JP3410223B2 - Health food and active oxygen scavenger containing rabudosine or its salt - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a health food and an active oxygen scavenger containing rhabdosine or a salt thereof as an active ingredient.

[0002]

2. Description of the Related Art "Enmeiso" is also known as "Hikiokoshi" and is a crude drug that is widely used in Chinese medicine and folk medicine. About the extract of this life-prolonging grass, gastric juice secretion inhibitory action,
Antitumor action, antibacterial action and the like have already been known, and their active ingredients and the like have been disclosed. Further, the addition of this extract to cosmetics is a known fact, and the cosmetics type permission standard (1993) permits the addition of various extracts as Hikikoshi extract to various cosmetics. Sho 63-141915] and tyrosinase inhibition (whitening) effect [Patent Document 3]
06] is disclosed.

"Rabdosin" means Rabdosia j
aponica [I.Agata et al., Chem.Pharm.Bul]
l. 36 (8), 3223-3225 (1988)] or Macrotomia euc
hroma [M. Nishizawa et al., Phytochemistry 29 (8), 2645-26
49 (1990)] is one of the ingredients contained in plants,
It has the following structure:

[Chemical 1] Although the structure of this rhabdosin and the method for purifying this substance from each plant are described in the above-mentioned literature, the physiological action of rhabdosin has never been known so far.

In recent years, it has been clarified that active oxygen radicals react with proteins, DNA or unsaturated fatty acids in the living body to cause cell damage and cause aging of the living body and various diseases. is there. Various diseases caused by these active oxygen radicals include aging, arteriosclerosis, inflammation, rheumatism, brain disease, diabetes, etc.
["Free radicals and medicine", edited by Kiyomi Kikukawa et al., Hirokawa Shoten, 187-311 (1991)]. Particularly, in atherosclerosis, low density lipoprotein (LDL) is associated with atherosclerosis.
It was clarified that peroxidation was involved, and probucol, a drug for treating hyperlipidemia, has high antioxidative activity.
[Noriko Noguchi, "Arteriosclerosis", Modern Medicine, 25 (10), 3339-33
P. 45 (1993)].

[0005]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention For the above-mentioned diseases thought to involve active oxygen radicals,
It has been confirmed that administration of an active oxygen radical scavenger or an antioxidant is effective, and administration of ascorbic acid (vitamin C), α-tocopherol (vitamin E), superoxide dismutase (SOD), etc. has been attempted. Although this has some effect,
(Kikawa Kiyomi et al. And Noguchi Noriko, supra), the effects of these radical scavengers are not yet sufficient. Also,
There are many problems with the stability of these compounds depending on the administration route, and it is desired to develop a safe, stable, and effective active oxygen radical scavenger that can withstand use in the prevention or treatment of various diseases. It is rare. The present inventors extensively sought an active oxygen radical scavenger capable of withstanding such a demand for herbal medicines, and conducted earnest research.

[0006]

As a result of this study, the extremely high effect of active oxygen radical scavenger was recognized on the extract of life-prolonging grass, which was not known as active oxygen radical scavenger or antioxidant until now. As a result of further research, it was found that rhabdosine, a component of the life-prolonging grass, present in this extract has extremely high active oxygen radical scavenging activity and antioxidant activity. Furthermore, by incorporating purified rhabdosin or a salt thereof or an extract containing a high concentration of rhabdosin, it is possible to use this extremely high active oxygen radical scavenging activity and antioxidant activity for the prevention and / or treatment of various diseases. The present invention has been completed.

That is, the present invention provides a health food and an active oxygen scavenger for preventing and / or treating various diseases caused by active oxygen, which contains rhabdosine or a salt thereof as an active ingredient. It is provided. Various diseases caused by active oxygen are as mentioned above, and therefore the active oxygen scavenger of the present invention is an anti-aging agent, anti-arteriosclerotic agent, anti-inflammatory agent, anti-rheumatic agent, anti-brain disease agent, anti-diabetic agent. Work as The health food and active oxygen scavenger of the present invention preferably contain rhabdosin or a salt thereof obtained by extraction from Enmeisou. In addition, the health food and active oxygen scavenger of the present invention can be prepared by blending a protracted herb extract containing a high concentration of rhabdosin.

Rabdosin can be obtained from a plant containing rhabdosin by the method described in the above-mentioned references by I. Agata et al. Or M. Nishizawa et al. For example,
By dipping and extracting leaves or whole plants of life-prolonging grass in water and / or ethanol at room temperature to reflux temperature,
A rhabdosine-containing extract is obtained. The extract is then washed and extracted with a suitable organic solvent (such as ethyl acetate) and the organic phase is further combined with a suitable column chromatography [size exclusion, adsorption and partition chromatography (water / developing solution as a developing solution). Purified rhabdosin can be obtained by fractionating with [preferably ethanol]].
By adding the purified rhabdosin thus obtained, the active ingredient, rhabdosin, can be added at a high concentration so that the desired effects can be sufficiently exhibited. Further, by properly selecting the extraction method, it is possible to obtain an extract containing rhabdosin in a higher concentration than other components. This extract may be further purified as described above, or may be directly added to the health food and active oxygen scavenger of the present invention.

Rabdosin can react with many inorganic and organic bases to form non-toxic salts. The health food and active oxygen scavenger of the present invention may contain such a non-toxic salt of rhabdosine. Examples of the base include inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide and potassium carbonate, and organic bases such as monoethanolamine, triethanolamine, arginine and histidine. Such a salt of rhabdosin can be prepared by a conventional method.

Examples of the health food of the present invention containing rhabdosine or a salt thereof include granules, tablet confectioneries, jellies, candy, beverages and the like, which can be eaten as necessary. As the active oxygen scavenger, ointments, patches and the like as external preparations, tablets, pills, powders, emulsions, liquids, syrups, suspensions, gelatin capsules etc. as internal preparations, etc. Further, forms such as injections and sprays can also be exemplified, and these can be administered by routes such as transdermal, oral, injection and intranasal.

The raw materials used in preparing the health food may be those commonly used in this field, such as lactose, dextrose, sucrose, sorbitol,
Examples thereof include mannitol, apple fiber, soybean fiber, meat extract, black vinegar extract, gelatin, corn starch, honey, animal and vegetable oils and fats, and polysaccharides.
Examples of the carrier used when preparing the active oxygen scavenger include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate alginate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, and cellulose derivatives. , Tragacanth, gelatin, syrup, methyl hydroxybenzoate, talc, magnesium stearate, water, mineral oil and the like.

The health food and active oxygen scavenger of the present invention may further contain lubricants, emulsifiers, suspending agents, antioxidants, preservatives, sweeteners and / or flavors, and the like. It may contain active ingredients (including water-soluble vitamins, oil-soluble vitamins, etc.). The health food and active oxygen scavenger of the present invention comprising such ingredients may be manufactured according to methods well known in the art.

Rabdosin or a salt thereof is effective over a wide dose range. Therefore, the daily dose of rhabdosine or a salt thereof is usually about 0.0 per kg of body weight.
It may be in the range of 1 to 1000 mg, preferably about 0.1 to 500 mg, more preferably about 1 to 300 mg.
This amount is eaten or administered in one or several divided portions. However, the actual dose will be determined with regard to the age, weight and response of the subject to be treated, the severity of the condition and the route of administration chosen.

[0014]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Example 1 Purification of Rhabdosin from Enmeisou 100g of Enmeisou (stem and leaf) 100% 80% ethanol
0 ml was added, homogenized at room temperature and filtered. The filtrate was concentrated under reduced pressure at 40 ° C. and dried. 100 ml of water to this
Redissolve by adding 10% H ₃ PO ₄
Adjusted to 3.0. The formed precipitate was removed by filtration to obtain 100 ml of a filtrate. The filtrate is ethyl acetate 100
The mixture was extracted 3 times with ml, and the ethyl acetate layer was concentrated under reduced pressure at 40 ° C. to obtain 6 g of ethyl acetate extract. To this ethyl acetate extract, add 18 ml of ethanol and dissolve, add 27 ml of water to this and stir well, then add ethanol: water (4: 6) 25
ml was added and left to stand. The mixture is filtered and the filtrate is Kie
It was subjected to selgel 60 silanisiert (Art 7719) column chromatography (developing solvent ethanol: water = 4: 6) to obtain a fraction containing rhabdosin at a high concentration. The fraction thus obtained was concentrated and then further separated on Sephadex LH-2.
It was subjected to 0 column chromatography (developing solvent ethanol: water = 4: 6) to obtain a crude rhabdosine fraction. Further, this was subjected to Toyopearl HW40 fine column chromatography (developing solvent ethanol: water = 4: 6), and the fraction containing rhabdosin was concentrated and dried under reduced pressure to obtain 45 mg of purified rhabdosin. In the above chromatography,
The fraction containing rhabdosin was confirmed by HPLC [apparatus Pharmacia LKB; column Pep RPC 5/5 (P
harmacia); solvent 2% acetic acid aqueous solution: acetonitrile = 8
5:15; flow rate 0.7 ml / min; detection wavelength UV313n
m]. Under this condition, the peak at an elution time of around 16 minutes was rhabdosin, and the fraction containing this peak was designated as the rhabdosin-containing fraction. The physical properties of the thus obtained purified rhabdosin are shown below, and these values were in agreement with the literature values. [α] ²⁵ _D : -78 ° (MeOH; c = 3.5) FAB-MS: m / z 719 [M + H] ⁺ , m / z 717 [M-
_{^{H] - λ max (MeOH)}} : nm (logε) 254 (4.20), 28
4 (4.00), 318sh, 346 (4.02)

Example 2 Activity of Rhabdosin The action of the purified rhabdosin obtained in Example 1 as an active oxygen radical scavenger was confirmed by the following three methods. A. According to ^{_{ESR · OH, O 2 - ·}} , spin trapping method is known which utilizes the ESR (electron spin resonance) as a specific and method for accurately quantify directly the ¹ O ₂ scavenging action of active oxygen radicals. That is, active oxygen species generated by an appropriate method are trapped with a spin trap agent such as DMPO,
This is a method for quantifying an ESR signal specific to each reactive oxygen species. A method is known in which an active oxygen radical scavenger such as labdosine is allowed to coexist in this measurement system, and the activity is measured by measuring the degree of signal attenuation.
["Free radicals and medicine" (supra), Chapter 2, "Methods for measuring free radicals", pages 103-120 (1991)]. As a result of performing such a measurement with respect to each active oxygen radical species of OH, O ₂ ⁻ , and ¹ O ₂ generated by each method described below, as shown in Table 1 below, It showed higher activity than vitamin E and vitamin C, which have been known so far as substances having a high scavenging effect on these reactive oxygen species.

The active oxygen radical scavenging activity was measured by the ESR spin trap method using DMPO as follows. Device : ESR spectrum is JES-RE1X (X-band) ESR spectrometer [JEOL Ltd. (Tokyo)]
Was measured at 22 ° C. Mn (II) ion doped into MgO as a standard substance [Digimarker: JEOL Ltd.]
Was used. Data analysis was performed by Macintosh LCII of Apple Computer. Specimens : Ascorbic acid, α-tocopherol, and rhabdosin. Experimental method : (1) The OH radical scavenging activity was measured by the Fenton reaction system. 0.01 mM FeSO ₄ (diluted with H ₂ O) 45 μl 25% DMPO (diluted with H ₂ O) 10 μl 10 mM Sample (diluted with H ₂ O or 1% SDS solution) X μl H ₂ O or 1% SDS solution 100-X μl 0.1 mM H ₂ (diluted with H ₂ O) O ₂ 45μl total volume 200μl solvent: water (Rabudoshin and ascorbic acid for) 1% SDS solution (for α- tocopherol) ₍₂₎ O 2 ^- · radical scavenging activity xanthine - xanthine It was measured with an oxidase system. 0.1M phosphate buffer (pH 7.4) X μl 10 mM Sample (phosphate buffer, diluted with 1% SDS solution) 100-X μl 2.0 mM hypoxanthine (diluted with phosphate buffer) 50 μl 5.5 mM DTPA (phosphate buffer) 30 μl 25% DMPO (diluted in phosphate buffer) 10 μl 0.272 Units / ml Xanthine oxidase (diluted in phosphate buffer) 50 μl Total volume 240 μl Solvent: 0.1 M phosphate buffer (pH 7.4) ( Rhabdosin and for ascorbic acid) 1% SDS solution (for α-tocopherol) (3) ¹ O ₂ scavenging activity was measured with a hematoporphyrin-UV-A irradiation system. 0.1 M Tris-HCl buffer (pH 8.0) 100-X μl 0.2 mM Hematoporphyrin (diluted with Tris-HCl buffer) 50 μl 10 mM Sample (diluted with Tris-HCl buffer) X μl 200 mM 2,2,6,6- Tetramethylpiperidone / HCl (diluted with Tris-HCl buffer) 50 μl Total volume 200 μl Solvent: 0.1 M Tris-HCl buffer (pH 8.0) (for rhabdosin and ascorbic acid) 1% SDS solution (for α-tocopherol) ) The above reagents were sequentially placed in a polycarbonate tube having a volume of 1.5 ml, stirred with a vortex mixer, and then ESR was added.
ESR transferred to a highly sensitive aqueous cell for measurement (Labotech)
It was fixed in the cavity of the device. The spectrum measurement was started 30 seconds after the stirring. ESR measurement conditions : magnetic field 336.5 ± 5 mT, modulation frequency 1
00 kHz, modulation width 0.1 mT, sweep time 2 minutes, response time 0.1 second, output 5 mW, amplification ratio 1000.

[0017]

TABLE 1: active oxygen radical scavenging activity IC ₅₀ (μM) Compound O ₂ ^- · scavenging action · OH scavenging action ¹ O ₂ scavenging action Rabudoshin 12.90 11.55 ± 3.95 414 Ascorbic acid 33.00 12.70 ± 4.33 105 ± 6 α-tocopherol 220.00 3900 435 ± 141

B. UV irradiation for skin fibroblasts
Inhibition of cell damage As a pathway of damage caused by ultraviolet rays on the body, O ₂ absorbs ultraviolet energy to generate reactive oxygen species, which react with biological constituents such as unsaturated fatty acids or DNA forming cell membranes. Proposed routes that cause disability
[Juichi Nishi, "Radical reaction of epidermal lipid peroxidation process by ultraviolet rays", Kurume Medical Society, 54 (11), 745-756 (199)
1); and Ryohei Ogura, “Light damage is enhanced in moist skin,” Nisshikai, 101 (11), 1285-1289 (199).
1)]. The effect of rhabdosin on the UV damage to the living body was examined using cultured skin fibroblasts. As a result, as shown in Table 2 below and FIGS. 1 and 2, the high inhibitory effect of rhabdosin was proved.

The inhibitory effect of UV cell damage on cultured skin fibroblasts was examined as follows. Experimental method : Cell: NB1RGB (normal human skin fibroblast) Medium: RITC80-7 (10% FBS) UV irradiation: UV-B (305 nm) FL20S ・ E-30 / U
V-RAD 10 METER 60 minutes (about 3.2 J / cm ² ) 5 minutes (about 0.3 J / cm ² ) Specimen: ascorbic acid, rhabdosine Operating procedure : Adjust NB1RGB to 8 × 10 ⁴ pieces / ml,
100 μl of each well was placed in a 96-well plate, and
Culturing was performed at 5 ° C. and 5% CO ₂ . After 48 hours, the wells were washed with Hank's buffer (no dye), 100 μl of sample adjusted to various concentrations with Hank's buffer was added, and then 6
UV irradiation was performed for 0 minutes or 5 minutes. Then, the wells were washed again with 100 μl of Hank's buffer, 200 μl of medium was added, and the cells were cultured at 37 ° C. under 5% CO ₂ for 48 hours. Then, the number of viable cells was measured by the neutral red measuring method. Analysis of experimental results :

## EQU1 ## A = [(OD _CW −OD _EW ) / OD _CW ] × 100 B = [(OD _TCW −OD _TEW ) / OD _TCW ] × 100 C = [(A−B) / A] × 100 A: UV irradiation Damage rate (%) B: UV damage rate (%) in the presence of scavenger C: Cell damage inhibition rate (%) CW: Control well (live cell count) EW: UV exposure well (live cell count) TCW: Sample treatment UV-irradiated control wells (live cell count) TEW: UV exposure wells treated with sample (live cell count)

[0020]

[Table 2] Table 2: Inhibitory effect of UV cell damage on cultured skin fibroblasts IC ₅₀ (mM) Compound UV irradiation dose (J / cm ² ) 3.2 0.3 Ascorbic acid 7.59 3.44 Rabdosin 0.52 0.27

C. Of unsaturated fatty acids by oxygen consumption measurement
Inhibition of peroxidation (chain-breaking action by scavenging lipid peroxyl radicals and organic radicals) The effect of rhabdosin to suppress biological damage caused by reactive oxygen species is the target of the first peroxidation reaction in the living body (especially on the skin surface). Squalene [Yoshiyuki Kono et al., Lipid peroxidation in human epidermis], J. Soc.
Cosmet. Chem. Jpn. 27 (1), pp. 33-40 (1993)] and methyl linoleate which is a methyl ester of linoleic acid which is a cell membrane constituent. As a result, Table 3 below
Also, as shown in Table 4, the high inhibitory effect of rhabdosin was proved.

The inhibitory effect of squalene and methyl linoleate on the peroxidation reaction was investigated by the oxygen absorption method as follows. Experimental method : Device: Dissolved oxygen concentration recorder GILSON OXYGRA
PH model 5/6 Specimens: Rabdosin, Ascorbic acid, α-Tocopherol, Probucol Initiator: 2,2'-Azobis (2-amidinopropane) dihydrochloride (AAPH) Procedure : (1) Triton X-100 An emulsified 4.87 mM squalene micellar aqueous solution was added to a dissolved oxygen recorder cell (volume: 1.5 ml).
To 15 mM with constant stirring at 50 ° C.
Aqueous solution of the sample adjusted to (labdosine, ascorbic acid)
Alternatively, 1 μl of a 10.6% Triton X-100 aqueous solution (α-tocopherol, probucol) of the sample (final concentration 10 μm)
M) Added. Next, 50 μl of 60 mM AAPH aqueous solution (final concentration: 0.2 mM) was added to the cell, and the recording of the dissolved oxygen concentration was immediately started. The results are shown in Table 3. (2) A micellar aqueous solution of 6.79 mM methyl linoleate emulsified with Triton X-100 was dissolved in a cell of a dissolved oxygen recorder (capacity 1.
5 ml) and keep it at 35 ° C with constant stirring for 1
Aqueous solution (labdosine, ascorbic acid) adjusted to 5 mM or 10.6% Triton X-100 aqueous solution of the sample
(α-tocopherol, probucol) 1 μl (final concentration
10 μM) was added. Next, 50 mM AAPH aqueous solution 50
μl (final concentration 0.2 mM) was added to the cell and recording of the dissolved oxygen concentration was started immediately. The results are shown in Table 4.

[0023]

[Table 3] Table 3: Inhibitory compound against peroxidation reaction of squalene Inhibition rate (%) Inhibition time (sec) Rabdosine 46.0 588 α-tocopherol 59.6 333 Ascorbic acid 72 163

[Table 4] Table 4: Inhibitory compound against peroxidation reaction of methyl linoleate Inhibition rate (%) Inhibition time (sec) Rabdosine 75.2 1770 Ascorbic acid 79.2 595 Probucol 28.6 970

[Equation 2] Inhibition rate (%) = (1-Rinh / Rp) x 100 Rinh: Oxygen consumption rate when each sample is added Rp: Oxygen consumption rate when no sample is added Inhibition time (seconds): Reaction start To Rinh = Rp

As described above, rhabdosine, which is a component contained in plants such as life-prolonging grass, has a high activity of directly eliminating reactive oxygen species from ESR.
It was shown to suppress the peroxidation reaction of unsaturated fatty acid, which is a biological component, with extremely high efficiency, and it was also shown to suppress the damage of cultured cells of the skin by UV. . Rhabdosin has long been known as an active oxygen radical scavenger or antioxidant, has a higher active oxygen radical scavenging ability than vitamin C and vitamin E used in cosmetics, health foods or pharmaceuticals, It was found that it has an antioxidant power superior to that of probucol, which has been proved to have high antioxidant power as a therapeutic drug for lipemia. Therefore, it was proved that rhabdosin is effective against various diseases caused by these active oxygen and can be an ingredient of excellent cosmetics, health foods or pharmaceuticals.

Example 3 Health Food A Using the purified rhabdosine obtained in Example 1, a health food having the following composition was prepared.

[Table 5] After mixing the respective materials in a fluidized granulator, water was sprayed for granulation, and the granules were obtained by drying at an inlet air temperature of 80 ° C.

Example 4 Health Food B Using the purified rhabdosine obtained in Example 1, a health food having the following composition was prepared.

[Table 6] The above materials were thoroughly mixed with 10 kg of water and then heated to 60 ° C. to form a paste. This is put into 50 kg of soybean oil that has been heated to 70 ° C. in advance, and a squid-type stirring blade
Mix well with a stirrer equipped with (2 sheets). After confirming that the paste was dispersed in soybean oil, the mixture was cooled to 5 ° C. and filtered to separate the produced particles. The obtained particles were washed with n-hexane and then dried to obtain microcapsules containing the above materials.

Example 5 Hard Gelatin Capsule A hard gelatin capsule is prepared using the following ingredients.

[Table 7]

Example 6 Tablets Tablets are prepared using the following ingredients.

[Table 8]

Example 7 Suspension A suspension is prepared using the following ingredients.

[Table 9] Component Usage Purified Rabdosin 50 mg Sodium carboxymethylcellulose 50 mg Syrup 1.25 ml Benzoic acid solution 0.10 ml Flavoring agent Suitable amount Coloring agent Suitable amount Pure water Total up to 5 ml

Example 8 An intravenous formulation is prepared using the following ingredients.

[Table 10]

As described above, purified rhabdosine was incorporated into health foods and active oxygen scavengers as an active ingredient for preventing and / or treating various diseases caused by active oxygen. When these health foods and active oxygen scavengers were applied to animals, good results were obtained.

[Brief description of drawings]

FIG. 1 is a graph showing the inhibitory effect of antioxidants on cell damage caused by UV.

FIG. 2 is a graph showing the inhibitory effect of antioxidants on cell damage caused by UV.

─────────────────────────────────────────────────── ─── Continuation of the front page (56) Reference JP-A-2-182160 (JP, A) JP-A-6-24937 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) A23L 1/30 A61K 31/235 A61K 35/78

Claims (4)

(57) [Claims] 1. A health food containing rhabdosin or a salt thereof obtained by extracting from Enmeisou. 2. The health food according to claim 1, which contains the life-prolonging grass extract containing a high concentration of rhabdosin. 3. An active oxygen scavenger containing rhabdosine or a salt thereof obtained by extraction from Enmeisou. 4. The active oxygen scavenger according to claim 3, which contains a life-saving plant extract containing Rhabdosin in a high concentration.