JPH0723391B2 - Ascorbic acid-geraniin conjugate - Google Patents

Description

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

Since ancient times, tannins have an astringent action, an intestinal regulating action, an oral cooling action, a blood pressure lowering action, a hemostatic action, a skin conditioning action, an anti-inflammatory action,
Many pharmacological effects such as a sunscreen effect and an antiaging effect have been discovered, and tannin has attracted attention as a useful physiologically active substance in recent years. Tannins are widely distributed in the plant kingdom, and typical examples thereof include green tea, black tea, Acacia catechu, Gobaishi, and Japanese ginger.
Although these have been widely used and prized before the existence of tannins was known, research in various fields has progressed in recent years, and the utility of these plants has been characterized mainly by the tannins contained in them. I knew that I could be attached. For example, dried leaf stems of Ganoderma lucidum are conventionally used as an anti-diarrheal drug, as an intestinal medicine, and are listed in the Japanese Pharmacopoeia, but the active ingredient is said to be tannins. As a result of research conducted by the researchers, geraniin, which is a tannin that is less astringent than these tannins and has excellent medicinal effects, has been isolated.
(T.Okuda, T.Yoshida and T.Hatano, J.Chem.Soc., Per
kin I, 1982.9.) Tannin has a number of excellent properties as described above and is an important raw material for pharmaceuticals, foods, cosmetics, etc. There was the problem of. That is, strong mucous membrane stimulating effect, easily colored or discolored over time, tannin is difficult to dissolve in water, tannin is unstable in an aqueous solution, and the like,
The improvement of these and the development of new medicinal components were desired.

Therefore, in view of such a present situation, the present inventors extensively studied tannin derivatives, and as a result of continuing diligent research, as a result of binding ascorbic acid and geraniin to a conjugate obtained in vivo, lipid peroxide suppressing effect and the like. It has been found that various excellent pharmacological effects of tannins can be solved, and that the above problems of tannin can be improved. The present invention is based on such findings.

The present invention will be described in detail below.

The present invention relates to an ascorbic acid-geraniin conjugate having a structure in which ascorbic acid and geraniin are bound.

The ascorbic acid-geraniin conjugate of the present invention is usually
Although it is obtained by binding ascorbic acid and geraniin by a chemical reaction or a microbial method, it can also be obtained by extracting from a plant such as Ganoderma lucidum. The above-mentioned ascorbic acid-geraniin conjugate is generally a white or pale yellow to yellow powder, is easily soluble in water, and is safe when eaten or applied to the skin. Ascorbic acid, which is a raw material used for producing the above-mentioned ascorbic acid-geraniin conjugate, is generally called vitamin C, and its chemical name is L-ascorbic acid (C6H806: 176.13). Geraniin, which is the other raw material, is a kind of tannin extracted from plants such as ginger ginger. In addition, a composition obtained by reacting powder of a plant such as ginger ginger directly with ascorbic acid together with other raw materials or a solvent, etc., a state in which geraniin contained in a plant such as ginger ginger is bound to ascorbic acid. Needless to say, this is included in the present invention.

Next, the method for producing the ascorbic acid-geraniin conjugate of the present invention will be described in detail.

The ascorbic acid-geraniin conjugate described above is made by known methods. For example, 1 to 2 moles of geraniin and 2 to 10 moles of ascorbic acid are added to a water-alcohol mixed solvent and mixed and stirred to uniformly dissolve or disperse. Warm and leave to react for several hours with stirring. Next, a method for obtaining an ascorbic acid-geraniin conjugate by isolating the ascorbic acid-geraniin conjugate from the obtained reaction solution using column chromatography or the like, distilling off the solvent under reduced pressure, and further drying under reduced pressure or freeze-drying. And so on. Here, what is important is that the reaction temperature is not too high, and when heated to a temperature of 60 ° C. or higher, decomposition of geraniin occurs,
I can't get what I want. Of course, ascorbic acid-geraniin conjugate of the present invention, ascorbic acid and geraniin, as long as they are bound by a chemical bond, regardless of the existence form, in particular, it is not limited to the above production method. Absent.

Next, in order to explain the present invention in more detail, the ascorbic acid-geraniin conjugate provided by the present invention will be described in detail below.

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

Example-1 Production of ascorbic acid-geraniin conjugate Geraniin 200 g was uniformly dissolved in 30 l of a water-methanol 9: 1 weight mixture, and 200 g of ascorbic acid was dissolved in 20 l of the solution, and the mixture was stirred. It should be uniform. Next, the pH is adjusted to 4 using a citric acid-dibasic sodium phosphate buffer, heated to 37 ° C., and left to react for 18 hours while slowly mixing and stirring. Next, this reaction solution was concentrated under reduced pressure, and the ascorbic acid-geraniin conjugate was isolated from the resulting concentrated solution using resin column chromatography (Toyopearl HW-40F), and the solvent was distilled off under reduced pressure and further coarsely pulverized. After drying under reduced pressure, 80 g of an ascorbic acid-geraniin conjugate was obtained. (Yield about 34%) The ascorbic acid-geraniin conjugate thus obtained is a white-white amorphous powder and has the following characteristic values.

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

 Hardly soluble in ethyl acetate.

 Insoluble in ether and non-polar solvents.

Melting point 360 ° C or more Specific rotation [α] ¹⁵ _D + 26 ° (C = 1, MeOH) Ultraviolet absorption spectrum λmax.MeOH, nm 223 (4.89), (logε) 282 (4.50) Infrared absorption spectrum νmax (KBr) cm ^-1 3430, 1800, 1750-1730, 1625 Thin layer chromatography (Cellulose TLC) Developing solvent 7% acetic acid aqueous solution Rf value 0.25 1 spot Confirmation of spots Ferric chloride gives gray blue, sodium nitrite-acetic acid gives orange yellow After that, it becomes grayish blue and then becomes yellowish brown.

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

Another embodiment is shown below.

Example-2 Ascorbic acid from ginger-
Extraction production of geraniin conjugate To 2 g of acetone-water (7: 3 V / V) mixed solution (2 l) was added to 500 g of dried aerial parts of Gensho ginger, and the mixture was thoroughly mixed and stirred using a mixer and then filtered. This operation is repeated twice, and the filtrate is collected, concentrated under reduced pressure at a temperature of 40 ° C. or lower, and water is added to make 500 ml. This solution is washed twice with 100 ml of ethyl ether to remove ether-soluble matter. Then use 150-200 ml of ethyl acetate each time from this solution
Extraction is performed 20 times, and ethyl acetate is distilled off from the obtained extract under reduced pressure to obtain 40 g of an extract. Next, this extract was dissolved in 400 ml of a water-methanol (7: 3V / V) mixed solution and allowed to stand for 2 to 3 days, the precipitated geraniin crystals were filtered off, and the filtrate was concentrated and then centrifuged. The target fraction was obtained using flow partition chromatography and further purified using resin column chromatography (Sephadex LH20) to obtain 0.25 g of ascorbic acid-geraniin conjugate. (Yield about 0.05%) The ascorbic acid-geraniin conjugate thus obtained was a white crystalline amorphous powder, and had substantially the same characteristic values as those of the ascorbic acid-geraniin conjugate obtained in Example 1. It was something that had.

Example 3 Ascorbic acid from ginger ginger-
Extraction production of geraniin conjugate To 2 g of acetone-water (7: 3 V / V) mixed solution (2 l) was added to 500 g of dried aerial parts of Gensho ginger, and the mixture was thoroughly mixed and stirred using a mixer and then filtered. This operation is repeated twice, and the filtrate is collected, concentrated under reduced pressure at a temperature of 40 ° C. or lower, and water is added to make 500 ml. This solution is washed twice with 100 ml of ethyl ether to remove ether-soluble matter. Next, n-butanol (saturated with water) was added to this solution for 200 m each time.
It is extracted 5 times using l, and n-butanol is distilled off from the obtained extract under reduced pressure to obtain 300 g of an extract. Next, the extract was dissolved in 300 ml of a water-methanol (7: 3 V / V) mixture,
After standing for 2-3 days, the precipitated geraniin crystals are filtered off, the filtrate is concentrated, the desired fraction is obtained by centrifugal countercurrent distribution chromatography, and further resin column chromatography (Sephadex LH20). Was used to obtain 0.30 g of an ascorbic acid-geraniin conjugate.
(Yield about 0.06%) The ascorbic acid-geraniin conjugate thus obtained was a white-white amorphous powder, and had substantially the same characteristic values as those of the ascorbic acid-geraniin conjugate obtained in Example 1. It was something that had.

Next, the above-mentioned ascorbic acid-geraniin conjugate is industrially applicable to many uses, and specifically added as a raw material component or active ingredient of medicines, foods, cosmetics, etc. to a target base. It is suitable for The compounding method may be the same as the known raw material component or additive component, and the compounding amount thereof is arbitrarily selected according to the intended use and the compounded product.

Next, with respect to the ascorbic acid-geraniin conjugate of the present invention (Example 1), in order to prove its superiority, the results of in vivo lipid peroxide inhibitory effect test using rat liver mitochondria 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 about 200 g were prepared, each of which was treated with an ascorbic acid-geraniin conjugate (group I), a catechin administration group (group II), and a blank (III). Group), and breeding was carried out for 5 days. Each administration group was injected intraperitoneally with a sample of 400 mg per 1 kg of body weight every day, and after fasting for 24 hours prior to the experiment, it was considered deadly due to decapitation, and the liver was taken out after blood removal and homogenized,
The centrifugation was repeated to obtain a mitochondrial fraction.

<Ultraviolet irradiation> In the mitochondrial fraction obtained as described above, a tris-hydrochloric acid buffer solution (PH = 0.15 mol / l solution 20 ml) was added.
7.4) is added to make a mitochondrial suspension so that the protein concentration becomes 4-5 mg / ml, put this in a petri dish with a diameter of 20 cm, and place it in ice water.
From a distance of, using the H-400P ultraviolet lamp for Toshiba lightening,
Ultraviolet irradiation of 5.58 × 10 ⁶ erg / cm ² was performed.

<Measurement of lipid peroxides> Measurement of mitochondrial lipid peroxides was performed using the thiobarbituric acid method (TBA method) and expressed in nmol (nanomolar) units as the number of molecules of malondialdehyde (MAD) per mg of protein. did. The protein was quantified by the Burette method using bovine albumin from Sigma as a standard.

As described above, the ascorbic acid-geraniin conjugate of the present invention is superior in the effect of suppressing lipid peroxides in vivo as compared with catechin which is a control product, and is widely used in the fields of cosmetics, pharmaceuticals, foods, chemical products and the like. It is possible.

Next, a stability test was conducted on the ascorbic acid-geraniin conjugate of the present invention, and the results are shown in Table-2. The less stable the sample, the more likely it is to color. The numerical values in the table are Gardner numbers, and the larger the value, the darker the color.

The test method is as follows.

<Sample> (a) Ascorbic acid-geraniin conjugate of Example-1 (b) Ascorbic acid-geraniin conjugate of Example-2 (c) L-ascorbic acid (control product) (d) Japanese Pharmacopoeia Genoshoko powder Genoshochotannin extracted from methanol (control) (e) Blank Stability test: 0.5 g of powdered sample and 4.5 g of propylene glycol each
What was uniformly dissolved or dispersed in the sample, weighed in a 4cmφ mini dish, left with a glass lid for the duration of the experiment at the window not exposed to direct sunlight, then water-methanol 8: 100 ml of a 2-weight mixture was added to make a solution, and the color strength was measured using a Gardner standard solution.

As described above, the ascorbic acid-geraniin conjugate of the present invention is superior in stability as compared with the control product, and does not cause discoloration or coloring when blended in cosmetics, medicines, foods, chemical products and the like. is there.

Next, in order to confirm the safety of the ascorbic acid-geraniin 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 by oral administration was performed using 6 groups of 1 dd mouse having a body weight of 13 to 19 g. As a sample, the ascorbic acid-geraniin conjugate of Example 1 was used, and 9.0 g / Kg of 10% added to a normal feed was orally administered, and life or death 72 hours later was determined. As a result, none died and no difference from the normal animal group was observed in the subsequent observation for 1 week.

(2) Subacute toxicity test 4.0 g / Kg 12 of the ascorbic acid-geraniin conjugate of Example-1 added to a normal feed in an amount of 10% using rats.
It was continuously administered for a week. As a result, no abnormalities were found in general symptoms, body weight feed intake, urinalysis, etc., and no abnormalities thought to be caused by the composition were observed in biochemical tests and pathological tests.

As described above, the ascorbic acid-geraniin conjugate of the present invention is highly safe when eaten or taken,
Further, it is also excellent in stability, and when considering its in vivo lipid peroxide suppressing effect and the like, it is an unprecedented one.
Further, in a clinical test, an effect of improving serum lipids and an effect of preventing obesity were confirmed although the number of cases was small.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI Technical display location A61K 7/48 9051-4C 31/70 ACN ADN 9454-4C

Claims (4)

[Claims] 1. An ascorbic acid-geraniin conjugate. 2. The ascorbic acid-geraniin conjugate obtained by binding ascorbic acid and geraniin.
Geraniin conjugate. 3. The ascorbic acid-geraniin conjugate according to claim (1), which is obtained by extracting the ascorbic acid-geraniin conjugate from Genzo ginger. 4. The ascorbic acid-geraniin conjugate according to any one of claims (1) to (3), wherein the ascorbic acid-geraniin conjugate has the following characteristic values. Solubility Easily soluble in water, methanol, ethanol and alcohols. Hardly soluble in ethyl acetate. Insoluble in ether and non-polar solvents. Melting point 360 ° C or more Specific rotation [α] ¹⁵ _D + 26 ° (C = 1, MeOH) Ultraviolet absorption spectrum λmax.MeOH, nm 223 (4.89), (logε) 282 (4.50) Infrared absorption spectrum νmax (KBr) cm ^-1 3430, 1800, 1750-1730, 1625 Thin layer chromatography (Cellulose TLC) Developing solvent 7% acetic acid aqueous solution Rf value 0.25 1 spot Confirmation of spots Ferric chloride gives gray blue, sodium nitrite-acetic acid gives orange yellow After that, it becomes grayish blue and then becomes yellowish brown.