RU2550954C2 - Method of human immunomodulation - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmaceutical industry, namely to means, possessive immunomodulating action. Method of human immunomodulation by application of live suspension of chlorella of strain Chlorella vulgaris IGF No C-111 with concentration 8-10 millions cell/ml in quantity 200 ml per day for adult human.

EFFECT: method makes it possible to increase human immunity effectively.

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Description

The invention relates to the pharmaceutical industry, and more specifically to a tool with immunomodulating effect.

At present, a large number of products have been developed and found wide practical application, including biologically active food additives (hereinafter BAA) containing various natural bioactive compounds.

Of particular importance in this spectrum are substances based on microalgae and, in particular, chlorella. Due to its unique natural composition and high concentration of biologically active substances, it compares favorably with higher plants (1).

Chlorella is an active producer of biomass with a large percentage of high-grade protein, fats, carbohydrates, vitamins ... in total more than 650 substances in concentrations far exceeding their content in traditional foods: meat, milk, vegetables and fruits.

Chlorella belongs to the category of “superfoods”, such as spirulina, barley and wheat greens, bee bread (bee products), propolis and royal jelly, which have gathered all the nutrients necessary for the embryos of the future life and ensuring the safe existence of all biological species (2 )

Today, there is a wide range of substances based on, as soon as chlorella, and in combination with other microalgae and components.

However, in all known substances and dietary supplements based on chlorella, only dry microalgae biomass or extracts and extracts from dry chlorella are used. This is primarily due to the properties of the chlorella strains themselves, used to obtain such substances. It is known that in the world for the production of dietary supplements the so-called soil strains of chlorella are used, which have low bactericidal properties and are not able to be stored in suspension. After culturing a suspension of chlorella soil strains, chlorella cells are separated from the culture medium, then the resulting paste is dried and dry microalgae biomass is obtained, which is used for the production of dietary supplements. In such cases, Chlorella culture medium is not used.

It is scientifically proven that a number of important and especially valuable biologically active substances formed in plants are able to maintain their properties only in a living cell. And vice versa, after heat treatment of plants, such substances undergo decomposition and lose their active properties. In addition, after drying the plant cell, many biologically active substances are not able to be stored for any long time, they undergo oxidation, decomposition and gradually lose their properties during their storage.

Another method of preparing medicines from medicinal plants and chlorella is based on the preparation of alcohol tinctures or extracts. At the same time, a sufficiently good sterility of the preparations is ensured without any heating, however, at the same time, some healing active ingredients may not be extracted from plants (3).

The best way out of this situation is to eat natural foods such as herbs, vegetables, fruits, etc., which have healing properties. However, such an opportunity, as a rule, is absent in most of the year, which requires processing and harvesting plants for future use. Plants can be dried, crushed, and consumer forms prepared in the form of finely ground powders, tablets and capsules (with the addition of minerals if necessary). However, in these cases, without additional measures to sterilize such dosage forms from natural raw materials, their ingestion is often unacceptable due to the possible microbial contamination of the feedstock (3).

It is known (4) that during photosynthesis, chlorella accumulates in its cells and releases a wide range of substances with pronounced biological activity into the culture medium, the biochemical composition of the biomass of chlorella cells (355 substances) and the culture medium of chlorella (312 substances) containing approximately the same amount and the composition of biologically active substances.

By changing the conditions for the cultivation of chlorella and the composition of the nutrient medium, one can purposefully change the content of the main components in the biomass of chlorella (4).

Numerous experiments with chlorella on animals in our country and abroad convincingly prove the great advantages and effectiveness of using a chlorella suspension compared to dry chlorella or paste (5, 6).

Obviously, from the point of view of the best conservation of the whole complex of biologically active substances synthesized by chlorella, it is necessary to use both the most living microalgae cell and all exometabolites released by chlorella into the culture medium during its photosynthesis. Only in this case, not only the best preservation of all biologically active substances is possible, but also the maximum effect of its use.

The main reason that the soil strains of chlorella are not able to be used in suspension is their lack of strong bactericidal properties that would be able to inhibit or stop the development of concomitant microflora during storage. For this reason, cells of soil strains of chlorella after cultivation are immediately separated from the culture medium and dried. The resulting dry powder is then typically tabletted and used, for example, as a dietary supplement.

Such powders based on dry chlorella cells have a number of significant disadvantages.

1. During the processing and drying of chlorella cells, a number of important biologically active substances are lost that can only be stored in a living chlorella cell.

2. The digestibility of the remaining biologically active substances in a dry cell of chlorella is much lower than in a living cell.

3. Do not use a wide range of biologically active substances that chlorella releases into the culture medium during its photosynthesis.

4. The long shelf life of dietary supplements based on dry chlorella cells inevitably leads to depletion and deterioration of the composition of biologically active substances due to their gradual oxidation and decomposition.

Thus, ready-made dietary supplements based on dry chlorella potentially in their biological value are an order of magnitude lower than the suspension of chlorella itself from which they are obtained.

An object of the present invention is to provide an immunomodulating agent based on living cells of the chlorella strain Chlorella vulgaris microalgae and exometabolites secreted by it into the culture medium, namely, a living suspension of chlorella.

To solve this problem, it is proposed to use the planktonic strain Chlorella vulgaris IGF No. C-111 (SU 1751981, C12N 1/12, 02/10/1997) or daughter strains of chlorella with high bactericidal qualities obtained on its basis. The strain Chlorella vulgaris IGF No. C-111 is the parent strain for strain BIN (RU 2192459, C12N 1/12, C02F 3/34 04/18/2001) and strain Parachlorella nurekis 1904 KIEG (301670, C12N 1/12, 05/19/2010). Both daughter strains are planktonic with high bactericidal properties. Chlorella vulgaris BIN, designed for biomass and wastewater treatment, Parachlorella nurekis 1904 KIEG is used to destroy blue-green algae, bacteria and fungi during algolization of water bodies. Both of them can be used as biomass producers, and therefore, as dietary supplements in the inventive suspension form, since they have the same high bactericidal properties as their parent strain IGF No. C-111.

The properties of planktonic strains of chlorella fundamentally differ from all previously known soil strains:

1. The ability to be stored only as a suspension in ordinary, non-sterile conditions due to its high bactericidal qualities.

Soil strains can be stored only on solid nutrient media on agar-agar, under special conditions and in compliance with sterility requirements when replanting them.

2. To be in a planktonic state or a state of hovering in a culture medium, both during cultivation and in a state of rest or storage, due to the presence of a weakly negative charge of microalgae cells.

Cells of soil strains do not have an electric charge, quickly settle to the bottom and require constant mixing during the entire cultivation time.

3. The cultivation of planktonic strains is carried out in a monoculture, while the presence of other types of microalgae and even other strains of chlorella is not allowed.

When cultivating soil strains of chlorella in a culture, other algae species and strains of chlorella are always present. In practice, several strains of chlorella are often cultured simultaneously.

4. Planktonic strains differ by an order of magnitude greater efficiency in their use, for example, in animal husbandry, the norms of feeding the suspension decreased ten times in comparison with soil strains with the same cell density in suspension and the same results.

5. Planktonic strains were much more unpretentious soil, when they are cultivated, both in relation to the conditions and designs of plants, and nutrient media.

With proper cultivation of planktonic strains of chlorella and maintaining a strong culture, due to pronounced bactericidal properties, it is possible to stabilize KMAFAnM indicators at the level of SanPin 2.3.2.1078-01 requirements for dietary supplements based on microalgae, in the complete absence of indicators for BGKP (coliform), pathogenic microflora , including salmonella, yeast and mold.

A method of cultivating planktonic strains of chlorella involves pouring nutrient medium into containers, inoculating the suspension with a strain, illuminating the culture container during microalgae growth, and maintaining the required suspension temperature. Microalgae cultivation is carried out in special plants for chlorella cultivators. They are containers made of transparent material (glass, plexiglass) in a metal frame with external and internal sources of artificial lighting, as well as with devices to automatically maintain a given temperature in suspension.

After cultivation and an increase in a given density of chlorella cells, the bacterial background in suspension remains normal for a long shelf life under normal conditions, and it does not require its preservation or freezing. Such high bactericidal properties are achieved due to the release of antibiotic substances into the culture medium by chlorella cells. Moreover, the antibiotic effects of such substances are pronounced in relation to only a wide range of pathogenic microflora and do not affect the beneficial intestinal microflora.

Example 1. A method of obtaining a dietary supplement

For the cultivation of microalgae based on the strain Chlorella vulgadis IGF N C-111 prepare a nutrient medium:

Ammonium nitrate - 0.2-0.4 g

Superphosphate, 10% solution - 0.3-0.5 ml

Iron chloride, 1% solution - 0.15 ml

Cobalt nitrate, 0.1% solution - 0.1 ml

Copper sulfate, 0.1% solution - 0.1 ml

Carbon dioxide solution (pH 3.0-5.0) - 5-10 ml

Tap water - 1000 ml

Beginning of chlorella cultivation. A nutrient medium with a temperature of about 30 ° C is poured into the chlorella cultivator in an amount of 80% of the volume of the tank and a suspension of the strain Chlorella vulgaris IGF N C-111 is introduced in an amount of 20% of the volume of the tank until the required initial cell density is reached (2 mln / l ) in culture. Then make a solution of carbon dioxide in an amount of 0.5-1.0% of the volume of the suspension of chlorella and include the lighting sources of the installation. In the process of further cultivation of chlorella, a carbon dioxide solution is injected every day in the form of a bacuspension (pH 3.0-5.0) once a day in an amount of 0.5-1.0% at the time of turning on the lamps of the installation. The cultivation of chlorella in the plant lasts four days until the required cell density in the culture is reached in the amount of 8-10 million / ml. The duration of illumination with culture lamps is 12-14 hours a day, the rest of the day, the lamps are turned off. The temperature in suspension during the cultivation process is maintained at 30 ° C. After four days of cultivation, the cell density in suspension reaches a value of 8-10 million / ml. Suspension of chlorella merges with the installation and in this form is ready to use as a dietary supplement.

Also, carbon dioxide can be supplied in the form of a mixture with air in the amount of 1-2% of the volume of air is supplied through the compressor and sprayers located at the bottom of the unit. The feed rate is 10-20 liters per 1 m ³ / min.

The finished suspension of chlorella may also subsequently thicken to obtain concentrated chlorella (KX) with a cell density of 20 to 1000 million / ml.

Example 2. A method of concentrating a suspension of chlorella.

Stage 1

According to the known method (SU No. 1091890), the initial suspension of chlorella is subjected to electroflotation, positively formed oxygen bubbles resulting from electrolysis of water, positively contribute to the ascent and concentration of biomass to the surface, then the biomass is removed into the receiver.

We found that when a culture medium or a suspension of chlorella is added to a highly concentrated biomass of chlorella in a certain proportion, sufficient to ensure cell density from 30 to 1000 million / ml in the resulting concentrate of chlorella biomass, it is possible to obtain a product - concentrated biomass of chlorella cells (CBH), which is already capable of being stored for at least 30 days under normal conditions.

In practice, this is achieved by the fact that the removal of foam from the surface of the suspension during electroflotation occurs simultaneously with a small amount of chlorella suspension, i.e. the foam is not taken in its pure form, but with a suspension of chlorella. Chlorella suspension has high bactericidal properties and acts as a preservative even with a 100-fold increase in cell density than in the original chlorella suspension.

The resulting purified Chlorella culture medium (CSC) is also a valuable biological product in which a wide complex of biologically active substances, exometabolites of chlorella cells (Chlorella, physiologically active substances and their use, is found dissolved. SS Melnikova, E.E. Manankina Institute of Photobiology, Academy of Sciences of the BSSR, Minsk, 1991).

Stage 2

To thicken the purified culture medium of chlorella (CSC) to values from 5 to 100 times, use the known reverse osmosis method or ultrafiltration.

The reverse osmosis method concentrates all substances in a purified culture medium (exometabolites of chlorella cells) and releases pure solvent (water) from the solution, with the exception of a certain amount of monovalent ions Na, K, Cl. Almost reverse osmosis reduces to thickening the purified culture medium of chlorella and obtaining a product of a concentrated culture medium of chlorella. No one has yet used this thickening method for the Chlorella culture medium. There is a wide variety of reverse osmosis plants today. Concentration is carried out at least an order of magnitude, and if necessary, 30-50 times. The storage period of KKSX is at least 3 months.

Concentration can be carried out by ultrafiltration. Using a pump, the culture medium under pressure is filtered from inside to outside of the hollow fibers of the UV module. Purified water passes through the porous walls of the hollow fibers and is then used as ordinary pure water. Particles of the Chlorella culture medium with a size of 0.01-0.1 μm remain inside the hollow fibers of the UV module. As a result of washing the fibers, the concentrated culture medium of chlorella is collected in the drive of the thickened culture medium.

The ultrafiltration method is simpler in its execution, but it allows you to concentrate only a certain range of substances of the Chlorella culture medium, the molecular sizes of which are in the range from 0.01 to 0.1 μm, which is its significant drawback.

Stage 3

To obtain KH mix KKSH and KBH. The number of CBH and CCC depends on the concentration (degree of thickening relative to the initial) of CBC and CCC. Can be mixed in various concentrations. If we keep in mind the maximum range of ratios of CBH and CCX in CC, in our case, CC will be a range of products, on the one hand, with a maximum concentration of chlorella cells and a minimum concentration of CCX (i.e., essentially CCC), on the other the concentration of chlorella cells and the maximum concentration of SCCS (i.e., in fact SCCX). For dietary supplements, the following range is obtained: cell density from 1 million / ml, up to 1000 million / ml (we will leave the minimum), and the liquid medium is KKSX with a thickening factor from 2 to 100 times.

In some cases, obtaining KX is advisable from one volume of the initial suspension, i.e. get and thickened biomass KBH, and thickened KKSX and then mix them. In this case, we can talk about the product, thickened relative to the original commodity suspension several times (10, 15, 20, 25, 30, etc.).

A suspension of chlorella with a cell density of 8-10 million / ml or concentrated chlorella (KX) with a cell density of 20 to 1000 million / ml can be stored under normal conditions at a temperature of 0 to 24 ° C for at least 45 days from the date of its receipt. During storage, chlorella cells gradually begin to settle to the bottom after a few days. Without the conditions for photosynthesis, chlorella cells gradually fall into a state of suspended animation, while the cells completely settle to the bottom within about one month. At the same time, the chlorella cells themselves remain in a living state and are ready at any time for photosynthesis. Therefore, before use, the suspension of chlorella must be shaken.

In order to increase the shelf life of a suspension of chlorella and concentrated chlorella (CH) up to 6 months, it is proposed to use carbon dioxide. The mass fraction of carbon dioxide for a suspension of chlorella is, as for lightly carbonated drinks (in accordance with GOST 28188) 0.20-0.30% inclusive. In this form, chlorella cells receive the required amount of carbon dioxide to breathe during storage in a tightly closed state.

Our studies on volunteer testers showed that the proposed live suspension of chlorella strain Chlorella vulgaris IGF No. C-111 with a concentration of 8-10 million cells / ml in an amount of 200 ml per day was a highly effective drug.

Example 3

A man, 43 years old, took a suspension of chlorella 200 ml per day for one month. Immunogram data are given before and after taking a suspension of chlorella:

Example 4

A man, 44 years old, took a suspension of chlorella 200 ml per day for one month. The immunogram data are shown before and after taking a live suspension of chlorella strain Chlorella vulgaris IGF No. C-111

The tests of examples 3 and 4 were carried out on the basis of the Penza Regional Center for AIDS Prevention, where studies were conducted to study the immunostimulating properties of the microalgae Chlorella vulgaris (strain IGF No. C-111) for immunological, biochemical and clinical parameters of people. As a result of studies, a stimulating effect on the phagocytosis system, the system of humoral immunity and cellular immunity in both examined patients was found. Both volunteers noted a steady improvement in their well-being from the first days of using the drug. The examined volunteers recorded significant improvements in the parameters of the immunogram during the observed month relative to the beginning of the experiment. In biochemical studies in both patients, normalization of most parameters occurred.

Thus, as a result of the studies, the immunostimulating effect of the suspension of microalgae Chlorella vulgaris strain IGF No. C-111 on the immunocompetent cells of both volunteers participating in the experiment was established.

Example 5

A group of eight volunteers: three men and five women. The age of men is from 43 to 45 years, the age of two women is 30 and 40 years old and three women are over 65 years old. One of the women aged 72 suffered two strokes before the experiment. The experiment was conducted on the basis of the Department of Immunology and Allergology of the Central Scientific Research Laboratory of the Penza Institute for Advanced Medical Studies.

Conclusion: On the basis of the Central Scientific Research Laboratory of the Penza Institute for the Advancement of Doctors, from March 21 to May 15, 2006, studies were conducted to study the immunostimulating properties of the microalgae Chlorella vulgaris (IGF strain No. C-111) on immunological, biochemical, and clinical parameters of people. Test volunteers in the amount of 8 people for a month took the studied microalgae at 100 grams 2 times a day. The studied parameters were taken before the experiment and after completion of the intake of this microalgae.

As a result of studies, a stimulating effect on the phagocytosis system in 3 patients, the humoral immunity system in 4 patients, and cellular immunity in 8 examined patients were found. In biochemical studies, normalization of most parameters occurred in all patients. A clinical blood test did not reveal any changes in the blood picture.

Thus, as a result of the studies, the immunostimulating effect of a live suspension of chlorella of the strain Chlorella vulgaris IGF No. C-111 on the immunocompetent cells of all participants in the human experiment was established.

Example 6

In the period from March 24 to May 15, 2008, studies were conducted on the basis of the Department of SFGOURUDN to study the effect on the human body of a food suspension of living microalgae Chlorella vulgaris microalgae (Strain IGF No. C-111). The immunostimulating properties and the effect on the immunological, biochemical and clinical parameters of people were analyzed. Test volunteers in the amount of 20 people (1) and 10 primates (2) for 15 days took the studied food suspension of microalgae 100 grams 2 times a day. The studied parameters were taken before the experiment and after completion of the intake of this microalgae. The list of studies included: immunological blood tests - general immunogram, cellular immunity, total IgE indicator, general diagnostic biochemical analysis. Additionally, group (1) analyzed: bioelectrography (assessment of autonomic functions), endoecological assessment of the degree of contamination of the body based on the specialized iris-prediction study “Iris-sigma”, assessment of visual functions and neurophysiological parameters using color campimetry, electrophysiological studies, subjective sensations and data objective examinations.

The studies were carried out jointly with the City Center for the Fight against AIDS and Infectious Diseases, the Eye Center with endoecological rehabilitation of the Oktyabrsky sanatorium, the Research Institute of Medical Primatology of the Russian Academy of Medical Sciences in Sochi.

As a result of studies, a stimulating effect on the phagocytosis system in 65% of patients, the humoral immunity system in 70% of patients, and cellular immunity in 84% of the examined patients were found. In biochemical studies, 80% of patients had normalization of most indicators. Improvements in vegetative functions and a decrease in the percentage of tissue, lymphatic and subcutaneous fat contamination, an improvement in visual functions, neurophysiological and electrophysiological indicators by an average of 15-25% were noted. A clinical blood test revealed an improvement in the blood picture, according to the following indicators: color indicator, platelets, lymphocytes, segmented, eosinophils, ESR in 68% of cases.

Thus, as a result of the studies, the immunostimulating effect of the living microalgae Chlorella vulgaris on the immunocompetent cells of all people participating in the experiment was established.

Example 7

Man, age 78. Takes a daily suspension of chlorella in an amount of at least 1.5 liters per day for 5 years, completely replacing it with water in its diet. Since the beginning of the use of a suspension of chlorella, he felt a significant improvement in health. Gradually got rid of a number of diseases, including the chronic nature of the digestive system, periodontal disease and colds. During the use of a suspension of chlorella, I have never consulted a doctor. Assesses his condition as a healthy 50 year old man.

According to our observations, the use of a suspension of chlorella in an amount of 200 ml per day in all volunteers already in the first week leads to a steady improvement in overall health. There is an improvement in digestion and normalization of the stool, a surge of strength and efficiency, sleep is shortened and recovery of the body after exercise is accelerated. Chlorella improves cerebral circulation and memory, the head begins to work clearly, blood pressure normalizes, and immunity strengthens. During the intake of chlorella, volunteers experienced a marked weakening of the symptoms of previously existing diseases.

The experience of using substances based on chlorella around the world proves a wide range of effects of this microalgae on the human body. An unsurpassed effect was found when using live chlorella of the strain Chlorella vulgaris IGF No. C-111 to relieve various endogenous and exogenous intoxications, alcohol hangover, in the treatment of constipation, peptic ulcer, allergies, arthritis, atherosclerosis, high cholesterol, hypertension, cardiovascular diseases, as well as an antiviral agent, etc. The general positive effect of chlorella suspension on the human body can be described as a significant and sustainable improvement in the quality of life.

Noticeable and steady improvements in general condition are observed during a certain long-term intake of dry chlorella. Obviously, substances based on dry chlorella cells certainly have a positive effect on the human body, since after isolating cells, drying or extraction, the composition of biologically active substances in them remains still quite high. However, in cases of the use of a live suspension of chlorella of the strain Chlorella vulgaris IGF No. C-111 by a person, such sustained improvements occur almost from the first week of administration. Such a positive result is achieved mainly due to the living form of the substance itself - a suspension of chlorella of the strain Chlorella vulgaris IGF No. С-111, which allows not only the best way to preserve the full spectrum of all biologically active substances in the microalgae cell and in the culture medium, but also contributes to maximum the digestibility of these substances by the human body.

Thus, for the first time, a living plant is proposed for human use in the form of a suspension of microalgae based on the planktonic strain of chlorella of strain Chlorella vulgaris IGF No. C-111, whose unique bactericidal properties allow the cells themselves to remain in suspension in this form for a long time under ordinary conditions without preservatives or freezing.

Chlorella suspension of strain Chlorella vulgaris IGF No. C-111 can be used both as a live suspension with a cell density obtained immediately after cultivation in an amount of 8-10 million / ml, and in a thickened form, with a cell concentration of 20 to 1000 million / ml

Bibliography

1. Mike Adams. Superfoods For Optimum Health: Chlorella and Spirulina.

2. Robert Atkins. Supplements: a natural alternative to drugs.

3. Patent RU (11) 2178660 (13) C2.

4. S.S. Melnikov, E.E. Manankin. Chlorella: physiologically active substances and their use. Minsk. “Science and Technology”, 1991

5. M.Ya. Salnikova, Chlorella - a new type of feed, Moscow, КО KOLOS ″, 1977

6. Muzafarov A.M., Taubaev T.T., Selyametov R.A., Chlorella and its use in animal husbandry, Tash., 1974.

Claims (1)

The method of human immunomodulation by taking a live suspension of chlorella strain Chlorella vulgaris IGF No. C-111 with a concentration of 8-10 million cells / ml in an amount of 200 ml per day.