WO2019093543A1 - Method for producing immunity-enhancing composition including enzyme-treated noni extract - Google Patents

Abstract

The present invention relates to a method for producing an immunity-enhancing composition including an enzyme-treated noni extract as an active ingredient. According to the present invention, an extract that can improve the production of immunity-related factors and thus has an immunity-enhancing effect may be provided. Moreover, as a health functional composition, the composition may be applied in various fields such as food products and the like.

Description

Method for preparing an immunostimulatory composition comprising an enzyme-treated noni extract

The present invention relates to a noni ( Morinda citrifolia ) extract and a preparation method thereof, and relates to a noni extract and a preparation method thereof capable of improving the immunity enhancing effect.

It is said that Morinda citrifolia is called Indian mulberry in India, Pajitian in China, painkiller tree in Caribbean, cheese fruit in Australia, and Nono in Tahiti island. Jiu) or the Pharaoh (巴 气 天) is called. Noni leaves, stems, flowers, fruits and seeds have been used in folk remedies and are recorded as the best natural remedy according to the ancient literature in the South Pacific. Indeed, the anthraquinone and serotonin in noni have been shown to help digestion, relieve pain, and help with hypertension and cancer.

Accordingly, there is a need for research on a method for more effectively improving the efficacy of the active substance contained in Noni compared with the noni extract obtained from water or an organic solvent such as ethanol or acetone.

It is an object of the present invention to provide a method for producing an immunoenhancing composition comprising an enzyme-treated noni extract and a method for producing the same.

In order to solve the above problems,

Obtaining a raw material from noni; and

And adding an enzyme-degrading enzyme to the raw material. The present invention also provides a method for producing an immunity enhancing composition comprising an enzyme-treated noni extract.

According to one embodiment, the antagonist may include one or more selected from the group consisting of peclyve, rohament, viscozyme, and cytolase.

According to one embodiment, the above-mentioned enzyme-degrading enzyme may be added at a concentration of 0.01 to 0.5 parts by weight based on 100 parts by weight of the raw material.

According to one embodiment, the treatment temperature of the enzyme is 20 to 80 ° C.

According to one embodiment, the present invention may further comprise a step of extracting, filtering and drying the product after the treatment with the enzyme.

According to another embodiment of the present invention, a noni extract, which is produced according to the above-described method, can be provided.

According to another embodiment of the present invention, a health functional food containing the extract may be provided.

Other details of the embodiments of the present invention are included in the following detailed description.

According to the noni extract and the preparation method thereof according to the present invention, the production of an immune-related factor can be improved, and thus an extract having an immunity-enhancing effect can be provided. In addition, it can be applied to various fields such as food as a health functional composition.

FIG. 1 is a schematic view of a method for producing an extract according to an embodiment of the present invention.

2 is a graph showing the results of cytotoxicity experiments.

FIG. 3 is a graph showing changes in macrophage-derived TNF-a according to the concentration of the extract. FIG.

FIG. 4 is a graph showing changes in TNF-a from rat serum according to the concentration of the extract. FIG.

FIG. 5 is a graph showing changes in mouse spleen-derived IFN-? -Related mRNA according to the concentration of the extract. FIG.

FIG. 6 is a graph showing changes in mouse spleen-derived TNF-.alpha. MRNA according to the concentration of extract. FIG.

FIG. 7 is a graph showing changes in IL-12-related mRNA from mouse spleen according to the concentration of the extract. FIG.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention is capable of various modifications and various embodiments, and specific embodiments are illustrated in the drawings and described in detail in the description. It is to be understood, however, that the invention is not to be limited to the specific embodiments, but includes all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

Hereinafter, the immunity enhancing composition comprising the enzyme-treated noni extract according to the embodiment of the present invention and a method for producing the same will be described in detail.

As used herein, the term " raw material " refers to a material that has been subjected to washing, hydrolysis, pulverization, and drying after removing the foreign matter after harvest, without being subjected to any other chemical treatment, extraction or filtration.

In the method for producing noni extract according to the present invention,

Obtaining a raw material from noni; and

And adding an enzyme-degrading enzyme to the raw material.

According to one embodiment, the noni matter can be obtained by hydrolysis, pulverization and lyophilization of noni. Specifically, the raw material can be obtained, for example, by pulverizing water without adding water to the noni, followed by extraction and filtration, and freeze-drying. In this case, freeze-drying can be carried out by preliminarily freezing and then drying to a moisture content of 5% or less. For example, after freezing at -40 ° C, 30 to 50 ° C, for example, at 40 ° C for 30 to 60 hours, For example, such that the moisture content is not more than 5%, for example, 3 to 5% for 48 hours.

For example, a pin mill, a disc mill, or the like may be used as the step of crushing the raw material, and the crushing method is not particularly limited as long as it is a general crushing method. Further, the amount of water in the hydrolysis step may be 2 to 50 times, for example, 5 to 20 times, based on the weight of noni.

According to one embodiment, the enzyme may include one or more selected from the group consisting of peclyve, rohament, viscozyme, and cytolase, but is not limited as long as it is a commonly used protease.

Peclyve can be generated by Aspergillus niger and can include peclyve CP, peclyve PR, or a combination thereof. peclyve CP may contain a pectin-lyase, a polygalacturonase, and a pectin-methyl-esterase. Also, peclyve PR may contain pectin-lyase, pectin-methyl-esterase. Peclyve may be provided in liquid form and has an optimal temperature range of 40 to 55 占 폚. It can also be used in diets and adjuvants, specifically for anti-oxidant and vitamin extraction.

Rohament can be produced by Trichoderma reesei and is a liquid fungal cellulase enzyme for the hydrolysis of starch-free polysaccharides. It is a cellulase enzyme that is used to hydrolyze starch-free polysaccharides. Cellulase, beta-glucan, And other enzymes that degrade plant cell wall polysaccharides such as silanes (xylan, xylan). The rohament can be processed in a distillation process and a mashing process.

Viscozyme can be produced by Aspergillus sp and can be produced by a variety of enzymes such as arabanase, cellulose, beta-glucanase, hemicellulose and xylanase , xylanase). The major enzyme activity of viscozyme is the activity of endo-β-glucanase which hydrolyzes 1,3 or 1,4-mers in β-D-glucan. Viscozyme is known to be an effective enzyme for extracting polyphenols from apples and the like.

Cytolase can be produced by Aspergillus niger and is an acid resistance pectinase and can be used as a pressing enzyme for depectinization and compaction in the processing of various fruit juices.

According to one embodiment, the enzyme of the present invention may be added in an amount of 0.01 to 0.5 parts by weight, for example, 0.1 to 0.3 parts by weight, based on 100 parts by weight of the raw material.

According to one embodiment, the treatment temperature of the said enzyme is 20 to 80 ° C, for example 40 to 60 ° C. When a powdered enzyme is used, it is necessary to warm and stir the enzyme so that the powder does not aggregate when the enzyme is melted, and it can be maintained at a temperature range that does not affect the activity of the enzyme.

In addition, the enzyme treatment can be carried out with stirring for 30 minutes to 10 hours, for example 1 to 5 hours.

According to one embodiment, the method may further include a step of extracting, filtering and drying the product after the treatment with the enzyme-degrading enzyme.

The conditions for extracting the product after the enzyme treatment can be 50 to 120 ° C, for example, 80 to 110 ° C for 1 to 10 hours, for example, 2 to 8 hours. The extraction method may include a solvent extraction method, a supercritical extraction method, an ultrasonic extraction method, a hot water extraction method, and the like.

The filtration and drying step may include, for example, a freeze-drying method, an evaporation concentration method, a reverse osmosis concentration method, a hot air drying method, a spray drying method, a membrane separation concentration method, and the like.

According to one embodiment, the drying step of the present invention may comprise a spray drying step. At this time, additives such as excipients and the like can be added. For example, dextrin can be added in an amount of 10 to 80%, for example, 30 to 60% based on the solid content. The additive is not particularly limited as long as it is a general food additive, and its content can be appropriately selected within a range that does not affect the activity and function of the composition.

The present invention also provides an immunoenhancing composition comprising an enzyme-treated noni extract as an active ingredient, which is produced according to the above-described method. In addition, a health functional food containing the composition can be provided.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Examples 1 to 5: Preparation of noni extract according to kinds of enzymes

In order to set the optimum enzyme, a composition was prepared by varying the type of the enzyme. First, Noni was hydrolyzed, ground and lyophilized to obtain a raw material. The obtained raw material was pulverized using a pin mill, a disc mill or the like, and water 10 times as much as the raw material was added. Thereafter, the enzyme-degrading enzyme as shown in Table 1 was added in an amount of 0.2 part by weight based on 100 parts by weight of the raw material, and the mixture was reacted at 50 DEG C for 2 hours. After the enzymatic reaction, the mixture was extracted by hot water extraction at 95 ° C. for 4 hours, concentrated under reduced pressure at a temperature of 50 to 60 ° C., and hot air temperature was 220 ° C. and exhaust air temperature was measured using a spray dryer (YOOJIN TECH / PSD- Was spray-dried at 120 ° C, and the moisture content was dried at a level of 3 to 5% to obtain a composition. A schematic diagram for obtaining the composition is shown in FIG.

division	enzyme
Example 1	Peclyve CP
Example 2	Peclyve PR
Example 3	Rohament CL
Example 4	Viscozyme L
Example 5	Cytolase PCL5

Experimental Example 1: Cytotoxicity analysis

To determine the cytotoxicity for each composition, MTT assay was used. First, mouse macrophage cell line RAW264.7 cells were cultured in 100 mm cell culture dishes at a density of 70-80% using RPMI 1640 medium containing 100 IU / ml penicillin and 100 ug / ml streptomycin and 10% FBS. The cultured mouse macrophage cell line RAW264.7 was adjusted to a concentration of 1 × 10 ⁶ cells / ml using RPMI 1640 medium, inoculated into a 96-well plate, and preincubated for 18 hours at 5% CO ₂ and 37 ° C. . The medium was then removed and the extracts were treated with media containing the concentrations of 200, 400, and 800 ug / mL, respectively. After 24 hours, 10 μl of MTT solution (stock concentration: 5 mg / ml) was added and an additional reaction was induced for 3 hours. 100 μl of DMSO was added to each well for reaction termination and formazan crystal dissolution. The cell viability was calculated from the OD value obtained by measuring the absorbance at 570 nm of the amount of MTT reduced to formazan, and the result is shown in Fig. As Comparative Example 1, an extract according to the same method as in Example 1 was used, except that the enzyme-degrading enzyme was not treated.

As shown in Fig. 2, it can be confirmed that there is no cytotoxicity in the experimental concentration range.

Experimental Example 2: Analysis of changes of macrophage-derived TNF-α according to extract concentration

In order to analyze the effect of the extract on the immune enhancement effect, the production of macrophage-derived TNF-α was measured. TNF is a cytokine that mediates and regulates innate immunity. TNF is secreted by antigen-stimulated T lymphocytes, NK cells, and mast cells, but since activated macrophages are the main producing cells, TNF production is measured as a factor for analyzing the immuno-promoting effect.

The mouse macrophage cell line RAW264.7 cultured in the same manner as in Experimental Example 1 was adjusted to a concentration of 1 × 10 ⁶ cells / ml using RPMI 1640 medium, inoculated into a 96-well plate, and cultured in 5% CO ₂ and 37% Lt; 0 > C for 18 hours. The medium was then removed and the extracts were treated with medium containing 200, 400 and 800 ug / mL of each extract. Twenty-four hours later, the supernatant was transferred to another 96-well plate in an amount of 100 μl, and the concentration of TNF-α was measured according to manufacturer's instructions using an enzyme-linked immunosorbent assay (ELISA) kit of R & D Systems. .

As shown in FIG. 3, it was confirmed that TNF-α production was the highest in the extract prepared by treating Peclyve CP.

Experimental Example 3: Analysis of changes in TNF-alpha derived from mouse serum according to the concentration of extract

In order to analyze the effect of the extract on the immunity enhancement, the production of TNF-α from rat serum was measured. First, the C57BL / 6 mice were sacrificed and the spleen was taken out. The splenocytes were picked and adjusted to a concentration of 2 × 10 ⁶ cells / ml using RPMI 1640 medium, and then inoculated into a 6-well plate. Were treated with medium containing 50, 100, and 200 ug / mL of extracts at 5% CO ₂ and 37 캜 for 18 hours, respectively. 100 μl of the supernatant was transferred to another 96-well plate, and the concentration of TNF-α was measured according to manufacturer's instructions using an enzyme-linked immunosorbent assay (ELISA) kit from R & D Systems. The results are shown in FIG.

As shown in FIG. 4, it can be confirmed that the TNF production is increased as the content of the extract increases.

Experimental Example 4: Analysis of IFN-γ-related mRNA changes according to extract concentration

In order to analyze the effect of the extract on the immunity enhancement, the production of IFN-y-related mRNA from rat spleen was measured. IFN-y is a cytokine that mediates and modulates adaptive immunity. It activates macrophages to remove microorganisms, promote the differentiation of sub-sensitized CD4 + T cells into TH1 cells, inhibit the differentiation into TH2 cells, and is produced in T lymphocytes and NK cells. First, the C57BL / 6 mice were sacrificed and the spleen was taken out. The splenocytes were picked and adjusted to a concentration of 2 × 10 ⁶ cells / ml using RPMI 1640 medium, and then inoculated into a 6-well plate. Were treated with medium containing 50, 100, and 200 ug / mL of extracts at 5% CO ₂ and 37 캜 for 12 hours, respectively. After removing the supernatant, total RNA was extracted using Trizol reagent. The extracted total RNA was amplified by real-time PCR using the first strand cDNA synthesis kit (Thermo scientific). The sense and antisense primer sequences of the target proteins used herein were prepared by reference to the existing literature, and GAPDH was used as a control gene. Specific sequences are shown in Table 2. PCR amplification was performed using SYBR Premix Ex Taq (Takara Bio) and real-time thermal cycler (Bio-Rad) according to the manufacturer's manual. The results are shown in Fig.

division	forward	reverse
IFN-y	5'-GCG TCA TTG AAT CAC ACC-3 '	5'-GGA CCT GTG GGT TGT TGA CC-3 '
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)	5'-CAA TGA ATA CGG CTA CAGCA-3 '	5'-AGG GAG ATG CTC AGT GTT GG-3 '

As shown in FIG. 5, it can be seen that the IFN-γ-related mRNA increases as the concentration of the extract increases.

Experimental Example 5: Analysis of TNF-α-related mRNA changes according to extract concentration

In order to analyze the effect of the extract on the immunity enhancement, the production of TNF-α-related mRNA from rat spleen was measured. The TNF-a nucleotide sequences used are shown in Table 3, and the sequence of GAPDH is shown in Table 2. The measurement was carried out in the same manner as in Experimental Example 4, and the results are shown in FIG.

division	forward	reverse
TNF-a	5'-AGC CCA CGT CGT AGC AAA CCA C-3 '	5'-ATC GGC TGG CAC CAC TAG TTG GT-3 '

As shown in FIG. 6, it can be confirmed that the TNF-α-related mRNA increases as the concentration of the extract increases.

Experimental Example 6: Analysis of IL-12-related mRNA changes according to extract concentration

In order to analyze the effect of the extract on the immunity enhancement, the production of IL-12-related mRNA from rat spleen was measured. IL-12 is a cytokine that mediates and regulates innate immunity. It plays an important role in initiating a series of reactions involving macrophages, NK cells, and T lymphocytes, which are involved in the removal of intracellular microorganisms. do. The used IL-12 base sequences are shown in Table 4, and the sequence of GAPDH is shown in Table 2. The measurement was carried out in the same manner as in Experimental Example 4, and the results are shown in FIG.

division	forward	reverse
IL-12	5'-ACA GCA CCA GCT TCT TCA TCA-3 '	5'-TCT TCA AAG GCT TCA TCT GCA-3 '

As shown in FIG. 7, it can be seen that the IL-12-related mRNA increases as the concentration of the extract increases.

As can be seen from the above results, according to the method for producing noni extract according to the present invention, immunity factors such as TNF, IL and IFN can be enhanced, and as a result, it can be expected that the immunity enhancing effect can be expected.

The above description is only illustrative of the technical idea of the present invention, and various changes and modifications may be made by those skilled in the art without departing from the essential characteristics of the present invention. It should be noted that the embodiments disclosed in the present invention are not intended to limit the scope of the present invention and are not intended to limit the scope of the present invention. The scope of protection of the present invention should be construed according to the following claims, and all technical ideas within the scope of equivalents should be construed as falling within the scope of the present invention.

Claims (7)

1. Obtaining a raw material from noni; and

   And adding an enzyme-degrading enzyme to the raw material, wherein the enzyme-treated noni extract is added to the raw material.
2. The method according to claim 1,

   Wherein the enzyme-degrading enzyme comprises at least one selected from the group consisting of peclyve, rohament, viscozyme, and cytolase.
3. The method according to claim 1,

   Wherein the enzyme-treated noni extract is added in an amount of 0.01 to 0.5 parts by weight based on 100 parts by weight of the raw material.
4. The method according to claim 1,

   Wherein the enzyme-treated noni-hydrolyzing enzyme has a treatment temperature of 20 to 80 ° C.
5. The method according to claim 1,

   Further comprising the step of extracting, filtering and drying the product after the treatment with the enzyme-degrading enzyme, wherein the enzyme-treated noni extract is used.
6. 8. An immunomodulating composition comprising an enzyme-treated noni extract, which is produced according to any one of claims 1 to 7.
7. The method according to claim 6,

   ≪ / RTI > wherein said composition comprises said composition.

Images