KR101495205B1 - A method of reducing of toxic substance in Korean aconite root - Google Patents

Abstract

The present invention relates to a method for reducing toxic substances of a chrysanthemum, comprising the steps of (S1) harvesting, drying and crushing chrysanthemum; (S2) steam-treating the ground chrysotile; (S3) cooling the vapor-treated chios; (S4) The culture broth obtained by mixing the chilled cherries at a weight ratio of Bacillus subtilis strain and Bacillus natto Sawamura at a weight ratio of 1: 2 to 2: 1 was added to the total volume of the choco (1) to 10% (w / v) to ferment the chococci. According to the method for reducing the choco toxin, it is possible to prevent the toxic substance aconitine Can be significantly reduced. Thus, it is anticipated that a chrysomophile having a beneficial effect of chrysanthemum and a greatly reduced toxic substance content can be obtained and usefully used in the art.

Description

[0001] The present invention relates to a method for reducing toxic substances in a microorganism,

More particularly, the present invention relates to a method for reducing the content of a toxic substance in a microorganism while maintaining an effective ingredient of a microorganism using a fermentation process, and a method for reducing the toxicity ≪ / RTI >

The Aconiti ciliare tuber is composed of the root and tail roots of the dicotyledonous plant, the buttercups family, the perennial plant belonging to the genus Chrysanthemum, (JE et al., J Korean Academic Journal, 6: 154 ~ 161, 1995), and as a medicinal product that mainly helps the body to warm up, , It is recorded that it is used for warming or heat dissipation. It has also been reported to be effective in the treatment of stroke, loss of sound due to cerebrovascular disease, and the like.

However, neonates contain toxic substances such as aconitine, hypaconitine, mesaconitine and deoxyaconitine, which can lead to side effects if used improperly Isao K. et al., Chem. Pharm. Bull. 30 (2): 758-761, 1982) and can exhibit toxic effects in very small amounts. Nevertheless, there is no specific regulation on the preparation of such a toxic substance, and there are many instances of acute poisoning due to the abuse of the toxic substance, since only the general effect of the toxic substance is known to the public without being overlooked (Choi DI 21: 309 ~ 311, 1992] and [Shi H. et al., J. Biol. < / RTI > , Zhougguo Zhong Yao Za Zhi 15: 174-177, 1990).

Neonatal symptoms such as systemic and intestinal sensory abnormalities, digestive system symptoms such as nausea and vomiting, and circulatory symptoms of various cardiac arrhythmia and hypotension are known to be characteristic clinical features of addiction due to choroid. In the present study, patients with ventricular tachycardia, ventricular tachycardia, ventricular tachycardia, and connective ischemic heart rhythm, were excluded. In most cases, the ventricular arrhythmia is a temporary, spontaneous ventricular arrhythmia but rarely causes ventricular fibrillation (Tai YT et al. , Cardiotoxicity after accidental herb induced aconite poisoning. Lancet 340: 1254-1256, 1992).

On the other hand, the composition of alkaloids such as aconitine is determined by the kind of aconitum used, the time of cultivation, the numerical method, etc. (Hsu HY et al., California: Oriental Healing Arts Institute, 368-371, (Pharmacopocia Committee. Beijing: People's Health Publishing House and Chemical Technology Press 26-28, 211-213, 1990).

Although the stable dose of aconitine is initially known to be 8 to 12 g, it can not be used in large quantities due to residual toxic substances, and the use amount of aconitine has been reduced to 5 to 3.0 g (Pepper K. et al ., Pfluggers Archiv 296: 328-336, 1976).

To remove these toxic substances, various methods have been reported. Among them, the most commonly used method is tapping. However, in the case of this method, there is a problem that a large amount of effective substances such as alkaloid are reduced due to water washing and heat treatment (Kim HK et al., Kor J. Pharmacogn. 33 (4): 296-300, 2002).

Therefore, the present inventors have been continuing research to develop a method for safely and safely using chococci by reducing the content of toxic substances, especially aconitine, in chococci while maintaining the useful effects of chococci. It was confirmed that the Bacillus subtilis strain suitable for fermentation of Fucoidan and the Bacillus subtilis strain were mixed at a certain ratio and used for the fermentation of the fermentation broth to have a considerable effect on the reduction of the concentration of aconitine, It was completed.

Accordingly, an object of the present invention is to provide a method for reducing the content of a toxic substance in a microorganism while maintaining the active ingredient of the microorganism.

Another object of the present invention is to provide a reduced amount of toxic substances obtained by the method.

In order to solve the above-mentioned technical problems, the present invention provides a method for reducing toxic substances of chrysanthemum comprising the following steps:

(S1) harvesting, drying and crushing the chrysanthemum;

(S2) steam-treating the ground chrysotile;

(S3) cooling the vapor-treated chios; And

(S4) The culture broth obtained by mixing the chilled cherries at a weight ratio of Bacillus subtilis strain and Bacillus natto Sawamura at a weight ratio of 1: 2 to 2: 1, 10% (w / v).

In order to solve the above technical problems, the present invention provides a reduced amount of toxic substances obtained according to the method.

As described above, according to the present invention, the content of aconitine, which is a toxic substance, can be significantly reduced without decreasing the active ingredient of chrysophyte, thereby obtaining a chrysomalant having a useful effect of chrysanthemum, So that it is expected to be useful in the art.

BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate exemplary embodiments of the invention and, together with the description of the invention, It should not be construed as limited.
Figure 1 schematically illustrates a method of reducing toxic substances in the ophiolumn.
FIG. 2 is a graph showing cytotoxicity results of HEK 293 of fresh ocher. FIG.
3 is a graph showing the cytotoxicity of HEK 293 of the present invention.
4 is a graph showing the cytotoxicity results of HepG2 against Vaccine oocyte.
FIG. 5 is a graph showing the cytotoxicity of HepG2 of the present invention product. FIG.
Fig. 6 shows the result of quantitative analysis of the high-performance liquid chromatography (HPLC) of the present invention.

A method of reducing toxic substances in a chrysoin according to the present invention is characterized in that it comprises the following steps:

(S1) harvesting, drying and crushing the chrysanthemum;

(S2) steam-treating the ground chrysotile;

(S3) cooling the vapor-treated chios; And

(S4) The culture broth obtained by mixing the chilled cherries at a weight ratio of Bacillus subtilis strain and Bacillus natto Sawamura at a weight ratio of 1: 2 to 2: 1, 10% (w / v).

The present invention relates to a method for fermenting chococci, which comprises culturing a mixture of a Bacillus subtilis strain and a Bacillus subtilis strain suitable for fermentation of chococci, It has a significant effect on the reduction of the content of cornitine.

In the method for reducing toxic substances in the chrysanthemum of the present invention, the steam oven steam treatment in step S2 may be performed by a conventional method known in the art, and such method is not particularly limited. For example, in the present invention, it is preferable to carry out the steam steam treatment for 30 to 60 minutes at a temperature of 80 to 90 DEG C, preferably 85 DEG C, to the ground chrysotile. By using the pulverized pulverizer used in the present invention, it is possible to maximize the steam steam effect, and through the steam treatment, it can be more effectively applied to the fermentation step for removing toxicity afterwards.

In the step (S3) of the present invention, it is preferable to cool the chrysanthemum at a temperature of 30 to 40 DEG C as a step of cooling the steam-treated chrysanthemum.

In step S4 of the present invention, the chilled oocyte is inoculated with a suitable fermentation strain such as a Bacillus subtilis strain and a Bacillus natto Sawamura strain as a Bacillus strain, At this time, the Bacillus subtilis strain and the Bacillus subtilis strain are preferably mixed at a weight ratio of 1: 2 to 2: 1, preferably 1: 1. The culture broth obtained by culturing the mixed strain may be added in an amount of 1 to 10% (w / v) based on the total volume of the broth to ferment the broth.

The fermentation using the strain is characterized in that the fermentation is carried out at a temperature of 30 to 40 ° C, preferably 35 to 37 ° C, for 4 to 9 days, preferably 2 to 5 days at a pH of 6 to 7.

Chloroforms obtained by fermentation of these strains are greatly beneficial when consumed by humans, because the content of toxic substances is greatly reduced and is safe and contains various other functional ingredients.

As described above, according to the present invention, the content of aconitine, which is a toxic substance, can be significantly reduced without decreasing the active ingredient of chrysophyte, thereby obtaining a chrysomalant having a useful effect of chrysanthemum, So that it is expected to be useful in the art.

In addition, the present invention provides a method of reducing the toxicity of a toxin by reducing the toxicity of the toxin.

The supernatant recovered after recovering the supernatant from the chios product obtained by the present invention by centrifugation, membrane separation apparatus or filtration method is used as a liquid agent itself, or is concentrated and lyophilized Granules and the like may be prepared and then formulated in a conventional formulation, for example, a liquid formulation, a hydrating powder, an emulsifiable concentrate, a tablet, a suspension, a powder and a water-dispersible granule according to a conventional method.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

≪ Example 1 >

The chrysotile was harvested, dried, ground and steam steamed at 85 ° C for 40 minutes.

Then, the steam-treated chios were cooled at a temperature of 35 ° C., and then Bacillus subtilis strain and Bacillus natto Sawamura strain were obtained, mixed at a weight ratio of 1: 1, and cultured. (W / v) based on the total volume of the chrysanthemum, and then fermented at a temperature of 35 ° C for 3 days to prepare a chorioallantoic material.

≪ Comparative Example 1 &

The cultured Bacillus subtilis strain was inoculated with 5% (w / v) of the total volume of the chococci in the harvested chrysanthemum and then fermented at a temperature of 35 ° C for 3 days to obtain a chombiotic product.

≪ Comparative Example 2 &

The chrysotile was harvested, dried, ground and steam steamed at 85 ° C for 40 minutes.

Then, the steam-treated chios were cooled at a temperature of 35 ° C., and the culture medium of Bacillus subtilis strain was inoculated in an amount of 5% (w / v) based on the total volume of the chococci. Then, fermentation was carried out for 3 days at a temperature of 35 ° C. .

≪ Comparative Example 3 &

Bacillus subtilis strains and Bacillus subtilis strains were harvested and mixed at a weight ratio of 1: 1. The culture broth was inoculated in an amount of 5% (w / v) to the total volume of chococci, Lt; / RTI > for 3 days.

≪ Comparative Example 4 &

The cultured Bacillus subtilis strain was inoculated with 5% (w / v) of the total volume of chrysanthemum and then fermented at 35 ℃ for 3 days.

<Test Example 1> Cytotoxicity test

(1) Measurement of proliferation effect of HEK293 and HepG2

HEK293, a human kidney cell line, and HepG2, a human liver cell line, were purchased from ATCC (American Type Culture Collection, USA) in order to perform a cytotoxicity test on the chiosetrophan prepared in Example 1 above. (Penicillin-streptomycin) (Gibco BRL., USA) with 10% fetal bovine serum (FBS, Gibco BRL., Grand Island, NY, USA) as an anti- (Dulbecco's modified Eagle Medium, DMEM, Grand Island, NY, USA) containing 5% CO ₂ at 37 ° C.

In order to investigate the effect of the present invention on the proliferation of macrophage cells, HEK293 cells were dispensed in a 96-well plate at 4 × 10 ³ cells / well and the herbal extracts were dissolved in DMSO and added at different concentrations. After incubation for 1 day, a tetrazolium salt CCK-8 kit (Cell Counting Kit-8, Dojindo Laboratories, Tokyo, Japan) was added and incubated for 4 hours. nm absorbance was measured. As a control group, we used a raw oenon which was not fermented on the chrysanthemum.

As shown in Equation (1), the absorbance ratio of the test group to the absorbance of the control well was calculated as%, and the average value of all wells was used.

The results are shown in FIG. 2 to FIG. 5, wherein FIG. 2 is a graph showing the cytotoxicity results of HEK 293 with Vaccine OO, and FIG. 3 is a graph showing cytotoxicity results of HEK 293 of the present invention . FIG. 4 is a graph showing cytotoxicity results of HepG2 against Vaccine oat, and FIG. 5 is a graph showing cytotoxicity results of HepG2 of the present invention.

As shown in FIGS. 2 and 3, no toxicity was observed in both the fresh and the fresh products, but it was found that the toxicity was significantly lowered at 1000 ㎍ / ㎖ than the fresh product.

In addition, as shown in Figs. 4 and 5, it can be seen that no toxicity was observed in both the raw och and the chrysoprotein.

(2) High Performance Liquid Chromatography (HPLC)

Quantitative analysis of aconitine, a toxic indicator substance of chrysanthemum, was carried out. 25 ml of methanol was added to 1 g of the sample obtained in Example 1 and sonicated for 60 minutes. This procedure was repeated twice, followed by concentration and dissolution in 6 ml of methanol. The results are shown in Fig.

As shown in FIG. 6, the content of aconitine, which is a toxic indicator substance of the chrysotrophin, is reduced by about 120 times as compared with that of fresh goat.

Following the above test, in order to compare the toxicity reduction performance of the present invention obtained in Example 1 with the chios products prepared in Comparative Examples 1 to 4, the degradation rate of the toxic substance, aconitine, was measured using HPLC . The analysis conditions of the HPLC system are as follows. The column was eluted with acetonitrile (CH ₃ CN: pH 3.0, 50 mM phosphoric acid, 6: 4, v / v) in a Capcell Pak C18 MG (460 x 250 nm ID, ) At a flow rate of 1.0 ml / min and a detector UV3001 UV detector at 235 nm. The results are shown in Table 1 below.

Aconitine Content (ppm) Decomposition rate of aconitine (%) Example 1 12 88 Comparative Example 1 67 33 Comparative Example 2 54 46 Comparative Example 3 73 27 Comparative Example 4 50 50

As shown in Table 1, the content of toxic substances in the choline products prepared according to the method of the present invention was 88%, which indicates that the decomposition ratio of aconitine was 88%. On the other hand, it was found that the degradation rate of aconitine was 50% at maximum in the case of the choline products prepared in Comparative Examples 1 to 4.

&Lt; Test Example 2 >

In order to measure the change of the useful component contained in the choroatocyst prepared in Example 1, the relative area ratio on the high performance liquid chromatography system was measured.

The relative area ratios on the high performance liquid chromatography system were determined as relative ratios assuming that the relative area ratios of the fresh rats (control rats) in the samples were 100%, and the results are shown in Table 2 below.

Relative area ratio (%) Control group 100 Example 1 96.4 Comparative Example 1 54 Comparative Example 2 66 Comparative Example 3 72 Comparative Example 4 71

As shown in Table 2, the components appearing on the chromatograph are mainly alkaloid components. As shown in Table 2, in the case of the inventive choline oxide prepared in Example 1, 96.4% I could. On the other hand, in the case of Comparative Examples 1 to 4, it was greatly reduced to 54 to 71%, indicating that not only toxic substances but also alkaloids and other useful substances were significantly reduced in content.

As described above, according to the present invention, the content of aconitine, which is a toxic substance, can be significantly reduced without decreasing the active ingredient of chrysophyte, thereby obtaining a chrysomalant having a useful effect of chrysanthemum, So that it is expected to be useful in the art.

Claims (6)

CLAIMS 1. A method for reducing toxic substances in chios characterized by comprising the steps of:
(S1) harvesting, drying and crushing the chrysanthemum;
(S2) steaming the pulverized chrysos at a temperature of 80 to 90 DEG C for 30 to 60 minutes;
(S3) cooling the steam-treated chios at a temperature of 30 to 40 DEG C; And
(S4) The cultured broth obtained by mixing the chilled cherries at a weight ratio of Bacillus subtilis strain and Bacillus natto Sawamura at a weight ratio of 1: 2 to 2: 1, 10% (w / v).
delete delete The method according to claim 1,
Wherein the Bacillus subtilis strain and the Bacillus natto Sawamura strain are mixed at a weight ratio of 1: 1 in the step (S4). The method according to claim 1,
Wherein the culturing is carried out at a temperature of 30 to 40 DEG C for 4 to 9 days at a pH of 6 to 7 in the step (S4). 6. A toxic component-reduced chios product produced according to any one of claims 1 to 5.

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