JP2015038043A - Composition for immunity activation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a low molecule composition or a composition containing an effective chemical component by establishing a method for isolating fractions of immunity activation activity in Carica papaya leaf extract and revealing the presence thereof, for the technical development of immunotherapy.SOLUTION: A composition for immunity activation containing, as an active ingredient, an immunity activation active ingredient obtained by subjecting a high-polarity component of Carica papaya leaf water-soluble extract to normal phase column chromatography with the mixed solvent of ethyl acetate and methanol can be utilized for medicines and foods.

Description

  The present invention relates to an immunostimulatory composition containing an immunostimulatory active ingredient obtained from papaya leaves as an active ingredient. More specifically, an immunostimulatory active ingredient obtained by subjecting the highly polar component of the water-soluble extract of papaya (Carica papaya) leaves to normal phase column chromatography using a mixed solvent of ethyl acetate and methanol is used as an active ingredient. The present invention relates to an immunostimulatory composition to be used, which relates to an immunostimulatory composition that can be used for pharmaceuticals and foods.

Immunotherapy is particularly active in the treatment of cancer (Non-Patent Document 1), and it is said that ipilimumab, an anti-CTL4 antibody, is effective in the treatment of progressive melanoma (Non-Patent Document 2). However, currently used for immunotherapy are biological products, which are generalized because they require administration in a way that infiltrates patients, require high medical costs, and have technical limitations. Not. In order to overcome this limitation and to make immunotherapy more effective and generalized, it is considered necessary to pioneer technology with compositions or chemical compounds containing chemical components effective by oral administration.
Immune cell therapy using T cells costimulated with an anti-CD3 / CD28 antibody is expected to treat cancer (Non-patent Documents 3-6). However, it is pointed out that it does not have sufficient applicability and effect, and it is still in a trial stage (Non-patent Document 7). To make immune cell therapy more effective, cultivate technologies such as T cell activation in the natural state, that is, signal activation by anti-CD3 / CD28 antibody-costimulation in vivo using low molecular weight compounds Is considered necessary.

  On the other hand, papaya (Carica Papaya) is now distributed throughout the tropics, and its leaves contain antioxidant vitamins C and tannins, as well as vitamins A, E, K, saponins, iron, etc. In addition, anti-inflammatory effects, parasite-control effects, and the like have been reported (Non-patent Document 8). Furthermore, a papaya leaf water-soluble extract has a tumor cell growth inhibitory effect (Patent Document 1), and this water-soluble extract has an effect of enhancing T cell activation by anti-CD3 / CD28 antibody costimulation. It is suggested that in vitro experiments with this extract suppress the production of IL-2, IL-4, etc., but enhance the production of cytokines that act as anti-tumors such as TNF-α, IFN-γ, IL-12 Has been reported (Non-patent Document 9). However, for practical use, it is necessary to establish a method for separating immunostimulatory active fractions and clarify the location of the active ingredients. There is also a report that regulatory T cells increase after ingestion of papaya fruit in healthy individuals and suppress cytokines such as IL-1β (Non-patent Document 10), but any component has not been studied.

JP 2008-214299 A

Topalian SL, Weiner GJ, Pardoll DM. Cancer immunotherapy comes of age. J Clin Oncol 2011; 29 (36): 4828-4836 Mellman I, Coukos G, Dranoff G. Cancer immunotherapy comes of age.Nature 2011; 480: 480-489 Lum LG, LeFever AV, Treisman JS, Garlie NK, Hanson JP Jr.Immune modulation in cancer patients after adoptive transfer of anti-CD3 / anti-CD28-costimulated T cells-phaseI clinical trial.J Immunother 2001; 24 (5): 408-419 Kalamasz D, Long SA, Taniguchi R, Buckner JH, Bonyhadi M. Optimization of human T-cell expansion ex vivo using magnetic beads conjugated with anti-CD3 and anti-CD28 antibodies. J Immunother 2004; 27 (5): 405-418 Bonylandi M, Frohlich M, Rasmussen A, Ferrand C, Grosmaire L, Robinet E, Leis J, Maziarz RT, Tiberghien P, Berenson RJ. In vitro engagement of CD3 and CD28 corrects T cell defects in chronic lymphotic leukemia .. J Immunol 2005 ; 174 (4): 2366-2375 Garlie NK, LeFever AV, Siebenlist RE, Levine BL, June CH, Lum LG.T cells coactivated with immobilized anti-CD3 and anti-CD28 as potent immunotherapy for cancer.J Immunother 1999; 22 (4): 336-345 Oelke M, Krueger C, Schneck J. Technological advances in adoptiv immunotherapy, Drugs Today 2005; 41 (1); 13-21 Hsu S. Green tea and the skin.J Am Acad Dermatol 2005; 52 (6): 1049-1059 Otsuki N, Dang NH, Kumagai E, Kondo A, Iwata S, Morimoto C. Aqueous extract of Carcia papaya leaves exhibits anti-tumor activity and immunomodulatory effects. J Ethnopharmacology 2010; 127: 760-767 Abdullah M, Chai PS, Loh CY, Chong MY, Quay HW, Vidyadaran S, Seman Z, Kandiah M, Seow HE.Carica papaya increases regulatory T cells and reduces IFN-γ + CD4 + T cells in healthy human subjects. Food Res 2011; 55 (5): 803-806

Immunotherapy is actively performed especially in the cancer field, but has not yet achieved sufficient effects and applicability. Biologics are used for immunotherapy, but it is considered necessary to pioneer technology using low molecular weight compounds and compositions containing them in order to overcome the limitations of immunotherapy. Immune cell therapy by T cell activation with anti-CD3 / CD28 antibody costimulation is expected to be effective, but it is still in the testing stage. To generalize this methodology, it is necessary to develop technologies such as stimulating signals activated by anti-CD3 / CD28 antibody costimulation in the body using compositions containing low molecular weight compounds or effective chemical components. it is conceivable that.
To date, it has been reported that water-soluble extracts of papaya leaves contain a composition that enhances the activation of T cells by anti-CD3 / CD28 antibody costimulation of human peripheral mononuclear cells. However, for practical use, it is necessary to establish a separation method for the fraction containing the immunostimulatory composition and to clarify the location.
In the present invention, a method for separating the immunostimulatory activity fraction in this papaya leaf extract is established, and its location is clarified to provide for the development of immunotherapy technology using a composition containing a low molecular weight or active chemical component. With the goal.

  In order to solve the problem, we performed column fractionation of water-soluble components of papaya leaves and established a method for separating components with immunostimulatory effects using cytokine production in human peripheral blood mononuclear cells with anti-CD3 / CD28 antibody costimulation as an index. The location of the active fraction was clarified. That is, the present inventors have found that an immunostimulatory active ingredient can be obtained by subjecting a highly polar component of a water-soluble extract of papaya leaves to normal phase column chromatography using a mixed solvent of ethyl acetate and methanol. It was. Accordingly, the present invention includes the following inventions (1) to (10).

(1) Contains as an active ingredient an immunostimulatory active ingredient obtained by subjecting a highly polar component of a water-soluble extract of papaya (Carica papaya) leaves to normal phase column chromatography using a mixed solvent of ethyl acetate and methanol. Composition for immunostimulation.
(2) The immunostimulation according to (1) above, wherein the highly polar component is a polar component obtained in a water phase fraction by partitioning extraction of a water-soluble extract of papaya (Carica papaya) leaves with water and butanol Composition.
(3) The above-mentioned immunostimulatory active component is a component that elutes the highly polar component by subjecting it to normal phase column chromatography using a mixed solvent having a volume ratio of ethyl acetate and methanol of 3: 1. The composition for immunostimulation according to 1) or (2).
(4) The immunostimulatory active ingredient is subjected to normal phase column chromatography, methanol is added to the ethyl acetate in a gradient to increase the polarity, and the volume ratio is ethyl acetate: methanol = 1: 0, 3: 1, Subsequently, when eluting at 1: 1, the composition for immunostimulation according to (3) above, which is a component that is mostly eluted at ethyl acetate: methanol = 3: 1.
(5) The composition for immunostimulation according to any one of (1) to (4) above, wherein the immunostimulatory active ingredient is a component containing a glycoside.
(6) The immunostimulatory active ingredient is a thin-layer chromatography (TLC) analysis, using a silica gel TLC plate, the developing solvent is ethyl acetate: methanol = 1: 1, Rf value is 0.2-0.8, anisaldehyde The composition for immunostimulation according to any one of (1) to (5) above, which is a component that develops brown or brown color by a coloring reagent.
(7) The composition for immunostimulation according to any one of (1) to (7) above, wherein the immunostimulatory active ingredient is a component showing a signal derived from sugar, olefin and aromatic ring in NMR analysis.
(8) The composition for immunostimulation according to any one of (1) to (7), wherein the immunostimulatory active component is a component having cytokine production enhancing activity.
(9) The composition for immunostimulation according to (8) above, wherein the cytokine is TNF-α, IFN-γ and IL-12.
(10) The composition for immunostimulation according to any one of (1) to (9) above, which is a pharmaceutical composition.
(11) The composition for immunostimulation according to any one of (1) to (9), which is a food composition.
(12) The immunostimulatory composition according to any one of (1) to (9) above, which is a health food, a functional food, a food for specified health use, a nutritional supplement or an enteral nutrition food.
(13) The composition for immunostimulation according to any one of (1) to (9) above, which is in the form of a drink, powder or tablet.

  The immunostimulatory active component of papaya leaves obtained in the present invention has the effect of enhancing T cell activation in human peripheral blood mononuclear cells upon anti-CD3 / CD28 antibody costimulation. Therefore, it is considered that the composition containing the immunostimulatory active ingredient of papaya leaves obtained in the present invention can be widely applied to the development of technology for performing immunotherapy such as cancer and immunodeficiency using chemical substances. Furthermore, it is thought that it can also contribute to the development of a technique for in vivo activation of immune cell therapy, which has previously been T cell activation by anti-CD3 / CD28 antibody costimulation in vitro. In the present invention, a method for separating an immunostimulatory fraction from a water-soluble extract of papaya leaves is established, and the location is clarified to enable the development of immunotherapy techniques using chemical substances. . Furthermore, since the immunostimulatory fraction is water-soluble, it is highly likely to be absorbed by oral administration. Since papaya is a food, the possibility of side effects is low. Therefore, in the application of the immunostimulatory composition derived from the water-soluble fraction of papaya leaves, it is considered that the hurdles are low in terms of whether or not oral administration is possible and the presence or absence of toxicity, which are the two major issues facing pharmaceutical development. Moreover, the composition containing the immunostimulatory active component of the papaya leaf obtained by this invention can also be used for the health food etc. for immunostimulating.

FIG. 1 shows a preparation scheme of a water-soluble extract from dried papaya leaves. 2A shows the column fractionation scheme (column fraction-1) of the papaya leaf water-soluble extract, B shows the TLC analysis result of each fraction, and C shows a clear component spot detected by TLC analysis. The immunostimulation assay results of the fractions are shown. FIG. 3A shows the column fractionation scheme (column fraction-2) of the papaya leaf water-soluble extract, B shows the TLC analysis result of each fraction (2F1 and 2F2), and C shows each fraction ( 2F1 and 2F2) immunostimulation assay results are shown. FIG. 4 shows the results of 1H-NMR analysis of the 2F2 fraction having immunoreactivity. FIG. 5 shows the HPLC measurement result of the 2F2 fraction having immunostimulatory activity. FIG. 6 shows the results of PDA analysis of the 2F2 fraction having immunostimulatory activity.

The present invention is described in detail below.
In the present invention, as an active ingredient, an immunostimulatory active ingredient obtained by subjecting a high-polarity component of a water-soluble extract of papaya (Carcia papaya) leaves to normal phase column chromatography using a mixed solvent of ethyl acetate and methanol is used. .
The water-soluble extract of papaya leaves is dried papaya (Carcia papaya) leaves, and the resulting dried product is put into, for example, ion-exchanged water and boiled for several hours, for example, 1 to 3 hours. A water-soluble extract can be obtained by maintaining the temperature at about 60 ° C. to 70 ° C., filtering the obtained water extract and concentrating the filtrate under reduced pressure.

  The highly polar component of the water-soluble extract can be obtained in the aqueous phase fraction by partition extraction of the water-soluble extract with water and butanol. Specifically, a water-soluble extract is dissolved in water or ion-exchanged water, and is partitioned and extracted with an equal volume of butanol to water or ion-exchanged water. More specifically, for example, 6.5 g of water-soluble extract is added. Dissolve in 100 ml of water or ion-exchanged water, perform partition extraction once with 100 ml of butanol, remove the less polar components, and concentrate the aqueous phase fraction as necessary to concentrate the highly polar components of the water-soluble extract. Can be obtained.

  Next, the immunostimulatory active component can be obtained by subjecting the highly polar component to normal phase column chromatography using a mixed solvent of ethyl acetate and methanol. Specifically, the high-polarity component is a normal phase column chromatography using a mixed solvent having a volume ratio of ethyl acetate and methanol of preferably 3 to 1: 1, more preferably 3: 1 and using silica gel or the like as a carrier. An immunostimulatory active component can be obtained from the component that elutes upon elution. More specifically, the high polarity component is subjected to normal phase column chromatography, and methanol is added to ethyl acetate in a gradient to increase the polarity, and the volume ratio is ethyl acetate: methanol = 1: 0, 3: 1. Then, eluting with 1: 1, an immunostimulatory active ingredient can be obtained from the ingredient eluting with ethyl acetate: methanol = 3: 1.

  The immunostimulatory active ingredient thus obtained is detected by anisaldehyde detection (Seigler DS, Pauli GF, Nahrstedt A, Leen R. Cyanogenic allosides and glucosides from Passiflora edulis and Carcia papaya. Phytochemistry 2002; 60: 873-882). It contains glycosides for color development. In the TLC analysis of the immunostimulatory active ingredient, a silica gel TLC plate (for example, silica gel TLC plate F254) was used, and the developing solvent showed an Rf value of 0.5-0.75 with ethyl acetate: methanol = 1: 1. Or it turns dark brown. In addition, the immunostimulatory active ingredient exhibits an enhancing effect on cytokine production. More specifically, for example, the effect of enhancing the production of TNF-α, IFN-γ and IL-12 in human peripheral blood mononuclear cells can be exhibited. The immunostimulatory active ingredient shows signals derived from sugars, olefins and aromatic rings in NMR analysis.

  An immunostimulatory composition containing an immunostimulatory active ingredient obtained from papaya leaves as an active ingredient can be used as a medicament for improving immune function. For use as a medicine, this immunostimulatory active ingredient is dried as necessary, and then used in the form of powders, tablets, etc. by conventional methods in addition to excipients normally used for powders, tablets, etc. be able to.

  The composition for immunostimulation of the present invention can also be used as health food, functional food, food for specified health use, nutritional supplement, enteral nutrition food, and the like. For example, commonly used formulations such as stabilizers, preservatives, coloring agents, fragrances, vitamins and the like are added to the immunostimulatory active ingredient, and drinks, powders, liquids, tablets, etc. are added in the usual manner. It can also be processed into forms such as powders and used as health foods, functional foods, foods for specified health use, dietary supplements, enteral nutritional foods, and the like.

  When the immunostimulatory active ingredient obtained from papaya leaves is used as a medicament for improving immune function, for example, the immunostimulatory active ingredient is converted to dry weight, and the amount of 1 mg to 1000 mg is usually 1 to 3 months per day. It is preferable to take it daily. Similarly, when edible as health foods, functional foods, foods for specified health use, dietary supplements, enteral nutritional foods, etc., the usual amount of 1 mg to 1000 mg per day (for 1 to 3 months) Preferably eaten daily.

Example 1
Preparation of water-soluble extract from papaya leaves According to the preparation scheme of water-soluble extract from dry papaya leaves shown in FIG. 1, water-soluble extract from papaya leaves was obtained by the following steps 1 to 3. .

Step 1: 25 g of dried papaya leaves were placed in 500 ml of ion-exchanged water and heated at 100 ° C. for 1 hour. Temperature control at 100 ° C. was performed using a silicon oil bath.
Process 2: Furthermore, the water temperature was kept at 80 ° C. and heated for 2 hours.
Step 3: The aqueous extract was filtered, and the filtrate was concentrated under reduced pressure to obtain about 6.5 g of a dry solid.

Example 2
Column separation-1 and immunostimulatory activity of water-soluble extract from papaya leaf Water solubility obtained in Example 1 according to column separation-1 and immunostimulatory activity of water-soluble extract of papaya leaf shown in Fig. 2 After obtaining a highly polar component from the extract, column separation was performed and the immunostimulatory activity of each fraction was measured.

  That is, 6.5 g of papaya leaf water-soluble extract was dissolved in 100 ml of ion-exchanged water and partitioned and extracted with butanol. As a result of concentrating the butanol extraction phase and the water extraction phase under reduced pressure, 1 g of butanol phase dried product and 5.5 g of aqueous phase dried product were obtained. 5.5 g of the dried aqueous phase was suspended in methanol, adsorbed on a reverse phase column (Wakogel (registered trademark) 100C18), and eluted with ethyl acetate: methanol (volume ratio = 1: 1). As a result, F1 (0.4 g), F2 (2.7 g), F3 (0.2 g), F4 (0.1), F5 (0.1 g) and a fraction 4 g eluted without separation were obtained. Separation was further advanced using a normal phase column for 4 g of the fraction that could not be separated. 4 g of this was suspended in methanol and adsorbed on a normal phase column (Wakogel C200), and then ethyl acetate: methanol (volume ratio = 3: 1), (volume ratio = 2: 1), (volume ratio = 1: Eluted in 1). As a result, F6 (0.5 g), F7 (0.1 g), and F8 (0.05 g) were obtained by elution with ethyl acetate: methanol (volume ratio = 3: 1). Elution with ethyl acetate: methanol (volume ratio = 2: 1) gave F9 (0.5 g), F10 (0.4 g), and F11 (0.13 g). F12 (0.11 g), F13 (0.6 g), and F14 (0.43 g) were obtained by elution with ethyl acetate: methanol (1: 1) (FIG. 2A).

  These fractions were separated by TLC and colored with phosphoric molybdate, and as a result, clear spots of components were confirmed in F1, F3, F6, F7, F10, and F11 (FIG. 2B). Therefore, cytokine production in these fractions was measured.

  Dilute the dried product of each fraction to a concentration of 0.1 g / 3 ml. Each fraction was added to isolated human peripheral blood mononuclear cells at two concentrations of 0.3 μl / 200 μl / well and 3 μl / 200 μl / well, and the amount of TNF-α produced was measured (n = 1). As a result, F7 had a strong TNF-α production action under the condition of sample addition amount 3 μl / 200 μl / well, and F6 also had a weak TNF-α production effect (FIG. 2C).

Example 3
Column separation-2 and immunostimulatory activity of water-soluble extract from papaya leaf Water solubility obtained in Example 1 along with column separation-2 and immunostimulatory activity of papaya leaf water-soluble extract shown in FIG. After obtaining a highly polar component from the extract, column separation was performed and the immunostimulatory activity of each fraction was measured.

  That is, in Example 2, since immunostimulatory activity was observed in the ethyl acetate: methanol (volume ratio = 3: 1) fraction in normal phase column chromatography, next, ethyl acetate: methanol (volume ratio) was obtained by normal phase column chromatography. = 3: 1), the eluted component was fractionated.

7.3 g of water-soluble extract from papaya leaves was dissolved in 100 ml of ion-exchanged water and partitioned and extracted with butanol. The butanol extraction phase and the aqueous phase were concentrated under reduced pressure to obtain 1.3 g of a butanol phase dry solid and 5.7 g of an aqueous phase dry solid. The aqueous phase dry solid was suspended in methanol and adsorbed on a normal phase column (Wakogel C200). After eluting the column sequentially with ethyl acetate (1.5L), ethyl acetate: methanol (volume ratio = 3: 1) (5L), ethyl acetate: methanol (volume ratio = 1: 1) (1.5L), the remaining adsorbed components Was eluted with 500 ml of water. As a result of concentrating each eluate under reduced pressure, ethyl acetate fraction (0.51 g), ethyl acetate: methanol (volume ratio = 3: 1) elution, 2F1 fraction (0.48 g) and 2F2 fraction (0.39 g), acetic acid An ethyl: methanol (1: 1) fraction (0.54 g) and a water-eluted fraction (3.76 g) were obtained (FIG. 3A).
Papaya (C.papaya) contains glycosides as components and uses anisaldehyde for detection (Seigler DS, Pauli GF, Nahrstedt A, Leen R. Cyanogenic allosides and glucosides from Passiflora edulis and Carcia papaya Phytochemistry 2002; 60: 873-882). Therefore, ethyl acetate: methanol (volume ratio = 3: 1) elution fractions 2F1 and 2F2 were separated by TLC to confirm the possibility of anisaldehyde color development. As a result, as shown in FIG. 3B, both 2F1 and 2F2 were detected with anisaldehyde.

  Next, the effect of 2F1 and 2F2 on cytokine production was examined. The concentrations of the 2F1 and 2F2 fractions were added to human peripheral blood mononuclear cells (2 × 105) under the conditions of 3 μl / 200 μl / well in the dilution performed in Example 2. As a result, the production of TNF-α, IFN-γ, and IL-12 was enhanced in human peripheral blood mononuclear cells added with 2F2 (n = 3).

Example 4
1H-NMR analysis of 2F2 fraction
After the 2F2 fraction was dissolved in heavy methanol (CD3OD), the solution was filtered through quartz wool, and the entire amount was transferred to a 5 mm diameter NMR sample tube to obtain a measurement sample. Measurements were made at 599.5 MHz with an INOVA 600 model (varian).
Since immunostimulatory activity was observed in the 2F2 fraction, 1H-NMR analysis of this fraction was performed. As a result, a strong sugar-derived signal at 3-4.2 ppm was shown as a feature of this fraction. In addition, a weak signal derived from saturated fatty acids was shown at 0.8-3.0 ppm, a weak signal derived from olefins at 5.0-6.0 ppm, and a weak signal derived from aromatic rings at 6.4-8.4 ppm (FIG. 4).

Example 5
HPLC analysis and PDA analysis of 2F2 fraction
Measurements were performed using an HP1100 HPLC system (Agilent Technologies Inc) and a Zorbax Sil 4.6x250 mm column (Agilent Technologies Inc). The mobile phase was (a) ethyl acetate / methanol = 900/100 (vol ratio) and (b) ethyl acetate: methanol = 100/900 (vol ratio), and the gradient conditions were vol of B at 0, 30, 45 minutes. % Performed at 0, 100, and 100. The results are shown in FIG.
PDA analysis of 272 nm retention time 9.9 min and 21.7 min peak was performed, and the results are shown in FIG.

In the present invention, a method for separating immunostimulated fractions from a water-soluble extract of papaya leaves by normal phase column chromatography was established and the location was clarified. In the 1H-NMR analysis of the immunostimulated fraction, a strong sugar-derived signal was detected. In the HPLC analysis, a large peak was observed at a retention time of 9.9 minutes and a small peak was observed at 21.7 minutes under gradient conditions. These peaks were shown to be formed from multiple peaks by PDA analysis. Therefore, the immunostimulating fraction separated by the method established according to the present invention contains a plurality of components, mainly components having a structure derived from sugar.
Based on the results of the present invention, materials can be widely provided for the development of technology for immunotherapy using low molecular weight compounds, and further for the development of technology for immune cell therapy that activates CD3 / CD28 signal in vivo.

Claims (13)

  Immunostimulation containing as an active ingredient an immunostimulatory active ingredient obtained by subjecting a highly polar component of a water-soluble extract of papaya (Carica papaya) leaves to normal phase column chromatography using a mixed solvent of ethyl acetate and methanol Composition.   The composition for immunostimulation according to claim 1, wherein the highly polar component is a highly polar component obtained in a water phase fraction by partitioning extraction of water-soluble extract of papaya (Carica papaya) leaves with water and butanol. .   The said immunostimulatory active component is a component which attaches | subjects the said high polarity component to normal phase column chromatography using the mixed solvent whose volume ratio of ethyl acetate and methanol is 3: 1, and is eluted. The composition for immunostimulation described in 1.     The immunostimulatory active ingredient is subjected to normal phase column chromatography, methanol is added to ethyl acetate in order to increase polarity, and the volume ratio is ethyl acetate: methanol = 1: 0, 3: 1, then 1: The composition for immunostimulation of Claim 3 which is a component which, when it elutes by 1, mostly elutes to ethyl acetate: methanol = 3: 1.   The composition for immunostimulation according to any one of claims 1 to 4, wherein the immunostimulatory active component is a component containing a glycoside.   In the thin layer chromatography (TLC) analysis, the immunostimulatory active component was a silica gel TLC plate, and the developing solvent was ethyl acetate: methanol = 1: 1, with an Rf value of 0.2-0.8, anisaldehyde coloring reagent. The composition for immunostimulation according to any one of claims 1 to 5, which is a component that develops brown or brown color.   The composition for immunostimulation according to any one of claims 1 to 7, wherein the immunostimulatory active component is a component showing a signal derived from sugar, olefin and aromatic ring in NMR analysis.   The composition for immunostimulation according to any one of claims 1 to 7, wherein the immunostimulatory active component is a component having cytokine production enhancing activity.   The composition for immunostimulation according to claim 8, wherein the cytokine is TNF-α, IFN-γ and IL-12.   The composition for immunostimulation according to any one of claims 1 to 9, which is a pharmaceutical composition.   The composition for immunostimulation according to any one of claims 1 to 9, which is a food composition.   The composition for immunostimulation according to any one of claims 1 to 9, which is a health food, a functional food, a food for specified health use, a nutritional supplement or an enteral nutrition food.   The composition for immunostimulation according to any one of claims 1 to 9, which is in the form of a drink, powder or tablet.