JP2003171296A - Superoxide scavenger - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new superoxide scavenger having excellent active oxygen scavenging property and safety. <P>SOLUTION: The superoxide scavenger uses the extract of Cinnamomum camphora Sieb. Glaucescens Alex. Braun as the active ingredient that is extracted in a solvent having ≤6 of dielectric constant and/or ≤4.4 of polar parameter, preferably selected from carbon dioxide, hexane, ethyl acetate, ethanol or their mixture. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel superoxide scavenger derived from H. camphorata.

[0002]

_2. Description of the Related Art Superoxide (O ₂ ⁻ ) is a molecule of oxygen.
It is one of active oxygen generated by one-electron reduction, and is mainly generated in mitochondria and phagocytes in vivo. In mitochondria, superoxide is constantly generated in the electron transport system existing in the inner membrane. On the other hand, phagocytes generate superoxide as a defense mechanism against the invasion of foreign substances such as bacteria and viruses into the living body. The superoxide generated here is usually eliminated by superoxide dismutase (SOD) present in the body, and is kept at a low level.

However, SO due to aging, stress, illness, etc.
Due to the decrease in D activity, the elimination of superoxide is insufficient, or the living tissue is injured due to inflammation, ischemia-reperfusion, excessive production of superoxide due to drugs, myocardial infarction, arteriosclerosis, diabetes, etc. , Causes cancer, stroke, cataract, rheumatism, hypertension and senile dementia. In particular, the skin is an organ in which superoxide is particularly likely to be generated because it is directly stimulated by environmental factors such as ultraviolet rays, and excessive superoxide generation and lipid peroxide generation are likely to occur, so that spots, freckles, etc. The production of the causative melanin pigment is promoted.

Attempts have been made to add SOD to foods, pharmaceuticals, cosmetics and the like in order to prevent the above-mentioned various disorders, but this enzyme is unstable to heat and is easily inactivated, and remarkably. There is a problem of being expensive.

Therefore, attempts have been made to find substances other than SOD, which have the same action as SOD, that is, a superoxide scavenging action. For example, a superoxide scavenger that extracts baicalein in yellow bud, a superoxide scavenger that uses condensed tannin separated and purified from Daihuang and brown have been proposed (Japanese Patent Laid-Open No. 64-50877). However, there is a problem in that only a small amount of Bakeralein extracted from yellow radish is obtained, and the condensed tannins extracted from Daihuang and Tea are not easily extracted and purified.

[0006] In addition, linalool and the like have been reported as compounds having an activity of eliminating superoxide in essential oils. [Krishnakantha TP, L
okesh BR, Indian J Bioche
m Biophys, 30 (2): 133-4 (1
993)] There is a problem that sufficient activity cannot be obtained.

Hosho is Lauraceae
It belongs to the genus Cinnamomum, is mainly distributed from southern China to southeastern Taiwan, and is also cultivated in Japan. Homaba leaves contain about 1% of essential oil component,
% Of which is linaool, and this essential oil is mainly used for perfume. However, other uses have not been heretofore known, and only a slight use as a tea beverage has been proposed (JP-A-10-8492).
2). In addition, first, the present inventors have proposed an “antioxidant” (Japanese Patent Application 2
000-359919), but the detailed effect as a superoxide scavenger was not known.

[0008] Then, as a result of earnest study, linalo
Since ol does not show strong activity, linalo
It was found that ol cannot explain the activity of the superoxide scavenger obtained in the present invention.

[0009]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a new superoxide scavenger having a scavenging effect on superoxide which has a harmful effect on foods and the living body.

[0010]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that
NAMOMUM camphora Sieb. gl
aucences Alex. It was found for the first time that the leaves of Braun) have an excellent superoxide scavenging action, and further it was found that the components extracted with a low-polarity solvent have a strong superoxide scavenging activity. That is, a novel superoxide scavenger can be obtained by containing a camphor leaf component extracted with a solvent having a dielectric constant of 6 or less or a polar parameter of 4.4 or less. The dielectric constant is a value indicating whether the electrolyte (ion-binding substance) is likely to become an ion in the solvent, and it can be said that the smaller the value of the dielectric constant, the lower the polarity of the solvent. In addition, the polar parameter is a parameter including a proton donating property, a proton accepting property, and a dipole contribution, and it can be said that the smaller the value, the lower the polarity of the solvent.

That is, the present invention provides a superoxide scavenger characterized by containing an extract of Rhododendron leaf as an active ingredient.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The superoxide scavenger referred to in the present invention has a function as an antioxidant as a quality preserving agent used in pharmaceuticals, foods, feeds, cosmetics, etc., or a superoxide scavenger in vivo. The present invention is characterized in that the ingredient of Hanamatsu leaf is used as an active ingredient.

No particular limitation is imposed on the place of production and the time of plucking for the camphor leaves used in the present invention. Further, the superoxide scavenger of the present invention may be used as it is, or as a dried product thereof, as it is, or after being processed into any form.
It is preferable to use the extract because it can be used for a wider range of purposes and has a high effect.

The camphor leaf of the present invention can be used as it is in a raw or dried state. In the case of using fresh Rhododendron leaf, it may be used as it is, or it may be used after being cut or used in a slurry form after being ground depending on the application. When the camphor leaves are dried and used, they can be dried by a known method such as shade drying, air drying, hot air drying, and freeze drying. The dried Rhododendron leaf may be used as it is, but may be shaped into chips, or may be crushed or ground to be used. Although not particularly limited, in consideration of extraction efficiency and the like, it is preferable to use dried leaves that are cut or roughly crushed to 5 mm or less.

The sucrose extract of the present invention can be extracted by any method such as heat extraction, continuous extraction, immersion extraction, countercurrent extraction and supercritical extraction.

In the present invention, a solvent having a relatively low polarity is used as the extraction solvent. That is, a solvent having a dielectric constant of 6 or less or a solvent having a polarity parameter of 4.4 or less is used. As the solvent having a dielectric constant of 6 or less, carbon dioxide (dielectric constant e = 1.6, the same applies hereinafter), hexane (1.
9), cyclohexane (2.0), diethyl ether (4.2), chloroform (4.7), ethyl acetate (6.0), and the like. As a solvent having a polar parameter of 4.4 or less, hexane can be used. (Polarity parameter P '=
0.1, the same below), tetrahydrofuran (4.0),
Examples include chloroform (4.1), ethanol (4.3), ethyl acetate (4.4) and the like. These solvents can be used as one kind or a mixed solvent of two or more kinds. Of these, carbon dioxide, hexane and ethyl acetate are particularly preferably used. In addition, ethanol can be used in terms of safety in use in foods.

The dielectric constant of the solvent can be measured by a known method, and a commercially available measuring device can be used. As for the polarity parameter of the solvent, the numerical value used as an index in liquid chromatography or the like can be applied, and the polarity parameter of the mixed solvent can be easily calculated by the following calculation formula.

P '= (Va × Pa + Vb × Pb) × 1 /
100 P ′ = polarity parameter of mixed solvent Va = volume of solvent a (%) Vb = volume of solvent b (%) Pa = polarity parameter of solvent a Pb = polarity parameter of solvent b

It is said that linalool, which is an essential oil component of scented camphor, has superoxide scavenging activity.
The superoxide scavenger of the present invention does not have linaool as an active substance, but has a component other than linaool as an active ingredient.

When solvent extraction is carried out in the present invention, it is preferable to use 5 to 100 parts by weight of the solvent for 1 part by weight of the camphor tree leaves. For example, when hexane is used, the amount of hexane is more preferably 10 to 50 parts by weight.

Further, the extraction temperature is 10 to 80 ° C.
Is preferable and 20-60 degreeC is more preferable. The extraction time is preferably several minutes to 2 hours,
More preferably, the stirring extraction is performed for 60 minutes.

In the present invention, the extraction liquid extracted from the Rhododendron japonica leaves may be made into an aqueous solution or a solid by removing the extraction solvent, if necessary. Further, when the extract is dried to form a solid, it can be dried by adding an excipient such as dextrin, or can be powdered by a known method such as spray drying or freeze drying. The obtained solid matter may be pulverized or ground to form a powder, and may be formed into a tablet, a tablet or the like by a known method.

The superoxide scavenger of the present invention can be added to medicines, foods, feeds and cosmetics.
For example, in foods, drinks such as coffee, tea, tea, fruit juice, soft drink, dairy products such as milk, butter, cheese, cream, miso soup, processed foods such as soup, nutritional supplements, syrups, edible oils, processed oils and fats. , Oils and fats such as margarine, mayonnaise, seasonings such as dressings, liquid or liquid foods such as jams, cooked rice, glutinous rice, bread, processed cereal products such as potato products, confectionery such as chocolate, candy and baked goods, It can be added to meat products such as ham and sausage, and solid foods such as sprinkles.

Antioxidants such as tocopherols,
A plurality of commercially available antioxidants such as ascorbic acid and sodium erythorbate may be used in combination. When used in combination, the ratio of syrup extract to antioxidant is 10:90 to 90 :.
10 is preferable and 50:50 to 80:20 is more preferable.

(Example 1) Production of superoxide scavenger from camphor leaves 20, 50, 99 v / v per 8 g of camphor leaves dried for 1 week
% Ethanol, ethyl acetate, and 160 g of hexane were added, the mixture was stirred at 60 ° C. for 1 hour, and filtered through a wire net. The obtained filtrate was subjected to centrifugal separation, and the solvent was distilled off from the supernatant under reduced pressure to obtain an extract.

(Example 2) Measurement of superoxide scavenging activity of scented camphor leaf extract The superoxide scavenging activity of the scented camphor tree extract prepared in the above Example 1 was measured. In addition, SOD test Wako (Wako Pure Chemical Industries) which is a kit of NBT reduction method was used for the measurement.

2.1, 1.0 of the extract obtained in Example 1
5, 0.525 mg / mL aqueous solution or 50% DMS
O aqueous solution (final concentrations of 0.1, 0.05, 0.
025 mg / mL) 0.1 mL, coloring reagent (0.1 M
Phosphate buffer solution (pH 8), 0.4 mmol / L xanthine, 0.24 mmol / L nitroblue tetrazolium) 1.0 mL was put into a test tube, and the temperature was kept constant at 37 ° C.,
Enzyme solution (xanthine oxidase (from buttermilk)
(0.049 unit / mL) 1.0 mL was added and stirred, and then reacted at 37 ° C. for 20 minutes. After 20 minutes, 2.0 mL of a reaction stop solution (sodium dodecyl sulfate 69 mmol / L) was added, and the absorbance at 560 nm was measured (this test: sample, E _S ). As a sample, a system in which a diluting solvent (water or a 50% DMSO aqueous solution) was added instead of the camphor extract (main test: blind, E _BL ), a blank solution (0.1 M phosphate buffer) was used instead of the enzyme solution. Solution (pH 8)) added (blinded: specimen blinded, ES _-BL ), diluted solvent instead of the sample, blank solution added instead of the enzyme solution (blinded: reagent blind, _EBL-BL ) was measured and the superoxide scavenging rate was calculated according to the following formula. Also,
(-)-Epigallocatech as a comparative control
in gallate and butylhydroxy
The superoxide scavenging activity was similarly measured for toluene (BHT).

The measurement procedure is shown in Table 1 below.

[0029]

[Table 1]

The results are shown in Table 2.

[0031]

[Table 2] Superoxide scavenging activity

As is clear from the results in Table 2, the lower the polarity of the extraction solvent, the higher the superoxide scavenging activity, and the hexane extract showed the strongest activity. This activity is (-)-epigal at 0.1 mg / mL.
It was almost the same as that of locatechin gallate. Furthermore, at lower concentrations, the hexane extract showed stronger activity. In addition, (-)-epigalloc
Since atechin gallate has to be isolated from green tea or the like, its purification is not easy and costly. On the other hand, the present invention is industrially advantageous because a sufficient effect can be obtained with a crude extract.

In order to confirm that the superoxide scavenging activity of the present invention is not derived from essential oil, the superoxide scavenging activity of linalool, which is the main component of the camphor essential oil, was measured. As a result, the superoxide scavenging activity of linalool was clearly lower than that of the extract of the solvent having the polar parameter of 4.4 or less, that is, 99% ethanol extract, ethyl acetate extract, and hexane extract in Table 2. It became a thing. Table 3 shows the measurement results of linalool's superoxide scavenging activity.

[0034]

Table 3 Superoxide scavenging activity of linalool

(Example 3) Soft drink water orange juice 20 parts, sucrose 15 parts, citric acid 0.2
Parts, 0.1 part of the extract obtained in Example 1, 0.1 part of fragrance, and 64.6 parts of water were added, and the mixture was sterilized by heating at 80 ° C. for 30 minutes and then cooled to give a soft drink.

[0036]

INDUSTRIAL APPLICABILITY The superoxide scavenger of the present invention is obtained from natural camphor leaves, has high safety,
Moreover, since it has a strong superoxide scavenging activity, it is useful for stabilizing pharmaceuticals, foods, cosmetics, etc. and erasing superoxide in vivo.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 39/06 A61P 39/06 C09K 15/34 C09K 15/34 F term (reference) 2B150 AB20 DD31 DD57 4B018 LB08 MD61 ME06 MF01 4C083 AA111 BB47 CC02 EE07 EE16 FF01 4C088 AB33 AC05 BA08 CA07 CA09 CA10 CA11 MA52 NA14 ZC02 ZC37 4H025 BA01

Claims (4)

[Claims] 1. A superoxide scavenger containing an extract of H. camphor as an active ingredient, which is obtained by extracting H. camphor leaves with a solvent having a dielectric constant of 6 or less. 2. A superoxide scavenger containing an extract of H. camphor as an active ingredient, which is obtained by extracting H. camphor leaves with a solvent having a polarity parameter of 4.4 or less. 3. The superoxide scavenger according to claim 1, wherein the solvent used for extraction is carbon dioxide, hexane or ethyl acetate. 4. Pharmaceuticals, foods, feeds and cosmetics comprising the superoxide scavenger according to claim 1 and / or 2.