JPH0434969B2 - - Google Patents

Description

[Detailed description of the invention]

[1] Object of the invention and industrial application field The present invention relates to a novel application (utilization) of a flavoid component (such as baicalein) contained in Scutellariae or an extract containing baicalein. That is, the above component according to the present invention contains active oxygen radicals: OR (O ^- ₂ , H ₂ O ₂ , OH・,
Therefore, it can be used as an active oxygen scavenger and remover when incorporated into formulations in the medical or cosmetic fields (including quasi-drugs). can. [2] Conventional technology (A) Active oxygen: Effect of OR Active oxygen (hereinafter abbreviated as OR for convenience) is
Research has continued into various reactions and their effects in living organisms, mainly involving the four types of reluminescence: O - 2 _, H ₂ O ₂ , OH・^, and chemiluminescence. For example, foreign enemies such as bacteria, viruses, and foreign substances
When it invades the living body, neutrophils, monocytes, and macrophages, which are phagocytes in the blood, begin phagocytosis against the invading foreign enemy. OR is produced in order to dissolve foreign substances that are ingested by the body. At this time, in addition to melting poor food, OR directly exerts a strong bacterial action against bacteria and foreign substances, serving as a means of self-defense for living organisms. However, in this case, if phagocytes are excessively stimulated, the production and secretion of OR and lysosomal enzymes will increase, resulting in various harmful effects on the living body. In other words, tissue damage caused by lysosomal enzymes has been known for a long time, but in this case, increased OR also causes high-grade tissue damage, and in recent years, diseases caused by OR grown in living organisms have been reported.
Many things have been revealed. For example, regarding the relationship between the skin and OR, the skin is an organ that comes into direct contact with the outside world and is therefore susceptible to the effects of these environmental factors. If OR is present in excess in the skin, it reacts with unsaturated fatty acids and increases lipid peroxides. In addition, formulations for cosmetics, external preparations, etc. contain fats and oils in their systems (formulations), so they are susceptible to the production of lipid peroxides due to the action of OR. These generated lipid peroxides trigger harmful effects such as arteriosclerosis, carcinogenesis, aging, membrane destruction, protein denaturation, and hemolysis.
For the skin, it causes inflammation, edema, necrosis, pigmentation, etc. To this end, we need to eliminate ORs that are deeply involved in each disease, that is, to eliminate ORs that are caused by excess ORs.
BACKGROUND OF THE INVENTION Excellent OR-capturing and removing substances are required for the treatment and prevention of diseases, and the search for such substances has continued. (B) Investigation of substances that capture and remove OR
The existence of SOD (superoxide dismutase), catalase, and glutathione peroxidase in plant organisms is known. For example, SOD is well known as an enzyme that specifically consumes O ^- ₂ in living organisms.
Like SOD and catalase, OR scavengers include tocopherols and the known antioxidants BHA and BHT. Further, as components derived from natural products, flavonoids such as the above-mentioned tocopherol (vitamin E), quercetin, rutin, campherol, myricetin, fuisetin, and morin are known. (c) Fields of application (utilization) of substances that capture and remove ORs Examples of the fields of application of substances that have the ability to capture and remove ORs include, for example, when used in cosmetics or medicinal topical liniments (including quasi-drugs). Acts as a preventive agent for skin aging. For example, when the skin is exposed to ultraviolet rays, the production of OR and even lipid peroxide increases, which at the same time promotes tissue damage and melanin formation, resulting in fine wrinkles and It is said that the appearance of age spots increases. Therefore, if the OR trapping remover is added to a prescription for external application, it will have a beautifying effect on the skin, but it should not be applied or rubbed on exposed areas, especially as it may cause stains and stains caused by sunlight. Prevents pigmentation such as freckles,
Moreover, it can be applied to the treatment thereof. Other diseases caused by excess OR include dermatitis herpetiformis, linear immunoglobulin bullous dermatosis, severe cement dermatitis, X-ray dermatitis, burns, trauma, solar dermatitis, Contact dermatitis, housewife's eczema, atopic dermatitis, eczema, Behchiet's disease, etc. are known.
It is known that these inflammations are deeply related to the production of prostaglandes as well as the production of lipid peroxides. In addition, in cases such as burns, if the increase in OR is suppressed, keloid formation will not develop, and
It is believed that symptoms can be alleviated to the extent that scarring occurs. In addition, other diseases accompanied by excessive production of OR include Kawasaki disease, which occurs within 5 days of onset;
It is known that myocardial infarction, aggravation of vascular disorders during brain trauma, damage and hardening of tissues such as the lungs and heart due to chemical substances, and exacerbation of diabetes.Furthermore, chronic stimulation by OR is said to be an important promoter of carcinogenesis. ing. Furthermore, autoimmune diseases that are exacerbated by an increase in OR are known to include cutaneous eczema, Crohn's disease, malignant rheumatoid arthritis, and ulcerative colitis. On the other hand, diseases in which the production of OR and the production of lipid peroxide are thought to have a close relationship include:
These include cataracts, pulmonary sclerosis, emphysema, bronchial damage, and as mentioned above, abnormal skin pigmentation symptoms such as age spots and freckles. The production of lipid peroxide is said to enhance the formation of melanin pigment, and it is also said that facial dyspigmentation may be caused by the production of OR directly promoting melanin formation. . Additionally, the spleen and small intestine are rich in xanthine oxidase. For this reason, tissue damage due to OR is likely to occur, and as a result, diabetes is likely to occur. Furthermore, in cases of vascular disorders caused by arteriosclerosis in the brain and heart (e.g., stroke, myocardial infarction), or during brain trauma, blood flow disturbances due to blood clots and rapid temporary atrophy of blood vessels can cause intravascular damage. ,
This results in an ischemic state, resulting in a state of oxygen deficiency. In this condition, xanthine dehydrogenase in the blood vessels changes to xanthine oxidase, which generates a large amount of OR in the blood flow within the blood vessels, causing fatal damage to the blood vessel walls. Furthermore, when looking at abnormalities of volujirin metabolism, for example, in the case of cutaneous vorujiria, vorujirin bodies increase, and in response to ultraviolet rays, large amounts of OR and lipid peroxide are produced by vorujirin bodies, and as a result, It is said to promote tissue damage and melanin formation, and in this case, OR production is known to act as a promoter of skin cancer. (D) Development status of OR scavenging and removal agents As mentioned in (A) to (c) above, substances with excellent OR scavenging and removal ability are said to be able to be used for the treatment and prevention of various diseases, and are used in a variety of ways. The development of new drugs is actively underway. And for example, SOD
Several applications have been proposed in this field. However, SOD is a very unstable substance,
Various stabilization methods have been considered in systems used in formulating cosmetics, which are difficult to formulate, and in order to exert their effects. For example, formulations using microencapsulation, ribosome formation, etc. have also been proposed. [3] Problems to be solved by the invention Most of the substances known so far that have the ability to capture and remove OR have no effect (action) on the metabolic system of cell membranes, or on the contrary, Many showed a tendency to slightly lower metabolism. However, suppression of phagocytic activity and metabolism, including OR production, is disadvantageous for the organism. That is, in the metabolic system of neutrophils and lymphocytes, it is desirable to have an OR trapping and removing substance that has an effect of enhancing the metabolic system. However, until now, such substances have been rare. The inventors compensated for the unstable shortcomings of SOD,
Furthermore, SOD is an enzyme that converts O ^- ₂ into O ₂ and H 2 O 2, and then converts H _{2 O 2 into H 2 O 2 by catalase} . There is a biochemical reaction mechanism between ₂ O and O ₂ , but here we will broadly discuss not only O - 2, but also O ^- ₂ .
We are looking for a substance that can remove H ₂ O ₂ , OH・, and chemiluminescence, and can also enhance the activity (metabolism) of each cell, including the activity of phagocytes, which is hardly found in other scavengers. was the theme for its development, and research was carried out diligently. In other words, the problem to be solved by the present invention is that among the many natural products, there is an effect of reducing (suppressing) the production of the four types of OR mentioned above, and at the same time, The condition is that the metabolism of each cell is not reduced (suppressed), and it has a scavenger effect (SOD-like effect) that can remove excess OR produced in the body. ) succeeded in discovering the substance. The inventor has continued to search steadily and steadily based on this purpose (theme). As a result, they discovered that an extract obtained from Scutellariae (scutellariae extract powder) has an excellent OR trapping and removal effect, leading to the present invention. Meanwhile, the inventors have already made the following table (Table 1)
As shown in the following, various applications have been developed for extracts obtained from Scutellariae, and the present invention has further expanded the field of application of Scutellariae.

[Table] [4] Structure of the Invention The present invention provides an OR trapping and removal agent containing baicalein or an extract containing baicalein obtained from Scutellariae (root of Scutellariae, a plant belonging to the Lamiaceae family). It consists of being used as a. Below, in order to specifically demonstrate the present invention, the test method, results, etc. will be shown and described in detail. (A) Identification of Components (Substances) As mentioned above, the OR trapping and removing agent according to the present invention is an extract derived from Scutellariae and is characterized by containing baicalein. flavonoid components,
Consists of extract mixed with other ingredients. As for the extraction method, it may be obtained by the methods shown in (a) to (e) in Table 1 above, or an extract obtained by the method shown below may be used. In addition, without being limited to these methods, extracts obtained based on other known extraction methods may be used,
In this case, it is essential that the obtained extract contains at least baicalein or baicalin (baicalin is a glycoside of baicalein) as a flavonoid. (B) Extraction method (manufacturing method) Add 600 ml of purified water to 100 g of Scutellariae powder and perform pretreatment immersion at 50° for 3 hours. After completing this operation, add 1.8 alcohol (ethanol or methanol, etc.) to the immersion liquid, immersion extraction,
Alternatively, extraction is performed under heating to obtain the filtrate. This filtrate contains nylon (bolyamide, cellulose,
After filtration, 2 parts of purified water are added to 1 part of the filtrate. As a result, a yellow extract powder is precipitated, and this is obtained and dried to obtain about 10 g of Scutellariae extract (Scutellaria scutellariae extract powder). (c) Characteristics of the extract The scutellariae extract obtained by the extraction method shown in (B) above is an extract that mainly contains baicalein, as shown in the following table (Table 2) and Figure 1. In addition, other substances such as scutellariae were also confirmed.
The figure is a chart obtained by high performance liquid chromatography, and the test method was carried out using the following conditions. "High performance liquid chromatography measurement conditions" Column: LS-410 (ODS), 4.6 mm x 25 cm. Eluent; acetonitrile 43: water 27: 0.6% phosphoric acid 30.
Flow rate: 1.0ml/min. Column temperature: 40℃. detection;
Ultraviolet wavelength 280nm.

[Table] (D) Action or Effect The action of the OR scavenging remover according to the present invention is as follows:
To specifically illustrate, the results obtained using the extract shown in (C) above (Scutellaria scutellariae extract powder) are shown in Tables 3 to 6 below, but first, according to the present invention, Its action or effect can be summarized as follows. This extract reduces all four types of OR produced not only in the cell system but also in the xanthine-xanthine oxidase system. In addition, it has a scavenger effect that removes excess OR produced in vivo without reducing the metabolism of each cell, including methyltransferase and phosphorivase _A2 . And its effect is powerful. In particular, H ₂ O ₂ was completely removed in both the cell system and the xanthine-xanthine oxidase system. In addition, the inhibitory effect on lipid peroxide formation was also observed. In other words, this extract is evaluated as an OR scavenging remover with an unprecedentedly strong cell activating effect, has immunostimulatory ability, and can be used in the prevention and treatment of various cancers, or in conjunction with other anticancer drugs. When used in combination, the side effects of these substances can be suppressed, and they can also be used as anticancer agents.

【table】

【table】

【table】

[Table] (E) Commentary on test methods related to action or effect Test methods related to Table 3: OR (O ^- ₂ , H ₂ O ₂ ,
To measure the effect of OH, chemiluminescence) on the human neutrophil system in relation to the production of active oxygen (OR), it was isolated from venous blood according to the method described in References (1) and (2) below. Based on zymosan-stimulated healthy human neutrophils (polymorph honuclear leukocytes: PMNs) treated with 1 mg/ml obsonin, Krebs Ringer's phosphate buffer containing 5% glucose (however, colorless Hank's solution was used for chemiluminescence measurements). ),
Prepare PMN culture solution. PMN is separated from the PMN-containing layer using the Ficoll-Hybaky gradient density method, and then the PMN-containing layer is mixed with the test serum and the molecular weight is 17.
Dilute _x104 with 6% dextran saline and lyse contaminated red blood cells with 0.876% ammonium chloride. Zymosan used for PMN stimulation is described in the literature (1).
According to ~(2), the test serum was previously treated with obsonin. Of OR, O ^- ₂ is reduced by cytochrome,
Measured with a Beckmann spectrometer. For H ₂ O ₂ , the degree of fluorescence reduction of scoboretin was measured using a fluorescence spectrophotometer (Hitachi MPF4 model). For OH, α-ketomethiobutyric acid (KMB) was added, and the production of ethylene gas was measured using a gas chromatograph (Hitachi). Chemiluminescence is performed under strict light shielding.
Measured using a liquid scintillation counter (Vaccard, Illinois, USA). Test method according to Table 4: For the xanthine-xanthine oxidase system OR production ability test, 0.1 ml of 13.5 mg hypoxanthine dissolved in 50 ml of physiological saline and 0.0 ml of 50 mM EDTA dissolved in 2.3 ml of physiological saline. Mix 0.5ml of 0.7μ xanthine oxidase, and 0.1ml of this mixed solution.
Further dilute and dissolve 2 ml in potassium phosphate buffer to produce OR. Measurement of O ^- ₂ , H ₂ O ₂ , OH・
It was carried out according to the measurement method shown in Table). (Literature on measurement methods) (1) Niwa et al.: Blood Vol. 64,
p.994-999 (1984) (2) Niwa et al.: Arch Dermatol (Arch
De rmatol) Vol. 121, p. 73-78 (1985) Test method according to Table 5: In the measurement of docosahexenoic acid lipid peroxide forming ability, after adding Scutellaria scutellariae extract powder and incubating for 20 hours, , a 1:1 mixture of 0.67% thiobarbituric acid (TBA):acetic acid was added, and 95
°C, heat for 45 minutes. After cooling with water, add n-butanol and centrifuge at 1250 x g for 10 minutes. Fluorescence spectrophotometer (Hitachi MPF4 model) using n-butanol layer
Measured by. Test method according to Table 6: Human neutrophil and lymphocyte cell membrane enzyme activity (activation) ability was measured by the following method. Preparation of microsomal fractions of neutrophil membranes and lymphocyte membranes Neutrophils and lymphocytes were separated from unperiphered blood of healthy individuals using Ficoll-Hypaque (manufactured by Wako Pure Chemical Industries, Ltd.), and then added to 0.25M sucrose. and treated with 24W ultrasonic waves for 10 seconds in ice water. Next, after centrifugation at 4°C, 14000xg for 10 minutes, the supernatant was ultracentrifuged at 4°C, 140000xg for 60 minutes to obtain a microsomal fraction as a precipitate. Floating on M-sucrose. Methyltransferase activity 100mM Tris-HCl (PH8.0), 0.1mM ethylene glycol-β-aminoethyl ether-N,
N,N′,N′-tetraacetic acid (EGTA) 50 μM S-
Adenosyl-L-[ ^3H -methyl]methionine ([ ³
H-SAM) 2μCi and microsomal fraction
Mix 200μg/protein and incubate at 37℃ for 30 minutes. Next, stop the reaction with 0.6ml of 0.25N hydrochloric acid,
After extracting the phospholipid components using chloroform,
[ ^3H -methyl] incorporated into membrane phospholipid components
, scintillation counter (cross card,
(Illinois, USA). Phospholibase A ₂ active microsomal fraction 200μg/protein
Suspended in 0.25M sucrose, 10μ (0.5μCi)
β-[ ^14C ]Arachidonyl PC (β-[ ^1-14C ]Arachidonyl-α'-stearyl-L-d-phosphatidylcholine) (Amersham, 59.3mCi/
nmol) and incubate at 30°C for 90 minutes.
The reaction was stopped by adding 1.5 ml of a chloroform-methanol (2:1) mixture at 4°C, and the chloroform layer was obtained by centrifugation at 2000 xg for 5 minutes. After drying the chloroform layer under reduced pressure at 30°C,
Add a chloroform-methanol (2:1) mixture, spot on TLC, and develop with a petroleum ether; ether: acetic acid (80:30:1) mixture. The separated β-[ ¹⁴ C]arachidonic acid is measured using a scintillation counter, and the free arachidonic acid is determined as a percentage using the following formula. (Formula) Separated β-[ ¹⁴ C] arachidonic acid / β-[ ¹⁴ C] arachidonic acid PC + isolated β-[ ¹⁴ C] arachidonic acid x 100 PC...Phosphatidylcholine (F) Consideration regarding action or effect Scutellariae Until now, there have been no reports that the components contained in Scutellariae and Scutellariae have an OR trapping and removal effect (SOD-like effect). As a result, the Scutellariae extract was found to be an excellent OR trapping and removing agent, with the results shown in Tables 3 to 5. Regarding the mechanism of action of Scutellaria scutellariae extract as an excellent OR scavenging remover,
Based on the results obtained, and with a few considerations, as shown in Table 6, we have investigated the methylation of the cell membranes of both neutrophils and lymphocytes, as well as phospholibase _A2 . It also increases markedly. Substances that exhibit such high values are extremely rare. This is because when methyltransferulase is activated, abduction, or translocation, of the inner and outer membranes occurs, resulting in a decrease in the viscosity of the cell membrane and an increase in fluidity. This subsequently activates phospholibase A ₂ and turns the arachidonic acid cascade, leading to the production of OR, the secretion of lysosomal enzymes, the promotion of migratory motility, oligophagy, etc., and the metabolic activity (reaction) of the cell. This is thought to have enhanced the In other words, Scutellariae extract powder has the effect of accelerating the metabolism of each cell, including phagocytes, and also has the effect of increasing the metabolism of each cell, including phagocytes.
It has the effect of removing OR. In other words, if we comprehensively evaluate this based on the results obtained in Table 6, we can see that the action of this extract is not only that it has a function as a simple OR scavenging remover, but also that A major feature of this product is that it is an OR trapping and removal agent with cell activation and immunostimulation abilities. [5] Effects of the invention Substances that act as scavengers for the production of OR have been known. but,
Most of them had no effect (reaction) on cell metabolism or caused a slight decrease in metabolic activity. On the other hand, it was found that the scutellariae extract according to the present invention exhibits remarkable effects on both functions, as shown in Tables 3 to 6. Substances that effectively function for both of these functions were extremely limited, but the present invention has made it possible to further expand the effective use of Scutellariae. When the present invention is applied to actual medicines or cosmetics, the dosage should be 10 ^-5 to 10 -5 if the data shown in Tables 3 to 6 are used.
A concentration of 10 ^-4 M is estimated to be the optimal effective concentration. According to this, not only the above-mentioned extract (scutellariae extract powder) but also the above-mentioned first
Scutellariae extracts obtained using the extraction methods shown in the table or other methods can also be used. However, in that case, it is essential to include baicalein as a flavonoid derived from Scutellariae, but other components derived from Scutellariae may also be included. In addition, it has very advantageous conditions in terms of formulation, namely, SOD is a known and proposed product aimed at trapping and removing OR.
Thermal stability and stability after dissolution in an aqueous system are markedly lacking, and various formulation methods such as ribosome formation and microencapsulation have been investigated as measures to solve this problem. However, due to the rapidity of SOD's responsiveness or the associated sustainability, it has not yet been possible to formulate a sufficient formulation. Therefore, there is currently a need for a substance that can replace SOD and has an OR capture and removal function, but until now there have been very few substances that can be used for this purpose. That is, in the present invention, as shown in the previous tables (Tables 3 to 5) and further Table 6, substances that have significant effects are further limited, and until now, stability in formulation has also been limited. However, it has been difficult to develop a practical OR scavenger remover. On the other hand, the extract according to the present invention obtained as a powder has excellent thermal stability and is stable for a long period of time if stored in a tightly closed container protected from light. Furthermore, regarding the stability in formulation (compounding), for example, according to the publication (a) shown in the previous table (Table 1), the system in the formulation is stable, and it is suitable for external application. It also has great merits, such as the fact that it can be easily incorporated and used in formulations (cosmetics, etc.). In addition, one important point in the formulation system is to formulate the formulation with a pH on the acidic side. That is, the extract according to the invention is a simple OR
It is not a trapping/removal agent, but has excellent cell activating ability as shown in Table 6, and its field of use is that it functions as a cell activator and also functions as an immunostimulant. . Therefore, its application is for diseases such as those shown in (A) to (B) in [2] above, as well as for the prevention and treatment of various cancers, and various drugs for the purpose of anti-aging. In addition, in the form of external application such as cosmetics, it is effective for preventing spots, freckles, and wrinkles, and for hair, it prevents hair loss, promotes hair growth, In particular, it is possible to prevent hair loss from occurring due to the administration of known anticancer drugs and other therapeutic agents. The inventor of the present invention selected many natural products (animals and plants) as starting materials and continued to search steadily, and finally developed an excellent OR scavenging agent that is unprecedented. , With the present invention as a trigger, from now on, baicalein has a flavone and flavonoid skeleton, which is a structural feature of baicalein.
It is expected that the development of OR scavenging and removal agents based on other plant-based or animal-based ingredients will become even more active, and it is thought that the effects they will bring will be significant.

[Brief explanation of drawings]

FIG. 1 shows that the scutellariae extract (scutellaria extract powder) obtained by the extraction method shown in (B) in [4] Structure of the Invention section in the detailed explanation section of the specification has the following properties: This chart is based on liquid chromatography. In Figure 1, A is baicalein and B is ougonin.

Claims (1)

[Claims] 1. An active oxygen scavenging and removal agent having cell activating ability and immunostimulating ability, which uses baicalein or an extract containing baicalein.