CN117137925A - A kind of nutritional preparation for preventing respiratory virus infection - Google Patents

Abstract

The invention provides a nutritional preparation for preventing respiratory tract virus infection, which comprises 3 '-sialyllactose (3' -SL) and 2 '-fucosyllactose (2' -FL), wherein the mass ratio of the 3 '-sialyllactose (3' -SL) to the 2 '-fucosyllactose (2' -FL) is 1:3.33-1:25. The invention can effectively inhibit the influenza A H1N1 virus, can be taken as food alone, can be added into common food, infant milk powder and health food, can be prepared into medicines with pharmaceutically acceptable carriers, and has the effect of effectively preventing and treating respiratory tract virus infection.

Description

A nutritional preparation for preventing respiratory viral infection

Technical Field

The present invention relates to the technical field of nutritional food, in particular to a nutritional preparation capable of preventing respiratory virus infection caused by influenza virus H1N1.

Background Art

Influenza (flu for short) is an acute respiratory infectious disease that breaks out with seasonal patterns. It is mainly caused by influenza viruses and is characterized by rapid transmission, strong infectivity, and severe pathogenicity. The pathogen of influenza is an RNA virus of the Orthomyxoviridae family. It is divided into three types: A, B, and C (or A, B, and C) based on the differences in matrix protein antigens and viral nucleoproteins. Hemagglutinin (HA) and neuraminidase (NA) are the main spike proteins on the envelope of influenza A viruses. The mutation of HA does not lead to the emergence of new subtypes, and mostly causes periodic or seasonal small-scale influenza; the mutation of NA will lead to the emergence of new subtypes, which often break out into large-scale influenza that spreads rapidly and affects a wide range when it is difficult to prevent. The Influenza A (H1N1) virus is the most widespread type of virus currently. This virus is a triple-rearrangement Influenza A virus complex, in which the parent gene fragments PB2 and PA are from avian influenza virus, MP, NP, HA, NA and NS are from swine influenza virus, and the PB1 involved in the rearrangement is a human influenza virus gene fragment. Because the Influenza A (H1N1) virus gathers the relevant gene fragments of avian influenza, swine influenza and human influenza viruses, it has undergone a qualitative change in its genetic material, making its virulence and ability to invade host cells significantly higher than that of the swine influenza virus parent, and it is inevitable that it will cause a global pandemic with rapid spread and high mortality rate.

After humans are infected with the H1N1 virus, the virus mainly replicates in the respiratory tract and lungs. The influenza virus can specifically bind to the α-2,6-galactosialic acid receptors (SAα2 and 6Gal) in the human upper respiratory tract and the α-2,3-galactosialic acid receptors (SAα2 and 3Gal) in avian cells, triggering the continuous transcription and replication of the virus, causing the spread of influenza between humans or poultry. Susceptible populations such as infants/children/pregnant women/elderly people may suffer from serious complications and sequelae after virus infection, posing a serious threat to life and health.

Influenza vaccination is currently one of the important means of preventing influenza, but influenza vaccines still have their shortcomings. The first is material limitations. The preparation of influenza vaccines must be able to replicate efficiently in chicken embryos. The process of producing influenza vaccines with chicken embryos is time-consuming and complicated, and cannot meet the needs of influenza outbreaks. The second is that the vaccine is more effective against the same subtype of virus infection, but less effective against different subtypes of virus infection;

Drugs that inhibit viral replication include oseltamivir, zanamivir and amantadine; Chinese herbal compound has a certain effect in improving the body's immunity and enhancing its own immune response to the virus. Drug preparations have shown different degrees of therapeutic effects in clinical practice, but several drugs still have drug safety issues and adverse reactions. In particular, there are few effective antiviral drugs for infants and children, and there are many problems with irregular use. Doctors strictly control the dosage and route of administration when using drugs, and the families of children with the disease are also cautious about the use of drugs. Among them, IFN-a currently has no nebulized inhalation preparation, and only injectable IFN-a is used as a nebulized inhalation preparation. This drug is currently "off-label use"; the adverse reactions of oseltamivir are relatively mild, but it can also cause adverse side effects on the gastrointestinal tract, liver, central nervous system, respiratory system, blood, skin, etc.; the main adverse reactions of arbidol include nausea, diarrhea, dizziness, and increased serum transaminase; ribavirin has serious adverse reactions. Due to its reaction in red blood cells, the subjective serious adverse reaction is hemolytic anemia. Within 1-2 weeks of oral administration, infants/children may experience decreased hemoglobin, decreased red blood cells, and decreased white blood cells, which seriously affect the child's own immunity.

Human milk oligosaccharides (HMOs) are the third largest solid substance in human milk after lactose and fat, with a content between 5 and 15 g/L. Currently, more than 200 types of HMOs have been identified. Although their structures are different, they are all composed of five monosaccharide structural units, namely D-glucose (Glc), D-galactose (Gal), N-acetylglucosamine (GlcNAc), L-fucose (Fuc) and N-acetylneuraminic acid (Neu5Ac). All HMOs molecules have a lactose residue (Galβ1-4Glc) at the reducing end, which can be extended by two different disaccharides, namely galactose β-1,3-N-acetylglucosamine, Galβ1-3GlcNAc or N-acetyllactose samine, Galβ1-4GlcNAc, with β-1,3 or β-1,6 bonds to form HMO backbones with different carbon chains, and further modified by different degrees of fucosylation (through α1-2 or α1-3 bonds) or sialylation (through α2-3 or α2-6 bonds) to form HMOs molecules with different structures. Studies have shown that HMOs have many important biological functions in the human body: regulating intestinal flora, antibacterial, reducing the risk of infection; regulating human immune function, reducing the risk of necrotizing enterocolitis, providing sialic acid to promote brain development and improve cognition, etc. However, at present, there has been no further research on the use of human milk oligosaccharides HMOs to mimic influenza virus H1N1 and prevent respiratory viral infections, nor has there been any similar nutritional food.

Summary of the invention

The object of the present invention is to provide a safe and effective nutritional preparation comprising human milk oligosaccharide components for preventing or treating H1N1 virus infectious respiratory diseases.

To achieve the above-mentioned purpose, the technical solution of the present invention is: a nutritional preparation for preventing respiratory viral infections, comprising 3'-sialyllactose (3'-SL) and 2'-fucosyllactose (2'-FL), wherein the mass ratio of 3'-sialyllactose (3'-SL) to 2'-fucosyllactose (2'-FL) is 1:3.33-1:25.

As an optimized implementation, the mass ratio of 3’-sialyllactose (3’-SL) to 2’-fucosyllactose (2’-FL) is 1:5-1:15.

As a specific implementation, the nutritional preparation for preventing respiratory viral infections can be used as a common food or a nutritional supplement or a combination of nutritional fortifiers in common food, infant formula milk powder, and health food, and the added amount of 3'-sialyllactose (3'-SL) and 2'-fucosyllactose (2'-FL) is ≥0.54%.

As a specific implementation, the nutritional preparation for preventing respiratory viral infections can be made into a medicine for treating respiratory viral infections with pharmaceutically acceptable carriers, excipients, and antioxidants, and the added amount of 3'-sialyllactose (3'-SL) and 2'-fucosyllactose (2'-FL) is ≥0.93%.

As a specific implementation, the product form of the nutritional preparation for preventing respiratory viral infection can be liquid or solid.

The preparation method of the present invention adopts dry mixing, dry-wet mixing or wet mixing process for mixing.

The research of the present invention shows that it has a significant mimicking effect on influenza virus H1N1, and compared with the prior art, it mainly has the following differences and effects.

The nutritional preparation provided by the present invention is derived from the nutritional components of breast milk, and has the same structure as breast milk. Its safety has been widely scientifically confirmed, and it is obviously different from general medicines.

The nutritional preparation combination for H1N1 influenza virus of the present invention can be taken for a long time or after infection to inhibit the damage of inflammation caused by influenza virus invasion to the human body;

The present invention screened out the best human milk oligosaccharide 3'-SL and 2'-FL, when the ratio is 1:10, it has a more effective effect of inhibiting the invasion of influenza A (H1N1) virus on cells;

The nutritional preparation provided by the present invention can be used as medicine, health food or ordinary food;

The nutritional preparation provided by the present invention can be in the form of liquid or solid, and can enter the human body to exert its effects through various forms such as oral administration, spraying, and buccal administration.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a comparison of the inhibitory effects of nine different dose combinations of two substances on three inflammatory factors;

Figure 2 shows the mRNA expression levels and differential scores of inflammatory factors in the 9 nutritional combinations during the anti-H1N1 virus process.

DETAILED DESCRIPTION

Example 1: Human milk oligosaccharides 3'-SL and 2'-FL are mixed in a mass ratio of 1:3.33-1:25 by dry mixing, dry-wet mixing or wet process to prepare a nutritional preparation for preventing respiratory viral infections.

Example 2: Human milk oligosaccharides 3'-SL and 2'-FL were mixed in a mass ratio of 1:10 to prepare a nutritional preparation for preventing respiratory viral infections.

Example 3: Mix human milk oligosaccharides 3'-SL and 2'-FL in a mass ratio of 1:10 and add them to infant formula powder, wherein the proportion of human milk oligosaccharides 3'-SL and 2'-FL in the mixture is ≥0.54%.

Example 4: 3'-sialyllactose (3'-SL) and 2'-fucosyllactose (2'-FL) are used as core materials, and porous starch, cyclodextrin, chitosan, etc. are used as wall materials to prepare microcapsule health products/drugs for preventing respiratory diseases. The added amount of 3'-sialyllactose (3'-SL) and 2'-fucosyllactose (2'-FL) is ≥0.93%, and the mass ratio of 3'-sialyllactose (3'-SL) and 2'-fucosyllactose (2'-FL) is 1:5-1:15.

Example

In order to screen the combined raw materials and proportions of the nutritional preparations and verify the effect of the simulated influenza virus H1N1, the present invention has conducted in-depth research.

Human milk oligosaccharides mainly include:

Fucosylated neutral oligosaccharides (2'-fucoylactose, 3'-fucoylactose, difucoylactose, lactose-N-fucopentose I, lactose-N-fucopentose II, lactose-N-fucopentose III, lactose-N-fucopentose V, lactose-N-fucohexose, lactose-N-difucohexose I, fucoylactose-N-hexose, fucosyllactose-N-neohexose I, fucosyllactose-N-neohexose II, difucosyllactose-N-hexose I, difucosyllactose-N-neohexose, difucosyllactose-N-neohexose I, difucosyllactose-N-neohexose II, fucosyllactose-p-lactose-N-hexose, trifucosyllactose-p-lactose-N-hexose I, etc.); Non-fucose neutral human milk oligosaccharides (lactose-N-tetraose, lactose-N-neotetraose, lactose-N-hexaose, lactose-N-neohexose, lactose-p-N-hexaose, lactose-p-N-neohexose, lactose-N-octaose, lactose-N-neooctaose, lactose-p-N-neooctaose, lactose-N-decaose, lacto-N-neodecaose, etc.);

Acidic oligosaccharides (3'-sialyllactose, 6'-sialyllactose, sialyllactose-N-tetraose a LSTc, sialyllactose-N-tetraose b LSTc, sialyllactose-N-tetraose c LSTc, disialyl-lactose-N-tetraose DSLNT, etc.).

2.1.1 Cells and viruses

The cell line (HEP-2) was purchased from ATCC. The influenza A (H1N1) virus was provided by the Chinese Center for Disease Control and Prevention and stored at -80 °C.

2.1.2 Experimental materials and reagents

Experimental materials: 2FL, 3SL, 6SL, LNT, LNnT. Weigh appropriate amounts of each substance and dissolve it in DMSO to a 20 mM stock solution. The H1N1 influenza virus liquid was passaged twice in chicken embryos, and the virus titer was determined to be ^2-7 .

96-well plates were purchased from Corning Costar (Cambridge, MA, USA); DMEM medium and fetal bovine serum (FBS) were purchased from Gibco, USA; double antibody (10,000 U/mL Penicillin-10,000 U/mL Streptomycin) was purchased from Beijing Solebao Biotechnology Co., Ltd. Phosphate buffered saline (PBS) and dimethyl sulfoxide (DMSO) were purchased from Sigma, USA.

Cell culture medium: DMEM medium containing 10% FBS and 1% double antibody;

Cell maintenance medium: DMEM medium containing 2% FBS and 1% double antibody.

2.1.3 Instruments

Cell culture incubator; optical microscope; clean bench, water bath, RT-PCR detector, Western Blot detection system.

1) Determination of TCID ₅₀ of H1N1 influenza virus

The influenza virus H1N1 cryopreserved solution was diluted 10-fold in DMEM medium without serum, and the diluted virus solution of each concentration was inoculated into a 96-well plate covered with monolayer cells in sequence, 100 μL/well, 6 replicates for each concentration, and a normal cell control group was set up. The 96-well plate with virus solution was adsorbed in a 37 ℃, 5% CO ₂ incubator for 2 h, and then the virus dilution solution was replaced with cell maintenance solution, and then placed in the incubator for further culture for 48 h. After that, the cell morphology was observed, the number of wells and the degree of lesions were recorded. When the cell control group was close to the normal morphology, the wells with the virus-infected cell lesion rate ≥50% were regarded as lesion wells, and the half cell culture infection dose (TCID ₅₀ ) of the virus was calculated according to the Reed-Muench method.

2) Experiment on cytotoxicity of nutrients

HEP-2 cells were revived and passaged 3-4 times. When the cells grew well, they were digested with trypsin and diluted to a concentration of 1.5×10 ⁵ cells/mL with complete culture medium. After mixing by pipetting, they were inoculated into 96-well plates at 100 μL/well and incubated in a 37°C, 5% CO ₂ incubator for 24 h. The test substances were diluted in culture medium in multiples (maximum concentration 200 μM), and each concentration was added to a 96-well plate at 100 μL/well. Each concentration was replicated in 3 wells, and a blank control group was set up. The cells were placed in a 37°C, 5% _CO2 incubator for 48 h, the cell morphology was observed, the supernatant was discarded, 10% CCK-8 solution was prepared with PBS, 100 μL was added to each well, and the cell absorbance was measured at a wavelength of 540 nm using a microplate reader after 1 h. The cell survival rate was calculated according to the formula: cell survival rate % = absorbance value of the drug group (A)/absorbance value of the cell control group (A) × 100%.

3) Detection of the effectiveness of nutritional composition against H1N1 virus

Normal cell control (negative control group), influenza virus control group (positive control group) and drug administration group were set up. Take the culture plate that has grown into a monolayer of cells, remove the culture medium, add 100 TCID ₅₀ virus solution (influenza virus control group and drug administration group) or culture medium (normal group), 100 µL/well, adsorb in a 37ºC, 5% CO ₂ incubator for 4 hours, remove the influenza virus solution, add 100 µL of the appropriate dose of compound, 3 replicates per concentration, repeat 3 times. After 48 hours, observe the cell morphology, discard the supernatant, prepare CCK-8 into a 10% solution with PBS, add 100 μL to each well, and measure the cell absorbance at a wavelength of 540 nm with an enzyme reader after 1 hour. According to the formula: Inhibition rate (%) = (drug average A value-virus control group average A value) / (cell control average A value-virus control group average A value) × 100%, calculate the drug's inhibition rate on virus-induced cell pathology.

4) Study on the mechanism of nutritional composition against H1N1 virus

Normal cell control (negative control group), influenza virus control group (positive control group) and drug administration group were set up (see the table below). Take the culture plate that has grown into a monolayer of cells, remove the culture medium, add 100 TCID ₅₀ of virus solution (influenza virus control group and drug administration group) or culture medium (normal group), 1.0 mL/well, adsorb in a 37ºC, 5% CO ₂ incubator for 4 hours, remove the influenza virus solution, add 1.0 mL of the appropriate dose of compound, 3 replicates per concentration, repeat 3 times. After 48 hours, take the supernatant and use ELISA kit to detect the content of inflammatory factors TNF-α, iNOS and IL6.

Normal cell control (negative control group), influenza virus control group (positive control group) and drug administration group were set up (see the table below). Take the culture plate that has grown into a monolayer of cells, remove the culture medium, add 100 TCID ₅₀ of virus solution (influenza virus control group and drug administration group) or culture medium (normal group), 1.0 mL/well, adsorb in a 37ºC, 5% CO ₂ incubator for 4 hours, remove the influenza virus solution, add 1.0 mL of the appropriate dose of compound, 3 replicates per concentration, and repeat 3 times. After 48 hours, remove the supernatant, wash the cells twice with pre-cooled PBS, extract total RNA with Trizol, and use real-time fluorescence quantitative PCR to detect the mRNA expression of inflammatory factors TNF-α, iNOS and IL6 after reverse transcription.

In this study, five types of human milk oligosaccharides were first selected, including one fucosyl neutral oligosaccharide (2’-FL), two non-fucosyl neutral oligosaccharides (LNT, LNnT) and two acidic oligosaccharides (3’-SL, 6’-SL). The cytotoxicity of nutrients was studied and it was found that the cell survival rate of the five nutrients was close to 100% regardless of whether they were administered at high or low concentrations, indicating that these substances had no significant effect on cell survival and were sufficiently safe.

The effects of these five nutrients in inhibiting viral cytopathic effects were further studied. The results are shown in Table 1. It can be seen from the table that compounds 3'-SL and 2'-FL have a significant inhibitory effect on H1N1 influenza virus-induced HEP-2 cell pathology, with IC50 values of 54.32±2.05 μM and 99.04±5.21 μM, respectively. The inhibition rates at different concentrations are shown in Table 2. The inhibition rates of other oligosaccharides, including fucosyl neutral and non-fucosyl neutral and other acidic oligosaccharides, were less than 50% at each concentration, indicating that the anti-H1N1 influenza virus effect was not obvious. After that, the two nutrients that significantly inhibited the pathogenicity of the virus to the cells were combined and the virus inhibition efficacy was studied again. The results showed that the inhibition rate of 2'-FL was found to be 75% after selecting a high concentration dose and 3'-SL was combined at a low concentration, which was higher than the inhibition rate of the two monomers at high concentrations (as shown in Table 2).

Based on the previous preliminary studies, the mechanism of action of 2’-FL and 3’-SL in inhibiting H1N1 influenza virus was further studied, and the specific implementation plan is shown in Table 3.

5. Test methods and test results

After influenza virus infects body cells, it activates the NF-κB pathway through the Toll-like receptor (TLR) pathway, causing an inflammatory response and leading to cell apoptosis. TLR activation stimulates antiviral responses and may lead to the secretion of a large number of proinflammatory mediators. In the immune response induced by inflammation, proinflammatory cytokines such as TNF-α and IL-6 play a key regulatory role. Tumor necrosis factor-a (TNF-α) is a pleiotropic cytokine produced by a variety of cells during inflammatory responses and immune regulatory stimulation, which can induce cell apoptosis. IL-6 is a strong activator of acute phase reactions and contributes to systemic and local inflammatory responses. Excessive IL-6 can induce a variety of chronic inflammatory diseases. NO free radicals are also crucial in inflammation and immune responses. It is synthesized by enzymes such as NOS (eNOS) and iNOS through the l-arginine pathway. Under normal physiological conditions, iNOS is dormant in dormant cells. However, under pathological conditions, it produces a large amount of NO and plays a dual role in chronic infection and inflammation. Reducing the production of NO may be an effective strategy for treating a variety of inflammatory diseases. This study first used the ELISA method to detect the production of two pro-inflammatory factors, and further used PCR to re-determine the mRNA expression levels of the inflammatory factors at the gene level.

It can be seen from Table 4 that the levels of proinflammatory factors TNF-a, IL-6 and iNOS in the normal control group were 59.67±6.67pg/mL, 246.39±8.24pg/mL and 2.87±0.09pg/mL, respectively, while the levels of the three in the model group after virus infection reached 144.11±1.92pg/mL, 417.62±6.46pg/mL and 5.14±0.25pg/mL, respectively. All three inflammatory factors increased significantly (as shown in Table 5), indicating that the model was successfully established. Through intervention with different doses of nutrients 2’-FL and 3’-SL at low, medium and high levels, it was found that the amount of proinflammatory factors was reduced (Table 4). However, after differential analysis, it was found that the 6 different concentrations of the two nutrients were very sensitive to the proinflammatory factor IL-6, and the amount of IL-6 was significantly lower than that of the model group (p≤0.001). As for the effect on TNF-a, the results showed that only the medium and high concentrations of 2’-FL produced significant differences (p＜0.05), and there was no significant difference between the low concentration of 2’-FL and the three concentrations of 3’-SL and the model group (p＞0.05). As for the production of iNOS, the results showed that there was a significant difference at the medium and high concentrations of 2’-FL and 3’-SL, respectively (p＜0.01), and there was no difference between the low concentrations and the model group (p＞0.05).

The results were better after combining the two nutrients. The difference analysis found that the IL-6 of the 9 groups after high, low and medium dose combinations was significantly different from the model group (p < 0.0001). As for TNF-a factor, except for the 3'-SL+2'-FL (HL) and 3'-SL+2'-FL (HH) combinations, which had no significant difference with the model group, the other 9 combinations all produced significant (p < 0.05) or extremely significant (p < 0.0001) differences; at the same time, except for 3'-SL+2'-FL (ML) among the 9 combinations, the iNOS production in the other 8 combinations was extremely different from the model group (p < 0.001). This shows that the effect of the combination of the two nutrients is stronger than any one of them alone in the infection of H1N1 virus to cells.

Furthermore, this study conducted a comparative analysis of the inhibitory effects of the nine combinations on the cytopathic effects of the H1N1 virus, with the normal cell group without any treatment as the control, and the analysis results are shown in Figure 1. As can be seen from the figure, the combination 3'-SL+2'-FL (MH) (Example 6) has the best inhibitory effect on the pathogenic effects of the H1N1 virus. Under its treatment, even after the initial viral infection, the amount of inflammatory factors generated in the cells was not significantly different from that of the control group cells that had not been treated with the virus (p>0.05).

In order to further confirm the inhibitory effect of nutrients on viruses, this study also used PCR to detect the mRNA expression of inflammatory factors at the gene level. Table 6 shows the mRNA expression of the three inflammatory factors in two nutrient monomers and different dose combinations. It can be seen from the table that the mRNA expression of inflammatory factors in the control group was very low, all about 1, and after the virus H1N1 infection, the mRNA expression of inflammatory factors TNF-a and IL-6 was found to be 100 times and 150 times that of the untreated ones, respectively, and the mRNA expression of the free radical iNOS was increased by about 9 times, indicating that the model was successful. After treatment with two nutrients, it was found that the mRNA expression of the three inflammatory factors also decreased significantly, especially in the 3'-SL+2FL (MH) group, the mRNA expression of the three inflammatory factors was low: TNF-a 48.52±1.53, IL-6 53.27±6.88 and iNOS 3.87±0.28, which is consistent with the results of the production of inflammatory factors. At the same time, one-way ANOVA difference analysis also confirmed that the 3’-SL+2FL (MH) group had the best effect, as shown in Figure 2.

Claims (8)

1. A nutritional formulation for preventing respiratory viral infection, comprising 3 '-sialyllactose and 2' -fucosyllactose, wherein the mass ratio of 3 '-sialyllactose to 2' -fucosyllactose is 1:3.33-1:25.

2. The nutritional formulation for preventing respiratory virus infection according to claim 1, wherein the mass ratio of 3 '-sialyllactose to 2' -fucosyllactose is 1:5-1:15.

3. nutritional formulation for the prevention of respiratory viral infections according to claim 1 or 2, characterized in that it can be applied as a common food or nutritional supplement combination in common food, infant formula, health food.

4. The nutritional preparation for preventing respiratory virus infection according to claim 3, wherein the addition amount of 3 '-sialyllactose and 2' -fucosyllactose in common food, infant formula and health food is not less than 0.54%.

5. A nutritional formulation for use in the prevention of respiratory viral infections according to claim 1 or 2, wherein the formulation is formulated with a pharmaceutically acceptable carrier to provide a medicament for the treatment of respiratory viral infections.

6. The nutritional formulation for preventing respiratory viral infection according to claim 1, wherein the amount of 3 '-sialyllactose and 2' -fucosyllactose added in the pharmaceutical product is not less than 0.93%.

7. A nutritional formulation for use in the prevention of infection by respiratory viruses according to claim 1 or claim 2 wherein 1 is in the form of a liquid or solid.

8. The nutritional formulation for preventing respiratory viral infection according to claim 1 or 2, wherein the formulation is prepared by dry blending, dry-wet blending or wet blending.