JPS62207289A - Bonded material of ascorbic acid and tannin - Google Patents

Abstract

NEW MATERIAL:A bonded material having the following properties. Appearance, white, pale yellow or yellow powder; solubility, easily soluble in water. EXAMPLE:Bonded ascorbic acid and geraniin of formula. USE:An agent suppressing lipid peroxide level in living body. Utilizable in the fields of medicine, food and cosmetic, etc. It has excellent safety and stability. PREPARATION:For example, 1-2mol of a tannin such as geraniin extracted from Geranium nepalense and 2-10mol of ascorbic acid are dissolved or dispersed in a mixture of water and alcohol and made to react with each other by heating at <=40 deg.C under stirring. The reaction mixture is treated by column chromatography to separate the objective material.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel ascorbic acid-tannin conjugate, which can be used in fields such as cosmetics, medicines, foods, and chemical products.

Since ancient times, tannins have had astringent, intestinal regulating, mouth-cooling, blood pressure-lowering, hemostatic, skin-conditioning, and anti-inflammatory effects.
Tannins have been discovered to have many pharmacological effects, such as sunscreen effects and anti-aging effects, and in recent years tannins have attracted attention as useful physiologically active substances. Tannins are widely distributed in the plant kingdom, and representative examples include green tea, black tea, Acacia japonica, Gobaishi, and Gennoshoko. These plants have been widely used and prized since before the existence of tannins was known, but in recent years research has progressed in various fields, and the benefits of these plants have been determined primarily by the tannins they contain. I have come to understand that it can be done. For example, the dried leaves and stems of Gennoshoko have long been used as an antidiarrheal and intestinal medicine, and are listed in the Japanese Pharmacopoeia, but their active ingredients are said to be tannins. As a result of research conducted by the inventors, geraniin, which is a tannin with less astringent taste than these tannins and has excellent medicinal properties, has been isolated. (T, 0kuda, T, Yoshida an
dT., Hatano, J., Che.
m, Soc,, Perkin I.

1982.9. ) As mentioned above, tannins have many excellent properties and are important as raw materials for medicines, foods, cosmetics, etc. However, there are several problems when actually incorporating them into products. That's what I did. In other words, tannins have a strong mucosal irritating effect, are easily colored or discolored over time, are difficult to dissolve in water, and are unstable in aqueous solutions. It was hoped that the development of

Therefore, in view of the current situation, the present inventors have extensively considered tannin derivatives and continued intensive research. As a result, the conjugate obtained by combining ascorbic acid and tannins has an in vivo lipid peroxide suppressing effect. They have discovered various excellent pharmacological effects such as, and have also found that the above-mentioned problems with tannins can be improved. The present invention is based on this knowledge.

The present invention will be explained in detail below.

The present invention relates to an ascorbic acid-tannin conjugate having a structure in which ascorbic acid and tannins are combined.

The ascorbic acid-tannin conjugate of the present invention is usually obtained by combining ascorbic acid and tannins through a chemical reaction or microbial method, but it can also be obtained by extracting it from plants such as Gennoshoko. It is. The above ascorbic acid-tannin conjugate is generally a white or pale yellow to yellow powder, and is easily soluble in water.
It is safe to eat or apply to the skin. The raw material ascorbic acid used in producing the above-mentioned ascorbic acid-tannin conjugate is generally called vitamin C, and its chemical name is L-ascorbic acid (C6H606: 17).
6.13>. In addition, known tannins can be used as the other raw material, and examples of such tannins include Ako, Akamegashiwa, Scutellariae, Gennoshoko, Waremokou, Asenyaku, Ishutori, Casuarina, Tannins used in the present invention include extracts of gobaica, green tea, black tea, ephedra, etc., and telkevin, keprinic acid, kepulic acid, malotusic acid, punicalagin, geraniin, alnuciin, catechin, shrimp catechin, gallocatechin, epigallocatechin, etc. It is included. Among the above tannins, geraniin extracted from Gennoshoko and Asenyaku tannin extracted from Asenyaku have excellent properties and are preferably used in the present invention. In addition, compositions obtained by reacting the powders of the herbal medicines mentioned above with ascorbic acid, either directly or together with other raw materials or solvents, etc., can also be used if the tannins contained in the herbal medicines are in a state of bonding with ascorbic acid. , is included in the present invention.

Next, we will discuss in detail the method for producing the ascorbic acid-tannin conjugate of the present invention.

The ascorbic acid-tannin conjugate of (e) above is produced by a known method. For example, add 1 to 2 moles of tannins and 2 to 10 moles of ascorbic acid to a water-alcohol mixed solvent, mix and stir to dissolve or disperse uniformly, and then heat to a temperature of 40°C or less, either as it is or by adding an acid or buffer solution. Heat and stir for several hours to react. Next, the ascorbic acid-tannin conjugate is isolated from the resulting reaction solution using column chromatography or the like, and the solvent is removed under reduced pressure.
The method includes rough drying under reduced pressure or freeze drying to obtain an ascorbic acid-tannin conjugate. The important thing here is not to make the reaction temperature too high; if it is heated to a temperature of 60° C. or higher, the tannins will decompose and the desired product will not be obtained. Of course, the ascorbic acid-tannin conjugate of the present invention is not particularly limited in its existing form as long as ascorbic acid and tannins are linked by a chemical bond, and is not limited to the above-mentioned production method. isn't it.

Next, in order to explain the present invention in more detail, an ascorbic acid-geraniin conjugate will be taken as an example of an ascorbic acid-tannin conjugate provided by the present invention, and will be described in detail below.

First, a production example of an ascorbic acid-geraniin conjugate is shown below.

Example 1 Preparation of ascorbic acid-geraniin conjugate Geraniin 200Q was uniformly dissolved in 3ON, a 9:1 mixture of water and methanol, and ascorbic acid 20Q was dissolved in this mixture.
A solution prepared by dissolving 0 g in 20 ρ of water was added and mixed and stirred to make it homogeneous. Next, the pH was adjusted to about 4 using a citric acid-dibasic sodium phosphate buffer, and then the mixture was heated to 37° C. and left to react for 18 hours while being slowly mixed and stirred.

Next, this reaction solution was concentrated under reduced pressure, and the resulting concentrated solution was subjected to resin column chromatography (Toyopearl HW-40F
The ascorbic acid-geraniin conjugate was isolated using the same method as above, and the solvent was removed under reduced pressure, followed by coarse pulverization and drying under reduced pressure to obtain 80 g of an ascorbic acid-geraniin conjugate. (yield
(approximately 34%) The ascorbic acid-geraniin conjugate thus obtained is a cheek-white amorphous powder and has the following characteristic values.

Solubility: Easily soluble in water, methanol, ethanol, and alcohols.

Slightly soluble in ethyl acetate.

Insoluble in ether, non-polar solvents.

Melting point 360”C or higher Specific rotation [α] +26° (C-1, Me
OH) Ultraviolet absorption spectrum λmax, MeOH, nm 223 (4,89
>, (109ε) 282 (4,50>Infrared absorption spectrum νmax (KBr)ca+ 3430,180
0゜1750-1730.1625 Thin layer chromatography (cellulose TLC) developing solvent 7% acetic acid aqueous solution Rf (a O, 251 spots Confirmation of spots Ferric chloride turns gray-blue, sodium nitrite-acetic acid turns orange-yellow and then gray It shows a blue color and later turns yellowish brown.

The structural formula of the ascorbic acid-geraniin conjugate estimated from hydrogen nuclear magnetic resonance spectrum, carbon nuclear magnetic resonance spectrum, etc. is represented by the following formula (1).

(Margin below) 0H C47H34032°5H20 An example of Haku is shown below.

Example 2 Preparation of ascorbic acid-shouko tannin conjugate Geraniin 200C1 of Example-1 is extract 200Q of ascorbic acid. 64 g of ascorbic acid-genoshoko tannin conjugate was obtained.

(The extract of Gennoshoko was extracted with methanol from Japanese Pharmacopoeia Gennoshoko powder.) Example-3
Production of ascorbic acid-acenyactanin conjugate Acenyactanin 200Q was mixed with water-methanol 8:2@
A solution of ascorbic acid 500c+ dissolved in 20.11 parts of water was added thereto and mixed and stirred to make it uniform. Next, the pH was adjusted to 4 using a citric acid-dibasic sodium phosphate buffer solution, and then heated to 37°C and heated to 18°C with slow mixing and stirring.
Leave it for some time to react. Next, this reaction solution was concentrated under reduced pressure, and the ascorbic acid-acenyactanin conjugate was removed from the resulting concentrated solution using polyamide column chromatography, the solvent was distilled off under reduced pressure, and the mixture was coarsely ground and dried under reduced pressure. Ascorbic acid-acenyactanin conjugate 5
4g was obtained. (The Japanese Pharmacopoeia Asenyaku powder was extracted with methanol.) (Yield: approx. 1
0%) Example 4 Extraction and production of ascorbic acid-geraniin conjugate from Shoko spp. Add 2N of acetone-water (7:3 V/V) mixture to 500 g of the above-ground part of dried Shoko spp. and mix thoroughly using a mixer. Mix, stir and filter.

Repeat this operation twice, collect the filtrate, and store it at 40°C.
Concentrate under reduced pressure at the following temperature, and add water to make 500 m.

This solution was washed twice with ethyl ether 100 to remove ether-soluble components. Next, this solution is extracted 20 times using 150 to 200 d of ethyl acetate each time, and ethyl acetate is distilled off under reduced pressure from the resulting extract to obtain extract 409. Next, this extract was mixed with a water-methanol (7:3 V/V) mixture of 400 dk:fglb, 2-3
The precipitated geraniin crystals were separated by filtration, and the filtrate was subjected to C-condensation. The desired fraction was obtained using centrifugal countercurrent partition chromatography, and then subjected to resin column chromatography ( 3ephadeX LH20) and ascorbic acid-geraniin conjugate 0.250ml.
I got it. (Yield: 0.05%) Example 5 Extraction and production of ascorbic acid-geraniin conjugate from Shoko root Add acetone-water (7:3 V/V) mixture 21 to 500 CI of the above-ground part of dried Shoko root. Thoroughly mix and filtrate using a mixer.

Repeat this operation twice, collect the filtrate, and store it at 40°C.
Concentrate under reduced pressure at the following temperature and add water to make 500 d.

This solution was washed twice with 100 mL of ethyl ether to remove ether-soluble components. Next, from this solution, n-butanol (water saturated) was added for 5 hours using 200 d each time.
The extract is extracted twice, and n-butanol is distilled off under reduced pressure from the resulting extract to obtain extract 3C1. Next, this extract was dissolved in 300 ml of water-methanol (7:3 V/V) i mixture,
After leaving to stand for 2 to 3 days, the precipitated geraniin crystals were filtered out, the filtrate was concentrated, and the desired fraction was obtained using centrifugal countercurrent partition chromatography, followed by resin column chromatography (Sephadex). Ascorbic acid-geraniin conjugate was purified using LH20>.
30 C1 (yield: about 0.06%) Next, the above-mentioned ascorbic acid-tannin conjugate can be used in many industrial applications, specifically in medicine, food, cosmetics, etc. It is suitable to be added to the target base as a raw material component or active ingredient.The method of blending can be the same as that for known raw material components or additive components, and the amount to be blended is determined according to the target base. It is arbitrarily selected depending on the purpose and compounded product.

Next, in order to prove the superiority of the ascorbic acid-tannin conjugate of the present invention (Example 1), an in vivo lipid peroxide suppression effect test was conducted using rat liver mitochondria. The results are shown in Table 1. .

At this time, catechin, which is said to have an effect of suppressing lipid peroxide in vivo, was used as a control product.

The test method is as follows.

Samples and animals> Three groups of 10 Wistar rats weighing 200 g were prepared, and these were divided into ascorbic acid-tannin conjugate administration group (group 1), catechin administration group (■ group), and blank (m group) and reared for 5 days. For each administration group, 400 m0 of the sample per body weight was injected intraperitoneally every day, and the animals were injected for 24 hours prior to the experiment, and the animals were sacrificed to death by decapitation. After blood removal, the livers were removed, homogenized, and centrifuged. Mitochondrial fractions were obtained by repeating the separation.

Ultraviolet irradiation> Potassium chloride 0.15 mol/l! was added to the mitochondrial fraction obtained as above. Add Tris-HCl buffer (PH = 7.4) containing 20 ml of solution until the protein concentration is between 4 and 4.
Prepare a mitochondrial suspension at a ratio of 5 m (7/d), place it in a 20 cm diameter petri dish, place it in ice water, and expose it to Toshiba Hikari H-400P ultraviolet light from a distance of 45 cm while keeping the temperature at O'C. Using a lamp, 5.58
Ultraviolet irradiation was performed in an amount of 106er(]/Cm2.

Measurement of lipid peroxide> Mitochondrial lipid peroxide was measured using the thiobarbic acid method (TBA method), and was expressed as the number of malondialdehyde (MAD) molecules per mg of protein.
Expressed in ol (nanomole) units. The protein concentration 1 was measured by the Goulet method using Sigma's bovine albumin as a standard.

Table 1 As shown above, the ascorbic acid-tannin conjugate of the present invention has an excellent in vivo lipid peroxide suppressing effect compared to the control product catechin, and is useful in fields such as cosmetics, medicine, food, and chemical products. It is widely used.

Next, a stability test was conducted on the ascorbic acid-tannin conjugate of the present invention, and the results are shown in Table 2. Samples with low stability tend to become discolored. The numbers in the table are Gardner numbers, and the larger the number, the darker the color.

The test method is as follows.

(a) Ascorbic acid-geraniin conjugate of Example-1 (b) Ascorbic acid-geraniin conjugate of Example-2 (c) Ascorbic acid-acenyactanin conjugate of Example-3 (ni) ) Ascorbic acid-geraniin conjugate of Example-4 (e) L-ascorbic acid (control amount) (f) Gennoshoko tannin control amount similar to that used in Example-2) (g) Blank stability test : 0.5 g of each powdered sample was uniformly dissolved or dispersed in 4.5 q of propylene glycol, and each was weighed into a 4 cm diameter mini-Petri dish, covered with a glass lid, and kept away from direct sunlight. During the experiment period, a water-methanol 8:2 mixture by weight of 10
0d was added to form a solution, and the color depth of this solution was measured using Gardner standard solution.

Table 2 As shown above, the ascorbic acid-tannin conjugate of the present invention has excellent stability compared to the control amount, and is used in cosmetics, medicines, etc.
It does not cause discoloration or coloring when added to foods, chemical products, etc.

Next, in order to confirm the safety of the ascorbic acid-tannin conjugate of the present invention, a toxicity test using animals was conducted, and the results are shown below.

Toxicity Test> (1) Acute Toxicity Test An acute toxicity test was conducted by oral administration using 1 group of 6 DD mouse mice weighing 13 to 19 SJ. The sample is Example-1
Orally administered 9.097 kg of ascorbic acid-geraniin conjugate added to normal feed at 10%.
Life or death was determined after 2 hours.

As a result, no animals were found to have died, and no difference from the normal animal group was observed during subsequent observation.

(2) Subacute toxicity test Using rats, 10% of the ascorbic acid-geraniin conjugate of Example-1 was added to normal feed, and the result was 4.09
/N5F was administered continuously for 12 weeks. As a result, general symptoms,
No abnormalities were observed in body weight, feed intake, urine tests, etc., and no abnormalities that could be attributed to the composition were observed in biochemical or pathological tests.

As described above, the ascorbic acid-tannin conjugate of the present invention is highly safe when eaten or taken, and
It also has excellent stability, and considering its in vivo lipid peroxide suppressing effect, etc., it is an unprecedented product. or,
In clinical trials, although the number of cases was small, it was confirmed to have an effect on improving serum lipids and preventing obesity.

Claims (7)

[Claims] (1) Ascorbic acid-tannin conjugate. (2) The ascorbic acid-tannin conjugate according to claim (1), wherein the ascorbic acid-tannin conjugate is obtained by combining ascorbic acid and tannins. (3) The ascorbic acid-tannin conjugate according to claim (1), which is obtained by extracting the ascorbic acid-tannin conjugate from a plant. (4) The ascorbic acid-tannin conjugate according to claim (1) or (3), which is obtained by extracting the ascorbic acid-tannin conjugate from Gennoshoko. (5) The ascorbic acid-tannin conjugate according to claim (1) or (3), which is obtained by extracting the ascorbic acid-tannin conjugate from Asenyaku. (6) The ascorbic acid-tannin conjugate according to claim (2), wherein the tannins are an extract of Gennosho extract or geraniin. (7) The ascorbic acid-tannin conjugate according to claim (2), wherein the tannins are Asenyaku extract.