JP2002371092A - New compound, extract of camellia flower containing the same, and application thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a protective agent of the gastric mucosa, an inhibitor of alcohol absorption, and an inhibitor of sugar absorption, containing a new compound as an active ingredient, because the new compound is included in an extract of a camellia flower, and the compound has activities for eliminating active oxygen, such as an SOD-like activities and DPPH radical-eliminating activities. SOLUTION: The extract of the camellia flower obtained by extracting the camellia flower with an aqueous alcohol or an aqueous solution of the aqueous alcohol contains at least a compound represented by formula (1) or formula (2). Besides these compounds, the protective agent of the gastric mucosa, the inhibitor of the alcohol absorption, and the inhibitor of the sugar absorption containing these compounds are also provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel compound, a camellia extract containing it, and their use.

[0002]

2. Description of the Related Art Conventionally, camellia has been used as a folk medicine for the treatment of hematemesis and intestinal bleeding blood (intestinal bleeding), and is also known to have a tonic effect. I have. In recent years, research on cosmetics for beautiful skin, such as conditioning and lightening, using components contained in camellia has been advanced. As the components of camellia flowers, leucoanthocyanin, anthocyanins, triterpene saponins, etc. are generally known, but the other components of camellia flowers and their medicinal properties are largely unknown, and further research is required. ing.

[0003]

According to the present invention, there is provided an extract obtained by extracting camellia flowers with an aqueous alcohol or an aqueous aqueous alcoholic solution, the extract comprising at least the following formula (1) or ( 2)

Embedded image A camellia extract containing the compound represented by the formula: is provided.
Further, according to the present invention, any one of the following formulas (1) to (6)

Embedded image Is provided. Further, according to the present invention, there are provided a gastric mucosa protective agent, an alcohol absorption inhibitor and a sugar absorption inhibitor, which contain the above camellia extract or at least one compound of formulas (1) to (6) as an active ingredient. Is done.

[0004]

DETAILED DESCRIPTION OF THE INVENTION Plant flowers that can be used to obtain the camellia flower extract of the present invention are theaceae (Theaceae).
It is not particularly limited as long as it is a plant flower belonging to, for example, Japanese camellia, snow camellia, honkon camellia, salwin camellia, yakushimatsubaki, suzuki,
Examples include various varieties of flowers such as uraku camellia, wabisque, cane camellia, foliage, Iwane squeezing, spring light, and taro crown. Above all, a flower of a camellia (Camellia Japonica L.) is preferable. These plants grow widely in Japan, China and the like, and any plants growing anywhere can be used. However, the components contained in the flowers of the Camellia plant are considered to have a slight difference in the amount and the type depending on the kind and the place of production of the Camellia plant as a raw material. It is preferable to use those cultivated in Japan in order to obtain. In addition, as a flower of a Camellia plant, any of a single species or a plurality of species of Camellia plants may be used. The flowers of the Camellia family usually include gaku pieces, petals, carpels, stamens, pistils, etc., and are preferably fresh.

The aqueous alcohol used in the present invention includes aliphatic alcohols having 1 to 3 carbon atoms, such as methanol, ethanol and propanol. Of these, methanol is preferred. Examples of the aqueous alcohol aqueous solution include an aqueous solution containing about 1 to 50% by weight of an aqueous alcohol.

According to the present invention, a camellia plant extract is obtained by extracting flowers of a camellia plant with aqueous alcohol or an aqueous aqueous alcohol solution. At this time, the aqueous alcohol or the like is about 1 to 10 times (weight), preferably 1 to 10 times, that of the flowers of the Camellia plant
It is appropriate to use about 5 times.

[0007] The extraction may be carried out by either cold soaking or hot soaking, but at a temperature of about 50 to 85 ° C, with or without shaking, the flowers of the above-mentioned Camellia plant are mixed with the above-mentioned solvent. It is appropriate to carry out this by immersing in water. When immersing under shaking, it is appropriate to carry out for about 30 minutes to 5 hours, and when immersing under non-shaking, 1 hour to 10 hours.
It is appropriate to carry out for about a day. The extraction process may be performed only once for the same camellia flower material, but is preferably performed twice or more.

[0008] The extract obtained above is preferably further extracted with n-butanol. At this time, for example,
Water, ethyl acetate and the like may be added together with n-butanol to perform partition extraction. Partitioned extraction can be performed in the same manner as the extraction with aqueous alcohol described above. In particular, the partitioning extraction is carried out at room temperature, with or without shaking, by adding n-butanol to an extract obtained with an aqueous alcohol or an aqueous aqueous alcohol solution or an extract concentrated as described later with 1 to 30 ( It is appropriate to add about (weight) times.
The extract is preferably concentrated after the flowers of the Camellia plant are extracted with aqueous alcohol or an aqueous aqueous alcohol solution or further with n-butanol. The concentration at this time is preferably performed at a low temperature or heating and under reduced pressure. Further, this concentration may be performed until the solution is dried, or may be filtered before concentration, and the filtrate may be concentrated to obtain an extract.

[0009] The obtained extract may be subjected to a purification treatment. As the purification treatment, a chromatography method, an elution method using an ion exchange resin, or the like can be employed alone or in combination. For example, the chromatographic method includes normal phase chromatography, reverse phase chromatography, high performance liquid chromatography, centrifugal liquid chromatography, column chromatography, thin layer chromatography, and the like, or a method in which any of them is combined. . Purification conditions such as a carrier and an elution solvent at this time can be appropriately selected according to various types of chromatography.
For example, in the case of thin layer chromatography, a water-methanol solvent can be used, and in the case of high performance liquid chromatography, a methanol-acetic acid solvent can be used.

When the aqueous alcohol (particularly methanol) extract or n-butanol extract obtained above is subjected to normal-phase and reverse-phase thin-layer chromatography, and further subjected to high-performance liquid chromatography, the results are shown above. Formula (1)-
Spots and peaks corresponding to the six new compounds of formula (6) can be obtained. As described above, the compounds of formulas (1) to (6) can be isolated from the camellia flower extract of the present invention by purifying the component showing the finally obtained peak.

[0011] The extract obtained by the method of the present invention comprises:
As will be described later, various effects such as a scavenging effect of active oxygen, a protective effect against ethanol-hydrochloric acid-induced gastric mucosal damage, a platelet aggregating effect, an inhibitory effect on blood glucose increase due to a glucose load, an inhibitory effect on alcohol absorption, an inhibitory effect on sugar absorption are recognized. And
In particular, it can be used as it is or as diluted with an appropriate medium as a gastric mucosa protective agent, alcohol absorption inhibitor or sugar absorption inhibitor. The extract of the present invention may be used as a health food as it is or diluted with an appropriate medium and in an amount that does not exhibit a pharmaceutical therapeutic effect, that is, an amount for maintaining health. The form of the extract in these cases may be any of preparations such as powders, granules, tablets, capsules or liquids.

Suitable vehicles include, for example, lactose, starch, dextrin, calcium phosphate, calcium carbonate, synthetic and natural aluminum silicates, magnesium oxide, dry aluminum hydroxide, magnesium stearate, sodium bicarbonate, dry yeast, zinc oxide ,talc,
Kaolin, powdered boric acid, zinc stearate, magnesium stearate, magnesium carbonate, precipitated calcium carbonate,
Examples include bismuth subgallate, potassium aluminum sulfate powder, glycerin, propylene glycol, fatty oil, ethylene glycol, polyethylene glycol, sorbitol, and surfactants. The above-mentioned various preparations can be produced by a known method widely used in the fields of medicine, health food and the like.

The amount of the extract or compound of the present invention to be used can be appropriately adjusted depending on the degree of concentration and purification, the purpose of use, the target disease such as treatment or prevention, the degree of the disease, the body weight, etc. , As an extract or with the above compound as an active ingredient, 1 μg to 1000
It is appropriate to give about 0 mg in two or three divided doses.

[0014]

EXAMPLES Examples of the camellia flower extract of the present invention, the novel compounds contained therein, and the effects thereof will be specifically described below.

(1) Extraction of Camellia Flower and New Compound Contained in Camellia Flower Extract FIG. 1 shows an outline of a method for extracting camellia flower. 6.0 kg of fresh camellia flowers (Camellia Japonica L.) collected from Izu Oshima were immersed in 6 liters of methanol (99.9%), and extracted under heating at 80 ° C. for 3 hours. This was repeated three times, the obtained methanol was combined, and the methanol was evaporated to obtain a methanol extract (480 g). Then
Ethyl acetate and water (1: 1) were added 1 liter each to 480 g of the obtained methanol extract at room temperature and shaken, and the obtained ethyl acetate layer and aqueous layer were separated and collected. This operation was repeated three times. Further, 3 liters of n-butanol (99.8%) was added to the obtained aqueous layer at room temperature and shaken, and the obtained n-butanol layer and aqueous layer were separated and collected. This operation was repeated three times. The solvent was evaporated from the n-butanol layer and the aqueous layer to obtain an n-butanol extract and an aqueous extract, respectively (121 g and 332 g). Normal-phase and reverse-phase thin-layer chromatograms of the methanol extract, ethyl acetate extract, n-butanol extract and water extract are shown in FIGS. 2 (a) and (b), respectively.

Subsequently, the obtained n-butanol extract was subjected to thin-layer chromatography using a SiO ₂ column to confirm the separation state, and the mobile phase was used as CHCl.
₃ - methanol - water 7: 3: 0.5 → 6: 4: 1 →
5: 5: 5 (v / v) and the composition ratio was sequentially switched and separated to obtain 13 fractions (FIG. 3 (a): normal phase,
(B): reverse phase). Of the 13 fractions obtained,
The fraction obtained as the fraction 5 was further confirmed by thin layer chromatography using an ODS column while confirming the separation state, and methanol (10, 3
0, 50, 60, 70, 100%) → CHCl ₃ -methanol-water (6: 4: 1 (v / v)) → methanol;
The composition ratio was sequentially changed to separate, and 12 fractions were obtained (FIG. 4 (a): normal phase, (b): reverse phase). 1 obtained
The fraction obtained as fraction 10 among the fractions of No. 2 was subjected to liquid chromatography (manufactured by Shimadzu Corporation:
HPLC device, liquid sending pump: LC-10AS, differential refractometer: RID-6A, YMC-PAC ODS column: 4
A12S05-2520WT SH-343-5, flow rate: 0.9 ml / min, pressure: 170-180 kgf / c
m ² ) and 1% aqueous methanol-acetic acid as a mobile phase were separated into two fractions, and the above formula (1)
And a compound of formula (2) were obtained (compound of formula (1): 264 mg and compound of formula (2): 175 mg).

The fraction obtained as the fraction 11 among the obtained 12 fractions is separated into three fractions in the same manner as described above, and the above formulas (3), (4) and (5) are obtained. (Equation (3))
Compound: 15.4 mg, Compound of Formula (4): 34.1
mg, and the compound of the formula (5): 13.6 mg). Further, of the 12 fractions obtained, one fraction was separated from the fraction obtained as Fraction 12 in the same manner as described above, except that a 1% aqueous solution of methanol—acetic acid and a 1% aqueous solution of CH ₃ CN—acetic acid were used as the mobile phase. Separation gave the above compound (6) (compound of formula (6): 9.9 m)
g). These chromatograms are shown in FIG. 5 ((a): normal phase,
(B): reverse phase). The physical properties of the compounds of formulas (1) to (4) are shown in FIGS. In addition, the determination of the structural formula of each compound of the formulas (1) to (4) was performed by the NMR method. FIGS. 10 to 14 show HPLC charts of the compounds of formulas (1) and (2), formula (3), formula (4), formula (5), and formula (6), respectively.

(2) Active oxygen scavenging activity As an active oxygen scavenging activity involved in various disease states such as aging and inflammation, an in vitro system was examined for SOD-like activity and DPPH radical scavenging activity by a xanthine oxidase method.

(I) Experimental method for SOD-like activity 3 mM xanthine, 3 mM EDTA, 0.15% bovine serum albumin (BSA), nitroblue tetrazolium
0.75 mM of (NBT) was added and mixed in 0.1 ml each. 2.5 ml of each concentration of the test substance was added thereto, preliminarily heated at 25 ° C. for 10 minutes, 0.1 ml of a xanthine oxidase solution was added, and the mixture was incubated at 25 ° C. for 20 minutes. The reaction was stopped by adding 0.1 ml of 6 mM CuCl ₂ and the absorbance at 560 nm was measured. The test substance was dimethyl sulfoxide (DMS
O) and then sequentially diluted with distilled water. In addition, measurement was performed using water to which 2.5 ml of the test substance was added as a control.

(Ii) Experimental method for DPPH radical scavenging activity 0.1 M acetic acid-sodium acetate buffer (pH 5.5)
1.0 ml, 2.0 × 10 ^-4 M 1,1-diphenyl-2-
Picrylhydrazyl (DPPH) radical 0.5ml,
0.5 ml of ethanol and 1.0 ml of test substance at each concentration
Were mixed and left at room temperature for 30 minutes, and the absorbance at 517 nm was measured. DPPH was dissolved in ethanol, and the test substance was dissolved in DMSO and then diluted with ethanol.

(Iii) Results As shown in Table 1, similar to the known antioxidants, the camellia methanol extract, the ethyl acetate and the n-butanol fraction exhibited active oxygen scavenging activity and radical scavenging activity. Was

[0022]

[Table 1]

(3) Gastric Mucosa Protective Effect The effect of Camellia extract and the compounds contained therein on acute gastric mucosal damage to rats is examined, and it is involved in hemostasis to demonstrate the therapeutic and preventive effects on gastrointestinal bleeding. Platelet aggregation activity was also examined. (I) Experimental method for protective effect against ethanol hydrochloride-induced gastric mucosal damage SD male rats (8-9 weeks old, around 250 g) were subjected to 24
After fasting for an hour, camellia methanol extract (125, 2
50 mg / kg) or n-butanol fraction (25, 50
mg / kg), and 1 hour later, 1.5 ml of 99.5% ethanol was orally administered. One hour later, the stomach was removed and the tissue was fixed with 1.5% formalin. Then, the length (mm) of the damage generated in the gastric mucosa was measured, and the total value was defined as the damage coefficient. Further, for the compounds of formulas (1) and (2), the length (mm) of the damage caused to the gastric mucosa was measured in the same manner as described above, and the result was used as the damage coefficient.

(Ii) Platelet Aggregation Activity Whole blood was collected from the auricular artery of a Japanese white male rabbit, and washed platelets (5 × 10 ⁶ cells / μl, 225 μl / assay) were prepared and incubated in the presence of 1 mM CaCl _2. Then, various concentrations of camellia flower extract (including fractions) were added thereto, and the platelet aggregation activity was measured by light transmittance using an aggregometer (Hematracer). In addition, the aggregation activity of 1 U / ml thrombin was determined by the aggregation rate of 1
00%. The platelet aggregation activity of the compounds of formulas (1) and (2) was also measured in the same manner as described above. In this case, the aggregation activity of 0.5 U / ml thrombin was defined as an aggregation rate of 100%.

(Iii) Results As shown in Table 2, the camellia methanol extract (250 mg / k)
g) and the n-butanol fraction (50 mg / kg)
Gastric mucosal damage was significantly suppressed. In addition, as shown in Table 3, the compound of formula (1) (20 mg / kg) and the compound of formula (2) (10, 20 mg / kg) were compared with those of ethanol-induced acute gastric mucosal injury model. Was significantly suppressed.

[0026]

[Table 2]

[0027]

[Table 3] Furthermore, as shown in Table 4, the methanol extract, the n-butanol fraction, and the ethyl acetate fraction showed strong platelet aggregating activity, and were used in clinically for hemostasis of gastrointestinal damage and trauma accompanied by hemorrhage. Platelet aggregation effect (0.5
(A complete aggregation is induced at a concentration of １1 U / ml). In addition, as shown in Table 5, the compounds of formulas (1) and (2) also exhibited platelet aggregation activity.

[0028]

[Table 4]

[0029]

[Table 5]

[0030] From this, methanol extract of camellia flower,
It was suggested that the n-butanol fraction and the compounds of formulas (1) and (2) have a strong gastric mucosal protective effect and can be applied to gastrointestinal disorders such as gastritis due to excessive drinking. In addition, it was shown to be useful for gastrointestinal disorders associated with bleeding (acute gastritis and acute colitis) due to its strong platelet aggregation activity.

(4) Inhibition of alcohol absorption (i) Experimental method Male Wistar rats (5-6 weeks old, body weight: 130)
g) was fasted for 20 to 24 hours, and then a methanol extract, an n-butanol fraction and an ethyl acetate fraction were orally administered, and 30 minutes later, 20% ethanol 5 ml / k.
g was administered orally. Blood was collected over time (0.5, 1, 2 hours), and the blood alcohol concentration was measured by the alcohol dehydrogenase-aldehyde dehydrogenase method.

(Ii) Results As shown in Table 6, camellia methanol extract (500 m
g / kg) and n-butanol fraction (125 mg / k
g) significantly reduced blood ethanol levels.

[0033]

[Table 6]

(5) Sugar absorption inhibitory activity (i) Experimental method of aldose reductase inhibitory action 0.1 M lithium sulfate, 1 mM DL-glyceraldehyde, enzyme fraction (from rat lens), test substance at each concentration, 135 mM containing 0.03 mM of the reduced form of nicotinamide amide adenine dimcleotide phosphate (NADPH)
After incubating a phosphate buffer (pH 7.0) at 30 ° C. for 30 minutes, the reaction was stopped by adding hydrochloric acid. This was treated with a strong alkali, heated at 60 ° C. for 20 minutes, returned to room temperature, and the produced NADP was quantified by a fluorescence method (excitation wavelength: 360 nm, measurement wavelength: 460 n).
m). The test substance used was dissolved in DMSO.

(Ii) Experimental method for inhibiting glucose absorption Wistar male rats (5-6 weeks old, 120-150)
g) after fasting for 20 hours, extract methanol extract
(500, 1000 mg / kg) or n-butanol fraction (250, 500
mg / kg) was orally administered. After 30 minutes glucose (0.5 g /
kg) was orally administered, blood was collected over time from the orbital vein, and the blood glucose concentration was measured by the glucose oxidase method.

(Iii) Results For aldose reductase, IC50 = 23.8 μg /
ml, an inhibitory effect was observed. In addition, as shown in Table 7, the methanol extract was not significant, but showed a tendency to suppress the increase in blood glucose level in glucose-loaded rats, although not as significant as shown in Table 7. The n-butanol fraction is
At a dose of 500 mg / kg, it showed a significant inhibitory effect on blood glucose elevation 30 minutes after glucose loading.

[0037]

[Table 7] From the above results, it was confirmed that the methanol extract of camellia and the n-butanol fraction can prevent postprandial hyperglycemia and are useful for preventing and improving diabetes.

[0038]

According to the present invention, the novel compound of formula (1) and / or (2) is contained in an extract obtained by extracting camellia flowers with aqueous alcohol or an aqueous alcohol solution. It was confirmed that. In addition, based on these components, it has SOD-like activity and DPPH radical scavenging activity; protective effect against ethanol-induced hydrochloric acid-induced gastric mucosal damage and platelet aggregation activity; alcohol absorption inhibitory activity; glucose absorption inhibitory activity and aldose reductase inhibitory activity. It has been confirmed that it is useful for the prevention and improvement of pathological conditions involving free radicals such as aging and arteriosclerosis, gastrointestinal disorders with bleeding (acute gastritis and acute colitis), and diabetes. .

Further, according to the present invention, the novel compounds represented by the formulas (1) to (6) are sources of various actions and effects of the above-mentioned camellia flower extract, and are used in medicines and health foods. Application development of is expected. Furthermore, according to the present invention, since the camellia extract has active oxygen scavenging activity such as SOD-like activity and DPPH radical scavenging activity, pathological conditions involving free radicals such as aging and arteriosclerosis. It is also effective for protection against ethanol-hydrochloric acid-induced gastric mucosal damage and for the prevention and treatment of gastrointestinal disorders (acute gastritis, acute colitis) accompanied by bleeding due to platelet aggregation activity. It has an inhibitory action, and it was recognized that it was useful for prevention and improvement of diabetes such as glucose absorption inhibitory action and aldose reductase inhibitory action. Therefore, it is possible to develop applications for medicines, health foods, and the like.

[Brief description of the drawings]

FIG. 1 is a schematic diagram illustrating a method for extracting camellia flowers according to the present invention.

FIG. 2 is a thin-layer chromatogram of each extract of camellia flowers of the present invention.

FIG. 3 is a thin-layer chromatogram of a butanol extract of camellia flowers of the present invention.

FIG. 4 is a thin-layer chromatogram of fraction 5 of a butanol extract of camellia flower of the present invention.

FIG. 5 is a thin-layer chromatogram showing a novel compound contained in a camellia flower extract of the present invention.

FIG. 6 is a view showing physical properties of the compound of the formula (1) of the present invention.

FIG. 7 is a view showing physical properties of the compound of the formula (2) of the present invention.

FIG. 8 is a view showing physical properties of the compound of the formula (3) of the present invention.

FIG. 9 is a view showing physical properties of the compound of the formula (4) of the present invention. It is a chart of HPLC.

FIG. 10 shows the HP of the compounds of formulas (1) and (2) of the present invention.
It is a chart of LC.

FIG. 11 is a HPLC chart of the compound of the formula (3) of the present invention.

FIG. 12 is a HPLC chart of the compound of the formula (4) of the present invention.

FIG. 13 is a HPLC chart of the compound of the formula (5) of the present invention.

FIG. 14 is a HPLC chart of the compound of the formula (6) of the present invention.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme Court ゛ (Reference) A61P 3/10 A61P 3/10 (72) Inventor Masahiro Kagawa 4-2-1 Oishi Ryumon 4-chome, Otsu City, Shiga Prefecture Inside Kano Shojuan (72) Inventor Teruki Ogushi 4-2-1 Oishi Ryumon 4-chome, Otsu City, Shiga Prefecture Inside (72) Inventor Yasunobu Asao 4-2-1 Oishi Ryumon 4-chome, Otsu City, Shiga Prefecture F-term (reference) 4K057 BB04 CC01 DD01 JJ52 4C086 AA01 AA02 AA03 AA04 EA10 GA16 GA17 MA01 MA04 NA14 ZA66 ZC51 4C088 AB45 AC03 BA09 BA10 BA13 BA32 CA05 CA06 CA14 MA02 MA52 NA14 ZA66 ZC

Claims (7)

[Claims] 1. An extract from which camellia flowers can be extracted with an aqueous alcohol or an aqueous aqueous alcohol solution, wherein the extract has at least the following formula (1) or (2): A camellia extract comprising a compound represented by the formula: 2. An extract obtained by further extracting butanol with the extract of claim 1, wherein the extract contains at least a compound represented by the formula (1) or (2). Flower extract. 3. One of the following formulas (1) to (6): A compound represented by the formula: 4. A gastric mucosa protective agent comprising the camellia flower extract according to claim 1 or 2. 5. A gastric mucosal protective agent comprising any one of the compounds according to claim 3. 6. An alcohol absorption inhibitor comprising the camellia flower extract according to claim 1 or 2. 7. A sugar absorption inhibitor containing the camellia flower extract according to claim 1 or 2.