyzed at a microscopic level using only the meristematic part of the A. cepa roots. To prepare the medications, the roots were subject to hydrolysis and simultaneous colouring in ceramic crucibles, in the aceto-orcein solution heated to a boil over the flame of a spirit lamp. Once cooled, the crucibles were left in the dye (Medvedeva et al., 2014) for 24-72 h at temperature of 4 0 С.
Three roots were used per each concentration and per each control, to prepare 3 squashed medications. Calculation was made of 1000 cells, with phases and chromosome aberrations marked, with 40x and 100x amplifications (with immersion) using the "Mikromed-1 microscope, var.1-20". Simultaneously with cells calculation, the photographs were taken with the help of the TOUPCAM 2.0 camera. In total, over 52000 cells were calculated.
To assess the root growth, new bulbs were placed in aqueous extracts of inflorescences and leaves of the similar concentrations and controls for 48 h, and the length of all the roots was measured. In total, 217 roots were measured (Fiskesjo, 1985;Wierzbicka and Antosiewicz, 1988;O'Hare et al., 1995). The mitotic index (MI) was calculated by using the following equations (Bakare et al. 2000): MI= the total number of dividing cells/the total cell number) × 100.

Statistical analysis
Statistical analyses were done using the program Statistica 8.0 and Microsoft Excel. The differences in the mitotic rate between the experimental and control groups (the negative control) were tested applying the Mann-Whitney non-parametric test. The significance level was taken as p≤0.05.

Results and discussion
The study presents the primary estimation which has been for the first time made over the genotoxicity of aqueous extracts of the P. weyrichii inflorescences and leaves because this plant contains a great amount of biologically active compounds, which means that it can be used in the development of medications and biological supplements. The flavonoid content in the inflorescences samples was equal to 5.9% of all the dried tissue (5.9mg ml), and in the leaves samples -4.4% (4.4 mg ml).
A 24-h experiment has revealed the root growth inhibition, root dying the colour of the solution, and root roughening in 20%, 50% and 100 % concentrations of aqueous extracts of inflorescences and 50% and 80% concentrations of aqueous extracts of leaves. The next day, the root growth was observed in 20% concentrations of the inflorescences aqueous extracts and in 50% and 80% concentrations of aqueous extracts of leaves, as well as sediment and slime on the bulb bottoms in 80% and 100% concentrations of aqueous extracts of inflorescences. In 96 h, all the roots died in 100% concentrations of aqueous extracts both of inflorescences and leaves (Fig. 2 a, b) except 20% concentration o aqueous extracts of inflorescences and 20% and 50% concentrations of aqueous extracts of leaves.
To estimate the toxicity, we measured the length of the roots because this is the indicator of the toxicity of the substances tested. This indicator is easily correlated with the microscopic data, and takes no time to be recorded (Fiskesjö, 1985;Sobrero and Ronco, 2004;Konuk et al., 2007).
The concentration-dependent inhibition of the root growth in the aqueous extract of inflorescences was observed after exposure in these extracts for 48 h (Fig.1).
In addition, the roots bending was observed in 20% and 50% concentrations of the inflorescences extracts after a 24 h exposure and in 50% and 80% concentrations of the leaves extracts after a 48-h exposure (Fig. 2 c, b). According to Levan (Levan, 1949), the phenomenon like this is due to the toxic effect of the substances. Of all the mitotic disorders, the most recurrent ones were bridges in telophase and anaphase, chromatin budding in MNs, chromosomes lagging in metaphase, chromosome fragments and their sticking. Also observed were cells-ghosts with damaged chromatin or enucleated cells, cells with apoptotic bodies, giant cells and cells with damaged membranes.
A factual, concentration-dependent, reduction in the MI compared to negative control was observed in the solutions of all the aqueous extracts of leaves and inflorescences (p0, 05) ( Table 1).
There was also the significant reduction in the MI in the aqueous extract of inflorescences compared to the aqueous extract of leaves.  In this study we used the Allium test because it is an express-test, and is effective and sensitive when used in biological monitoring. It allows us to assess the environmental pollution, toxicity of different compounds, particles, physical factors and plant extracts (Levan, 1938 . The Allium test allows us to assess the cyto-and genotoxicity of the factors of different nature, not spending a lot of physical and economical resources (Teixeira et al., 2003), to avoid solving the problems connected with ethical use of plant objects; it provides with large amounts of data and the results are correlated with those obtained on cell lines . It should be also noted that, in comparison with other plant objects used in genotoxicity tests (Smirnova et al., 2012;Bonea and Bonciu, 2017;Daphedar and Taranath, 2018), the chromosomes and cells of A. cepa L. are rather great in size, which makes it easy, using primitive equipment, to count phases and mitotic disorders and assess even some changes in cells (e.g., membrane breakdown).
In testing, a number of parameters are taken into account, which allows us to get a distinct cyto-and genotoxicity pattern. These parameters are as follows: the mitotic index (MI) and a number of changes in genetic material which are classified and in scientific literature into 2 categoriesclastogenic (chromatid fragments, MNs, ring chromosomes, bridges); aneugenic (chromosomes 'sticking', C-mitosis, nucleus buds, (Sharma et al., 1990, Kurás, 2004) giant cells appearance)). These changes are related to disruption of the DNA molecule or chromosomes breakdown, to mitotic spindle breakdown and finalizing the cytokinesis. There is a separate category of turbagenic changes, which include laggard chromosomes, vagrants chromosomes (Bonciu et al., 2018).
The MI is an indicator of cell division, and the index reduction is related to mitodepressive effect of the substances tested (Akinboro and Bakare, 2007; Sharma and Vig, 2012), which was observed in the study when the concentrations of aqueous extracts of leaves and inflorescences increased. It is due to the intervention into the mitotic cycle and indicates the possible cytostatic and cytotoxic effect. The decrease in the mitotic cell activity can be related to inhibition of the DNA synthesis in the S-phase (El-Ghamery et al., 2000) or to blocking in the G2-phase of the cell cycle, which results in finalizing the entry of a cell into mitosis (Bruneri, 1971;Christopher and Kapoor, 1988;Sudhakar et al., 2001).
The similar effect of plant extracts has been already described as a concentration-dependent decrease in the MI in both the studies of the different, potentially medicinal and useful in industry plants, and those which are well known in medicine and production (Oyedare et  concentrations (the roots were long-term hydrolyzed) can be related to a high content of tanning agents, including also tannins contained in a great amount in P. weyrichii. In studying the plant aqueous extracts genotoxicity, the most well-known and often observed are bridges in the anaphase and telophase, lagging chromosomes, chromatin budding or breakdown in the inter-phase, MNs and C-mitosis, as a variant of breakdown or spindle inhibiting in the metaphase and disorder in microtubes functioning alongside with sticking and lagging chromosomes and fragments (Fiskesjo, 1988;Pesnya et al., 2011;Prajitha and Thoppil, 2016;Costalonga et al., 2017: Madić et al., 2017Bibi et al., 2019). In this study we have observed, practically, all types of effects. The breakdowns of chromosomes in meristematic cells in the aqueous extracts of P. weyrichii are seen in Fig. 4, disorders are seen in Fig. 5. The mitotic phases normal in the control (distilled water), are presented in Fig.3.
The bridges in the anaphase ( Fig. 4 (4, 11) can appear in the translocation process and in uneven exchange due to the presence of dicentric chromosomes, as well as due to disintegration between chromosomes and chromatids, followed by their joining (El-Ghamery et al., 2000), which results in chromosome mutations at the structural level (Devi and Thoppil, 2016).
The sticking of chromosomes occurs as a result of chromatin defect and is considered to be an irreversible process resulting in the cell death ( Fig. 4 (13,14) (Pawlowski et al., 2012).
The apoptotic cells and cells with the damaged membranes ( Fig. 5) are observed in 50% and 100% concentrations of aqueous extracts of inflorescences after a 24-and 18-h exposure, respectively. It can be supposed that apoptosis, as the mechanism of the programmed cell death has proven to be a reaction to a stress impact, and the damage of the cell membranes was induced by membrane ferments or by decrease in the cellulose content (Sultan and Celik, 2007;. The deviations in mitosis include also elongated cells, giant cells and cells-ghosts that appear due to cytoskeleton damage in the interphase, as well as deformed nuclei and bulged chromatin, which appear in spindle and cytokines inhibiting (Mushtaq et al., 2019). For instance, giant cells and cells-ghosts ( Fig. 4 (10) and Fig. 4 (18) were observed in positive control of 1% hydrogen peroxide after a 48-72 h exposure and in 100% concentration of the aqueous extract of leaves after a 24-h exposure, and nucleus-free cells were observed in 50% concentration of aqueous extract of leaves after a 24-h exposure.
The nuclear buds (Fig. 4 (1) appear due to displacement or bulging of the genetic material from aneuploid cells (Fernandes at al., 2007).
In (Korovkina et al., 2020), the extract of P. weyrichii inflorescences is characterized by high antioxidant activity and possesses a great amount of phenol and other compounds if compared to the leaves extract, which confirms the results obtained by this study.

Conclusion
The study of aqueous extracts is carried out for the first time with inflorescences and leaves of P. weyrichii, and we believe that it contributes to understanding of the effect of natural extracts of potential medicinal plants on living organisms, as well as to the assessment of their toxicity and genotoxicity. The results of the study show that aqueous extracts of inflorescences and leaves of P. weyrichii of 50%, 80% and 100% concentrations have a mitodepressive effect on the cells of the root meristem of A. cepa inhibit the root growth and cause chromosome destruction. The mitodepressive effect in aqueous extracts of inflorescences was more intensive than that in aqueous extracts of leaves because the average value of the MI in aqueous extracts of leaves was 2-3 times higher. The authors suppose that these effects are due to a higher content of flavonoids and other compounds in inflorescences and due to a greater antioxidant activity, to the synergetic or antagonistic effect produced by the substances contained. This idea is to be studied further. It is necessary to go on working at selection of concentrations, qualitative and quantitative chemical analysis of the plant studied, which could serve as a source of biologically active compounds.

Disclosure statement
No potential conflict of interest was reported by the authors.