Pharmacognostic Study of Collection and Study of its Hepatoprotective Activity

Previously the anti-inflammatory activity,5 antioxidant activity (AOA) of aqueous extracts of the herbal formulation and its components in vitro.6 and the chemical composition of volatile compounds of essential oil7 were studied. Water soluble substances of cowberry leaves, pepper mint leaves, fruits of rose and low cudweed herb were found to make the most significant contribution to the AOA of the herbal formulation.6


INTRODUCTION
According to statistics for 2014-2018, in the Siberian Federal District the number of patients per 100 thousand people (with a diagnosis established for the first time in their lives) with liver disease is 21 -36% higher and with fibrosis and cirrhosis it is higher by 33-36% than the all-Russian indicators. 1 The cold climatic conditions in Siberia, the poor standard of the population living and the traditional use of fatty foods and alcohol leading to liver diseases are the reasons of this situation. In the treatment of liver diseases, herbal drugs are widely used, which are advantageously distinguished from synthetic ones by a softer, sparing effect on the patient's body. 2,3 Today, the list of herbal hepatoprotectors allowed in Russia for the treatment of liver diseases is not large and includes several items (hepabene, sibektan, maksar, karsil, silymarin, etc. 4 ), while the problem of effective therapy is far from being resolved. Herbal formulations are the most affordable and cheapest drug form and remain popular among the population.
Previously the anti-inflammatory activity, 5 antioxidant activity (AOA) of aqueous extracts of the herbal formulation and its components in vitro. 6 and the chemical composition of volatile compounds of essential oil 7 were studied. Water soluble substances of cowberry leaves, pepper mint leaves, fruits of rose and low cudweed herb were found to make the most significant contribution to the AOA of the herbal formulation. 6 The purpose of this work was to establish parameters required for quality control of the herbal formulation based on pharmacognostic studies and to determine its hepatoprotective effect on combined liver damage induced by tetracycline and ethanol.
The raw materials were crushed separately for the herb formulation preparation: rhizomes and roots of elecampane and spiny eleuterococcus, cowberry leaves -up to a particle size of 3 mm, pepper mint leaves -5 mm, low cudweed herb -7 mm, rose and hawthorn fruits -2 mm, as documented in the State Pharmacopoeia of the Russian Federation. 8 10 g of the herb formulation were sieved through a sieve with a diameter of 0.18 mm for screening vegetable dust, 25-30 fragments were collected for each component of the herbal formulation and glued to a disk with a diameter of 25 mm in dry form for analysis using Hitachi TM 1000 electron microscope.
The study of phenolic composition and the quantitative determination of eleutheroside B in herbal formulation were carried out on a HPLC (Gilson, France) and manual injector (Rheodyne 7125, USA) with following computer processing of the results (Multichrom program for Windows). As the stationary phase there was used a metal column (4.6 x 250 mm) filled with Chromasil C18; particle size -5 microns. As a mobile phase there was used methanol-water -concentrated phosphoric acid (400: 600: 5). The analysis was carried out at room temperature. The rate of eluent was 0.8 mL/min. The duration of the analysis was 50 min. Detection for phenolic composition determination was carried out with the use of UV detector (Gilson UV/VIS, 151) at 254 nm and for quantitative determination of eleutheroside B at 266 nm wavelength. Identification of individual substances was carried out by comparing retention times of peaks in the chromatogram of the tested solution with those in the standard solution chromatogram. The quantitative ratio of identified substances in the tested samples was estimated according to peaks square with the use of the internal normalization method.

Sample preparation for HPLC analysis
Herbal formulation was crushed to particles passing through a sieve with an opening size of 1 mm. Precisely weighed amount (≈ 2.0 g) of crushed herbal formulation was put into round bottomed flask (V = 100 mLl) and 20 mL of 70% ethyl alcohol was added; the flask was heated at reflux on a water bath for 1 hour after boiling of the alcohol mixture. After cooling the mixture was filtered through a paper filter into measuring flask (V = 25 mL) and then ethanol was made up the volume (solution A). At the same time the series of 0.05% solutions of standard samples of phenolic compounds, namely, the solution of rutin, quercetin, luteolin, gallic acid, caffeic acid, chlorogenic acid, hyperoside, ferulic acid, neochlorogenic acid, chicoric acid, epicatechin, epigallocatechin gallate, eleutheroside B were prepared in 70 % ethanol. The tested solutions and solutions of standard substances in volume of 50 μL each were chromatographed according to the above method.
Quantitative determination of the content of the main groups of biologically active substances (BAS) was carried out using the methods of the State Pharmacopoeia of the Russian Federation XIV edition: eleutheroside B -by HPLC; arbutin -by chromatospectrophotometric method; essential oils -by the hydrodistillation method with a Clevenger apparatus; tannins and organic acids in terms of malic acid -by titrimetric method; flavonoids, anthocyanins, ascorbic acid, carotenoids in terms of ß-carotene -by spectrophotometric method. 8 The modified anthron-sulfur method and сoefficient of the conversion to monosaccharide (glucose -358, galacturonic acid -214) were used for the quantity determination of the polysaccharides group composition. 9 The pharmacological studies were carried out in accordance with the Federal Law of the Russian Federation "On Circulation of Medicines", "Guidelines for Preclinical Trials of Medicinal products". The experiments were carried out on 84 non-linear rats of both sexes with an initial body weight of 180-200 g. Animals were received from the Federal State-Funded Scientific Institution "Scientific Center for Biomedical Technologies" of the Federal Medical and Biological Agency of Russia and kept in vivarium with free access to food and water. Pharmacological studies were carried out in accordance with the Order of the Ministry of Health of the Russian Federation No. 199n of April 1, 2016 "On approval of the Rules of Good Laboratory Practice" and in accordance with GLP. 10 The studies were approved by the local Bioethics Committee (Protocol No. 2 of February 15, 2017).
A combined liver damage in rats was simulated by intragastric administration of tetracycline hydrochloride dissolved in Tween-80 (1:10) at a dose of 0.5 g/kg animal weight. 3 hours after tetracycline administration the 40% water solution of ethanol was administered to rats at the dose 12,5 mL/kg once a day for 7 days.
Herbal formulation infusion (1:10) was prepared according to the method of the State Pharmacopoeia 8 for oral administration (infusion dry residue -31.2 ± 1.2 mg/mL) and was administered to white rats once a day for 7 days at the dose 0.1 mL/ kg, that corresponds to 312 ± 12 mg/kg of extractive substances. The interval between administration of the herbal formulation infusion, tetracycline and ethanol was 3-5 hours. Cholosas (herbal hepatoprotective and choleretic drug) in the form of syrup registered in the Russian State Register of Medicines 4 was chosen as a standard drug at the dose of 0.1 mL/kg. Animals of the control group received the same amount of purified water according to a similar scheme.
The functional state of the liver of animals was evaluated in the dynamics of the development of damage on the 7th day from the beginning of the experiments. The hepatoprotective effect of herbal formulation and the reference drug was estimated by the activity of enzymes in serum -alanine aminotransferase (ALT) and aspartate aminotransferase (AcT) and by the content of total bilirubin, cholesterol and alkaline phosphatase activity in blood serum. The intensity of lipid peroxidation (LPO) was evaluated by the content of malonic dialdehyde (MDA) in the liver tissue homogenate. The state of the antioxidant defense of the body was evaluated by the level of catalase activity in blood serum.
In order to study the choleretic effect of herbal formulation, white rats received the single dose of 0.1 mL/kg infusion. Bile samples were taken from an anesthetized animal (sodium thiopental, 45 mg / kg) through a polyethylene cannula inserted into the common bile duct. The samples were collected every hour during 4 hours. The choleretic activity of this herbal formulation was estimated by the rate of secretion and the amount of total excreted bile, by the level of bilirubin, bile acids and cholesterol.
Antitoxic liver function was evaluated by the duration of hexenal sleep in white rats. Hexenal was administered to animals intraperitoneally once a day at a dose of 70 mg / kg of rat weight.
Analytical chemical data were statistically processed using Microsoft Excel 2010 application programs in accordance with the State Pharmacopoeia of the Russian Federation XIV edition (OFS 1.1.0013.15) 8 , and pharmacological experiments were performed using Statistica Software program version 6.0 (USA). Data were expressed as an average value (M ± m), the significance of differences in average values between groups was evaluated using Student's t-test (for independent groups, differences were considered significant at P ≤ 0.05).

RESULTS
The most significant anatomical features for each component of herbal formulation were revealed as a result of a microscopic study of large particles of the herbal formulation (particle size 2-7 mm), (Figure 1).
Thus, the detection of 1-2 anatomical specific features for each component of the herbal formulation in micropreparations is sufficient to identify the components of the herbal formulation and determine its authenticity.
Ten compounds were identified in 70% ethanol extract of herbal formulation using the HPLC method (Table 1 and Figure 2).
Gallic, isoferulic and chicoric acids, catechin, eleutheroside B, luteolin are dominated in 70% ethanol extract of the herbal formulation. The data on the quantitative content of biologically active substances (BAS) are given in Table 2. The total flavonoid content is estimated based on the spectrophotometric method using aluminum chloride, that forms stable complexes with flavonoids in acidic solution 8 . The UV spectrum of the 70% ethanol extract of the herbal formulation has maximum absorption at 330 sh., 280 ± 2 and 218 nm and minimum absorption at 262 and 308 nm, maximum absorption of complexes of flavonoids with aluminum chloride in the UV spectrum of the herbal formulation (λ max 399 ± 2 nm) coincides with that of standard sample of luteolin (λ max -400 nm) (Figure 3).
The relative error of the average and individual results of the quantitative determination of flavonoids, arbutin, ascorbic acid, eleutheroside B, tannins does not exceed 5% (Table 3), that is consistent with the requirements of the State Pharmacopoeia of the Russian Federation XIV edition 8 .
For standardization of the herbal formulation, taking into account the obtained experimental data, the content of the following active substances is regulated, namely, essential oils -at least 0.30%; flavonoids in terms of luteolin -not less than 1.0%; ascorbic acid -not less than 0.5%; tannins -not less than 3.0%; arbutin -not less than 0.4%; eleutheroside B -not less than 0.01%.
The results of pharmacological studies of the herbal formulation are presented in tables 4 -7. It has been found that tetracycline and ethanol initiate LPO processes in the liver, as evidenced by an increase in the formation of MDA by 48%.
The intensification of LPO processes causes a decrease in catalase activity by 31.2% compared with indicators of intact animals (
The herbal formulation eliminates the prooxidant effect of tetracycline and ethanol: the MDA content in the liver tissue decreased by 56.4% in the experimental group on the 7th day of the experiment. The activity of serum catalase is increased by 32%, that contributes to the increase in the activity of the endogenous antioxidant system of the body ( Table 4).
The activity of enzymes of hepatic origin in the blood under the influence of BAS of herbal formulation is reduced by 23 and 14%.

Table 4: Effect of herbal formulation infusion on biochemical indicators in white rats with combined liver damage induced with tetracycline and ethanol.
Note: Here and elsewhere below asterisk * denote that the differences between control and experimental groups are significant at P < 0,05.
The administration of tetracycline in combination with ethanol inhibits the bile formation function of the liver, the content of cholates in the bile decreases by 58,5%, bilirubin -by 8%, cholesterol -by 15%, while the total amount of bile decreases by 16% on the 7th day of observation (Table 5, 6).
The herbal formulation has more pronounced choleretic effect compared to Cholosas: when using this herbal formulation infusion, the rate of bile secretion increases within 1-3 hours, and Cholosaswithin 1-2 hours. The concentration of the main bile ingredients increases against this background: bile acids by -72,7%, bilirubin -by 11,7%, cholesterol -by 21,1% (Table 6). Thus, the infusion of the herbal formulation has pronounced hepatoprotective and choleretic activity in the experiment on rats.
The improvement in the antitoxic function of the liver in white rats is noticed against the background of the administration herbal formulation infusion, as evidenced by a reduction in the duration of hexenal sleep in white rats by 25% as compared to the control on day 7 (Table 7).

DISCUSSION
Based on the studies carried out, the presence and content of the main BAS in the collection and its hepatoprotective activity were established.
Synergistic or antagonistic reactions of BAS to cell signal systems during inflammatory reactions and oxidative stress are usually present in the mechanism of action of herbal remedies. The protective effect of the herbal formulation in combined liver damage can be achieved due to the hepatoprotective and choleretic activity of eleutherosides, sesamine, isofraxidin of the spiny eleuterococcus 11 and BAS of the low cudweed 12 . Flavonoids, tannins have hepatoprotective activity 13 , phenylpropanoids of the hawthorn fruits exhibit cytotoxic activity against liver carcinoma cells inducing apoptosis 14 .
Also, sesquiterpenoids, alantolactone of the elecampane 15-16 having anti-inflammatory effects; BAS of spiny eleuterococcus 11, 17 modulating the immune response in inflammation 11 and BAS of cowberry leaves possessing antitoxic activity 18 play an important role in achieving the pharmacological effect of the herbal formulation.
Oxidative stress plays a key role in the pathogenesis of alcoholic and toxic liver damage 19 , natural antioxidants are important in its therapy. The antioxidant activity of herbal formulation established previously in vitro 6 was confirmed in animal experiment in this work: herbal formulation infusion reduced the intensity of the LPO process, that significantly contributed to an increase in the activity of the endogenous antioxidant defense of the body, the catalase activity in the experimental group increased and exceeded that of the control group ( Table 4). AOA of herbal formulation is realized through the AOA of phenolic compounds of the low cudweed herb 20 , hawthorn 21 and rose fruits [22][23] , elecampane 24 and spiny eleuterococcus rhizomes and roots 25 , proanthocyanidins and flavonoids of cowberry leaves 26,27 , flavones, flavanones, phenolic acids 28 and essential oil of pepper mint leaves 29 .
Thus, the studied herbal formulation in the experiment on animals with liver damage induced tetracycline and ethanol has the pronounced hepatoprotective and choleretic effect due to the presence of phenolic antioxidants that have proven antioxidant, hepatoprotective, antiinflammatory and choleretic properties.

CONCLUSION
Eleutheroside B, catechin, epigallocatechin gallate, luteolin, chlorogenic, chicoric, caffeic, ferulic, gallic and isoferulic acids were identified in the 7-component herbal formulation using the HPLC method. The herbal formulation was standardized for the content of active substances: essential oils -not less than 0.30%; flavonoids in terms of luteolin -not less than 1.0%; ascorbic acid -not less than 0.5%; tannins -not less than 3.0%; arbutin -not less than 0.4%; eleutheroside B -not less than 0.01%.
The multicomponent herbal formulation has the antioxidant and choleretic effect, stimulates regenerative processes in the liver and is not inferior in terms of hepatoprotective activity to the reference drug -Cholosas in the model of combined liver damage induced tetracycline and ethanol in rats. The obtained results argue the possibility of using the 7-component collection in prevention and complex treatment of liver diseases.