Composition for immunomodulation and use thereof

Abstract

The present invention relates to a composition for immunomodulation and its use, comprising Lactobacillus paracasei strain LT12, β-glucan and bovine colostrum powder. Also provided is the use of the composition for preparing an immunomodulatory pharmaceutical composition or dietary supplement, wherein the composition can enhance the expression of IL-6, IL-10, TNF-α and TGF-β1.

Description

Technical Field

[0001] The present invention relates to compositions for immunomodulation and uses thereof. Background Art

[0002] Most influenza viruses or pathogenic bacteria infect and colonize when the immune system is low, and then cause a cytokine storm to eliminate the pathogens, causing the immune system to overreact and cause inflammation. When the immune system is overloaded, it causes damage to organ tissues (such as pneumonia). Therefore, both excessive and insufficient immunity can cause disease. The immune system focuses on balance. When foreign objects such as microorganisms and viruses invade, it can enhance immunity to resist; when allergenic factors induce cytokine storms, it can reduce immunity to alleviate inflammatory symptoms. Only in this way can the two-way immune regulation keep the immune system in a balanced state and maintain health.

[0003] In the early stage, the human immune system mainly uses innate immunity as the first line of defense. The innate immune response can non-specifically identify and act on pathogens, directly fight pathogens to resist foreign infections, and is a rapid anti-infection action. Sentinel innate immune cells are responsible for activating innate and adaptive immune responses to pathogens (adaptive immunity), and macrophages are the key indicators of host defense through pathogen phagocytosis. The activation of different signaling pathways leads to two types of macrophages with different functions: pro-inflammatory (classically activated macrophage, M1) and anti-inflammatory (alternatively activated macrophage, M2). Polarized M1 and M2 macrophages can reversibly functionally redifferentiate into anti-inflammatory or pro-inflammatory macrophages, respectively. The role of M1 macrophages is to secrete pro-inflammatory cytokines (cytokines) and chemokines (chemokines), mainly IL-6, IL-12 and TNF-α, and present antigens to participate in immune responses, which helps drive antigen-specific Th1 cell inflammatory responses. In addition, the polarization of the M1 phenotype is considered to be crucial for effective antiviral immune responses in the lungs. M2 macrophages mainly secrete anti-inflammatory cytokines such as arginase-I, interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), which have the effects of reducing inflammatory responses, promoting tumor growth and immunosuppression. In short, M1 and M2 macrophages can transform into each other in a specific microenvironment.

[0004] In terms of maintaining good health, preventing a low immune system from causing viral infection is the first line of defense. Nowadays, many people in society take health supplements as a way to prevent disease infection or allergies, but many products currently on the market either enhance immunity or fight allergies, etc., so there is still a need to develop products with bidirectional immune regulation effects. Summary of the invention

[0005] The present invention unexpectedly found that the combination of Lactobacillus paracasei strain LT12 (LT12), Beta-Glucan (BG) and Bovine Colostrum powder (BCP) can effectively induce the production of multiple cytokines and show synergistic and multiplying effects. Therefore, the present invention provides a composition with multifunctional multiplying immunomodulatory effects and its use.

[0006] On the one hand, the present invention provides a composition for immunomodulation, comprising Lactobacillus paracasei strain LT12 (LT12), Beta-Glucan (BG) and Bovine Colostrum powder (BCP), wherein the combination of the three provides a multifunctional accelerative immunomodulatory effect, particularly enhancing the production of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10) and / or transforming growth factor Beta 1 (TGF-β1).

[0007] In one embodiment of the present invention, the composition of the present invention, expressed in weight percentage, is mainly composed of 0.5% to 20% of Lactobacillus paracasei strain LT12 (LT12), 0.5% to 25% of β-glucan (Beta-Glucan, BG) and 0.05% to 10% of bovine colostrum powder (Bovine Colostrum powder, BCP) as the main active ingredients.

[0008] In one embodiment of the present invention, the composition of the present invention, expressed in weight percentage, is mainly composed of 1% to 10% of Lactobacillus paracasei strain LT12 (LT12), 1% to 12% of β-glucan (Beta-Glucan, BG) and 0.05% to 5% of bovine colostrum powder (Bovine Colostrum powder, BCP) as the main active ingredients.

[0009] In one embodiment of the present invention, the composition of the present invention, expressed in weight percentage, is mainly composed of 1% to 5% of Lactobacillus paracasei strain LT12 (LT12), 1% to 6% of β-glucan (Beta-Glucan, BG) and 1% to 4% of bovine colostrum powder (Bovine Colostrum powder, BCP) as the main active ingredients.

[0010] In one embodiment of the present invention, the composition of the present invention, expressed in weight percentage, is mainly composed of 2% to 4% of Lactobacillus paracasei strain LT12 (LT12), 4% to 5% of β-glucan (Beta-Glucan, BG) and 1% to 2% of bovine colostrum powder (Bovine Colostrum powder, BCP) as the main active ingredients.

[0011] In one embodiment of the present invention, the β-glucan is yeast β-1,3 / 1,6-glucan.

[0012] On the other hand, the present invention provides a pharmaceutical composition for immunomodulation, comprising a pharmaceutically acceptable carrier, Lactobacillus paracasei strain LT12 (LT12), Beta-Glucan (BG) and Bovine Colostrum powder (BCP), wherein the combination of the three provides a synergistic effect of enhancing the production of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10) and / or transforming growth factor Beta 1 (TGF-β1).

[0013] In another aspect, the present invention provides a dietary supplement for immunomodulation, comprising an edible carrier, Lactobacillus paracasei strain LT12 (LT12), Beta-Glucan (BG) and Bovine Colostrum powder (BCP), wherein the combination of the three provides an enhancement of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10) and transforming growth factor-β1.

[0014] (Transforming Growth Factor Beta 1, TGF-β1) produces a synergistic effect.

[0015] In yet another aspect, the present invention provides use of the composition of the present invention for preparing an immunomodulatory pharmaceutical composition or a dietary supplement.

[0016] According to the present invention, the composition does not affect macrophage survival rate.

[0017] It should be understood that the foregoing invention content and the following embodiments are only exemplary and illustrative, and are not intended to limit the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Shows the growth status of Lactobacillus paracasei strain LT12 in MRS medium.

[0019] Figure 2 Shows the effects of single ingredients and compound compositions on the survival rate of macrophage RAW 264.7.

[0020] Figure 3 The effects of single ingredients and compound compositions on the secretion of various cytokines by macrophage RAW 264.7 are shown. Figure 3 Part A shows the effect on TNF-α secretion; Figure 3 Part B shows the effect on IL-6 secretion; Figure 3 Part C shows the effect on TGF-β1 secretion; Figure 3 Part D shows the effect on IL-10 secretion. DETAILED DESCRIPTION

[0021] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0022] As used herein, the terms "a" or "an" refer to a quantity of one or at least one.

[0023] The invention provides a composition with multifunctional immunomodulatory effect and use thereof. The composition comprises three components: Lactobacillus paracasei strain LT12 (LT12), beta-glucan (BG) and bovine colostrum powder (BCP).

[0024] The Lactobacillus paracasei strain LT12 (abbreviated as LT12) used in the present invention is a novel strain developed by the applicant, and has applied for and obtained patents in many countries, such as U.S. Patent No. 8,372,392 and European Patent No. 2,338,977, which are incorporated into this case as references. As shown in the above patents, LT12 can stimulate human peripheral blood mononuclear cells (PBMC) to secrete a large amount of IFN-γ, and can also effectively inhibit allergic reactions. Analysis using a cytokine antibody array chip shows that LT12 can simultaneously promote the expression of multiple cytokines, including IL-1, IL-6, IL-10, MCP-2 and TNF-α, indicating that LT12 has the function of coordinating the immune system and bidirectional immune regulation. The strain LT12 was deposited at the Agricultural Research Culture Collection (International Depositary Authority, 1815 N. University Street, Peoria, Ill., 61601, USA) on September 21, 2009 in accordance with the Budapest Treaty, with the deposit number NRRL-B50327.

[0025] The term "β-glucan" (β-Glucans, abbreviated as BG) used herein refers to a class of β-D-glucose polysaccharides, which usually form a linear backbone with 1-3 β-glycosidic bonds. β-glucan molecules can also have branched glucose side chains connected to other positions on the D-glucose backbone, such as β-1,3 / 1,6-glucan, which has 1-6 side chains. The β-glucan used in the present invention can be obtained from any source, such as β-D-glucose polysaccharides naturally present in grains, bacteria and fungal cell walls. In one embodiment of the present invention, the β-glucan is β-1,3 / 1,6-glucan, and in one embodiment, it is β-glucan derived from yeast, such as yeast β-glucan or yeast β-1,3 / 1,6-glucan.

[0026] The term "colostrum" as used herein refers to the first milk produced by mammals after giving birth, which is different in composition from mature milk. The "cow colostrum powder" used in the present invention refers to the dry powder of cow colostrum, wherein cow colostrum refers to the first form of milk produced by the mammary gland of a dairy cow just after giving birth to a calf.

[0027] The term "composition" as used herein refers to a product produced by mixing or combining more than one active ingredient. The composition can be prepared by mixing multiple active ingredients with a pharmaceutically acceptable or edible carrier according to the relevant preparation methods commonly used in the field of the present invention.

[0028] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of the administered active ingredient or compound that is sufficient to alleviate the disease or symptoms being treated to a certain extent. The result can be a reduction and / or alleviation of the signs, symptoms or causes of the disease, or any other desired change in the biological system. For example, an "effective amount" for therapeutic use refers to the amount of the composition of the present invention required to provide a clinically significant effect on regulating immunity. In any individual case, an appropriate "effective" amount can be determined using dosing techniques, such as dose escalation.

[0029] According to the present invention, the composition of the present invention includes Lactobacillus paracasei LT12, β-glucan and bovine colostrum powder. According to the examples, the composition has the ability to enhance the expression of IL-6, IL-10, TNF-α and TGF-β1, but does not affect the survival rate of macrophages.

[0030] The immunomodulatory effect of the composition of the present invention is evaluated by the following methods: the cell survival rate is determined by MTT test (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay) to analyze the safety and effective concentration of the composition for cells, and the immunomodulatory effect of the composition of the present invention is analyzed by enzyme-linked immunosorbent assay (ELISA), including TNF-α and IL-6 (pro-inflammatory cytokines) related to inflammation and TGF-β1 and IL-10 (anti-inflammatory cytokines) related to anti-inflammation. The present invention has found that high concentrations of LT12, BCP and BG, either alone or in combination, will not cause damage to macrophage RAW 264.7 when acting on cells for 4 to 24 hours, but can induce a large amount of IL-6 cytokine secretion, and the concentration of cytokine also increases with time, in a proportional relationship, and the composition increases the induction amount by about 25 to 75 times compared to 8 to 24 hours without stimulation; it increases the induction amount by 4 to 40 times compared to other groups alone or in combination of two phases, and has a significant additive effect. In addition, the induction of TNF-α can also increase the secretion amount by about 1 to 10 times compared to other groups, and also has an additive effect. In the determination of anti-inflammatory cytokine TGF-β1, the composition of the present invention can increase the induction amount by about 20 to 30 times in a short period of time (2 to 4 hours) compared to the absence of stimulation; it increases the induction amount by 1 to 20 times compared to BG and BCP or other groups in combination of two phases, showing a significant additive effect.

[0031] In general, the composition designed with LT12, BG and BCP as the main ingredients has the effect of inducing immunity and has a significant effect on inducing IL-6 secretion. It can also induce Th1 and Th2 related immune cell hormones at the same time, achieving a two-way regulation effect. Considering the possible pathogenic mechanism of the virus, the composition of the present invention has enhanced immune efficacy, and the resistance of the user can be naturally increased. The above results show that the composition of the present invention is a comprehensive immune regulation and promotion product with both anti-allergic and enhanced immune activity, which is suitable as a daily dietary supplement to prevent bacteria from invading.

[0032] The present invention is a preparation example of the composition for efficacy experiments. The preparation example of the composition of the present invention is expressed in weight percentage, and is composed of about 3.13% of Lactobacillus paracasei strain LT12 (LT12), about 4% of β-glucan (Beta-Glucan, BG) and about 1.5% of bovine colostrum powder (Bovine Colostrum powder, BCP) as the main active ingredients, and contains a dietary acceptable carrier.

[0033] The present invention is further illustrated by the following examples, which are to be construed as merely illustrative and not limitative of the remainder of the invention in any way.

[0034] Example

[0035] Example 1: Growth status of Lactobacillus paracasei LT12

[0036] The growth curve of LT12 is shown in Table 1 and Figure 1 , dilute the activated lactic acid bacteria strain LT12 to 10 1 CFU / mL was used for the first 4 hours of culture, and the culture was in the lag phase. After 6 hours of culture, the culture increased by 1 log CFU / mL (log phase) every two hours. After 22 hours, the culture entered the stationary phase and did not reach the death phase at 36 hours, indicating that the bacteria in this period can be used for subsequent experiments. Therefore, the number of bacteria designed for this experiment was 10 3 , 10 5 , 10 7 , 10 8 and 10 9 According to the growth curve results, lactic acid bacteria culture solutions cultured for 8 hours (3.46±0.01log CFU / mL), 14 hours (5.56±0.12log CFU / mL), 20 hours (7.34±0.08log CFU / mL), 22 hours (8.65±0.06log CFU / mL) and 36 hours (9.15±0.09log CFU / mL) were selected for subsequent experiments.

[0037] Table 1: Growth of lactic acid bacteria LT12 in MRS medium

[0038]

[0039]

[0040] Note: 5x10 3=3.70Log CFU / mL; 5x10 5 =5.70Log CFU / mL; 5x10 7 =7.70Log / CFU / mL

[0041] Example 2: Toxicity test of single components and compound compositions on the survival rate of macrophage RAW 264.7

[0042] First, the toxicity of single components and compound composition samples at specific concentrations to macrophage RAW264.7 cells was determined. Figure 2 ) It can be found that BCP and LT12 can stimulate the proliferation of macrophages RAW264.7, while BG causes the cells to proliferate within 4 to 12 hours, but slightly reduces the number of surviving cells at 24 hours. The composition of the present invention also does not show cytotoxicity. Overall, whether it is a single prescription or a compound combination, there is no cytotoxicity, and the survival rate is more than 90%. Not only does it not cause macrophage damage, but some of them can even slightly promote their proliferation.

[0043] Example 3: Immunomodulatory effects of single components and compound compositions on macrophage RAW 264.7

[0044] After the toxicity test, ELISA was used to measure the cytokine content of macrophage RAW264.7. Pro-inflammatory cytokines (TNF-α, IL-6) and anti-inflammatory cytokines (TGF-β1, IL-10) that play an important role in the early stage of immune response were measured. The results are as follows Figure 3 shown.

[0045] like Figure 3 As shown in Part A, within 24 hours, the longer the stimulation time, the higher the induced TNF-α content. Compared with the non-stimulated group (control group), the highest multiples of stimulation induction for each sample were 9.59 times after 24 hours of LT12 stimulation, 1.93 times after 24 hours of BCP stimulation, and 7.96 times after 12 hours of BG stimulation. Among them, the induction amount of BG stimulation no longer increased after 24 hours, and it is speculated that the optimal action time of this dose is 12 hours. If compared with 12 hours as the benchmark point, BG has the best ability to induce TNF-α secretion, followed by LT12, and BCP is relatively weak. The formula group with BG added reached the highest induction amount at 12 hours, and compared at the 12-hour time point, the compound composition containing three ingredients had the highest TNF-α induction amount (14.81 times), with a multiplying effect ( Figure 3 Part A of the .

[0046] IL-6 and TNF-α have similar trends, and both increase induction with the increase of stimulation time. Compared with the unstimulated group (control group), the highest multiples of induction for each sample were 2.70 times after LT12 stimulation for 24 hours, 7.53 times after BCP stimulation for 24 hours, and 5.56 times after BG stimulation for 12 hours. Among them, the induction amount of BG stimulation no longer increased after 24 hours, and it is speculated that the optimal action time of this dose is 12 hours. If compared with 24 hours as the benchmark point, BCP has the best ability to induce IL-6 secretion, followed by BG, and LT12 is relatively weak. Except for the LT12+BG group, the compound combination groups all reached the highest IL-6 induction amount at 24 hours, and compared with other groups, the compound combination containing three components increased by 26.76 times in 8 hours and reached a maximum value of 72.92 times at 24 hours, with a significant additive effect ( Figure 3 Part B of the

[0047] Figure 3 The results of Part C show that although the secretion of TGF-β1 also shows a trend of increasing with the increase of stimulation time, it can be induced to increase by the maximum times in a short time (4 hours) compared with the non-stimulated group (control group). Compared with the non-stimulated group (control group), the highest multiples of stimulation induction of each sample were 35.26 times after 4 hours of LT12 stimulation, 3.25 times after 4 hours of BCP stimulation, and 1.97 times after 24 hours of BG stimulation. If compared with 24 hours as the benchmark point, LT12 has the best ability to induce TGF-β1 secretion, followed by BCP, and BG is relatively weak, and in particular, the induction amount of LT12 alone is much higher than that of each group. The group of the compound composition also increased the most times in 4 hours, and compared with other combinations, the compound composition containing three components has the highest TGF-β1 induction amount (30.51 times), which has a multiplying effect.

[0048] In terms of IL-10, Figure 3 Part D shows that compared with the non-stimulated group (control group), the induced IL-10 content did not increase significantly with the extension of stimulation time. Compared with the non-stimulated group (control group), the highest multiples of each sample stimulated individually were 1.11 times after 12 hours of LT12 stimulation, 1.13 times after 12 hours of BCP stimulation, and 1.61 times after 12 hours of BG stimulation. Overall, each single-ingredient sample at a fixed dose did not significantly enhance the secretion of IL-10 by RAW264.7. The formula group reached the highest induction amount at 12 hours, and compared with other combinations, the compound combination formula containing three ingredients had the highest IL-10 induction amount (2.36 times).

[0049] In summary, the composition of the present invention is a dietary supplement designed for immunomodulation based on three main ingredients, LT12, BCP and BG, which can effectively induce the production of TNF-α, IL-6 and TGF-β1, indicating that it has an immunomodulatory effect, and if the best effect is to be achieved, none of them can be missing, indicating that there is a synergistic or additive effect between the three ingredients. Overall, the formula design of the present invention has the potential to help immunomodulation and is suitable for daily and long-term consumption to achieve a preventive and protective effect.

Claims

1. A composition for immunomodulation, comprising a Lactobacillus paracasei strain ( Lactobacillus paracasei )LT12, β-glucan and bovine colostrum powder, wherein the ratio combination of the three provides a multifunctional accelerative immune regulation effect, wherein the Lactobacillus paracasei strain LT12 is preserved in the Agricultural Research Service of the United States, and the preservation number is NRRL-B50327.

2. The composition according to claim 1, wherein the main active ingredients are mainly composed of 0.5-20% of Lactobacillus paracasei strain LT12, 0.5-25% of β-glucan and 0.05-10% of bovine colostrum powder, expressed in weight percentage.

3. The composition according to claim 2, which is mainly composed of 1-10% of Lactobacillus paracasei strain LT12, 1-12% of β-glucan and 0.05-5% of bovine colostrum powder as the main active ingredients, expressed in weight percentage.

4. The composition according to claim 3, which is mainly composed of 1-5% of Lactobacillus paracasei strain LT12, 1-6% of β-glucan and 1-4% of bovine colostrum powder as the main active ingredients, expressed in weight percentage.

5. The composition according to claim 4, wherein the main active ingredients are 2-4% of Lactobacillus paracasei strain LT12, 4-5% of β-glucan and 1-2% of bovine colostrum powder, expressed in weight percentage. The composition according to claim 1 , wherein the β-glucan is β-1,3 / 1,6-glucan.

7. A pharmaceutical composition for immunomodulation, comprising a pharmaceutically acceptable carrier and a Lactobacillus paracasei strain ( Lactobacillus paracasei )LT12, β-glucan and bovine colostrum powder, wherein the combination of the three provides a synergistic effect of enhancing the production of tumor necrosis factor-α, interleukin-6, interleukin-10 and transforming growth factor-β1, wherein the Lactobacillus paracasei strain LT12 is deposited in the Agricultural Research Service of the United States with the deposit number NRRL-B50327.

8. A dietary supplement for immunomodulation, comprising a dietary acceptable carrier and a strain of Lactobacillus paracasei ( Lactobacillus paracasei )LT12, β-glucan and bovine colostrum powder, wherein the combination of the three provides a synergistic effect of enhancing the production of tumor necrosis factor-α, interleukin-6, interleukin-10 and transforming growth factor-β1, wherein the Lactobacillus paracasei strain LT12 is deposited in the Agricultural Research Service of the United States with the deposit number NRRL-B50327.

9. Use of the composition according to claim 1 in the preparation of an immunomodulatory dietary supplement.

10. Use of the composition according to claim 2 in the preparation of an immunomodulatory dietary supplement.

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