The bile acid composition analysis of the lipophilic complex of animal bile

Using gas chromatography, the qualitative composition and the quantitative content of bile acids in the lipophilic complexes of animal bile was studied. As a result of the studies, 6 steroids were identified in the lipophilic complexes of bile, 5 of which are bile acids. It was defined that cholic and deoxycholic acid predominate in all the studied objects, while their amount depends on the used raw materials.


The purpose of the study
The aim of the study was to study the bile acid composition of lipophilic complexes (LC). The objects of the study were bile samples obtained from bile of cattle (BBt) and pigs (BSs).

Materials and methods
Determination of bile [4,5,8,9,11] acids in the LC of animal bile (Bos taurus L., Sus scrofa domesticus L.) were carried out by the method of gas-liquid chromatography (GLC) on polar stationary phases with previous methylation. The allocating and registration of bile acids was carried out on a gas chromatograph "Chrome-5" on a metal column 2.6 m long, filled with a sorbent "Chromaton-super" with 5% oV-17. The analysis of samples of free bile acids was carried out in an isothermal mode at 200 °C and heating of the flame-ionization detector -250 °C. The speed of the carrier gas of high purity nitrogen is 50 ml/min, hydrogen -30 ml/min, air -300 ml/min.
A 300 mg bile sample was placed in a 25 ml flask, 6 ml of Folch solution (chloroformmethanol 2:1) was added for the dissolution of lipophilic components of bile and separation from protein in the dry fraction of bile. The contents were mixed and heated on a water heater at 50 °C for 5 minutes. Samples with a dry fraction were centrifuged at 3 thousand rpm for 10 minutes. 10 ml of 20% NaOH solution ware added to the solutions and hydrolysis was carried out at 150 °C for 30 minutes. The test tubes were cooled, 5 ml of ethyl acetate was added, thoroughly mixed, and after retention, the upper phase was sampled, transferred to a 40 ml reaction tube, evaporated to a dry residue on an water heater under nitrogen. 0.1 ml of trimethyltrichloroselene solution was added to the dry residue in order to convert it into an inert compound. To replace the active groups of bile acids, the sample was subjected to additional methylation with 5% sulfuric acid in methanol at 80 °C for 30 minutes. After cooling the samples, 3 ml of purified water was added, intensively shaken, 5 ml of a mixture of chloroform-hexane-methanol was added in a ratio of 10: 10:1, thoroughly mixed and after retention, the upper phase was selected, transferred to a centrofuge tube, in which the solution was evaporated to 0.5-2 ml, for insertion into a gas chromatograph. Qualitative analysis was carried out by comparing the retention time of bile acids with known samples. The quantitative content is calculated by the formula: where: Cx is the concentration of bile acid in the complex, mg; Cst is the concentration of standard bile acid, mg; Ssample is the area of the peak of bile acid in the complex, sq.mm; Sst is the peak area of standard bile acid, sq.mm.

Results and discussion
The results of the study of the steroid composition of animal bile and LC bile are shown in Tables 1-2 and Fig. 1. The content of bile acids in the BSs is 1.36 times higher than in the BBt and cholesterol is 1.49 times higher, which indicates the prospects of using BSs to produce bile acids.
For bile LC, methylation was carried out without adding a Folch solution and centrifugation, and began with the addition of 5 ml of ethyl acetate and 10 ml of 20% NaOH solution, then followed the procedure described above. The results of the analysis are presented in Table 2. The quantitative determination showed that the highest content of bile acids is in the LC of BBt -1.22 times more than in the LC of BSs, while the cholesterol content in the LC of BBt is 1.24 times less than in the LC of BSs. As can be seen from Table 2, as a result of the study, 6 steroid compounds were identified in the bile LC, of which 5 belong to bile acids. It was found that cholic and deoxycholic acid predominate in all the studied objects, while there is more cholic acid in LC BBt and more deoxycholic acid in LC BSs.
Comparing the content of cholesterol and bile acids in native bile (Table. 1) and the obtained LC (Table. 2), it was found that the concentration of bile acids increases about 5.45 times for BBt and 3.28 times for BSs, and cholesterol decreases by 1.9 times. The results of the comparative analysis are presented in the diagram (Fig. 1).
The technology for obtaining LC bile also makes it possible to obtain a glycoprotein complex (GPC) and a water residue of bile (WRB), for the creation on their basis of new drugs with established pharmacological activity.
By methods of liquid-liquid extraction and chromatography in the complexes of animals bile the presence of 137 compounds was established: bile acids (45), fatty acids (20), amino acids (17), among them -9 essential, 14 fatty acids, phospholipids (13), cholesterol (1), porphyrins (16), enzymes (6), macro-and micronutrients (19). By two-dimensional chromatography in the LC of bile revealed 75 substances which by chromatographic mobility, fluorescence under UV-light and the colouring with specific reagents were referred to steroids (46), phospholipids (13), porphyrins (16). Chinese medicine has incorporated the use of bear gall bladders and bile for over 1300 years. The Pharmacopoeia of the People's Republic of China (1977) indicates that bear gall bladder is useful for cleansing heat symptoms, calming the liver and brightening the eyes. It is also used for treating convulsions, conjunctivitis and pharyngolaryngitis. Pharmacological and clinical studies have confirmed the therapeutic effects of bear bile, especially of its components, ursodeoxycholic acid (UDCA) and chenodeoxycholic acids (CDCA), for the treatment of gallstones and liver diseases. Pig bile has also been found to have antiinflammatory, nticonvulsant and analgesic effects, and can prolong the survival time of mice under hypoxic conditions. The bile of mammals, as well as of other animal species, results from metabolic processes in the liver. Bile consists of approximately 86% water and 14% dry matter. The most important components of the dry matter are free and conjugated bile acids.
The antimicrobial activity of LC in concentration 0,1% against Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis and Candida albicans and antibacterial activity of WRB BBt towards Staphylococcus aureus and Candida albicans were established. Pharmacological researches were conducted in vivo and in vitro. It was established that bile GPC helps absorption of fats, activate lipase in digestion and stimulates peristalsis in experimental animals.
The WRB has expressed antihypoxic action and stabilizes the membranes. LC showes pronounced hepatotropic and antioxidant effects. WRB in a dose of 12 ml/kg, LC -20 mg/kg, GPC -25 ml/kg at 30-day intragastric introduction do not have toxic effects on the peripheral blood picture and morpho-functional state of the internal organs in the subacute experiment. Complexes can be recommended as medications to improve digestion, in violation of cerebral and peripheral circulation, redox metabolism in tissues, at hepatitis, as a tonic, stabilizing agent in adverse effects.

Conclusions 5
1. The qualitative composition was studied by gas chromatography and the quantitative content of bile acids and cholesterol in native bile and LC was established.