KR20100002796A - A method of separation to sulforaphene from baemuchae and the use of sulforaphene - Google Patents

Abstract

PURPOSE: A method for isolating sulforaphene from x Brassica raphanus is provided to suppress generation and growth of x Brassica raphanus, prevent and treat diseases including gastritis, gastric ulcer, or gastric cancer. CONSTITUTION: A method for isolating sulforaphene of chemical formula 1 from x Brassica raphanus comprises: a step of adding x Brassica raphanus with distilled water to hydrolyze at 35-40°C for one to five hours; a step of centrifuging hydrolysate to collect supernatant and extracting through liquid-liquid extraction; a step of removing moixture and compressing; and a step of fractioning active ingredient. A pharmaceutical formulation for preventing and treating Helicobacter infection-relating diseases contains the sulforaphene of chemical formula 1. The pharmaceutical formulation is used in the form of tablet or capsule.

Description

A method of separation to sulforaphene from Baemuchae and the use of sulforaphene}

The present invention relates to a process for the preparation of sulfolafens isolated from broth and its use.

The gastric mucosal layer that protects the stomach is susceptible to damage from a variety of factors. Bacteria [Suzuki, such as a typical attack factor NSAIDs (NSAID), such as acid, alcohol, aspirin, Helicobacter pylori (Helicobacter pylori). M. and S. Miura, Nippon Rinsho, 15 (12), 3154-3158, 1993], stress-induced microcirculation disorders and hypotension of gastric mucosa [Tadaoki Mizuno, Yokohama Med. Bull. , 38 (3,4), 87-97, 1987]. Due to these factors, when the mucosal layer is damaged, inflammation is accompanied by erosion, redness, bleeding, and edema. In addition, the duodenum, which is directly connected to the stomach, may cause inflammation and duodenal ulcer by similar factors.

On the other hand, in order to treat inflammation and ulcers of the stomach and duodenum caused by these attack factors, a drug having the effects of inhibiting gastric acid secretion, inhibiting proliferation of Helicobacter pylori, promoting mucus secretion, promoting epithelial cell regeneration, and anti-inflammatory . Currently, the most representative gastritis and gastric ulcer drugs are H2 antagonists and proton pump inhibitors, which have the effect of inhibiting gastric acid secretion, and have proven excellent effects in clinical practice. [J Int Med Res 17 (suppl) 9A, 1989 Meth Find Exp Clin Pharmacol 11 (suppl 1) 87, 1989; N Eng J Med 323: 1672, 1990. However, these gastric acid secretion inhibitors are problematic for high recurrence rate after the end of the administration (Drug Intell Clin Pharm 21: 493, 1987, Gut 30: 449, 1989 Yale J Biol Med 65: 649, 1992).

Mucosal protective agents, such as promoting normal regeneration of mucosal tissues to resist the re-attack of gastritis-inducing factors to reduce the high gastritis recurrence rate, a disadvantage of fast-acting gastric acid secretion inhibitors, are an important part of the treatment of gastritis . Currently, drugs such as rebamipide and sofalcone have been developed as gastric mucosa protective agents, but generally have shortcomings, high doses, and need to be taken for a relatively long time.

Baemuchae used in the present invention is a new vegetable grown by interlacing cabbage and radish, research on this is currently being actively made.

On the other hand, isothiocyanates of cruciferous crops are already known as sulforaphane (sulforaphane), which has been previously separated from broccoli, and it is known from many documents that the sulforaphane has an anticancer effect in Helicobacter bacteria and other body organs. It has been reported.

However, there is no report on the study of separating the sulfolafen (Formula 1) structurally different from the sulforaphane (Formula 2) from the Baemuchae as in the present invention.

[Formula 1]

[Formula 2]

Therefore, the present inventors succeeded in separating sulforaphene, which is an isothiocyanate material different from sulforaphane, from Baewoochae, and the content of sulforaphene does not decrease as the growth is extended, unlike sulforaphane. The present invention was completed by confirming. In addition, the sulfolafen has been shown to inhibit the production and growth of Helicobacter bacteria.

Accordingly, an object of the present invention is to provide a method for separating sulforaphene from baemulchae.

Another object of the present invention is to provide a composition for preventing and treating diseases related to Helicobacter infection, which contains sulfolafen as an active ingredient.

Another object of the present invention is to provide a health food containing sulfolafen as an active ingredient.

In order to solve the above problems, the present invention

1) hydrolyzing Baekchae with distilled water at 35-40 ° C. for 1-5 hours

2) centrifuging the hydrolyzate and extracting the supernatant by liquid-liquid extraction;

3) removing water of the extract and then concentrating under reduced pressure

4) fractionating the active substance by solid phase extraction for the purpose of single identification of the extract

Characterized in that the method for separating the sulforaphene from the baewoochae made, including.

In another aspect, the present invention is characterized by the composition for the prevention and treatment of diseases related to Helicobacter infection containing sulfolafen as an active ingredient.

In another aspect, the present invention is characterized by a health food containing sulfolafen as an active ingredient.

The present invention will be described in more detail as follows.

The present invention is to isolate the sulforaphene (sulforaphene) from Baemuchae , reveals the effect of inhibiting the production and growth of Helicobacter pyroli bacteria involved in causing the gastritis, gastric ulcer and gastric cancer The present invention relates to a method for isolating sulfolafenes from broth and to the use of sulfolafenes.

The method for extracting sulfolafen from the Baemuchae according to the present invention will be described in detail below.

First, after homogenizing the Baekmuchae with distilled water, it is hydrolyzed for 1 to 5 hours at 35 ~ 40 ℃. The baewoochae contains all of the roots, maturation or shoots of the leaves, but try to extract by performing the sampling by each part. In this case, the chemical structure of isothiocyanates, which is known to have an anticancer effect under neutral conditions, is hydrolyzed under neutral conditions, while hydrolysis under neutral conditions is the most hydrolyzed glucosinolates. It is preferable to produce a lot, and if the hydrolysis temperature and time is out of the above range, the yield of the material to be separated is insufficient, and the anti-cancer property is not known or does not have an effect during prolonged hydrolysis. And isothiocyanate, which is decomposed into a compound and has a known anticancer effect, is rapidly reduced.

The hydrolyzate is centrifuged at 6000-10000 rpm for 20-30 minutes to obtain a supernatant, followed by liquid-liquid extraction with a solvent having a very low affinity for water. As the extraction solvent of the target material, dichloromethane or chloroform is preferable.

Water of the solvent extract is removed by adding sodium sulfate and then concentrated under reduced pressure at 30 ~ 40 ℃ using a nitrogen pressure reducer.

In addition, in order to fractionate sulfolafen and such nonpolar substances for easy analysis, the extract eluted with methanol is concentrated under reduced pressure at 40 to 50 ° C. through a solid phase extraction method. The hydrolyzate was extracted with an organic solvent and then collected through a solid phase extraction method to obtain a very pale yellow green or pale yellow substance, which was identified as sulfolafen.

It was confirmed that the isolated sulfolafen inhibits the production of Helicobacter bacteria, it can be prepared a drug for the prevention and treatment of diseases related to Helicobacter infection containing it as an active ingredient. The disease associated with Helicobacter infection may include gastritis, gastric ulcer, gastric cancer, and the like.

The medicament is prepared by a conventional manufacturing method and formulated into tablets, capsules, etc. Among them, a combination of lactose, microcrystalline cellulose, magnesium stearate, and the like, which are used as bases in the manufacture of tablets, and sulfolafen 2 to 10: 1 When used in proportions of the present invention, a tablet having an activity for preventing and treating diseases related to Helicobacter infection can be prepared.

In the manufacture of such a drug, it can be used as an active substance itself, but can also be prepared as a powder, granules, capsules, etc. by mixing with a pharmaceutically acceptable carrier, forming agent, diluent, etc. Do. In addition, the sulfolafen according to the present invention can be changed according to body absorption, weight, age of the patient, sex, health status diet, administration time, administration method, excretion rate, severity of the disease and the like. In general, it is preferable to administer about 0.05 to 1 mg of sulfolafen per 1 kg of body weight. In addition, the formulated unit dosage form may be administered several times at regular intervals or by using a specialized dosage method according to the judgment of an expert who monitors or observes the administration of the drug as needed and the needs of the individual.

In addition, the present invention includes a health food containing sulfolafen as an active ingredient.

Health foods are foods made by adding sulfolafen to food materials such as beverages, teas, spices, gums and confectionery, or by encapsulating, powdering, and suspensions. Meaning, unlike the general medicine has the advantage that there is no side effect that can occur when taking a long-term use of the drug as a food raw material.

Since sulfolafen according to the present invention has an excellent effect of inhibiting the production of Helicobacter bacteria, it is very useful for preventing and treating related diseases such as gastritis, gastric ulcer or gastric cancer.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example 1 Separation and Structure Verification of Sulforaphene

30 g of distilled water was added to 5 g of Baekchae, homogenized for 1 minute, hydrolyzed at 37 ° C for 3 hours, centrifuged at 8000 rpm for 20 minutes, and extracted with dichloromethane three times. It removed and the extract was obtained. This was concentrated under reduced pressure at 35 ° C. using a nitrogen depressurizer to 2 ml, followed by solid phase extraction using a sep-pak silica cartridge and eluted with 3 ml of methanol. 0.3 ml was used for analysis by concentration under reduced pressure at 45 ℃ using a nitrogen pressure reducer.

The extract was quantitatively and qualitatively analyzed using HPLC and GC-Mass equipped with an evarporative light scattering detector (ELSD). The conditions of the equipment are as follows.

First, HPLC was used for Gilson's 305 series, and the analytical column was equipped with Nucleosil 100-5C18 (150 × 4.6 mm) from Macherey-nagel. The isocratic mobile phase solvent method was used and the composition of the solvent was determined by weight ratio of water: acetonitrile: tert-butyl alcohol = 8: 1: 1 with 5 mM ammonium acetate. . The flow rate of the HPLC pump was 1 mL / min and the temperature of the column oven was maintained at 40 ° C. The condition of the detector was that the gas flow rate was 2 mL / min, and the temperature of the detector was maintained at 50 ° C., so that the gain was adjusted to 1.

GC-Mass assay conditions for qualitative analysis were adjusted using an HP5970 detector mounted on the HP5890. The column was equipped with a silica capillary column (DB-5, 0.25 × 30 mm) and the oven temperature of GC was raised by 10 ° C. per minute from 50 ° C. to 250 ° C. The temperature of the injector was maintained at 250 ° C. and the mobile phase flowed helium gas at 0.8 mL / min. E.I. ranged from 70 eV and mass scan ranged from 40 to 500 m / z.

In particular, 5 g of samples collected only from cotyledons and hypocotyls from 7 to 11 days after sowing were cultivated to obtain 650 ㎍ (yield 13%) of light green or pale yellow substance containing sulfolafen. . As a result of structural analysis by HPLC and GC-MS, the material was analyzed by sulfolafen represented by the following Chemical Formula 1 [FIG. 1 and FIG. 2].

[Formula 1]

Comparative Example 1: Separation of Sulforaphane

From broccoli Vertelli et al [Davide V., Maria P., Daniela B. and Agar M. 1998. Separation by solid phase extraction and quantification by reverse phase hplc of sulforaphane in broccoli., Food Chem. 63: 417-421.] Was isolated from the formula (2) 120 ㎍ (yield 12%) per gram of sulforaphane in vivo.

[Formula 2]

Test Example 1: Comparison of Contents

The purpose of this study was to determine the growth days with the highest yields by comparing the sulforaphane and sulforaphene contents of each plant growth stage.

First, Baemuchae and broccoli seeds were grown in a container containing wet tissue, respectively, and watered twice a day at 25 ° C. and 15 hours of light conditions. Sampling was carried out by 5, 7, 9, 11, 14 days from the date of tooth growth, and the growth days of five shoots were separated. The material was extracted from the cotyledons and hypocotyls, which are edible parts except roots.

The sulfolafen obtained by this extraction was an average of 147 µg.

The following Table 1 and Figures 3 to 6 show the change in the content of isothiocyanate material during the growth of broccoli, buds, young plants, and mature bodies of each of the bark.

TABLE 1

As shown in Figures 3 to 6 and Table 1, the sulforaphane isolated from broccoli was confirmed that the content is reduced during the growth of the plant, the sulfolafens isolated from the baemuchae did not decrease during the growth of the plant Did.

This is in contrast to the tendency that the sulfolafens produced by plants in order to overcome external disturbances such as pests and herbivore attacks according to the growth stages of plants grow and the sulforaphane content decreases like broccoli. Maintaining the content and in terms of the intake of antioxidants means that the extraction yield and efficacy is very good material.

Test Example 2: Confirmation of the inhibitory effect of the production of Helicobacter bacteria

In order to know the efficacy of sulfolafens that inhibit or inhibit the growth of Helicobacter pylori, experiments were conducted in which antibiotics amoxicillin and metronidazole, which inhibit Helicobacter pylori growth, were compared with sulfofen and known sulforaphane. Table 2 compares the antibiotic activity of sulfolafen and other treatments in the inhibition of growth of Helicobacter pylori specific strains. Table 2 demonstrates that the pylori strain-specific sulfolafens have efficacy equivalent to or greater than existing antibiotics.

TABLE 2

Test Example  3

Helicobacter pylori strains were grown in broth liquid medium at 37 ° C. in mass, and the bacteria were collected and resuspended in PBS. Rats were treated with food (food) and water intake for 12 hours and then treated with pylori 10 ⁹ CFU / ml by administering 40 μl of Helicobacter pylori dissolved in PBS. Animals were anesthetized with metapan and sacrificed to directly culture the infection of the gastric membrane, followed by urease test [Y. Tokunaga et al, J Gastroenterol hepatol (2000) 12: 617-621] confirmed the infection of Helicobacter pylori. After confirmation, the extracted sulfolafen and other antibiotics were orally administered 20 mg twice a day. The following table 3 shows that rats infected with sulfofen penetrate the actual Helicobacter pylori improvement of gastric mucosa infection by urease test, and sulfofen is more effective than antibiotics in animals infected with Helicobacter pylori. Demonstrates or similar.

TABLE 3

Test Example 4: Oral Administration of Rats Acute Toxicity

Acute toxicity test was performed using 6-week-old SPF SD rats as follows.

Sulforaphene was dissolved in two animals per group and administered orally at a 400 mg / kg dose. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed. Hematological and hematological examinations were performed. Necropsy was performed to observe abdominal and thoracic organ abnormalities. As a result, no significant clinical symptoms or dead animals were noted in all animals treated with the test substance, and no toxic changes were observed in weight changes, blood tests, blood biochemical tests, and autopsy findings. Therefore, sulfolafen according to the present invention did not show a toxic change to 400 mg / kg in all rats and the minimum lethal dose (LD ₅₀ ) of oral administration was determined to be a safe substance more than 400 mg / kg.

Formulation Example 1 Preparation of Tablet

Tablets for oral administration using the sulfolafen of the present invention in the following composition were prepared using a wet granulation method and a dry granulation method.

[Furtherance]

20 mg of sulfolafen, 10 mg of hard silicic anhydride, 2 mg of magnesium stearate, 50 mg of microcrystalline cellulose, 25 mg of sodium starch glycolate, 113 mg of corn starch, proper amount of ethanol anhydride.

Formulation Example 2: Preparation of Transdermal

The transdermal drug was prepared using the sulfolafen of the present invention in the following manner.

20 mg sulfolafen, 1.3 g sodium polyacrylate, 3.6 g glycerin, 0.04 g aluminum hydroxide, 0.2 g methyl paraben, 14 g water.

Formulation Example 3: Beverage Preparation

After dissolving 500 mg of sulfolafen in an appropriate amount of water, vitamin C as an auxiliary component, citric acid, sodium citrate, and oligosaccharides as an auxiliary component were added, and an appropriate amount of sodium benzoate was added as a preservative, followed by adding water to make 100 ml. To prepare a beverage composition. At this time, taurine, myo-inositol, folic acid, pantothenic acid, etc. may be added alone or together.

FIG. 1 compares the separation pattern of sulfolafen and standard material of baewoochae separated by high performance liquid chromatography [A: standard material; B: sulfolafen isolated from pear seed].

Figure 2 compares the results of mass spectrometry of sulforaphene and the standard separated from the mussels [A: standard; B: sulfolafen isolated from pear seed].

Figure 3 shows the change in the content of sulforaphane during the growth of broccoli sprouts.

Figure 4 shows the change in the content of sulfolafen during the growth of baebuchae sprout.

Figure 5 shows the change in the content of sulforaphane during the growth of broccoli young plants.

Figure 6 shows the change in the content of sulfolafen during the growth of young plants of Baekchae.

Claims (3)

1) Step of hydrolyzing Ba Brassica ( x Brassica raphanus ) at 35 ~ 40 ℃ for 1 ~ 5 hours by adding distilled water 2) centrifuging the hydrolyzate and extracting the supernatant by liquid-liquid extraction; 3) removing water of the extract and then concentrating under reduced pressure 4) fractionating the active substance by solid phase extraction for the purpose of single identification of the extract Method for separating the sulforaphene (sulforaphene) represented by the following formula (1) from Baemuchae, characterized in that consisting of. [Formula 1]

According to claim 1, wherein the sulforaphene (sulforaphene) is characterized in that it is effective for diseases related to Helicobacter infection. The method of claim 2, wherein the disease associated with H. pylori infection is gastritis, gastric ulcer or gastric cancer.

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