JP4477285B2 - Anti-inflammatory agent, PGE2 production inhibitor, IL-1α production inhibitor and IL-6 production inhibitor - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an anti-inflammatory agent, PGE ₂ The present invention relates to a production inhibitor, an IL-1α production inhibitor, and an IL-6 production inhibitor.
[0002]
[Prior art]
In general, inflammation is a defense reaction of a living body that attempts to repair and regenerate the damaged site when an invasion that causes some harm to the living body is applied or damage occurs. This defense reaction suppresses the spread of tissue and cell damage, eliminates the cause of damage, and tries to repair the tissue, but on the other hand, excessive pain, swelling, itching, fever, etc. occur, Symptoms that are unfavorable to the living body may occur, for example, damage may be amplified in reverse.
Therefore, it is necessary to suppress excessive inflammation to reduce the patient's pain and accelerate the treatment of damage, and as an inflammation suppression means, administration of an anti-inflammatory agent having an anti-inflammatory effect is performed.
[0003]
[Problems to be solved by the invention]
However, anti-inflammatory agents that have been developed so far have excellent effects, but most of them are known to have a strong physiological action in addition to the intended anti-inflammatory action.
[0004]
Accordingly, an object of the present invention is to provide an anti-inflammatory agent that has an anti-inflammatory action and at the same time has high safety.
[0005]
[Means for Solving the Problems]
For the first time by the inventors, carnosic acid, carnosol, as well as Novara extraction Thing is PGE which has an excellent anti-inflammatory action and is a chemical mediator of inflammatory reaction in particular ₂ (Prostaglongin E ₂ ) And IL-1α (interleukin-1α) and IL-6 (interleukin-6), which are inflammatory cytokines, were found to be effective and at the same time safe for the living body. Was completed.
[0006]
That is, the anti-inflammatory agent of the present invention includes carnosic acid, carnosol, as well as Novara extraction Thing It contains at least 1 sort chosen from these. According to the present invention, carnosic acid, carnosol, as well as Novara extraction Thing PGE containing at least one selected from ₂ Production inhibitors, IL-1α production inhibitors, and IL-6 production inhibitors are provided.
[0007]
The primrose used in the anti-inflammatory agent of the present invention belongs to the genus Praia (Copper jade), and is a fertile perennial plant.
The extract of primroses is obtained by drying primroses, chopping them, immersing them in water, a hydrophilic organic solvent such as ethanol, or a mixture thereof, and filtering the resulting liquid. The extract can be divided into a foliage part, a fruit part, a hairy root part and the like, and a foliage part and a fruit part are particularly preferably used.
Rosemary extract, Sakura extract, Cycad extract, Novara extract, Strawberry extract, Yukinoshita extract, Kudzu extract, Lemongrass extract and Yukiyanagi extract can be obtained in the same manner as Primrose extract. . Carnosic acid and carnosol are also isolated from rosemary.
[0008]
Primrose extract, rosemary extract, carnosic acid, cherry extract, cycad extract, carnosol, nobara extract, strawberry extract, saxifrage extract, kudzu extract, lemongrass extract and snow willow extract in anti-inflammatory agents The blending amount of at least one selected from the products is not particularly limited, but is preferably 0.0001 to 20.0% by weight, more preferably 0.0005 to dry solid weight based on the total amount. 5.0% by weight.
When the blending amount is less than 0.0001% by weight, the effect of the present invention cannot be sufficiently obtained. On the other hand, when the blending amount exceeds 20.0% by weight, the improvement of the effect commensurate with the increase is not recognized.
[0009]
The anti-inflammatory agent of this invention can be made into dosage forms, such as lotions, emulsions, creams, ointments, packs, etc. as a skin external preparation.
[0010]
In the anti-inflammatory agent of the present invention, a dye, an antiseptic, a surfactant, a fragrance, a pigment and the like can be appropriately blended.
[0011]
【Example】
Hereinafter, the present invention will be described in more detail based on examples. However, the present invention is not limited to these examples. In the following preparation examples and examples, “%” means “% by weight” unless otherwise specified.
[0012]
Preparation Example 1
[Preparation of primrose extract]
Primrose was obtained by mass growth in a foliage culture system. The obtained primrose was naturally dried, divided into a foliage part, a fruit part and a hairy root part, each cut into small pieces, immersed in 50% ethanol aqueous solution or water overnight, and filtered with a filter paper to obtain an extract.
[0013]
[Fractionation of primrose extract]
The 50% ethanol extract of the obtained foliage part was concentrated by an evaporator until it became paste-like. The same amount of water and hexane was added to the concentrated extract, and the mixture was distributed using a separatory funnel.
The aqueous layer was separated into a precipitate and a supernatant by centrifugation, the precipitate was concentrated to dryness, dissolved or dispersed in 50% ethanol, 50% ethanol was distilled off, and freeze-dried. The hexane layer was dissolved in 50% ethanol after hexane was distilled off, and the ethanol was distilled off and then lyophilized.
[0014]
Example 1
(PGE of primrose extract) ₂ Production suppression effect)
[Creation of multilayered epidermal cells (liquid layer culture method)]
Frozen normal human epidermis cells (primary, neonatal foreskin derived: NHEK (F) CASCADE, tertiary culture) grown semi-confluent using HuMedia-KG2 (Kurabo) in a 96-well culture plate (IWAKI) at 60,000 cells / well). After culturing for 24 hours and confirming that the cells are adhered, 1.2 mM Ca is used to promote differentiation of epidermal cells. ²⁺ The medium was changed to K110 type II additive containing K + (K110 II (+), Kyokuto). 1.2 mM Ca ²⁺ / K110II (+) was cultured for 7 days to obtain stratified epidermal cells. The medium was changed every 2 days.
[0015]
PGE of primrose extract ₂ The production inhibitory effect was confirmed by the following experiment.
The medium of the obtained stratified epidermal cells is removed by aspiration to obtain 1.2 mM Ca. ²⁺ The cells were washed with 200 μl of / K110II (−) and preincubated with 200 μl of the same medium for 2 hours. Thereafter, the medium was removed by suction and irradiated with UVB (ultraviolet B region). The UVB lamp used was TOSHIBA FL20SE, and the irradiation device used was Delmale irradiation device (Eisai). Irradiation was started about 1 hour after the lamp was turned on. Irradiation intensity is 0.5mW / cm ² Adjust the height to be 50mJ / cm ² Of UVB. During the irradiation, the lid of the microplate was removed.
1.2 mM Ca containing primrose extract (water extract and 50% ethanol extract) collected from the foliage and fruit after UVB irradiation ²⁺ / K110II (−) 200 μl (Primrose concentration: 1 μg / ml, 10 μg / ml, 100 μg / ml) was added and cultured. After culturing for 24 hours, the supernatant was recovered and PGE in the supernatant was recovered. ₂ PGE ₂ Quantification was performed using EIA system (BIOTRAK). A micropleat reader EL312e (BIO-TEK INSTRUMENTS INC) was used for the absorbance measurement.
The results are shown in FIG.
In FIG. 1, A shows the results relating to the 50% ethanol extract of the foliage part, B the water extract of the foliage part, C the 50% ethanol extract of the fruit part, and D the result relating to the water extract of the fruit part.
[0016]
As apparent from FIGS. 1A and 1B, the 50% ethanol extract of primrose leaves is PEG when the primrose extract is not added. ₂ Was about 20 ng / ml medium, but the concentration of primrose extract decreased to about 17 ng / ml medium at 10 μg / ml, and to about 10 ng / ml medium at 100 μg / ml. The water extract of primrose stem leaves is PEG when the primrose extract is not added. ₂ Was about 20 ng / ml medium, but the concentration of primrose extract decreased to about 15 ng / ml medium at 10 μg / ml and to about 10 ng / ml medium at 100 μg / ml. That is, primrose leaves and stems are both 50% ethanol extract and water extract PGE. ₂ It had a production inhibitory effect.
Similarly, as is clear from C and D, 50% ethanol extract of the fruit part is PEG when the primrose extract is not added. ₂ Production of about 21 ng / ml medium was reduced to about 16 ng / ml medium at a concentration of 100 μg / ml, and when the water extract was not added with the primrose extract, ₂ Of about 21 ng / ml medium was reduced to about 18 ng / ml medium at a concentration of 100 μg / ml. ₂ It had a production inhibitory effect.
In particular, the activity of the extract of the foliage is about 50% inhibition at a concentration of 100 μg / ml, and compared with the case where the extract of primrose is not contained. ₂ The production suppression effect was very excellent.
“Ng / ml medium” means PGE produced in 1 ml of medium. ₂ Production amount.
[0017]
Example 2
(PGE of water fraction and hexane fraction of 50% ethanol extract of primrose leaves ₂ Production suppression effect)
50% ethanol extract of primroses (SAL700), 50% ethanol extract of primroses-aqueous layer fraction (SAL701), 50% ethanol extract of primroses-aqueous layer precipitation (SAL702) And 50% ethanol extract-hexane fraction (SAL703) of primrose leaves and primroses at concentrations of primroses of 25 μg / ml, 50 μg / ml, 100 μg / ml, and 200 μg / ml in the same manner as in Example 1. ₂ The production suppression effect was tested.
The results are shown in FIG.
In FIG. 2, A shows the results for SAL700, B for SAL701, C for SAL702, and D for SAL703.
[0018]
As is apparent from FIG. 2, the 50% ethanol extract of primrose leaves is an aqueous fraction and PEG when the primrose extract is not contained. ₂ Was about 6 μg / ml, but the concentration of primrose decreased to about 3.5 μg / ml at 100 μg / ml and to about 2 μg / ml at 200 μg / ml. In addition, in the aqueous layer precipitation, when the primrose extract is not included, PEG ₂ Was about 5 μg / ml, but the concentration of primrose decreased to about 3 μg / ml at 200 μg / ml. Also, in the hexane fraction, when primrose extract is not included, PEG ₂ Was about 7.5 μg / ml, but the concentration of primrose decreased to about 4 μg / ml at 200 μg / ml. That is, it is PGE whether it is a water fraction or a hexane fraction. ₂ It had a production inhibitory effect. Especially the water fraction showed a high effect.
[0019]
Example 3
(PGE of three kinds of flavonoids in primrose extract ₂ Production suppression effect)
Three kinds of flavonoids isolated from the extract of primrose (linarin, diosmin and luteolin 7-O-glucoside) were treated in the same manner as in Example 1 to obtain flavonoid concentrations. PGE at 0.1 μg / ml, 1 μg / ml, 10 μg / ml, 100 μg / ml ₂ The production suppression effect was tested.
The results are shown in FIG.
In addition, in FIG. 3, A is a linalin, B is a diosmin, C shows the result regarding luteolin 7-O-glucoside.
[0020]
As is apparent from FIG. 3, linalin is PEG when not added. ₂ Was about 6 μg / ml, but the linalin concentration was about 5 μg / ml at 0.1 μg / ml, about 4 μg / ml at 1 μg / ml, about 3 μg / ml at 10 μg / ml, 100 μg / Ml is reduced to about 1.5 μg / ml. ₂ Was reduced to about 2 μg / ml at 10 μg / ml, about 1 μg / ml at 100 μg / ml, and no addition of luteolin 7-O-glucoside. , PEG ₂ Was about 7 μg / ml, but the concentration of luteolin 7-O-glucoside was about 5 μg / ml at 1 μg / ml, about 3 μg / ml at 10 μg / ml, and about 0.5 μg at 100 μg / ml. / Ml. In other words, all three flavonoids used in the test had a PGE concentration of 1/10 to 1/20 lower than SAL701 of 50% ethanol extract of stems and leaves. ₂ It was confirmed that it has a production inhibitory effect.
[0021]
Example 4
(IL-1α production inhibitory effect of primrose extract)
Frozen normal human epidermal cells (NHEK (F) CASCADE, tertiary culture) grown semi-confluent using HuMedia-KG2 (Kurabo) were seeded in 96-well culture plates (IWAKI) at 60,000 cells / well. After culturing for 24 hours and confirming that the cells are adhered, 0.6 mM Ca containing primrose extract ²⁺ The medium was changed with / K110II (+) and cultured for 72 hours. During the 48 hours, the medium was replaced with the same medium. After culturing for 72 hours, the medium was changed with the same medium containing primrose extract and 50 μg / ml LPS, and culturing was continued for 1 hour to stimulate the epidermal cells with LPS. The culture supernatant after stimulation was collected, and the amount of IL-1α in the supernatant was quantified using (h) IL-1α ELISA system (BIOTRAK).
The results are shown in FIG.
In FIG. 4, A shows the results regarding 50% ethanol extract of the foliage part, B shows the 50% ethanol extract of the fruit part, and C shows the result concerning the hairy root ethanol extract.
[0022]
As is apparent from FIG. 4, when using a 50% ethanol extract of the foliage, the inhibition rate is about 25 pg / ml and 50% at a concentration of 0.5 μg / ml and about 20 pg / ml and 60% at a concentration of 5 μg / ml It was the inhibition rate. In addition, the 50% ethanol extract of the fruit part was about 30 pg / ml at a concentration of 0.5 μg / ml and 5 μg / ml, which was a 50% inhibition rate. The hairy root ethanol extract was about 25 pg / ml at a concentration of 0.5 μg / ml and 5 μg / ml, which was 60% inhibition.
[0023]
Example 5
(IL-6 production inhibitory effect of primrose extract)
Mouse macrophage RAW264.7 was transferred to a 24-well culture plate (IWAKI) at 1.0 × 10 ^Five Cells / well were seeded. As the medium, DMEM containing 10% by volume FBS (10% by volume FBS / DMEM) was used and cultured for 24 hours. After confirming the state of the cells, the medium was removed by aspiration, 450 μl of 10 vol% FBS / DMEM containing primrose extract was added, and subsequently stimulated with 50 μl of LPS (Liposaccharide, Sigma). After 24 hours of cell stimulation, the culture supernatant was collected, and the amount of IL-6 in the supernatant was measured using (m) IL-6 ELISA system (BIOTRAK).
The results are shown in FIG.
In FIG. 5, A shows the results for 50% ethanol extract of the foliage part, B shows the 50% ethanol extract of the fruit part, C shows the water extract of the fruit part, and D shows the ethanol extract of the hairy root part.
[0024]
As apparent from FIG. 5, in the case of the 50% ethanol extract of the foliage part, an effect was recognized in a concentration-dependent manner, and the inhibition rate was 57% at about 10 ng / ml at a concentration of 1 μg / ml. In the fruit part 50% ethanol extract and water extract, an inhibition rate of about 25% was obtained at 0.05 to 1 μg / ml. The hairy root ethanol extract showed an effect in a concentration-dependent manner at 0.05 to 1 μg / ml.
[0025]
Example 6
(Cytotoxicity test)
In order to examine the effect of primrose extract on cell growth, alamarBlue Assay was performed. That is, after removing the culture supernatant, 1.2 mM Ca containing 10% alamarBlue ²⁺ 200 μl of / K110II (+) was added and incubated for 2 hours. The absorbance at 550 nm and 630 nm of the supernatant was measured. The numerical values are shown assuming that the absorbance of the primrose extract-free group is 100%.
The results are shown in FIG.
In FIG. 6, A is a 50% ethanol extract of the foliage, B is a water extract of the foliage, C is a 50% ethanol extract of the fruit, D is a water extract of the fruit, and E is a hairy root. The ethanol extract and F show the results for the hairy root water extract.
[0026]
As is clear from FIG. 6, cytotoxicity and cell proliferation action were not confirmed at 1 to 100 μg / ml in the extracts of shoots, fruits and hairy roots of primrose.
[0027]
Preparation Example 2
[Preparation of rosemary extract]
Rosemary stems and leaves were naturally dried, chopped, immersed in 50% aqueous ethanol overnight, and filtered through filter paper to obtain an extract.
[0028]
Example 7
(Pose of rosemary extract ₂ Production suppression effect)
PGE according to Example 1 with 50% rosemary ethanol extract ₂ The production suppression effect was tested. The results are shown in FIG.
[0029]
As is apparent from FIG. 7, rosemary 50% ethanol extract is PGE when it does not contain the rosemary extract. ₂ The amount produced was about 7 ng / ml medium, and the rosemary extract concentration 0.5% solution was reduced to about 6 ng / ml medium and 1.0% solution was reduced to about 5 ng / ml medium. Therefore, rosemary 50% ethanol extract is PGE ₂ It had a production inhibitory effect.
[0030]
Example 8
(IL-1α production inhibitory effect of rosemary extract)
Using a 50% rosemary ethanol extract, the IL-1α production inhibitory effect was tested according to Example 4. The results are shown in FIG.
[0031]
As can be seen from FIG. 8, the rosemary 50% ethanol extract had an IL-1α production of about 50 pg / ml when the rosemary extract was not included, and rosemary extract concentration of 0. The 1% solution was reduced to about 25 pg / ml and the 1.0% solution was reduced to about 20 pg / ml. Therefore, the rosemary 50% ethanol extract had an IL-1α production inhibitory effect.
[0032]
Example 9
(IL-6 production inhibitory effect of rosemary extract)
The IL-6 production inhibitory effect was tested according to Example 5 using a rosemary 50% ethanol extract. The results are shown in FIG.
[0033]
As can be seen from FIG. 9, the rosemary 50% ethanol extract had a rosemary extract concentration of 0. 23 ng / ml when the rosemary extract was not included. The 5% solution was reduced to about 19 ng / ml, the 1.0% solution to about 17 ng / ml, and the 2.0% solution to about 6 ng / ml. Therefore, the rosemary 50% ethanol extract had an IL-6 production inhibitory effect.
[0034]
Preparation Example 3
(Preparation of carnosic acid)
Rosemary was extracted with ethyl acetate, and the extract was shaken and distributed with ether and 1N sodium hydroxide solution. The sodium hydroxide layer was separated, neutralized with 2N hydrochloric acid, and extracted with ether. The ether extract was passed through silica gel chromatography (benzene-acetone (10: 1)) and recrystallized with benzene to deposit a colorless powder.
The remaining benzene solution excluding the precipitated colorless powder was treated with polyamide, dissolved in a small amount of benzene, and recrystallized by adding hexane to obtain colorless needle crystal carnosic acid.
[0035]
Example 10
(Carnosic acid PGE ₂ Production suppression effect)
PGE according to Example 1 using carnosic acid ₂ The production suppression effect was tested. The results are shown in FIG.
[0036]
As is clear from FIG. 10, carnosic acid is PGE when it does not contain carnosic acid. ₂ The production amount was about 7 ng / ml medium, and the carnosic acid concentration was reduced to about 3.5 ng / ml medium with a 10 μM (μmol / l, hereinafter the same) solution and about 2 ng / ml medium with a 10 μM solution. Carnosic acid is therefore an excellent PGE ₂ It had a production inhibitory effect.
[0037]
Example 11
(Inhibitory effect of carnosic acid on IL-1α production)
Using carnosic acid, the IL-1α production inhibitory effect was tested according to Example 4. The results are shown in FIG.
[0038]
As is clear from FIG. 11, when carnosic acid was not included, the IL-1α production amount was about 50 pg / ml when the carnosic acid was not included, and the carnosic acid concentration was about 20 pg / ml with a 10 μM solution. To about 12 pg / ml and 40 μM solution to about 10 pg / ml. Therefore, carnosic acid had an excellent IL-1α production inhibitory effect.
[0039]
Example 12
(Inhibitory effect of carnosic acid on IL-6 production)
Using carnosic acid, the IL-6 production inhibitory effect was tested according to Example 5. The results are shown in FIG.
[0040]
As is clear from FIG. 12, when carnosic acid was not included, IL-6 production amount was about 23 ng / ml, but carnosic acid concentration was about 20 ng / ml with 10 μM solution and 20 μM solution. To about 15 ng / ml and 40 μM solution to about 5 ng / ml. Therefore, carnosic acid had an excellent IL-6 production inhibitory effect.
[0041]
Preparation Example 4
[Preparation of cherry extract]
A cherry 50% ethanol extract was obtained in the same manner as in Preparation Example 2 using the stems and leaves of the cherry.
[0042]
Example 13
(PGE of cherry extract ₂ Production suppression effect)
PGE according to Example 1 using 50% ethanol extract of cherry ₂ The production suppression effect was tested. The results are shown in FIG.
[0043]
As is apparent from FIG. 13, the cherry 50% ethanol extract is PGE when it does not contain the cherry extract. ₂ Production was reduced from about 7.5 ng / ml medium to about 7 ng / ml medium with 0.5% solution of cherry extract and about 5.5 ng / ml medium with 1.0% solution. . Therefore, cherry 50% ethanol extract is PGE ₂ It had a production inhibitory effect.
[0044]
Example 14
(IL-1α production inhibitory effect of cherry extract)
Using a cherry 50% ethanol extract, an IL-1α production inhibitory effect was tested according to Example 4. The results are shown in FIG.
[0045]
As is clear from FIG. 14, the cherry extract with 50% ethanol had a production of IL-1α of about 50 pg / ml when the cherry extract was not included. To about 40 pg / ml and 2.0% solution to about 30 pg / ml. Therefore, the cherry 50% ethanol extract had an IL-1α production inhibitory effect.
[0046]
Example 15
(IL-6 production inhibitory effect of cherry extract)
Using a cherry 50% ethanol extract, an IL-6 production inhibitory effect was tested according to Example 5. The results are shown in FIG.
[0047]
As apparent from FIG. 15, the cherry extract with 50% ethanol had a IL-6 production amount of about 22 ng / ml when the cherry extract was not included. To about 17 ng / ml and 2.0% solution to about 11 ng / ml. Therefore, the cherry 50% ethanol extract had an IL-6 production inhibitory effect.
[0048]
Preparation Example 5
[Preparation of cycad extract]
A cycad 50% ethanol extract was obtained in the same manner as in Preparation Example 2 using the stems and leaves of cycad.
[0049]
Example 16
(PGE of cycad extract ₂ Production suppression effect)
PGE according to Example 1 using 50% ethanol extract of cycad ₂ The production suppression effect was tested. The results are shown in FIG.
[0050]
As can be seen from FIG. 16, cycad 50% ethanol extract is PGE when it does not contain cycad extract ₂ The production amount was about 2 ng / ml medium, and the cycad extract concentration 1.0% solution was reduced to about 1 ng / ml medium and the 2.0% solution was reduced to about 0.8 ng / ml medium. Therefore, cycad 50% ethanol extract is PGE ₂ It had a production inhibitory effect.
[0051]
Example 17
(Inhibitory effect of cycad extract on IL-1α production)
Using a cycad 50% ethanol extract, the IL-1α production inhibitory effect was tested according to Example 4. The results are shown in FIG.
[0052]
As can be seen from FIG. 17, the 50% ethanol extract of cycad, when the cycad extract was not included, showed that the IL-1α production was about 49 pg / ml. To about 40 pg / ml. Therefore, the cycad 50% ethanol extract had an IL-1α production inhibitory effect.
[0053]
Example 18
(Inhibitory effect of cycad extract on IL-6 production)
Using a cycad 50% ethanol extract, an IL-6 production inhibitory effect was tested according to Example 5. The results are shown in FIG.
[0054]
As can be seen from FIG. 18, the cycad 50% ethanol extract had a cycad extract concentration of 0.5% when IL-6 production was about 23 ng / ml when the cycad extract was not included. To about 20 ng / ml and 2.0% solution to about 9 ng / ml. Therefore, the cycad 50% ethanol extract had an IL-6 production inhibitory effect.
[0055]
Preparation Example 6
(Preparation of carnosol)
Rosemary was extracted with ethyl acetate, and the extract was shaken and distributed with ether and 1N sodium hydroxide solution. The sodium hydroxide layer was separated, neutralized with 2N hydrochloric acid, and extracted with ether. The ether extract was passed through silica gel chromatography (benzene-acetone (10: 1), recrystallized with benzene, and the resulting colorless powder was further recrystallized with methanol to obtain colorless needle crystal carnosol.
[0056]
Example 19
(Carnosol PGE ₂ Production suppression effect)
Using carnosol, PGE according to Example 1 ₂ The production suppression effect was tested. The results are shown in FIG.
[0057]
As is clear from FIG. 19, carnosol is PGE when it is not included. ₂ The production amount was about 7 ng / ml medium, which was reduced to about 3.5 ng / ml medium with a 10 μM carnosol concentration solution. Carnosol is therefore an excellent PGE ₂ It had a production inhibitory effect.
[0058]
Example 20
(Inhibitory effect of carnosol on IL-1α production)
Using carnosol, the IL-1α production inhibitory effect was tested according to Example 4. The results are shown in FIG.
[0059]
As is apparent from FIG. 20, when carnosol does not contain carnosol, IL-1α production was about 50 pg / ml, compared with carnosol concentration of about 21 pg / ml with 10 μM solution and about 16 pg with 20 μM solution. / Ml. Therefore, carnosol had an excellent IL-1α production inhibitory effect.
[0060]
Example 21
(Inhibitory effect of carnosol on IL-6 production)
Using carnosol, the IL-6 production inhibitory effect was tested according to Example 5. The results are shown in FIG.
[0061]
As is clear from FIG. 21, when carnosol does not contain carnosol, IL-6 production was about 23 ng / ml, compared with carnosol concentration of about 19 ng / ml with 10 μM solution and about 17 ng with 20 μM solution. To about 11 ng / ml with a 40 μM solution. Therefore, carnosol had an excellent IL-6 production inhibitory effect.
[0062]
Preparation Example 7
[Preparation of Novara extract]
A Novara 50% ethanol extract was obtained in the same manner as in Preparation Example 2, using the stems and leaves of Novara.
[0063]
Example 22
(Pole of Novara extract) ₂ Production suppression effect)
PGE according to Example 1 using Novara 50% ethanol extract ₂ The production suppression effect was tested. The results are shown in FIG.
[0064]
As is apparent from FIG. 22, Novara 50% ethanol extract is PGE when it does not contain the Novara extract. ₂ The amount produced was about 7 ng / ml medium, and the Novara extract concentration 0.1% solution was reduced to about 5.5 ng / ml medium and 0.5% solution to about 3 ng / ml medium. Therefore, Novara 50% ethanol extract is PGE ₂ It had a production inhibitory effect.
[0065]
Example 23
(IL-1α production inhibitory effect of Novara extract)
Using a Novara 50% ethanol extract, an IL-1α production inhibitory effect was tested according to Example 4. The results are shown in FIG.
[0066]
As can be seen from FIG. 23, when the Novara 50% ethanol extract did not contain the Novara extract, the IL-1α production amount was about 50 pg / ml. To about 42 pg / ml and 0.5% solution to about 36 pg / ml. Therefore, Novara 50% ethanol extract had an IL-1α production inhibitory effect.
[0067]
Example 24
(IL-6 production inhibitory effect of Novara extract)
Using a Novara 50% ethanol extract, an IL-6 production inhibitory effect was tested according to Example 5. The results are shown in FIG.
[0068]
As is clear from FIG. 24, the Novara 50% ethanol extract had a IL-6 production amount of about 23 ng / ml when the Novara extract was not included. Was reduced to about 18 ng / ml, 1.0% solution to about 14 ng / ml, and 2.0% solution to about 4 ng / ml. Therefore, Novara 50% ethanol extract had an IL-6 production inhibitory effect.
[0069]
Preparation Example 8
[Preparation of strawberry extract]
A strawberry 50% ethanol extract was obtained in the same manner as in Preparation Example 2, using the strawberry foliage.
[0070]
Example 25
(PGE of strawberry extract ₂ Production suppression effect)
PGE according to Example 1 using 50% ethanol extract of strawberry ₂ The production suppression effect was tested. The results are shown in FIG.
[0071]
As is clear from FIG. 25, the 50% ethanol extract of strawberry is PGE when it does not contain the strawberry extract. ₂ The production amount was about 7.5 ng / ml medium, but the strawberry extract concentration 1.0% solution was reduced to about 6 ng / ml medium, and the 2.0% solution was reduced to about 4.5 ng / ml medium. . Therefore, strawberry 50% ethanol extract is PGE ₂ It had a production inhibitory effect.
[0072]
Example 26
(IL-1α production inhibitory effect of strawberry extract)
Using a 50% ethanol extract of strawberry, the IL-1α production inhibitory effect was tested according to Example 4. The results are shown in FIG.
[0073]
As is clear from FIG. 26, when the strawberry 50% ethanol extract does not contain the strawberry extract, the IL-1α production amount was about 50 pg / ml. To about 35 pg / ml and 1.0% solution to about 32 pg / ml. Therefore, the strawberry 50% ethanol extract had an IL-1α production inhibitory effect.
[0074]
Example 27
(IL-6 production inhibitory effect of strawberry extract)
Using a Notigo 50% ethanol extract, an IL-6 production inhibitory effect was tested according to Example 5. The results are shown in FIG.
[0075]
As apparent from FIG. 27, the strawberry 50% ethanol extract had a IL-6 production of about 25 ng / ml when the strawberry extract was not included. To about 20 ng / ml and 2.0% solution to about 8 ng / ml. Therefore, the strawberry 50% ethanol extract had an IL-6 production inhibitory effect.
[0076]
Preparation Example 9
[Preparation of the extract of Yukinoshita]
Using the foliage of Yukinoshita, a 50% ethanol extract of Yukinoshita was obtained in the same manner as in Preparation Example 2.
[0077]
Example 28
(PGE of Yukinoshita extract ₂ Production suppression effect)
PGE according to Example 1 using 50% ethanol extract of Yukinoshita ₂ The production suppression effect was tested. The results are shown in FIG.
[0078]
As is clear from FIG. 28, the 50% ethanol extract of Yukishinoshita is PGE when the Yukinoshita extract is not included. ₂ The yield was reduced from about 2 ng / ml medium to about 0.6 ng / ml medium with a 0.5% solution of the Yukinoshita extract and about 0.5 ng / ml medium with a 1.0% solution. . Therefore, Yukinoshita 50% ethanol extract is PGE ₂ It had a production inhibitory effect.
[0079]
Example 29
(IL-1α production inhibitory effect of Yukinoshita extract)
Using a 50% ethanol extract of Yukinoshita, the IL-1α production inhibitory effect was tested according to Example 4. The results are shown in FIG.
[0080]
As can be seen from FIG. 29, when the 50% ethanol extract of Yukishinota does not contain the Yukinoshita extract, the yield of IL-1α was about 50 pg / ml. To about 40 pg / ml and 1.0% solution to about 35 pg / ml. Therefore, the 50% ethanol extract of Yukinoshita had an IL-1α production inhibitory effect.
[0081]
Example 30
(IL-6 production inhibitory effect of Yukinoshita extract)
Using a 50% ethanol extract of Yukinoshita, the IL-6 production inhibitory effect was tested according to Example 5. The results are shown in FIG.
[0082]
As is clear from FIG. 30, the 50% ethanol extract of Yukishinota had a IL-6 production amount of about 23 ng / ml when the Yukinoshita extract was not included. To about 18 ng / ml and 2.0% solution to about 14 ng / ml. Therefore, Yukinoshita 50% ethanol extract had an inhibitory effect on IL-6 production.
[0083]
Preparation Example 10
[Preparation of Kudzu Extract]
Using the scraps and leaves of the scrap, a 50% scrap extract of ethanol was obtained in the same manner as in Preparation Example 2.
[0084]
Example 31
(PGE of waste extract ₂ Production suppression effect)
Using PGE 50% ethanol extract, PGE according to Example 1 ₂ The production suppression effect was tested. The results are shown in FIG.
[0085]
As can be seen from FIG. 31, the waste 50% ethanol extract is PGE when it does not contain the waste extract. ₂ The production amount was about 2.3 ng / ml medium to about 1.2 ng / ml medium with a 0.5% solution of waste extract concentration and about 1 ng / ml medium with a 1.0% solution. Reduced to about 0.7 ng / ml medium with 0% solution. Therefore, the 50% ethanol extract of waste is PGE ₂ It had a production inhibitory effect.
[0086]
Example 32
(Inhibitory effect of waste extract on IL-1α production)
A test for inhibiting IL-1α production was carried out in accordance with Example 4 using a 50% waste ethanol extract. The results are shown in FIG.
[0087]
As can be seen from FIG. 32, when the waste extract of 50% ethanol was not contained, the IL-1α production amount was about 50 pg / ml at a waste extract concentration of 1.0%. It was reduced to about 40 pg / ml at 2.0% to about 42 pg / ml. Therefore, the waste 50% ethanol extract had an IL-1α production inhibitory effect.
[0088]
Example 33
(Inhibitory effect of waste extract on IL-6 production)
A test for the inhibitory effect on IL-6 production was carried out in accordance with Example 5 using an extract of 50% ethanol. The results are shown in FIG.
[0089]
As can be seen from FIG. 33, in the case of the 50% ethanol extract, when the waste extract is not contained, the IL-6 production amount was about 23 ng / ml. To about 19 ng / ml and 2.0% solution to about 11 ng / ml. Therefore, the waste 50% ethanol extract had an IL-6 production inhibitory effect.
[0090]
Preparation Example 11
[Preparation of lemongrass extract]
Lemongrass 50% ethanol extract was obtained in the same manner as in Preparation Example 2 using the stem and leaves of lemongrass.
[0091]
Example 34
(Lemongrass extract PGE ₂ Production suppression effect)
PGE according to Example 1 using Lemongrass 50% ethanol extract ₂ The production suppression effect was tested. The results are shown in FIG.
[0092]
As is clear from FIG. 34, the lemongrass 50% ethanol extract is PGE when it does not contain the lemongrass extract. ₂ The amount of production was about 2.5 ng / ml medium reduced to about 2 ng / ml medium with 0.5% lemongrass extract concentration solution and about 1.4 ng / ml medium with 2.0% solution. It was. Lemongrass 50% ethanol extract is therefore PGE ₂ It had a production inhibitory effect.
[0093]
Example 35
(IL-1α production inhibitory effect of lemongrass extract)
Using a lemongrass 50% ethanol extract, an IL-1α production inhibitory effect was tested according to Example 4. The results are shown in FIG.
[0094]
As is clear from FIG. 35, when the lemongrass 50% ethanol extract did not contain the lemongrass extract, the IL-1α production amount was about 50 pg / ml. The 5% solution was reduced to about 43 pg / ml, 1.0% to about 33 pg / ml, and 2.0% to about 24 pg / ml. Therefore, the lemongrass 50% ethanol extract had an IL-1α production inhibitory effect.
[0095]
Example 36
(IL-6 production inhibitory effect of lemongrass extract)
Using a lemongrass 50% ethanol extract, an IL-6 production inhibitory effect was tested according to Example 5. The results are shown in FIG.
[0096]
As is clear from FIG. 36, the lemongrass 50% ethanol extract had an IL-6 production of about 23 ng / ml when the lemongrass extract was not included. The 0% solution was reduced to about 17 ng / ml and the 2.0% solution was reduced to about 14 ng / ml. Therefore, the lemongrass 50% ethanol extract had an IL-6 production inhibitory effect.
[0097]
Preparation Example 12
[Preparation of snowy willow extract]
A snowy willow 50% ethanol extract was obtained in the same manner as in Preparation Example 2 using the foliage of the snowy willow.
[0098]
Example 37
(PGE of snow willow extract ₂ Production suppression effect)
PGE according to Example 1 using a willow 50% ethanol extract ₂ The production suppression effect was tested. The results are shown in FIG.
[0099]
As is clear from FIG. 37, the willow 50% ethanol extract is PGE when it does not contain the willow extract. ₂ The yield was reduced from about 2.9 ng / ml medium to about 2.5 ng / ml medium with 0.5% solution of snow willow extract and about 2 ng / ml medium with 2.0% solution. . Therefore, the willow 50% ethanol extract is PGE ₂ It had a production inhibitory effect.
[0]
Example 38
(IL-1α production inhibitory effect of snow willow extract)
A test of IL-1α production inhibitory effect was conducted in accordance with Example 4 using a snowy willow 50% ethanol extract. The results are shown in FIG.
[0]
As is clear from FIG. 38, in the case of the snow willow 50% ethanol extract, when the snow willow extract is not contained, the IL-1α production amount was about 50 pg / ml. Was reduced to about 30 pg / ml and 2.0% to about 27 pg / ml. Therefore, the snowy willow 50% ethanol extract had an IL-1α production inhibitory effect.
[0]
Example 39
(IL-6 production inhibitory effect of snow willow extract)
The test of IL-6 production inhibitory effect was conducted according to Example 5 using a snowy willow 50% ethanol extract. The results are shown in FIG.
[0]
As is clear from FIG. 39, in the case of the willow 50% ethanol extract, the amount of IL-6 produced was about 23 ng / ml in the case where the willow extract was not included. To about 20 ng / ml and 2.0% solution to about 13 ng / ml. Therefore, the snowy willow 50% ethanol extract had an IL-6 production inhibitory effect.
[0]
Example 40
Squalene, cetylisooctanoate and microcrystalline wax are dissolved by heating, and then clay mineral and POE glycerol triisostearate (surfactant) are added, adjusted to 70 ° C, and uniformly dispersed and dissolved to obtain an oily gel. It was.
Next, a primrose extract of a predetermined concentration was dissolved in purified water, adjusted to 70 ° C., and slowly added into the oily gel with sufficient stirring.
After uniformly mixing with a homomixer, the mixture was deaerated and filtered, and then cooled to 30 ° C. to obtain a cream. The blending ratio of each component is as follows.
[0]
Composition ratio (wt%)
Squalene 20
Cetyl isooctanoate 8.5
Microcrystalline wax 1
Clay mineral 1.3
POE glycerol
Triisostearic acid ester 0.2
Primrose extract 0.01
Water remaining
[0]
Example 41
Using the cherry extract, a cream having the following composition was obtained.
Composition ratio (wt%)
Squalene 20
Cetyl isooctanoate 8.5
Microcrystalline wax 1
Clay mineral 1.3
POE glycerol
Triisostearic acid ester 0.2
Sakura extract 0.01
Water remaining
[0]
Example 42
Using the cycad extract, a cream having the following composition was obtained.
Composition ratio (wt%)
Squalene 20
Cetyl isooctanoate 8.5
Microcrystalline wax 1
Clay mineral 1.3
POE glycerol
Triisostearic acid ester 0.2
Cycad extract 0.1
Water remaining
[0]
Example 43
A cream having the following composition was obtained using Novara extract.
Composition ratio (wt%)
Squalene 20
Cetyl isooctanoate 8.5
Microcrystalline wax 1
Clay mineral 1.3
POE glycerol
Triisostearic acid ester 0.2
Novara extract 0.1
Water remaining
[0]
Example 44
Using the strawberry extract, a cream having the following composition was obtained.
Composition ratio (wt%)
Squalene 20
Cetyl isooctanoate 8.5
Microcrystalline wax 1
Clay mineral 1.3
POE glycerol
Triisostearic acid ester 0.2
Strawberry extract 0.1
Water remaining
[0]
Example 45
Using the Yukinoshita extract, a cream having the following composition was obtained.
Composition ratio (wt%)
Squalene 20
Cetyl isooctanoate 8.5
Microcrystalline wax 1
Clay mineral 1.3
POE glycerol
Triisostearic acid ester 0.2
Yukinoshita extract 0.1
Water remaining
[0]
Example 46
Using the kuzu extract, a cream having the following composition was obtained.
Composition ratio (wt%)
Squalene 20
Cetyl isooctanoate 8.5
Microcrystalline wax 1
Clay mineral 1.3
POE glycerol
Triisostearic acid ester 0.2
Scrap extract 0.1
Water remaining
[0]
Example 47
A cream having the following composition was obtained using the lemongrass extract.
Composition ratio (wt%)
Squalene 20
Cetyl isooctanoate 8.5
Microcrystalline wax 1
Clay mineral 1.3
POE glycerol
Triisostearic acid ester 0.2
Lemongrass extract 0.1
Water remaining
[0]
Example 48
A cream having the following composition was obtained using a snowy willow extract.
Composition ratio (wt%)
Squalene 20
Cetyl isooctanoate 8.5
Microcrystalline wax 1
Clay mineral 1.3
POE glycerol
Triisostearic acid ester 0.2
Snowy willow extract 0.1
Water remaining
[0]
【The invention's effect】
According to the present invention, an anti-inflammatory agent having high anti-inflammatory activity and at the same time, PGE ₂ Production inhibitors, IL-1α production inhibitors, IL-6 production inhibitors and antiallergic agents can be provided.
[Brief description of the drawings]
FIG. 1 PGE of primrose extract ₂ The graph which shows a production inhibitory effect.
FIG. 2 PGE fraction of primrose extract ₂ The graph which shows a production inhibitory effect.
[Fig. 3] PGE of flavonoids derived from primrose ₂ The graph which shows a production inhibitory effect.
FIG. 4 is a graph showing the IL-1α production inhibitory effect of primrose extract.
FIG. 5 is a graph showing the IL-6 production inhibitory effect of primrose extract.
FIG. 6 is a graph showing the effect of primrose extract on human epidermal cell growth.
FIG. 7: PGE of rosemary 50% ethanol extract ₂ The graph which shows a production inhibitory effect.
FIG. 8 is a graph showing the IL-1α production inhibitory effect of a 50% rosemary ethanol extract.
FIG. 9 is a graph showing the IL-6 production inhibitory effect of a 50% rosemary ethanol extract.
FIG. 10: PGE of carnosic acid ₂ The graph which shows a production inhibitory effect.
FIG. 11 is a graph showing the inhibitory effect of carnosic acid on IL-1α production.
FIG. 12 is a graph showing the inhibitory effect of carnosic acid on IL-6 production.
FIG. 13: PGE of Sakura 50% ethanol extract ₂ The graph which shows a production inhibitory effect.
FIG. 14 is a graph showing the inhibitory effect of cherry 50% ethanol extract on IL-1α production.
FIG. 15 is a graph showing the IL-6 production inhibitory effect of a cherry 50% ethanol extract.
FIG. 16: PGE of 50% ethanol extract of cycad ₂ The graph which shows a production inhibitory effect.
FIG. 17 is a graph showing the IL-1α production inhibitory effect of 50% ethanol extract of cycad.
FIG. 18 is a graph showing the IL-6 production inhibitory effect of a 50% ethanol extract of cycad.
FIG. 19: Carnosol PGE ₂ The graph which shows a production inhibitory effect.
FIG. 20 is a graph showing the inhibitory effect of carnosol on IL-1α production.
FIG. 21 is a graph showing the inhibitory effect of carnosol on IL-6 production.
FIG. 22: PGE of Novara 50% ethanol extract ₂ The graph which shows a production inhibitory effect.
FIG. 23 is a graph showing the IL-1α production inhibitory effect of Novara 50% ethanol extract.
FIG. 24 is a graph showing the IL-6 production inhibitory effect of Novara 50% ethanol extract.
FIG. 25: PGE of 50% ethanol extract of strawberry ₂ The graph which shows a production inhibitory effect.
FIG. 26 is a graph showing the IL-1α production inhibitory effect of strawberry 50% ethanol extract.
FIG. 27 is a graph showing the IL-6 production inhibitory effect of strawberry 50% ethanol extract.
FIG. 28: PGE of 50% ethanol extract of Yukinoshita ₂ The graph which shows a production inhibitory effect.
FIG. 29 is a graph showing the IL-1α production inhibitory effect of a 50% ethanol extract of Yukinoshita.
FIG. 30 is a graph showing the IL-6 production inhibitory effect of a 50% ethanol extract of Yukinoshita.
FIG. 31: PGE of 50% ethanol extract of waste ₂ The graph which shows a production inhibitory effect.
FIG. 32 is a graph showing the IL-1α production inhibitory effect of a 50% waste ethanol extract.
FIG. 33 is a graph showing the IL-6 production inhibitory effect of a 50% waste extract of ethanol.
FIG. 34: PGE of lemongrass 50% ethanol extract ₂ The graph which shows a production inhibitory effect.
FIG. 35 is a graph showing the IL-1α production inhibitory effect of a lemongrass 50% ethanol extract.
FIG. 36 is a graph showing the IL-6 production inhibitory effect of a lemongrass 50% ethanol extract.
FIG. 37: PGE of 50% ethanol extract of snow willow ₂ The graph which shows a production inhibitory effect.
FIG. 38 is a graph showing the IL-1α production inhibitory effect of a willow 50% ethanol extract.
FIG. 39 is a graph showing the IL-6 production inhibitory effect of a willow 50% ethanol extract.

Claims (4)

Carnosic acid, an anti-inflammatory agent characterized by containing the carnosol and Novara extract or al least one selected. Carnosic acid, carnosol, and PGE ₂ production inhibitor characterized by containing at least one selected Novara extract or al. Carnosic acid, carnosol, and IL-l [alpha] production inhibitor characterized by containing at least one selected Novara extract or al. Carnosic acid, carnosol, and IL-6 production inhibitor characterized by containing at least one selected Novara extract or al.

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