JP3432529B2 - Intestinal composition - Google Patents

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an intestinal composition. More specifically, the present invention relates to an intestinal composition effective for improving the intestinal environment by lowering the pH in the human intestine, that is, the pH of feces, and improving human health. [0002] Conventionally, it has been difficult to observe bacteria in the human intestine. However, remarkable progress of anaerobic culture technology in recent years has led to much research on human intestinal flora. As a result, intestinal bacteria have a deep relationship with the pH of human feces as a host, volatile short-chain fatty acids, putrefaction products as malodorous components, and various enzyme activities, and affect human physiological conditions. It has been revealed. [0003] These intestinal bacteria are involved in the metabolism of various substances and endogenous substances derived from foods and drugs, and are important for human nutrition, physiological functions, infectious immunity, carcinogenesis, aging, drug efficacy, and the like. (Tomoashi Mitsuoka "The World of Intestinal Bacteria", Bunsosha, Tokyo, 1980; DJ Hent
ges "Human Intestinal Micr
ofola in Health and Dise
case "Academic Press, New Y
ork, 1983). [0004] Among these intestinal bacteria, useful bacteria such as Bifidobacterium bacteria and Lactobacillus bacteria which are advantageous in protection of human infection, nutrition, and suppression of harmful bacterial growth, and Clostridium There are harmful bacteria related to carcinogenesis, liver disease, arteriosclerosis, hypertension, spontaneous infections and the like, such as genus bacteria. In addition, some harmful bacteria may enter into organs that cause opportunistic infections and cannot grow when healthy, causing sepsis,
It often results in undesirable situations such as endocarditis, tumors of the brain, liver and lungs, cystitis, vaginitis. [0005] By the way, it has been reported that these harmful bacteria do not easily grow when the pH in the intestine becomes acidic. For example, since the pH of feces of infants is as low as 4.5 to 5.5, it is known that the useful bacteria Bifidobacterium spp. Become the most dominant, while the harmful bacteria Clostridium spp. (Tomota Mitsuoka, "Story of Intestinal Bacteria", Iwanami Shoten, Tokyo, 1978). Thus, intestinal pH
That is, if the pH of feces is inclined to the acidic side, it is possible to correct the abnormality of intestinal bacteria and to improve the flora. [0006] If the pH in the intestine is inclined toward the acidic side, intestinal peristalsis is promoted, and bowel movement is promoted, thereby improving constipation. The persistence of constipation not only impairs nutritional intake or imposes a heavy burden on the psyche, but also causes diseases such as hypertension, stroke, diabetes, and hemorrhoids. However, there is no example of such a composition for intestinal treatment aimed at lowering fecal pH, and there is only one example of using a Lactobacillus bacterium as a lactic acid bacterium preparation, but gastric acid and bile before reaching the intestine. Affected, it is difficult to colonize the intestine. Furthermore, it is known that catechins in green tea selectively suppress the growth of Clostridium bacteria in the intestine in a test tube (Japanese Patent Publication No. 2-295923).
However, it is unclear in practice whether catechins in green tea exert intestinal effects such as improvement of constipation due to a decrease in pH in vivo. [0007] Lowering the pH of the intestine, ie, the pH of feces, improves the intestinal flora and constipation, ie, purifies the intestine and prevents various diseases. It is important for the treatment to be useful for the treatment and to safely lower the intestinal pH for the human body. An object of the present invention is to provide an intestinal composition. Means for Solving the Problems The inventors of the present invention have conducted intensive studies using the decrease in fecal pH as an index, and as a result, they have been found to be contained in plants of the Camellia family, especially in tea that we are drinking everyday. It has been found for the first time that the polyphenol compound to be used lowers the pH of feces, and the present invention has been completed. The polyphenol compound of the present invention is
(+)-Catechin, (+)-gallocatechin, (+)-gallocatechin gallate, (-)-epicatechin, (-)-
Epicatechin gallate, (-)-epigallocatechin gallate and seven polyphenol analogs of (-)-epigallocatechin gallate. The polyphenol compound of the present invention can be obtained by treating an extract of tea water or alcohol with an ultrafiltration membrane and a reverse osmosis membrane. Goods can be used. The method for preparing the polyphenol compound used in the present invention is as follows. First, tea is extracted at room temperature with a sufficient amount of water or alcohol. After extraction,
The residue is separated by a known method to obtain an extract. The solvent is removed from the extract, water is added to the residue to dissolve, and the residue is passed through an ultrafiltration membrane having a molecular weight cutoff of 6,000 to 10,000. The liquid having passed through the ultrafiltration membrane can be purified to a desired purity by removing the solvent by a usual concentration and drying method.
% Or more of purified powder of polyphenols. To further increase the purity, an adsorbent that selectively adsorbs polyphenols is used. It can be easily prepared by a conventional adsorption separation method, for example, a method using an organic adsorbent, an inorganic adsorbent, activated carbon, an anion exchange resin, a carbohydrate coagulant, a protein coagulant, a hydrophobic resin, etc. Purity can be increased to 70% or more. Alternatively, the tea is extracted at room temperature with a sufficient amount of water or alcohol. After the extraction, the residue is separated by a known method to obtain an extract. The solvent was removed from the extract, water was added to the residue, and the mixture was dissolved.Then, the mixture was partitioned with hexane, chloroform and ethyl acetate in that order, and the hexane-soluble fraction, chloroform-soluble fraction and ethyl acetate-soluble fraction were separated. obtain. The partitioning with hexane and chloroform in this operation can be omitted depending on the conditions such as the degree of coloring and viscosity of the water or alcohol extract, but in order to increase the purity of the ethyl acetate-soluble fraction, the partitioning with hexane and chloroform is necessary. It is desirable to implement As the alcohol used for the extraction, lower alcohols such as methyl alcohol, ethyl alcohol, n-propyl alcohol, isopropyl alcohol and butyl alcohol are preferable from the viewpoint of operability and extraction efficiency. Further, the fraction obtained by the adsorption separation method or the ethyl acetate-soluble fraction obtained above is subjected to silica gel chromatography to obtain chloroform-methyl alcohol (20: 1, V / V) and chloroform-methyl. Seven types of compounds can be obtained by sequentially eluting with a solvent of alcohol (10: 1, V / V). Further, if necessary, the mixture is further subjected to Sephadex LH-20 and eluted with a suitable solvent such as methyl alcohol, whereby
Alternatively, recycle HPLC (manufactured by Nippon Kagaku Kogyo, LC-
908, GS-320 column, solvent methyl alcohol)
By using the above, seven kinds of compounds having higher purity can be obtained. The preparation of the polyphenol compound from the tea leaves described above is not particularly limited,
When used for foods, the former preparation method is preferred. For details, see the patent filed earlier (Japanese Patent Laid-Open No. 2-6499).
Is disclosed in detail. When the obtained polyphenol compounds are used in the present invention, they can be used alone or as a mixture of two or more kinds, and further, a crude extract containing polyphenol can be used. The product of the present invention exerts its effects upon oral ingestion. The form of use can be in the form of tablets, granules, capsules, syrups, drinks or various foods. The dose of the product of the present invention to humans is 0.00. g to 0.03 g / Kg body weight is preferable, and the effect is weak at a dose of less than 0.00011 g / Kg body weight. It is not preferable because it can be considered. It is unknown what effect the polyphenol compound, which is the active ingredient of the present invention, lowers the pH in the intestine, in other words, the pH in feces, but it is not known, but the bacteria of the genus Bifidobacterium or Lactobacillus in the intestine. It is presumed that as a result of these useful bacteria producing organic acids such as acetic acid and propionic acid, the pH of feces decreases. Hereinafter, the present invention will be described in detail with reference to Examples and Test Examples. Example 1 About 1 liter of water is added to 1 kg of commercially available green tea, and the mixture is stirred and heated to 80 ° C.
For 3 hours. The extract obtained by filtration was concentrated to dryness to obtain 350 g of a hot water extract of green tea (38% purity as a mixture of polyphenol compounds). Example 2 To 350 g of the hot water extract obtained in Example 1, 8 liters of water was added and dissolved, and the mixture was sequentially partitioned with hexane and chloroform. 10 L of ethyl acetate was added to the aqueous layer after the distribution, and the mixture was vigorously stirred and allowed to stand. The ethyl acetate layer was separated, and the ethyl acetate was distilled off, followed by drying to obtain 70 g of an ethyl acetate-soluble fraction (polyphenol compound). 74.5% purity as a mixture). The content of total polyphenol compounds in the ethyl acetate-soluble fraction was 74.5
% Of each polyphenol compound, (+)-catechin 3.5%, (+)-gallocatechin 14.8%, (+)-
Gallocatechin gallate 11.6%, (-)-epicatechin 7
%, (-)-Epicatechin gallate 4.6%, (-)-epigallocatechin gallate 15.0% and (-)-epigallocatechin gallate 18.0%. Example 3 10 g of the ethyl acetate-soluble fraction obtained in Example 2 was subjected to silica gel chromatography (solvent, chloroform-methyl alcohol, 20: 1, 10: 1, v / v), and Sephadex LH- 20 (solvent, methyl alcohol), recycled HPLC (LC-908, GS-3 manufactured by JASCO)
By sequentially using 20 columns and the solvent methyl alcohol), 0.3 g of (+)-catechin, 1.22 g of (+)-gallocatechin and 0.9 of (+)-gallocatechin gallate were used, respectively.
g, 0.5 g of (-)-epicatechin gallate, 0.38 g of (-)-epicatechin gallate, 1.2 g of (-)-epigallocatechin gallate and 1.5 g of (-)-epigallocatechin gallate were obtained. Example 4 50 g of the ethyl acetate-soluble fraction obtained in Example 2 was heat-sterilized, and then hydroxypropyl methylcellulose was used as an excipient.
450 g and 10 g of stearic acid as a lubricant were added and the mixture was tableted to give 200 tablets. Example 5 50 g of the ethyl acetate-soluble fraction obtained in Example 2 was sterilized by heating, and then 0.4 g per capsule in a Japanese Pharmacopoeia capsule (# 1).
It filled and obtained 100 capsules. Embodiment 6 FIG. Glucose 528 g, fructose 85.4 g,
Water was added to 15.8 g of powdered citric acid, 11.2 g of sodium citrate, 1.3 g of calcium lactate, 1.3 g of magnesium chloride, 13.2 g of powdered natural flavor, 5.5 g of vitamin C and the ethyl acetate-soluble fraction obtained in Example 2. The volume was adjusted to 11 liters, filled in 100 ml aliquots in dry heat sterilized 110 ml brown bottles, sealed with an aluminum cap, and then sterilized at 120 ° C. for 30 minutes to obtain 100 drinks. Example 7 1 kg of commercially available green tea was extracted with 20 liters of hot water at 85 ° C. while stirring for 30 minutes, and the tea leaves were removed by filtration to obtain a 17 liter extract. This solution was passed through an ultrafiltration apparatus (DDS, membrane type GR-81PP, molecular weight cut off 6000) to obtain 15 liters of passing solution. 5 liters of water was added to the concentrated residue, and the same operation was carried out to obtain 6 liters of the passing solution. The two solutions were combined and concentrated by a reverse osmosis membrane (manufactured by DDS, membrane type HC-50) to 1 liter to obtain a polyphenol compound having a purity of 35% (233 g as a polyphenol compound). Example 8 The concentrated product obtained in Example 7 was adsorbed on an adsorption resin (Duolite).
S-876 (manufactured by Sumitomo Chemical Co., Ltd.) and adsorbed, washed with deionized water, eluted with 50% ethanol, and concentrated under reduced pressure to evaporate ethanol to form a concentrated aqueous solution. After freeze-drying by the usual method, purity 74.5
% Polyphenol compound was obtained. The component composition of the obtained polyphenol compound was (+)-catechin 3.5
%, (+)-Gallocatechin 14.8%, (+)-gallocatechin gallate 11.6%, (-)-epicatechin 7%, (-)
-Epicatechin gallate 4.6%, (-)-epigallocatechin 15.0% and (-)-epigallocatechin gallate 1
8.0%. Example 9 Mixture 10 of the polyphenol compound obtained in Example 7
g of silica gel chromatography (solvent, chloroform-methyl alcohol, 20: 1, 10: 1, V /
V), recycled HPLC (LC-90 manufactured by Nippon Kagaku Kogyo Kogyo)
8, GS-320 column, solvent methyl alcohol) in order to obtain (+)-catechin 0.3 each.
g, (+)-gallocatechin 1.22 g, (+)-gallocatechin gallate 0.9 g, (-)-epicatechin 0.5%,
0.38 g of (-)-epicatechin gallate, 1.2 g of (-)-epigallocatechin gallate and 1.5 g of (-)-epigallocatechin gallate were obtained. Example 10 The mixture of polyphenol compounds obtained in Example 7
g after heat sterilization, 450 g of hydroxypropyl methylcellulose as an excipient and 10 g of stearic acid as a lubricant.
g were added and the mixture was compressed to give 200 tablets. Example 11 50 g of a mixture of the polyphenol compounds obtained in Example 7
After heat sterilization, 0.4 g per capsule was filled in capsules (# 1) of Japan Pharmacy to obtain 100 capsules. Example 12 528 g of glucose, 85.4 g of fructose, 15.8 g of powdered citric acid, 11.2 g of sodium citrate, 1.3 g of calcium lactate, 1.3 g of magnesium chloride, 13.2 g of powdered natural flavor, vitamin C
Water was added to 5.5 g of the polyphenol compound mixture obtained in Example 7 to make 11 liters, and the mixture was sterilized by dry heat.
100 ml each was filled in a ml brown bottle, sealed with an aluminum cap, and then sterilized at 120 ° C. for 30 minutes to obtain 100 drinks. Test Example 1 Single Dose Toxicity Test Each group consisted of 10 ddy mice, and each group was treated with a polyphenol compound suspended in physiological saline at a constant temperature (23 ± 1).
° C) and constant humidity (55 ± 5%) under the condition of Richfield Wilcocsonson (Litchfield).
-Wilcoxon) result of obtaining LD ₅₀ by method was females 3.1 g / Kg, males 5 g / Kg or more. Test Example 2 Cytotoxicity test MA104 cells (monkey kidney cells) were cultured in 1.2 × 10 ⁵ cells
BHKcell containing 10% FCS to be / tube
It was added to the medium (without adding antibiotics). Then, the polyphenol compound was added at 5 μg / ml, 1 μg / ml and 0.5 μg / ml.
g / ml, and cultured at 37 ° C. for 4 days to examine cell proliferation. As a result, the growth curve was similar to that of the control containing only physiological saline, and no cytotoxicity was observed. Test Example 3 Reverse mutation test Ames (Am) targeting reversion test from histidine auxotrophy to non-auxotrophy in Salmonella (S. typhimurium)
es) The test was performed. Direct tests and metabolic activation tests were performed using Salmonella typhirium TA100 and Salmonella typhirium TA98 as test bacteria. As a result, no increase in the number of mutant colonies was observed in the direct test and the metabolic activation test, and the cells were determined to have no mutagenicity (negative). Test Example 4 Clinical study: Fecal samples were collected twice from a total of eight healthy males and four females during a normal dietary control period.
Thereafter, the above-mentioned polyphenol compound was ingested at a rate of 1.2 g / day for 28 days using the capsules prepared in Example 5, and four times of feces were collected on the 12th, 14th, 26th and 28th days. . Thereafter, the intake of the polyphenol compound was stopped, and feces were collected twice on the 12th and 14th days after the stoppage. When feces were collected eight times in total, the pH of feces was measured with a pH meter. Table 1 shows the results. Table 1 shows that the polyphenol compound was administered before administration, during administration-1 (day 12 and day 14), and during administration-
The results are shown for 2 (day 26 and day 28) and four periods post-dose. In addition, since sampling was performed twice in each period, the pH indicates an average of 16 in total. [Table 1] Test Example 5 Clinical study: Fecal samples were collected twice from a total of eight healthy males and four females during a normal dietary control period.
Thereafter, using the tablets prepared in Example 10, the above-mentioned polyphenol compound was ingested in an amount of 1.2 g / day for 28 days, and the 12th, 14th, 26th, and 28th day of 4
The feces were collected. Thereafter, the intake of the above-mentioned polyphenol compound was stopped.
The feces were collected. When feces were collected eight times in total, the pH of feces was measured with a pH meter. Table 1 shows the results.
Shown in Table 2 shows that the polyphenol compound was administered before administration, during administration-1 (day 12 and day 14), and during administration-
The results are shown for 2 (day 26 and day 28) and four periods post-dose. In addition, since sampling was performed twice in each period, the pH indicates an average of 16 in total. [Table 2] As evident from Tables 1 and 2, the polyphenol compounds significantly reduced human fecal pH. The polyphenol compound as the active ingredient of the present invention can efficiently reduce the pH of feces,
It is extremely effective for intestinal purification such as improvement of human intestinal flora and constipation. Moreover, since this component is a tea component that has been used for drinking since ancient times, its safety is extremely high, and it can be supplied in large quantities, so the present invention contributes to the promotion of human health However, it is large.

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Kim Takezo 9-5 Akabori Shinmachi, Yokkaichi City, Mie Prefecture Taiyo Chemical Co., Ltd. (72) Inventor Nagataka 9-5 Akabori Shinmachi, Yokkaichi City, Mie Prefecture Taiyo Chemical (56) References “Food and Science”, 1989, Vol. 31, No. 12, pp. 110-117 (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 31/353 A61K 35/78 C07D 311/62 BIOSIS (STN) CAPLUS (STN) MEDLINE (STN) REGISTRY (STN) EMBASE (STN)

Claims (1)

(57) [Claims] [Claim 1] (+)-Catechin, (+)-gallocatechin, (+)-gallocatechin gallate, (-)-epicatechin, (-)-epicatechin gallate, ( A fecal pH lowering composition comprising one or more compounds selected from the group of polyphenol compounds consisting of-)-epigallocatechin and (-)-epigallocatechin gallate.