Introduction
The normal microflora (microbiota) of the gastrointestinal tract (GIT) is a complex ecosystem, including more than 500 species of microorganisms [1].
The total weight of the microbiota is about 3 kg. The microbiota of the gastrointestinal tract supports a significant number of biochemical processes and is compared in importance to the liver. Some researchers even call the microbiota a special “microbial organ,” since the microbiota is involved in immunostimulation, the synthesis of B vitamins and vitamin K, the regulation of motility and other functions of the gastrointestinal tract, the synthesis of short-chain fatty acids (SCFA) and many other processes [2]. Characteristic changes in the microflora profile play a significant role not only in the pathogenesis of gastrointestinal diseases, but also diseases of other organs and systems [3]. The quantitative and qualitative composition of the colon microflora serves as a sensitive indicator of human health and is a “preclinical” marker of homeostasis disorders [4].
In the industry standard “Patient Management Protocol. Intestinal dysbiosis” (OST 91500.11.0004 dated 06/09/2003) normal flora is defined as the qualitative and quantitative ratio in individual organs and systems of various populations of microbes that maintain the biochemical, metabolic and immune balance of the macroorganism, necessary for maintaining health. Intestinal dysbiosis, in turn, is defined as a clinical and laboratory syndrome characterized by changes in the qualitative and/or quantitative composition of the normal intestinal flora, translocation of its representatives to unusual biotopes, metabolic and immune disorders, accompanied by clinical symptoms in some patients [5].
The microflora of the gastrointestinal tract is very sensitive to the effects of such external factors as taking antibiotics, alcohol, psychological and physical stress, radiation, macro- and microelement composition of food [6]. Negative effects of antibiotic exposure include decreased SCFA synthesis, decreased hepatic blood flow, decreased calcium absorption, and damage to the gastrointestinal mucosa [7].
Dietary factors that negatively affect gastrointestinal microflora include: • sulfur compounds (sulfates, sulfites, etc.), which are often used as preservatives for dried fruits, vegetables, apples, packaged juices, flour, most alcoholic products and many medications [ 8]; • a diet high in protein (undigested protein is a source of toxic metabolites such as ammonia, amines, phenols, sulfides, indoles) [9]; • a high-carbohydrate diet with an excess of simple sugars, which increases the enzymatic activity of bacteria and increases the concentration of bile acids, which leads to a slower transit time through the intestines [10].
Treatment of dysbiosis can be carried out with probiotics, i.e. by directly “replanting” the patient with living, active flora. Lactic acid bacteria and bifidobacteria are the most common probiotics (WHO Expert Consultation, 2001) [11].
It should be noted that the bifido- and lactoflora strains taken by a particular patient, as a rule, do not correspond to the unique innate profile of the strains of this patient. The latter makes it necessary to personalized the prescription of specific microflora strains to the patient, which is practically extremely difficult to implement. As a result of the lack of genetic compatibility between the administered strains and the strains of the patient's body, side effects develop (for example, sepsis may occur in children) or the effectiveness of probiotic therapy is extremely reduced and the therapy does not have long-term effects.
The second important direction in the treatment of intestinal dysbiosis is to take into account the fact that different sets of nutrients are needed to feed positive and pathogenic flora. Prebiotics are substances of natural or synthetic origin that selectively stimulate the growth and/or enzymatic activity of one or more types of normal flora. Prebiotics usually include dietary fiber, oligosaccharides (inulin), and lactulose. Taking prebiotics creates a breeding ground for the growth of positive flora [12].
This paper briefly presents the results of metabolomic modeling of one of these drugs (Hilak forte, Teva Pharmaceutical Industries Ltd., Israel), for which there is significant experience in effective clinical use.
In the presented systematic analysis, the following issues are consistently considered: • features of symbiotic and pathogenic microflora; • changes in the microflora profile associated with dysbacteriosis; • molecular factors influencing the survival of microflora; • connection between stress hormones and biofilm formation; • comparative metabolomic analysis of positive and negative flora of the gastrointestinal tract, indicating the basis of the molecular action of the drug Hilak forte; • experience of clinical use of the drug Hilak forte.
Symbiotic and pathogenic microflora
The human intestine contains at least 500 species of microorganisms. It is customary to distinguish between permanent (obligate) microflora, predominant in the number of colony-forming units (CFU) and limited in species composition, and transient (opportunistic) microflora, characterized by diversity, but inferior to obligate flora in quantitative composition.
The obligate microflora of the duodenum and jejunum is dominated by streptococci, lactobacilli and veillonella; in the microflora of the ileum - Escherichia coli and anaerobic bacteria. The large intestine is characterized by a high degree of contamination, which is determined by associations of anaerobic and facultative anaerobic microorganisms: bifidobacteria (108–109 CFU/g of colon contents), lactobacilli (106–108 CFU/g), Escherichia (106–108 CFU/g) and enterococci (105–106 CFU/g) [14].
Normomicroflora is involved in the formation of colonization resistance, i.e., it ensures the prevention of colonization of the gastrointestinal tract by aggressive opportunistic microorganisms (for example, Clostridium difficile and Candida albicans, hemolyzing Escherichia coli, lactose-negative enterobacteria, etc.). Healthy microflora is involved in the processes of detoxification of xenobiotics, including synthetic pharmaceuticals. The fundamental difference between the metabolism occurring in the intestines is that the processes of hydrolysis and reduction dominate in it, while in the liver, oxidation processes dominate.
Changes in microbiota profile associated with dysbiosis
To choose the optimal course of treatment for dysbiosis, it is necessary to diagnose the stage of this disease.
According to the bacteriological classification of I.B. Kuvaeva and K.S. Ladodo (1991), there are four phases of dysbiosis:
I – latent phase of dysbacteriosis [4]. There are no clinical manifestations of dysbacteriosis. There is a decrease in the amount of positive microflora (bifidobacteria, lactobacilli, symbiotic Escherichia coli) several times compared to the norm.
II – starting phase of dysbacteriosis. Functional digestive disorders include constipation or, conversely, sporadic loose stools of a greenish color with an unpleasant odor and a shift in pH to the alkaline side. Plasma-coagulating staphylococci, Proteus, and fungi of the genus Candida multiply.
III – phase of aggression of aerobic flora. Intestinal dysfunction with disorders of motility, enzyme secretion and absorption, frequent, loose stools (often green), decreased appetite, and deterioration of well-being. There is a characteristic increase in the content of aggressive microorganisms (Staphylococcus aureus and Proteus - up to 108, hemolytic enterococci, replacement of full-fledged Escherichia coli with bacteria of the genera Klebsiella, Enterobacter, Citrobacter, etc. is observed).
IV – phase of associative dysbacteriosis. Digestive system disorders, body weight deficiency, pale skin, loss of appetite, frequent greenish stools with a pungent odor and an admixture of mucus (sometimes blood). Vegetation of enteropathogenic serotypes of Escherichia coli, Salmonella, Shigella and other pathogens of acute intestinal infections is characteristic; clostridia may multiply. Starting from the second stage, active growth of colonies of pathogenic microorganisms occurs.
Molecular factors influencing the survival of microflora
There have been no systematic studies on differences in the metabolism of positive and pathogenic microflora of the gastrointestinal tract. Bacteriological studies have identified various molecular factors that may influence the survival of positive flora. These factors include, in particular, the content of pyrophosphate in the bacterial nutrient medium, the resistance of microorganisms to bile acids, the influence of endogenous regulators such as histamine and catecholamines, and the influence of waste products of a certain type of bacteria.
Exogenous pyrophosphate (part of the drug Hilak forte) causes an increase in the growth of E. coli (by 25–35%) even at low levels of glucose in the nutrient medium in vitro and in vivo. Increasing pyrophosphate levels in E. coli culture media modulates the synthesis of more than 20 proteins in bacterial stress response mechanisms. These processes improve the ability of E. coli to utilize carbon-containing nutrients and survive in the intestine [15].
Bile acids significantly regulate the survival of gastrointestinal microflora. For example, an analysis of the sensitivity to bile of 195 enterobacteria in culture showed that the resistance of bacteria to bile decreases in the following order: Shigella > Salmonella > Klebsiella > Providencia. The level of sensitivity to bile determines the ability of enterobacteria to colonize the bile ducts and proximal parts of the digestive tract [16].
Histamine, a known neurotransmitter and regulator of inflammation, makes a certain contribution to the bacteria-host interaction through the so-called. two-component signaling systems regulating SCFA metabolism and bacterial chemotaxis [17]. In the experiment, histamine antagonists also contributed to a decrease in the number of E. coli colonies [18]. This result is very important from a practical point of view. It is traditionally believed that frequently used antihistamines do not affect the state of intestinal microflora. Therefore, when taking antihistamines, additional support for the microbiota should be provided with prebiotic drugs (inulin, Hilak forte, etc.).
. Two-component bacterial signaling systems exert the influence of histamine, glucose and other metabolites on the vital activity of intestinal microflora (using the example of E. coli) [18].
In addition to histamine, molecules such as acetoacetate, polyamines, lipids and glucose also influence the life activity of bacteria through two-component bacterial signaling systems (Fig. 1). Acetoacetate or spermidine initiates activation of the histidine kinase protein AtoS (“first component”), which activates the “second component” of AtoC by phosphorylation of amino acid residues Asp55 and His73. Histamine and calcium have a regulatory effect on this process, regulating the transport of nutrients across the membrane and thereby supporting the survival of positive E. coli strains [18].
Stress hormones – catecholamines and the formation of biofilms by gastrointestinal microflora
It is important to note that the gastrointestinal microflora responds to changes in the levels of stress hormones - adrenaline and norepinephrine. Experiencing acute stress in patients (F45.0) reduces the level of lactobacilli and bifidobacteria within several days (p < 0.001) [19]. At the same time, norepinephrine, adrenaline and dopamine stimulate the division of typical Escherichia coli [20].
The effects of stress hormones on the intestinal microflora are associated with the most important feature of bacterial survival - the formation of the so-called. bacterial films (eng. biofilms). Bacterial films are colonies of bacteria, the survival of which is extremely enhanced due to active cooperation between individual microorganisms (so-called quorum signaling). In a biofilm, bacteria interact with each other and with the surface of the gastrointestinal epithelium. These “clumped” bacterial cells often surround themselves with so-called. a matrix of extracellular polymeric substances – DNA, proteins, polysaccharides [21].
Bacteria capable of forming biofilms have receptors for specific quorum signaling molecules. Of particular interest are the signaling molecules N-acylhomoserine lactones and autoinducer-2, produced by the gastrointestinal microflora. N-acylhomoserine lactones (sometimes also called autoinducers type 1 - AI-1) differ in the length of the R-group side chain, containing chains 4–18 carbon atoms long. N-acylhomoserine lactones with a long R-chain of more than 4 carbon atoms are biologically active, with a longer R-chain corresponding to a more stable signal molecule and a higher affinity of the signal molecule for the corresponding receptor [22, 23]. By binding N-acylhomoserine lactones, the SdiA receptor protein activates the transcription of genes that accelerate bacterial cell division processes for biofilm formation [24].
It is known that Hilak forte contains waste products of typical E. coli, which have mechanisms of response to N-acylhomoserine lactones (including a special transport protein), while, for example, C. difficile, Staphylococcus aureus, C. albicans do not have such mechanisms. Moreover, certain N-acylhomoserine lactones not only promote the survival of typical E. coli, but also inhibit the growth of pathogenic flora. For example, 3-oxo-12-homoserine lactone reduces the virulence of S. aureus by reducing exotoxin secretion and expression of the virulence genes sarA and agr [25]. Certain homoserine lactones also inhibit the development of fungi of the genus Candida [26].
Autoinducer-2 (AI-2), a furanosyl borate ester, is also one of the main quorum signaling molecules in various bacterial species [27]. In general, the molecular mechanism of action of AI-2 can be described as follows. The AI-2 molecule specifically binds to the Lsr-type transport protein and is transported inside bacterial cells, where it undergoes phosphorylation by the LsrK kinase protein. Phospho-AI-2 then binds to the LSRR transcriptional repressor receptor, which is separated from the promoter of the lsr operon (gene group), which leads to the initiation of transcription of the lsr genes. These genes include the luxS gene, encoding the protein of the same name, which in turn plays an important role in the processing of S-adenosyl-L-methionine with the formation of AI-2 as a by-product [28].
Microorganisms of the intestinal flora differ in the presence of quorum signaling systems mediated by the signaling molecule AI-2. For example, the main gene of this signaling system, the luxS gene, was found in the genomes of the positive flora E. coli and Lactobacillus acidophilus, but not in the genomes of Streptococcus faecalis and the yeast-like fungi C. albicans. Therefore, AI-2 will promote the survival and growth of E. coli, L. acidophilus and will not affect the survival of S. faecalis or C. albicans.
Elevated levels of catecholamines are perceived as stress factors by both humans and their microbiota. Catecholamines directly stimulate biofilm formation. Norepinephrine has the most intense effect on the microbiota and leads to the production of the AI-2 molecule by the E. coli bacterium [29]. Other environmental factors that influence the formation of biofilms are high concentrations of tryptophan (high levels of tryptophan, on the contrary, prevent the formation of biofilms by E. coli [30] and certain, currently not yet identified, peptides secreted by bacteria. Since treatment of the bacterial growth medium proteinase K significantly reduced the degree of bacterial aggregation into biofilms, which indicates the important role of protein components in the formation of microbiota biofilms [31].
Thus, analysis of the genomes of the studied representatives of the gastrointestinal tract microbiota showed that the genes responsible for the synthesis and cellular response of microorganisms to biofilm growth stimulators (AI types 1 and 2) are present in such representatives of the microbiota as E. coli and L. acidophilus Therefore, it can be assumed that autoinducers that promote the survival of bacterial films of the microbiota are present in germ-free extracts of the waste products of these microorganisms.
Comparative metabolomic analysis of positive and negative intestinal flora and the basis of the molecular action of the drug Hilak forte
It is known that extracts of waste products of a certain type of bacteria stimulate the growth of certain bacteria. When comparing 9 cultures of bifidobacteria and 18 opportunistic microorganisms in vitro isolated from patients with intestinal dysbiosis, it was shown that bifidoflora reduced the levels of growth factors, anti-lysozyme activity and the formation of biofilms of Klebsiella pneumoniae, Staphylococcus aureus, C. albicans [32].
Separate studies of the molecular components included in the extracts of waste products of a number of bacteria have been carried out. Liquid and gel chromatography of low molecular weight exometabolites of the culture liquid of E. coli strain M-17 showed that the stimulating activity of the extract is partly due to glutamic and succinic acids [33]. However, a systematic analysis of the composition of such extracts of bacterial activity has not been carried out.
This section presents the results of our analysis based on metabolome models of several typical representatives of the gastrointestinal tract microflora. Note that the terms “genome”, “proteome”, “metabolome” mean the totality of all molecules of a certain type found inside a particular organism. The genome is the collection of all the genes of an organism. The gene expression processes of the genome lead to the formation of a set of all corresponding proteins (proteome). Protein enzymes support certain chemical reactions, which result in the formation of various metabolites that form the body's metabolome.
Thus, the metabolome is the entire array of metabolites or the so-called. small molecules of a biological system. Obviously, if the genome of a particular organism is known, it becomes possible to determine its proteome. Further, establishing the functions (so-called annotation) of proteome proteins makes it possible to model the body’s metabolome. Data of this kind are presented in numerous bioinformatic databases, among which the KEGG [34], MetaCyc/BioCyc [35] and Reactome [36] databases should be especially noted.
Data on the metabolomes of various organisms are extremely important information for the development and analysis of the molecular effects of drugs [37]. In the present study, the method of comparative metabolomic analysis [37] was applied to various representatives of the intestinal microbiota in order to establish the range of possible mechanisms of molecular effects of the drug Hilak forte. Obviously, an effective and safe prebiotic drug should support the survival of positive intestinal flora and inhibit (or at least not support) the growth of negative flora. Since Hilak forte contains part of the metabolome of bacteria corresponding to positive microflora, comparison of the metabolomes of these microorganisms with the metabolomes of bacteria of negative flora indicates the most likely molecular pathways through which the therapeutic effect of the drug is achieved. Summary information on the studied metabolomes is presented in Table. 1.
As can be seen from the data presented in table. 1, the metabolomes of different bacterial species have a significant number of common metabolites. For example, of the 1230 metabolites found in the 4 positive flora metabolome models, 396 molecules were included in each of the 4 metabolomes. In the case of pathogenic flora, out of 970 molecules of 3 metabolomes, 350 were included in each of the studied metabolomes.
Escherichia coli (E. coli) is a gram-negative rod-shaped bacterium, a component of normal intestinal flora. E. coli synthesizes vitamin K [38] and prevents the growth of pathogenic microorganisms in the intestine [39]. Pathogenic strains of E. coli (for example, serotype O157:H7, etc.) are not typical for the microbiota. Fecal enterococcus (Enterococcus faecalis, S. faecalis) is part of the microbiota, playing an important role in ensuring colonization resistance of the gastrointestinal mucosa. Pathogenic strains of E. faecalis that are not typical for the microbiota are the causative agent of various infections, including nosocomial infections. L. acidophilus and L. helveticus are species of bacteria of the genus Lactobacillus that ferment lactose to lactic acid and survive in the digestive tract at a lower pH value (pH = 4.5 or less), i.e. in more acidic environments. Lactobacillus acidophilus is used as probiotics, the effect of which is largely due to the lactic acid they produce. Lactic acid provides a highly acidic environment and creates unfavorable conditions for the life of many pathogenic and opportunistic bacteria (staphylococci, Proteus, enteropathogenic Escherichia coli), the optimum growth of which is in the more alkaline pH range (pH = 6.7 or more). It is interesting to note that the role of the metabolome of Lactobacillus bacteria extends beyond the gastrointestinal tract. In particular, L. helveticus secretes tripeptides that reduce high blood pressure through inhibition of angiotensin-converting enzyme [40].
As a result of a comparative metabolomic analysis of the bacteria described above with the metabolomes of C. difficile, S. aureus and C. albicans, more than 90 metabolites were identified that were found in representatives of the studied positive flora (i.e. E. coli, S. faecalis, L. acidophilus, L. helveticus) and at the same time absent from the studied representatives of C. difficile, S. aureus, C. albicans. The main groups of these metabolites are given in table. 2.
Thus, comparative metabolomic analysis showed that positive microflora contains metabolites necessary for the synthesis and processing of vitamins (vitamins B6, B2, K), supports the reactions of processing bile acids, neutralizing certain metabolites (processing of oxalates, conversion of amines into urea, synthesis of glutathione) , synthesis of succinic acid and biosynthesis of SCFAs. The metabolomes of these representatives of the microbiota contain a number of specific sugars (for example, the prebiotic melibiose, i.e., the disaccharide of galactose and glucose). The presence of these molecules in extracts of waste products will accelerate the growth of positive flora without stimulating the growth of pathogenic flora.
For example, metabolites associated with the biosynthesis of vitamin B6 (pyridoxal derivatives, etc.) produce vitamin B6 not so much for the needs of the host organism, but to maintain the metabolic activity of the microbiota itself. According to the present analysis, vitamin B6 derivatives such as pyridoxal phosphate are part of more than 70 enzymes important for E. coli, such as glutamate decarboxylase, tryptophanase, serine hydroxymethyl synthetase, cysteine desulfurase, alanine racemase (metabolism of amino acids), porphyrinobilinogen deaminase (metabolism bile acids), adenylosuccinate synthase, 4-aminobutyrate synthase (SCFA metabolism), and the downstream selenium homeostasis enzyme, Se-cysteine synthetase. As a cofactor of a protein involved in the transport and processing of maltose, pyridoxal phosphate directly determines the ability of E. coli to process maltose and is therefore required for the survival of this and other bacteria in the microbiota [41].
. Results of a comparative metabolomic analysis of the studied microorganisms of the intestinal microflora (We emphasize that these metabolites are part of the metabolomes of positive flora and are absent in the metabolomes of C. difficile, S. aureus, C. albicans).
It is important to note that our data (Table 2) clearly illustrates the correctness of considering the microbiota as a specific “microbial organ” involved in the regulation of the metabolism of vitamins B6, B2, K in the host organism. Moreover, the comparisons sometimes found in the literature of this “microbial organ” with the liver are also very justified: for example, positive flora significantly contributes to the processing of bile acids, thereby complementing one of the most important functions of the liver.
An extremely interesting result of this study is the establishment of the relationship between the metabolome of the most important representatives of the microbiota and oxalate metabolism. It is known that excess oxalates in the body leads to diseases such as urolithiasis, arthritis, and potentiates the development of atherosclerosis due to the maintenance of chronic inflammation of the endothelium of blood vessels. At the same time, the oxalate anion is a very acceptable source of dietary carbon for microbiota bacteria [42], which transform excess oxalate, which is toxic to the host organism, into carbon dioxide.
In bacteria of positive flora, this process is carried out by the enzyme formyl-coenzyme A transferase and the Mg2+-dependent enzyme oxalyl-coenzyme A decarboxylase. This process depends on at least three micronutrients: pantothenic acid (vitamin B5, necessary for the synthesis of coenzyme A), thiamine diphosphate (vitamin B1) and magnesium. Since oxalate transformations are one of the important carbon sources for bacteria of positive flora, the survival of the microbiota largely depends on the nutritional supply of the body with vitamins B1, B5 and magnesium.
According to the present analysis (Table 2), an important difference between the microbiota metabolomes and the metabolomes of pathogenic microflora is the presence of metabolites associated with the biotransformations of inorganic selenium. Any microorganism needs the amino acid selenocysteine, which is necessary for the synthesis of more than 10 selenoproteins. However, of the microorganisms examined in this study, only representatives of positive microflora are able to synthesize selenocysteine from inorganic selenium.
Selenocysteine biosynthesis is supported by two enzymes: selenide-water dikinase and selenocysteine synthetase. Selenide-water dikinase is a magnesium-dependent enzyme that synthesizes an important intermediate product, the selenophosphate molecule (HO3PSe), from inorganic selenide (Se-2), ATP and water. The vitamin B6-dependent enzyme selenocysteine synthetase transforms selenophosphate and serine into selenocysteine. The resulting selenocysteine is incorporated into bacterial selenoproteins such as ydfZ, selA, selB, etc. Thanks to these enzymes, E. coli and other representatives of the microbiota can use inorganic selenide directly from the environment.
The comparative analysis of metabolomes also allows us to formulate promising directions for research into the composition of the drug Hilak Forte. For example, the presence of selenide ion and other selenium derivatives in the metabolomes of E. coli, S. faecalis, L. acidophilus, L. helveticus suggests a fairly high selenium content in the preparation Hilak forte. Therefore, it is very promising to study the content of selenium and other trace elements in this preparation using adsorption mass spectrometry. The presence of biotransformation products of various vitamins (B1, B2, B6, K, carotenoids) in the metabolomes of these microorganisms suggests the importance of studying the vitamin profile of the drug.
Clinical experience with the use of the drug Hilak forte as confirmation of the results of metabolomic analysis
This drug for the treatment of dysbiosis is based on an extract of metabolic products of such representatives of the microbiota as E. coli DSM, S. faecalis DSM, L. acidophilus DSM, L. helveticus DSM. The drug contains SCFAs, which ensure restoration of the affected intestinal microflora [43]. In addition, the drug contains significant amounts of phosphoric, citric and lactic acids, which help normalize the pH and the growth of positive gastrointestinal microflora. The drug promotes the rapid restoration of positive intestinal microflora disturbed during the use of antibiotics or irradiation, and is indicated for dyspepsia, constipation, diarrhea, flatulence, gastroenteritis, allergic skin diseases and acute intestinal infections.
Clinical work carried out by Russian and foreign researchers has shown the high efficiency and safety of using the drug in clinical practice. The drug Hilak forte is used in the prevention of dysbiosis after antibiotic therapy [44], is an effective means for maintaining microbiocenosis and preventing the development of dysbiosis [45], is effective as an adaptogen for acute respiratory infections in children [46], and is successfully used by patients with irritable bowel syndrome [47– 49]). When using Hilak forte, normalization of the levels and ratios of SCFAs was noted [50], it helps to accelerate recovery from acute intestinal infection [51, 52]. The use of Hilak forte is also effective for patients with irritable bowel syndrome: after only 2 weeks of taking the drug, partial or complete regression of subjective complaints, normalization of the pH and microbial spectrum of feces were observed [53, 54].
Of particular note are the results of a comprehensive randomized study of the effectiveness of the drug [55] in a group of 84 patients (36–74 years) with functional constipation lasting 1.5–27.0 years (average 8 years). Patients of group 1 (main, n = 32) received Hilak forte (40–50 drops 3 times a day for 4 weeks) as part of complex therapy. In addition to a significant improvement in clinical symptoms, the bacteriological study of feces of patients with degree II of intestinal dysbiosis carried out in this work revealed positive dynamics when taking Hilak Forte: during the treatment, fungi of the genus Candida and Proteus disappeared, and the amount of hemolyzing form of E. coli decreased. Therapy with Hilak forte leads to certain changes in the profile of short-chain acids: the proportion of acetic acid significantly decreases (p < 0.001), the proportions of butyric and propionic acids increase (p < 0.01) [55]. In general, clinical studies have shown that Hilak Forte actually has a differentiated effect on the survival of positive and pathogenic microflora, i.e., it promotes the survival of positive intestinal microflora and the elimination of pathogenic flora, having a clear healing effect on the metabolome of the “microbial organ.”
Molecular mechanisms of action of components of germless microbiota extracts.
Indications for use of the drug Hilak and Hilak Forte
Violation of the physiological flora of the small and large intestines (during and after treatment with antibiotics or sulfa drugs, radiation therapy), chronic gastrointestinal diseases; maldigestion syndrome, dyspepsia; diarrhea, flatulence, constipation; gastroenterocolitis, colitis; dysfunctions of the gastrointestinal tract caused by climate change; hypo- and antacid conditions, including during pregnancy; allergic skin diseases, urticaria (endogenously caused and chronic eczema); after acute intestinal infections (salmonellosis, etc.), including in infants.
Use of the drug Hilak and Hilak Forte
Drops are taken orally before or during meals in a sufficient amount of liquid (except milk) 3 times a day: adults and children over 12 years of age - 40-60 drops per dose; children over 2 years of age - 20–40 drops per dose; children under 2 years of age - 15–30 drops per dose. After the condition improves, the daily dose can be halved. The duration of treatment is usually 2–4 weeks. The need for longer use is determined by the doctor.
special instructions
During treatment you should follow a number of recommendations:
- Do not drink Hilak Forte with milk, dairy or fermented milk products
- Do not take antacids, as they neutralize the acids that make up Hilak Forte.
- An open bottle should be stored for no more than 1.5 months.
- The drug does not require special storage conditions. It is enough to adhere to the temperature regime – from +4 to +25 degrees Celsius.
