Patent Description:
The present invention relates to a composition for modulating immunity, in particular for enhancing the expression of interleukin-<NUM>.

For the infection pattern of most influenza viruses or pathogenic bacteria, the pathogens infect and colonize a host when the host has a weakened immune system. In order to eliminate the pathogens after the infection, the host may have cytokine storm, which is an immune overreaction that causes inflammation and leads to organ and tissue damage (such as pneumonia) when the immune system is overloaded. Therefore, an immune system that is too strong or too weak may cause diseases. It is important to have a well-balanced immune system, so that the immune system can be boosted to fight against foreign objects such as microorganisms and viruses during the infection, and the immune system can be reduced to alleviate inflammation during cytokine storm caused by sensitizing factors. Such two-way immunomodulation can make the immune system balanced and maintain good health.

In the early stage, innate immunity is mainly the first line of defense against invading pathogens in the human immune system. The innate immune responses, which are a rapid anti-infective action, non-specifically identify and act on pathogens, directly fight against pathogens to resist external infections. The sentinel innate immune cells are responsible for activating the innate and adaptive immunity to pathogens. Macrophages are a key indicator of host defense through pathogen phagocytosis. Activation through different signaling pathways results in 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 be reversibly functionally redifferentiated into pro-inflammatory and anti-inflammatory macrophages, respectively. The role of M1 macrophages is to secrete pro-inflammatory cytokines and chemokines, mainly IL-<NUM>, IL-<NUM> and TNF-α, present antigens, and thus participate in the immune response and help drive antigen-specific Th1 cell inflammatory reaction. In addition, the polarization of the M1 phenotype is considered to be essential for an effective antiviral immune response in the lungs. M2 macrophages mainly secrete Arginase-I, IL-<NUM> and TGF-β and other anti-inflammatory cytokines, which have the function of reducing inflammation, and contributing to tumor growth and immunosuppressive function. In short, M1 and M2 macrophages can transform into each other in a specific microenvironment.

To maintain good health, how to prevent a weakened immune system that causes virus infection is the first line of defense that should be strictly guarded. Nowadays, dietary supplements are one of the common methods to prevent the occurrence of infectious disease and allergies. However, most dietary supplements are focus on one-way immunomodulation, either enhancing immunity or reducing allergies. <CIT> discloses a composition comprising lyophilized bovine colostrum powder and yeast beta-glucan exerting synergistic effects for improving immunity. <CIT> discloses a nutritional product comprising geta-glucan amd colostrum freeze-dried powder having synergistic effect. <CIT> discloses a lactoferrin powder for enhancing immunity comprising colostrum and yeast beta-glucan. Wei et al, disclose synergistic effects of Lactobacillus rhamnosus with bovine colostrum on immunity (<NPL>).

Accordingly, it is desirable to develop a product with two-way immunomodulatory effects.

It is unexpectedly found in the present invention that the combination of the three functional ingredients, LT12, BG and BCP, can efficiently induce the production of TNF-α, IL-<NUM>, and TGF-β1, suggesting the synergistic or additive effects among the three ingredients. Accordingly, provided are compositions for modulating immunity which provides multi-functional synergistic effects, and the use thereof.

Accordingly, one aspect of the present invention is to provide a composition for modulating immunity, comprising Lactobacillus paracasei LT12 (LT12), β-glucan (BG), and Bovine Colostrum Powder (BCP) at a ratio to enhance the expression of interleukin-<NUM> (IL-<NUM>).

According to the present invention, the composition consists essentially of <NUM>%-<NUM>% of Lactobacillus paracasei strain LT12 (LT12), <NUM>%-<NUM>% of β-glucan ( Beta-Glucan, BG) and <NUM>%-<NUM>% of Bovine Colostrum powder (BCP) as active components, in a percentage by total weight of the composition.

In a particular embodiment of the present invention, the composition the composition consists essentially of <NUM>%-<NUM>% of Lactobacillus paracasei strain LT12 (LT12), <NUM>%-<NUM>% of β-glucan ( Beta-Glucan, BG) and <NUM>%-<NUM>% of Bovine Colostrum powder (BCP) as active components, in a percentage by total weight of the composition.

In a particular embodiment of the present invention, the composition the composition consists essentially of about <NUM>% of Lactobacillus paracasei strain LT12 (LT12), about <NUM>% of β-glucan ( Beta-Glucan, BG) and about <NUM>% of Bovine Colostrum powder (BCP) as active components, in a percentage by total weight of the composition.

In one embodiment of the invention, the β-glucan is yeast β-<NUM>,<NUM>/<NUM>,<NUM>-glucan.

In one embodiment of the invention, the composition further comprises a pharmaceutically acceptable or edible carrier.

Another aspect of the present invention is to provide a pharmaceutical composition for modulating immunity and immune strengthening activities for preventing the infection of any pathogen, comprising a pharmaceutically acceptable carrier, and a therapeutically effective amount of the above-defined composition.

Another aspect of the present invention is to provide a dietary supplement for modulating immunity, comprising a dietary acceptable carrier, and the above-defined composition.

According to the present invention, the composition does not affect the viability of macrophage.

It is to be understood that the following detailed description is exemplary and explanatory only and shall not be restrictive of the invention. The invention shall be defined by the appended set of claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs.

As used herein, the term "a" or "an" means one or more than one (that is, at least one) of the grammatical object of the article, unless otherwise made clear in the specific use of the article in only a singular sense.

The present invention provides a composition for modulating immunity, comprising three single ingredients: Lactobacillus paracasei LT12 (LT12), β-glucan (BG) and Bovine Colostrum Powder (BCP), and the use thereof.

Lactobacillus species constitute a significant component of the human and animal microbiota at a number of body sites, such as the digestive system, and the female genital system. Among Lactobacillus species, a new strain of Lactobacillus paracasei, LT12 (hereinafter LT12), was developed and has been granted patents in many countries, e.g. <CIT> and <CIT>. The studies showed that LT12 could stimulate human peripheral blood mononuclear cells (PBMCs) to secrete a large amount of IFN-γ and effectively inhibited allergic reactions as well. The results of the cytokine antibody array analysis showed that LT12 could simultaneously enhance the expression of multiple cytokines, including IL-<NUM>, IL-<NUM>, IL-<NUM>, MCP-<NUM> and TNF-α, indicating that LT12 has effects of coordinating immune system and two-way immunomodulation. In accordance with the Budapest Treaty, the strain LT12 used in this disclosure was deposited with the American Agricultural Research Culture Collection, International Depositary Authority, <NUM> N. University Street, Peoria, Ill. , <NUM>, USA, on September <NUM>, <NUM>, with its deposit number NRRL-B50327.

As used herein, the term "β-Glucans" or "BG" refers to a group of β-D-glucose polysaccharides typically forming a linear backbone with <NUM>-<NUM>β-glycosidic bonds. β-glucan molecules can also have branching glucose side-chains attached to other positions on the main D-glucose chain, for example, β-<NUM>,<NUM>/<NUM>,<NUM>-glucan having <NUM>-<NUM> side-chains. In the present invention, the β-glucans can be derived from any resources available, for example, β-glucans naturally occurring in the cell walls of cereals, bacteria, and fungi. One example of the β-glucans is β-<NUM>,<NUM>/<NUM>,<NUM>-glucan. In one example of the invention, the beta-glucan made from yeast; for examplesuch as yeast β-glucan or yeast β-<NUM>,<NUM>/<NUM>,<NUM>-glucan.

Colostrum is the first milk produced post-partum by mammals and is compositionally distinct from mature milk. The term "Bovine Colostrum Powder" as used herein is the dried powder of bovine colostrum, wherein the bovine colostrum is the first form of milk produced by the mammary glands of a cow immediately following delivery of the newborn calves.

The term "composition" as used herein, refers a product that results from the mixing or combining of more than one active ingredient. It can be prepared by mixing multiple active ingredients with a pharmaceutically acceptable or edible carrier, which can be prepared according to any commonly used or standard methods in the art.

The terms "effective amount" or "therapeutically effective amount," as used herein, refer to a sufficient amount of an agent or a compound being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an "effective amount" for therapeutic uses is the amount of the composition as disclosed herein required to provide a clinically significant effect on modulating immunity. An appropriate "effective" amount in any individual case may be determined using known techniques, such as a dose escalation study.

One aspect of the present invention is to provide a composition for modulating immunity, comprising Lactobacillus paracasei LT12, β-glucan, and Bovine Colostrum Powder. The composition consists essentially of <NUM>%-<NUM>% of LT12, <NUM>%-<NUM>% of BG and <NUM>%-<NUM>% of BCP as active components, in a percentage by total weight of the composition. It was confirmed that the composition of the invention enhanced the expression of IL-<NUM>, but did not affect the viability of macrophage.

To conduct the efficacy experiment, one example of the composition of the invention was prepared, which consists essentially of about <NUM>% of LT12, about <NUM>% of BG and <NUM>% BCP as active components, in a percentage of total weight of the composition.

An immunomodulatory effect of the composition is evaluated by the following methods. MTT assay (<NUM>-(<NUM>,<NUM>-Dimethylthiazol-<NUM>-yl)-<NUM>,<NUM>-diphenyltetrazolium bromide) was used to determine the cell survival rate and analyze the safety and effective concentration of the composition to the cells. Enzyme-linked immunosorbent assay (ELISA) was used to analyze the immunomodulatory effects of the composition, including TNF-α and IL-<NUM> (pro-inflammatory cytokines) related to inflammation and TGF-β1 and IL-<NUM> (anti-inflammatory cytokines) related to anti-inflammatory.

In present invention, it is unexpectedly found that the composition can induce the secretion of IL-<NUM> cytokine in a large amount, and the concentration of the cytokine also increases with time, in a proportional relationship. Also, compared with the control (no stimulation), the composition increases the induction amount by about <NUM>~<NUM> times in <NUM>~<NUM> hours; compared with groups of single ingredient and combinations of two ingredients, the composition increases the induction amount by about <NUM>~<NUM> times, indicating the significantly synergistic effects of the composition. In addition, compared with other groups, the composition can increase the secretion of TNF-α by about <NUM>~<NUM> times, which also indicates the synergistic effects of the composition. Furthermore, in the analysis of the anti-inflammatory cytokine, TGF-β1, compared with the control (no stimulation), the composition increases the induction amount by about <NUM>~<NUM> times in a short period of time (<NUM>~<NUM> hours); compared with BG, BCP, and groups of combinations of two ingredients, the composition increases the induction amount by about <NUM>~<NUM> times, indicating the significantly synergistic effects of the composition. The composition consists essentially of LT12, BG and BCP as active components, which synergistically induces and modulates immunity. The composition of the invention has an extremely significantly synergistic effect on inducing IL-<NUM> secretion, and simultaneously induce Th1 and Th2 related cytokines, indicating that the formula has the best two-way immunomodulatory effects among all the test groups, including the groups containing single component or the combinations of two components. Considering the possible pathogenic mechanism of virus, the composition of the present invention was also confirmed to enhance immune effects and. The present invention is a comprehensive product for immunomodulation promotion with anti-allergic and immune strengthening activities, which is suitable as a daily dietary supplement to prevent the infection of any pathogen.

The present invention is further illustrated by the following examples, which are provided for the purpose of demonstration rather than limitation.

The growth curve of LT12 is shown in both Table <NUM> and <FIG>. Activated Lactobacillus paracasei LT12 was diluted to <NUM><NUM> CFU/mL for cultivation. For the first four hours, the growth of the bacteria was in the lag phase, and from <NUM> hours after the initiation of cultivation, the bacteria numbers increased one log CFU/mL about every two hours (log phase). The growth of the bacteria entered the stationary phase at <NUM> hours and has not reached the death phase at <NUM> hours. The results indicate that the bacteria in this time period (stationary phase) can be used for subsequent experiments. Based on the growth curve, bacteria culture for <NUM> hours (<NUM>±<NUM> log CFU/mL), <NUM> hours (<NUM>±<NUM> log CFU/mL), <NUM> hours (<NUM>±<NUM> log CFU/mL), <NUM> hours (<NUM>±<NUM> log CFU/mL), and <NUM> hours (<NUM>±<NUM> log CFU/mL) were collected for the subsequent experiments, which need <NUM><NUM>, <NUM><NUM>, <NUM><NUM>, <NUM><NUM>, and <NUM><NUM> CFU/mL bacteria for the experiment design.

First of all, it was confirmed that the samples at the certain concentrations did not have a toxic effect on macrophage RAW264. Based on the results of the cytotoxicity test, it was found that both BCP and LT12 could stimulate the proliferation of macrophages RAW264. <NUM>, while BG made the cells proliferate within <NUM> to <NUM> hours, but slightly reduced the cell viability at <NUM> hours. In addition, the formula of the present invention did not show cytotoxicity to macrophages RAW264. Generally speaking, neither single ingredients nor compound combinations show toxic effect, and the survival rates of macrophages RAW264. <NUM> in all of the groups are more than <NUM>%. These single components and compound combinations do not cause macrophage damage, and some of them can even slightly promote the proliferation of macrophage (<FIG>).

After confirming that the formula does not have toxic effect on macrophages RAW264. <NUM>, content of cytokines was determined by ELISA. The pro-inflammatory factors (TNF-α, IL-<NUM>) and anti-inflammatory factors (TGF-β1, IL-<NUM>) that play important roles in the initial stage of the immune response were determined. The results are as shown in <FIG>.

As shown in <FIG>, within <NUM> hours, the longer the stimulation time, the higher the TNF-α content induced. Compared with the non-stimulation group (Control), the highest folds of TNF-α content induced by each single ingredient were the <NUM>-fold increase after <NUM> hours of LT12 stimulation, the <NUM>-fold increase after <NUM> hours of BCP stimulation, and the <NUM>-fold increase after <NUM> hours of BG stimulation, respectively. There was no increase in induction amount of TNF-α after <NUM> hours of BG stimulation, and it is inferred that the best action time of BG at this concentration is <NUM> hours. When compared the induction at the <NUM>-hour time point, BG has the best ability to induce TNF-α secretion, followed by LT12, and BCP has relatively weak ability of induction. The groups of compound combinations containing BG had the highest induction amount of TNF-α at <NUM> hours. When compared the induction at the <NUM>-hour time point, the compound combination containing all the three ingredients had the highest induction amount (<NUM> folds) and had a synergistic effect.

IL-<NUM> has a similar trend to TNF-α, as shown in <FIG>, and the induction amount of both cytokines increased as the stimulation time increased. Compared with the non-stimulation group (Control), the highest folds of IL-<NUM> content induced by each single ingredient were the <NUM>-fold increase after <NUM> hours of LT12 stimulation, the <NUM>-fold increase after <NUM> hours of BCP stimulation, and the <NUM>-fold increase after <NUM> hours of BG stimulation, respectively. There was no increase in induction amount of IL-<NUM> after <NUM> hours of BG stimulation, and it is inferred that the best action time of BG at this concentration is <NUM> hours. When compared the induction at the <NUM>-hour time point, BCP has the best ability to induce IL-<NUM> secretion, followed by BG, and LT12 has relatively weak ability of induction. The groups of compound combinations, except the group of LT12 + BG, had the highest induction amount of IL-<NUM> at <NUM> hours. Compared with other group, the compound combination containing all the three ingredients induced a <NUM>-fold increase of IL-<NUM> secretion after <NUM> hours of stimulation, and had the highest induction amount (<NUM> folds) after <NUM> hours of stimulation, showing a significantly synergistic effect.

The result in <FIG> shows that although TGF-β1 secretion also increased as the stimulation time increased, compared with the non-stimulation group (Control), the highest induction fold of TGF-β1 secretion was reached in a short period of time (<NUM> hours). Compared with the non-stimulation group (Control), the highest folds of TGF-B1 content induced by each single ingredient were the <NUM>-fold increase after <NUM> hours of LT12 stimulation, the <NUM>-fold increase after <NUM> hours of BCP stimulation, and the <NUM>-fold increase after <NUM> hours of BG stimulation, respectively. When compared the induction at the <NUM>-hour time point, LT12 has the best ability to induce TGF-β1 secretion, followed by BCP, and BG has relatively weak ability of induction. Specifically, the secretion amount of TGF-β1 induced by the single ingredient LT12 is much higher than other single ingredients. The highest induction fold of TGF-β1 secretion induced by the compound combinations also reached at <NUM> hours. Compared with other compound combinations, the compound combination containing all the three ingredients induced the most TGF-β1 secretion (<NUM> folds), showing a synergistic effect.

In terms of IL-<NUM>, <FIG> shows that compared with the non-stimulation group (Control), the content of IL-<NUM> did not significantly increase as the stimulation time increased. Compared with the non-stimulation group (Control), the highest folds of IL-<NUM> content induced by each single ingredient were the <NUM>-fold increase after <NUM> hours of LT12 stimulation, the <NUM>-fold increase after <NUM> hours of BCP stimulation, and the <NUM>-fold increase after <NUM> hours of BG stimulation, respectively. The highest induction fold of IL-<NUM> secretion induced by the compound combinations was reached at <NUM> hours. Compared with other compound combinations, the compound combination containing all the three ingredients induced the most IL-<NUM> secretion (<NUM> folds). Generally speaking, each single ingredient and their combinations at a certain concentration do not significantly increase IL-<NUM> secretion in RAW264.

Claim 1:
A composition comprising Lactobacillus paracasei LT12 (LT12), β-glucan (BG) and Bovine Colostrum Powder (BCP), wherein the composition consists essentially of <NUM>%-<NUM>% of LT12, <NUM>%-<NUM>% of BG and <NUM>%-<NUM>% of BCP as active components, in a percentage by total weight of the composition, and wherein the composition enhances the expression of interleukin-<NUM> (IL-<NUM>).