KR101369445B1 - Extraction method of Artemisia capillaris Thumb for increasing anti-inflammation activity - Google Patents

Abstract

The present invention relates to a method of extracting Artemisia capillaris Thumb optimized to increase anti-inflammatory activity. The present invention also provides a pharmaceutical composition or a functional food composition for improving, treating or preventing inflammation of the extract of Injin mugwort, which has been enhanced according to the extraction method, and the extract of Injin mugwort as an active ingredient.

Description

Extraction method of Artemisia capillaris Thumb for increasing anti-inflammation activity

The present invention relates to a method for extracting Artemisia capillaris Thumb optimized for enhancing anti-inflammatory activity. The present invention also relates to a jinjin mugwort extract having an enhanced anti-inflammatory activity extracted according to the extraction method and a pharmaceutical composition or food composition for enhancing the anti-inflammatory activity comprising the same.

Mugwort is an edible plant native to Korea, and is a food resource rich in protein, fat, fiber, saccharide calcium, phosphorus, vitamins A, B, and C. It has been widely consumed as foods such as herbs, mugwort, porridge, and mugwort rice cakes since ancient times. Among injin (茵蔯) Artemisia is a perennial plant type deciduous shrub belonging to the genus Artemisia Asteraceae, raw dangssuk, aedang mugwort, sacheolssuk (Artemisia capillaries THUNB.), Artillery, Artemisia iwayomogi ), Artemisa sacrorum subsp.Kitamura, Artemisa sacrorum subsp. There is manshuria. vestita Kitamura). In particular, young leaves are called starch or heat, and they have been traditionally used in the private sector as edible, medicinal, and single medicine because of their unique fragrances and medicinal properties. Injin mugwort is a young shoot of the perennial herbaceous perennial plant of Asteraceae. It is widely used in hepatitis, cirrhosis, liver cancer, and is also used in diseases such as soap, gallbladder stones. The pharmacological action is excellence of bile secretion promoting action, hepatic function protecting action, especially hepatocyte regeneration action. Lipid degradation, coronary artery dilation and blood pressure lowering effect, fever, diuretic, antibacterial effect is also seen. Injin mugwort has the effect of lowering heat and eliminating moisture, and it is known to be effective in treating the symptoms of moist jaundice and urine. It is known to reduce and improve liver function, antimutagenic effect, antibacterial effect.

However, there is no patent document that discloses a method for enhancing the anti-inflammatory effect of such jinjin mugwort, and in particular, a document disclosed about the extraction method of jinjin mugwort that can maximize the anti-inflammatory effect has not been reported yet.

Republic of Korea Patent Publication No. 10-2007-0042405 Republic of Korea Patent Publication No. 10-0528313

Therefore, the technical problem to be achieved by the present invention is to find out the extraction conditions of the optimized Injin mugwort to enhance the anti-inflammatory activity, to provide a method for extracting the Injin mugwort having an anti-inflammatory activity enhancing effect.

The present invention also provides an extract of Injin mugwort with enhanced anti-inflammatory activity according to the extraction method of Injin mugwort.

In addition, the present invention provides a pharmaceutical composition or food composition for enhancing anti-inflammatory activity, which contains such a extract of jinjinjin as an active ingredient.

In order to achieve the above technical problem, the present invention is to improve the anti-inflammatory activity, characterized in that the extraction for 4.5 to 7.5 hours at an extraction temperature of 53 to 73 ℃ ethanol aqueous solution of 42 to 66% by volume as an extraction solvent Provides a method of extracting jinjin jin.

The present inventors used an ethanol aqueous solution having a concentration of 42 to 66% by volume of ethanol as the extraction solvent, and the extract of Injin mugwort extract using the extraction conditions of the extraction temperature of 53 to 73 ℃ and the extraction time of 4.5 to 7.5 hours The present invention was confirmed by the fact that it is very effective in enhancing the inflammatory activity.

Phosphorus mugwort used in the present invention is preferably used after drying the harvested phosphorus mugwort leaves.

The concentration of ethanol is preferably 42 to 66% by volume, more preferably 52 to 56%, and even more preferably 54% in the ethanol aqueous solution, which is an extract solvent used in the extract of Injin mugwort of the present invention.

In the phosphorus mugwort extracting method of the present invention, the extraction temperature is preferably in the range of 53 to 73 ° C, more preferably in the range of 61 to 65 ° C, even more preferably 63 ° C.

Extraction time in the jinjin mugwort extraction method of the present invention is preferably 4.5 to 7.5 hours, more preferably 5.5 to 6.5 hours, even more preferably 6.0 hours.

The extract of Injin mugwort according to the present invention is not the best extraction condition in terms of simple yield or total phenolic compound content, but surprisingly, Injin mugwort extract according to the present invention in terms of production amount of NO and PGE ₂ associated with anti-inflammatory activity. The condition was the most optimal. These results indicate that the extraction conditions for enhancing the anti-inflammatory effect of Injin mugwort extract are substantially different from those for increasing the yield or the content of total phenolic compounds.

The present invention also provides an extract of Injin mugwort with enhanced anti-inflammatory activity extracted according to the extraction method, and provides a pharmaceutical composition or a functional food composition for enhancing anti-inflammatory activity, which contains such extract as an active ingredient.

Functional foods that can be used the extract of the jinjinjinjin of the present invention, for example, drinks, gums, tea, vitamin complexes, dietary supplements, breads, confectionery, candy, etc., but the present invention is limited to these kinds of products It doesn't happen.

That is, the composition for treating or preventing inflammation prepared under the extraction conditions according to the present invention may be prepared in the form of medicines and functional foods. Such medicaments and functional foods may include pharmaceutically acceptable excipients or additives. The compositions of the present invention may be administered alone or in admixture with any convenient vehicle, excipient, or the like, and such dosage forms may be single-dose or multiple-dose formulations.

The pharmaceutical or functional food containing the composition of the present invention may be a solid preparation or a liquid preparation. Solid preparations include, but are not limited to, powders, granules, tablets, capsules, suppositories, and the like. Solid form preparations may include, but are not limited to, excipients, flavoring agents, binders, preservatives, disintegrants, lubricants, fillers, and the like. Examples of the liquid preparation include water, a solution such as a solution of propylene glycol, a suspension, an emulsion, and the like, but not limited thereto, and it can be prepared by adding a suitable coloring agent, a flavoring agent, a stabilizer, a tackifying agent and the like.

For example, powders may be prepared by simply mixing a jinjin wormwood extract prepared under specific extraction conditions, which is an active ingredient of the present invention, with a suitable pharmaceutically acceptable excipient such as lactose, starch, microcrystalline cellulose. Granules extract of Injin mugwort of the present invention; Suitable excipients which are pharmaceutically acceptable; And a suitable pharmaceutically acceptable binder such as polyvinylpyrrolidone and hydroxypropylcellulose, followed by wet granulation using a solvent such as water, ethanol or isopropanol or dry granulation using a compressive force . Further, the tablet may be prepared by mixing the above granule with a suitable pharmaceutically acceptable salt such as magnesium stearate and then tableting it using a tablet machine.

The composition of the present invention may be administered orally or parenterally (for example, intramuscular injection, intraperitoneal injection, intravenous injection, infusion, subcutaneous injection, implant), inhalant, nasal administration agent, , Rectal, sublingual, transdermal, topical, and the like, but is not limited thereto. May be formulated into suitable dosage unit formulations, including those conventionally used and non-toxic, pharmaceutically acceptable carriers, additives, vehicles according to the route of administration. Depot formulations that are capable of sustained release of the drug over a period of time are also within the scope of the present invention.

In order to achieve the object of the present invention, the inflammation of the extracts prepared according to the method of the present invention may be administered from about 0.0001 mg / kg to about 10 g / kg, about 0.001 mg / kg to about 1 g / day A daily dose of kg is preferred. However, the dosage may vary depending on the degree of purification of the extract of Injin mugwort, the condition of the patient (age, sex, weight, etc.), the severity of the condition being treated. For convenience, the total daily dose may be divided as needed and divided into several doses throughout the day.

The present invention provides a production method for producing an extract of jinjin jinja excellent in anti-inflammatory activity, that is, extraction conditions. In addition, the present invention provides a pharmaceutical composition or a functional food composition for enhancing the anti-inflammatory activity, the anti-inflammatory activity of the extract extracted according to the extraction method and the anti-inflammatory activity containing the extract as an active ingredient.

1 is a graph showing three-dimensional superimposing the extraction conditions with the minimum NO production and the extraction conditions with the minimum PGE ₂ production.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

Extract of Injin mugwort according to different extraction conditions

Artemisia capillaris Thumb was used after drying the ground leaves of Injin mugwort obtained from Cheju National University and grinding them using a grinder.

The pulverized phosphorus mugwort leaves were extracted according to the extraction temperature, the concentration of the ethanol as the extraction solvent and the extraction time as shown in Table 1.

Example No. Extraction condition Extraction temperature (캜) EtOH concentration (%) Extraction time (hr) One 42 20 3 2 78 20 3 3 42 80 3 4 78 80 3 5 42 20 9 6 78 20 9 7 42 80 9 8 78 80 9 9 30 50 6 10 90 50 6 11 60 0 6 12 60 100 6 13 60 50 One 14 60 50 11 15 60 50 6 16 60 50 6 17 60 50 6 18 60 50 6 19 60 50 6 20 60 50 6

<Evaluation of yield>

The total extraction yield of the sample extracted for each extraction condition as in Examples 1 to 20 was measured as follows. 10 mL of the extract was taken, evaporated to dryness at 105 ° C., and weighed. The extraction yield (%, total extraction yield) was expressed as a percentage of the amount of the raw material (building amount) used in the preparation of the extract.

Yield measurement results according to the extraction conditions in each of Examples 1 to 20 are shown in Table 2 below, it was measured in the range of 13.37 ~ 42.75%.

As shown in Table 2 below, the yield of the extract of Injin mugwort was higher as the ethanol concentration of the extraction solvent was lower and the extraction temperature was higher, indicating that the extraction temperature was most affected.

<Measurement of Total Phenolic Compound Content>

The total phenolic compound content of the extract was determined by modifying the Folin-Ciocalteau method. 500 μL of 1N Folin-Ciocalteau's phenolic reagent and 10 mL of 2% Na ₂ CO ₃ were added to 500 μL of each extract, and the mixture was allowed to stand at room temperature for 1 hour and then absorbance was measured at 750 nm. At this time, the total phenolic compound content in the sample was determined from the calibration curve obtained by using gallic acid.

As a result of measuring the content (mg / 100g) of the total phenolic compounds according to the extraction conditions in each of Examples 1 to 20, it was measured in the range of 59.59 to 74.26 mg / 100g.

As shown in Table 2 below, the total phenolic compound content was found to increase as the ethanol concentration and extraction time increased, and the total phenolic compound content had the greatest effect on the extraction time and ethanol concentration on the extraction solvent. Appeared to be receiving.

<NO generation measurement>

The NO concentration was determined by measuring the nitrite concentration in the culture medium using the Griess regent. After dispensing RAW 264.7 cells into a 96 well plate at 1 × 10 ⁵ cells / well, incubating for 24 hours, pretreatment at 30 μg / ml of extract, and treating lipopolysaccharide (LPS, lipopolysaccharide) (1 ug / mL) after 2 hours. And incubated for 24 hours. The cell culture supernatant (100 uL) was placed in a 96-well plate, and an equal volume of Griess regent was added thereto. After reacting at room temperature for 10 minutes, the absorbance was measured at 540 nm using an ELISA microplate reader. NO concentration in the culture was determined using a standard curve for each concentration of sodium nitrite.

As a result of measuring the NO yield (%) according to the extraction conditions in each of Examples 1 to 20 was measured in the range of 57.79 ~ 87.21% as shown in Table 2 below.

As shown in Table 2, the amount of NO produced was found to decrease as the concentration of the extraction solvent is lower the higher the extraction temperature and the extraction time.

<Measurement of PGE ₂ Production>

RAW 264.7 cells were dispensed into 6 well plates with a cell number of 1 × 10 ⁶ cells / ml and incubated for 24 hours in a 37 ° C., 5% CO ₂ incubator. After exchanging with fresh RPMI 1640 medium, the cells were pre-treated at 30 μg / ml of the extract, incubated for 2 hours, and then incubated for 24 hours with LPS (1 ug / mL). And the separated by the cell culture was centrifuged for 3 minutes at 5,000 rpm the supernatant using the PGE ₂ enzyme immunoassay (EIA) kits (R & D Systems, Abington, UK) was measured both PGE _2.

PGE ₂ production amount (%) according to the extraction conditions in each of Examples 1 to 20 was in the range of 53.21 ~ 86.69% as shown in Table 2 below.

As shown in Table 2, the production of PGE ₂ decreased as the concentration of the extraction solvent was lower and the extraction temperature and extraction time were higher, which was similar to the NO production.

Example No. yield
(%) Total phenolic compounds
(mg / 100g) LPS induced
NO production amount (%) LPS induced
PGE ₂ Production (%) One 18.49 59.59 80.47 73.38 2 24.29 67.41 83.32 78.81 3 19.22 67.18 87.21 86.69 4 25.79 69.45 69.34 67.11 5 19.45 63.18 70.38 74.38 6 26.61 69.48 85.08 86.69 7 20.03 70.37 69.50 68.32 8 29.64 72.22 72.50 73.38 9 19.60 66.35 78.66 75.15 10 42.75 74.26 63.18 63.41 11 14.27 60.22 80.68 71.65 12 13.37 63.18 83.84 75.15 13 20.60 66.84 75.92 69.01 14 25.93 69.41 71.36 66.71 15 21.01 67.22 57.79 53.21 16 21.64 66.83 59.40 54.14 17 21.21 68.23 58.41 54.36 18 21.14 68.32 58.77 54.75 19 21.40 66.12 60.48 53.97 20 21.37 69.05 58.72 54.36

Based on the experimental results shown in Table 2, the extraction conditions, that is, extraction temperature, ethanol concentration and extraction time to minimize NO and PGE ₂ production for the anti-inflammatory activity enhancement effect was evaluated. For reference, the extraction conditions for optimizing yield and total phenolic compound content were also evaluated.

<Establishing Optimal Extraction Conditions by Creating Regression Formulas>

On the basis of the extraction conditions and evaluation results used in Examples 1 to 20 it was intended to establish the optimum extraction conditions. For response surface analysis, SAS (statistical analysis system) program was used.

As independent variables, the extraction temperature (X ₁ ), ethanol concentration (X ₂ ) and extraction time (X ₃ ) of the sample were set in five steps of -1.68, -1, 0, 1 and -1.68 as shown in Table 3 below. .

Extraction condition Independent variable Argument value -1.682 -One 0 One 1.682 Extraction temperature (캜) X ₁ 30 42 60 78 90 EtOH concentration (%) X ₂ 0 20 50 80 100 Extraction time (hr) X ₃ One 3 6 9 11

The dependent variable (Y _n ) affected by the independent variable (X _i ) is the quality factor of the extract, yield (Y ₁ ), total polyphenol content (Y ₂ ), NO production (Y ₃ ) and PGE ₂ production (Y ₄ ) and these measurements were repeated three times and the average value was used for the regression analysis. Using the results obtained in Examples 1 to 20 (Table 2), the reaction surface regression analysis was performed to obtain an optimum extraction condition, and each dependent variable as shown in Table 4, that is, yield, total phenolic compound content, Regression equations for NO production and PGE ₂ production were obtained.

reaction Second order Polynomials R ² Significance level yield Y _Y = 43.300079-1.067861X ₁ + 0.151573X ₂ -1.009167X ₃ + 0.010386X ₁ ² + 0.000741X ₁ X ₂ -0.002181X ₂ ² + 0.010185X ₁ X ₃ + 0.001944X ₂ X ₃ + 0.059906X ₃ ² 0.9089 0.0004 Total phenolic compounds Y _TP = 54.381526-0.143614X ₁ + 0.416293X ₂ + 0.191777X ₃ + 0.003459X ₁ ² -0.002315X ₁ X ₂ -0.002197X ₂ ² -0.004491X ₁ X ₃ + 0.000417X ₂ X ₃ + 0.037325X ₃ ² 0.9303 0.0001 LPS derived NO production Y _NO = 166.746113-1.795566X ₁ -0.476033X ₂ -12.006566X ₃ + 0.013512X ₁ ² -0.007505X ₁ X ₂ + 0.009401X ₂ ² -0.075766X ₁ X ₃ -0.008632X ₂ X ₃ + 0.595208X ₃ ² 0.9062 0.0005 LPS
Induced PGE ₂ Production Y _P = 173.194583-2.523157X ₁ -0.289006X ₂ -11.023825X ₃ + 0.019932X ₁ ² -0.007467X ₁ X ₂ + 0.008825X ₂ ² -0.072940X ₁ X ₃ -0.029131X ₂ X ₃ + 0.660889X ₃ ² 0.9123 0.0003

The optimum extraction conditions and quality characteristic values for each variable are estimated and shown in Table 5.

The calculated extraction conditions of Table 5 above were almost similar to the optimized extraction conditions obtained through the practical implementation in Examples 1-20.

Specifically, in yield, the R ² value of the response surface regression equation for the result was 0.9089, and the significance was recognized at the significance level within 5% (Table 4). The predicted steady point was the saddle point, which was estimated to have an extraction temperature of 89.66 ° C, an ethanol concentration of 50.85% and an extraction time of 39.24% at 6.74 hr.

In addition, the R ² value of the regression equation for the result of measuring the total phenolic compound content of the extract according to the extraction conditions was 0.9303, which was recognized at a significance level within 5% of significance (Table 4). The predicted peak of the total phenolic compound content was the saddle point, and the maximum value of 74.68 mg / 100 g was estimated when the extraction temperature for the sample was 89.62 ° C, the ethanol concentration of the solvent was 49.05%, and the extraction time was 6.78 hr.

In addition, in the NO production amount, R ² of the response surface regression equation for the result was 0.9062, and the significance was recognized at the significance level within 5% (Table 4). Inflammatory reactions caused by bacterial inflammatory lipopolysaccharides (LPS) produce pro-inflammatory mediators, which are produced by inducible nitric oxide synthase (iNOS). Prostaglandin E ₂ (PGE ₂ ) produced by nitrogen (NO) and cyclooxygenase-2 (COX-2). After treatment with the extract of Injinho, the amount of NO produced in the culture medium of LPS-stimulated cells was measured, and the predicted normal point was the minimum point. The extraction temperature was 64.55 ℃, and the ethanol concentration of the extraction solvent was 54.60%. At 6.46 hr, the time was found to be 58.55%, the lowest NO production.

In PGE ₂ production, R ² of the response surface regression equation for the results was 0.9123, and the significance was recognized at a significance level of less than 5% (Table 4). Inflammatory reactions involve various mediators such as cytokines, prostaglandin E2 (PGE ₂ ), lysosomal enzymes, and free radicals. Stimulation such as) activates the nucleic acid factor-κB (NF-κB), a transcription factor of the inflammatory response, resulting in the expression of cyclooxygenase-2 (COX-2), which produces excess PGE ₂ and causes inflammation. . PGE ₂ Produced in Culture of LPS-Stimulated Cells after Treatment of Injinho Extract As a result of measuring the amount, the predicted normal point is the minimum point. When the extraction temperature is 60.00 ℃, the ethanol concentration of the solvent is 50.00%, and the extraction time is 6.00 hr, PGE ₂ The yield was 53.94%, the lowest value.

From these results, in order to establish the optimum extraction conditions for the anti-inflammatory effect of the present invention, it was possible to superimpose the reaction surface of the NO and PGE ₂ production amount to set the range of the optimum extraction conditions (see Fig. 1). The results are shown in Table 6 below, extraction temperature 53 ~ 73 ℃, ethanol concentration 42 ~ 66% and extraction time 4.5 ~ 7.5 hours was the optimum extraction conditions for the anti-inflammatory effect.

Extraction condition Preferred range Optimal value Extraction temperature (캜) 53-73 63 EtOH concentration (%) 42-66 54 Extraction time (hr) 4.5 ~ 7.5 6.0

Example 21 Extract of Injin mugwort

The leaves of Injin mugwort were extracted according to the optimized extraction conditions of Injin mugwort identified through Examples 1 to 20. That is, after extracting phosphorus mugwort with extraction temperature of 63 ° C., ethanol aqueous solution with 54% by volume of ethanol and extraction time of 6.5 hr, NO and PGE ₂ production were measured. The experiment was repeated three times to calculate the measured value, the results are shown in Table 7 below.

NO production (%) PGE ₂ Production amount (%) 57.79 ± 0.82 53.08 ± 0.30

As shown in Table 7, when extracting the leaves of the jinjinjin in optimized extraction conditions through the experiment was excellent in terms of NO production and PGE ₂ production.

Table 8, NO and PGE ₂ production amount as in Example 1 to 20 the regression equation obtained from the calculated amount of NO in the above Example 21, obtained through actual implementation and PGE ₂ The production amounts are shown in comparison.

NO production (%) PGE ₂ Production amount (%) Forecast
(A) Experimental value
Example 21 (B) B / A × 100
(%) Forecast
(A) Experimental value
Example 21 (B) B / A × 100
(%) 58.53 57.79 ± 0.82 98.33 53.98 53.08 ± 0.30 97.54

As shown in Table 8, the NO and PGE ₂ production and the regression equations obtained through the practical implementation in Example 21 and NO and PGE ₂ It can be seen that the production amount is substantially similar.

Extraction conditions in the extract of Injin mugwort of the present invention is an extraction method optimized anti-inflammatory effect. The present invention provides a pharmaceutical composition or functional food composition for inflammation improvement, treatment or prevention containing the extract of Injin mugwort extracted according to this extraction method as an active ingredient. Phosphorus mugwort extract according to the present invention is expected to be useful in the art because it can greatly enhance the anti-inflammatory activity.

Claims (11)

A method of extracting phosphorus mugwort for enhancing anti-inflammatory activity, which is characterized in that the extract of phosphorus mugwort is extracted for 5.5 to 6.5 hours at an extraction temperature of 61 to 65 ° C. using an aqueous ethanol solution having an ethanol concentration of 52 to 56% by volume. According to claim 1, Injin mugwort is an extraction method, characterized in that the dried after grinding the Injin mugwort leaves. delete The method of claim 1, wherein the concentration of ethanol is 54%. delete The method of claim 1, wherein the extraction temperature is 63 ℃. delete The extraction method according to claim 1, wherein the extraction time is 6 hours. Extract from the extract method according to any one of claims 1, 2, 4, 6 and 8, jinjin mugwort extract with enhanced anti-inflammatory effect. A pharmaceutical composition for treating or preventing inflammation comprising the extract of Injin mugwort of claim 9. A functional food composition for improving inflammation, comprising the extract of Mugwort of claim 9.

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