KR20210013482A - Method for Extracting Active Ingredient of Herb Medicine and Composition for Enhancing Activity of Macrophage Prepared therby - Google Patents

Abstract

The present invention relates to a novel method for extracting active ingredients of herbal medicines using plant cell wall degrading enzymes and an herbal composition for plant having an effect of enhancing macrophage activity representing the innate immune system prepared by the method. More specifically, the method comprises the steps of: preparing one type or a plurality of types of dried plant herbal medicine pulverized product (A); treating the pulverized product with a plant cell wall degrading enzyme including at least one of cellulase, hemicellulase and pectinase (B); and inactivating the enzyme (C). In addition, the present invention relates to the herbal composition having a macrophage activity enhancing effect prepared by using the extraction method.

Description

[Method for Extracting Active Ingredient of Herb Medicine and Composition for Enhancing Activity of Macrophage Prepared therby]

The present invention relates to a method for extracting an active ingredient of a new plant herbal medicine using a plant cell wall degrading enzyme, and a plant herbal composition having an effect of enhancing macrophage activity representing an innate immune system produced by this method.

Crude drugs are a kind of pharmaceuticals, and refer to pharmaceuticals or pharmaceutical materials that have been subjected to simple processing such as drying, slicing, or powdering of natural products with known biological efficacy. Herbal medicines have minerals such as forget-me-not, gypsum, and yeongsa, and animals such as musk, ungdam, and bongmil, but most of them are vegetable.

In oriental medicine, various herbal medicines are dried and used in combination according to the prescribed ingredients and contents.According to the specific manufacturing method, herbal medicines are also referred to as Tangje (湯劑, Jeonje煎劑), acupuncture (浸劑), powder (粉劑), and pills. It is divided into (丸劑) and tanje (炭劑). Among them, Tangje is a juice obtained by hot water extraction in the so-called'dalim' and is used in almost all types of herbal medicine.

In plants used as herbal medicines,'biologically active ingredients' exist in the cell or in the cell wall. That is, in the dried herbal medicine, the active ingredient exists in the hard and dense cell wall made of macropolysaccharides such as cellulose, pectin, and hemicellulose , or between the cell wall and the cell wall . The'decoction' for the manufacture of hot water is a process of separating the active ingredient from the cell wall (hot water extraction) by loosening and disintegrating the hard and dense cell wall of a dried herbal medicine with high temperature hot water. At this time, some cell wall components are pyrolyzed to extract pectin or hemicellulose itself, or immunoactive polysaccharides in a modified form during the physical extraction process, thereby contributing to the increase of the efficacy of the herbal medicine. Protein is not an active ingredient in bath medicine because the protein is denatured in the process of drying plant tissues and decoction at high temperatures.

The process of making herbal herbal medicines has three problems: First, the weakening of hot water usually requires a lot of time and cost because it has to be treated in hot water of 90°C or higher for 3 to 6 hours. Second, it is known that the medicinal efficacy and pharmacological response differ depending on the dosage and decoction time of medicinal herbs.Since the size, material, and control characteristics of the medicinal herbs are very diverse, it is difficult to maintain the uniformity of the quality of the resultant'Pyeongyak'. . Third, for example, in the case of herbal medicines with volatile active ingredients (cinnamon, mint, gyeji, gwakhyang, cheongho, sesin, baekdugu, chodugu, danhyang, mokhyang, fennel, gamguk, etc.), considerable active ingredients volatilize over time at high temperatures. Become and disappear. In order to solve this problem, a method of adding a volatile herbal medicine to the rear end of the decoction has been attempted, but this is complicated in the process and is not easy to apply, especially in mass production.

Registered Patent 10-1774564 relates to a method of preparing a ginseng-derived polysaccharide fraction having immune function enhancing activity by sequentially treating ginseng with amylase and cellulase and obtaining only polysaccharides that precipitate and separate with ethanol. Registered Patent 10-1774566 relates to a method of obtaining a polysaccharide fraction having immune function enhancing activity by treating barley leaves with pectinase and separating and separating polysaccharides with ethanol. Registered Patent 10-1868507 relates to a cosmetic composition for antioxidant, anti-inflammatory, and wrinkle improvement obtained by treating graviola powder with cellulase and pectinase, and separating it. Although there is no mention of'polysaccharide', it is undergoing a precipitation and separation process with ethanol. It can be seen that the final product is'polysaccharide'.

These conventional techniques are to produce functional polysaccharides by treating plant tissues with degrading enzymes, and to purely separate them.In the first place, various physiologically active ingredients (even if their structure and function are not clearly identified) are used. I am not interested in extracting efficiently.

On the other hand, as research results show that foods using various substances derived from herbal medicines compensate for the effects of environmental hormones mixed (residue) in the environment and food, or reinforce the biological defense system against diseases by activating the immune system, Industrialization of functional foods or medicinal foods is drawing attention. For example, health functional foods using herbal resources, health supplements, natural colors and natural flavors, tea and medicinal liquor, sauces and spices as food additives added during cooking are attracting attention. In line with this trend, it is also timely to provide some general-purpose herbal materials widely used in various herbal medicines as materials for their safety and medicinal effects are sufficiently known and easy to collect or cultivate so that they can be used as health functional foods or health supplements. will be.

Registered Patent 10-1774564 Registered Patent 10-1774566 Registered Patent 10-1868507

It is an object of the present invention to provide a method for extracting biologically active ingredients from herbal medicines more moderately and more efficiently than the conventional'decoction method'.

In addition, an object of the present invention is to provide a composition with enhanced macrophage activity enhancing effect representing the innate immune system prepared according to such an active ingredient extraction method.

The present invention for achieving the above object

(A) preparing a pulverized product of one type or a plurality of types of dried herbal medicines; (B) treating the pulverized product with a plant cell wall degrading enzyme containing at least one of cellulase, hemicellulase, or pectinase; It relates to a method for extracting an active ingredient from a plant herbal medicine comprising (C) inactivating the enzyme.

In addition, the present invention relates to a plant-based herbal composition having an effect of enhancing macrophage activity prepared using the above extraction method.

As described above, according to the present invention, it is possible to obtain a plant herbal extract of uniform quality in an economical manner compared to the conventional decoction method.

In addition, according to the present invention, since extraction is performed at a relatively low temperature compared to the decoction method, it is possible to preserve volatile active ingredients.

In addition, according to the present invention, by adopting a unique extraction method, it is possible to provide a herbal composition having excellent macrophage activity enhancing effect representing the innate immune system by utilizing universally inexpensive plant herbal medicines.

1 is a diagram showing that the composition according to the present invention is not toxic to macrophages.
2 to 5 are diagrams showing the ability of macrophages to produce activators according to an example composition of the present invention, respectively.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings and prior experiments and examples. However, these drawings and embodiments are only examples for easily explaining the content and scope of the technical idea of the present invention, thereby not limiting or changing the technical scope of the present invention. It will be obvious to those skilled in the art that various modifications and changes are possible within the scope of the technical idea of the present invention based on these examples.

(1) Method of extracting active ingredients from herbal medicines

As described above, the present invention was derived to improve the conventional decoction method of extracting the active ingredient of a plant herbal medicine existing inside the dry and hard and dense plant cell wall or between the cell wall and the cell wall with hot water at high temperature. That is, in the present invention, the active ingredient present in the cell wall is effectively dismantled by partially decomposing the hard and dense cell wall under moderate conditions by treating the plant cell wall degrading enzyme with macropolysaccharides such as cellulose, pectin, and hemicellulose constituting the plant cell wall. It relates to an extraction method comprising the following steps to be extracted.

(A) preparing a pulverized product of one or more types of dried vegetable herbal medicine

(B) treating the pulverized product with a plant cell wall degrading enzyme containing at least one of cellulase, hemicellulase, or pectinase.

(C) inactivating the enzyme.

According to the present invention, an active ingredient may be extracted from a single plant herbal medicine and used as it is, or may be used by mixing the extracted active ingredients in an appropriate ratio. Furthermore, it is possible to extract the active ingredient (a mixture of) at once in a state in which the vegetable herbal medicine is mixed in an appropriate ratio in advance for each material.

Since the present invention is based on an enzymatic reaction, the extraction efficiency will increase as the pulverized product of the substance to be extracted (vegetable herbal medicine) is finer, but if it is too fine, the pulverization cost and difficulty in removing the residue later when necessary. It is good to adjust the size accordingly. In the following examples, the pulverized material was 100 mesh, but the optimum particle size may be changed.

Since the step of treating with cell wall degrading enzyme goes through an'enzymatic reaction' process, it is necessary to appropriately select and determine the type of enzyme, the ratio of enzyme and substrate (pulverized product) and moisture, reaction conditions such as temperature and pH, etc. In the following examples, the enzyme treatment was performed in a specific environment, but it will be natural that each variable can be appropriately changed and adjusted according to circumstances. In order to terminate the enzyme reaction, the enzyme is deactivated, for example, by heating. As described above, according to the present invention, the active ingredient of the herbal medicine is effectively extracted by enzymatically decomposing and dismantling the cell wall components. At the same time, it can be inferred that enzymatically degraded pectin or hemicellulose itself, or immunoactive polysaccharides in a modified form in the process of being degraded by the enzymes, will also be produced more than in the conventional decoction method.

By drying the composition obtained through the method of extracting the active ingredient in this way, it may be used as a solid phase such as scattered ash or pills, and it may be used as a bath medicine by removing the residue and separating the extract.

As can be seen from the following examples, the plant herbal composition obtained according to the extraction method according to the present invention has far superior macrophage activator production capacity compared to the composition (control) prepared by the conventional decoction method, which in turn has the present invention. It shows that the herbal composition by is superior to that of the conventional decoction method. Although the production yield of the composition according to the present invention is similar to or less than that of the control, the fact that the composition according to the example has a remarkably excellent ability to produce an immune activator, is that the immune-active active ingredient contained in the cell wall of a vegetable herbal medicine is extracted with conventional hot water. It is interpreted as because it is extracted at a much higher concentration than (decoction method). In other words, it seems that this is because the immunologically active active ingredient present in the cell wall of the raw material plant herbal medicine is extracted much more efficiently and at a higher ratio than the conventional hot water extraction method.

(2) vegetable herbal composition

In addition, the present invention relates to largely two types of plant-based herbal compositions having an immune function activating function by utilizing the above-described method for extracting active ingredients of plant-based herbal medicines.

The first composition is 4-6 parts by weight of Angelicae, 2.5-3.5 parts by weight of Chungoong, 1.5-2.5 parts by weight of Cornus milk, Galgeun and Baekchul, respectively, Licorice, Baek Peony, Bokryeong, Sanyak, Cinnamon, and Health respectively 0.8-1.2 parts by weight It is a solid or liquid composition obtained using water as a raw material.

The second composition contains 4-6 parts by weight of Angelicae, 2.5-3.5 parts by weight of Chungoong, 1.5-2.5 parts by weight of Hasu-O, Galgeun, Sasam, Sanyak, respectively, Licorice, Bokryeong, Rowan, and Health 0.8-1.2 parts by weight of mixed pulverized products, respectively. It is a solid or liquid composition obtained as a raw material.

In the following examples, the specific manufacturing process for the liquid of the two compositions is described. The liquid composition is obtained by separating the liquid part from the solid composition, so the solid composition contains all of the liquid composition (components and properties of) as it is.

Both of the liquid compositions thus obtained were not toxic to macrophages at a concentration of 50 to 100 μg/mL, and thus were not toxic to the human body.

Macrophages are representative immune cells of the innate immune system and are not only important for the removal of foreign antigens by phagocytosis, but also perform important functions involved in the regulation of important immune responses of the acquired immune system and various immune systems. As a result of confirming whether the two liquid compositions according to the present invention contribute to macrophage activation, they showed significantly higher macrophage activity compared to the control (extract by the decoction method by the conventional method). More specifically, the composition according to the present invention has the ability to produce cytokines such as nitric oxide (NO) and interleukin (IL)-6, IL-12 and tumor necrosis factor (TNF)-α, which are known as macrophage activating factors. Compared to, it was significantly superior (see Examples).

The characteristics of each activator are as follows.

NO is a radical produced from activated macrophages by inducible nitric oxide synthase (iNOS) in large quantities and is involved in removing foreign invading substances or cancer cells. TNF-α is an important cytokine that is secreted by activated macrophages to regulate immune cells, and functions to induce suicide of modified cells or to inhibit tumorigenesis and viral replication. IL-6 is a cytokine that is secreted by activated macrophages and affects acquired immunity such as proliferation of B cell lines and secretion of antibodies. IL-12 is a cytokine that is secreted from activated macrophages and promotes maturation and differentiation of natural killer cells (NK cells) and helper T cells.

Therefore, it was confirmed that the composition according to the present invention can activate the immune function of the human body.

[Example]

1. Preparation of vegetable herbal liquid composition

The herbal medicines according to Table 1 below were pulverized to prepare particles of 100 mesh or less, and 20 times (w/v) of distilled water was added to prepare a raw material mixture composition 1 and composition 2.

For each of the compositions 1 and 2, 0.1% (w/w) of the enzyme was added to the herbal composition, as shown in Table 2 below, and reacted for 12 hours under the conditions shown in Table 3 while stirring. Subsequently, the enzyme was deactivated by heating, and then centrifuged (9,000× g , 5°C, 30 minutes) to recover the supernatant. This state corresponds to the vegetable herbal liquid composition according to the present invention, but for economical efficiency, the liquid composition may be obtained by a conventional filtering method instead of a centrifugation method.

The sources and treatment conditions of each enzyme are listed in Table 3.

Each of the compositions 1 and 2 in Table 1 above was boiled at 95°C until the volume of the mixture of water and herbal medicines was halved, and the first supernatant and the residue were separated, and 20 times (w/v) distilled water was added to the residue at the same temperature. Boil at and cut in half, and then the second supernatant was separated and the first and second supernatant were combined to prepare controls 1 and 2.

On the other hand, when enzyme treatment is combined with the hot water extraction process, the synergistic effect is In order to confirm whether or not, a composition treated with the enzymes and conditions in Tables 2 and 3 was prepared again in each of Controls 1 and 2, and the following characteristics were analyzed, showing similar or inferior results to Controls 1 and 2. In other words, enzymatic treatment after hot water extraction did not show any positive effects. Therefore, detailed descriptions related thereto are omitted.

2. Characterization of the vegetable herbal liquid composition

Each liquid composition obtained in Examples was concentrated to about 10 brix, dialyzed (dialysis membrane 12 kDa), concentrated to about 10 brix, and lyophilized.

(1) Yield comparison

The yield of Controls 1 and 2 extracted with hot water was higher on average than in the Examples. This is interpreted as because, as described above, in the Example, the residue was removed after the enzyme treatment once, and the extract was obtained, but in the control group, extraction was performed twice and then the residue was removed.

(2) Toxicity and proliferative activity of the composition against macrophages

In order to confirm whether the composition according to the embodiment of the present invention exhibits toxicity to macrophages, the survival rate of macrophages after culturing macrophages with the composition was measured using the Ez-Cytox reagent (see FIG. 1). In the figure, Saline represents the physiological saline solution that does not contain the composition, and the gray bars and the black bars are when 50 and 100 μg/mL compositions are added, respectively (the same is true for all of the drawings below).

As can be seen from the figure, the compositions according to the examples did not show toxicity at concentrations of 50 and 100 μg/mL, but rather, it was confirmed that macrophage proliferation was promoted by 1.24 to 1.36 times compared to the saline control group at both treatment concentrations. there was. The compositions according to the examples were not only non-toxic like controls 1 and 2, but also an effect of promoting the proliferation of macrophages than the control group was observed.

(3) Measurement of macrophage activity of the composition

In order to confirm whether the composition according to an embodiment of the present invention promotes macrophage activation, the production of representative activated cytokines secreted from macrophages was analyzed by treating the composition on macrophages recovered from the peritoneal cavity of ICR mice.

① Experiment method

ICR mice were intraperitoneally injected with 5% thioglycollate (TG) medium to induce differentiation of macrophages through leaching of monocytes into the peritoneal cavity, and 3 to 4 days after TG injection, RPMI-1640 medium containing fetal bovine serum (FBS) was injected intraperitoneally. Thus, the differentiated macrophages leached into the abdominal cavity were recovered.

Dispense 200 μL (1×10 ⁶ cells/mL) of macrophage suspension into each well of a 96 well plate and incubate at 37°C in a CO ₂ incubator to form a monolayer of macrophages attached to the wells. Unadsorbed cells were removed by washing with phosphate buffered saline (PBS). A composition diluted to a certain concentration was added to the adsorbed macrophages and cultured again at 37° C. in a CO ₂ incubator.

After 24 hours of macrophage culture, the culture supernatant was transferred to another well plate, and Ez-Cytox solution was added to measure macrophage proliferation and toxicity by the composition, while a macrophage culture solution was used to determine whether the sample was activated with macrophages. The concentrations of cytokines such as IL-6, IL-12, and TNF-α, which are activating factors produced in phagocytes, were measured by ELISA method according to the manufacturer's instructions, and nitric oxide (NO), a radical compound, was measured by the Griess method.

The measured levels of production of IL-6, IL-12, TNF-α, and NO are shown in FIGS. 2 to 5, respectively. It is p<0.05 between the saline group and the examples.

② Analysis result: IL-6 (see Fig. 2)

IL-6 produced significantly better than the control group 1 (saline level) and control group 2 (316.3 pg/mL in 100 μg/mL sample), and the higher the composition concentration, the higher the production of IL-6. At concentrations of 50 and 100 μg/mL, Examples 2-1 (927.1 and 6593.5 pg/mL), 2-3 (896.9 and 4317.9 pg/mL), and 2-4 (519.1 and 3904.5 pg/mL) produced particularly high production. Indicated.

③ Analysis result: IL-12 (see Fig. 3)

IL-12 showed little activity compared to physiological saline (4.0 pg/mL) in Controls 1 and 2 (4.3 to 4.8 pg/mL), and Example 1 at both concentrations (50 and 100 μg/mL). 1 (5.5 and 6.9 pg/mL), 1-2 (5.2 and 5.0 pg/mL), 1-3 (6.0 and 7.9 pg/mL), 1-4 (4.3 and 5.4 pg/mL), 2-2 ( 6.0 and 7.2 pg/mL) also showed some superiority compared to the physiological saline group. However, Example 2-1 (6.7 and 12.8 pg/mL), 2-3 (6.8 and 10.5 pg/mL), and 2-4 (.5.3 and 9.1 pg/mL) were significant compared to Controls 1 and 2 and saline. It showed excellent activity.

④ Analysis result: TNF-α (see Fig. 4)

TNF-α was a low content in Controls 1 and 2 (173.8 to 2028.7 pg/mL), but Examples showed very superior activity, except for Example 1-4. Particularly, at both concentrations, Example 2-3 (2453.5 and 4208.4 pg/mL) had remarkably excellent TNF-α production capacity.

⑤ Analysis result: NO (see Fig. 5)

Looking at the NO production ability, Controls 1 and 2 (1.0 to 2.0 μM) showed little activity compared to the saline solution (1.1 μM), but at both concentrations, the examples showed a very productive ability of about 2 to 20 times more than this. Showed. Example 2-1 (10.6 and 19.6 μM), Example 2-3 (13.0 and 18.0 μM), and Example 2-4 (6.3 and 19.8 μM) exhibited remarkably good activity at both concentrations, especially Example 2-3 showed the highest NO production at a concentration as low as 50 μg.

3. Conclusion

In conclusion, all the compositions according to the examples of the present invention did not show toxicity to macrophages, so safety was confirmed.

In addition, the compositions according to the examples are mostly compared to Controls 1 and 2 obtained by conventional decoction in terms of the ability to produce cytokines such as IL-6, IL-12 and TNF-α, which are immune activating factors of macrophages, and NO, which is a radical compound. Showed a remarkably excellent effect in

Therefore, it was confirmed that the plant herbal medicine extraction compositions prepared according to the extraction method according to the present invention have superior immune activity enhancing function compared to the conventional hot water extraction (decoction method) compositions.

This is interpreted as because the cell walls of the hard and dense plant-based herbal medicines of the present invention are rapidly and efficiently disassembled loosely than those of the decoction method, and the active ingredients of the herbal medicines present inside the cells or between the cell walls are extracted rapidly and in large quantities. do. In addition, it can be reasonably expected that the immune-active components present in plant cells or cell walls are partially hydrolyzed by enzymes and modified to extract useful components rather than general hot water extraction.

Claims (6)

(A) preparing a pulverized product of one type or a plurality of types of dried herbal medicines;
(B) treating the pulverized product with a plant cell wall degrading enzyme containing at least one of cellulase, hemicellulase, or pectinase;
(C) inactivating the enzyme;
A method for extracting an active ingredient of a plant herbal medicine comprising:
The method according to claim 1,
After the step (C), the step of separating the extract by removing the residue of the pulverized plant herbal medicine is added.
(A) 4-6 parts by weight of Angelica Angelica, 2.5-3.5 parts by weight of Chungoong, 1.5-2.5 parts by weight of Cornus milk, Galgeun, Baekchul, respectively, Licorice, Baek Peony, Bokryeong, Sanyak, Cinnamon, respectively 0.8-1.2 parts by weight of mixed crushed product Preparing;
(B) treating the pulverized product with a plant cell wall degrading enzyme containing at least one of cellulase, hemicellulase, or pectinase;
(C) drying the treated pulverized product; a solid plant composition having a macrophage activity enhancing effect, characterized in that prepared by a method comprising.
(A) 4-6 parts by weight of Angelica Angelica, 2.5-3.5 parts by weight of Chungoong, 1.5-2.5 parts by weight of Cornus milk, Galgeun, Baekchul, respectively, Licorice, Baek Peony, Bokryeong, Sanyak, Cinnamon, respectively 0.8-1.2 parts by weight of mixed crushed product Preparing;
(B) treating with a plant cell wall degrading enzyme containing at least one of the cellulase, hemicellulase, or pectinase;
(C) deactivating the enzyme;
(D) removing the residue of the treated pulverized product to separate the extract; a liquid vegetable herbal composition having an effect of enhancing macrophage activity, characterized in that it is prepared by a method comprising.
(A) 4 to 6 parts by weight of Angelica, 2.5 to 3.5 parts by weight of Chungoong, 1.5 to 2.5 parts by weight of each of Hasuoh, Galgeun, Sasam, Sanyak, Licorice, Bokryeong, Rowan, and Health respectively 0.8 to 1.2 parts by weight step;
(B) treating the pulverized product with a plant cell wall degrading enzyme containing at least one of cellulase, hemicellulase, or pectinase;
(C) drying the treated pulverized product; a solid plant composition having a macrophage activity enhancing effect, characterized in that prepared by a method comprising.
(A) 4 to 6 parts by weight of Angelica, 2.5 to 3.5 parts by weight of Chungoong, 1.5 to 2.5 parts by weight of each of Hasuoh, Galgeun, Sasam, Sanyak, Licorice, Bokryeong, Rowan, and Health respectively 0.8 to 1.2 parts by weight step;
(B) treating the pulverized product with a plant cell wall degrading enzyme containing at least one of cellulase, hemicellulase, or pectinase;
(C) deactivating the enzyme;
(D) removing the residue of the treated pulverized product to separate the extract; a liquid vegetable herbal composition having an effect of enhancing macrophage activity, characterized in that it is prepared by a method comprising.

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