Histological research of hepatoprotective activity of tablets Lavaflam in rats with subchronic hepatitis

Materials of research. Experimental pharmacological studies were performed on the white rats weighing 180–220 g. The study was carried out in the Central Research Laboratory (CRL) of the National University of Pharmacy, Kharkіv, Ukraine (Certificate No 023/13 of 05.03.2013) according to the methodological recommendations of the State Expert Center of the Ministry of Health of Ukraine. The rats were divided into four experimental groups: the first group – intact control; the second group – positive pathology – the animals were injected TChM; the third group – animals, received TChM and Lavaflam; the fourth group – animals, received TChM and reference drug Сarsil.

Hepatobiliary system diseases are medical and social problem at the present time as the diseases affect people of all countries and are common to all social strata and to all age groups. According to the current data by WHO hepatobiliary pathology has 30 % of the planet population [1]. In Ukraine the incidence of hepatitis has increased by 76.6 % over the past 10 years and prevalence -in 2.2 times. Mortality has doubled over the past 20 years, and 60% of patients are people of working age [1]. Hepatoprotective drugs are used for hepatobiliary pathology treatment regardless of the etiology. At the same time, the preparations of plant origin are played a significant role in this process. These preparations are with following herbs: Cynara scolymus, Silybum marianum, Helichrysum arenarium.
A new combined herbal drug Lavaflam was developed under the direction of Doctor of pharmaceutical sciences, associate professor Bobrytskaya L. at the Department of Industrial Technology of Drugs in the National University of Pharmacy, Kharkiv (Ukraine). 1 tablet of Lavaflam contains 50 mg of Flamin and 20 mg of Lavender oil, medical form is tablets 0.6 grams № 50 ( Table 1) [3]. Lavaflam will be produced by the pharmaceutical company "Zdorovye" (Kharkіv, Ukraine). Analysis of the literature shows hepatoprotective properties of biologically active substances (flavones, flavonols, flavonones and chalcones, oxycinnamic acids, terpene compounds, glycosides, polysaccharides, alkaloid of coumarin etc.), which are the parts of Lavaflam tablets, and their wide application in hepatology [4]. Lavaflam is predicted to have choleretic, antiseptic, antispasmodic, antibacterial, anti-inflammatory effects, to improve the detoxification function of liver, normalize of gallbladder tone, increase biliary tract peristalsis, increase cholagogue activity.
So it made sense to study hepatoprotective properties of Lavaflam tablets and to determine a possibility to use it as a hepatoprotector.
Pharmacological study of the Lavaflam hepatoprotective properties was conducted as a preclinical study on the model of subchronic hepatitis in rats induced by tetrachloromethane (TChM).
The purpose of the study was histological evidence of the drug Lavaflam hepatoprotective properties in the experimental model of subchronic hepatitis.
Research objectives: 1. Development of experimental model of subchronic hepatitis in rats.
2. Comparison of hepatoprotective properties of Lavaflam and Carsil preparations on the basis of liver cells, portal vein, hepatic artery, hepatic venules, arterioles, sinusoidal hemocapillaries, bile duct epithelium histological examination.
Object of study: hepatoprotective properties of Lavaflam tablets.
Methods of research: pharmacological, morphological, histological, methods of mathematical statistics.

Materials of research
Experimental pharmacological studies were performed on the white rats weighing 180-220 g. The study was carried out in the Central Research Laboratory (CRL) of the National University of Pharmacy, Kharkiv, Ukraine (Certificate № 023/13 of 05.03.2013) according to the methodological recommendations of the State Expert Center of the Ministry of Health of Ukraine [5]. Rats that are included in the study were grown in the vivarium and before the experiment they were acclimated in the room for research during seven days. All studies were performed accordingly with the requirements of the European convention for the protection of vertebrate animals used for experimental or other scientific purposes [6].
Study of hepatoprotective activity of Lavaflam tablets was performed on the model of experimental subchronic hepatitis in rats caused by the intragastrically introduced TChM in the form of 50 % oil solution in dose of 0.4 ml/100 g of animal weight during 4 days by dint of metal probe [7].
The drug Lavaflam and the reference drug Сarsil (Sopharma, Bulgaria, tablet 22.5 mg, series F45522) that were used for experimental subchronic hepatitis treatment, were studied on the next day after the pathology modeling. Lavaflam and Carsil tablets were administered prophylactically intragastrically once a day for a week prior to the onset of pathology development formation. In the last days of the experiment (7 days) the introduction was continued in the treatment regimen -intragastrically, once a day, for 4 days and 3 days after the formation of pathology. In general, the course of the studied drugs application was 14 days.
The rats were divided into four experimental groups, 10 animals in each: the first group -intact control; the second group -positive pathology -the animals were injected TChM; the third group -animals, received TChM and Lavaflam in a dose of 50 mg/kg body weight of the rats; the fourth group -animals, received TChM and reference drug Сarsil in a dose of 50-5 mg/kg body weight of the rats. The doses of Lavaflam and the reference drug Сarsil were calculated on the coefficients of species stability by Yu. R. Rybolovlev [8]. All the experiments were carried out according to the requirements of the EC Directions 86/609/EEC November 24, 1986 concerning care and use of laboratory animals.
Histological assessment of the liver morphological state was performed. Histological material was fixed in 10 % solution of neutral formalin and it was embedded in celloidin paraffin. Liver slices were stained with hematoxylin and eosin. Micropreparations were observed with the microscope Granum, microphotographs of images were taken using a digital video camera Granum DSM 310. Photos were processed on a computer Pentium 2.4GHz using the program Toup View.
The degree of liver damage in comparison with the groups of rats -intact control (the first group of animals) and positive pathology (the second group of rats) was taken into account to assess microscopic changes in the liver. Semi-quantitative assessments of the pathological process and the influence of Lavaflam on it were made on the liver slices. The following signs were evaluated: severity of fatty dystrophy, necrotic manifestations and existence of tissue pattern violation. Sokolovsky's method was taken as the basis for semi-quantitative visual assessment of histochemical changes.
While applying the method of mathematical statistics, the significance level of p < 0.05 was accepted. In order to obtain conclusions when comparing statistical samples of relative variables, after the single-factor analysis of variance (or the Kruskal-Wallis criterion for data not subject to the normal distribution), differences were found between the experimental groups; the Newman-Keils criteria, the Student's criteria for multiple comparisons or Mann-Whitney criterion, for nominal data -Fisher's exact criteria were used. For the mathematical calculations the standard statistical software package Statistica 6.0 was used [9].

Results and discussion
Microscopic examination of experimental animals' liver was performed after autopsy. In animals of intact control (the first group of rats) the liver had not expressive a typical structure segmental pattern of tissue. The segments borders were determined by triads, these zones were narrow. Endothelium conditions of the portal vein, hepatic artery, bile duct terminal branches, which are parts of triads, and other blood vessels, were normal. The radial direction of the hepatic plate system was not disturbed. Hepatocytes had a typical shape and size, distinct cell membranes. The nuclei of cells were oval and centrally located; they contain basically one, at least 2 nucleoli. Cell cytoplasm was uniformly colored; it did not contain any visible corpuscles on the optical level. The content of dual-core cells was normal. The cells that were in some distribution phases were not found in the micropreparate. Sinusoidal capillaries were moderately dilated, sometimes with erythrocytes. The lymphoid cells number in the capillaries was moderate. The condition of stellate reticuloendotheliocytes (Kupffer cells) was normal (Fig. 1).
Explicit violation of the liver parenchyma was found in the rats of the second group (positive pathology group) 3 days after the last TChM administration. The changes were localized in centrolobular and periportal zones of the segments. The destructive zones size varied from 1/2 to 2/3 of hepatic lobules. Sometimes these zones merged with each other (Fig. 2).
We observed discomplexation, hepatic plate's radial direction loss, scattered unicellular necrosis of hepatocytes, pronounced fat and protein degeneration of the cells. Manifestations of inflammatory response were minimal. Terminal hepatic veins were dilated; however sinusoidal capillaries were often compressed and poorly visible. Fatty degeneration in the destruction zones was mainly of medium and microvesicular character, in places small fat cysts were seen. Small groups of activated Kupffer cells were observed among necrotized hepatocytes (Fig. 3).
In the restricted parts of the lobules (the intermediate zone of the liver acini), the hepatic parenchyma was relatively retained. Hepatocytes of such zones were often swollen with blurred cell borders and fine cytoplasmic vacuolization. The radial direction of cells was not clear. There was a noticeable decrease in dual-core cells. At the same time, manifestations of regenerative processes in the form of moderate anisonucleosis were observed (Fig. 4). In the group of animals that received TChM and Lavaflam (the third group of rats) we observed significant improvement of the hepatic parenchyma structural organization in most animals. Destruction zones are clearly reduced (Fig. 5).
Actually, it has been noted no protein degeneration of hepatocytes, fat dystrophy was reduced, there were fine vesicles, they do not violate cell integrity and fatty cysts were absent. Necrosis was in a small number of cells. Microcirculation was improved (Fig. 6).
Hepatocytes on unchanged parts of the segments kept the radial direction in hepatic plates, they were morphologically more complete. The pool of dual-core cells was enlarged. We observed polymorphism of cells and nuclei and a lot of polyploid cells. They had an increased in size nucleus and cytoplasm, and hyperchromias were observed in theman increase in the amount of chromatin in the nucleus (Fig.  7). This indicated a physiological regeneration of the liver. Moreover, the increase in the number of binuclear cells was regarded as an increase in their adaptive capabilities as they synthesize more protein than single-nucleated cells.
In the group of animals with induced subchronic hep-atitis by TChM after administration of the reference drug Сarsil (the fourth group of animals) there was a more noticeable fluctuation in the prevalence of the hepatic lobules parenchyma pattern zones of disturbance. Hepatocytes had middle-vesicular fatty degeneration in destructive zones. Protein dystrophy was not found in all animals. Necrotic cell changes were minimal. Outside the zones of hepatocytes destruction were morphologically more complete. Swelling of the cells was absent, cell membranes were distinct and cytoplasm vacuolation was not expressed. It was found that anisonucleosis was moderate, binuclear cells were noticeable in various zones of lobules in sufficient quantity. Vascular disorders, cellular response were not expressive in all animals (Fig. 8).
It has been revealed that Lavaflam significantly reduced the zones of destruction, the severity of hepatocytes fatty degeneration and necrotic manifestations. According to the expressive influence on necrotic manifestations, Lavaflam is not inferior to the drug of comparison Carsil and ahead of it in its effect on the destruction zones prevalence, and shows a tendency to reduce changes in lipid metabolism ( Table 2).