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Regulatory Mechanisms
in Biosystems
Regulatory Mechanisms
in Biosystems
ISSN 2519-8521 (Print) ISSN 2520-2588 (Online) Regul. Mech. Biosyst., 2019, 10(4), 497-506 doi: 10.15421/021973
Effect of alcohol tincture of Aralia elata on the organism of rats and their gut microbiota against the background of excessive fat diet
V. V. Brygadyrenko*, M. A. Lieshchova**, M. V. Bilan**, N. M. Tishkina**, A. V. Horchanok**
*Oles Honchar Dnipro National University, Dnipro, Ukraine **Dnipro State Agrarian and Economic University, Dnipro, Ukraine
Brygadyrenko, V. V., Lieshchova, M. A., Bilan, M. V., Tishkina, N. M., & Horchanok, A. V. (2019). Effect of alcohol tincture ofAralia elata on the organism ofrats and their gut microbiota against the background of excessive fat diet. Regulatory Mechanisms in Biosystems, 10(4), 497-506. doi-10.15421/021973
Pharmacological effects of the medicinal plant Aralia elata (Miq.) Seem. 1868 (Araliaceae) are related to presence of over 150 secondary metabolites, including flavonoids, sterols, polysaccharides, terpenoid saponins and terpenoid acids, though the main biologically active substances of A. elata are saponins and flavonoids. Some clinical tests and experimental studies have proved the influence of A. elata on the organism through increase in physical work capacity, exerting an anti-stress effect against a broad range of harmful factors, including stress from cold, immobilization, ultraviolet radiation and low pressure. The literature also reports the anti-ulcer, anti-secretory, antifungal, anti-tumour, and antimicrobial activity of preparations from this plant. In our laboratory experiment, we determined that ethanolic tincture of A. elata has no effect on the intensity of growth of body weight of young rats against the background of excessive content of fat in their diet. Excessive fat-feeding of male rats leads mostly to disorders in the functioning of the liver and development of steatosis. This was accompanied by reduction in relative mass of the liver, increase in activity of hepatic enzymes, dysproteinemia, increase in the level of bilirubin and decrease in the level of urea. Additional use of 0.1% ethanol led to impaired functioning of the kidneys, reduction of their relative mass, signs of dehydration, increase in the level of creatinine and total calcium in the blood. Use of ethanolic tincture of A. elata mitigates negative excess of fat, is accompanied by normalization of indices of mass of the organs, less notable dysproteinemia, impairment in the level of creatinine, glucose, urea, cholesterol, bilirubin and total calcium. Ethanolic tincture of A. elata has a low immunosuppressive action, against the background of a high fat diet it leads to increase in the amount of typical Escherichia coli, decrease in Enterococcus spp. and Enterobacter spp., significant decrease and in high concentrations (0.1% ethanolic tincture of A. elata) elimination of bacteria of Clostridium and Klebsiella genera, and also various yeast fungi in the intestine. In the examined male rats, against the background of excess of fat in the diet, no serious changes in the composition of the normal gut microbiota (Bifidobacterium spp., Lactobacillus spp., Proteus spp., Staphylococcus spp., Candida spp.) was observed, nor were any lactose-negative enterobacteria (Citrobacter genus) found. Perspectives of further research include determining histological, histochemical and immune-histological changes in the organs of laboratory animals under the effect of ethanolic tincture of A. elata following excessive accumulation of fat.
Article info
Received 15.09.2019 Received in revised form
17.10.2019 Accepted 18.10.2019
Oles Honchar Dnipro National University, Gagarin av., 72, Dnipro, 49010, Ukraine. Tel.: +38-050-93-90-788. E-mail: brigad@ua.fm
Dnipro State Agrarian and Economic University, Serhii Efremov st., 25, Dnipro, 49600, Ukraine. Tel.: +38-056-268-54-17. E-mail:
lieshchova.m.c@dsau.dp.ua
Keywords: relative mass of the organs; increase in the body weight; gut microbiota; microbiome; high-fat diet.
Introduction
The widely known medical plant Aralia elata (Miq.) Seem. 1868 (commonly called Japanese anjelica tree, also known as Manchurian aralia tree Aralia mandshurica Rupr. et Maxim.) of the Araliaceae family is a tree or bush (up to 10 m in height), distributed in the North-East regions of China, Japan, Korea and the Far East of the Russian Federation. Traditionally, for medical purposes, roots of the plant are used, more rarely - leaves and bark. Currently, roots of A. elata are used in classic pharmacology of many countries. They are processed into alcoholic tinctures and tablets, or included in the composition of some complex preparations. Tinctura Araliae is an ethyl tincture (70%), standardized, available at the pharmacy on prescription and is recommended for oral administration at a dose of 0.75-1.00 mL twice a day. Saparal tablets are pure extract of roots of A. elata, each containing 50 mL of mixture of ammonia salts of aralosides A, B and C. The preparation is for oral administration in the dose of one tablet twice a day. Arfazetin is a mixture of different types of plants, including root of Aralia (15%); it is recommended as a hypoglycemic preparation (Shikov et al., 2016). Pharmacological effects of the plant are related to the presence of over 150 secondary metabolites, including
flavonoids, sterols, polysaccharides, terpenoid saponins and terpenoid acids (Guo et al., 2009; Wang et al., 2011; Clement & Clement, 2015). However, the main biologically active substances of A. elata are saponins and flavonoids (Zhang & Song, 2012; Zhang et al., 2013, 2018; Wang et al., 2014). In their studies, Saito et al. (1993), out of 11 sapo-nins in the leaves of A. elata, studied the most active ones, those which reduced the aspartate aminotrasferase (AST) and alanine aminotrasfe-rase (ALT) production by hepatocytes during CCl4-induced hepatitis in rats (Saito et al., 1993). Furthermore, saponins extracted from leaves of A. elata had significant anti-tumour action in humans with mammary gland cancer - both in vivo and in vitro (Li et al., 2019). Today, complex spectral analysis has distinguished new triterpene saponins (con-gmusaponin I, II, III, congmuyenoside V, congmuyenoside VI), their inhibiting activity towards enzymes a-glucosidase, protein tyrosine phosphatase 1B (PTP1B) and neuroprotective effects have been determined (Zhang et al., 2018a, b).
Medicinal plants with complex compositions of saponins, flavono-ids and alcoloids can cause side-effects or synergic effects over chemical interactions in the organism of animals and humans (Saad, 2006). Therefore, for confirming safety of chemical substances from A. elata
for humans, a number of toxicological surveys on experimental animals have been performed, the toxicity and allowable doses of medical preparation for humans have been determined (Li et al., 2015, 2016). On mice, during acute peroral delivery of alcohol tincture (70%), LD50 was determined to equal 1.25 g/kg (Turova, 1974). According to the data by Pivo-varova & Lesiovskaya (2003), for commercial alchohol tincture (70%) the root of Aralia (1 part) : alcohol (5 parts) in subcutaneous injection equaled 6.3 ± 0.7 mL/kg. The chronic toxicity of alcohol tincture of A. elata was studied for rats which received it in injections during four weeks in daily doses of 0.25, 0.50 and 1.00 g per 1 kg of body weight. These doses did not lead to lethal effect or any clinical manifestations, also no changes in the internal organs were found during autopsy, nor changes in biochemical and morphological parameters of blood and urine analyses (Jin et al., 2006). While determining acute toxicity of alcohol extract from leaves of A. elata in rats which received peroral introduction of doses of 1.00, 2.15, 4.64 and 10.00 g/kg, Li et al. (2015) behavioural disorders and toxic syndrome were observed with lethal outcome in 14 days. Peroral introduction of those doses also caused various side-effects, and mortality increased in relation to increase in the dose of the preparation (Li et al., 2015).
Despite lack of knowledge on the mechanism of action, some clinical tests and experimental studies have confirmed that use of A. elata preparations increase physical working capability and have an antistress effect against a broad range of harmful stress factors, including stress from cold, immobilization, ultraviolet radiation, low atmospheric pressure. This phytoadaptogen affects the central nervous system (CNS), reproductive, immune, respiratory and digestive systems, optimizes the course of metabolic syndrome, showing hypolipidemic and anti-diabetic effects by changing blood coagulation (Shikov et al, 2016; Lee et al., 2019).
Tincture of A. elata is a familiar adaptogen, therefore a major part of its effects on the organism is related to general properties of Aralia-containing preparations. They are regulators of metabolism, which increase the ability of the organism to adapt to various stress factors of the environment and prevent damage caused by stresses (Panossian et al., 1999). Phytoadaptogens in small and average doses stimulate CNS, and in higher doses have a sedative effect (Montiel-Ruiz et al., 2012; Panossian et al., 2012). Data on the influence of A. elata on CNS are contradictory. A stimulating effect on the nervous system was induced by intragastric introduction of alcohol tincture of A. elata. During a course of injections, Saparal and Tinctura Araliae preparations increased general moving and exploring activity of animals in the "Open field" test, most notable changes occurred following consumption of alcohol tincture of the plant. Emotional status of male rats in all experimental groups was higher than in the control, and decrease in cosmetic activity in experimental groups was probably related to increase in general moving activity of the animals (Kharin et al., 2011). Extract of A. elata, given to mice intraperitoneally (in the dose of 10, 30 and 100 mg/kg) 30 minutes before taking pentobarbital prolonged time of narcosis, confirming the inhibitory effect on CNS. However, high doses of the extract (100 mg/kg) in groups of female mice somewhat reduced this time (Ahumada et al., 1991). Introduction of alcohol extract of Aralia root (0.5 g/kg) and sodium 5,5-diethylbarbiturate (0.15 mg/g) at the same time reduced time of narcosis by 38% (Brekhman, 1963).
Adaptogenic effect of A. elata (commercial and non-commercial forms of alcohol tincture) in cases of hypobaric hypoxia has been determined on mice (Pospelova & Barnaulov, 2000). Dilatometric surveys (Stjopin et al., 2019) also revealed high antiradical activity of alcohol tincture of the plant, by these indicators, it was inferior only to tincture of Eleutherococcus senticosus (Rupr. & Maxim.) Maxim.
Anti-ulcer and anti-secretory properties of extract from Aralia were studied with animal models (laboratory rats) with peptic ulcer disease. Preliminary treatment with the plant (50 mg/kg intraperitoneally) reduced the frequency and severity of lesions of the glands of the stomach (caused by cold stress), significantly decreased secretory volume of the stomach (by 40%), increased pH of the stomach and decreased acid reflux by 81%o (Hernandez et al., 1988). Studies on laboratory mice with non-alcohol steatohepatitis caused by diet with high content of fat, confirmed the healing effect of arazolides from A. elata, isolated from the plant's roots (Luo et al., 2015). Molecular mechanisms of anti-inflam-
matory and anti-apoptotic action have been studied and general protective effect of saponins of A. elata was proved in cases of damage to endothelial cells of blood vessels on the example of endothelium of the umbilical vein of a human, affected by necrosis factor-a (TNF-a) (Zhou et al., 2018).
A number of researchers found anti-tumour efficiency of active substances isolated from A. elata. An in vitro experiment has revealed that processing of cell culture of cancer cells (human kidney cancer cell lines GRC-1 and 786-O) with different doses of araloside A significantly reduced their vitality and increased the number of Tunnel-positive cancer cells in the processed cell culture (Yu, 2011). Against tumour cells of humans (A549), the substances isolated from an alcohol extract of leaves of A. elata also exerted cytotoxicity (Kuang et al., 2013).
Li et al. (2019) have determined anti-cancer activity and pharmaceutical safety of alcohol extract from leaves of A. elata. The preparation has demonstrated high cytotoxicity for different lines of tumour cells in vitro, and also anti-tumour activity in vivo on mice with mammary gland cancer. The extract significantly downregulated the growth of tumour and induced apaptosis of MCF-7 cells. In that experiment, also the safety of the Aralia preparation for CNS and cardio-vascular system of mice and dogs was studied. The mice with mammary gland cancer, which were treated using the extract from leaves of A elata, were observed to have no changes in the behaviour, moving activity and coordination of movement. There were no changes in the cardiovascular system in dogs. Frequency of heartbeat, parameters of electrocardiogram (interval of PR, interval of PR, duration of P and QRS peaks), frequency of breathing, respiratory volume, temperature of the body and arterial pressure showed no significant differences between the experimental and control groups (Li et al., 2019).
Use of medicinal plants can be important for prevention of human diseases induced by intestinal microorganisms: over change in their number, composition of communities and production of potentially harmful metabolites (Ahn et al., 1994). Extract from roots of Aralia has antifungal effect against Pyricularia grisea and antibacterial activity towards gram-positive bacilli.
Peroral treatment of women with non-diabetic obesity, who received low-calorie diet, with a preparation which contained extracts from A elata and Engelhardia chrysolepis (Juglandaceae) led to decrease in the total body weight and mass of fat, reduction of perilipin in adipocytes and content of triglycerides in blood plasma, stimulation of activity of hormone-sensitive lipase (Abidov et al., 2006). We should note the influence of high-fat diet on development of obesity, hepatic steatosis, type 2 diabetes, and also pathogenesis of dysbiotic syndrome (Cani et al., 2007; Cho et al., 2009).
According to the classification of toxicity, the substance with LD50 equaling 0.5 to 5.0 g/kg perorally is low-toxic (Loomis & Hayes, 1996). Surveys by Li et al. (2015) have revealed that for extract of Aralia this parameter equals 3.16 g/kg for females and 5.84 g/kg for males. In experiments aimed at determination of toxicity of preparations of Aralia the maximum tolerance of rats equaled 1.00 g/kg for females and 2.15 g/kg for males. Therefore, use of alcohol tincture of the plant in one way or the other will affect the growth and development of animals, especially following disorders in metabolism.
The objective of this study was to determine the general effect of different doses of alcohol tincture of roots of A. elata on the growth and development of young laboratory rats and their gut microbiota against the background of excessive fat content.
Materials and methods
This study was performed in correspondence to the principles described in the guidance on maintaining and using experimental animals, according to ethical norms formulated in the order No 3447-IV from 21.02.2006 "On protection of animals against cruel treatment" and approved by the local ethics committee of the Dnipro National Agrarian-Economic University (Dnipro, Ukraine). The young of non pedigree laboratory male rats weighing 50 ± 10 g were maintained in normal conditions: 12-day cycle "day - night", free access to food and water (Lieshchova et al., 2019). Diet of all animals had excessive fat content (3600 kcal/kg) due to addition of sunflower oil (Table 1).
Table 1
Diet of laboratory animals
Characteristic of diet, unit of measurement
Value of characteristic
Metabolic energy, kcal/kg Dry matter, % Crude protein, % Crude cellulose, % Fat, %
Carbohydrate, % Insoluble ash in HCl, %
Other ingredients
Corn, %
Full-fat soybean, % Sunflower seed meal, % Wheat middling, % Alfalfa meal, % Meat bone meal, % Molasses, % Vegetable oil, % Limestone, % Dicalcium phosphate, % NaCl, %
Vitamin-mineral premixes, % Ca, %
P, %
3,600.0 82.0 22.3 7.2 13.5 39.7 0.9
33.0 39.0 7.0 1.5 3.0 2.0 1.0 10.0 2.0 1.0 0.45 0.25 1.20 0.60
Over the recent years, A. elata, as a valuable medical introducent, has been broadly used in botanical gardens of Ukraine (Buida & Konia-kin, 2019). We prepared alcoholic tincture using roots of the plant (34 year old roots were used), collected in the Botanical Garden of Oles Honchar Dnipro National University (Khromykh et al., 2018). The roots were cut into pieces of 4-5 cm length, rinsed in cold water, dried during two months to stable mass in the room temperature out of direct sunlight,
Table 2
Characteristic of experimental groups of male rats
fragmented using secateurs to 2-3 mm pieces, left to dissolve for 20 days in the temperature of 20 °C in 70% ethyl alcohol in the ratio (by mass) of 1 part of dry roots to 5 parts of ethyl alcohol, the obtained tincture was filtrated, achieving its 10% concentration using 70% ethyl alcohol.
Four groups of animals were formed with 7 animals in each one: the first group had free access to clean water, the second group - free access to 0.1% ethanol, the third group - to 0.1% ethanolic tincture of A. elata, the fourth group - to 0.01% ethanolic tincture of A. elata. The experiment lasted for 35 days (Table 2).
During the study, the animals were recorded as having changes in the body weight, amount of water and fodder consumed. After the experiment, the animals were subjected to euthanasia by drawing blood from the heart under narcosis (80 mg/kg of ketamine and 12 mg/kg of xylazine, intraperitoneally). After dissection, we visually evaluated the condition of the internal organs (heart, liver, thymus, spleen, stomach, thin and large intestines, kidneys, testicles, breastbone) for presence of pathological changes, weighed on scales with accuracy to 1 mg with following determination of index of the mass of the organs.
Blood samples taken during euthanasia were used for biochemical morphological surveys. Biochemical parameters were determined using an automatic analyzer Mura 200 (Italy) and a set of reagents High Technology (USA), PZ Cormay S.A. (Poland) and Spinreact S.A. (Spain). Total protein was determined using the biuret method; globulins and protein coefficient - calculational method; albumins - by reaction with Bromocresol green, activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) - by kinetic method based on the optical test of Warburg; alkaline phosphatase - enzymic method with n-nitrophenilphosphate; glucose - glucose-oxidase method. Number of erythrocytes and leukocytes in stabilized blood of the rats was determined using an automatic hematologic analyzer BC-2800Vet and Mind-ray. For the leukogram, blood smears were prepared using the Pappenheim method and stained with Giemsa stain.
Group
Feeding
Watering
Average amount of ethanol drunk by one animal, mg/day
Average amount of ethanol drunk by one animal, mg/kg of body weight per day
Average amount of dry substances extracted from A. elata, consumed by one animal, mg/day
Average amount of dry substances extracted from A. elata, consumed by one animal, mg/kg of body weight per day
Average amount of water in the feeding mixture, drunk by one animal, g/day
Free access to
Conlrol, H2O fodder with excessive content of fat Free access to clean water 0.0 0.0 0.00 0.00 25.2
Conlrol, 0.1% elhanol _ " _ Free access to 0.1% ethanol instead of water 26.8 258.1 0.00 0.00 26.8
0.01% elhanolic " Free access to ethanolic tincture of 2.7 26.5 0.27 2.60 27.5
tinclure of A. elata A. elata (0.01%) instead of water
0.1% elhanolic Free access to ethanolic tincture of 22.0 243.0 2.41 23.21 24.4
tincture of A. elata A. elata (0.1%) instead of water
In compliance with asepsis rules, the content of the rats' intestine was extracted from the rectum, directly after the animals were killed. For the study, 1 g of feces were stirred in a mortar with 9 mL of sterile physiologic solution (10-1 concentration) and then 8 serial ten-time dilutions were performed to obtain concentration of 10-9 g of feces per 1 mL of solution. These dilutions were inoculated to elective growth media and incubated in a thermostat at the temperature of 37 °C. The estimation was made after 24-72 h, determining colony-forming units per gram of excrement (CFU/g) by counting the grown colonies.
For identifying and differentiating colonies, we studied morphological features, tinctorial, cultural and enzymic properties of microorganisms. By bacterioscopy of the stained smears, using generally accepted methods, we conducted visual confirmation of the studied microorganisms, and with mycological surveys - identified species of fungi.
The data were analyzed using Statistica 8.0 program (StatSoft Inc., USA). The tables demonstrate the results as x ± SD (x ± standard deviation). Differences between the values of the control and experimental groups were determined using the Tukey test (with consideration of Boniferroni's correction), where the differences were considered significant at P < 0.05.
Results
Over the 35-day experiment the animals increased their body weight from 54.7 ± 10.8 to 152.8 ± 11.5 g, that is an individual rat increased its body weight on average by 98.1 g - almost by three times. In the conditions of excessive content of fats in the food, the impact of alcohol reliably slowed the growth of rats (to the level of 78.4% of the control group). Replacing solution of ethyl alcohol with 0.01% or 0.1% tincture of A. elata caused no significant changes compared with the group of animals which consumed solution of ethyl alcohol (Table 3). Consumption of liquid in all four groups of animals significantly did not differ, changing on average in the range of25.7-29.1 g per animal per day.
While having excessive content of fat in their food and consuming alcohol, the rats had a reliable decrease in the weight of the heart (to 77.8% of the values of the control group, Table 4). It is interesting that addition of equivalent amount of Aralia tincture instead of alcohol reduced notability of changes in the heart of animals. Consumption of 0.1% of solution of alcohol instead of drinking water against the background of excessive calorie content in the diet caused decrease in relative mass of the liver to 75.5%i of the same value in the animals of the control group. Replacing
ethyl alcohol with an equivalent amount of alcohol tincture of Aralia consumption of both ethyl alcohol and alcohol tincture of different con-caused reliable increase (by 21.0% compared with the control group) of centration. Relative mass of the breastbone and the spleen in different relative mass of the liver. Relative mass of the lungs decreased during variants ofthe experiments did not significantly change (Table 4).
Table 3
Change in the body weight and fodder consumption of young male rats under the impact of addition of ethanolic tincture of A. elata to their ration (x ± SD, n = 7, duration of experiment - 35 days)
Parameter Control, H2O Control, 0.1% ethanol Control (0.1% etianol) compared to the control (H2O), % EUanolic tincture ofA. elata, 0.01% Ethanolic tincture ofA. elata (0.01%) compared to the control (H2O), % Ethanolic tincture ofA. elata, 0.1% Ethanolic tincture ofA. elata (0.1%) compared to the control (H2O), %
Change in body weight, g/day 3.29 ± 0.07a 2.58 ± 0.21b 78.4 2.64 ± 0.13b 80.3 2.90 ± 0.20b 88.3
Consumption of liquid 26.7 28.3 106.0 29.1 109.0 25.7 96.1
by animals, g/day
Note: different letters indicate values which reliably differed one from another within one line of the table according to the results of comparison using the Tukey test with Bonferroni correction.
Table 4
Change in relative mass of the organs (%) of male rats under the influence of addition to their ration of ethanolic tincture of A. elata (x ± SD, n = 7, duration of experiment - 35 days)
Control Ethanolic tincture Ethanolic tincture
Organ Control, H2O Control, 0.1% ethanol (0.1% etianol) compared to the control (H2O), % Ethanolic tincture ofA. elata, 0.01% ofA. elata (0.01%) compared to the control (H2O), % Ethanolic tincture ofA. elata, 0.1% ofA. elata (0.1%) compared to the control (H2O), %
Heart 0.519 ± 0.066a 0.404 ± 0.029b 77.8 0.417 ± 0.041b 80.3 0.492 ± 0.056ab 94.8
Liver 5.77 ± 0.46a 4.35 ± 0.43b 75.5 4.78 ± 0.68b 83.0 6.98 ± 0.56c 121.0
Lungs 1.073 ± 0.053a 0.720 ± 0.183b 67.1 0.869 ± 0.041b 81.0 0.756 ± 0.164b 70.5
Breast bone 1.45 ± 0.16a 1.61 ± 0.19a 110.4 1.60 ± 0.24a 109.9 1.56 ± 0.28a 107.4
Thymus 0.560 ± 0.043a 0.437 ± 0.287* 78.0 0.305 ± 0.023b 54.5 0.217 ± 0.050c 38.7
Spleen 0.442 ± 0.033a 0.422 ± 0.036a 95.5 0.477 ± 0.193a 107.9 0.407 ± 0.051a 92.1
Stomach 0.790 ± 0.106a 0.706 ± 0.091a 89.3 0.814 ± 0.082a 103.0 0.969 ± 0.107ab 122.7
Small intestine 6.65 ± 0.45a 5.14 ± 0.21b 77.3 6.45 ± 0.87a 97.1 6.57 ± 0.85a 98.9
Colon 1.82 ± 0.13a 1.57 ± 0.05b 86.0 1.37 ± 0.22b 75.2 1.72 ± 0.18ab 94.5
Right kidney 0.375 ± 0.048a 0.399 ± 0.035a 106.3 0.416 ± 0.024a 111.0 0.447 ± 0.038a 119.1
Left kidney 0.398 ± 0.053a 0.374 ± 0.018a 93.9 0.428 ± 0.036a 107.6 0.460 ± 0.042a 115.6
Testis 0.651 ± 0.050a 0.705 ± 0.050a 108.4 0.742 ± 0.115a 114.0 0.774 ± 0.191a 118.9
Note: see Table 3.
A significant decrease was noted in the relative mass of the thymus following excessive content of fat in food and while consuming Aralia tincture: to 54.5% and 38.7% of the values of the control group for 0.01%i and 0.1%o Aralia tinctures respectively (Table 4).
Consumption of 0.1%o tincture of Aralia instead of water, against the background of excessive content of fats in food, resulted in increase in relative mass of the stomach of rats by 22.7%. Increase by 8.5% and 21.6% in relative mass of the thin and large intestines occurred while consuming tincture of Aralia, compared with the group which consumed equivalent amount of alcohol. Consumption of tincture of Aralia led to an unreliable increase in the relative mass of the kidneys (by 15.6% and 19.1%o for the left and right kidneys respectively). Also, at the level of tendency (unreliably), an increase was seen in relative mass of the testicles: by 19.8%o compared with the control group of rats (Table 4).
Consumption of 0.1% of ethanol instead of water resulted in a reliable increase by 26.9% in concentration of protein in blood, and replacement of ethanol with alcohol tincture of Aralia contributed to restoration of the concentration of protein in the blood to the normal level (Table 5). Similar changes were characteristic for concentration of albumins. Consuming ethyl alcohol following excessive consumption of fats caused reliable increase by 34.4% in concentration of globulins in the blood of rats, and with alcohol tincture of Aralia made this effect was less notable. Protein coefficient reliably decreased by 19.2% compared with the control group with consumption of 0.1% tincture of Aralia. Ethyl alcohol increased the content of creatinine in blood of animals by 30.9%, and Aralia tincture made this less notable. Consumption of alcohol reduced activity of asparatate aminotransferase and alanine aminotransferase to 65.0%o and 72.3% of the values of the control group, and tincture of Aralia restored the values of activity of these enzymes to the levels of the control group. The De Ritis ratio and concentration of total bilirubin in blood in all four groups of animals reliably did not differ. Activity of alkaline phosphate reached minimum values during consumption of 0.1% ethanolic tincture of A. elata, equaling 44.2% compared to the control.
Concentration of glucose in the blood of the rats fell to 85.6% of the level of the control group during consumption of solution of ethyl alcohol instead of water and to 91.6% of the control during consumption of 0.1% ethanolic tincture of A. elata (Table 5).
While drinking tincture of Aralia the concentration of calcium in the blood ofthe animals had a tendency towards growth (by 9.9-10.6%), and non-organic phosphorus reliably fell (to 67.5-75.0% of the values of the control group). Ratio of Ca/P during consumption of tincture of A. elata increased by 45.3% and 67.4% for the groups of animals which drank 0.01% and 0.1% ethanolic tincture of A. elata respectively (Table 5).
Apart from this, a strong decrease should be mentioned for the concentration of cholesterol to the level of 58.5% ofthe concentration of the control group during intake of 0.1%o ethanolic tincture ofA. elata (Table 5).
Drinking 0.1% Aralia tinicture instead of water increased the concentration of hemoglobin in the blood of rats by 10.8% (Table 6). Similar changes were observed also for hematocrit (increase by 12.5%) and the amount of erythrocytes (increase by 6.9%). No reliable changes were seen in the erythrocyte sedimentation rate, concentration of thrombocytes, eosinophiles and lymphocytes in the blood of animals in all the variants of the experiment. Concentration of monocytes in the rats did not decrease under the impact of alcohol compared with the control group of animals which consumed different concentrations of ethanolic tincture ofA. elata (Table 6). We should emphasize that drinking Aralia tincture doubles the percent of the content of band-form leukocytes (to 200.0% and 213.3% ofthe values of the control group, Table 6).
Analysis of the studies of microbial composition of feces of the examined animals which received alcohol tincture of roots of A. elata in concentrations of 0.1% and 0.01% following a high fat diet during the experiment revealed that the level of representatives of normal micro-biota (Bifidobacterium spp., Lactobacillus spp., Proteus spp., Staphylococcus spp., Candida spp., and also some lactose-negative enterobac-teria (Citrobacter genus) in the experimental groups was close to the control (Fig. 1-3).
Table 5
Change in biochemical parameters of blood of males of rats under effect of addition
to their ration of ethanolic tincture of A. elata (x ± SD, n = 7, duration of experiment - 35 days)
Parameters Control, H2O Control, 0.1% ethanol Control (0.1% ethanol) compared to the control (H2O), % Ethanolic tincture of A. elata, 0.01% Ethanolic tincture ofA. elata (0.01%) compared to the control (H2O), % Ethanolic tincture of A elata, 0.1% Ethanolic tincture ofA. elata (0.1%) compared to the control (H2O), %
Total protein, g/L 69.7 ± 4.2s 88.4 ± 2.2b 126.9 73.0 ± 6.0a 104.8 73.6 ± 3.7a 105.6
Albumins, g/L 43.3 ± 3.9s 53.0 ± 2.0b 122.3 42.8 ± 2.1a 98.8 42.0 ± 1.3a 96.9
Globulins, g/L 26.3 ± 0.5s 35.4 ± 2.4b 134.4 30.2 ± 4.2* 114.7 31.6 ± 2.6* 120.0
Protein coefficient, U 1.63 ± 0.09s 1.50 ± 0.11a 91.8 1.42 ± 0.15ab 86.9 1.32 ± 0.07b 80.8
Urea, mmol/L 6.10 ± 0.22a 6.12 ± 1.07* 100.3 4.66 ± 0.87b 76.4 6.28 ± 0.98ab 103.0
Uric acid, mg/100 g 11.4 ± 0.4a 11.5 ± 1.6ab 100.8 8.9 ± 1.7b 77.7 12.0 ± 1.9s5 105.3
Creatinine, ^mol/L 64.3 ± 3.9a 84.2 ± 3.2b 130.9 71.2 ± 7.1c 110.7 71.2 ± 5.8c 110.7
AST, U/L 216±29a 140 ± 39b 65.0 231±30a 106.9 231±19a 107.1
ALT, U/L 84.3 ± 8.1a 61.0 ± 5.5b 72.3 95.0 ± 12.8a 112.7 90.2 ± 13.8a 107.0
De Ritis ratio (AST/ALT), U 2.57 ± 0.25a 2.34 ± 0.74a 91.2 2.26 ± 0.43a 88.1 2.66 ± 0.46s 103.6
Alkaline phosphatase, U/L 1564±192a 1057±85b 67.6 1548±378a 99.0 691±103c 44.2
Total bilirubin, ^mol/L 5.90 ± 0.33a 5.30 ± 0.62a 89.8 5.80 ± 0.41a 98.3 4.96 ± 1.10s 84.1
Glucose, mmol/L 6.33 ± 0.34a 5.42 ± 0.37b 85.6 6.02 ± 0.42ab 95.1 5.80 ± 0.76ab 91.6
Total calcium, mmol/L 2.57 ± 0.39a 3.28 ± 0.28b 127.8 2.82 ± 0.19ab 109.9 2.84 ± 0.15ab 110.6
Non-organic phosphorus, mmol/L 4.00 ± 0.24a 3.58 ± 0.47ab 89.5 3.00 ± 0.18b 75.0 2.70 ± 0.28b 67.5
Ratio of Ca/P 0.633 ± 0.125a 0.946 ± 0.199ab 149.4 0.920 ± 0.075b 145.3 1.060 ± 0.120b 167.4
Cholesterol, mmol/L 1.300 ± 0.163a 1.060 ± 0.080ab 81.5 1.140 ± 0.196ab 87.7 0.760 ± 0.196b 58.5
Note: see Table 3.
Table 6
Change in general analysis of blood and leukogram of male rats under effect of addition to their ration of ethanolic tincture of A. elata (x ± SD, n = 7, duration of experiment - 35 days)
Control Ethanolic tincture of A. elata, 0.01% Elhanolic tincture Ethanolic tincture of A. elata, 0.1% Ethanolic tincture
Parameter Control, H2O Control, 0.1% ethanol (0.1% ethanol) compared to the control (H2O), % ofA. elata (0.01%) compared to the control (H2O), % ofA. elata (0.1%) compared to the control (H2O), %
Hemoglobin, g/L 123.3 ± 7.7ab 123.4 ± 7.9s5 100.1 115.4 ± 10.3s 93.6 136.6 ± 7.6b 110.8
Hematocrit, % 39.7 ± 0.2s5 39.6 ± 3.1s5 99.9 36.8 ± 3.0s 92.7 44.6 ± 3.6b 112.5
Erythrocytes, 1012/L 7.57 ± 0.21s5 7.32 ± 0.70s5 96.7 6.62 ± 0.72s 87.5 8.09 ± 0.675 106.9
erythrocyte sedimentation rate (ESR), mm/h 1.0 ± 0.0a 1.0 ± 0.0s 100.0 1.0 ± 0.0s 100.0 1.0 ± 0.0s 100.0
Thrombocytes, 109/L 358±44a 409±66s 114.4 323 ± 41s 90.2 349 ± 98s 97.6
Leukocytes, 109/L 9.6 ± 0.6a 10.7 ± 2.5s5 111.5 11.8 ± 1.2b 123.3 11.4 ± 1.3s5 118.8
Leukocytic formula
Eosinophils, % 1.0 ± 0.0a 0.7 ± 0.5s 66.7 1.0 ± 0.0s 100.0 1.0 ± 0.0s 100.0
Neutrophils, %: - young - band 0a 3.0 ± 3.6s5 0a 3.2 ± 1.2s 106.7 0a 6.4 ± 2.4s5 213.3 0a 6.0 ± 1.3b 200.0
- with segmented nuclei 18.3 ± 3.9a 23.2 ± 1.7s5 126.5 17.6 ± 5.8s 96.0 18.2 ± 2.4s 99.3
Lymphocytes, % 73.3 ± 0.9a 71.0 ± 1.9s 96.8 73.0 ± 8.0s 99.5 71.2 ± 2.3s 97.1
Monocytes, % 4.25 ± 1.26a 1.33 ± 0.52b 31.4 2.00 ± 0.89s5 47.1 3.67 ± 1.03s 86.3
Note: see Table 3.
Table 7
Number of microorganisms (Lg CFU/gram of feces) in four groups of rats,
to whose diet alchohol tincture of the roots of A. elata was added (x ± SD, n = 7, duration of the experiment was 35 days)
Gut microbiota Control, Control, Ethanolic tincture Ethanolic tincture
H2O 0.1% ethanol ofA. elata, 0.01% ofA. elata, 0.1%
Bifidobacterium spp. 9.33 ± 0.58 9.40 ± 0.89 9.00 ± 1.00 8.80 ± 0.84
Lactobacillus spp. 8.33 ± 0.58 8.40 ± 0.55 8.80 ± 0.45 8.40 ± 0.55
Escherichia coli typical 8.00 ± 0.00 5.36 ± 3.03 6.46 ± 3.66 7.40 ± 0.55*
Enterococcus spp. 7.33 ± 0.55 4.52 ± 2.65 4.39 ± 2.50# 2.00 ± 2.74#
Proteus spp. 3.03 ± 0.50 3.94 ± 0.37 3.25 ± 1.82 3.30 ± 1.85
Enterobacter spp. 2.00 ± 0.00 0.80 ± 1.10 1.26 ± 1.16 0.86 ± 1.18#
Citrobacter spp. 1.43 ± 1.25 2.79 ± 1.57 1.27 ± 1.74 1.64 ± 1.52
Klebsiella spp. 0.00 ± 0.00 0.40 ± 0.89 0.00 ± 0.00* 0.00 ± 0.00*
Clostridium spp. 1.33 ± 1.15 1.20 ± 1.10 0.40 ± 0.89 0.00 ± 0.00*#
Staphylococcus spp. 3.53 ± 0.68 1.86 ± 1.84 3.17 ± 0.74 2.19 ± 1.30
Candida spp. 2.75 ± 0.18 3.10 ± 0.30 2.34 ± 1.43 3.18 ± 0.68
Notes: * - P < 0.05 comparison with the groups where the liquid for the animals was ethanol, # -animals was water.
P < 0.05 compared with the groups where the liquid for the
During the experiment, compared with the control, no reliable changes in the composition of the gut microbiota were seen in the group of rats which had alcohol introduced into their diet. Because alcohol can be one ofthe main factors affecting the composition of gut microbiota, we should
note that adding alcohol tincture and ethanol to high-fat diet contributed to decrease in the number of typical E. coli (Table 7). However, this parameter was reliably higher (by one hundred times) after high concentrations were drunk (0.1%) and by ten times after low concentrations of tincture of
roots of A. elata weie drunk (0.01%), by contrast to presence of E. coli, detected in animals which had been given ethanol.
Fig. 1. Drops of neutral fat in the native preparations of feces of rats of the control group which drank water: bar - 10 ^m
The animals of the experimental groups were observed to have decrease in the number of bacteria of genus Enterococcus by 100 and 10,000 times, Enterobacter - by 100 times compared with the animals in the control group, which received no alcohol. The number of bacteria of Enterococcus and Enterobacter genera in the composition of lumen microbiota of the rectum of the rats in the experimental groups (and especially in animals which drank tincture of Aralia in concentration of 0.1%) was close to their amount in the feces of animals which drank ethanol (Table 7). Adding alcohol tincture of Aralia to the diet in low dosage caused (at the level of tendency) decrease in the amount of bacteria of the Clostridium genus in the intestine of rats, and tinctures of Aralia in high concentrations led to their elimination, with similar results for Klebsiella spp. (Table 7).
On the 5th day of cultivation in the Sabouraud agar, we observed growth of mold colonies, different by size and shape, which differed by height and colour of the mycelium (Fig. 3). One animal which drank alcohol tincture of roots of A. elata in high concentration (0.1%) was observed to have 4 x 105 CFU/g; two animals which received the tincture in the concentration of 0.01% - 2-4 x 105 CFU/g and four animals of the control group which received 0.1% ethanol or water - growth of 2-4 x105 CFU/g. The survey of micromorphology of the fungi identified the following species: Aspergillus flavus, A. niger, Acremoniella atra, Alternaria sp.,Mucor sp. (Fig. 4).
Fig. 2. Microscopic image of feces of rats of the control group, which received water: a - Gramm staining, b - Zlatogorov staining, white arrows - spherical forms, grey arrows - rod-shaped bacterium, black arrow - spore-forming forms; bar - 5 ^m
Fig. 3. Growth of different colonies of mold in Sabouraud agar, 5th day at 26 °C: natural size
a
o
vJÊÖS^ifSä-.
Fig. 4. Micromorphology of fungi: a - final conidiophore heads with sterigmata and unfallen conidia of Aspergillus flavus, b - sterigmata of Aspergillus niger, arranged in two layers; c - aerial mycelium of Acremoniella atra, some conidiophores have rounded young conidia; fallen oviform, colourless (young) and brown conidia are notable; d - conidia of Alternaria spp.: mature conidia with transverse and longitudinal septa, round at the base, and pointed at the end, with a short conidiophore at the base; young conidia are elongated with transverse septa; e - rounded sporanges ofMucor spp., empty and full with sporangiospores; bar - 10 ^m
Discussion
In the experiment, against the background of excessive content of fat in the diet of rats, we observed no significant increase in the mass of animals. In the control, gain of body weight of animals did not exceed 3.29 g/day, and was much lower during consumption of alcohol and different doses of alcohol tincture of Aralia. Increase in the animals' body weight to a large extent depends on the content of carbohydrates rather than fat. The weight of the rats given a high fat diet in the experiment changed insignificantly compared with the control (usual diet), and addition of fructose to the diet led to reliable increase in the body
weight already in the third week of the experiment compared both with the control group and animals which received excessive content of fat in the fodder (Ozkan & Yakan, 2019). Study for subchronic toxicity of the tincture in the doses of 60, 180 and 540 mg/kg on the rats during 12 weeks with the following 4-week period of recovery showed that the preparation caused no gross changes in consumption of fodder by animals. However, with consumption of high doses of the tincture, body weight of male rats increased slowly compared with the control during the period of recovery (Li et al., 2015). Therefore, reduction of tempi of growth of animals whose diet had high content of fats, in our opinion, to a larger degree depended on the alcohol.
a
c
e
Mass of the organs is often used as a sensitive indicator for evaluating the toxic impact of different substances, including medical (Balogun et al., 2014; Varcholyak & Gutyi, 2019). Relative mass of the organs of animals following the diet with excessive content of fat changed both under the influence of alcohol and alcohol tincture of Aralia. Relative mass of the heart was reliably lower in all the variants of the experiment, and most significantly differed from the control with consumption of pure 0.1% ethanol. The high-fat diet itself did not lead to changes in the relative mass of the heart of animals, it remained within reference values of the norm for the corresponding age group (Abrashova et al., 2013). Relative mass of the liver along with biochemical blood parameters are indicators which can characterize functional condition of the organism. Against the background of excess of fat in the diet, the animals were observed to have a reliably low index of liver mass, which can be explained by development of hepatic steatosis as a result of which lipid drops accumulate in hepatocytes and the organ becomes lighter. When ethanol and low concentrations of Aralia were added to the diet, the process exacerbated, and during use of 0.1% alcohol tincture, by contrast, the mass of the organ significantly increased compared both with the control and other variants of the experiment, which can indicate the hepatoprotective activity of the plant. On the model of hepactic steatosis, in oleic acid-induced HepG2 cells, Hwang et al. (2015) observed a general medical effect of the extract of A. eleta. The substances of this plant have been proved to reduce accumulation of lipids in the liver and significantly decrease the expression of genes with lipolytic activity, increase expression of genes with lipolytic activity in HepG2 cells, and also rapidly increase consumption of glucose (Hwang et al., 2015). The study by Luo et al. (2015) on laboratory rats has demonstrated that treatment of non-alcohol hepatic steatosis caused by excessive-fat diet using aralosides from roots ofA. eleta in different doses (75, 150 mg/kg per day, intragastric) over 12 weeks led to decrease in activity of ALT and AST in the blood. Also, the animals were seen to have a decrease in the expression of protein and mRNA of anti-inflammatory cytokines (IL-6, MCP-1, TNF-a), but concentration of total lipids in blood was constant. At the histological level, decrease in steatosis of hepatocytes was detected, and immunohistologically the aralosides were shown to regulate apoptosis and expression of proteins of inflammation, reducing phosphorylation of IREE-12, JNK, IkB and the subsequent activation of NF-kB p65 (Luo et al., 2015).
With use of alcohol tinctures of Amlia in different concentrations, and also when using ethanol, decrease in relative mass of the lungs was observed, with this organ showing normal parameters against the background of excess of fat in the diet. Relative mass of the spleen, kidneys and testicles was lower than the reference values in all groups of animals, and during the use of Amlia tincture the reduction was much less notable. Decreases in the mass of thymus (by 16%), spleen (by 19%) and adrenal glands (by 33%) were observed in mice subjected to long (18 h) immobilizing stress during simultaneous injection of Aralia tincture (0.1 mg/kg, perorally, five times a day), confirming the anti-stress property of this preparation (Chernyak & Titova, 2014).
Hematological and biochemical parameters are the main diagnostic criteria in clinical practice. Values of some hematological and biochemical markers indicate an unfavourable effect of the medicines on organs and systems (Young & Friedman, 2001). Standard markers of normal function of the liver are biochemical blood parameters: AST, ALT, alkaline phosphatase, amount of albumins (Yu et al., 2012). The function of the kidneys is examined by such biochemical markers as creatinine, urea and the level of uric acid in the blood (Jia et al., 2014), whereas the functions of the heart are indicated by activity of creatinine kinase (Ba-koma et al., 2013). Li et al. (2015) have determined that different doses of extract of A. elata have no effect on the system of the blood of rats. In our research, both high-fat diet and addition of ethanol and alcohol tinctures of Amlia led to changes in blood parameters. Against the background of excess of fat in the diet during use of ethanol, the animals were observed to have signs of dehydration and abnormal functioning of the liver and kidneys. When using alcoholic tinctures of Amlia these disorders were less notable. Compared with the control group, the animals of the experimental groups were observed to have notable hyper-proteinemia and dysproteinemia, mostly due to hyper albumins against
the background of hypoglobulemia. Decrease in the level of globulins can indicate immunosuppressive effect, and by this fact Li et al. (2015) explain the change in this parameter of rats' blood during intake of 60, 180 and 540 mg/kg doses of extract of A. elata. In our experiment, in the experimental group of the animals, decrease in globulins was minimum, and with use of alcohol and alcohol tincture of A. elata reliably increased, but did not reach reference values of the healthy animals (Abrashova et al., 2013).
The immunosuppressive action of this preparation can be indicated also by condition of the main organ of the immune protection - thymus, the mass of which was reliably lower in the groups of animals that received 0.01% and 0.1% ethanolic tincture of A. elata. In the thymus, stem cells of the bone marrow transform into T-lymphocytes, undergoing antigen-independent proliferation and differentiation (Carrio & Lopez, 2013). In determining chronic activity of the extract of Aralia on rats, a significant reduction of absolute and relative mass of the thymus was observed in the group of animals with high dose and only at the end of the treatment, but pathohistological analysis showed no changes (Li et al, 2015).
As is known, the same preparation, depending on dose, can have either therapeutic or toxic effect. Presumably, the effect on the immune system is one of the mechanisms of anti-tumour action of the extract of root ofA. elata (Yu, 2011; Li et al., 2019).
The high-fat diet caused increase in the activity of the main hepatic enzymes (AST and ALT) compared with reference values for this age group of rats. However, consumption of alcohol reduced the activity of enzymes practically to the norm, and alcohol tinctures of Amlia in different concentrations intensified this process. By activity of enzymes in blood (alanine aminotransferase (ALT), gamma-glutamiltranspeptidase (GGT), serum alkaline phosphatase (SAP)), a hepatobiliar toxic effect of the extract of the root of Amlia in different doses (0.16, 1.50 and 3.00 g/kg) was determined on swine. By the end of the experiment (60th day), regardless of the dose, the level of ALT and GGT significantly increased, which together with increase in the level of lymphocytes and polymorphonuclear neutrophils in the portal and periportal regions indicates subclinical hepatitis (Burgos et al., 1997).
The level of glucose in blood of the animals given a high-fat diet increased, and during use of Amlia tincture insignificantly reduced, but without reaching reference values (Abrashova et al., 2013). High hypo-glycaemic activity of metabolites ofA. elata was observed in the experiment for identifying tolerance to sacharose. At the same time, the rats were earlier perorally given oleic acid and oligoglycosides) (100 mg/kg), 30 min before being given glucose. These metabolites displayed high hypoglycaemic activity (Yoshikawa et al., 1993, 1996).
Qualitative and quantitative composition of gut microbiota and multi-cellular intestinal parasites can change depending on the compound of food, age, taking medical preparations (especially antibiotics), specifics of diet, diseases of infectious and non-infectious origin (Guinane & Cotter, 2013; Boyko & Brygadyrenko, 2016; Zazharskyi et al., 2019). The effect of a high-fat diet on development of obesity, hepatic steatosis, type 2 diabetes, and also pathogenesis of dysbiotic syndrome should also be mentioned (Cani et al., 2007; Cho et al., 2009). During the analysis of the results of microbiological study of feces of the animals in our experiment against the background of excess of fat in diet, we observed no serious changes in the composition of the normal gut microbiota (Bifidobacterium spp., Lactobacillus spp., Proteus spp., Staphylococcus spp., Candida spp.), and some lactose-generating bacteria (Citrobacter genus).
Wolf & Lorenz (2012) have determined that in the process of obesity, the ratio of Firmicutes : Bacteroidetes increases, ending with overall increase in the population of Clostridium. The latter, acting as more effective carbohydrate metabolizers, extract more energy from food, allowing it to be used more efficiently. This additional energy, if not spent, will ultimately be stored as fat deposits (Wolf & Lorenz, 2012). Similar results were obtained by the researchers who in experiments on animals determined that high-fat diet led to increased content of bacteria of the Firmicutes phylum and reduction of Bacteroidetes (Turnbaugh et al., 2008a). At the same time, data of the studies in humans vary. Some confirm this pattern (Ley et al., 2006; Turnbaugh et al., 2008b), whereas others found no significant difference (Duncan et al., 2008). Also, some surveys revealed increase in the number of Bacteroidetes in
relation to Firmicutes in cases of obesity (Schwiertz et al., 2010). Hassan et al. (2018) determined that due to use of the high fat diet, the number of Bacteroidetes decreased and proportion of Firmicutes and Cyanobac-teria increased in the cecum of the examined animals.
In our studies, in rats fed a high fat diet the consumption of 0.1% ethanol significantly did not change the amount of lumen gut microbiota compared with the animals which received water. Jakovlev et al. (2017) indicate that alcohol intoxication is accompanied by significant disorders in homeostasis, changes in protein, carbohydrate, lipid, mineral metabolisms, and an important role in these processes is played by dysbiotic shifts of the normal gut microbiota. In the experiments with rats subjected to alcohol intoxication over two months, disorders in the main symbiotic relations were seen, with increase in the transitory conditionally-pathogenic microbiota. A significant decrease in the amount of Escherichia of Clostridium in the intestinal content was observed. However, the content of enterococci did not change, and the number of Staphylococcus sapro-phyticus increased (Yakovleva & Mikhaylova, 2012). Chronic endogenous alcohol intoxication in rats of young and mature experimental groups was accompanied by change in the balance in the biocenosis of the intestine towards pathogenic flora, and the most notable shift of the biocenosis towards pathogenic flora was seen in the animals of the adult age group (Knyshova et al., 2016).
Against the background of fat diet and use of ethanolic tincture of A. elata in our experiment, the amount of typical Escherichia coli was 100 times higher at high concentration (0.1%) and 10 times higher at low concentration (0.01%o) of tincture of roots of A. elata, by contrast to animals that received ethanol. In the animals of the control group, a reliable 100 and 10,000 times decrease in the number of Enterococcus spp., and 100 times decrease in Enterobacter spp. was observed, compared with the animals that received no alcohol. We observed 1,000 times reduction of the number of microorganisms of Clostridium in the animals given 0.01% tincture, and 0.1% tincture of A. elata roots led to their elimination. After consumption of A. elata, the rats were observed to have no Klebsiella spp. Also we observed resistance against growth of pathogenic fungi in the intestinal content of animals which received ethanolic tincture of A. elata in high concentration (0.1%). Similar anti-fungal action of the extract from Aralia continentalis (125 mg/L and higher) was confirmed by Oh et al. (2016), when rice sheath blight was reduced by no less than 16%.
Conclusions
Ethanolic tincture of A. elata has no effect on the intensity of growth of body weight of young rats against the background of excessive fat content in the fodder. Excessive fat diet leads mostly to disorder in hepatic function and development of steatosis, accompanied by reduction of its mass, increase in the activity of hepatic enzymes, dyspro-teinemia, increase in the level of bilirubin and decrease in urea level. Additional use of 0.1%o ethanol caused disorder in function of kidneys -decrease in their relative mass, signs of dehydration, increase in the creati-nine level and total calcium in blood. Ethanolic tincture of A. elata alleviates the negative effect of excessive fat, leads to normalization in the indices of mass of the organs, less notable dysproteinemia, change in the level of creatinine, glucose, urea, cholysterol, bilirubin, and total calcium.
Ethanolic tincture of A. elata has a low immunosuppressive action, which manifests in decrease in the mass of thymus, especially with use of high dose of the tincture (0.1%) and hypoglobulinemia, while total amount of lymphocytes in blood does not change.
Against the background of high-fat diet, ethanolic tincture of A. elata led to increase in the number of typical E. coli, decrease in Enterococcus spp. and Enterobacter spp., significant decrease and high concentrations (0.1%) caused elimination of microorganisms of Clostridium and Kleb-siella genera and various fungi.
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