KR20030032483A - Extracts of cacao bean and husk with antiinflammatory effects - Google Patents

Abstract

PURPOSE: Provided is a solvent fraction of cacao beans and cacao hulls exhibiting strong inhibition activity against inflammation caused by bacterial endotoxin and various skin inflammation-inducing materia. Therefore, pharmaceuticals and food additives containing the solvent fraction are useful for the prevention and treatment of inflammation. CONSTITUTION: An antiinflammatory agent and a food additive having antiinflammatory activity characteristically contains cacao beans and cacao hulls as an effective ingredient. For an example, 1 part by weight of a fraction of cacao beans is extracted in 6 parts by weight of 50% acetone at 60deg.C for 5hr under refluxing and centrifuged at 8,000rpm at 4deg.C for 30min., and the residue is extracted one time by the same method. The extracts are filtered with Whatman No. 41 paper and subjected to absorption column chromatography with a 20% aqueous ethanol solution.

Description

Extracts of cacao bean and husk with antiinflammatory effects

The present invention relates to cacao beans or soybean skin fractions having an inhibitory effect on inflammation, and more particularly cacao beans having excellent inhibitory effects against inflammation caused by endotoxins and various dermatitis causing substances secreted by bacteria. Or soybean skin fractions.

Cacao, the raw material of chocolate and cocoa, has the scientific name Theobroma cacao L, meaning divine food. The origin of cacao is estimated to be from Central America to the northern part of South America, and has been cultivated for a long time in BC and has been treated with corn as a very valuable food. It is settled as a typical sign food. Cacao beans, which are used as raw materials for chocolate, vary depending on the country of origin, but are not only rich in dietary fiber, but also contain significant amounts of polyphenols.

On the other hand, cacao beans are shelled out during the production of chocolate so that nibs are used as raw materials for chocolate or cocoa, and shells (about 15% by weight of the total cacao beans) that are produced as by-products are almost discarded. In other words, the world produces about 2.5 million tons of cacao beans, about 400,000 tons of cacao beans are being discarded, and according to the 1990 Customs Statistics Korea, about 600 tons of cacao beans It is known that it is being discharged as waste. However, like cacao beans, such cacao beans are not only rich in dietary fiber, but also contain a significant amount of polyphenols.

Thus, methods have been devised to exploit the functionality of polyphenols in cacao beans. Polyphenols of various structures have been shown to inhibit the activity of leukemia, prostate cancer, colon cancer, kidney cancer, lung cancer, breast cancer and also inhibit topoisomerase II. It has been claimed to have a patent for the structure and use of these polyphenols (Korean Patent Application No. 1998-707952).

However, the inventors of the present invention are inexpensive since they use soybean peel polyphenol extracts obtained as by-products during chocolate production, long before the patents on the structure and the physiological activity of the polyphenols of cacao beans, which are claimed above, are filed. And polyphenols are mainly composed of polymers such as procyanidin A and procyanidin C-1, and thus have high enzymatic inhibitory activity and thus inhibit the activity of glucosyltransferase, a decay enzyme. In fact, the cacao bean husk extract was prepared, and chewing gum containing the same was prepared [Korean Patent No. 45179, 1991; U.S. Patent 4908212, 1990. Recently, patents have been developed by increasing the content of polyphenols to prepare cacao bean husk fractions which further enhance the inhibitory activity of glucosyltransferases (US Pat. No. 6159451, 2000).

As mentioned above, cacao beans and bean husks contain various physiological activities such as dietary fiber, polyphenols of various structures, and phenolic acid, methylxanthines such as deobromine and caffeine. As a result, the specific structures of the polyphenols do not actually exhibit physiological activity, and the separation and commercial use of only these specific structures may be too expensive to use. Therefore, it would be practical to use fractions that greatly enhanced several physiological activities by a simple separation process as already registered in the patent (US Pat. No. 6,159,451, 2000).

Inflammation is a bacterium-derived abscess that actively infiltrate tissues with purulent fungi that white blood cells devour, resulting in the death of white blood cells, tissues damaged by bacteria, and cell debris, which accumulate in the tissues. The pathological state of the place, and the anti-inflammatory effect is to inhibit the growth of invading bacteria by antibacterial agents, or to activate the macrophage (macrophage) that phages the foreign matter accumulated in the abscesses to enhance the ability to digest and excrete them Abscess is the process of treatment. In general, an inflammatory response is a defense process of a living body that attempts to repair and repair the damaged area when an invasion that causes some organic change in a cell or tissue of the living body is applied. Therefore, such a series of reactions include local blood vessels, various tissue cells of body fluids, immune-involved cells, and the like.

To date, anti-inflammatory agents have been shown to inhibit the biosynthesis of prostaglandins activated by physicochemical and physiological stimuli from the cell membranes of damaged tissues or leukocytes inoculated by chemotactic factors in acute inflammation. (Moyazawa, K and Mikami, T., Japan J. Pharmacol., 38, 199, 1985). However, these drugs have a therapeutic effect on the symptoms of acute inflammation, but in chronic inflammation such as rheumatoid arthritis, only the primary inflammation is reported, and immunological therapeutic effects are not expected. Recently, researches on arachidonic acid have been actively conducted on hydroxyeicosatetraenoic acid (HETE), leukotriene B4, and leukotriene C4, which are substances produced by lipoxygenase. This has been shown to have some effect on chronic inflammation (Huskisson, ED et al., Inflammation, Mechanism and Treatment, MTP, Lancaster, England, p.55, 1980).

Therefore, it is necessary to study the substances that can be safely ingested without additional purification process, including natural plant extracts, can inhibit inflammation, and can be easily used in food.

However, there are no reports on the inhibitory effect of cacao beans and cacao bean husk extracts on inflammation caused by bacterial endotoxins and inflammation-causing substances.

Therefore, while the inventors have studied the physiological activity of cacao beans and soybean hulls in various angles, the present inventors have shown that the fractions have an excellent inhibitory effect on inflammation caused by bacterial endotoxin and inflammation causing substances in animal experiments. The invention was completed.

Accordingly, an object of the present invention is to provide an anti-inflammatory agent containing cacao beans or bean husk fraction as an active ingredient.

1 is a graph showing the mouse mortality rate by the endotoxin of bacteria, LPS (lipopolysaccharide) when orally administered cacao beans fractions to mice.

2 is a graph showing the mouse mortality rate by LPS, which is a bacterial endotoxin when orally administered cacao bean husk fraction to mice.

Figure 3 is a graph showing the inhibition rate of ear inflammation in rats by oral treatment of cacao bean fraction in rats, TPA (12- O- tetradecanoylphorbol-13-acetate) which is a dermatitis-inducing substance.

4 is a graph showing the inhibition rate of ear inflammation in rats by TPA, a dermatitis-inducing substance, when cacao bean skin fractions were orally treated in rats.

FIG. 5 is a graph showing the ear inflammation inhibition rate of rats by croton oil, a skin inflammation-causing substance, when cacao bean fractions were orally treated in mice.

FIG. 6 is a graph showing the inhibition rate of ear inflammation in rats by croton oil, an inflammation-causing substance, when cacao bean skin fractions were orally treated in mice.

FIG. 7 is a graph showing the inhibition rate of ear inflammation in rats by the disinfectant dithranol when the cacao bean fraction was orally treated in mice.

Figure 8 is a graph showing the inhibition rate of ear inflammation in rats by disinol, an inflammation-causing substance, when cacao bean skin fractions were orally treated in rats.

Figure 9 shows the inhibition rate of ear inflammation in rats by TPA, a skin irritant, when oral administration of cacao beans and bean husk fractions having anti-inflammatory activity and EGCG (epigallocatechingallate) of green tea, which is one of anti-inflammatory substances present in food, This is a graph comparing.

The present invention is characterized by an anti-inflammatory agent containing cacao beans or bean husk fraction as an active ingredient.

The present invention will be described in detail as follows.

Cacao beans or soybean peel fraction according to the present invention is useful for preventing and treating inflammation by showing excellent inhibitory effect against inflammation caused by endotoxin and various dermatitis causing substances secreted by bacteria.

Accordingly, the present invention includes a drug or a food additive containing the cacao beans or soybean peel fraction as an active ingredient.

Remove the cocoa butter from cacao beans according to the present invention 40 parts by adding 50% acetone, 50% ethanol or 50% methanol [Duksan, Korea] to 1 part by weight of the remaining fraction (cacao) or cacao beans peel 40 After stirring under reflux for 4-6 hours at a temperature of ˜70 ° C., the supernatant obtained by centrifugation of the extract was collected and the remaining residue was repeatedly extracted. The extract was then filtered and dried to obtain a cacao bean or soybean extract. The extract is adsorbed onto a styrenic porous resin, fractionated using 20% ethanol, and then 60% ethanol is added to obtain a solvent fraction.

When the fraction is orally administered to rats, it significantly lowers the mortality rate of rats caused by injection of LPS, an endotoxin of inflammation-causing bacteria, and is also caused by TPA, disranol, and croton oil, which cause inflammation. Significantly suppresses inflammation

On the other hand, the present invention comprises a drug or a food additive containing cacao beans or soybean skin fraction as an active ingredient, the method of producing the extract drug or food additives according to a known method.

When prepared as a pharmaceutical, cacao beans or bean husk fractions may be used as such, but may be mixed with pharmaceutically acceptable carriers, forming agents, diluents, etc., to give powders, granules, capsules or injectables. It is also possible to manufacture. In addition, cacao beans according to the present invention has been used for food from ancient times, there is no particular restriction on the dosage of the fraction, body absorption, weight, age of patients, sex, dietary conditions, administration time, administration method, excretion rate And the severity of the disease. In general, cacao beans or bean husk fractions are preferably administered at about 0.1 to 10 mg per kg body weight. Therefore, the drug containing the active ingredient of the present invention is to be prepared in consideration of the effective amount range, and the unit dosage form formulated in this way according to the judgment of experts and the needs of the individual to monitor or observe the administration of the drug as needed Specialized medications can be used or administered at regular intervals.

On the other hand, when manufacturing as a food additive can be prepared by including the extract of cacao beans (or bean peel) of the present invention in food materials such as beverages, gum, confectionery.

Drugs and food additives containing the cacao beans or soybean peel fraction as described as an active ingredient is excellent for the prevention and inhibition of inflammation.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples.

Example 1: Preparation of Fractions Having Inhibitory Effect from Cacao Beans

<1-1> Preparation of Extracts Having Inhibitory Effect by Solvent Extraction

After removing cocoa butter from cacao beans, 6 parts by weight of 50% acetone [Duksan, Korea] aqueous solution was added to 1 part by weight of the remaining fraction (cacao), and the mixture was stirred and refluxed at a temperature of 60 ° C. for 5 hours. The supernatant obtained by centrifugation [Vision, Korea] for 30 minutes at rpm, 4 ° C. was collected, and the remaining residue was repeatedly extracted in the same manner. The extract thus obtained was filtered through filter paper (Whatmann 41), and then freeze-dried to obtain a cacao bean extract.

<1-2> Preparation of Fractions Having Inhibition of Inflammation by Adsorption Column Chromatography

From the extract obtained in the above <1-1>, a fraction was prepared by adsorption column chromatography with reference to a previously patented method [US Pat. No. 6159451, 2000]. That is, it was adsorbed on the hydrophobic filler XAD-4 [Sigma, USA] or HP-20 [Mitsbish, Japan], washed with 20% ethanol, and the remaining fractions were fractionated with 60% ethanol again.

Example 2 Preparation of Fractions Having Inflammatory Effect from Cacao Bean Peel

<2-1> Preparation of an extract having inhibitory effect from cacao bean husk by solvent extraction

6 parts by weight of 50% acetone [Duksan, Korea] aqueous solution was added to 1 part by weight of cacao beans, and the mixture was stirred under reflux for 5 hours at a temperature of 60 ° C., and then the extract was centrifuged at 8,000 rpm, 4 ° C. for 30 minutes [Vision , Korea], the supernatant obtained was collected, and the remaining residue was repeatedly extracted once in the same manner. The extract thus obtained was filtered through filter paper (Whatmann 41), and then freeze-dried to obtain a cacao bean peel extract.

<2-2> Preparation of Fractions Having Inhibitory Effect of Inflammation by Adsorption Column Chromatography

Fractions were prepared by adsorption column chromatography from the extract obtained in the above <2-1> with reference to a method of patent application [US Patent No. 6159451, 2000]. That is, it was adsorbed on the hydrophobic filler XAD-4 [Sigma, USA] or HP-20 [Mitsbish, Japan], washed with 20% ethanol, and the remaining fractions were fractionated with 60% ethanol again.

The following experiments were carried out to evaluate the anti-inflammatory efficacy of the cacao beans or soy bark fraction obtained as described above.

Example 3: Inhibitory Activity of Inflammation Caused by Bacterial Endotoxin

The inhibitory activity against inflammation caused by bacterial endotoxin of the fractions obtained in Examples 1 and 2 was carried out according to the following known method [Yang et al., J. Nutr., 128: 2334-2340, 1998]. It was.

First, 25 to 30 g of BALB / C mice were prepared per group and fasted for 12 hours before the experiment, and the samples obtained by the above method were orally administered to the mice at a concentration of 1 g / kg. On the other hand, the control group was orally administered only the same amount of phosphate solution. After 30 minutes of anesthesia with xylazine hydrochloride, after 5 minutes, 40 mg / kg of LPS was intraperitoneally administered. Survival was measured every 2 hours until 30 hours. The degree of anti-inflammatory was measured by the difference in survival numbers for the control group.

The cacao beans and bean husk fractions are shown in Figures 1 and 2 to test the inhibitory effect on the mortality of mice caused by LPS, which is a bacterial endotoxin and inflammation-inducing substance. As shown in Figures 1 and 2, the mortality decreases of 32% and 36% were observed at 30 h, respectively, when orally administered cacao beans and soybean skin fractions to mice at a concentration of 1 g / kg respectively. In particular, at 12 hours, the mortality rate was 44% and 48%, respectively.

Example 4: Inhibitory Activity of Inflammation Induced by TPA

Inhibitory activity of inflammation caused by TPA of the fractions obtained in Examples 1 and 2 was determined by the following known methods [Romay C., et al., Inflamm. Res. 47: 334-338, 1998].

First, 8 rats per group of 25-30 g BALB / C mice were prepared and fasted 12 hours before the experiment, and the samples obtained by the above method were orally administered at respective concentrations. After 30 minutes of anesthesia with xyrazin hydrochloride, another 5 minutes later TPA was applied to both ears at 25 μg per ear. After 5 hours the right and left ears were cut out with a 6 mm punch and weighed. The degree of anti-inflammatory was measured by the difference in weight with respect to the control. (Figs. 3 to 9 are shown as averages, and statistical significance is determined by the ANOVA method [Ott, RL, An introduction to statistical methods and according to the two-tailed Dunnett's t-test at the significance level * P <0.05, ** P <0.01. data analysis, 1997].

3 and 4 show the results of experiments on the inhibitory effect on inflammation caused by TPA, which is an inflammation-causing substance of cacao beans and bean husk fractions. As shown in Figures 3 and 4, the cacao bean and soybean peel fractions showed an inhibition rate of 62% and 64% when administered orally to rats at a concentration of 0.5 g / kg, respectively, and administered orally to mice at a concentration of 1 g / kg. Inhibition rate of 72% and 68%, respectively, showed similar inhibitory efficacy.

Example 5 Inhibitory Activity of Inflammation Caused by Croton Oil

The inhibitory activity of inflammation caused by croton oil of the fractions obtained in Examples 1 and 2 was determined by the following known method [Clementi G., et al., Life Sci. 57: 193-197, 1995; Heiman A. S., et al., Steroids 62: 491-499, 1997.

Similar to the above method, samples were administered orally at respective concentrations. After 30 minutes of anesthesia with xyrazin hydrochloride, again 5 minutes later croton oil was applied to both ears at 20 μl per ear. After 5 hours the right and left ears were cut out with a 6 mm punch and weighed. The degree of anti-inflammatory was measured by the difference in weight with respect to the control.

5 and 6 show the results of experiments on the inhibitory effect of inflammation caused by croton oil, an inflammation-causing substance of cacao beans and soybean hull fractions. As shown in Figures 5 and 6, the cacao bean and soybean hull fractions showed an inhibitory rate of 67% and 53% when administered orally to rats at a concentration of 0.5 g / kg, respectively, and orally to rats at a concentration of 1 g / kg. Similar inhibitory efficacy was shown by showing inflammatory inhibition rates of 77% and 71%, respectively. As shown in Figures 5 and 6, cacao beans and soy bark fractions showed a strong inflammatory inhibitory activity of 77% and 68% when orally administered to mice at a concentration of 1 g / kg, respectively.

Example 6: Inhibitory Activity of Inflammation Caused by Disranol

The inhibitory activity of sepsis caused by dislanol of the fractions obtained in Examples 1 and 2 was determined by the following known methods [Viluksela M., et al., Arch. Dermatol. Res. 283: 262-268, 1991; Blazso G., et al., Prostaglandins 50: 161-168, 1995].

Similar to the above method, samples were administered orally at respective concentrations. After 30 minutes of anesthesia with xyrazin hydrochloride, another 5 minutes later dislanol was applied to both ears at 50 μg per ear. After 5 hours the right and left ears were cut out with a 6 mm punch and weighed. The degree of anti-inflammatory is measured by the difference in weight relative to the control.

7 and 8 show the results of experiments on the inhibitory effect on the inflammation caused by dislanol, an inflammation-causing substance, of the cacao beans and bean husk fractions. As shown in Figs. 7 and 8, cacao bean and soybean skin fractions showed strong inflammatory inhibitory activity of 82% and 78% when administered orally to rats at a concentration of 1 g / kg, respectively, and at a concentration of 0.5 g / kg, respectively. Inflammation inhibition rates were 51% and 59%.

As described above, oral administration of the cacao beans and bean husk fractions showed a strong inhibitory activity of inflammation caused by TPA, croton oil, dislanol. In addition, by observing that inflammation is inhibited as the concentration of the treated sample is gradually increased, it can be seen that the inflammation is dependent on the concentration of cacao beans and soybean skin fractions administered orally.

As a result of the above experiment, the fractions of cacao beans and soybean hulls showed strong anti-inflammatory effect when orally administered to rats.

Example 7: Preparation of Tablets

10 g of active ingredients

70 g of lactose

15 g of crystalline cellulose

5 g of magnesium stearate

Total amount 100 g

The tablets were prepared by direct tableting method after mixing the ingredients listed above finely. The total amount of each tablet is 100 mg, of which the active ingredient content is 10 mg.

Example 8: Preparation of Powder

10 g of active ingredients

50 g of corn starch

40 g of carboxy cellulose

Total amount 100 g

A powder was prepared by crushing and mixing the ingredients listed above. 100 mg of powder was put into the hard capsule to prepare a capsule.

Example 9: Toxicity Test

Toxicity experiments were performed on cacao beans or bean hull fractions of the present invention as follows. Specifically, the fractions were dissolved in dimethylsulfoxide (DMSO), diluted with water, and then administered to each mouse (10 mice per group) 1 g / kg and observed for 7 days, but no rats died.

Example 10

Chewing gum was prepared in a conventional manner by combining 20% by weight of gum base, 76.9% by weight of sugar, 1% by weight of perfume, and 2% by weight of water, and 0.1% by weight of cacao beans or legume fractions obtained in the above examples.

Example 11

Candy was prepared in a conventional manner by combining 60% by weight of sugar, 39.8% by weight of starch syrup, and 0.1% by weight of fragrance, and 0.1% by weight of cacao beans or bean husk fraction obtained in the above examples.

Example 12

Force 1st grade 25.59 wt%, gravity 1st grade 22.22 wt%, white sugar 4.80 wt%, salt 0.73 wt%, glucose 0.78 wt%, palm shortening 11.78 wt%, ammo 1.54 wt%, sodium bicarbonate 0.17 wt%, sodium bisulfite 0.16 wt% , Rice flour 1.45 wt%, Vitamin B₁0.0001 wt%, Vitamin B₂ 0.0001 wt%, Milk flavor 0.04 wt%, Water 20.6998 wt%, Whole milk powder 1.16 wt% Substitute powder 0.29 wt%, calcium phosphate 0.03 wt%, Spray salt Biscuits were prepared in a conventional manner by combining 0.29% by weight and 7.27% by weight of spray oil with 1% by weight of cacao beans or legume fractions obtained in the above examples.

Example 13

0.26% by weight of honey, 0.0002% by weight of thioctoamide, 0.0004% by weight of nicotinic acid, 0.0001% by weight of riboflavin hydrochloride, 0.0001% by weight of pyridoxine hydrochloride, 0.001% by weight of inositol, 0.002% by weight of orthoic acid and 98.7362% by weight of water The beverage was prepared by the conventional method by combining the cacao beans or bean husk fraction obtained in the example.

Example 14

65.18% pork, 25% poultry, 3.5% starch, 1.7% soy protein, 1.62% salt, 0.5% glucose and 1.5% glycerin and 1% cacao beans or bean husk fractions obtained in the above examples. Sausage was prepared by combining the conventional method.

Example 15

55% by weight of spirulina, 10% by weight of guar gum enzyme, 0.01% by weight of vitamin B ₁ hydrochloride, 0.01% by weight of vitamin B6 hydrochloride, 0.23% by weight of DL-methionine, 0.7% by weight of magnesium stearate, 22.2% by weight of lactose and 1.85% by weight of corn starch And 10% by weight of cacao beans or bean husk fraction obtained in the above Example was prepared a tablet-type health supplement in a conventional manner.

Example 16

Chitooligosaccharide 11.26 wt%, Garlic powder 0.2 wt%, Ginkgo biloba extract powder 0.2 wt%, Beta-carotene (30% suspension) 0.9 wt%, Alpha tocopherol 1.2 wt%, Lecithin 1.2 wt%, Refined palm oil 4.5 wt%, Hest 1.6 Weight%, soybean oil 18.994 weight%, gelatin 37.83 weight%, glycerin 16.51 weight%, ethyl vanillin 0.09 weight% titanium dioxide and 0.076 weight% food coloring and 0.44 weight% cacao beans or soybean skin fractions obtained in the above examples. By blending to prepare a capsule-type dietary supplement in a conventional manner.

Comparative example

In order to compare the activity of this fraction with EGCG (epigallocatechingallate) of green tea, which is a generally well known anti-inflammatory substance, it was tested in the same manner as in Example 4. As shown in Figure 9, the fractions of cacao beans and soybean hulls showed very good efficacy compared to EGCG, a pure anti-inflammatory material.

As a result, it was found that cacao beans and legume fractions by the method used in the present invention have strong anti-inflammatory activity in animal experiments.

As described above, the cacao beans or soybean peel fraction according to the present invention has an excellent inhibitory effect on inflammation when administered orally, when used as an inhibitor of inflammation, by bacterial endotoxin and external inflammatory agents In addition to effectively suppressing the inflammation that occurs, only anti-inflammatory active fractions can be formulated using solvent extraction and simple adsorption column chromatography, which can be used in the food industry. The production cost can be lowered compared to the preparation, and the economic efficiency can be improved by recycling a large amount of cacao beans.

In addition, since the solvent and column chromatography used as food grade, it can be safely ingested without a separate purification process to remove toxicity. Therefore, it is possible to produce processed foods by adding the cacao beans or bean husk fraction, it can be expected to have the effect of inhibiting inflammation by the ingestion of such processed foods.

Claims (2)

Anti-inflammatory agent containing cacao beans or bean husk fraction as an active ingredient. A food additive having an anti-inflammatory action, characterized in that it contains cacao beans or a legume fraction.