KR20060017912A - Compound for improving neuralgia containing dibenzo-p-dioxine derivatives extracted from marin plants and articles comprising thereof - Google Patents

Abstract

The present invention relates to a neuralgia-improving product containing a neuroprotective active ingredient through inflammation suppression extracted from seaweed plants, more specifically, dibenzo-p-dioxine derivatives isolated from seaweeds The present invention relates to a neuralgia improving agent having a neuronal cell protective activity as an active ingredient and a product containing the same.

The neuralgia improving agent of the present invention can be used for the treatment of neuralgia by economically and efficiently containing the neuronal protective active ingredient isolated from marine edible plants, and has no advantage for human beings for a long time in the form of drugs or foods. It is expected that the range of application will vary.

Dibenzo-p-dioxin, neuralgia, anti-inflammatory, LPS, Cytokine, TNF

Description

Compound for improving neuralgia containing dibenzo-para-dioxin derivatives extracted from seaweeds and products comprising the same {Compound for improving neuralgia containing dibenzo-p-dioxine derivatives extracted from marin plants and articles comprising

1 is a graph showing a high performance liquid chromatogram of crude extracts of Ecklonia cava and rhubarb mixtures.

The present invention relates to a neuralgia-improving agent containing a neuroprotective active ingredient through inflammation suppression extracted from seaweed plants and to a product comprising the same, more specifically, dibenzo-p-dioxin (dibenzo-p- The present invention relates to a neuralgia improving agent having a neuronal protective activity using an active ingredient of dioxine derivatives and a product containing the same.

Neuralgia symptoms are mainly caused by nerve inflammation, nerve compression due to aging or chronic inflammation of bones, muscles, and blood vessels around the nerves, but the clinical diagnosis and treatment goals are ambiguous. There is no special treatment other than the prescription, and these anti-inflammatory drugs have only temporary relief, and serious problems of toxicity and side effects when taken in the long term. Therefore, it can be said that it is desirable to reduce the pain by inhibiting inflammation while protecting nerve cells from aging or oxidative stress using natural ingredients with no side effects. Known as a natural ingredient capable of such action include green tea extract or grape seed extract or EGCG contained in them (Mandel S, Weinreb O, Amit T, Youdim MB. J Neurochem. 2004 Mar; 88 (6): 1555 69), Resveratrol (Zhuang H, Kim YS, Koehler RC, Dore S. Ann NY Acad Sci. 2003 May; 993: 276-86; discussion 287-8), Quercetin (Ha HJ, Kwon YS, Park SM , Shin T, Park JH, Kim HC, Kwon MS, Wie MB.Biol Pharm Bull. 2003 Apr; 26 (4): 544-6). However, the inflammatory inhibitory activity of these components is generally weak, and the effect of clear neuralgia improvement in actual human application has not been proven.

Therefore, in the present invention, as a result of the research to solve the above-mentioned problems, a polyphenolic substance consisting of dibenzo-p-dioxine as the basic skeleton present in brown algae, which is a marine edible plant, Compared with the known natural ingredients, it shows a remarkably superior neuronal cell protective activity and anti-inflammatory action, and in human application, it has been shown to significantly reduce neuropathy and remarkably improve the quality of daily life. Completed with

Accordingly, an object of the present invention is to provide a neuralgia-improving agent comprising a dibenzo-p-dioxin derivative having a neuronal protective action and an anti-inflammatory effect isolated economically and efficiently from brown algae, which is a marine edible plant.

Another object of the present invention to provide a product comprising the neuralgia improving agent of the present invention.

A product containing a neuralgia improving agent containing a neuroprotective agent through inflammation suppression extracted from a seaweed plant for achieving another object of the present invention is provided in the form of food or medicine.

Looking at the present invention in more detail as follows.

As described above, in the present invention, dibenzo-p-dioxine derivatives isolated from marine edible brown algae have been found to not only protect neurons but also have anti-inflammatory action as a cooperative mechanism. Neuralgia improving agents including dibenzo-p-dioxin derivatives have been found to significantly reduce neuralgia in the elderly and significantly improve the quality of life without side effects.

Neuralgia improving agent containing a neuronal protective action and inflammation inhibitory ingredient extracted from the seaweed plant of the present invention for achieving the object of the present invention is a compound represented by the formula 1 to 10 having a neuronal protective activity isolated from algae From 0.1 to 99.9% by weight of one or more dibenzo-p-dioxin derivatives selected from.

[Formula 1] [Formula 2]

[Formula 3] [Formula 4]

[Formula 5] [Formula 6]

[Formula 7] [Formula 8]

[Formula 9] [Formula 10]

In the above formula, R is hydrogen, alkyl of 1 to 10 carbon atoms, alkenyl of 2 to 10 carbon atoms, phenyl, phenylalkyl of 7 to 10 carbon atoms, alkanoyl of 1 to 10 carbon atoms, hydroxy phenyl or dihydroxyphenyl group And preferably R is hydrogen.

The dibenzo-p-dioxin derivatives represented by Chemical Formulas 1 to 10 may be included in an amount of 0.1 to 99.9 wt% in at least one or a mixture of two or more in the neuralgia improving agent of the present invention. In the case of a composition in which the compound is used in a mixture of two or more, 0.1 to 6% by weight of the dibenzo-p-dioxin derivative represented by the formula (1), and 5 to 60 of the dibenzo-p-dioxin derivative represented by the formula (2) % By weight, 1 to 30% by weight of the dibenzo-p-dioxin derivative represented by the formula (3), 0.5 to 20% by weight of the dibenzo-p-dioxin derivative represented by the formula (4), dibenzo represented by the formula (5) 0.1 to 10% by weight of the -p-dioxin derivative, 0.5 to 15% by weight of the dibenzo-p-dioxin derivative represented by the formula (6), 0.1 to 5 to the dibenzo-p-dioxin derivative represented by the formula (7). % By weight, 0.1 to 5% by weight of the dibenzo-p-dioxin derivative represented by the formula (8), 0.1 to 10% by weight of the dibenzo-p-dioxin derivative represented by the formula (9), dibenzo represented by the formula (10) -p-dioxin derivatives are preferably mixed in the range of 0.1 to 12% by weight, The compositions selected are configured to mix with each other within the range of the composition ratios described above to achieve 100% by weight. The content is a range in consideration of the effective dose showing the neuronal protective activity and inflammation inhibitory activity of each of the dibenzo-p-dioxin derivatives, and if outside the above range tends to decrease the neuronal cell protection and inflammation inhibitory effect.

The brown algae used to extract the dibenzo-p-dioxin derivative in the present invention is Eisenia bacilli ( Eisenia) bicyclis ), Eisenia arborea , Esenia des Maresiodes desmarestioides), kids Senia Gala par Janice (Eisenia galapagensis), every child Senia Sony (Eisenia masonii ), Ecklonia kurome ), Ecklonia cava), Eccles Catalonia stall Ronnie Ferraro (Ecklonia stolonifera), Eccles Catalonia Maxima (Ecklonia maxima), Eccles Catalonia radiah other (Ecklonia radiata ), Ecklonia bicyclis ), Ecklonia biruncinate), Eccles Catalonia Bush the day the lease (Ecklonia buccinalis), par S. Tips for (Ecklonia in Catalonia Eccles South caepaestipes ), Ecklonia exasperta), Eccles Catalonia Paz Tee Hunger other (Ecklonia fastigiata), Eccles Catalonia Breda bipseu (Ecklonia brevipes ), Ecklonia arborea , Ecklonia latifolia latifolia), Eccles Village Tea Catalonia (Ecklonia muratii), Eccles Catalonia radio Xhosa (Ecklonia radicosa , Ecklonia richardiana , and Ecklonia wrightii , preferably Eisenia bicyclis ), Ecklonia cava , Eclonia kurome kurome ) and Eclonia stolonifera .

The dibenzo-p-dioxin extraction method from the brown algae is carried out by a conventional organic solvent extraction method. The organic solvent is methanol, ethanol, ethyl acetate, acetonitrile, acetone, water, and mixtures thereof, preferably water / ethanol. Optionally, the extraction process may be repeated two or more times to increase the yield. The residues and solvents contained in the extracts are removed using conventional separation and concentrating means such as centrifuges and rotary evaporators, and may be subjected to additional separation and purification if higher purity is required, but without additional processing. The extract can be used economically advantageous.

Neuralgia improving agents of the present invention can be prepared in the form of pharmaceuticals and foods. At this time, the neuralgia improving agent of the present invention is contained in a pharmaceutically effective amount, the effective amount of the dibenzo-p-dioxin derivative or a mixture thereof as an active ingredient is selected and used in the range of 0.1 to 99.9% by weight of the total neuralgia improving agent, It is preferably 20 to 70% by weight for ease of preparation of the formulation, ease of administration and the like.

Neuralgia improving agent of the present invention can be used in addition to the above-mentioned active ingredient, excipients commonly used in the art. Excipients include diluents and flavoring agents, such as polyols (mannitol, sorbitol, sucrose, etc.), crumbled or swelling agents (such as sodium croscarmellose, starch and derivatives, cellulose and derivatives), lubricants (such as magnesium stearate), Fluids (such as aerosil 200) can be used, and also, jujube extract, grape extract, kelp extract, fructose, fructose glucose liquid sugar, starch, royal jelly, liquid sugar, edible carrot powder, refined honey, refined sucrose Osse, skim milk powder, plum extract, sugar powder, green tea powder and the like can be used.

The improver of the present invention is prepared in any pharmaceutically acceptable form when formulated as a medicament and formulated into preparations such as injections, liquids, pills, tablets, capsules, powders and suspensions according to conventional methods in the pharmaceutical art. And a tablet, a capsule, or a liquid.

In addition, the improver of the present invention is not toxic to the human body can be used as a health supplement because it can be ingested in the long term. When prepared as a health functional food it is possible to include the form contained in pills, tablets, capsules, beverages or bakery, but is not limited thereto.

The improver of the present invention is effective to take 1 mg to 1000 mg / kg, preferably 1 to 100 mg / kg daily, and the method of ingestion is not particularly limited, but oral administration is preferred.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited thereto.

Example 1

Seaweeds ( Ecklonia cava ) and rhubarb ( Eisenia bicyclis ), which are collected from offshore, are first washed with distilled water to remove debris and dried in the shade before being crushed. 500 g of algae consisting of the algae (350 g of Ecklonia cava and 150 g of rhubarb) are extracted under reflux for 2 hours in order to elute the active substance by using 20 times the amount of 10% alcoholic solvent. This process was repeated twice. Then, the residue was filtered off and the solvent extract was concentrated under reduced pressure using a rotary evaporator. The concentrate was suspended in 20% of 95% ethanol, extracted three times and filtered, and the extract was concentrated under reduced pressure to give a crude extract.

Examples 2-11

The crude extract prepared in Example 1 was loaded on 15 times the amount of silica gel, and then concentrated by distilling the dibenzo-p-dioxin derivative using a mixed solvent of ethyl acetate / acetone (volume ratio 9/1), followed by 0.2. Filtration with a um membrane filter paper was loaded on high performance liquid chromatography. In high-performance liquid chromatography, the column was HP ODS Hypersil column, distilled water and methanol as solvent, and the solvent supply was linear gradient over 15 minutes from 15% to 70% methanol at 1.0 ml / min flow rate. Dibenzo-p-dioxin derivatives were separated into 10 singles. Table 1

Examples 12-16

The samples of Examples 2 to 11 and their mixtures were prepared in various compositions as shown in Table 1. (See Table 1)

Comparative Examples 1 to 5

EGCG, resveratrol, quercetin, green tea extract, and grape seed extract, which are known as neuroprotective agents through inflammation, were used as controls. Table 1

Example 17

Neuronal protective activity against inflammatory responses induced by LPS

IMR-32 neuroblastoma cell line was purchased from ATCC, USA. IMR-32 was incubated in a medium containing 10% calf serum in Minimum Essential Medium (MEM). Each sample was added to 10 mg / ml in dimeltyl sulfoxide (DMSO), which was diluted to 1000: 1 using culture medium. The final DMSO concentration was 0.1%, which had no effect on cell growth or apoptosis. IMR-32 cell line was loaded at a concentration of 2 × 10 ⁵ cells / ml in a 96 well plate by 180 μl, and after 24 hours, the cells were adhered well to the bottom of the well plate. After treatment to a final concentration of 1ug / mL, each sample was treated to a final concentration of 10㎛ / ㎖ reacted for 24 hours, and XTT assay (XTT assay) was performed as follows. 5 ml of 1 mg / ml XTT and 0.1 ml of 0.383 mg / ml N-methyl dibenzopyrazine (PMS) were dissolved in a 37 ° C. water bath, mixed, and then mixed with an XTT labeling mixture. Made. 50 μl / well was added to the cells treated with the mixture and developed for 3 to 4 hours, and the optical density (OD) was measured at 450 nm. Vehicles without LPS as a control and vehicle with LPS as a negative control were used. The cytoprotective effect was determined by the following equation and summarized in Table 3.

Cytoprotective effect (%) = 100 x (ODs -ODp) / (ODn-ODp)

ODs: OD values for the groups treated with LPS and samples

ODp: OD value of group treated with LPS only

ODn: OD value of untreated LPS group

As a result, the dibenzo-p-dioxy derivatives and the composition consisting of them showed significantly better cytoprotective activity than the comparative group.

Example 18

Inflammatory cytokine production inhibitory effects:

TNF-α, IL-1β and IL-6 are proinflammatory cytokines that are known to trigger neuronal destruction by inducing inflammatory responses. Accordingly, the effect of inhibiting these of each sample was studied.

Rat macrophages (RAW 264.7) were cultured in minimally essential culture medium (DMEM) containing 10% calf serum and 1% penicillin / streptomycin. Culture conditions were 37 ℃, 5% CO2, saturated humidity. The cell line was loaded into a 96 well plate at a concentration of 2 × 10 ⁵ cells / ml, and after 24 hours, the cells were adhered well to the bottom of the well plate, and LPS (E. coli, serotype 055 : B5) was treated to a final concentration of 1 ug / mL, and each sample was treated to a final concentration of 10 占 // mL and allowed to react for 18 hours. The concentrations of TNF-α, IL-1β and IL-6 were determined by ELISA method, and the inhibition rate was determined using the following equation.

% Inhibition = 100 x (Cp-Cs) / (Cp-Cn)

Cs: cytokine concentrations in groups treated with LPS and samples

Cp: cytokine concentration in LPS-treated group

Cn: Cytokine Concentration in Untreated LPS Groups

In the cells not treated with LPS, there was no expression of cytokine. The concentrations of cytokine (pg / mL) of the LPS-treated and sample groups were summarized in Table 2.

Inhibition rate of each component is summarized in Table 3 by the formula.

As a result, the dibenzo-p-dioxy derivatives and compositions thereof were shown to be remarkably superior to the comparative group in the effect of inhibiting the expression of the cytokine, an inflammation-inducing factor.

Example 19

Single Toxicity Test in Rats

Five-week-old SD rats were used for testing after seven days of acclimation. Free access to water and feed was maintained at 23 ± 1 ° C, 55 ± 5% humidity, and a 12-hour light / dark cycle. Example 1 The dosages of the compositions were 2000, 667, 222, and 0 mg / kg / day, divided into 10 male and female groups per group, and observed and necropsied for 2 weeks after a single oral administration. The result is as follows.

1. Mortality: No mortality was observed in the male and female controls and the test substance administration group during the observation period.

2. General symptoms: During the observation period, no general symptoms due to administration were observed.

3. Body weight change: During the observation period, weight gain inhibition was observed in males of the test substance group compared with the control group at 7 and 14 days (p <0.05).

4. Gross autopsy findings: No significant gross lesions were observed in autopsy results.

As a result of the above, the lethal dose of the sketch upon oral single administration using rats was judged to be 2,000 mg / kg or more, respectively.

Example 20

4-week repeated dose toxicity test in mice

Five-week-old ICR mice were used for testing after seven days of acclimation. Free access to water and feed was maintained at 23 ± 1 C, 55 ± 5% humidity, and a 12-hour contrast cycle. The composition of Example 1 was dosed at 200, 67, 22 and 0 mg / kg / day, divided into 10 male and female groups per group, and repeated oral administration for 4 weeks. Test items and results were as follows.

(1) No dead animals associated with administration of the test substance were observed.

(2) No change of general symptoms related to administration of test substance was observed.

(3) No change in body weight was found in both male and female.

(4) No abnormalities related to the administration of the test substance were recognized in all the male and female groups in the feed and water intake.

(5) In the ophthalmologic examination, no abnormalities were observed in all groups of male and female.

(6) In urinalysis, no toxicological abnormalities were observed in all male and female groups.

(7) No toxicological changes were observed in the hematological examination by the administration of test substance in all groups of male and female.

(8) No toxicological changes related to the administration of test substance were observed in blood biochemical tests.

(9) At autopsy findings, no abnormal findings were observed in all male and female treatment groups.

(10) No significant change in organ weight was observed in all groups of male and female.

(11) Histopathological examination showed no toxicological changes due to the administration of the test substance.

In view of the above results, no toxicological changes were observed by repeated oral administration of 4 weeks in rats. Therefore, the NOEL of this test substance was judged to be 200 mg / kg / day or more.

Example 21

Preparation of Liquid Preparation

Liquid formulations and placebos that promote absorption in the body and have no rejection were prepared as follows.

Jujube extract (36brix, 15ml), grape extract (12brix, 65ml), the extract of Example 1 (0.5 g), kelp extract (1.0g), was mixed for 1 hour at room temperature, and then packaged into a polyethylene pouch. (Capacity 80mL)

Placebo was to have the same composition and packaging except for the ingredients of Example 1. The sample and placebo could not distinguish taste.

Example 22

In order to confirm the neuralgia relaxation effect in the human body using the food containing the component of the present invention was tested under the conditions of Table 4a, the results are shown in Table 4c and Table 4d.

There were no statistically significant differences in age, sex, height, weight and symptoms at week 0 between the sample and placebo groups. (P> 0.1)

In the overall symptom evaluation of the subjects, the placebo group showed a slight tendency of improvement at 2 weeks (P = 0.479) and 4 weeks (P = 0.070) compared to week 0, but there was no significance. On the other hand, the experimental group showed significant improvement of 15% (P = 0.051) at 2 weeks and 32% (P <0.001) at 4 weeks. Results of weekly comparison between test group and placebo group (Table): There was no significant difference between the test group and the placebo group at week 0, but there was a significant difference at week 2 and 4, and the difference was greater at week 4. .

In the visual analogue scale of Pain, the placebo group showed a slight tendency of improvement at 2 weeks (P = 0.361) and 4 weeks (P = 0.102) compared to week 0, but there was no significance. On the other hand, in the experimental group, there was no significant improvement at Week 2 but improved 21% (P = 0.109). At Week 4, there was a significant improvement of 39% (P = 0.002). Results of weekly comparison of test group and placebo group (Table), there was no significant difference between the test group and the placebo group at week 0 (P = 0.087), but there was a significant difference between the 2nd and 4th week. It became bigger at 4 weeks.

As shown in the results of the above examples, the dibenzo-p-dioxin derivatives isolated from various brown algae which are marine edible plants according to the present invention are harmless to the human body as polyphenolic substances and are remarkably superior to known natural components. It is expected to provide economical and effective functional foods and medicines for neuralgia improvement, having protective and anti-inflammatory activity.

Claims (6)

Characterized in that it contains 0.1 to 99.9% by weight of one or more dibenzo-p-dioxin derivatives selected from compounds represented by the following formulas 1 to 10 having neuronal protective activity through inflammation inhibition isolated from seaweeds A beverage or bakery food comprising a pharmaceutically effective amount of a neuralgia improving agent containing a neuroprotective agent extracted from a seaweed plant. [Formula 1] [Formula 2]

[Formula 3] [Formula 4]

[Formula 5] [Formula 6]

[Formula 7] [Formula 8]

[Formula 9] [Formula 10]

(In the above formula, R is hydrogen, alkyl of 1 to 10 carbon atoms, alkenyl of 2 to 10 carbon atoms, phenyl, phenylalkyl of 7 to 10 carbon atoms, alkanoyl of 1 to 10 carbon atoms, hydroxy phenyl or dihydroxyphenyl Qi.) The food product according to claim 1, wherein R of the compound represented by Chemical Formulas 1 to 10 is hydrogen. According to claim 1, wherein the neuralgia improving agent is dibenzo-p-dioxin derivative represented by the formula 1 of 0.1 to 6% by weight, dibenzo-p-dioxin derivative represented by the formula 2 of 5 to 60% by weight, 1 to 30% by weight of the dibenzo-p-dioxin derivative represented by the formula (3), 0.5 to 20% by weight of the dibenzo-p-dioxin derivative represented by the formula (4), 0.1 to 10% by weight of the formula represented by the formula (5) Dibenzo-p-dioxin derivative, 0.5 to 15% by weight of the dibenzo-p-dioxin derivative represented by the formula (6), 0.1 to 5% by weight of the dibenzo-p-dioxine derivative represented by the formula (7), 0.1 to 5% by weight of the dibenzo-p-dioxin derivative represented by the formula (8), 0.1 to 10% by weight of the dibenzo-p-dioxin derivative represented by the formula (9), 0.1 to 12% by weight of the formula (10) Dibenzo-p-dioxin derivatives, or mixtures thereof mixed with each other in the range of the above-described weight ratio Food and characterized. The method of claim 1, wherein the seaweed is Eisenia bicyclis ), Eisenia arborea), kids Senia des Mares Tio Rhodes (Eisenia desmarestioides), kids Senia Gala par Janice (Eisenia galapagensis ), Eisenia masonii , Ecklonia kurome ), Eclonia cava , Eclonia stolonifera stolonifera), Eccles Catalonia maxima (Ecklonia maxima), Eccles Catalonia radiah other (Ecklonia radiata ), Ecklonia bicyclis , Eclonia Byronsinate biruncinate , Ecklonia in buccinalis), Eccles S. Tips for Catalonia car wave (Ecklonia caepaestipes), Eccles Catalonia exciter SARS peota (Ecklonia exasperta ), Ecklonia fastigiata ), Eclonia Brebbi ( Ecklonia brevipes ), Ecklonia arborea ), Ecklonia latifolia , and Eclonia mutifolia muratii), Eccles Catalonia radio Kosa (Ecklonia radicosa), Eccles Catalonia retarder Diana (Ecklonia richardiana ) and Ecklonia wrightii . According to claim 1, wherein the neuralgia improver active ingredient is jujube extract, grape extract, kelp extract, fructose, fructose glucose sugar, starch, royal jelly, liquid sugar, edible carrot powder, purified honey, purified sucrose, degreasing Powdered milk, plum extract, sugar powder, green tea powder, thinner, flavoring agent. Food comprising one or more excipients selected from crumbled formulations, swelling agents, lubricants and flow agents. The food product according to claim 1, wherein the daily intake of the neuralgia improving agent is 0.1 to 100 mg / kg.

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