RU2629331C1 - Drug with immunomodulatory effect - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: drug with an immunomodulatory and antiviral effect, which is an extract of the herbaceous saltwort [Salsola laricifolia], obtained by extraction of dry herbaceous saltwort [Salsola laricifolia] with 30-80% aqueous solution of polyhydric alcohol at 60-90°C; using the extractant:dry grass ratio of 1:(0.003-0.6), respectively.

EFFECT: drug has a pronounced immunomodulatory and antiviral effect, effectively increases the nonspecific resistance of the mammalian organism.

10 cl, 9 dwg, 67 tbl, 55 ex

Description

FIELD OF THE INVENTION

The invention relates to medicine and veterinary medicine, in particular to the field of development of new effective herbal remedies-immunomodulators.

State of the art

It is known that the value of immunomodulators is currently increasing in clinical medicine and veterinary medicine. Immunomodulators, allowing to implement an adequate immune response to a pathogenic agent, providing treatment without the use of toxic chemotherapeutic agents, are perceived as the most promising way of pharmaceutical development. Recovery and stimulation of the immune response is of great importance not only for infectious diseases, but also in oncology, because stopping the immunosuppression caused by chemotherapy and the tumor itself, significantly increases the chances of patients to recover. For these reasons, in recent years, a large number of immunomodulating drugs have been introduced into the clinic, characterized either by an immunoprotective effect, or to some extent stimulating some immune functions (phagocytosis, the activity of T-killers, natural killers, etc.). Most of the preparations are xenobiotics, of which cycloferon (meglumine acridone acetate), tiloron (amixin), glutoxim, levamisole, polyoxidonium can be mentioned [Radar Drug Encyclopedia, 8th ed., Ed. Yu.F. Krylova, M., 2001], leacadine [Radar Encyclopedia of Medicines, M., 2004], Galavit, Kagocel, immunofan [Radar Encyclopedia of Medicines, M., 2007], Diucipon [RU 2292892, publ. 02/10/2007], sulphiphone [RU 2136668, publ. 09/10/1999], ingavirin [Vidal Handbook, 17th ed., M., 2011], thymogen [M.D. Mashkovsky, Medicines, 16th ed., M., 2012] and others. A common disadvantage of this category of substances is an unbalanced effect on the complex immune system, which often causes severe side effects (agranulocytosis, autoimmune diseases, etc.). Xenobiotics, on the other hand, often have hepatotoxicity. More safe are drugs of natural origin - tinctures of Leuzea, Eleutherococcus, Rhodiola rosea, ginseng, Echinacea (Immunal), standardized extract of potato seedlings (Panavir), etc. The most widely used immunomodulators are extracts and tinctures of Echinacea, which, in fact, belong to them tinctures of leuzea, eleutherococcus, Rhodiola rosea, ginseng, etc. are classified as adaptogens). In 1996, for example, in the USA, echinacea preparations were the most popular among herbal remedies [Bertram G. Katzung, Basic and Clinical Pharmacology, 2nd ed., Vol. 2, 2008]. Therapists do not remain without the attention of therapists in Echinacea in Russia [Radar Encyclopedia of Medicines, M., 2007; V.Yu. Martov, A.N. Hams, Medicines in the practice of a doctor, 2nd ed., M., 2010; Vidal Handbook, 17th ed., M., 2011; M.D. Mashkovsky, Medicines, 16th ed., M., 2012], with each year their nomenclature is expanding on the market [Russian Radar, 8th ed., Ed. Yu.F. Krylova, M., 2001; Radar of Russia, M., 2007]. In an in vitro experiment, E. purpurea juice increased the production of human phagocytes of interleukins-1, -6, -10 and tumor necrosis factor-α, increased the activity of natural killers and the level of antibody-dependent cytotoxicity. Echinacea extracts also show anti-inflammatory effects associated with inhibition of cyclooxygenase [Bertram G. Katzung, Basic and Clinical Pharmacology, 2nd ed., T. 2, 2008]. In the clinic, their use significantly reduces the severity of symptoms of acute respiratory infections and the timing of recovery. Given the very low toxicity and relatively rare side effects, the interest in the development of immunomodulators based on juice or extracts of plants of the genus Echinacea is very high. Only recently, a large number of drugs and dietary supplements have been created for use in medicine (RU 2468810 publ. 10.12.2012, RU 2137490 publ. 09/20/1999, RU 2182011 publ. 10.05.2002, EP 0464298 publ. 08.01.1992, etc. ) and veterinary medicine (CN 102716164 publ. 10.10.2012, RU 2525426 publ. 08/10/2014, RU 2356557 publ. 05.27.2009, etc.). However, drugs from plants of the genus Echinacea far from solve the problem of safe effects on the immune system. Firstly, the effect of these drugs is unbalanced, they can aggravate autoimmune diseases [Bertram G. Katzung, Basic and Clinical Pharmacology, 2nd ed., T. 2, 2008], and their use is contraindicated in immunodeficiency states (AIDS, cancer) autoimmune diseases of any etiology, tuberculosis and other nosologies, in which patients just need safe immunomodulators. Secondly, no significant increase in disease resistance was found with prophylactic use [Bertram G. Katzung, Basic and Clinical Pharmacology, 2nd ed., T. 2, 2008; M.D. Mashkovsky, Medicines, 16th ed., M., 2012]. Thirdly, most Echinacea-based drugs contain a large amount of ethanol, which limits their use in pediatrics and is dangerous when taken with drugs that have an inhibitory effect on alcohol dehydrogenase. Fourth, echinacea is a plant of the family Asteraceae, and allergic reactions relatively often develop to these plants [Bertram G. Katzung, Basic and Clinical Pharmacology, 2nd ed., T. 2, 2008], incl. and quite severe - anaphylaxis, attacks of bronchial asthma.

A number of drugs with high immunomodulating and antiviral activity based on peptidoglycans and acidic glycans isolated from the growing shoots of various plants have been developed [RU 2195308 publ. 12/27/2002, RU 2151600 publ. 06/27/2000, RU 2028303 publ. 02/09/1995, RU 2003110010 publ. 10/20/2004]. The drug “Panavir” was introduced into medical practice, which has established itself as a safe and effective immunomodulator [M.D. Mashkovsky, Medicines, 16th ed., M., 2012]. The disadvantage of these drugs is a narrow and limited effect on the immune system and the need to use injection in a hospital.

Many complex phytopreparations with an immunomodulatory effect based on components have been proposed, for which a certain effect on the immune system is known - plants of the genus Echinacea, buckthorn buckthorn, licorice, aloe, chaga, lewsea, Aralia manchurian, ginseng, lemongrass, cat's claw, rhodiola rosiola RU 2516932 publ. 05/20/2014, RU 2510277 publ. 03/27/2014, CN 103933097 publ. 07/23/2014, UA 49851 publ. 05/11/2010, RU 2401121 publ. 10.10.2010, RU 2236244 publ. September 20, 2004, RU 2438694 publ. 01/10/2012, RU 2525426 publ. 08/10/2014, CN 103877437 publ. 06/25/2014, KR 20140003866 publ. 01/10/2014, CN 101828703 publ. 09/15/2010, RU 2516886 publ. 05/20/2014, RU 2129433 publ. 04/27/1999, CN 101361774 publ. 02/11/2009, US2012039918 publ. 02.16.2012, RU 2496510 publ. 10.27.2013, RU 2393871. publ. 07/10/2010, CN 103860906 publ. 06/18/2014, RU 2283657 publ. 09/20/2006 and others.]. The disadvantage of these drugs is the variable clinical effect and the extreme complexity of standardizing multicomponent herbal remedies.

The closest to the drug we offer is the development of Mongolian scientists [MN2031, issued September 27, 2002] - the Salimon preparation, which is an aqueous-ethanol extract of the herb larch leafwort (Salsola laricifolia). Salimon showed good efficacy in the treatment of cancer patients (in combination with other drugs and procedures) [MN2031, issued September 27, 2002; Mongolyn anagaah uhaan, 2001, 1 (114), Shineserhuu budargani darhlal sergeeh uldliyn sudalgaa; Mongolyn anagaah uhaan, 2002, 1 (118), “Salimon emiyn emnil zuin sudalgaany dun”; Eruul Mandijn Shinjleh Uhaan, 2013, 9 (3) (25), Shineserhuu budargana “Salsola laricifolia Turcz. Ex Litv” urgamlyn havdryn esreg urlidlyn sudalgaa], as well as a significant increase in non-specific resistance to 2013 , 9 (6) (28), Zarim urgamlyn niyelmel nayrlagyn darhlaa zugrutulyah uldlylyn sudalaa; Mongolyn anagaah uhaan, 1995, 4 (93), Emijn bodisyn immun, deserted by wildlyn erchimzhilt, Telvees Hamaarahui membranes]. The disadvantage of this drug is the unsatisfactory efficiency of extraction of highly polar polysaccharides and glycosides with water-ethanol mixtures. In addition, the use of ethanol in the production of medicines entails certain economic costs and organizational difficulties, as a result of which the product becomes unprofitable or unavailable at a price for the consumer. The storage, transportation and sale of SFCs is also associated with great difficulties due to the fire hazard of such products and the strict regulation of the circulation of alcohol-containing products in Russia. In particular, the use of alcoholic extracts for the manufacture of dietary supplements and preparations for young children is not permitted. In veterinary medicine, the use of herbal preparations based on alcohol extracts is also difficult due to the negative reaction of animals to the strong and pungent smell of ethanol.

Attempts have been made to use purely water extracts (decoctions, infusions) of larch leaf horticulture in an experiment [Mongolyn anagaah uhaan, 1995, 4 (93), Emijn bodysyn immun immunological zylidn uldldijn erzhimzhilt, Telvees Hamaarahui membranes; P. Bolormaa "Shineserhuu Budargana, Huurmag Bulchirheit Ortuzyn immunotropic Uldlyn pharmacologists", diss. for a job. account.doc. sciences], however, in this case, the preparations were less effective and not stable during storage and were not found in practice.

Disclosure of invention

The aim of the development was to create a highly effective and safe phytopreparation immunomodulator from a plant that is not part of the family Asteraceae, which increases the non-specific resistance of mammals and birds to diseases of various etiologies that do not contain ethyl alcohol.

The invention is implemented by the fact that the proposed drug with immunomodulating and antiviral effects, increasing the nonspecific resistance of the organism of warm-blooded animals to bacterial and viral infections, which is an extract of grass larch leafwort (Salsola laricifolia), obtained by extraction of dry grass of larch leafwort (Salsola 3080) % aqueous solution of polyhydric alcohol at 60-90 ° C; using the ratio of extractant: dry grass equal to 1: (0.003-0.6), respectively.

It turned out to be unexpected that the extraction of dry grass of the larch solyanka (Salsola laricifolia) with aqueous solutions of polyhydric alcohols such as 1,2-propylene glycol and / or glycerin, and / or erythritol, and / or xylitol, and / or sorbitol, and / or mannitol, and / or dulcite and / or inositol, or mixtures thereof, is more effective than extraction with an aqueous-ethanol solution or water (see table 1).

Since aqueous solutions of polyhydric alcohols have a high viscosity, it is advisable to carry out extraction at moderately elevated temperatures (60-90) ° C; at a temperature below 60 ° C, the solution is too viscous and is retained by meal, which dramatically reduces the yield of the product, at a temperature above 90 ° C, the destruction of biologically active components of the extract is possible (example 4). A study of the kinetics of extraction showed that under these conditions the equilibrium concentrations of indicator components (flavonoids, oxycoumarins, polysaccharides) are established after 4-10 hours depending on the temperature and extractant, while when using 30-50% ethanol as an extractant at room temperature equilibrium concentrations are established after 3-5 days (examples 14, 31).

A 30-80% aqueous solution of polyhydric alcohol is used as an extractant for dry grass. When the concentration of polyhydric alcohol is less than 30%, the extraction efficiency of polyphenolic compounds sharply decreases, at a higher concentration than 80%, the viscosity increases, which prevents the separation of the obtained extract from meal by filtration and reduces the yield of the finished product (example 1).

Amount of extractant: dry grass of larch leaf solyanka is optimal in a weight ratio of 1: (0.2-0.6). An increase in the amount of dry grass over 0.6 parts by weight is not technically feasible due to the properties of the grass; the porosity and moisture capacity of even compressed dry grass is such that the amount of extractant does not fill the volume to the upper level of the grass “package”, and therefore, part of the loaded grass is not subjected to extraction. For extraction, you can take less than 0.2 weight parts of grass, up to 0.003 as when obtaining a veterinary preparation, however, this reduces the efficiency of the process, increases energy costs, and problems arise with standardization of the extract (see example 30).

Extraction at a given ratio of extractant to dry grass can be carried out both by one procedure and by multiple extraction with small portions of extractant, until a specified ratio is achieved. Both methods have their advantages, but the resulting extracts do not differ significantly either in composition (content of signal components) or biological activity, and relate to well-known methods for producing phytoextracts [Minina S.A., Kaukhova I.E. / Chemistry and technology of phytopreparations: a training manual. M .: GEOTAR-Media. 2009.560C. ISBN 978-5-9704-0878-0], for this reason we did not consider it necessary to introduce the indicated technological nuances into the claims. A one-time procedure is implemented quickly, but it requires unacceptably large capacity during the industrial sale of equipment; multiple extraction is long, but allows the use of equipment of acceptable size (examples 12, 14, 26, 36-38); in this case, the necessary performance of the hardware circuit is achieved by the consistent use of “batteries” of extractors of acceptable sizes [Minina SA, Kaukhova I.E. / Chemistry and technology of phytopreparations: a training manual. M .: GEOTAR-Media. 2009.560C. ISBN 978-5-9704-0878-0]. When polyhydric alcohols with low solubility in cold water but sufficient solubility under extraction conditions (60-90 ° C) were used for the preparation of the extractant, it turned out to be unexpected that some polyhydric alcohols crystallize upon cooling of the extract, while they practically do not contain phytoextract components (see .example 20, 23, 24). High-purity polyhydric alcohol is easily separated by filtration and can be used for subsequent extraction. The extract thus concentrated with a reduced content of polyhydric alcohol and a high concentration of active components, is stable during storage, has a pleasant, not sugary-sweet taste characteristic of polyhydric alcohols.

Under the proposed extraction conditions, there are practically no extractive substances left in the meal, the proposed method for producing a medicinal product allows the maximum extraction of biologically active substances with minimal loss of valuable components. This method of obtaining highly concentrated extracts of larch leaf solyanka grass has not been found in the available literature. Moreover, the extract obtained when using an aqueous solution of polyhydric alcohol as an extractant showed a significantly higher immunomodulating activity according to the tests used than alcohol extracts (see examples 14. 43. 44), at the same time, they were acceptable for different types of animals ( example 7-9, 14, 37, 38), not flammable, applicable in pediatrics and in other cases where the use of alcoholic extracts is limited

The results of extracts of larch leaf solyanka (Salsola laricifolia) under various conditions with different dry grass: extractant ratios are shown in table 2.

Medicinal products for medical and veterinary use, developed on the basis of extracts of larch solanum (Salsola laricifolia), have been tested for toxic properties. The results of the study of acute toxicity and toxicometry of the drug obtained according to example 28, allow it to be attributed to class V of practically non-toxic drugs [N. Hodge et al. Clinical Toxicology of Commercial Products. Acute Poisoning Ed. IV, Baltimore, 1975, 427 p.] And low-hazard substances (IV hazard class according to GOST 12.1.007-76) (see example 41). Identical toxicometry results were obtained for the veterinary preparation prepared according to example 29 (see example 42).

The pharmacological activity of the medicament according to the invention was studied in a model for the treatment of acute influenza pneumonia in mice (see example 39, 43). As the comparison drugs used the well-known drug from Echinacea purpurea "Immunal", as well as the drug made according to example 31 - prototype. All the studied drugs had little effect on the intensity of the reproduction of the influenza virus in the lung tissue, the prototype drug had no effect at all, and in the case of Immunal and the experimental drug, the virus titer was lower, at the level of 83.8% of the control; although this difference cannot be attributed to reliable, nevertheless, there is a tendency to believe that there is a mild direct inhibition of the multiplication of the influenza virus. The clinical effects were significantly more pronounced. So, the degree of lung damage when using the drug according to example 28, was more than 2 times lower than that of the control group, and significantly lower than in the group using the prototype. Morphological analysis of the lung parenchyma also confirmed a twofold decrease in damage to lung structures. At the same time, the antioxidant activity of the lung tissue increased by 2.5 times, and the level of peroxidation products decreased as compared with the control, which indicates the activation of protective and reparative processes under the influence of the experimental drug. The therapeutic effect was manifested both in the bronchial structures and in the alveoli, epithelial hyperplasia in both cases was almost 3 times lower than the control, inflammatory infiltration decreased 2 times, in these parameters the drug according to the invention significantly exceeded the prototype and comparison drug. The result of the study of the adrenal cortex showed that all three studied drugs approximately inhibited the stress-induced changes in the cells of the adrenal cortex; the cells were 2 times larger than the cells of control animals.

The prophylactic activity of the preparation obtained in Example 28 was studied for influenza infection (see Example 44). The effect was significantly higher than in the case of the prototype drug obtained in example 31, and is comparable with the effect of the preparation “Immunal”.

An optimal immune response is achieved only through the interaction of the humoral and cellular parts of the immune system. B cells are responsible for the humoral link of acquired immunity, that is, they produce antibodies, while T cells are the basis of the cell link of a specific immune response. Under the action of the developed drug, an increase in the B-cell immunity of animals was observed; an increase in the number of antibody-forming cells in the spleen of experimental animals in response to the administration of a thymus-dependent antigen significantly increased the stimulation index by 2.5 times compared with the control, similarly increased titers of agglutinins in blood serum by almost 4 times (see examples 12, 45-47 ) Strengthening of the T-cell immunity occurred due to an increase in the number of autologous rosette-forming cells (auto-ROCK) in both absolute and relative terms and amounted to 2 and 10 times, respectively. The results of the tests indicate a significant activation of T- and B-cell immunity under the influence of an experimental drug, which indicates the immunomodulating effect of the drug.

The prophylactic and therapeutic use of the developed drug for bacterial respiratory diseases of puppies was studied. In the control groups, despite the use of antibiotics, mortality was 15%. With the preventive use of the experimental drug, there was no mortality; in the event of the onset of the disease, the dogs quickly recovered. In the complex therapy of treatment with the drug, mortality, although there was, was reduced. The convalescence period compared with the control was 2 times shorter (see example 48).

In cases of herpes infection of cats, accompanied in severe cases by a mortality rate of approximately 30% and a rehabilitation period in surviving animals for more than 1.5 months, the use of the drug eliminated the death of animals and shortened the recovery time. The use of the drug in the initial stage of the disease reduced the time of convalescence to 7-9 days (see example 49).

The drug was highly effective after surgery in dogs and cats, eliminating the occurrence of complications and shortening the recovery time (see example 50, 51).

It is known that the use of antibiotics and chemotherapy is always accompanied by severe immunosuppression, this lengthens the recovery period and contributes to complications. The use of the preparation according to the invention allowed to quickly normalize the hematological and biochemical parameters of the blood of animals, which significantly reduced the time of convalescence (see example 52).

The big problem in veterinary medicine of small domestic animals is the viral enteritis of cats, a highly contagious disease, accompanied by the mass death of kittens and young animals. There are no specific preparations for the treatment of viral enteritis in cats. The use of the developed drug for prophylactic and therapeutic purposes allowed to avoid mortality and reduce the recovery period of animals (see example 53).

In dogs, severe diseases of the gastrointestinal tract with severe dysbiosis are often observed, accompanied by pathological changes in the immune system. These conditions, although of a bacterial nature, are very difficult to treat. The introduction of the developed drug to the complex of therapeutic measures made it possible to halve the period of convalescence and quickly normalize the immunological status of animals. So, in the absence of clinical manifestations of the disease after treatment, the control group of animals showed a low immunoregulatory index (CD4 + / CD8 +) of 1.38, a decrease in the function of phagocytes, etc .; in animals treated with an experimental agent, in the same period of time, all the studied parameters were within normal limits, which confirms the immunomodulating effect of the proposed drug. The drug effectively increased non-specific resistance in productive animals (example 37); had a significant immunomodulating effect when growing broilers (example 38). Comparison of native extracts obtained using various polyhydric alcohols (Example 40) revealed that the biological effect in all cases differed very slightly, with some correlation between the polarity of the extractant and the manifestation of an immunomodulating effect. The same thing was found in the experiment of example 2.

The developed drug allows for the effective treatment of immunosuppressive conditions, while the adequate immune response is restored and the recovery time is reduced (see example 52, 54, etc.). The proposed method for the treatment of immunosuppressive conditions consists in a long-term, until normalization of immune functions, oral administration of the developed drug, can be used both in the form of monotherapy and accompanied by the use of other therapeutic agents - antibiotics, vitamins, antispasmodics, etc. (see example 48, 49, 53, 54).

The extract of the larch leaf solyanka (Salsola laricifolia) obtained by extracting the herb with aqueous solutions of polyhydric alcohols can be used, in accordance with the General Pharmacopoeia Monograph 1.4.1.0018.15 within a day after receipt, or for a slightly longer time when stored at low temperature, in general, these phyto-extracts can potentially support the growth of microorganisms, therefore, long-term storage under normal conditions is impossible; although some polyhydric alcohols (propylene glycol, xylitol) exhibit their own bacteriostatic effect and extracts with their use remain relatively long (examples 37, 38). Nevertheless, the main risk factor for the instability of alcohol-free phytoextracts is the possible growth of microorganisms (bacteria and fungi), which is unacceptable in itself, because microorganisms produce toxins, and vegetative microorganisms also quickly destroy biologically active substances of phytoextracts. To prevent the growth of microorganisms, pharmaceutically acceptable preservatives are introduced into phytoextracts - sorbates, benzoates, benzyl alcohol, parabens, sulfites, etc., most of these preservatives are significantly more effective in a slightly acidic environment. It should be emphasized that, as was discovered during the development process, extracts of larch leafwort with aqueous solutions of polyhydric alcohols for long-term storage (which ensures the convenience of using the drug in practice) require stabilization - namely, maintaining a pH in the region of maximum stability of biologically active substances (the interval was experimentally determined pH values 3.5-5.0), blocking the undesirable oxidation processes of flavonoids, lignans and other components of the phenolic structure, and canned bacteriostats Comrade. Food grade non-toxic acid: succinic, malic, acetic, lactic, phosphoric, propionic, gluconic, etc., can be taken as weak non-toxic acids, convenient for stabilizing the pH, without changing the biological properties of the drug, however, we have chosen to implement the invention ascorbic acid is preferred since it also has antioxidant properties, stabilizing biophenols (which simplifies the composition and production technology), has a pleasant taste, inexpensive and is available on the market. As an antioxidant, also without changing the biological properties of the drug, any of the known food antioxidant stabilizers can be used: esters of ascorbic acid and its salts, propyl gallate, ionol, tert-butyl hydroxy anisole, sulfites and others. However, compared with ascorbic acid, they have some drawbacks, namely, they require the additional use of acid to regulate the pH, often give an unpleasant taste, and poorly and slowly dissolve in aqueous solutions of polyhydric alcohols. With prolonged use, some of them have undesirable toxic effects. For example, ionol and tert-butylhydroxy anisole may show hepatotoxicity, and sulfites are relatively allergenic and dangerous for patients with bronchial asthma [special instructions for intravenous and intramuscular administration of Metadoxil and No-spa preparations on www.webvidal.ru] [E226, E227, E228 www.procook.ru/articles.php?do=68], [Hugot D., Causeret J., Leclerc J. Effets d I'ingest on de sulfites sur l'excretion du calcium chez Ie rat . Ann. Biol. Anim. Biochim. Biophys., 5, 53-59 (1965)], [Harmful substances in industry. Inorganic and Organoelement Compounds: A Handbook. / Ed. prof. N.V. Lazareva, ed. 7th break and add. - L .: Chemistry, 1977, p. 65], [Rehm H.J., Wittmann N. Beitrag zur Kenntnis der antimikrobiellen Wirkung der schwefligen Saure. II. Mitteilung. Die Wirkung der dissoziierten und undissoziierten Saure auf verschiedene Mikroorganismen. Z. Lebensin. Unters. Forsch., 120 (6), 465-478 (1963)].

Similarly, any known sufficiently effective food preservative can be used as a bacteriostatic preservative: sorbic and benzoic acids, their salts, benzyl alcohol, benzyl benzoate, nisin, natamycin, parabens, phenoxyethanol and others. Sorbic and benzoic acid, and partly benzyl alcohol, are rated as one of the safest preservatives, in effective doses they do not affect the taste, and at the same time, they are affordable on the market. The minimum values of acceptable concentrations of preservatives (0.01% wt. Sorbates and benzoates, 0.014% benzyl alcohol) are taken from the condition that the bacteriostatic effect is effective against detectable resistant microorganisms - contaminants (examples 6, 10, 25); the upper permissible levels are limited by the effect on the organoleptic properties of the drug, as well as in accordance with TP / TS standards.

To obtain a stable drug, it is not essential which specific stabilizer and preservative is used. In our opinion, the optimal stabilizer is ascorbic acid, which has a pleasant taste, vitamin activity, and is an antioxidant, but this does not exclude the use of other carboxylic acids as stabilizers. With its help, the pH value is easily maintained in the region of maximum chemical stability of phytoextract components - from 3.5 to 5.0. In the same pH range, food preservatives are most effective (see Example 10). At pH greater than 5.0, there is a rapid destruction of oxycoumarins and flavonoids (hydrolysis, oxidation), which reduces the biological effect during storage of the drug. At a pH of less than 3.5, the drug becomes unacceptable in terms of organoleptic properties (sour taste). The optimal pH is usually achieved when the concentration of ascorbic acid in the range from 0.1 to 3.05 mass. % and depends on the parameters of phyto-raw materials.

количествах, чем предложено, нецелесообразно по экономическим причинам и, как обнаружено, сказывается отрицательно на органолептических свойствах экстракта (сильно раздражает слизистую поверхность гортани). Известные в качестве бактериостатиков-стабилизаторов для «фитоэкстрактов» эфиры п-оксибензойной кислоты («парабены») также могут быть использованы, но они менее приемлемы ввиду известных побочных эффектов и неприятного вкуса. Определение эффективных концентраций консервантов приведено в примере 6.As bacteriostatic preservatives, benzoic acid or its pharmaceutically acceptable salts can be used as safe in a concentration of (0.01-0.5) mass. % and / or sorbic acid or its pharmaceutically acceptable salts in a concentration of (0.01-0.6) mass. %, and / or benzyl alcohol in a concentration of (0.014-0.6) mass. % When the concentration of preservatives is less than 0.01%, the drug does not withstand the declared shelf life (2 years) in microbiological purity (see examples 10, 25). The use of preservatives in

in quantities than suggested, it is inappropriate for economic reasons and, as found, affects the organoleptic properties of the extract (strongly irritates the mucous surface of the larynx). The esters of p-hydroxybenzoic acid (“parabens”), known as bacteriostatic stabilizers for “phytoextracts”, can also be used, but they are less acceptable due to known side effects and unpleasant taste. The determination of effective concentrations of preservatives is shown in example 6.

The target additives are preservatives and stabilizers, it is technically simple to add to the finished extract, with the receipt of a product that has the proper production for the industrial production of the drug, stability - 2 years or more during natural storage (examples 8, 9). However, in this case, during forced shutdowns of the extraction process (see example 13), an emergency may occur - a rapid growth of microflora in the extractor, which leads to economic losses. To eliminate this phenomenon, target additives are better added to the extractant (example 14-26) or during the extraction process (example 5-7), the latter is convenient if the extractor is equipped with a mixing device and means for controlling the pH level.

It should be noted that targeted additives naturally affect the stability of the drug, but do not affect the biological effects (see example 7). In this case, the organoleptic properties of the product are most favorably affected, which is important for commercial use, primarily ascorbic acid and partly citric, while other stabilizers (for example, acetic acid) impair the organoleptic properties (taste, smell) (see examples 7, 8).

As follows from the studies, already very small amounts of extractives from the larch solyanka (Salsola laricifolia) have a pronounced immunomodulating effect (examples 3). Moreover, the effect in the field of effective doses of extractive substances practically does not increase (examples 3, 39); although even in very large doses the extracts of the larch leaf hodgepodge do not show toxic (example 3) and sensitizing (example 55) effects, for economic reasons, given that the larch leaf hodgepodge (Salsola laricifolia) is an endemic plant of the Mongolian Gobi and relatively expensive raw materials, it is advisable to minimize the quantities used phyto-raw materials. Technically, you can immediately obtain extracts with a minimally effective concentration of extractives (example 14), but from the point of view of organizing an economically viable production of a phytopreparation for both veterinary and medical use, it is advisable to obtain a concentrated extract, then standardize by dilution to certain concentrations of signal components (example 27 ) continue to be used for the manufacture of specific preparations, dietary supplements and feed additives, introducing the required amount of pharmaceutically acceptable diluent. As the latter, you can use both purified water (example 39) and solutions of polyhydric alcohols (examples 27-29), which provides the necessary organoleptic properties (examples 39) of the preparations, in addition, saline, solutions can be used as a pharmaceutically acceptable diluent Ringer and others. In this case, it is necessary to provide effective concentrations of preservatives that inhibit the growth of microorganisms (see example 6) and stabilizers that ensure the pH of the drugs in the field of stability and acceptable rganoleptic properties - from 3.5 to 5.0 (examples 7, 8).

Thus, a highly effective and safe phytopreparation immunomodulator from a plant not belonging to the family Compositae has been developed, which increases the nonspecific resistance of mammals and birds to diseases of various etiologies, suitable for use in oncology, not containing ethyl alcohol, which makes it possible to use it in veterinary medicine and for the treatment of young children, and also eliminates the known difficulties in handling drugs containing high concentrations of ethanol, giving toxicity, narcotic properties and fire hazard.

A brief description of the drawings.

In FIG. Figure 1 shows the dynamics of the release of flavonoids during the extraction of larch leaf solyanka (Salsola laricifolia) with propylene glycol solutions. Flavonoids were determined by the SF method by staining the aluminum complex in terms of rutin, where: 1 - extraction using 80% propylene glycol solution; 2 - extraction using a 50% propylene glycol solution; 3 - extraction using a 30% propylene glycol solution; 4 - extraction using 10% propylene glycol solution; 5 - extraction using pure propylene glycol (100%).

In FIG. Figure 2 shows the dynamics of the release of oxycoumarins upon extraction of the larch leaf solyanka (Salsola laricifolia) with propylene glycol solutions. Oxycoumarins were determined by the SF method in terms of fraxetin, where: 1 - extraction using 80% propylene glycol solution; 2 - extraction using a 50% propylene glycol solution; 3 - extraction using a 30% propylene glycol solution; 4 - extraction using 10% propylene glycol solution; 5 - extraction using pure propylene glycol (100%).

In FIG. Figure 3 shows the dynamics of the release of polysaccharides upon extraction of the larch leaf solyanka (Salsola laricifolia) with propylene glycol solutions. Polysaccharides were determined by the SF method, where: 1 - extraction using 80% propylene glycol solution; 2 - extraction using a 50% propylene glycol solution; 3 - extraction using a 30% propylene glycol solution; 4 - extraction using 10% propylene glycol solution; 5 - extraction using pure propylene glycol (100%).

In FIG. Figure 4 shows the dynamics of the release of oxycoumarins upon extraction of the larch leaf solyanka (Salsola laricifolia) with solutions of polyhydric alcohols. Oxycoumarins were determined by the SF method in terms of fraxetin, where: 1 - extraction with a 30% xylitol solution; 2 - extraction with a 30% sorbitol solution; 3 - extraction with a 30% erythritol solution; 4 - extraction with a 30% propylene glycol solution; 5 extraction with a 30% glycerol solution.

In FIG. Figure 5 shows the dynamics of the release of oxycoumarins upon extraction of the larch leaf solyanka (Salsola laricifolia) depending on the temperature of extraction. Oxycoumarins were determined by the SF method in terms of fraxetin, where: 1 - extraction at 105 ° C; 2 - extraction at 90 ° C; 3 - extraction at 60 ° C; 4 - extraction at 45 ° C.

In FIG. Figure 6 shows the dynamics of changes in the content of oxycoumarins in extracts of larch soliswort (Salsola laricifolia) depending on the pH level during accelerated aging (40 ° C). Oxycoumarins were determined by the SF method in terms of fraxetin, where: 1 - pH 3.5; 2 - pH 5.0; 3 - pH 6.0; 4 - pH 7.0; 5 - pH 8.0.

In FIG. 7 shows a sample of the extract of larch solyanka (Salsola laricifolia) without targeted additives on the 4th day of storage under natural conditions. There is a continuous growth of mushrooms (medium No. 2).

In FIG. Figure 8 shows a sample of the extract of larch solyanka (Salsola laricifolia) without targeted additives on the 4th day of storage under natural conditions. Separate fungal colonies are observed (medium No. 1).

In FIG. 9 shows a hardware diagram of the industrial production of larch leaf solyanka (Salsola laricifolia) extract. P1 - enameled reactor with a working volume of 2 m ³ , Df (P) 2 - enameled percolator with a working volume of 0.2 m ³ , P3 - enameled reactor with a working volume of 0.2 m ³ , P4 - enameled reactor with a working volume of 2 m ³ , P6 - enameled reactor with a working volume of 2 m ³ at a design pressure of 6 atm.

The implementation of the invention

Example 1 (different concentrations of extractant, propylene glycol)

In a thermostat-shaker 1TZh-0-03 with a pre-heated coolant up to 80 ° C (triethylene glycol), 5 volumetric flasks with a volume of 1 liter are placed, each of which contains 30 g of grass of chopped larch solyanka (Salsola laricifolia); the following extractant in an amount of 1 kg is placed in the flasks - in flask No. 1 - 10% propylene glycol solution, in flask No. 2 - 30% propylene glycol solution, in flask No. 3 - 50% propylene glycol solution, in flask No. 4 - 80% propylene glycol solution, in flask No. 5 - dry (humidity 0.11% according to K. Fisher) propylene glycol. Extraction is carried out, a sample is taken periodically every 30 minutes and the content of oxycoumarins in terms of fraksetin is determined by spectrophotometric method, the content of flavonoids by coloring of the aluminum complex in terms of rutin and polysaccharides by the SF method. Extractive substances are extracted into the extractant, the concentration of signal components increase. Based on the obtained analysis results, kinetic extraction curves are constructed (Figs. 1-3), from which it can be seen that equilibrium is established after some time, and further extraction stops. To test the effectiveness of the obtained samples of extracts as an immunomodulating agent used a test based on the study of non-specific resistance of animals to influenza virus. In the experiment we used white outbred mice weighing 15-20 g. In total, seven groups of experimental animals were formed (each group included 20 animals):

1. The control group receiving water;

2. The experimental group receiving the extract of larch leaf solyanka obtained by extracting the herb with 10% propylene glycol solution;

3. The experimental group receiving the extract of larch leaf solyanka obtained by extracting the herb with a 30% propylene glycol solution;

4. The experimental group receiving the extract of larch leaf solyanka obtained by extracting the herb with a 50% propylene glycol solution;

5. The experimental group receiving the extract of larch leaf solyanka obtained by extracting the herb with 80% propylene glycol solution;

6. The experimental group receiving the extract of larch leaf solyanka obtained by extracting the grass with dry propylene glycol;

7. The group received the drug Immunal.

The drugs were administered to animals intragastrically at a dose of 100 mg / kg 2 times a day for 15 days, the comparison drug was administered in the same dose. Animals in the control group received an equivalent amount of distilled water. On the 16th day after the start of the experiment, the animals were infected with an intranasally pathogenic strain of influenza virus A / Pr / 8/34 (H0N1) in a volume of 0.05 ml per mouse; the lethal dose of the virus was determined in a preliminary experiment by titration of the virus in mice, followed by the calculation of LD50. The results of the experiment were evaluated on day 5, recording the mortality of mice in the control and experimental groups. To determine the protection index (IZ), the protection coefficient (CI) of mice was calculated using the preparation

The protection index was calculated as follows:

It can be seen from the table that the propylene glycol extracts of larch leaf solanum (Salsola laricifolia) at an extractant concentration of 30% to 80% showed significantly greater efficacy in identical doses than the well-known herbal preparation Immunal. Dry leaf propylene glycol solyanka extract did not show any biological activity.

Example 2 (different polyols, 30%)

The experiment was carried out under conditions similar to experiment 1, but 30% aqueous solutions of propylene glycol, glycerin, erythritol, xylitol, sorbitol were taken as extractant; extraction is carried out at 80 ° C. Similarly to the previous experiment, the amount of oxycoumarins is determined as the signal components by the SF method in terms of fraxetin, and samples are taken once every 30 minutes. The concentration of oxycoumarins naturally increases to an equilibrium value, and then does not change, which indicates the cessation of extraction (see Fig. 4). The obtained extracts are hot filtered on Schott funnels, cooled, and used as an immunomodulating agent in the experiment; while the samples are stored in a refrigerator at a temperature of +4 to + 5 ° C, and before the introduction of the animals are heated to room temperature. As a comparison drug using the well-known drug "Immunal".

The experiment used 35 white mice of the Balb / C line [Vivarium FSBI NTSBMT FMBA of Russia] weighing 20-25 g. Animals were divided into 7 groups; preparations were administered intragastrically through a probe 2 times a day, at a dose of 100 ml per kg (analogously to example 1). The preparations were given to animals for 2 days, after which the blood count of lymphocytes was determined by flow cytometry (the content of CD4 + T, CD8 + T cells in peripheral blood was measured using a Monoclonal antibody to CD4, CD8 mouse (Exbio-Praha) , Czech Republic), a peripheral blood erythrocyte lysing solution - Excellyse-I (Exbio-Praha, Czech Republic) and a flow cytometry autoanalyzer FACS-Apogee Universal-50) containing subpopulations of T-lymphocytes CD4 + and CD8 +. The results of the experiment are shown in table 4. The introduction of the animal extracts of larch leaf solanum (Salsola laricifolia) significantly influenced certain immunological parameters, while the spectrum of changes was significantly different from the experience using the well-known preparation "Immunal", but there was no significant difference between the experiments using different polyhydric alcohols is detected.

Example 3 (different ratio of grass-extractant)

The grass of the larch leaf solyanka (Salsols laricifolia) is ground in a Retsch SM 300 knife mill with a 0.5 mm sieve of 800 rpm.

In 2-liter volumetric flasks, 1 g of ground dry grass of larch solyanka (Salsola laricifolia) (sample No. 1), 3 g of dry ground grass (sample No. 2), 30 g of dry ground grass (sample No. 3), 300 g of dry ground herbs (sample No. 4), 600 g of dry ground grass (sample No. 5), 800 g of dry ground grass (sample No. 6). 1 kg of 80% propylene glycol heated to 80 ° C is poured into the flasks and placed in a thermostat cabinet, previously heated to 80 ° C; at 80 ° C, the samples withstand 12 hours. Then the neck of the flask is tied with a filter cloth (Art. 56202-BT) and inverted, draining the extract into pre-weighed cylinders; the procedure is carried out in a heating cabinet. After stopping the flow of the extract, the obtained samples are weighed and the content of oxycoumarins is determined by the SF method in terms of fraxetin, and then the effect on the immunological parameters of animals is determined analogously to example No. 2. The results of the experiment are shown in table 5.

Example 4 (different extraction temperature)

In a thermostat-shaker 1TZh-0-03 with a coolant pre-heated to a predetermined temperature, place a 1 L volumetric flask into which 300 g of cut dry grass of larch leaf solyanka (Salsola laricifolia) and 1 kg of a 50% sorbitol solution are placed, include mixing and lead extraction, taking samples for analysis every 30 minutes. In the samples, oxycoumarins are determined by the SF method in terms of fraxetin, and flavonoids by coloring of the aluminum complex by the SF method in terms of rutin. The experiment is successively repeated at temperatures of 45 ° C, 60 ° C, 90 ° C and 105 ° C; extraction is carried out to establish an equilibrium state according to the concentration of oxycoumarins. The extracts obtained are filtered off, cooled and used to determine the effect on the immunological parameters of animals similar to experiment 2. The results of the experiment are shown in table 6 and in FIG. 5. It can be seen from them that at a temperature below 60 ° C the extraction slows down significantly and losses of the extract due to retention on the meal increase; at temperatures above 90 ° C, destruction of signal components is detected, and the resulting extract exhibits reduced immunomodulatory activity.

Example 5 (Effect of pH (upon extraction) on stability + extract)

For the experiment, a preservative buffer mixture is preliminarily prepared, for which 1 kg of purified water is loaded into a 2-liter glass equipped with a fluoroplastic stirrer, then 100 g of potassium sorbate and 100 g of sodium benzoate are introduced with stirring, then 450 g of citric acid are suspended. Anhydrous sodium carbonate is slowly added to the suspension with stirring, while carbon dioxide is released and the mass foams. A total of 460 g of sodium carbonate was added, achieving a stable pH of 10 ± 0.1. The resulting citrate mixture is a syrupy clear liquid, stable during storage.

In a four-necked round-bottomed flask with a volume of 4 L, equipped with a fluoroplastic stirrer, thermometer and electrodes for measuring pH, load 1 kg of purified water, with the stirrer switched on, load 1 kg of sorbitol in portions, mix until dissolved and heated, the mass is heated to 70 ° C. 20 ml of the citrate mixture are added to the solution, then ascorbic acid is added in small portions to a pH of 3.5. Continuing mixing, 600 g of cut dry grass of larch leaf solanum (Salsola laricifolia) is loaded into the extractant; the observed increase in pH is stopped by the addition of small amounts of ascorbic acid. The extraction process is carried out until the cessation of the growth of the content of signal components - oxycoumarins, determined by the SF method; this happens after 4.5 hours. The suspension is filtered from cake in a funnel with a porous glass plate, the resulting filtrate is analyzed and used in further studies.

Similarly receive samples of the extract with pH values of 5, 6, 7, 8; these samples are tested for stability according to OFS.1.1.0009.15. The results of the stability study are shown in table 7 and FIG. 6.

From the results obtained, it is seen that extracts of larch leaf solyanka obtained at a pH of 3.5-5.0 and preserving these values are stable when stored at 40 ° C for 6 months, which is equivalent to 2 years of storage under natural conditions. At higher pH values, the extracts are less stable, and at pH greater than 7, the composition changes rapidly. Stayed 6 months at 40 ° C, the samples were checked for their effect on immunological parameters in animals (analogously to example 2); the results are shown in table 8.

From the obtained data it is seen that samples with pH≥7 ceased to exhibit specific immunomodulatory activity after storage.

Example 6 (determination of effective concentrations of preservatives)

The determination of effective concentrations of preservatives is carried out according to OFS.1.2.4.0011.15. As a culture medium, a sterilized extract of larch leaf solyanka (Salsola laricifolia) is used. To obtain it, in a 4-liter three-necked flask equipped with a paddle stirrer, a thermometer and an electrode for pH measurement, 1 l of purified water is loaded, 1 kg of sorbitol is added with stirring, then heated to 80 ° C and 300 g of cut dry grass of larch leaf solyanka are loaded (Salsola laricifolia); The pH was adjusted to 4 by the addition of ascorbic acid. During the extraction process, the pH value slightly increases, ascorbic acid is added as necessary, maintaining a pH value of 4.0 ± 0.1; after 8 hours of extraction, the hot suspension is filtered on a filter with a porous glass plate. The filtrate in a closed container is sterilized with γ-radiation according to OFS.1.1.0016.15 at Megarad LLC, a sterile extract is used for the cultivation of test microorganisms. As the latter, G (+) spore-forming facultatively anaerobic bacillus with high resistance to sorbates was used, isolated from non-propotective action-acquired dietary supplement syrup.

The sterilized γ-radiation extract of larch leaf solyanka with the addition of preservatives was placed in sterile vials. Widespread and safe potassium sorbate, sodium benzoate, and benzyl alcohol at the concentrations indicated in Table 7 were used as preservatives. A suspension containing the selected test microflora in the amount necessary to achieve a cell concentration of 102 CFU / ml was added to each sample. The actual initial concentration of microorganisms in the test samples was determined immediately after contamination. Contaminated samples were kept in a thermostat protected from light at a temperature of 25.0 ° C for 28 days. On the 3rd, 14th and 28th day after inoculation of the samples, the number of viable microorganisms was determined, making inoculation by the deep method on Wednesday No. 1. The test results are presented in table 9.

The table shows that all the preservatives used in the selected concentrations showed high efficiency. During the first three days, a decrease in the level of contamination of samples compared to the initial concentration by an Ig value (CFU / ml) of more than 1 is shown. Subsequently, the level of contamination continued to fall. In some cases, there was a slight increase in the level of CFU, but this increase did not exceed 0.5 Ig CFU, which, in accordance with OFS.1.2.4.0011.15, allows us to neglect such a slight increase and state that the growth of microorganisms in the samples is not observed and preservatives show a high effectiveness in the claimed concentrations. Thus, preservatives are effective in concentrations of 0.25-40.0 g / l in the case of sorbates (potassium sorbate), 0.25-40.0 g / l in the case of benzoates (sodium benzoate) and 0.25-0.5 g / l in the case of benzyl alcohol.

Example 7 (Different stabilizers during extraction)

In a three-necked round-bottomed flask with a volume of 4 l, equipped with a fluoroplastic paddle stirrer, thermometers and electrodes for measuring pH, as well as a thermostatically controlled bath with triethylene glycol as a heat carrier, 1 kg of purified water is added and stirring is included. Water is heated to 80 ± 5 ° C and 1 kg of sorbitol is sprinkled in portions; when the sorbitol dissolves, the temperature decreases. The solution of sorbitol is heated to 80 ° C and first sprinkled with 0.5 g of potassium sorbate, 300 g of grass dry chopped larch solyanka (Salsola laricifolia). Then ascorbic acid is added in small portions to a stable pH of 4 ± 0.1; during the extraction process, the pH value increases slightly, while ascorbic acid is added. Samples are taken periodically and the content of oxiscumarines is determined by the SF method in terms of fraxetin; after 8.5 hours of extraction, the change in the concentration of oxycoumarins ceases. The hot suspension is filtered through a filter with a porous glass plate and cooled to room temperature. Similarly, extracts are obtained using as stabilizers succinic acid, lactic acid, malic acid, acetic acid, citric acid, tartaric acid, grape acid, gluconic acid, fumaric acid. Similarly, extracts with the same stabilizers are obtained in an increased amount, providing a pH value of 3.0-3.2. All samples obtained are analyzed for the content of signal components (oxycoumarins, flavonoids, polysaccharides), as well as organoleptic properties (taste, smell) and eatability by different species of animals are determined (table 10). After that, the obtained samples are stored in a thermostat at 40 ± 1 ° C for 6 months (which corresponds to 2 years of natural storage), after which the content of signal components is determined and tested by organoleptic parameters and eatability by different species of animals.

The results are shown in table 10, it can be seen from them that the stability of the extracts does not depend reliably on the stabilizer used, however, the organoleptic parameters significantly depend on the stabilizer. The highest organoleptic parameters and eatability were provided by ascorbic and citric acids. Samples using carboxylic acids as stabilizers with known significant biological activity (ascorbic, succinic acid) were studied on the effect on the level of non-specific resistance to the influenza virus; As a comparative sample, an extract without additives of stabilizers was used. The results are shown in table 10, which shows the absence of significant effects on the biological activity of the extracts of the hodgepodge of larch leaf stabilizers used.

Organoleptic evaluation of the samples was performed by volunteers (5 women, 5 men), who evaluated the sample in the range of 0-10 points; points were summed up, the average value was calculated.

The eatability of the samples by different animal species was determined as follows. To 10 g of an animal acceptable for this type of animal and habitual food (boiled chicken for dogs and cats, grain of wheat for rodents) add 1 g of the sample. In the control experiment, a 50% sorbitol solution was used as an additive. Before the test, the animal is deprived of food for 8-10 hours, with free access to water. When testing the animal in a separate box, they offer a test sample and a control, while containers with feed with additives are placed at a distance of 0.5 m from each other. The test result is evaluated as follows:

+100 points animal eagerly eats food with sample +50 points animal sniffs feed, licks 0 points animal prefers control - 50 points animal avoids prototype

The scores obtained are summarized and the average value for 5-10 animals is calculated. Outbred cats, dogs, rats, rabbits are used as test animals.

It is known that some carboxylic acids - intermediates of the Krebs cycle or with vitamin activity, can have a significant effect on the immune system of animals. To determine the effect of the stabilizers used, a test was used based on the study of non-specific resistance of animals to the influenza virus. In the experiment carried out similarly to the procedure described in example 1, white outbred mice weighing 15-20 g were used. The following experimental groups were formed:

1. The control group receiving a 50% solution of sorbitol;

2. The experimental group receiving extract of larch leaf solyanka, stabilized with ascorbic acid;

3. The experimental group receiving the extract of larch leaf hodgepodge, stabilized with succinic acid;

4. The experimental group receiving extract of larch leaf solyanka, stabilized with malic acid;

5. The experimental group receiving extract of larch leaf solyanka stabilized by fumaric acid;

6. The experimental group receiving unstabilized extract of larch leaf solyanka.

The preparations were administered to animals intragastrically using a probe at a dose of 500 mg / kg 2 times a day for 15 days, on day 16 after the start of the experiment, the animals were infected with an intranasally pathogenic influenza strain A / Pr / 8/34 (H0N1) in the amount of 0.05 ml per mouse; the lethal dose of the virus was determined in a preliminary experiment by titration of the virus in mice, followed by the calculation of LD50; the results of the experiment were evaluated on day 5, recording the mortality of mice in the control and experimental groups. Similarly to experiment 1, the protection coefficient (SC) and the protection index (FR) were calculated. In this experiment, an increased dose of the extract was used, suggesting a greater likelihood of manifestation of the effects of biologically inert stabilizers.

From table 11 it is seen that the effectiveness as an immunomodulating agent stabilized by various carboxylic acids and unstabilized extract is almost identical.

Example 8 (stabilizers when added to the finished product)

In an enameled reactor with an anchor stirrer, equipped with a temperature sensor, with a bottom fungal valve D _y 50, 10 l of purified water is loaded from the measuring device. By circulating the coolant through the jacket, the water is heated to 80 ° C, at this temperature 10 kg of sorbitol is loaded, when the sorbitol dissolves, the temperature decreases significantly. The solution is heated to 80 ° C, load 3 kg of dry cut grass of larch solyanka (Salsola laricifolia) and at this temperature the extraction is carried out for 8 hours, after which the suspension is poured into a polymer container and transferred to a suction filter. After filtering the hot extract from the meal, the filtrate is cooled to room temperature, 10 g of potassium sorbate are added to it and divided into samples of 0.5 kg; each sample is acidified with a pharmaceutically acceptable carboxylic acid: ascorbic, citric, succinic, lactic, malic, acetic, tartaric, grape, gluconic and fumaric acids to a pH of 4.0 ± 0.1. Samples analyze and determine the organoleptic properties analogously to example 7 (the results are presented in table 12); then stored at 40 ° C in a thermostat cabinet for 6 months (equivalent to 2 years of natural storage), after which the analysis and testing are repeated. The results of the experiment are shown in table 12, from them it is seen that the introduction of stabilizers after the extraction process does not differ from the introduction of stabilizers in the extraction process.

Example 9 (stabilization of the finished extract - different pH values)

The experiment is carried out similarly to experiment 8; in the obtained unstabilized extract taken in an amount of 1000 ml each sample, add a citrate mixture (see example 5) and ascorbic acid so that the obtained samples of extracts have a pH value of 8; 7; 6; 5; four; 3.5; 3.0; 2.5. Samples are analyzed for the content of signal components tested by organoleptic parameters and animal eatability, as in example 7 (table 13); after 6 months of storage at 40 ° C, analysis and testing are repeated. From the results of the experiment (see table 14), it can be seen that extracts at pH greater than 5 are chemically unstable, and samples with pH less than 3.5 have low palatability and extremely low animal eatability, due to the pronounced acidic taste of such samples and irritation of the mucous membranes the oral cavity.

Example 10 (stabilization of the finished extract - effect on microbiological stability)

In an enameled reactor with an anchor stirrer, equipped with a temperature sensor, with a bottom fungal valve ДУ50, 10 l of purified water is loaded from the measuring device. By circulating the coolant through the jacket, the water is heated to 60 ° C, at this temperature 10 kg of sorbitol is loaded, after dissolving the sorbitol and raising the temperature to 60 ° C, 3 kg of dry cut grass of larch leafwort (Salsola laricifolia) is loaded into the reactor with stirring. Extraction is carried out by periodically taking samples in which the amount of oxycoumarins is calculated in terms of fraxetin by the SF method. When the concentration of oxycoumarins in the extract stops growing, which occurs after 10.5 hours, the suspension is poured into a polymer container, filtered on a suction filter. Get 8.58 kg of extract with the following parameters:

1. Description: dark brown unclear syrup with a characteristic pleasant fruity odor.

2. The total content of flavonoids by the SF method (aluminum complex) in terms of rutin 0.102%

3. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method in terms of fraxetin 0.97%

4. The content of polysaccharides by the SF method of 2.05%

5. Microbiological purity:

- total number of aerobic bacteria - 1.2 * 10 ² CFU (norm - not more than 10 ⁴ CFU)

- the total number of mushrooms is less than 10 CFU (norm - not more than 10 ² CFU)

- Salmonella - absent

- Escherichia coli - absent

- enterobacteria - absent

- St. Aureus - absent.

The extract is divided into samples of 500 g with the addition of potassium sorbate as a preservative, in amounts as indicated in table 14, and ascorbic acid as a stabilizer until the desired pH level is reached. Samples were kept at room temperature for 6 months and checked for oxycoumarins by the SF method. in terms of fraksetin, the content of flavonoids by coloring the aluminum complex by the SF method in terms of rutin, the content of polysaccharides by the SF method and microbiological indicators.

As can be seen from table 14, the extract at pH greater than 5 is unstable; at pH less than 3.5 is not applicable for organoleptic properties.

Example 11 (extraction with sugar solutions)

In a flask with a volume of 3 l of a Buchi Rotovapor R215 rotary film evaporator, 300 g of grass of dry cut larch solyanka (Salsola laricifolia), which has the following parameters, is placed:

1. Description - cut grass with a rigid structure, greenish-yellowish gray fragments measuring 8-25 mm × 0.2-1.3 mm.

2. Humidity (by drying) 5.3%.

3. Ash content of 7.9%.

4. The amount of oxycoumarins, SF method in terms of fraksetin 0.99%.

5. mass fraction of tannins in terms of tannin in indigosulfonic acid 3.53%.

6. Mass fraction of polyphenolic compounds 2.94%.

7. Microbiological purity

The number of aerobic bacteria 8.2 * 10 ² CFU

The number of mushrooms 8.4 * 10 CFU

Esherichia coli - absent

Pseudomonos spp - missing

Salmonella - absent

Staphylococcus - absent

Enterobacteriaceae - absent.

The extractant is prepared as follows: honey (GOST R 54644-2011, manufacturer of the Honey Valley LLC) is analyzed for water content by the K. Fischer method, considering the rest as sugars, an aqueous solution with a sugar content of 50% in an amount of 500 g is prepared, added to the extractant 0.5 g of potassium sorbate and is added to the grass through a funnel. The bath is heated to 80 ° C; fix the flask, set the rotation to 80 rpm and carry out the extraction for 4 hours. After that, the hot suspension is poured onto a preheated filter with a porous glass plate, filtered under vacuum with 300 mm Hg, the meal is squeezed out under a pressure of 0.1 kg / cm ² , which is ensured by the pressure (under its own weight) of the fluoroplastic rod (also heated to 80 ° C) ) for meal during the filtration process. Get 751 g of the extract. Similarly, extraction is carried out using a 50% sorbitol solution and a 50% glucose solution. The extracts obtained are analyzed similarly to previous experiments, and the effect on non-specific resistance to the influenza virus is studied analogously to example 1. The results are shown in table 15, they show that the extracts obtained using sorbitol solution, glucose solution and honey solution do not differ significantly in immunomodulating effects, while in extracts using sugar it is not possible to reliably determine the signal components by the applicable method, and the use of selective HPLC-MS methods is very expensive This is a requirement and requires the development of the methods themselves.

Example 12 (extraction with a mixed extractant (erythritol, sorbitol, xylitol); stabilizer - two acids)

To carry out the extraction process, an enameled percolator with a calculated volume of 120 l, equipped with a heating jacket, a thermowell, a filter field and a fungal valve on the lower slope, and an enameled reactor with a volume of 63 l, with an anchor stirrer having a heating jacket, a thermowell with a temperature sensor and a lower one are used descent with fungal valve.

For extraction, dry cut grass of larch solyanka (Salsola laricifolia) is used, which corresponds to the Mongolian standard MNS-4403-05; The parameters of the material taken for extraction are as follows:

1. Description - cut grass with a rigid structure, greenish-yellowish gray fragments measuring 5–30 mm × 0.2–1.5 mm;

2. Smell - specific, pleasant;

3. Taste - sour;

4. Humidity (by drying) - 3.9%;

5. Mineral impurities (insoluble in 10% hydrochloric acid) - 0.5%;

6. Ash content 9.2%;

7. The amount of oxycoumarins, SF method, in terms of fraxetin 2.92%;

8. The amount of flavonoids, according to the coloring of the aluminum complex, by the SF method, in terms of rutin, 0.203%;

9. Mass fraction of polyphenols 3.11%.

60 kg of cut dry grass of larch solyanka (Salsola laricifolia) is loaded into the percolator. 92.5 kg of purified water are charged into an enameled reactor. Turn on the mixer and coolant circulation in the reactor jacket, turn on heating. Upon reaching 60 ° C, 35 kg of erythritol are charged through the hatch in the reactor, then 80 kg of sorbitol are added in portions, 32.5 kg of xylitol are loaded after complete dissolution, mixed until dissolved and heated to 80 ° C. The hot extractant is transferred to the percolator in an amount of 100 kg, extraction is carried out for 8 hours at 90 ° C, after which the extract is poured into a polymer container through the filter field of the percolator. 21.88 kg of extract is obtained. To the meal remaining in the percolator, 75 kg of extractant are poured and extraction is repeated for 12 hours, when draining, 62.77 kg of syrup extract is obtained. The remaining 75 kg of extract agent is loaded into the meal, the extraction process takes 16 hours. After draining, 65.85 kg of extract is obtained.

All three extracts are combined in a 200 L polymer barrel, mixed with a stock; add 0.5 kg of potassium sorbate and again mix the stock until the granules are completely dissolved. After this, while stirring, add 5 kg of ascorbic acid, the pH of the extract after stirring 5.8. Succinic acid is added in small portions while stirring with a stock until a stable pH of 4.0 is reached - a total of 6.32 kg of succinic acid is charged for this purpose. After loading all the components receive 184.2 kg of extract with the following parameters:

1. Description - thick dark brown syrup with a pleasant fruity odor, transparent;

2. The density of 1.26 g / cm ³ ;

3. pH 4.1;

4. The total content of oxycoumarins by the SF method, in terms of fraxetin 0.39%;

5. The total content of flavonoids by the SF method, according to the color of the aluminum complex, in terms of rutin, 0.056%;

6. Microbiological purity:

- the number of aerobic bacteria 2.2 * 10 ² CFU;

- the number of mushrooms is less than 10 CFU.

After 6 months of storage under natural conditions, the parameters of the extract are as follows:

1. Description - a thick transparent dark brown syrup with a pleasant fruity odor;

2. The density of 1.22 g / cm ³ ;

3. pH 4.15;

4. The total content of oxycoumarins in terms of fraxetin (SF method) 0.36%;

5. The total content of flavonoids, according to the color of the aluminum complex, by the SF method, in terms of rutin, 0.052%;

6. Microbiological purity:

- the number of aerobic bacteria 1.4 * 10 ² CFU;

- the number of mushrooms is less than 10 CFU.

The study of the effect of the obtained extract on antibody formation was studied by the method of local hemolysis and in the hemagglutination reaction during immunization of mice with sheep erythrocytes. In the experiment, white outbred mice weighing 15-20 g were used. To determine the change in the number of antibody-forming cells (AOK) in the spleen, the experimental extract was orally administered to the individuals of the experimental group at a dose of 0.2 ml / kg simultaneously with intraperitoneal immunization with ram red blood cells (2% suspension) at a dose of 0.5 ml / mouse. The control group obtained the extract was not used. The study of changes in the amount of AOK was performed on the 4th day after immunization - the spleen was removed and lymphocytes were obtained. Around each antibody-forming cell, a hemolysis zone appears, which is visible on the agar as a transparent dot. The stimulation index (IS) was calculated as the ratio of the number of AOK of the experimental group to AOK in individuals of the control group:

The results are presented in table 16, from which it is seen that the introduction of the extract leads to the activation of the transformation of B-lymphocytes into AOK as compared with the control, which was expressed in an increase in the number of AOK in the spleen. A 2.7-fold increase in the stimulation index in individuals of the experimental group indicates a stimulating effect of the extract on antibody production and an increase in the humoral immune response; an increase in the AOK content of B-lymphocyte precursors is associated with an activation of the functional activity of T-lymphocytes.

To determine the change in antibody titers in blood serum, the obtained extract at a dose of 0.2 ml / kg was administered to mice of the experimental group with simultaneous intraperitoneal immunization with 2% suspension of sheep erythrocytes. The control group did not receive the extract, but was immediately immunized. The reaction is based on the ability of the agglutinins contained in the blood serum of immunized animals to agglutinate sheep red blood cells. The titers of agglutinins in the blood serum of animals and IP were counted on the 7th day after immunization. The results are presented in table 17, which shows that on the 7th day after immunization in animals of the experimental group, serum agglutinin titers increased to 7.4 (log ₂ ), while in individuals of the experimental group the level was 1.9 (log ₂ ). Thus, a single application of larch leaf solyanka extract is accompanied by an increase in the immune response to the thymus-dependent antigen, and there is no suppression by T-suppressors of B-lymphocyte activity against the T-dependent antigen. Larch leaf solyanka extract has a stimulating effect on the humoral immune response, as evidenced by an increase in the stimulation index to 3.89.

A study was also conducted of the action of the extract of larch solyanka on the cellular part of the immune response by determining the delayed-type hypersensitivity reaction (HRT) and determining the kinetics of autosocket-forming cells (auto-ROCK).

When determining the delayed-type hypersensitivity, the individuals of the experimental group were injected with a 0.2 ml / kg larch leaf solyanka extract with simultaneous intraperitoneal immunization with a 2% suspension of ram red blood cells. On the 5th day, a resolving dose of ram erythrocytes was introduced - 10% suspension in a volume of 0.02 ml into the pads of the right paws. A physiological saline in the same volume was injected into the collanteral paw as a control. Individuals in the control group underwent a similar procedure without the use of larch leaf solyanka extract. After 24 hours, the inflammation index (VI) was determined by the following formula:

, где:

where:

O is the mass of the experimental paw;

K is the mass of the control paw.

The results are presented in table 18.

It can be seen from the results that the use of the extract induces HRT induction, which is caused by the infiltration of the inflammatory focus by cells, and not by a vascular reaction. The use of larch leaf solyanka extract caused an IV shift of 29.4%. Strengthening HRT indicates the ability of the extract to stimulate the production of cell response mediators - lymphokines.

When studying the dynamics of autoimmune rosette-forming cells, it was found that the use of the solyanka extract of larch-leaf kinetics of auto-ROCK was characterized by an increase in both the absolute and relative number of rosette-forming cells. The maximum values of the readings were observed on the 10-15th day (see table 19).

Example 13 (the growth of microorganisms in the percolator when the process is forced to stop)

The process is carried out analogously to example 12. At the first discharge, 22.86 kg of extract are obtained (parameters, see table 20). After loading the second portion of the extractant, the process is stopped for external reasons and the heating of the percolator is stopped; after 14 days, include heating, after heating the percolator and holding for 6 hours, the extract is drained; at the same time, the pressure in the percolator rises to 2.2 atm, discharge is accompanied by the formation of foam, at the end of the discharge of gas with an unpleasant odor. The results of the analysis and testing are presented in table 20, from which it can be seen that with a prolonged forced delay in the extraction process and provided that there are no preservatives and stabilizers in the extractant, microflora growth is observed.

Example 14 (mixed stabilizer, comparison of economic indicators and activity with the prototype)

For the experiment, we use the herb of the larch leaf solyanka (Salsola laricifolia) with the following parameters:

1. Description - cut grass with a rigid structure, greenish-yellowish fragments of 4 ÷ 35 × 0.2 ÷ 1.6 mm in size;

2. Taste - sour, fruity;

3. Smell - specific, pleasant;

4. Humidity (by drying) 4.2%;

5. Ash content of 7.6%;

6. The amount of oxycoumarins by the SF method in terms of fraxetin 2.98%;

7. The amount of flavonoids by coloring the aluminum complex, SF method, in terms of rutin 0.355%.

Analogously to example 12 in an enameled reactor with a volume of 63 l, equipped with an anchor stirrer, thermowell, jacket for heating, a 50% sorbitol solution is prepared and heated to 80 ° C. In the percolator, 3 kg of grass of larch leafwort (Salsola laricifolia) are loaded and extraction is carried out repeatedly in portions of 100 l for 10 hours each procedure. In total, the extraction process is repeated 10 times, the resulting extract is poured into a polymer container with a volume of 1 m ³ , mixed with a circulation pump. Continuing mixing, add to the total extract 0.5 kg of potassium sorbate, 5 kg of ascorbic acid and then in small portions of citric acid, a total of 2.4 kg of citric acid is added. A total of 1,004.9 kg of extract is obtained with the following parameters:

1. Description - yellow solution with a pleasant fruity odor, pleasant sour taste;

2. pH 4.1;

3. The density of 1.22 g / cm ³ ;

4. The content of oxycoumarins in terms of fraxetin, SF method 0,00876%.

In the same scheme, extraction is carried out with a 30% ethanol solution according to US Pat. MN2031. For this, 60 kg of dry larch leaf grass, ground to particles less than 0.5 mm, are loaded into a percolator with moderate compaction. Extraction is carried out by percolation with a 30% ethanol solution; however, one ectraction procedure takes 3 days. The result is 156 l (ρ = 0.952 g / cm ³ ), or 148 kg of extract with the following parameters:

1. The density of 0.952 g / cm ³ ;

2. The amount of oxycoumarins in terms of fraxetin using the SF method is 0.0112%;

3. The dry residue of 1.1%.

These samples are tested for eating by different species of animals and for influencing nonspecific resistance to the influenza virus, similarly to example 1. The results are shown in tables 21 and 22, which show that with a comparable concentration of signal components, sorbitol extract is noticeably more active as an immunomodulator, to note that in the case of using an aqueous solution of polyhydric alcohol as an extractant, the specific consumption of phyto-raw materials is significantly lower than when using an ethanol solution, the process is more It is odd and explosion proof; in addition, in the “eatability” test, animals avoid samples with alcohol extract.

Example 15 (1,2-propylene glycol, 60 ° C)

1000 ml of purified water, 1000 g of 1,2-propylene glycol, 10 g of ascorbic acid and 5 g of potassium sorbate are placed in a 2000 ml glass reactor. The mass is stirred and heated to 60 ° C. In a hot water-propylene glycol solution, chopped grass of larch leaf solyanka is portioned, densifying as it is wetted to a total load of 600 g. Extraction is carried out at 60 ° C, sampling every hour, in which the content of oxycoumarins and oxycoumarins glycosides is determined by spectrophotometric (SF) method in in terms of fraxetin, until the cessation of changes in their concentration. After 8 hours, the concentration of oxycoumarins ceases to increase, the process is completed. The gravity extract is discharged from the reactor onto a glass funnel with a porous plate, filtered under vacuum. Obtain 1160 g of a dark brown extract with the following parameters:

1. Description. Dark brown turbid relatively low viscosity syrup with a characteristic pleasant fruity odor, with a slight sediment.

2. pH 4.2

3. The density of 1.1 g / cm ³

4. Heavy metals according to GF XIII - less than 0.001%

5. The total content of flavonoids by the SF method (aluminum complex) in terms of rutin 0.14%

6. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method in terms of fraxetin 0.92%

7. The content of SF polysaccharides by the method of 3.3%

8. The content of ascorbic acid 0.77% (titration method)

9. The content of sorbic acid 0.22% (by GLC)

10. Microbiological purity:

- the total number of aerobic bacteria is 1.1⋅10 ² CFU (normal - not more than 10 ⁴ CFU)

- the total number of mushrooms is less than 10 CFU (norm - not more than 10 ² CFU)

- Salmonella - absent

- Escherichia coli - absent

- enterobacteria - absent

- St. Aureus - absent.

After two years of storage of samples in vivo, packaged in 100 ml glass bottles, the parameters of the extract are as follows:

1. Description. Dark brown transparent relatively low-viscosity syrup with a characteristic pleasant fruity odor, a thin dark film at the bottom of the bottles.

2. pH 4.3

3. The density of 1.1 g / cm ³

4. The total content of flavonoids SF method (aluminum complex) in terms of rutin 0.135%

5. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method in terms of fraxetin 0.91%

6. The content of SF polysaccharides by the method of 3.29%

7. The content of ascorbic acid 0.71% (titration method)

8. The sorbic acid content of 0.18% (by GLC)

9. Microbiological purity:

- the total number of aerobic bacteria is less than 10 CFU

- total number of mushrooms - less than 10 CFU

- Salmonella - absent

- enterobacteria - absent

- St. Aureus - absent.

Thus, the extract obtained is stable when stored in vivo for two years.

Example 16 (extraction with a 50% sorbitol solution at 90 ° C) 600 g of crushed dry grass of larch leafwort are placed in a 2000 ml glass reactor. Separately, 1 kg of sorbitol in 1 liter of water is dissolved in a glass beaker with stirring and heating to 60-70 ° C (sorbitol dissolves with a significant decrease in temperature). 5 g of potassium sorbate and 10 g of ascorbic acid are added to a hot sorbitol solution. The resulting solution is poured into the grass in the reactor, the mass is heated with a thermostat to 90 ° C and maintained at this temperature for 6 hours, taking samples every half hour to determine the content of the sum of oxycoumarins and glycosides of oxycoumarins. After 4 hours, the concentration of oxycoumarins ceases to increase. The extract is drained by gravity from the reactor to a glass funnel with a porous plate, filtered under a small vacuum. Receive 1726 g (1380 ml) of the extract with the following parameters:

1. Description. Dark red-brown syrup with a slight sediment, unclear, has a characteristic pleasant fruity smell.

2. pH 3.9

3. The density of 1.25 g / cm ³

4. Heavy metals according to GF XIII - less than 0.001%

5. The total flavonoid content by spectrophotometric (SF) method (with aluminum complex) in terms of rutin 0.099%

6. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method in terms of fraksetin 1.03%

7. The content of oxycoumarins and glycosides of oxycoumarins (HPLC method):

- fraksetin 0.44%

- fraxidine 0.023%

- isofraxidine 0.13%

- scopoletin 0.08%

- fraksidin 8-o-β-D-glucopyranoside 0.01%

- scopolin 0.01%

- fraxin 0.06%

8. The content of SF polysaccharides by the method of 1.99%

9. The content of ascorbic acid 0.91% (titration method)

10. The content of sorbic acid 0.2% (by GLC)

11. Microbiological purity:

- the total number of aerobic bacteria is less than 10 CFU

- total number of mushrooms - less than 10 CFU

- Bacillus cereus - less than 10 CFU

- Salmonella - absent

- Escherichia coli - absent

12. The content of manganese ions of 0.0001% (method of atomic emission spectroscopy with inductively coupled plasma)

13. Boron content 0.0002% (inductively coupled plasma atomic emission spectroscopy)

14. Flavonoid content (HPLC method):

- rutin 0.026%

- campferol 0.032%

- apigenin 0.009%.

After two years of storage of samples in vivo, the parameters of the extract are as follows:

1. Description. Dark red-brown syrup, transparent, has a characteristic pleasant fruity smell, at the bottom of the bottles a thin black film - sediment.

2. pH 4.01

3. The density of 1.25 g / cm ³

4. The total content of flavonoids SF method (aluminum complex) in terms of rutin 0,095%

5. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method in terms of fraksetin 1,025%

6. The content of oxycoumarins and glycosides of oxycoumarins (HPLC method):

- fraksetin 0.43%

- fraksidin 0.02%

- isofraxidine 0.13%

- scopoletin 0.08%

- scopolin 0.01%

- fraxin 0.06%

7. The content of SF polysaccharides by the method of 2.0%

8. The content of ascorbic acid 0.82% (titration method)

9. The content of sorbic acid 0.2% (by GLC)

10. Microbiological purity:

- the total number of aerobic bacteria is less than 10 CFU

- total number of mushrooms - less than 10 CFU

- Bacillus cereus - absent

- Salmonella - absent

- Escherichia coli - absent.

Thus, the obtained sample during storage for two years practically did not change in parameters and remained suitable for use.

Example 17 (1,2-propylene glycol 30%)

The experiment is carried out analogously to example 16, but to prepare the extractant 300 g of 1,2-propylene glycol, 5 g of ascorbic acid and 2.5 g of calcium sorbate are mixed, water is added up to 1 kg. The extraction is carried out at 70 ° C, the equilibrium concentration of extractives in the liquid phase is reached after 9.5 hours. 715 g of extract is obtained with the following parameters:

1. Description. Dark brown muddy syrup with a characteristic pleasant fruity odor, foams, when standing, a slight light precipitate forms.

2. pH 4.2

3. The density of 1.025 g / cm ³

4. The total content of flavonoids by the SF method (by coloring the aluminum complex) in terms of rutin 0.25%

5. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method in terms of fraksetin 0.61%

6. The content of SF polysaccharides by the method of 2.88%

7. Microbiological purity:

- the total number of aerobic bacteria is 0.9⋅10 ³ CFU (norm is not more than 10 ⁴ CFU)

- the total number of mushrooms is less than 1.8⋅10 CFU.

After two years of storage of the sample in vivo in a bottle of dark glass, the parameters of the extract are as follows:

1. Description. Dark red-brown transparent syrup with a characteristic pleasant fruity odor, with a small layer of light sediment at the bottom of the container.

2. pH 4.7

3. The density of 1.05 g / cm ³

4. The total content of flavonoids SF method (aluminum complex) in terms of rutin 0.16%

5. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method in terms of fraksetin 0.33%

6. The content of SF polysaccharides by the method of 1.86%

7. Microbiological purity:

- the total number of aerobic bacteria is less than 10 CFU

- the total number of mushrooms is less than 10 CFU.

Thus, the use of an extractant - 30% 1,2-propylene glycol under the stated conditions allows to obtain an extract of the required quality and stable during storage.

Example 18 (sorbitol + xylitol + erythritol, 80% solution)

In a glass reactor with a volume of 2000 ml is placed 600 g of cut grass of larch leaf hodgepodge. Separately, 400 g of purified water is charged into a heat-resistant glass beaker placed in a bath with triethylene glycol and equipped with a stirrer. The stirrer and heating are turned on; upon reaching a temperature of 90-95 ° C, 1 kg of sorbitol, 400 g of xylitol and 200 g of erythritol are loaded. The mixture is stirred while heating to 100 ± 5 ° C until the components are completely dissolved, 5 g of sorbic acid and 15 g of ascorbic acid are added. The hot solution is carefully poured into a reactor with cut grass, placed in a thermostat with a set temperature of 90 ° C. The extraction is carried out until the growth of the concentration of the sum of oxycoumarins in the liquid phase ceases, the equilibrium state is established after 8 hours. To carry out the filtration, a glass funnel with a large-pore plate is heated in an oven at 100 ° C; in a glass beaker prepare 1 kg of hot solution for washing the meal, loading 200 g of purified water, 100 g of erythritol, 200 g of xylitol and 500 g of sorbitol and heating the mixture to 100 ° C.

The heated glass funnel is installed on a Bunsen flask, the contents of the reactor are quickly transferred to the funnel, filtered, the reactor is washed with the prepared solution, the compacted meal is washed with it and squeezed until the flow of the extract stops. After filtration and cooling of the filtrate to room temperature, 1899 g of extract is obtained with the following parameters:

1. Description - a thick dark brown transparent syrup, with a pleasant fruity odor, with a pleasant sweet and sour taste;

2. The density of 1.26 g / cm ³

3. Viscosity 22850 centipoise;

4. pH 4.4 (pH of a 10% aqueous solution of 4.1);

5. The total content of SF flavonoids by the method (by coloring the aluminum complex) in terms of rutin 0.18%;

6. The total content of oxycoumarins and glycosides of oxycoumarins in terms of fraksetin 0.86%;

7. The content of polysaccharides by the SF method is 2.77%;

8. The content of ascorbic acid by titration 1.26%;

9. Microbiological purity

- The total number of aerobic bacteria is less than 10 CFU;

- The total number of mushrooms is less than 10 CFU.

The sample is Packed in glass bottles of dark glass of 100 ml and stored in vivo for two years. After storage, the parameters of the extract are as follows:

1. Description - a thick dark brown transparent syrup with a pleasant fruity odor, with a pleasant sour-sweet taste. In individual bottles at the bottom, single large crystals are found.

2. The density of 1.26 g / cm ³ ;

3. Viscosity 21990 centipoise;

4. pH 4.5 (pH of a 10% aqueous solution of 4.2);

5. The total content of SF flavonoids by the method (by coloring the aluminum complex) in terms of rutin 0.17%;

6. The total content of oxycoumarins and glycosides of oxycoumarins in terms of fraksetin 0.82%;

7. The content of SF polysaccharides by the method of 2.78%;

8. The content of ascorbic acid by titration of 1.12%;

9. Microbiological purity

- The total number of aerobic bacteria is less than 10 CFU;

- The total number of mushrooms is less than 10 CFU.

Thus, the obtained extract, when stored under natural conditions for two years, is stable. Single xylitol crystals precipitated in separate bottles do not affect the consumer properties of the syrup.

Example 19 (xylitol + 1,2-propylene glycol, stabilizers 0.1%, preservatives 0.01%)

In a 2000 ml glass reactor placed in a thermostat, 600 g of cut grass of larch leaf solyanka are loaded. 700 g of xylitol, 700 g of 1,2-propylene glycol are added to a heat-resistant glass, 2 g of ascorbic acid and water up to 2 kg are added, the mixture is heated in a water bath with stirring until the components are completely dissolved. The resulting solution is carefully poured into the grass in the reactor, the mass is heated to (80 ± 5) ° C and maintained at this temperature, periodically determining the total content of oxycoumarins and glycosides of oxycoumarins, until their equilibrium concentration in the liquid phase stops growing. The extraction process under these conditions takes 8 hours. The mass together with the meal is transferred to a glass funnel with a large-pore filter plate and filtered under vacuum, squeezing the meal until the flow of the extract stops. 1088 g of extract is obtained. To the extract, having a pH of 4.6, add 0.1 g of sorbic acid and 0.1 g of benzoic acid and mix with a fluoroplastic rod. In this case, the pH drops to 3.5 and the extract brightens a little. The parameters of the obtained extract are as follows:

1. Description. Dark brown transparent syrup with a characteristic pleasant fruity odor.

2. pH 3.5

3. The density of 1.22 g / cm ³

4. The total content of flavonoids SF method (aluminum complex) in terms of rutin 0.22%

5. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method in terms of fraxetin 0.59%

6. The content of SF polysaccharides by the method of 1.98%

7. The content of ascorbic acid 0.11% (titration method)

8. The content of sorbic acid of 0.005% (by GLC)

9. Microbiological purity:

- total number of aerobic bacteria - 5⋅10 ² CFU

- the total number of mushrooms is 2.8⋅10 CFU.

The extract is packaged in 100 ml glass bottles and stored under normal conditions for two years. After storage, the parameters of the extract are as follows:

1. Description. Dark brown syrup with a red tint with a characteristic pleasant fruity odor, without sediment.

2. pH 3.8

3. The density of 1.22 g / cm ³

4. The total content of flavonoids SF method (aluminum complex) in terms of rutin 0.237%

5. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method in terms of fraksetin 0.53%

6. The content of SF polysaccharides by the method of 1.95%

7. The content of ascorbic acid 0.1% (titration method)

8. Microbiological purity:

- the total number of aerobic bacteria is 1.2⋅10 ² CFU

- the total number of mushrooms is -1.1⋅10 CFU.

Thus, the content of ascorbic acid stabilizer 0.1% and preservatives of sorbic and benzoic acids 0.01% is sufficient to stabilize the product.

Example 20 (mannitol, the extract is stable for three years)

In a glass reactor with a volume of 2000 ml is placed 600 g of grass larch leafwort. Separately, in a glass beaker equipped with a stirrer and a water bath, 1.2 kg of mannitol in 800 ml of purified water is dissolved with stirring and heating to (90-95) ° C. 5 g of sorbic acid, 5 g of benzoic acid and 10 g of ascorbic acid are added to the hot solution. The resulting hot solution poured grass in the reactor. The reactor is placed in a thermostat with a set temperature of 90 ° and maintained until the growth of the concentration of the sum of oxycoumarins in the liquid phase ceases (4 hours). The mass is quickly transferred to a pre-heated glass funnel with a large-pore plate and quickly filtered under vacuum, avoiding foaming of the filtrate (which is achieved by adjusting the vacuum level). Receive 1208 g (1210 ml) of the extract, which upon cooling begins to crystallize. The extract is kept for a day, then the precipitated precipitate of mannitol is filtered off (it is used in subsequent experiments). 585 g of extract is obtained. The parameters of the resulting product are as follows:

1. Description. Dark brown transparent syrup with a characteristic pleasant fruity odor, pleasant sour, moderately sweet taste.

2. pH 4.1

3. The density of 1.08 g / cm ³

4. The total content of flavonoids SF method (aluminum complex) in terms of rutin 0.28%

5. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method in terms of fraxetin 1.24%

6. The content of oxycoumarins and glycosides of oxycoumarins (HPLC method):

- fraksetin 0.538%

- fraxidine 0.0278%

- isofraxidine 0.162%

- scopoletin 0.051%

- fraksidin 8-o-β-D-glucopyranoside 0.018%

- scopolin 0.016%

- fraxin 0.089%

7. The content of SF polysaccharides by the method of 3.8%

8. The content of ascorbic acid 1.88% (titration method)

9. Microbiological purity:

- the total number of aerobic bacteria is 1.88⋅10 ³ CFU (normal - not more than 10 ⁴ )

- the total number of mushrooms is 5.2⋅10 CFU.

The extract is packaged in 100 ml dark glass glass bottles and stored under natural conditions for two years. After storage, the parameters of the extract are as follows:

1. Description. Dark brown transparent syrup with a characteristic pleasant fruity odor, pleasant sour, moderately sweet taste.

2. pH 4.2

3. The density of 1.08 g / cm ³

4. The total content of flavonoids SF method (aluminum complex) in terms of rutin 0.27%

5. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method in terms of fraksetin 1.22%

6. The content of polysaccharides SF method of 3.7%

7. The content of ascorbic acid 1.68% (titration method)

8. Microbiological purity:

- the total number of aerobic bacteria is less than 10 CFU

- the total number of mushrooms is less than 10 CFU.

After storing the samples for three years in vivo, the product parameters are as follows:

1. Description. Dark brown transparent syrup with a characteristic pleasant fruity odor, pleasant sour, moderately sweet taste.

2. pH 4.2

3. The density of 1.08 g / cm ³

4. The total content of flavonoids SF method (aluminum complex) in terms of rutin 0.26%

5. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method in terms of fraksetin 1.21%

6. The content of SF polysaccharides by the method of 3.68%

7. The content of ascorbic acid 1.62% (titration method)

8. Microbiological purity:

- the total number of aerobic bacteria is less than 10 CFU

- total number of mushrooms - less than 10 CFU

9. The content of mannitol syrup 20.8% (HPLC method with a refractometric detector).

Thus, the product obtained by extraction of larch leaf solyanka grass with hot saturated mannitol solution and subsequent concentration due to crystallization of the excess polyol upon cooling is stable under natural conditions for 3 years.

Example 21 (inositol 50%)

In a glass reactor with a volume of 2000 ml, placed in a thermostat with triethylene glycol, 600 g of cut dry grass of larch leafwort are loaded. Separately, in a heat-resistant glass beaker placed in a bath and equipped with a stirrer, 1 kg of inositol (EP 7.0) in 1 kg of purified water is dissolved with stirring and heating to 100 ° C. 10 g of sorbic acid and 30 g of ascorbic acid are added to the hot solution, mixed and poured onto the grass in a reactor. The extraction is carried out at 90 ° C until the concentration of the sum of oxycoumarins and oxycoumarins glycosides ceases to increase. After 5 hours, the increase in the concentration of signal components stops. The syrup is filtered off from the meal using a glass filter with a large porous plate. As a result of the experiment, 1686 g of extract is obtained with the following parameters:

1. Description - a dark red-brown, moderately viscous syrup, with a characteristic pleasant fruity odor;

2. pH 3.6;

3. The density of 1.19 g / cm ³ ;

4. The total content of SF flavonoids by the method (by coloring the aluminum complex) in terms of rutin 0.12%;

5. The total content of oxycoumarins and glycosides of oxycoumarins in terms of fraxetin 0.89%;

6. The content of SF polysaccharides by the method of 1.69%

7. The content of ascorbic acid by titration of 0.86%;

8. Microbiological purity

- The total number of aerobic bacteria -1.8 * 10 ² CFU;

- The total number of mushrooms is 3.8 * 10 CFU.

After two years of storage of the sample in vivo, its parameters are as follows:

1. Description - dark red-brown syrup with a slight sediment with a pleasant fruity odor;

2. pH 3.8;

3. The density of 1.19 g / cm ³ ;

4. The total content of SF flavonoids by the method (by coloring the aluminum complex) in terms of rutin 0.11%;

5. The total content of oxycoumarins and glycosides of oxycoumarins in terms of fraksetin 0.86%;

6. The content of SF polysaccharides by the method of 1.66%;

7. The content of ascorbic acid by titration of 0.69%;

8. Microbiological purity

- The total number of aerobic bacteria is less than 10 ² CFU;

- The total number of mushrooms is less than 10 CFU.

Thus, the extract of leafy leaf solyanka obtained using a 50% aqueous solution of inositol is stable when stored under natural conditions for two years. The use of inositol with a vitamin and adaptogenic activity of polyhydric alcohol can deepen the clinical effect of the drug.

Example 22 (glycerin 70%)

The experiment is carried out analogously to example 15, but for the preparation of the extractant, 1400 g of dry glycerol (hch), 22 g of ascorbic acid, 10 g of potassium sorbate and 6 g of sodium benzoate are mixed, 600 ml of purified water are added. The mass is mixed and heated using a thermostat to 90 ° C. Loaded in portions cut grass of larch solyanka. The equilibrium concentration of extractives in the liquid phase is reached after 4 hours. Receive 1404 g (1180 ml) of the extract with the following parameters:

1. Description. Turbid orange-brown syrup with a characteristic pleasant fruity odor.

2. pH 3.9

3. The density of 1.19 g / cm ³

4. Heavy metals according to GF XIII - less than 0.001%

5. The total content of flavonoids SF method (aluminum complex) in terms of rutin 0.16%

6. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method in terms of fraxetin 0.68%

7. The content of polysaccharides SF method 1.99%

8. The content of ascorbic acid 1.17% (titration method)

9. The content of sorbic acid 0.493% (by GLC)

10. Microbiological purity:

- total number of aerobic bacteria - 1.5⋅10 ² CFU

- total number of mushrooms - 1.5⋅10 CFU

- Salmonella - absent

- Escherichia coli - absent

- enterobacteria - absent

- St. Aureus - absent.

The syrup is packaged in glass bottles of 1 liter of dark glass and stored in vivo for two years. After storage, the parameters of the extract are as follows:

1. Description. Transparent dark brown extract with a pleasant odor, at the bottom of the package a small amount of light sediment.

2. pH 4.2

3. The density of 1.18 g / cm ³

4. The total content of flavonoids SF method (aluminum complex) in terms of rutin 0.17%

5. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method in terms of fraxetin 0.63%

6. The content of SF polysaccharides by the method of 1.91%

7. The content of ascorbic acid 1.09% (titration method)

8. The sorbic acid content of 0.46% (by GLC)

9. Microbiological purity:

- the total number of aerobic bacteria is 1.1⋅10 ² CFU

- total number of mushrooms - 2.2⋅10 CFU

- Escherichia coli - absent

- Salmonella - absent

- enterobacteria - absent

- St. Aureus - absent.

Thus, the obtained extract is stable during storage for two years.

Example 23 (Dulcite + glycerin, 50% solution)

The extraction is carried out similarly to experiment 21, but to prepare the extractant, 1 kg of purified water, 240 g of glycerol, 760 g of dulcite, 4 g of sorbic acid, 1 g of calcium benzoate and 30 g of ascorbic acid are mixed. The extraction is carried out at 90 ° C, the increase in the concentration of the sum of oxycoumarins and glycosides of oxycoumarins stops 9 hours after the start of extraction. The filtrate is maintained under natural conditions for 5 days, while part of the dulcite crystallizes. The resulting suspension is filtered, 1210 g of extract is obtained. Product parameters are as follows:

1. Description - transparent dark brown, not very viscous syrup, with a characteristic pleasant fruity odor, sweet and sour taste;

2. The density of 1.08 g / cm ³ ;

3. pH 3.9;

4. The total content of flavonoids by the SF method (by coloring the aluminum complex) in terms of rutin 0.19%;

5. The total content of oxycoumarins and glycosides of oxycoumarins in terms of fraxetin 0.64%;

6. The content of tannins in terms of tannin 1.51%;

7. The content of polyphenolic compounds is 1.06%;

8. The content of Quaternary ammonium bases (betaines) in terms of glycine betaine - 0,0178%;

9. The content of SF polysaccharides by the method of 2.19%;

10. The content of ascorbic acid by titration of 1.24%;

11. Microbiological purity

- The total number of aerobic bacteria -2.8 * 10 ² CFU;

- The total number of mushrooms is 5.2 * 10 CFU.

After two years of storage in vivo, the extract has the following parameters:

1. Description - transparent dark brown, moderately viscous syrup, with a characteristic pleasant fruity odor, pleasant sweet and sour taste;

2. The density of 1.08 g / cm ³ ;

3. pH 4.1;

4. The total content of flavonoids by the SF method (by coloring the aluminum complex) in terms of rutin 0.175%;

5. The total content of oxycoumarins and glycosides of oxycoumarins in terms of fraxetin 0.625%;

6. The content of tannins in terms of tannin 1.49%;

7. The content of polyphenolic compounds 1.11%;

8. The content of Quaternary ammonium bases (betaines) in terms of glycine betaine is 0.0112%;

9. The content of SF polysaccharides by the method of 2.17%

10. The content of ascorbic acid by titration of 0.98%;

11. Microbiological purity

- The total number of aerobic bacteria is less than 10 CFU;

- The total number of mushrooms is less than 10 CFU.

Thus, the resulting syrup is stable when stored under natural conditions for two years.

Example 24 (xylitol 50%)

The experiment is carried out analogously to example 16, but in the preparation of the extractant, xylitol in the same amount is used instead of sorbitol. 1330 ml of extract is obtained, which, after cooling to room temperature and holding for one day, partially crystallizes. The crystals are filtered on a filter with a porous glass plate, squeezing until the flow ceases to drip. Get 320 g of very large crystals, almost containing no extractive substances of larch leaf solyanka and which are xylitol according to the results of the analysis. As a result of the experiment, 1380 g (1140 ml) of the extract are obtained. The parameters of the resulting product are as follows:

1. Description. Very dark red-brown syrup, unclear, with a pleasant fruity smell.

2. pH 4.1

3. The density of 1.21 g / cm ³

4. Heavy metals according to GF XIII - less than 0.001%

5. The total content of flavonoids SF method (aluminum complex) in terms of rutin 0.18%

6. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method in terms of fraksetin 1.12%

7. The content of oxycoumarins and glycosides of oxycoumarins (HPLC method):

- fraksetin 0.482%

- fraxidine 0.0251%

- isofraxidine 0.144%

- scopoletin 0,084%

- fraksidin 8-o-β-O-glucopyranoside 0.0106%

- scopolin 0.0115%

- fraxin 0.065%

8. The content of SF polysaccharides by the method of 2.2%

9. The content of ascorbic acid 0.918% (titration method)

10. The content of sorbic acid 0.22% (by GLC)

11. The content of flavonoids (HPLC method):

- rutin 0.047%

- campferol 0.059%

- apigenin 0.016%

15. The content of manganese ions 0.00012% (method of atomic emission spectroscopy with inductively coupled plasma)

12. Boron content 0.00022% (inductively coupled plasma atomic emission spectroscopy)

13. Microbiological purity:

- the total number of aerobic bacteria is less than 10 CFU

- total number of mushrooms - less than 10 CFU

- Bacillus cereus - absent

- Salmonella - absent

- Escherichia coli - absent.

After two years of storage of samples in vivo, the parameters of the extract are as follows:

1. Description. Dark red-brown syrup, transparent, has a characteristic pleasant smell (fruity), at the bottom of the bottles a thin black film - sediment.

2. pH 4.2

3. The density of 1.21 g / cm ³

4. The total content of flavonoids SF method (aluminum complex) in terms of rutin 0.16%

5. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method in terms of fraksetin 1.08%

6. The content of polysaccharides SF method 2.2%

7. The content of ascorbic acid 0.91% (titration method)

8. The content of sorbic acid 0.21% (by GLC)

9. Microbiological purity:

- the total number of aerobic bacteria is less than 10 CFU

- total number of mushrooms - less than 10 CFU

- Bacillus cereus - absent

- Salmonella - absent

- Escherichia coli - absent.

Thus, the resulting sample is stable during storage for two years. Xylitol crystallized upon receipt of the extract is used without further processing in subsequent experiments.

Example 25 (erythritol, preservatives less than 0.01%)

The experiment is carried out analogously to example 16, but erythritol in the same amount is used as the polyhydric alcohol in the preparation of the extractant, and 0.1 g of potassium sorbate is charged. 1439.9 g (1210 ml) of the extract are obtained. The parameters of the extract are as follows:

1. Description. Dark brown with a red tint, a muddy foaming syrup with a pleasant characteristic fruity odor.

2. pH 4.2

3. The density of 1.19 g / cm ³

4. Heavy metals according to GF XIII - less than 0.001%

5. The total content of flavonoids SF method (aluminum complex) in terms of rutin 0.16%

6. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method in terms of fraksetin 0.89%

7. The content of SF polysaccharides by the method of 2.8%

8. The content of ascorbic acid 0.89% (titration method)

9. The content of sorbic acid of 0.005% (by GLC)

10. Microbiological purity:

- total number of aerobic bacteria in 1 ml - 2⋅10 ² CFU

- the total number of mushrooms in 1 ml is less than 10 CFU

- Bacillus cereus in 1 ml - absent

- Salmonella in 10 ml - absent

- Escherichia coli in 1 ml - absent

- enterobacteria in 1 ml - less than 10 (normal - not more than 102)

- St. Aureus in 1 ml is absent.

The extract is packaged in dark glass bottles of 100 ml. After six months of storage of samples in vivo, the experiment was terminated due to obvious damage to the sample. Its parameters are as follows:

1. Description. Turbid dark brown transparent syrup with an unpleasant musty odor, sour taste.

2. pH 3.5

3. The total content of flavonoids by the SF method (aluminum complex) in terms of rutin is not detected;

4. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method in terms of fraxetin 0.26%;

5. The content of SF polysaccharides by the method of 0.5%

6. The content of ascorbic acid 0.15% (titration method)

7. Microbiological purity:

- aerobic bacteria 10 ⁵ CFU;

- mushrooms 5⋅10 ² CFU.

Thus, the obtained erythritic extract of larch leaf solyanka containing less than 0.01% preservatives is not stable during storage.

Example 26 (Industrial production of larch leaf solyanka extract)

For the process, an enameled percolator with a displacement of 200 l is used, equipped with a heating jacket, thermowell and fungal valve on the lower slope, and an enameled reactor with an anchor stirrer having a heating jacket, thermowell with thermal sensor and lower trigger with fungal valve.

For extraction, the cut dry grass of the larch leaf solyanka corresponding to the Mongolian standard MNS-4403-05 is used. The parameters of the material taken for extraction are as follows:

1. Description - cut grass with a rigid structure, greenish-yellowish gray fragments 8–25 mm × 0.2–1.3 mm in size;

2. Smell - specific, pleasant;

3. Taste - sour;

4. Humidity (by drying) - 5.2%;

5. Impurities of organic foreign substances - 0.16%;

6. Mineral impurities (sand, rock) - 0.4% (impurities insoluble in 10% hydrochloric acid);

7. Parts rotten or damaged by fungi - 0.22%;

8. Ash content - 8.4%;

9. The amount of oxycoumarins, SF method in terms of fraxetin 0.96%;

10. Substances extracted with water - 9.0%;

11. Mass fraction of tannins in terms of tannin in indigosulfonic acid 3.49%;

12. Mass fraction of polyphenolic compounds 2.83%;

13. Heavy metals according to GF XIII - less than 0.001%;

14. Microbiological purity:

- aerobic bacteria 7 * 102 CFU;

- mushrooms 6 * 10 CFU;

- Escherichia coli - absent;

- Pseudomonas spp - absent;

- Salmonella - absent;

- Staphylococcus - absent;

- Enteobacteraceae - absent.

55 kg of cut dry grass of larch solyanka are loaded into the percolator. 92.5 kg of purified water are charged into an enameled reactor. Turn on the mixer and the circulation of the coolant in the jacket for heating. When reaching 30 ° C in the mass through the hatch load 35 kg of 1,2-propylene glycol (pharmacopeia), then in portions of 80 kg of sorbitol. After complete dissolution of sorbitol, 32.5 kg of xylitol are charged, then stabilizers and preservatives - 7.31 kg of ascorbic acid, 2.72 kg of benzyl alcohol, 0.47 kg of potassium sorbate. All components are mixed when heated to 90 ° C and completely dissolved.

100 kg of extractant is drained through the lower slope of the reactor and transferred to a percolator. The coolant temperature is set at 85 ° C, the start of extraction is considered to be the moment the temperature reaches 80 ° C in the mass. Periodically, a sample is taken from the mass and the content of the amount of oxycoumarins in terms of fraxetin is determined. The concentration of the signal component slowly increases and after 8 hours it stops growing, which is considered the end of the process. The hot extract through the filter field is poured into a receiving tank, get 25.47 kg of extract with the following parameters:

1. Description - a thick dark brown, almost black syrup with a characteristic pleasant odor;

2. Density - 1.21 g / cm ³ ;

3. pH 4.2;

4. The total content of SF flavonoids by the method (by coloring the aluminum complex) in terms of rutin - 0.189%;

5. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method in terms of fraksetin - 0.633%;

6. The content of tannins in terms of tannin 1.44%;

7. The content of polyphenolic compounds is 1.02%;

8. The content of Quaternary ammonium bases (betaines) in terms of glycine betaine 0.0162%;

9. The content of SF polysaccharides by the method of 2.62%;

10. The content of ascorbic acid by titration by 2.33%;

11. The content of sorbic acid by GLC 0.17%;

12. The content of benzyl alcohol by GLC 1.06%;

13. The content of 1,2-propylene glycol by GLC 13.8%;

14. Microbiological purity:

- aerobic bacteria 9.9 * 10 ² CFU;

- mushrooms 9.3 * 10 CFU;

- Escherichia coli - absent;

- Pseudomonas spp - absent;

- Salmonella - absent;

- Staphylococcus - absent;

- Enteobacteraceae - absent.

To the wet meal remaining in the percolator, 75 kg of extractant are charged from the reactor and the process is repeated similarly to the above. The cessation of growth in the concentration of the signal component is detected after 12 hours, which is considered the end of the extraction process. The extract is poured as described above, and 59.70 kg of syrup are obtained with the following parameters:

1. Description - a thick dark red syrup with a characteristic pleasant fruity odor;

2. Density - 1.22 g / cm ³ ;

3. pH 3.9;

4. The total content of flavonoids by the SF method (according to the coloring of the aluminum complex) in terms of rutin - 0.172%;

5. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method in terms of fraksetin - 0.575%;

6. The content of tannins in terms of tannin 1.32%;

7. The content of polyphenolic compounds is 0.926%;

8. The content of Quaternary ammonium bases (betaines) in terms of glycine betaine 0.0259%;

9. The content of polysaccharides by the SF method 2.38%;

10. The content of ascorbic acid by titration 2.91%;

11. The content of sorbic acid by GLC 0.19%;

12. The content of benzyl alcohol by GLC 1.09%;

13. The content of 1,2-propylene glycol by GLC 14.1%;

14. Microbiological purity:

- aerobic bacteria 1.2 * 10 CFU;

- mushrooms 1.1 * 10 CFU;

75 kg of extractant from the reactor are again charged to the meal and the extraction is repeated. The equilibrium concentration of the signal component is reached after 16 hours, after which the extract is drained. Get 61.80 kg of syrup with the following parameters:

1. Description - dark red syrup with a characteristic pleasant fruity taste;

2. Density - 1.23 g / cm ³ ;

3. pH 3.8;

4. The total content of SF flavonoids by the method (by coloring the aluminum complex) in terms of rutin - 0.11%;

5. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method in terms of fraksetin - 0.337%;

6. The content of tannins in terms of tannin - 0.857%;

7. The content of polyphenolic compounds of 0.61%;

8. The content of Quaternary ammonium bases (betaines) in terms of glycine betaine 0,0096;

9. The content of polysaccharides by the SF method is 1.56%;

10. The content of ascorbic acid by titration of 2.35%;

11. The content of sorbic acid by GLC 0.175%;

12. The content of benzyl alcohol by GLC 1.05%;

13. The content of 1,2-propylene glycol by GLC 13.75%;

14. Microbiological purity:

- aerobic bacteria - less than 10 CFU;

- mushrooms - less than 10 CFU.

Thus, as a result of extraction, 146.97 kg of extract is obtained with the amount of oxycoumarins in terms of fraxetin of more than 0.3% (0.337-0.633%), suitable for obtaining medical preparations, dietary supplements and veterinary preparations. After extraction, wet meal remains in the amount of 158.03 kg, containing on average about 20% sorbitol, about 9% xylitol, more than 2% ascorbic acid, and a number of other animal components useful for the body - antioxidants, polysaccharides, vitamins, which suggests its possible use as a feed additive in animal husbandry.

Example 27 (Standardization of Syrups)

In an enameled reactor with an anchor mixer, equipped with a jacket, a thermowell with a temperature sensor, a lower descent with a fungal valve, 100 kg of purified water is loaded. When the mixer is turned on and the heated coolant is supplied to the apparatus jacket, 100 kg of food sorbitol are loaded in portions through the hatch. After complete dissolution and heating of the solution to 80 ° C, 0.5 kg of potassium sorbate and 1.5 kg of ascorbic acid are loaded into the reactor. The solution is stirred until the components are completely dissolved, and then poured into a polymer container.

The products obtained in experiment 26 are loaded into the reactor:

1. 25.47 kg of syrup with a total content of oxycoumarins of 0.633% in terms of fraxetin;

2. 59.70 kg of syrup with a total content of oxycoumarins of 0.575%;

3. 61.80 kg of syrup with a total content of oxycoumarins of 0.337%.

Then, 90 kg of solution from the polymer container is loaded into the reactor, stirred for 30 minutes at 60 ° C and poured into a polymer container. Get 237 kg of standardized syrup with the following parameters:

1. Description - a dark brown-burgundy transparent syrup with a pleasant fruity smell;

2. Density - 1.23 g / cm ³ ;

3. pH 3.7;

4. The total content of SF flavonoids by the method (by coloring the aluminum complex) in terms of rutin - 0,092%;

5. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method in terms of fraksetin 0.30%;

6. The content of tannins in terms of tannin 0.71%;

7. The content of polyphenolic compounds 0.502%;

8. The content of Quaternary ammonium compounds (betaines) in terms of glycine betaine is 0.0082%;

9. The content of polysaccharides by the SF method is 1.18%;

10. The content of ascorbic acid by titration of 1.88%;

11. Microbiological purity:

- the number of aerobic bacteria 1.3 * 10 ² CFU;

- the number of mushrooms 3.3 * 10 CFU.

The parameters of the sample after storage under natural conditions in a polymer container for two years are as follows:

1. Description - a dark brown-burgundy transparent syrup with a pleasant fruity smell;

2. Density - 1.23 g / cm ³ ;

3. pH 3.8;

4. The total content of SF flavonoids by the method (by coloring the aluminum complex) in terms of rutin - 0.091%;

5. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method in terms of fraksetin 0.30%;

6. The content of tannins in terms of tannin 0.72%;

7. The content of polyphenolic compounds 0.50%;

8. The content of Quaternary ammonium compounds (betaines) in terms of glycine betaine is 0.0078%;

9. The content of polysaccharides by the SF method 1.19%;

10. The content of ascorbic acid by titration of 1.74%;

11. Microbiological purity:

- the number of aerobic bacteria is less than 10 CFU;

- the number of mushrooms is less than 10 CFU.

Thus, the extract, which is convenient for practical use (production of medical and veterinary preparations, production of dietary supplements), standardized for signal components, is stable when stored under natural conditions in a polymer container for two years. Similarly, extracts according to examples 15-25 can be standardized, which ensures their practical use.

Example 28 (Obtaining a medicinal product for medical use)

In the reactor, analogously to example 27, 150 kg of a stabilized sorbitol solution (50%) are prepared, then 30 kg of a standardized extract are added with the stirrer turned on (as in example 27). The components are mixed for an hour, poured into a polymer barrel and transferred to the packaging. Packing is carried out in glass bottles of dark glass of 100 ml. Product parameters are as follows:

1. Description - reddish-brown syrup with a pleasant characteristic fruity odor, sour-sweet taste;

2. Density - 1.23 g / cm ³ ;

3. pH 3.7;

4. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method in terms of fraxetin 0.05%;

5. The content of SF polysaccharides by the method of 0.20%;

6. The content of ascorbic acid by titration of 0.78%;

7. Microbiological purity:

- the number of aerobic bacteria is less than 10 CFU;

- the number of mushrooms is less than 10 CFU.

After storing the samples in vivo for two years, their parameters are as follows:

1. Description - a reddish-brown syrup, transparent, with a pleasant characteristic smell of fruit, a sour-sweet taste with a hint of fruit compote;

2. Density - 1.23 g / cm ³ ;

3. pH 3.8;

4. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method in terms of fraxetin 0.05%;

5. The content of SF polysaccharides by the method of 0.19%;

6. The content of ascorbic acid by titration of 0.73%;

7. Microbiological purity:

- the number of aerobic bacteria is less than 10 CFU;

- the number of mushrooms is less than 10 CFU.

Thus, the extract of the larch leaf solyanka for medical use with the content of the signal component of 0.05% is stable when stored under natural conditions for two years. This product was used in studies of the acute toxicity of a medicinal product, the prevention and treatment of pneumonia (see example 41, 43, 44).

Example 29 (Obtaining a veterinary drug)

187.9 kg of purified water are loaded into the reactor, with the stirrer turned on, 10 kg of sorbitol, 100 g of ascorbic acid and 20 g of potassium sorbate are loaded, then 2 kg of the extract according to Example 27. The mass is stirred for 30 minutes and poured into a polymer container, then transferred to packaging . Packed in bottles of 100 ml of dark transparent plastic, a label is glued to each bottle. Product Parameters:

1. Description - yellow solution;

2. pH 4.8;

3. Density at 20 ° C 1.039 g / cm ³ ;

4. The content of the amount of oxycoumarins SF method in terms of fraksetin - 0,003%;

5. The content of SF polysaccharides by the method of 0.012%.

This product is used for veterinary clinical research (see example 42, 45-54).

Example 30 (dry grass-extractant ratio 0.003: 1)

100 kg of purified water is loaded into the enameled reactor, the mixer is turned on, the coolant is supplied to the apparatus jacket and heated to 80 ° C; in the process of heating at 40-50 ° C, 10 kg of xylitol, 200 g of ascorbic acid, 20 g of potassium sorbate and 90 kg of 1,2-propylene glycol are loaded; at 60 ° C, 0.6 kg of larch leaf grass containing the amount of oxycoumarins in terms of on fraksetin 0.99%. After reaching a temperature of 80 ° C in the mass, the extraction process is carried out until a constant concentration of signal components in the liquid phase is established; after 2.5 hours, the concentration of signal components reaches a stable level of 0.0028%. Extraction on this is considered complete. The hot extract is filtered to remove oil cake on a Druk filter, cooled with stirring and packaged in 100 ml polymeric bottles of dark plastic. Receive 1785 packages. Product parameters at the time of packaging are as follows:

1. Description - yellow solution with a pleasant fruity odor;

2. pH 4.5;

3. Density at 20 ° C 1.12 g / cm ³ ;

4. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method in terms of fraxetin 0.0028%;

5. The content of SF polysaccharides by the method of 0.0128%.

After two years of storage of samples in vivo, the parameters of the syrup are as follows:

1. Description - yellow solution with a pleasant fruity odor;

2. pH 4.4;

3. Density - 1.13 g / cm ³ ;

4. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method in terms of fraxetin 0.0029%;

5. The content of SF polysaccharides by the method of 0.013%.

Thus, the product for veterinary use, obtained by direct extraction, is stable when stored under normal conditions for two years. However, the energy consumption during extraction per unit of product is two orders of magnitude higher than when obtaining a concentrated extract with subsequent dilution to the required level of concentration of the amount of oxycoumarins.

Example 31 (prototype)

18.75 L of ethanol with a concentration of 96.0% was charged into the n / a reactor and purified water was added up to 54.7 L.

The stalks and leaves of the leafy leaf solyanka (Salsola laricifolia) are ground in a cutting mill to particles less than 0.5 mm. 12 kg of crushed grass is loaded into a maceration tank, and 12 liters of diluted alcohol are moistened to swell, incubated for 4 hours. The swollen raw materials are transferred to a percolator, they are loaded with 16 liters of diluted alcohol and insisted with frequent agitation for 3 days. After that, slowly percolate, after draining again add 26 l of diluted alcohol to the percolator and insist 3 days, then plums from two percolations are mixed and kept in a cool place for 24 hours, then filtered. The filtrate is analyzed, packed in 50 ml bottles of dark glass. In general, 30 l of extract (600 bottles of 50 ml) are obtained with the following parameters:

1. The amount of oxycoumarins SF method of 0.0179%;

2. The ethanol content of 31.0%;

3. The dry residue of 1.25%.

This product obtained in accordance with MN2031 (Mongolian patent) was used in comparative experiments (see example 43, 44).

Example 32 (aqueous extract)

In a glass reactor placed in a thermostat bath, 2 l of purified water, 5 g of ascorbic acid and 2.5 g of potassium sorbate are placed. Turn on the thermostat, heat the bath and the contents of the reactor to 90 ° C. In a hot extractant, in portions, as they are filled, 600 g of cut grass of larch leaf solyanka are loaded over an hour. The extraction process is carried out, periodically determining the content of the sum of oxycoumarins and glycosides of oxycoumarins; their concentration is slowly growing and after 24 hours it stops at the level of 0.055%. The hot extract is drained and filtered through a glass filter with a large porous plate. Obtain 1180 g of an aqueous extract, after cooling, having the following parameters:

1. Description - dark brown, slightly viscous, sour taste extract with a pleasant fruity odor;

2. Density - 1.0 g / cm ³ ;

3. pH 4.4;

4. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method in terms of fraxetin 0.055%;

5. The content of individual oxycoumarins and glycosides of oxycoumarins by HPLC:

- fraxidine 0.00047%;

- isofraxetin 0.00347%;

- scopoletin 0.00121%;

- fraksetin 0.0184%;

- scopolin 0.0070%;

- fraxin 0.00357%;

- fraxidine 8-o-β-D-glucopyrazonide 0.0007%;

6. The content of SF polysaccharides by the method of 1.92%;

7. The content of ascorbic acid 0.24% (titration method);

8. Microbiological purity:

- aerobic bacteria 8.2 * 10 ³ CFU;

- mushrooms 9.1 * 10 CFU.

After 3 months, the experiment was terminated due to obvious damage to the sample.

Its parameters:

1. Description - a turbid liquid of a dark color, sour taste, with an unpleasant rotten odor, on the surface a mucous film;

2. pH 3.8;

3. The total content of SF oxycoumarins by the method in terms of fraxetin is not detected;

4. The content of ascorbic acid is not detected;

5. Microbiological purity:

- aerobic bacteria - continuous growth;

- mushrooms - continuous growth.

Thus, the aqueous extract of larch leaf solyanka, despite the presence of preservatives and stabilizers, is not stable during storage. When extracted with water, the recoverable amounts of biologically active components are significantly less than under the conditions proposed according to the invention, which is most likely due to the preservation of the membrane structure of plant cells in a purely aqueous medium, which prevents extraction.

Example 33 (Obtaining biologically active additives for use in humans)

In an enameled reactor with a volume of 1 m ³ equipped with an anchor stirrer, 950 L of purified water, 45 kg of standardized extract (according to Example 27), 5 kg of ascorbic acid, 500 g of potassium sorbate, 50.0 g of calcium sorbate, 10 g of benzoate are loaded with stirring. sodium. Stirred for an hour, carbonized and Packed in polyethylene terephthalate containers for food from dark plastic with a volume of 0.5 l. Get 2000 units of packaged product, with the parameters at the time of packaging:

1. Description - yellow solution with a brownish tint with a pleasant smell, sweet and sour taste

2. Density at 20 ° C 1.041 g / cm ³

3. pH 4.2

4. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method of 0.0125%

5. The content of polysaccharides by SF method 0,055%

6. The content of ascorbic acid by titration of 0.52%

7. Microbiological purity

- The number of aerobic bacteria 110 CFU

- The number of mushrooms is less than 10 CFU.

After storing the product for 3 years, the following parameters:

1. Description - yellow with a brown tint solution with a pleasant fruity smell, sweet and sour taste, a thin dark film is found at the bottom of the packages

2. Density at 20 ° C 1.039 g / cm ³

3. pH 4.3

4. The total content of oxycoumarins SF method of 0.011%

5. The content of polysaccharides by SF method 0,045%

6. The content of ascorbic acid by titration of 0.44%

7. Microbiological purity

- The number of aerobic bacteria is less than 100 CFU

- The number of mushrooms is less than 10 CFU.

Example 34 (obtaining a veterinary preparation for productive animals)

In an enameled reactor with a volume of 0.5 m ³ equipped with an anchor stirrer, 395 l of purified water is charged through a measuring device, with the stirrer switched on, 1 kg of ascorbic acid, 20 g of potassium sorbate and 40 g of sodium benzoate are loaded through a hatch, then from a portable container of 4 kg of extract as in example 27. The mass is stirred for 30 minutes and transferred to the packaging. Packed in polyethylene canisters with a volume of 5 l, get 80 canisters of the product with the following parameters at the time of packaging:

1. Description - yellow solution with a pleasant smell, sour taste

2. pH 4.9

3. Density at 20 ° C 1.011 g / cm ³

4. The content of oxycoumarins SF method in terms of fraxetin 0.003%

5. The content of SF polysaccharides by the method of 0.011%.

After 2 years of storage of the product in the indicated container, the product parameters are as follows:

1. Description - yellow solution with a characteristic odor, sour taste, a slight dark film at the bottom of the cans

2. pH 4.9

3. Density at 20 ° C 1.010 g / cm ³

4. The content of oxycoumarins SF method in terms of fraxetin 0.0028%

5. The content of SF polysaccharides by the method of 0.010%.

Thus, the product parameters for 2 years of storage changed slightly and did not go beyond the permissible limits.

Example 35 (obtaining an extract in the absence of preservatives and stabilizers in the extractant)

In 2 glass reactors with a volume of 2 l each, 500 g of larch leaf solyanka grass is placed. Separately, 2.4 kg of mannitol is dissolved in a 5 L glass beaker equipped with a stirrer and a water bath, in 1.6 L of purified water heated to 90 ° C. The reactors are placed in a thermostat bath, the temperature is set at 90 ° C and the grass is poured with the prepared extractant. The extraction process is carried out until the growth of the concentration of the sum of oxycoumarins in the liquid phase stops. The mass is quickly transferred to a heated funnel with a glass porous filter plate, and filtered under vacuum, avoiding foaming of the filtrate. Obtain 2.58 l of the filtrate extract, which is divided into two portions of 1.29 l; 5 g of sorbic acid, 5 g of benzoic acid and 10 g of ascorbic acid are added in one serving. The solutions were kept at room temperature, then precipitated mannitol crystals were filtered off. As a result of the experiment, two samples of the extract are obtained:

With target additives, 591 g of extract are obtained with the following parameters:

1. Description - dark brown transparent syrup with a characteristic pleasant fruity odor, pleasant sour, moderately sweet taste;

2. pH 4.2;

3. density 1.08 g / cm ³ ;

4. The total content of flavonoids by the SF method (aluminum complex) in terms of rutin 0.31%;

5. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method in terms of fraksetin 1.18%;

6. The content of polysaccharides by SF method 3.9%;

7. The content of ascorbic acid 1.84% (titration method);

8. Microbiological purity:

The total number of aerobic bacteria is 1.56 * 10 ² CFU;

The total number of mushrooms is 4.8 * 10 CFU.

A sample without targeted additives, 584 g of extract with the following parameters are obtained:

1. Description - a very dark brown syrup with a characteristic smell of dried fruits, sweet taste;

2. pH 6.1;

3. density 1.07 g / cm ³ ;

4. The total content of flavonoids by the SF method (aluminum complex) in terms of rutin 0.29%;

5. The total content of oxycoumarins and glycosides of oxiscoumarins by the SF method in terms of fraksetin 1.09%;

6. The content of polysaccharides by SF method of 4.1%;

7. The content of ascorbic acid (titration method) 0.15%;

8. Microbiological purity:

The total number of aerobic bacteria is 1.81 * 10 ³ CFU;

The total number of mushrooms is 8.3 * 10 CFU.

Then daily samples are taken from both samples and the microbiological purity of the products is determined, as well as the pH value and the signal component — the sum of oxycoumarins. The results are presented in table 23. From the results it is seen that in the absence of target additives, the extracts obtained are unstable, and microorganisms multiply rapidly in them (Fig. 7, 8).

Nevertheless, when using a water-mannitol extractant, the extract obtained is stable for at least a day, which allows you to carry out all the necessary technological procedures, including introduce preservatives and stabilizers to obtain the medicine according to claim 1; and also allows you to use it for its intended purpose without targeted additives at home, in a hospital, etc., i.e. when there is no need for packaging and long-term storage of the medicinal product according to claim 1.

Example 36 (Industrial production of the extract without the addition of stabilizers / preservatives in the extractant)

Dietary supplement for humans

The process is carried out on the hardware circuit shown in FIG. 9. For the process, an enamelled reactor P1 is used to prepare the extractant with a stirrer and a jacket, equipped with a strain gauge; enameled percolator Df (P) 2 with a working volume of 200 l, equipped with a heating jacket, false fluoroplastic bottom, thermowell and fungal valve on the lower slope; P3 enameled reactor for collecting the filtrate extract and introducing target additives with a stirrer, a shirt, a hatch, a fungal valve on the lower slope and a strain gauge; enameled reactor P4 for averaging and standardization of the obtained extract equipped with a jacket, stirrer and bottom fungal valve; enameled mernik M5 for dosing the extract in the preparation of dietary supplement; P6 enameled reactor for a design pressure of 6 atm for the preparation of dietary supplements and carbonization, equipped with a stirrer, a jacket, a carbon dioxide bubbler and a lower fungal valve.

As a raw material for extraction, dry grass of larch leafwort (Salsola laricifolia) is used, which corresponds to the Mongolian standard MNS-4403-05; The parameters of the raw materials taken are as follows:

1. Description - cut grass with a rigid structure, greenish-yellowish gray fragments measuring 8-25 mm × 0.2-1.3 mm.

2. Humidity (by drying) 5.3%.

3. Ash content of 7.9%.

4. The amount of oxycoumarins, SF method in terms of fraksetin 1.99%.

5. mass fraction of tannins in terms of tannin in indigosulfonic acid 3.53%.

6. Mass fraction of polyphenolic compounds 2.94%.

7. Microbiological purity

The number of aerobic bacteria 8.2 * 10 ² CFU

The number of mushrooms 8.4 * 10 CFU

Esherichia coli - absent

Pseudomonos spp - missing

Salmonella - absent

Staphylococcus - absent

Enterobacteriaceae - absent.

60 kg of cut dry grass of larch solyanka (bulk density 0.367 g / cm ³ ) is loaded into the percolator. In an enameled reactor P1 (2 m ³ ) load 1000 kg of purified water, include a stirrer, heat the water to 80 ° C. Then load 500 kg of sorbitol and mix until completely dissolved. After that, 250 kg of xylitol and 250 kg of inositol are charged and mixed until completely dissolved. The extractant is stored in P1 with stirring and a temperature of 80 ± 5 ° C.

100 kg of extractant is drained through the lower outlet from the reactor P1 into the percolator Df (P) 2. The coolant temperature is set at 85 ° C, the start of extraction is considered the moment the temperature reaches 80 ° C in the phyto-mass. Periodically, a sample is taken from the mass and the content of the amount of oxycoumarins in terms of fraxetin is determined. The concentration of the signal component increases slowly, after 8.5 hours it stops growing, which is considered the end of the process. At the reactor P3, a stirrer is switched on and the hot extract is drained into the reactor P3 through a filter field Df (P) 2. The P3 reactor is equipped with a strain gauge, which allows you to determine the weight of the drained extract. At the first extraction, 22.8 kg of extract is obtained. Through the loading hatch, 10 g of potassium sorbate are loaded into the P3 reactor, mixed, a sample is taken to determine the pH; pH 5.8. Ascorbic acid is added in portions until a pH of 5.0 is reached; this is achieved by adding 16 g of ascorbic acid. The content of oxycoumarins in the extract is 0.688%, and flavonoids in terms of rutin 0.178%. The extract from reactor P3 is transferred to reactor P4 with the stirrer operating. In reactor P4, the extract was cooled to room temperature with stirring.

In the percolator Df (P) 2 to wet meal, 72 kg of extractant from reactor P1 are loaded in portions. The extraction process is carried out similarly to that described during the first extraction, the equilibrium is established after 9.5 hours. Pour 62.5 kg of the extract into the P3 reactor, add 28 g of potassium sorbate and ascorbic acid to a pH of 5.0. The extract from reactor P3 is transferred to reactor P4 with the stirrer operating. In reactor P4, the extract was cooled to room temperature with stirring.

In a similar manner, a third and subsequent extraction is carried out until the extractant is completely consumed (final extractant: dry grass ratio = 1: 0.03). As a result, in the reactor P4 receive 1940 kg of syrup extract with the following parameters:

1. Description - a thick dark brown reddish syrup with a characteristic pleasant fruity smell, sweet-sour taste.

2. The density of 1.21 g / cm ³

3. pH 5.0

4. The total content of flavonoids in terms of rutin 0.0091%

5. the total content of oxycoumarins and glycosides of oxycoumarins by the SF method in terms of fraksetin 0.027%

6. Microbiological purity

The number of aerobic bacteria 1.5 * 10 ² CFU

The number of mushrooms is less than 10 CFU.

To prepare a food product - a biologically active additive that strengthens the immune system and reduces the incidence of acute respiratory infections, in a 2 m ³ enameled P6 reactor equipped with an anchor stirrer and a thermowell, 1981 l of purified water, 0.4 kg of ascorbic acid, 0.01 kg are loaded with stirring citric acid, 0.5 kg of potassium sorbate, 0.1 kg of calcium sorbate, 0.02 kg of sodium benzoate.

Using a measuring device, 7.74 kg of syrup extract (6.4 L) is loaded into the reactor, stirred for 1 hour, then cooled to 5 ± 1 ° C, carbonized by feeding food carbon dioxide through a bubbler. The resulting solution is Packed in polyethylene terephthalate containers for food products from dark plastic with a volume of 0.5 l. 3978 units of packaged product are obtained, with parameters at the time of packaging:

1. Description - yellow solution with a brown tint with a pleasant fruity odor, sweet and sour, with a “compote” hue of taste

2. Density at 20 ° C 1.002 g / cm ³

3. pH 4.2

4. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method is 0.0001%

5. The content of ascorbic acid 0.02% (titration method)

6. Microbiological purity:

The number of aerobic bacteria 2 * 10 ² CFU

The number of mushrooms is less than 10 CFU.

After storing the product for 2 years, the product parameters are as follows:

1. Description - yellow solution with a brownish-reddish tint with a pleasant fruity odor.

2. pH 4.3

3. The total content of oxycoumarins and glycosides of oxycoumarins SF method 0,00009%.

4. The content of ascorbic acid is 0.018% (titration method).

5. Microbiological purity:

The number of aerobic bacteria is less than 10 CFU

The number of mushrooms is less than 10 CFU.

A dietary supplement based on the extract of larch leaf solyanka has a pleasant taste, stable during storage for 2 years and is designed to reduce the incidence of acute respiratory infections in the autumn-winter period, diarrhea in the summer period and other nosologies associated with functional immunity deficiency.

Example 37 (extractant without target additives, 30% xylitol)

Feed additive for productive animals, stable extract without targeted additives.

The process is carried out on the hardware circuit described in example 36.6 kg of cut grass of larch leaf solyanka is placed in the percolator Df (P) 2. An extractant is prepared in reactor P1 by dissolving 600 kg of xylitol in 1400 kg of purified water. From reactor P1, 100 kg of extractant are loaded into the percolator Df (P) 2; extraction is carried out at 80 ° C (temperature in the percolator) until a constant level of signal components is achieved - oxycoumarins in terms of fraxetin, this occurs after 5.5 hours. Then the extract from the percolator Df (P) 2 is poured into the reactor P3 and after measuring the weight, volume and sampling in the reactor P4. During the first extraction, 85.8 kg of extract is obtained with the content of oxycoumarins 0.0733% and flavonoids 0.0218%, with a pH of 5.8 and a density of 1.109 g / cm ³ . 1 kg of this extract is taken in a bottle of dark glass, stored under natural conditions, taking samples daily, in which the pH, the content of oxycoumarins in terms of fraksetin and flavonoids in terms of rutin are determined.

For the next extraction, extractant from reactor P1 is taken in an amount equal to the obtained extract, i.e. 85.8 kg; extraction is carried out under the same conditions. After 7 hours, the level of signal components ceases to change, which indicates the completion of extraction. Thus, the extraction of the loaded grass is repeated until the prepared extractant is exhausted. The extracts without introducing the target additives are poured into reactor P3, and then transferred to reactor P4, in which the temperature of 5 ° C ± 1 ° C is maintained by circulating the refrigerant through the jacket; draining the extracts is carried out with the stirrer turned on. During cooling, part of xylitol crystallizes from the extracts, the suspension is stirred under the indicated conditions until the extraction process is completed and the extractant is completely consumed. A total of 1992 kg of extract is obtained. In the reactor P4, 1 kg of benzoic acid is loaded, the mass is heated to room temperature (the precipitated xylitol and the loaded benzoic acid are dissolved), then ascorbic acid is added in portions to the extract to pH 4.9, for this purpose 1.2 kg of ascorbic acid is charged . The resulting product is Packed in 5 l polyethylene canisters of 5 kg, get 398 canisters.

Product parameters at the time of packaging:

1. Description: brownish-yellow syrup, with a characteristic fruity odor, pleasant sweet and sour taste;

2. The density of 1.108 g / cm ³ (at 20 ° C);

3. pH 4.9;

4. The content of oxycoumarins and glycosides of oxycoumarins, in terms of fraxetin 0.00315%, spectrophotometric method;

5. The content of flavonoids in terms of rutin spectrophotometric method of 0.00122%;

6. The content of ascorbic acid by titration method 0,063%;

7. Microbiological purity:

The number of aerobic bacteria 5.8 * 10 ² CFU;

The number of mushrooms 2 * 10 CFU;

Escherichia coli in 1 g - not detected;

Salmonella in 10 g is absent;

Bacillus cereus - absent.

After two years of storage under natural conditions, the product parameters are as follows:

1. Description: brownish-yellow syrup, with a characteristic fruity odor, pleasant sweet and sour taste;

2. The density of 1.108 g / cm ³ (at 20 ° C);

3. pH 4.8;

4. The content of oxycoumarins in terms of fraxetin, SF method 0,00312%;

5. The content of flavonoids in terms of rutin, spectrophotometric method of 0.00121%;

6. The content of ascorbic acid by titration of 0.059%;

7. Microbiological purity:

The number of aerobic bacteria 4.3 * 10 CFU;

The number of mushrooms is 1.2 * 10 CFU.

It has been experimentally established that the main risk factor for the instability of larch leaf solyanka extracts is the growth of aerobic, mainly spore-forming bacteria, the spores of which are present in the initial phyto-raw material. At the same time, the first extract is enriched with spores as much as possible. This problem is solved by the introduction of preservatives in an amount of 0.01-1.7 mass. % and a stabilizer to a pH of 5-3.5, i.e. to those values at which food preservatives are most effective; however, some polyhydric alcohols have their own bacteriostatic activity, preventing the growth of microorganisms, although not for a very long time, but this allows you to carry out all the necessary technological procedures to obtain the finished drug. For these reasons, the stability of the extract in the absence of target additives was tested for the first spore-enriched first extract from a phyto-raw material load, the results are shown in table 24. In addition to the content of phytoextract signal components, the content of oxalic acid was also determined as one of the main metabolites of vegetative microorganisms and fungi ( by GLC after methylation with diazomethane). It can be seen from the obtained experimental data that, in the absence of target additives, the extract obtained by extraction with a 30% xylitol solution is stable for two weeks even at room temperature.

The resulting product was used as a feed additive to increase the non-specific resistance of animals in pig farming. The experiment was carried out on two groups of sows of 15 goals, selected for approximately the same weight, without obvious pathology. Pregnant uterus in the experimental group was given with water a solution of a feed additive in an amount of 0.1 ml per 1 kg of body weight twice a day; after farrowing, the supplement was not given. The results are shown in table 25.

It is evident from experience that the introduction of an immunomodulating feed additive into the diet of pregnant uterus based on the extract of larch leaf solyanka significantly increases the nonspecific resistance of both uterus and piglets, which is expressed in a significant increase and normalization of the physiological parameters of animals.

Example 38 (stability of the extract obtained without the use of preservatives / stabilizers, within the declared pH, preparation for poultry farming)

The process was carried out on the hardware circuit described in example 36.

The parameters of the experimentally tested batch of larch leaf solyanka grass (Salsola laricifolia) (batch 011515):

1. Description - cut grass with a rigid structure, greenish-yellowish moist fragments of 5-30 × 0.1-0.5 mm in size;

2. Smell - specific, pleasant;

3. Taste - sour, fruity;

4. Humidity (by drying) 6.1%;

5. Mineral impurities (insoluble in 10% hydrochloric acid) - 0.6%;

6. Parts that are rotten or damaged by fungi are not detected;

7. Ash content 8.1%;

8. The amount of oxycoumarins, SF method in terms of fraxetin 3.88%;

9. The amount of flavonoids in terms of rutin - 1.3%;

10. Mass fraction of tannins in terms of tannin on indigosulfonic acid 5.68%;

11. Mass fraction of polyphenolic compounds 4.42%;

12. Heavy metals according to GFHSh less than 0.001%;

13. Arsenic content by atomic emission method - not detected;

14. The cadmium content of the atomic emission method is 0.001 mg / kg;

15. Lead content by atomic emission method 0.004 mg / kg;

16. Mercury content by atomic emission method - not detected;

17. The copper content of the atomic emission method of 0.8 mg / kg;

18. microbiological purity:

The total number of aerobic bacteria is 8.2 * 10 ² CFU;

The total number of mushrooms is 5.8 * 10 CFU;

Escherichia coli - absent;

Salmonella - absent;

Staphylococcus - absent;

Enterobacteriaceae - absent.

60 kg of cut grass of larch solyanka (Salsola laricifolia) is placed in a percolator with a seal, the extractant is prepared by mixing in a reactor P1 160 kg of 1,2-propylene glycol and 40 kg of purified water. The extractant is heated to 80 ° C with stirring, poured through the lower descent half (100 kg) and transferred to a percolator. The process is carried out at 90 ° C until the change in the concentration of signal components ceases, this occurs after 3.5 hours. The extract is poured into the reactor P1, obtained during the first extraction of 35.8 kg of extract with the following parameters:

1. Description - dark brown syrup with a characteristic pleasant odor with a slight sediment.

2. pH 6.2.

3. The density of 1.09 g / cm ³ .

4. Heavy metals according to GFIII III less than 0.001%.

5. The total content of flavonoids by the SF method in terms of rutin is 0.16%.

6. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method in terms of fraksetin 1.09%.

7. The content of polysaccharides by the SF method is 1.9%.

8. Microbiological purity:

The total number of aerobic bacteria is 1.8 * 10 ² CFU. The total number of mushrooms is less than 10 CFU. From this sample, 5 samples of 1 l were taken to study the stability of the extract at different pH values.

The extractant residue is poured to the meal from the reactor P1 and the extraction is repeated under the same conditions, equilibrium is reached after 6.5 hours; the extract is drained and receive 84.5 kg; in a reactor P3, both portions are mixed and 115.3 kg of extract are obtained with the following parameters:

1. Description - dark brown syrup with a characteristic pleasant odor, with a slight sediment.

2. pH 6.5.

3. The density of 1.088 g / cm ³ .

4. Heavy metals for GF XIII less than 0.001%.

5. The total content of flavonoids by the SF method in terms of rutin is 0.13%.

6. The total content of oxycoumarins and glycosides of oxycoumarins in terms of fraxetin 1.048%.

7. The content of polysaccharides by the SF method is 1.95%.

1.5 m ³ of purified water is charged into reactor P4; with the stirrer switched on, 115.3 kg of the combined extract are poured from reactor P3; reactor P3 is washed twice with 150 L of purified water, which is drained into reactor P4. With continued stirring, 0.2 kg of potassium sorbate is loaded into the reactor, then ascorbic acid is added in portions until a stable pH of 3.5 is reached; this requires 61.3 kg of pharmacopoeial ascorbic acid (with a basic substance content of 99.5% in this series). Then the purified water is brought to a volume of 2 m ³ , and Packed in polymer cans with a volume of 20 l (100 units). The resulting product at the time of release has the following parameters:

1. Description - a yellow solution with a clear fruity odor, pleasant sour taste.

2. pH 3.5.

3. The density of 1.017 g / cm ³

4. Heavy metals for GF XIII less than 0.001%.

5. The total content of flavonoids by the SF method for coloring the aluminum complex of the complex, in terms of rutin, is 0.0075%.

6. The total content of oxycoumarins and glycosides of oxycoumarins by the SF method, in terms of fraksetin, 0.0604%.

7. The content of ascorbic acid by titration is 3.07%.

8. The content of polysaccharides by SF method of 0.112%.

9. Microbiological purity:

The total number of aerobic bacteria 1.2 * 10 ² CFU

The total number of mushrooms 1.5 * 10 CFU

Escherichia coli - absent

Pseudomonas spp - missing

Salmonella - absent

Staphylococcus - absent.

An experiment on the effect of the drug on indicators of nonspecific immunity in broiler chickens was carried out in a vivarium of a poultry farm on broiler chickens of the Cobb 500 cross. In the experiment used 2 groups of chickens with 35 goals each - control and experimental. The experimental group was given daily a preparation with drinking water at a dose of 1 ml / kg of bird weight, in the control group, a 3% solution of ascorbic acid was given in the same amount.

Both solutions were given once daily for 112 days. The study used methodological recommendations [Quality assessment of feed, organs, tissues, eggs and poultry. The methodical guide for livestock laboratories under the general editorship of the academician of the Russian Academy of Agricultural Sciences Fisinina et al. / Sergiev Posad. 2010. VNITIP. 120 S.], [Methodology for research on the technology for the production of eggs and poultry meat under the general editorship of Lukashenko B.C. et al. / Sergiev Posad. 2015. VNITIP. 104 С.ISBN 978-5-980-20-154-8], [Feed, feed additives, biologically active substances for agricultural poultry Ponomarenko Yu.A. et al. / M.: Printing House of the Agricultural Academy. 2009.665C. ISBN 978-5-85941-323-2], [Diseases of poultry and farm birds edited by Kalenka B.U. and others. 10th edition in 3 volumes / M .: Aquarium Print. 2011. ISBN 978-5-4238-0080-2], the results of the study are shown in tables 27 and 28.

Thus, the preparation based on the extract of larch leaf solyanka when growing broilers significantly increased the non-specific resistance of the bird, which was reflected in the normalization of immunological parameters (Tables 26 and 27) and the safety of the livestock; in the experimental conditions, the safety increase was 14.3% compared with the control.

To study the stability of extracts in the absence of special additives (preservatives and stabilizers) and with the introduction of additives to different pH values, selected samples from the first extract are used (see above), because it was experimentally found that the first extracts during the extraction process contain the largest number of spores of microorganisms and, accordingly, the risk of insufficient stability is higher compared to the second and subsequent extracts.

The extract was taken as the first sample without the addition of target additives; to the next four samples, potassium sorbate was added in an amount of 0.1 g (sample 1 kg), while the pH increased to 6.8. Ascorbic acid was added to samples 3, 4, and 5 to a pH of 5.5, 3.5, and 3.0, respectively. Samples were stored under natural conditions, samples were taken in the first week every day, then once a week.

Samples were stored under natural conditions, samples were taken in the first week every day, then once a week. The results of changing the parameters of the extracts are shown in table 28.

From the results of an experiment on the stability of the larch leaf solyanka extract, it can be seen that, in the absence of targeted additives, the extract is suitable for the preparation of a drug for almost four weeks. The introduction of potassium sorbate without pH-stabilizing stabilizers does not protect against the growth of microorganisms under experimental conditions, but as the pH decreases caused by microorganisms, the effectiveness of the preservative increases and becomes sufficient. Extracts with a pH of 5.5 to 3.0 are stable during storage, however, a sample with a pH of 3.0 due to its extremely acidic taste is hardly acceptable when used.

Example 39 (the effect of diluent on the biological activity of the claimed drugs)

In a two-liter glass equipped with a stirrer, 1 l of purified water is placed, heated to 60 ° C and 500 g of sorbitol are loaded in portions, after dissolving 330 g of xylitol and 330 g of erythritol.

In a two-liter glass reactor, placed in a thermostat, load 600 g of cut grass of larch leaf solyanka and pour the prepared extractant. Set the temperature of the thermostat 90 ° C, the countdown of the start of extraction from reaching the set temperature in the thermostat. With an interval of 30 minutes, samples of the extract are taken and the content of oxycoumarins is determined by the SF method in terms of fraxetin. The equilibrium concentration is established after 8.5 hours, after which the reactor is taken out, the extract is drained. As a result of the experiment, 1158 g of extract is obtained with the following parameters:

1. Description - thick dark brown transparent syrup with a pleasant fruity odor, sweet taste;

2. The density of 1.24 g / cm ³ ;

3. pH 6.6;

4. The total content of flavonoids by the SF method (by coloring the aluminum complex) in terms of rutin 0.164%;

5. The total content of oxycoumarins and glycosides of oxycoumarins in terms of fraksetin 0.722%;

6. The content of polysaccharides by the SF method of 3.02%.

Samples of the drug are prepared from the extract obtained as follows: the sample of the syrup taken with a magnetic stirrer is dissolved in the selected diluent (purified water, 5% sorbitol solution or 50% sorbitol solution), preservatives are added, then the pH value is adjusted carefully by the introduction of ascorbic acid to 4, 0 ± 0.5 (at 20 ° C), after which it is adjusted with the appropriate diluent to the desired weight.

Sample No. 1 - 0.6 g of the extract is dissolved in 500 g of purified water, 1.5 g of potassium sorbate, 1.5 g of sodium benzoate are dissolved with stirring and adjusted to pH = 4.0 ± 0.5 with the addition of crystalline ascorbic acid; adjusted with purified water to 600 g;

Sample No. 2 - is prepared similarly to Example No. 1, but instead of purified water use a 5% sorbitol solution;

Sample No. 3 - prepared similarly to Example No. 1, but instead of purified water use a 50% sorbitol solution;

Sample No. 4 - 1.5 g of the extract 1.5 g is dissolved in 500 g of purified water, 1.5 g of potassium sorbate, 1.5 g of sodium benzoate are dissolved with stirring and adjusted to pH = 4.0 ± 0 by adding ascorbic acid. 5; adjusted with purified water to 600 g;

Sample No. 5 - prepared similarly to Example No. 4, but instead of purified water use a 5% sorbitol solution;

Sample No. 6 - prepare similarly to sample No. 4, but instead of purified water use a 50% sorbitol solution;

Sample No. 7 - 300 g of the extract is dissolved in purified water, 1.5 g of potassium sorbate, 1.5 g of sodium benzoate are dissolved with stirring and adjusted with ascorbic acid to pH = 4.0 ± 0.5; adjusted with purified water to 600 g;

Sample No. 8 - prepared similarly to Example No. 7, but instead of purified water use a 5% sorbitol solution;

Sample No. 9 - prepared similarly to Sample No. 7, but instead of purified water use a 50% sorbitol solution;

Sample No. 10 - 1.5 g of potassium sorbate, 1.5 g of sodium benzoate are dissolved in 600 g of the extract with stirring, and adjusted to pH = 4.0 ± 0.5 with the addition of ascorbic acid.

The obtained samples are studied for the manifestation of pharmacological activity similarly to experiment 3. The comparison drug was taken the well-known drug "Immunal drops for oral administration", which is an alcohol solution of the juice of the herb Echinacea purpurea (Echinacea purpurea (L.) Moench) /

Materials and methods

In the experiment, C57BL16 mice were used; to reproduce the pathological process, the influenza virus A / PR / 8/34 (H1N1), obtained from the Museum of the Research Institute of Influenza RAMS and adapted to mice, was used. The therapeutic effect of the drugs was evaluated by the intensity of virus reproduction in the lungs after intranasal infection of experimental and control mice under light ether anesthesia at a dose of 0.01 LD ₅₀ with the influenza virus in a volume of 0.05 ml per mouse. The drugs began to be administered intragastrically from the 3rd day after infection at a dose of 1300 mg / kg 2 times a day for 10 days. A control group of mice received distilled water.

A total of 12 experimental groups were formed (each group of 25 animals):

1. Animals without treatment (control group);

2. Animals treated with Immunal;

3. Animals treated with the drug "Sample No. 1" (see above);

4. Animals treated with the drug "Sample No. 2" (see above);

5. Animals treated with the drug "Sample No. 3" (see above);

6. Animals treated with the drug "Sample No. 4" (see above);

7. Animals treated with the drug "Sample No. 5" (see above);

8. Animals treated with the drug "Sample No. 6" (see above);

9. Animals treated with the drug "Sample No. 7" (see above);

10. Animals treated with the drug "Sample No. 8" (see above);

11. Animals treated with the drug "Sample No. 9" (see above);

12. Animals treated with the drug "Sample No. 10" (see above).

The virus content in the lungs was determined 96 hours after infection by titration of a 10% pulmonary suspension in a culture of the chorioallantoic membrane of chicken embryos (V. Ilyenko, “Methods for testing and evaluating the antiviral activity of influenza virus. Guidelines,” 35 pp., St. Petersburg, 1977 ) The titer of the virus was determined by Reed and Mench and expressed in units. TID ₅₀ (tissue infectious doses).

The degree of lung damage was assessed by the presence of hemorrhagic foci and expressed as a percentage of the affected lobes.

Animals were sacrificed on day 14 after infection, the lungs were removed and used for biochemical and pathomorphological studies. The level of lipid peroxidation was determined, which was estimated by the content of malonic aldehyde in the lung tissue homogenate. The antioxidant activity of the tissue was evaluated based on the ability of the lung tissue homogenate to inhibit the oxidation of egg yolk phospholipids induced by ferrous iron.

The nature of the pathological process was evaluated by macroscopic and microscopic changes. Macroscopically evaluated the frequency of detection of lesions of the lungs. The intensity of the viral lesion of the respiratory epithelium, the intensity of the toxic manifestations of influenza, as well as the activity and nature of inflammatory cellular reactions were microscopically determined.

For histological studies, the lungs and adrenal glands of animals were fixed in a 10% solution of neutral formalin, dehydrated in alcohols of increasing concentration and embedded in paraffin; paraffin sections were stained with hematoxylin-eosin and examined using a microscope.

The results of the study.

The studied preparations under the experimental conditions did not significantly affect the intensity of the multiplication of influenza virus in the lungs of mice of the experimental groups as compared with the control, however, a certain dose-dependent (depending on the extract content in the experimental preparation) titer reduction suggests a direct antiviral effect. In animals infected intranasally with the influenza virus and not receiving treatment, a typical influenza bronchopneumonia developed in the lungs, 3 days after infection, signs of a specific lesion were found both in the trachea and bronchi, and in the alveoli. In the thickness of the epithelium, polymorphic white blood cells migrated, undergoing massive decay there. Peribronchial connective tissue acquired swelling and was infiltrated by polynuclear cells. As a result, these processes led to interstitial edema and a decrease in the lumen of the alveoli, interalveolar connective tissue was abundantly infiltrated by free-cell elements. The use of preparations based on extracts of larch leaf solyanka reduced the level of lung damage 2-3 times compared with the control, while in cases of preparations 6-12 corresponding to the claimed composition, the protective effect was relatively the same and significantly higher than in the case of example 43-45 (the content of the extract is less than the claimed), and in the case of example 42 (the well-known preparation "Immunal"). Preparations based on extracts of larch solyanka have a pronounced effect on the antioxidant systems of the lung tissue, reducing the level of peroxidation and significantly, significantly increasing the antioxidant activity of lung tissue, which can be attributed to the effects of oxycoumarins, which are known effective antioxidants. In relation to these effects, a clear dose dependence is detected.

Thus, preparations based on the extract of larch solyanka, at a concentration of the native extract from 0.24 to 99.85% (in the latter case, the native extract with targeted additives, but without dilution) show a pronounced protective and therapeutic effect on the model of influenza infection. A decrease in the concentration of the native extract in the drug below 0.24% (up to 0.1%) significantly reduces the therapeutic effect. The composition of the diluent solution does not significantly affect the biological effects of the drug.

Example 40 (The effectiveness of drugs according to the invention in the case of using various polyhydric alcohols to obtain the extract)

The effectiveness of the drugs according to the invention obtained by extracting the herb of the larch leaf in aqueous solutions of polyhydric alcohols, in the case of using various polyhydric alcohols, was compared by prophylactic activity on the model of influenza infection. In the experiment I used C57B L / 16 mice, which have reduced T-cell immunity and are therefore highly sensitive to the influenza virus. A total of 8 experimental groups of animals were formed, 30 animals per group. For the experiment, preparations with a concentration of extracts of 99.89 masses were used. %

The drugs were administered to mice intragastrically at a dose of 1000 mg / kg 2 times a day for 10 days; the control group received a similar amount of distilled water. On day 11 after the start of drug administration, mice were infected intranasally under mild ether anesthesia with a strain of the virus A / PR / 8/34 (H0N1) in a volume of 0.05 ml per mouse. The lethal dose of the virus was determined in a preliminary experiment by titration of the virus in mice, followed by the calculation of LD50. The results of the experiment were evaluated on day 6, recording the mortality of mice in the control and experimental groups. The evaluation of the test results was carried out on the basis of the following indicators:

- Comparison of the percentage of dead mice in the control and experimental groups;

- determination of the protection index of the test drug

where the protection coefficient was calculated by the formula

In the control group, the virus caused almost 90% death of animals. The introduction of drugs significantly, at times, reduced the mortality rate of mice and increased the protection index. All preparations based on extracts of larch leaf solyanka turned out to be close in effectiveness, the effect slightly depended on the polyhydric alcohol used in the extraction.

Thus, the drug according to the invention exhibits the claimed activity when using various polyhydric alcohols for extraction, with an insignificant difference in effects. There is a correlation between the severity of the biological effect and the content in the extracts of signal determinants - oxycoumarins and polysaccharides.

Example 41 (Study of acute toxicity of a medicinal product)

Materials and methods

The determination of acute toxicity indices included experiments on nonlinear white rats weighing 160-180 g of age 13-14 weeks and white mice weighing 18-20 g of age 8-9 weeks of both sexes. Rats and mice came from the nursery RAMS "Rappolovo" of the Leningrad region.

Recorded indicators: mortality, time of death of animals, symptoms of poisoning, daily monitoring of general condition and behavior, weighing, consumption of feed and water, autopsy and macroscopic description of dead and all surviving animals at the end of the study (euthanasia was carried out by overdose of ether), assessment of local irritant effect , determination of mass coefficients of internal organs.

The preparation obtained in example 28 was administered to white rats and mice of both sexes (6 animals of the same sex) intragastrically through an atraumatic metal probe in increasing doses according to Litchfield-Wilcoxon. The follow-up period was 14 days.

The investigated dose range was 5000, 10000 and 15000 mg / kg. High doses were achieved by repeated injections of the drug with an interval of 30 minutes. Control animals received similar volumes of distilled water at intervals of 30 minutes (maximum solvent volume).

Research results

In neither of the experimental groups, nor in mice, nor in rats, death was noted. The general condition and behavior of animals was as usual.

Tables 32 and 33 show the mass coefficients of the internal organs of mice and rats, averaged over groups. There were no significant differences between the groups.

Thus, according to necropsy, acute intragastric administration of the experimental drug did not cause macroscopic changes in the internal organs, endocrine system and brain of experimental white mice and rats, and was not accompanied by inflammatory changes or irritation of the mucous membranes of the stomach and intestines.

The results of toxicometry, necropsy data made it possible to classify the experimental drug as class V of practically non-toxic drugs (N. Hodge et al. Clinical Toxicology of Commercial Products. Acute Poisoning. Ed. IV, Baltimore, 1975, 427 p). The condition of the animals after acute administration of the drug testified to the good tolerance and safety of the experimental agent in doses exceeding the maximum therapeutic (200 mg / kg, based on 14 g (11.4 ml) per day for a person with an average weight of 70 kg) at times.

In addition, the results of toxicometry made it possible to attribute the experimental preparation to low-hazard substances (IV hazard class according to GOST 12.1.007-76).

Example 42 (Study of acute toxicity of a veterinary drug)

Materials and methods

In the experiment to determine acute toxicity, white mice weighing 18-20 g were used. Each group consisted of 6 animals. The drug obtained in example 29 was injected into the stomach through an atraumatic metal probe in increasing doses: 0.3; 0.4; 0.5; 0.6; 0.7 and 0.8 ml / mouse or 15; twenty; 25; thirty; 35 and 40 ml / kg. The drug was administered to animals on an empty stomach, i.e. at least 4 hours after feeding. The animals were monitored for 14 days, taking into account the clinical picture of intoxication and animal mortality by day.

Research results

The clinical picture of intoxication of animals exposed to the drug is not expressed. Animals of the experimental group were active, mobile and did not differ in behavior from the control. The coat of animals was smooth and neat, no bloody secretions were observed.

During the postmortem examination of these animals, no signs of pathology of the internal organs were noted. The stomach and intestines were of normal filling and consistency of contents. No hemorrhagic inflammation of the gastrointestinal tract was noted. Macroanatomical changes in the liver, kidneys and spleen were not detected.

As a result, the drug was practically non-toxic. It was not possible to determine lethal doses (LD ₁₆ , LD ₅₀ and LD ₈₄ ). The maximum dose administered was 40 ml / kg (41.5 g / kg) of live weight (see table 34).

The data obtained indicate that the experimental preparation under acute experience when introduced into the stomach belongs to the IV class of toxicity (according to GOST 12.1.007-76).

Example 43 (Study of pharmacological activity)

Evaluated the effectiveness of the drugs obtained according to example 28 and example 31 (prototype) in the treatment of influenza infection. The comparison drug was the preparation “Immunal drops for oral administration”, which is an alcoholic solution of the juice of the herb Echinacea purpurea (Echinacea purpurea (L.) Moench).

Materials and methods

Nonlinear white mice weighing 25-30 g were used in the experiment. To reproduce the pathological process, the influenza virus A / PR / 8/34 (HINI) was used, obtained from the Museum of the Research Institute of Influenza of the Russian Academy of Medical Sciences and adapted to white mice.

The therapeutic effect of the drugs was evaluated by the intensity of virus reproduction in the lungs after intranasal infection of experimental and control mice under light ether anesthesia at a dose of 0.01 LD ₅₀ (lethal dose) of influenza virus in a volume of 0.05 ml per mouse. The drugs began to be administered iv with the 3rd day after infection at a dose of 1300 mg / kg 2 times a day for 10 days. A control group of mice (without treatment) received distilled water.

In total, 4 experimental groups were formed (each group included 20 animals):

1. Animals without treatment (control group).

2. Animals infected and treated with Immunal.

3. Animals with infection and treatment with the drug obtained by the prototype (example 31).

4. Animals with infection and treatment with the drug obtained according to the invention (example 28).

The virus content in the lungs was determined 96 hours after infection by titration of a 10% pulmonary suspension in a culture of the chorionallantoid membrane of chicken embryos [Ilyenko V.I. Test methods and evaluations of the antiviral activity of chemical compounds against influenza virus. Methodical instructions. 35 p., St. Petersburg, 1977]. Virus titer was determined by Reed and Mench and expressed in Ig TID ₅₀ (tissue infectious doses).

The degree of lung damage was assessed by the presence of hemorrhagic foci and expressed as a percentage of the affected lobes.

Animals were sacrificed on day 14 after infection, the lungs were removed and used for biochemical and pathomorphological studies. The level of lipid peroxidation was determined, which was estimated by the content of malonic aldehyde in the lung tissue homogenate.

The antioxidant activity of the tissue was evaluated based on the ability of the lung tissue homogenate to inhibit the oxidation of egg yolk phospholipids induced by ferrous iron.

The nature of the pathological process was evaluated by macroscopic and microscopic changes. Macroscopically evaluated the frequency of detection of lesions of the lungs. Microscopically determined the intensity of a specific viral lesion of the respiratory epithelium, the intensity of toxic manifestations of influenza, as well as the activity and nature of inflammatory cellular reactions.

For histological studies, the lungs and adrenal glands of animals were fixed in a 10% solution of neutral formalin, dehydrated in alcohols of increasing concentration and embedded in paraffin. Paraffin sections were stained with hematoxylin-eosin and examined under a light microscope.

The reliability of the results was evaluated using Student's criterion.

Research results

The studied drugs had practically no effect on the intensity of the multiplication of influenza virus in the lungs of mice of the experimental groups compared with the control. The degree of lung damage on the 14th day after infection, which was assessed visually by the presence of foci of pneumonia, ranged from 17% (control group) to 8.2% (experimental group 4). However, there were no significant differences in the infectious titers of the influenza virus of animals of all groups. Although there was a downward trend in titer in group 4, which suggests the direct antiviral effect of the experimental drug (see table 36).

In animals infected intranasally with the influenza virus and not receiving treatment, typical influenza bronchopneumonia developed in the lungs. 3 days after infection, signs of a specific lesion were found both in the trachea and bronchi, and in the alveolar sections of the lungs. They consisted of destructive changes in the respiratory epithelium, accompanied by the formation of numerous basophilic and eosinophilic influenza inclusions in the cytoplasm. In the thickness of the epithelium and the gaps of the airways, polymorphonuclear leukocytes migrated, undergoing massive decay there. Peribronchial connective tissue was edematous and infiltrated by polynuclear cells. The pathological process in the respiratory departments led to interstitial edema and a decrease in the lumen of the alveoli, interalveolar connective tissue in such places was infiltrated by free-cell elements.

The use of those studied for the drug according to the invention was 2 times lower than in the control and significantly lower than in the case of known drugs. It is interesting to note that the level of lipid peroxidation for all experimental groups did not differ much, however, the antioxidant activity of the lung tissue in the case of the proposed drug was 2.5 times higher than in animals of the control group, and significantly higher than in animals treated with drugs comparison (see table 36).

On the 14th day, a part of the surviving animals in the lungs formed extensive foci of chronic lesions in the form of persistent hyperplasia and hypertrophy of the bronchiolar epithelium, leading to clogging of the lumens of the bronchi with hypertrophic cells and dense neutrophilic exudate, infiltration of the lung stroma with lymphoid macrophage epilepsy and neutrophilia, and neutrophilia epiphilia, and overgrowths. These changes are typical of the late stages of influenza pneumonia caused by toxigenic strains of the influenza virus.

The intensity of lung lesions was evaluated using a four-point system: 1 - segmental lesions, 2 - lobar lesions, 3 - total lung lesions, 4 - total lesions of both lungs. Similarly, the prevalence of hyperplasia of the epithelium of the bronchi and alveoli was estimated.

Oral administration of drugs, starting from the acute phase of the development of influenza infection (from 3 days), had a beneficial effect on the dynamics of pathomorphological processes in the bronchopulmonary tissue of infected animals (see table 37).

Histological analysis of lung sections showed that in groups of animals that were injected with drugs, in the foci of chronic lesion, airiness somewhat increased due to the spread of atelectasis, expansion of the lumens of the bronchi and their release from exudate. Previously, the metaplasized sections of the respiratory departments were aerial and lined with squamous epithelium. Inflammatory infiltration of the stroma in such areas was insignificant.

To study the possible mechanism of the therapeutic effect of drugs for histological studies, animal adrenal glands were used.

As a result of the studies, a significant increase in the size and vacuolization of the adrenal cortex cells of experimental animals was found, three zones were clearly distinguished: the subcapsular glomerular zone, represented by 3-4 rows of small cells with a light cytoplasm and a centrally located nucleus; a beam zone, the cells of which were much larger and polymorphic and contained, along with granular material, small vacuoles; the mesh zone located on the border with the adrenal medulla, represented by smaller cells with granular cytoplasm (see table 38).

findings

The effect of drugs on the development of experimental influenza infection in the treatment regimen had a protective effect, which manifested itself in a significant decrease in the intensity of the manifestation of pathomorphological characteristics of chronic influenza pneumonia (the intensity of lung damage, hyperplasia of the bronchial and alveolar epithelium, inflammatory infiltration). The therapeutic effect is probably non-specific and is partly mediated by the hormonal effect of glucocorticosteroids, since during the experiments a stimulating effect of the drugs on the cells of the adrenal cortex was established.

Studies have shown the effectiveness of the drug obtained in example 28, in comparison with the prototype (the drug obtained in example 31) and the comparison product "Immunal" as an immunomodulator in the treatment of viral infection, which allows us to recommend it for the prevention of possible pulmonary complications with influenza.

Example 44 (Study of prophylactic activity)

Evaluated the effectiveness of the drugs obtained according to example 28 and example 31 (prototype), for the prevention of influenza infection. The comparison drug was Immunal Drops for oral administration.

Materials and methods

White outbred mice weighing 12-15 g were used in the experiment. A total of 4 experimental groups were formed (each group included 20 animals):

1. Control group (animals without prophylaxis).

2. Experienced (animals that received the drug "Immunal").

3. Experienced (animals that received the drug prepared according to the prototype - example 31).

4. Experienced (animals that received the drug prepared according to the invention - example 28).

The drugs were administered to mice intragastrically at a dose of 1300 mg / kg 2 times a day for 10 days. The comparison drug was administered similarly at the same dose. A control group of mice received distilled water. On the 11th day after the start of drug administration, the mice were infected intranasally under mild ether anesthesia with the influenza strain A / Pr / 8/34 (H0N1) pathogenic for them in a volume of 0.05 ml per mouse. The lethal dose of the virus was determined in a preliminary experiment by titration of the virus in mice, followed by the calculation of LD ₅₀ .

The results of the experiment were evaluated on day 5, recording the mortality of mice in the control and experimental groups.

The evaluation of the test results was carried out on the basis of the following indicators:

- Comparison of the percentage of dead mice in the control and experimental groups;

- determining the protection index of the test drug.

To determine the protection index (IZ), the multiplicity or coefficient of protection (CI) of mice was calculated by the preparation.

The calculation of the protection index (IZ) was carried out as follows:

Research results

In the control group, the virus caused 80% death of mice. Administration to animals reduced the mortality rate of mice and increased the protection index.

The experimental drug obtained in example 28 was significantly more effective than the prototype (the drug obtained in example 31) and comparable in activity with the comparison drug “Immunal” in the prevention of viral infection (see table 39).

Example 45 (Study on the accumulation of antibody-forming cells in mice - B-cell unit of the immune system)

Materials and methods

The effect of the experimental preparation obtained in Example 29 on the integral responses of the humoral immunity of mice — the ability to produce antibody-forming cells (AOKs) in response to the administration of a thymus-dependent antigen — ram erythrocytes (EB) — was evaluated. The study was performed on hybrid mice (CBAxC57B1 / 6) F1 weighing 19 ± 0.4 grams. In each group, there were 10 mice. In total, 2 groups were formed:

1. The control group (animals immunized with EB receiving saline).

2. Experienced (animals immunized with EB treated with the experimental drug).

To determine the amount of AOK in the spleen, the mice were orally administered at a therapeutic dose (0.2 ml / kg) simultaneously with the intraperitoneal immunization of their EBs at a dose of 5 × 10 ⁶ in a volume of 0.5 ml / mouse, physiological saline was administered to control animals and also immunized their eB. Immunization was carried out once, the experimental drug was given daily in a therapeutic dose. On the 4th day, the spleens were removed, lymphocytes were obtained, and the study was performed by the method of local hemolysis in agarose gel, the amount of AOK in the spleens was determined. The number of AOKs was counted in each sample among 200 lymphocytes. The stimulation index (IS) was determined as the ratio of the number of AOK in the experiment to the number of AOK in the control. IP 1.5 and above - an indicator of the stimulation of immunity, below 0.7 - immunosuppression.

results

An increase in the number of antibody producers in the spleen of mice of the experimental group indicated the stimulating effect of the experimental drug on antibody genesis. The introduction of the drug led to activation of the transformation of B-lymphocytes into AOK as compared with the control, which was expressed in a statistically significant increase in the number of AOK in the spleen. IP increased 2.5 times, i.e. the drug enhanced the humoral immune response in animals (see table 40). The increase in the content of AOK is mediated by the activation of the functional activity of T-lymphocytes.

Example 46 (Study of antibody titers in mouse serum - B-cell unit of the immune system)

The change in antibody titers in the blood serum of mice was determined with the introduction of the experimental preparation obtained according to example 29. The reaction is based on the ability of agglutinins contained in the blood serum of immunized animals to “glue” EB. It is known that substances with a stimulating effect cause an increase in the concentration of antibodies in the blood serum compared to the control.

Materials and methods

Agglutinin titers were determined in the microvariant of a direct hemagglutination reaction. The study was performed on hybrid mice (CBAxC57B1 / 6) F1 weighing 20 ± 0.7 grams. In each group, there were 10 mice. In total, 2 groups were formed:

1. The control group (animals immunized with EB receiving saline).

2. Experienced (animals immunized with EB receiving an experimental drug).

To determine the change in agglutinin titers, the test drug was administered orally to the mice in a therapeutic dose (0.2 ml / kg) simultaneously with the intraperitoneal immunization of their EBs at a dose of 5-108 in a volume of 0.5 ml / mouse, physiological saline was administered to control animals and they were also immunized EB. With a single immunization, the test drug was administered daily for the entire observation period (7 days). Daily, starting from the 3rd day after immunization, blood was taken from the subclavian vein in mice of both groups to obtain serum and determine the titer of anti-erythrocyte antibodies.

results

On the 8th day, the titers of agglutinins in the blood serum of mice of the experimental group increased 3.8 times. Thus, the experimental drug had a stimulating effect on the humoral immune response (see table 41).

The use of the test drug was accompanied by an increase in the immune response to the thymus-dependent antigen (EB). Apparently, in this case there was no suppression of T-soup by springs of B-lymphocyte activity. Humoral antibody production to a thymus-dependent antigen (EB) is due to the cooperative interaction of B-lymphocytes and the T-helper population of immunocompetent cells (Petrov R.V., Cheredeyev A.I., 1974).

Example 47 (Study of the number of autorezeta cells in mice - T-cell immunity)

Tests to study the effect of immunomodulators on the T-cell link of the immune response include determining the change in the number of autologous rosette-forming cells (auto-ROCK).

According to Marian E. et al. (1984) and Leskova V.P. (1984) auto-ROCKs are post-thymic cells - precursors of T-lymphocytes, which, under the influence of serum thymic factor and interleukin-2, turn into mature helper cells and are a kind of reserve of the immune system.

Materials and methods

The study used 50 CBA mice weighing 25-28 grams. 2 groups were formed (10 animals in the control group, 40 in the experimental group):

1. The control group (immunized animals that did not receive the experimental drug).

2. Experienced (immunized animals treated with the experimental drug).

All mice were immunized once with EB at a dose of 5-108 in a volume of 0.3 ml / mouse. The animals of the experimental group were orally administered the test drug obtained in example 29 daily at a therapeutic dose (0.2 ml / kg), control animals received saline. Studies were carried out on 5, 10, 15 and 20 days after giving the drug. The spleen was removed from the euthanized animals, homogenized, a cell suspension was prepared, it was treated with a 5% EB suspension, and the number of rosetting cells was determined by standard methods.

results

Throughout the experiment, there was a statistically significant increase in both the absolute and the relative number of rosette-forming cells in the spleens of mice of the experimental group. The maximum values were reached on the 10-15th day of drug administration - 18.3 and 17.4% (80.8 and 71.8 cells / μl), respectively, while the relative and absolute number of auto-ROCK recorded in animals in the control, did not differ significantly during the whole experiment and amounted to 1.0-1.9% (30.5 and 36.3 cells / μl). The number of auto-ROCKs on the 10th day of the experiment in absolute terms increased by more than 2 times, and in relative terms by almost 10 times (see table 42).

Example 48 (Prevention and treatment of dog respiratory diseases)

On the basis of Balashikhaisky SBBZH in the period from March 10 to July 30, 2010, the drug was tested, obtained according to example 29, in dogs.

In the spring, respiratory diseases of newborn puppies are observed. Fatal outcome is usually 15%.

The drug was used for the prevention and treatment of respiratory diseases in puppies. Analyzes revealed the presence of colibacilli, salmonella, protea.

As a preventive measure, the experimental preparation was given to 18 dogs twice a day for 15-20 days. The drug was administered orally at 1 ml per 5 kg of animal weight. In case of illness, dogs were given antibiotics and vitamins. From 4-5 days from the start of drug therapy, the clinical signs of the disease disappeared, motor activity and appetite appeared. All animals recovered. Lethal outcomes are not fixed.

For therapeutic purposes, the experimental drug was given to 19 puppies who became ill with dyspepsia and bronchopneumonia on 3-5 days of life. Clinical signs of the disease were expressed as follows: sick puppies were lying, the condition was depressed, difficulty breathing, fever, refusal to feed. For treatment, tetracycline antibiotics were administered to animals. At the same time, an experimental preparation was given twice a day for 25 days. Signs of the disease disappeared by 4-5 days from the start of treatment. The use of the drug helped to reduce the course of treatment by 2 times. But in this group there were 2 fallen puppies.

The use of the drug for prophylactic and therapeutic purposes has improved the safety of puppies.

Example 49 (Treatment of feline infectious diseases)

From June 20 to August 30, 2010, on the basis of the Federal State Institution of the Stupino District SBBJ, the therapeutic efficacy of the drug obtained in Example 29 was tested on cats in the treatment of infectious diseases.

The experiment was carried out on an experimental group of 13 cats of various breeds and ages with different periods of herpesvirus infection. The diagnosis was made on the basis of epizootic, laboratory and clinical data. The experimental drug was given at a dose of 1 ml per 5 kg of animal weight twice a day for 21 days in combination with other drugs traditionally used to treat herpes virus infection: antibiotics (ampicillin), sulfanilamides (biseptol), antihistamines (diphenhydramine).

A control group of 9 cats was treated with antibiotics, vitamins, and other symptomatic treatments and rehabilitation.

The timing of the onset of clinical recovery in the experimental group depended on the stage of the disease. In the initial stage (1-3 days), the drug had a persistent positive effect and the disease clinically ended after 7-9 days with a subsequent rehabilitation period. In the later stages of the disease, the disease lasted up to 3 weeks. All animals recovered. Allergic reactions and side effects on the introduction of an experimental drug were not observed. In the control group, the disease lasted from 2-6 weeks. Recovery was observed in no more than 30% of cats.

Thus, in combination with specific drugs, the experimental drug proved to be a good treatment for herpesvirus infection in cats, which made it possible to increase the effectiveness of the therapy and significantly reduce the recovery time.

Example 50 (Use after surgery)

On the basis of the Babushkinskoy district veterinary clinic, from January to April 2010, clinical trials of the drug obtained according to example 29 were carried out.

The drug was used after surgery on dogs and cats. Under the experiment were 12 dogs and 17 cats. Dogs after castration and cats after ovariogisterectomy were given an experimental preparation for 25-27 days twice a day at a dose of 1 ml per 5 kg of animal weight. Additionally, no medication was prescribed. In experimental animals no further complications were observed.

An experimental drug was recommended as a prophylactic that enhances the immune system of domestic animals.

Example 51 (Application after surgery in cats)

We conducted an experiment on the basis of the Voskresenskaya district SBJ from June 5 to August 25, 2010 to test the therapeutic efficacy of the drug obtained in Example 29 on cats after undergoing surgical operations.

In total, 11 cats of different ages and breeds were under the experience. The experimental preparation was given with food at a dose of 1 ml per 5 kg of animal weight twice a day for 27 days according to indications. Experimental animals more easily tolerated the postoperative period, were more active compared to control animals that did not receive the drug. The drug was well tolerated, side effects were not manifested.

Thus, the experimental drug was recommended in practice in the postoperative period in cats.

Example 52 (Use in immunodeficiency cats)

On the basis of the shelter for homeless animals SGOF “Zoozashchita plus” Serpukhov district of the Moscow region, a test of the drug obtained in example 29 was performed on 13 cats of different sex, age and breed with signs of immunodeficiency after chemotherapy treatment and antibiotic therapy.

Animals with similar clinical signs were divided into 2 groups. The first group (10 animals) was administered an experimental preparation at a dose of 1 ml per 5 kg of animal weight orally twice a day for 21-27 days; the second group (3 animals) served as a control and did not receive the drug. The effectiveness of the drug was evaluated on the basis of hematological and biochemical blood parameters.

The use of an experimental drug contributed to the effective treatment and normalization of hematological and biochemical blood parameters.

The developed drug was a good tool for treating the immunodeficiency state after chemotherapeutic treatment and antibiotic therapy in cats, which made it possible to increase the effectiveness of the therapy and significantly reduce the recovery time.

Example 53 (Prevention and treatment of gastrointestinal diseases of cats)

Clinical trials of the preparation obtained in Example 29 were carried out on the basis of the Shelter Zoozashchita Plus Shelter in the City of Serpukhov, Moscow Region, from April to October 2010. The tests were performed to prevent and treat gastrointestinal diseases of cats (enteritis).

For prophylactic purposes, the drug was administered orally to 12 cats twice a day, 1 ml per 5 kg of animal weight for 21 days. Clinical signs after the transmission of viral infections ended in 15-19 days with a full recovery.

For therapeutic purposes, the drug was used on 10 cats after the onset of diarrhea at 20 days of age. Anamnesis of the disease: animals had symptoms of diarrhea, they refused to feed, were depressed. The experimental drug was administered orally from the first day of the onset of signs of the disease at a dose of 1 ml per 5 kg of animal body weight for 25 days. At the same time, sick cats received antibiotics according to the instructions. On the 3rd day from the start of the drug, the cats showed motor activity, appetite, signs of diarrhea remained insignificant.

The use of the drug for prophylactic and therapeutic purposes contributed to the absence of mortality and a more rapid restoration of animals to physiological norms.

Example 54 (Treatment of gastrointestinal diseases of dogs)

Materials and methods

The study was carried out on dogs admitted to the clinic with clinical signs of pathology of the gastrointestinal tract (GIT) (diarrhea, unformed stools, flatulence - i.e. with manifestations of clinical and laboratory dysbacteriosis syndrome). When assigning studies to animals, a complete diagnostic examination was carried out, including laboratory tests: hematological, coprological, biochemical and instrumental (X-ray and ultrasound studies of the gastrointestinal tract). In parallel, immunological studies and bacteriological analysis were carried out, which included the rubbing of the selected microflora to antibiotics. Infectious diseases were excluded with the help of ELISA tests of LLC Hema and VetExpert.

Biochemical studies were performed on a Mindray VA-88A biochemistry analyzer using diagnostic systems from Olveks Diagnostic. Ultrasound diagnostics was performed on a portable ultrasound scanner of the NT1 PU-2200V and Mindray DP-6900 brands using microconvex sensors with a frequency of 3.5-8 MG. X-ray studies were carried out on an X-ray apparatus RUM-20 and 12P6 (Russia) by methods generally accepted in radiology.

A coprogram and a study for the presence of helminth eggs and protozoa oocysts were carried out according to standard methods.

The MTT test was carried out according to the following procedure: isolated lymphoid pool (from patients) from whole blood was incubated in 500 μl (2 * 10 ⁸ ) in eppendorf tubes at 37 ° C for 48 hours. Each tube with cell suspensions at the end of the incubation was centrifuged for 10 min at 1000 d.

Re-dissolve the resulting pellet in 500 μl of MTT solution and incubate for one hour. After incubation, the cells were redissolved in 500 μl of DMSO, 200 μl of suspension was taken from each tube and placed in the wells of a 96-well flat-bottom plate. The optical density readings were read on a Multiscan Ascent Thermo (Scientific) plate reader (Bernas T., Dobrucki J.W. 2000).

The lysosomal cationic test was carried out according to the generally accepted method (Menshikov V.V. 1999).

Phagocytic activity and figocytic number were determined by the method (Pasteur E.U., Ovod V.V., Pozur V.K. 1989).

The determination of lymphocyte subpopulations was carried out by the method of rosette formation according to the generally accepted technique (Petrov I.V. et al. 1984).

Microbiological studies were carried out according to standard protocols (Labinskaya A.S. 1978).

The formation of groups was carried out according to similar clinical signs and physiological parameters (see table 13). 2 groups were formed: experimental and control, 5 individuals of both sexes in each. The treatment regimen for the control group included the following:

1. Dietary fractional nutrition

2. Enterodesum or enterosgel - inside

3. Cleansing enema with enterodesis

4. Phys. solution or glucose 5% iv

5. Gammavit 0.5 ml per kg s / c

6. Lactobifadol, Vetom, Sainex, etc. (what is in the pharmacy) - inside

7. Antispasmodics according to indications

8. Hemostatic drugs according to indications

9. Antibiotics for enterocolitis.

The experimental group in the treatment regimen additionally included the drug obtained in example 30 (twice a day during feeding at a dose of 1 ml per 5 kg of weight for 27 days).

All animals showed leukocytosis, red blood counts (erythrocytes, hemoglobin and hematocrit) were within normal limits (see table 44).

In animals, hepatopathy and, apparently, inflammatory processes of the small intestine were clearly expressed. This was indicated by an increase in the activity of enzymes such as ALT and AST. Activation of amylase and lipase enzymes could indicate involvement in the pancreas. And an increase in alkaline phosphatase activity indicated that intestinal mucosa was also involved in the inflammatory process. In addition, an increase in the concentration of total protein due to the globulin fraction could indicate that the inflammatory process in the body occurred with the activation of conditionally pathogenic microflora (see table 45).

Coprological studies also showed impaired pancreatic function and the presence of inflammatory processes in the intestine (see table 46).

In diseased animals, an increase in the pool of B-lymphocytes was detected. In addition, there was a shift in the immunoregulatory index towards immunosuppression (see table 47)

During the recovery period, all animals in the control group showed a gradual restoration of blood counts after the therapeutic effect (see table 48, 49).

Positive dynamics was also observed during coprological studies (see table 50).

Immunological studies showed that after treatment, the subpopulations of lymphocytes as a percentage were within normal limits, but the immunoregulatory index was below normal, on average, indicating the prevalence of immunosuppression mechanisms in the body (see table 51).

As further rehabilitation procedures were carried out, a gradual increase in nonspecific immunity factors was noted, such as phagocytic activity, respiratory activity, cationic protein accumulation, which indicated favorable dynamics of the development of the healing process (see table 52).

Similarly to animals of the control group, all animals of the experimental group showed leukocytosis, indicators of red blood (erythrocytes, hemoglobin and hematocrit) were within normal limits (see table 54).

In the animals of the experimental group, the biochemical blood parameters were similar to those of the animals of the control group, which indicated hepatopathy and inflammatory processes in the small intestine (see table 55).

Coprological studies also showed impaired pancreatic function and the presence of inflammatory processes in the intestine (see table 56).

In diseased animals, an increase in the pool of B-lymphocytes was detected, as well as activation of the T-helper and suppressor immunity. In addition, there was a shift in the immunoregulatory index towards immunosuppression (see table 57).

During the recovery period, all animals of the experimental group showed a gradual restoration of blood counts after the therapeutic effect (see table 58, 59).

Positive dynamics was also observed during coprological studies (see table 60).

Immunological studies showed that after treatment, the immunoregulatory index and subpopulations of lymphocytes as a percentage were within normal limits (see table 61). As the control method of treatment was carried out, a gradual increase in nonspecific immunity factors was noted, such as phagocytic activity, respiratory activity, and cationic protein accumulation, which indicated favorable dynamics in the development of the healing process (see table 62).

The picture of the biocenosis of the microflora of the large intestine of sick animals is presented in table 63.

To correct the prescribed drug treatment, the isolated strains of E. coli and staphylococcus were tested for sensitivity to antibiotics (hemolytic and plasma coagulating activity) (see table 64).

findings

There was a reduction in the treatment time of animals from the experimental group. Animals of the control group were treated on average 10 days, and experimental - 5-6, which is associated with activation of the immune system. Normalization of blood immunological parameters during the recovery period occurred faster in the experimental group, because immunosuppression was observed in the control, which was expressed in a relatively low content of T-helper lymphocytes and, accordingly, a lower immunoregulatory index (see table 66).

When comparing nonspecific protection factors in animals of the experimental group, there was a more intense activation of the phagocytic immunity link as compared to the control, which was expressed in an increase in phagocytic activity by 10% and phagocytic number of neutrophils by 1.4 times on 10 days. In addition, the phagocytic cells of the experimental group showed an increase in respiratory activity by 1.14 times (see table 52, 62).

Thus, an experimental drug during clinical trials has shown itself to be an effective and safe immunomodulating drug.

Example 55 (study of allergenic properties and local irritating effects on mucous membranes)

Materials and methods

The effect of the experimental preparation obtained in Example 29 on the integrated models of the allergic response (conjunctival and intradermal test, mast cell degranulation reaction) in response to the use of the drug was evaluated. The study was conducted on guinea pigs.

Conjunctival tests on day 12 after intradermal sensitization at a dose of 0.2 ml / kg did not differ from the corresponding indices in the control individuals. Changes in the vascular pattern of the conjunctiva of the eyes after 15 minutes and 24 hours were weakly expressed.

Similar results were obtained in animals after 10 and 20 cutaneous applications of the drug at a dose of 0.2 ml / kg. The skin after 10 and 20 times the application of the drug remained without visible changes during the entire observation period. Skin reactions — skin color (absence of hyperemia) and skinfold thickness (absence of infiltration) after 10 and 20 multiple applications did not differ from those in control animals.

Application of the drug into the ear of pre-intradermally sensitized guinea pigs did not cause skin reactions of immediate and delayed hypersensitivity after 15 minutes, 24, 48 and 72 hours.

Mast cells obtained from the peritoneal fluid of intact white rats were used to test the blood serum of sensitized and control guinea pigs in an indirect degranulation reaction.

The percentage of degranulated mast cells in experimental animals did not differ significantly from that of control individuals and was below 10%, which corresponds to a negative assessment of the test.

To determine allergic reactions in the blood, the preparation of Example 29 was administered to animals intramuscularly at a dose of 0.2 ml / kg. Calculation of the absolute number of eosinophils in the blood showed that their number in individuals of the control and experimental groups did not statistically significantly differ (table 67).

Thus, based on the studies, it was shown that the intramuscular injection of the drug according to example 29 at a dose of 0.2 ml / kg does not lead to allergization of the animal.

Claims (10)

1. A drug with immunomodulating and antiviral action that increases the nonspecific resistance of the organism of warm-blooded animals to bacterial and viral infections, which is an extract of the herb of the larch leaf [Salsola laricifolia] obtained by the extraction of the dry grass of the larch leaf [Salsola laricifolia] multi-hydrated solution of 30-80% alcohol at 60-90 ° C, using the ratio of extractant: dry grass equal to 1: (0.003-0.6), respectively. 2. The drug according to claim 1, characterized in that 1,2-propylene glycol and / or glycerol and / or erythritol and / or xylitol and / or sorbitol and / or mannitol and / or mannitol are used as the polyhydric alcohol or dulcite and / or inositol. 3. The drug according to claim 1, characterized in that stabilizers and preservatives are added during extraction, while stabilizers are added until a pH of from 3.5 to 5.0 is reached, and preservatives in an amount of 0.01-1.70 wt.% . 4. The drug according to claim 3, characterized in that a pharmaceutically acceptable carboxylic acid is used as stabilizer. 5. The medicine according to claim 4, characterized in that ascorbic acid and / or succinic acid and / or lactic acid and / or malic acid and / or acetic acid and / or acetic acid are used as pharmaceutically acceptable carboxylic acids. citric acid and / or tartaric acid and / or grape acid and / or gluconic acid and / or fumaric acid. 6. The drug according to p. 3, characterized in that as preservatives use benzoic acid or its pharmaceutically acceptable salts at a concentration of 0.01-0.5 wt.% And / or sorbic acid or its pharmaceutically acceptable salts at a concentration of 0, 01-0.6 wt.% And / or benzyl alcohol in a concentration of 0.014-0.6 wt.%. 7. The drug according to claim 1, characterized in that stabilizers and preservatives are added after extraction, while stabilizers are added until a pH of from 3.5 to 5.0 is reached, and preservatives in an amount of 0.01-1.70 wt.% . 8. The drug according to claim 7, characterized in that the pharmaceutically acceptable carboxylic acid is used as stabilizers. 9. The medicine according to claim 8, characterized in that ascorbic acid and / or succinic acid and / or lactic acid and / or malic acid and / or acetic acid and / or acetic acid are used as pharmaceutically acceptable carboxylic acids. citric acid and / or tartaric acid and / or grape acid and / or gluconic acid and / or fumaric acid. 10. The drug according to p. 7, characterized in that as preservatives use benzoic acid or its pharmaceutically acceptable salts at a concentration of 0.01-0.5 wt.% And / or sorbic acid or its pharmaceutically acceptable salts at a concentration of 0, 01-0.6 wt.% And / or benzyl alcohol in a concentration of 0.014-0.6 wt.%.

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