KR20110121487A - Medical plant extract having anti-oxident and anti-inflammatry activity and method of production thereof - Google Patents

Abstract

PURPOSE: An oriental medicine material extract containing eugenol derivatives and a method for preparing the same are provided to ensure antioxidation, anti-aging, and anti-inflammation. CONSTITUTION: A method for preparing steam distillation extract of oriental medicine materials comprises: a step of drying herb medicines and adding distilled water; a step of adding ether and isolating with a steam distillation device; and a step of decompressing. A functional composition for antioxidation or antiaging contains the extract as an active ingredient. A functional composition for anti-inflammation contains the extract as an active ingredient. The extract contains eugenol derivatives as an active ingredient. The eugenol derivative is methyleugenol or acetyleugenol.

Description

Medicinal plant extract having anti-oxident and anti-inflammatry activity and method of production

The present invention relates to extracts of herbal medicines having anti-oxidant, anti-aging and anti-inflammatory activities at the same time. The present invention relates to extracts of herbal medicines containing eugenol-like substances with physiological activities such as eugenol and methyleugenol and acetyleugenol.

It has been found that oxidation in vivo by free radicals or free radicals causes aging and disease. Free radicals or free radicals cause lipid and sugar oxidation, protein denaturation, and DNA transfer in the human body, resulting in biological cell membranes and gene damage, causing aging, disease, and other cancers. Therefore, the search for a substance having a free radical or active oxygen scavenging ability has been actively conducted (John M. et. Al., 1994). Korean Patent No. 10-439012 is a method of extracting antioxidant and anti-aging active substances from the bark tree, and the leaves of the bark tree are extracted with hot water at 70-99 ° C. for 15 minutes, separated by primary color, lyophilized to provide powder, and then dissolved with methanol. Disclosed is a technique for separating and identifying 4-hydroxy-3-methoxy benzoic acid and the like from a methanol soluble extract obtained by concentrating the filtered filtrate under reduced pressure at 38 ° C.

In addition, Korean Patent No. 10-499455 discloses a method for extracting the antioxidant Monacollin K which antagonizes 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reducing agent from the fungus of the genus Monascus.

In addition, free radicals generated from metabolites of active oxygen species such as superoxide, H ₂ O ₂ , and OH, which are biological compounds that promote the aging of human cells, tissues, and organs, can be denatured in vivo, furthermore, protein cell membranes and Antioxidants or anti-aging agents which inhibit oxidative function of DNA and inhibit their oxidative function include vitamin C, vitamin E, natural compounds such as N-acetyl-L-Cysteine, S-al1rlyl-L-Cysteine and oltipraz PBN (α-phenyl-Nt-butyl nitrone) has been reported.In Korean Patent No. 10-535266, ginseng is ground and extracted with ethanol, and then lyophilized extract is better than crust, tea, phosphorus, cloves and celery extract. It is disclosed that it has antioxidant and anti-aging effects.

Inflammation of the human body, on the other hand, is a bacterium-induced abscess, where white blood cells invade and proliferate Pseudomonas aeruginosa, resulting in the death of white blood cells and the destruction of damaged tissue cells. Refers to a pathological condition. In addition, the anti-inflammatory action refers to inhibiting the growth of invading bacteria by antibiotics or antibacterial agents, or to promoting the function of digesting and excreting them by activating the macrophage that phages foreign substances accumulated in the abscess.

In general, the inflammatory response is a biological defense process that attempts to repair and repair the damaged area when exposed to an invasion that causes organic changes in cells or tissues. Therefore, these defense mechanisms include local blood vessels, various tissue cells of body fluids and immune cells.

Anti-inflammatory agents are mainly drugs that inhibit the biosynthesis of activated prostaglandins by stimulating from the cells of damaged tissues, the cells involved in inflammation, or the membranes of white blood cells (Moyazawa et, al., 1985). . However, these drugs were mainly effective in treating venus inflammatory disease, but in chronic inflammation such as rheumatoid arthritis, only palliative treatment effect was not expected. Therefore, in recent years, arachidonic acid is produced by lipoxy genase (HETE), leukotriene B _4, leukotriene C ₄ and the like are actively studied. Korean Patent No. 10-447948 discloses extracts, hexane fractions, chlorform fractions and butane fractions as anti-inflammatory activity and anti-arteriosclerosis activity as an inhibitory effect on body body and nitric oxide (NO). . In addition, Korean Patent No. 10-441565 discloses the anti-inflammatory effect of the dried buckwheat or buckwheat husk caused by the ethanol extract TPA.

However, until now, no herbal extracts with eugenol derivatives having physiological activities such as antioxidant, anti-aging and anti-inflammatory have been studied.

Accordingly, an object of the present invention is to provide an herbal extract having Eugenol derivatives and simultaneously having antioxidant, anti-aging and anti-inflammatory activity.

The above object of the present invention is to select a herbal medicine containing Eugenol derivatives, and then fermented to natural fermentation, and then added with distilled water, extracted with ether, concentrated under reduced pressure to obtain a steam distillate, which is cooled and stored for antioxidant, anti-aging and anti-inflammatory effects. Was achieved by evaluating

Hereinafter, specific examples of the present invention will be described with reference to Examples.

The steam distillate of the herbal medicine of the present invention contains eugenol derivatives and has excellent physiological activities such as antioxidant, anti-aging and anti-inflammatory.

1 is a diagram illustrating a process of preparing a steam distillate using the herbal medicine of the present invention.
Figure 2 is a photograph showing a distillation apparatus of the steam distillate for producing a bioactive material using the herbal medicine of the present invention.
Figure 3 is a graph showing the 5-LO and COX-2 inhibitory effect of the steam distillate for each herbal medicine raw material of the present invention.
Figure 4 shows the GC-MS Spectra of the flavor component of the clove constituting the present invention.
Figure 5 shows the GC-MS Spectra of eight herbal distillates (A) of Comparative Example and 10 herbal herb vapor distillates (B) of the present invention.
Figure 6 shows the GS-MS Spectra of eight herbal distillates (A) of Comparative Example and 10 kinds of herbal distillates (B) of the present invention.
7 is a graph showing the antioxidant activity of eight herbal distillates of Comparative Examples and the herbal distillates of ten herbs of the present invention.

The steam distillate containing Eugenal derivative of the present invention was prepared as shown in FIG.

In other words, the steam distillate of the present invention, unlike <Comparative Example> was added in addition to sensinju, Mokhyang, Cheongung, Angelica, Clove, Gobon, Peppermint, Apricot, and further added to the Shinsin, Sangbaekpi and prepared as in the following examples.

Herbal medicine used materials and products of Sky Lake Co., Ltd.

<Examples>

Co., Ltd. purchased from Sky Lake Co., Ltd., Mokyang, Cheongung, Dongguk, Clove, Gobon, Peppermint, Love Leaf, Sessin, Sangbaekpi as shown in Fig. Distilled water was added through a heating tank and a distillation unit, followed by heating.

After drying, fermented 500 g of 10 kinds of Chinese herbal medicines aged naturally fermented, 500 ml of distilled water was added, and 250 mL of ether was added to another water extract using an underwater distillation apparatus (Figure 2) for 4 hours, and the ether obtained was concentrated under reduced pressure and concentrated with nitrogen. The solution was obtained, cooled, stored and used for the evaluation of antioxidant, anti-aging and anti-inflammatory efficacy.

Comparative Example

In the above examples, herbal distillates were obtained by the same method using 10 conventional herbal medicines except for ten medicinal herb and sesame and baekbaekpi.

  Experimental results (Table 1), the yield of the traditional herbal distillate of the eight herbs was the highest clove was 16,340.3 mg of 100 g of dry matter, 428.0 mg, Gobon 419.0 mg, Cheongung 352.4 mg, Mint 227.0 mg, Angelica 196.0 mg, Angelica juju In order of 105.6 mg, the leaves were the lowest as 56.0 mg.

Experimental Example 1 Specific Biological Activity

Antioxidant activity

Antioxidant activity of clove and hardwood, which had a high yield of water vapor distillation among eight herbal medicines, was added to 2 mL of 0.1 mM DPPH solution according to the method of Tagashira M. and Ohtake Y. et al. (1998), and 0.1 mL of distillate was vortexed at room temperature. After reacting for 10 minutes, the absorbance at 517 nm was measured. DPPH radical scavenging activity was calculated using the following equation, and the value was expressed as IC ₅₀ value (concentration showing 50% inhibitory activity).

DPPH radical scavenging activity (%) = (absorbance of 1-sample addition / absorption of no addition) x 100

As a result, DPPH radical scavenging activity of cloves and high bones, which had high yield of water vapor distillation, was higher in cloves (IC ₅₀ = 8.85 μg / mL) than in the originals (IC ₅₀ = 1.25 mg / mL). In addition, the clove showed a higher capture activity than the natural antioxidant α-tocopherol (12.32 μg / mL) (Table 2).

Anti-aging activity

Anti-aging activity was determined by modifying the method of Masamoto et al., Et al. (2003) as follows. Tyrosinase coenzyme solution was used by dissolving mushroom tyrosinase (1380 u / mL) in 0.1 M sodium phosphate buffer (pH 6.8). It was. Enzyme activity was measured in 500 μL of 0.1 M sodium phosphate buffer, 450 μL of distilled water, 50 μL of extract, and 50 μL of tyrosinase. Then, 500 μL of 2.5 mM L-DOPA was mixed and reacted at 37 ° C. for 1 minute. Absorbance was measured at. The tyrosinase inhibitory activity was the same as the DPPH radical scavenging activity calculation method, and the value was expressed as IC ₅₀ value (concentration showing 50% inhibitory activity).

Were a result, 8 kinds of crude drugs of the yield was high cloves and tyrosinase inhibitory activity of gobon the steam distillate cloves _{(IC 50 = 160.4 μg / mL} ) The inhibitory activity is larger than gobon _{(IC 50 = 20.36 mg / mL} ), Clove showed lower inhibitory activity than the natural antioxidant ascorbic acid (66.62 μg / mL) (Table 3).

Anti-inflammatory action (5-LO and COX-2 inhibitory activity)

Cell culture: Murine macrophage cell line RAW 264.7 cells were obtained from Korean Cell Line Bank (KLCB), and Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin / streptomycin) was added. Subcultures were performed every 2 to 3 days in a 37 ° C incubator with 5% CO ₂ . Cells were injected into 96 well plates (1 × 105 cells / well), adhered to the cells, and 100 ng / mL of lipopolysaccharide (LPS) was added and cultured for 24 hours.

5-LO Inhibitory Activity Measurement: The 5-LO inhibitory activity was measured using a lipoxygenase inhibitor screening assay kit. After 10 μL of the sample, 90 μL of 5-LO (220 units / mL) and 10 μL of 1 mM arachidonic acid were added and reacted at room temperature for 5 minutes. Then, 100 μL of chromogen was added and reacted at room temperature for 5 minutes, followed by ELISA. Absorbance was measured at 490 nm using an autoreader. Nordihydroguaiaretic acid (NDGA) was used as a positive control and inhibition rate was calculated.

Determination of COX-2 Inhibitory Activity: COX-2 inhibitory efficacy was tested using a COX inhibitor screening assay kit. Hemo 10 μL, enzyme 5 μL, buffer 950 uL was added to 20 μL of the sample. After reaction at 37 ° C, 10 μL of arachidonic acid was added and reacted at 37 ° C for 2 minutes to activate COX-2. After adding the color source of μL and reacted for 90 minutes at 25 ℃, the absorbance was measured at 420nm using an ELISA autoreader. At this time, indomethacin, one of nonsteroidal anti-inflammatory drugs (NSAIDs), was used as a positive control, and the inhibition rate was calculated.

As a result of experiment (Fig. 3), the 5-LO inhibitory efficacy of the steam distillates of the eight herbal medicines showed the highest clove with 86.15% of clove at the concentration of 25 μg / mL, followed by the head (66.48%) and Angelica (64.27%). The cloves were similar to the activity at 5 μg / mL of the NDGA positive control. The highest inhibitory effect of COX-2 was at the concentration of 25 μg / mL, the highest at the concentration of 91.94%, and at the concentration of clove, Angelica (89.46%), and juju (88.70%), the clove was 5 μg of indomethacin. Similar to activity at / mL concentration. Therefore, among the eight herbs, clove could be judged to have the highest anti-inflammatory effect.

Experimental Example 2 Analysis of Flavor Components in Cloves by GC-MS

First, the fragrance components of the clove were extracted by two methods using SDE and SPME. SDE was extracted during the steam distillation. In the case of SPME, put the cloves into a vial and heat the sample at 270 ℃ for 5 minutes, and then 1 minute. The fragrance was adsorbed and extracted by injecting a sample into the device for 1 minute, and extracted clove fragrance components were analyzed using GC-MS as shown below (Table 4).

As a result of the experiment (Fig. 4), among the eight herbs, the main flavor component of the clove excellent in antioxidant, anti-aging and anti-inflammatory action was identified as eugenol, which is thought to have contributed greatly to the bioactivity of the clove.

Experimental Example 3 Comparison of Physiological Activities of Three Eugenol Derivatives

Antioxidant and anti-aging activity of the three eugenol derivatives were the same as in <Experimental Example 1>, and the anti-inflammatory activity was measured according to the method of Block et al. (1988). In other words, 20 μL of extract and 20 μL of soybean 15-lipoxygenase (1 nM, final concentration) were added to 2 mL of 0.1 M Tris-HCl buffer (pH 8.5) for 5 minutes and linoleic acid (110 μM, final concentration) dissolved in ethanol. After the addition of μL to the substrate was reacted for 1 minute and the absorbance was measured at 234 nm. The 15-soybean lipoxygenase inhibitory activity was the same as the DPPH radical scavenging activity calculation method, and the value was expressed as IC50 value (concentration showing 50% inhibitory activity).

 As a result, we compared eugenol and its derivatives, methyl-eugenol and acetyleugenol, which were identified as the main bioactive substances in cloves. Soybean 15-lipoxygenase inhibitory activity was 2-5 times greater than eugenol and acetyleugenol (Table 5).

Experimental Example 4 Preparation of High-Quality Herbal Distillates Containing Sessin, Flavor Components, and Physiological Activities

Comparison of Aroma Components and Contents of Existing and Inventive Novel Herbal Distillates

The fragrance components of the existing herbal distillate and the new herbal distillate containing sessin from Sky Lake were analyzed using GC-MS.

Experimental results (Fig. 5), the main fragrance component of the existing herbal distillate and steam distillate was eugenol, the fragrance components of the new herbal distillate and steam distillate added sessin and baekryepi differs between eugenol, methyleugenol and acetyleugenol . The eugenol content of the existing herbal distillate was 82.66 ppm, the eugenol content of the new herbal distillate was 119.17 ppm, and the contents of methyleugenol and acetyleugenol were 6.5 ppm and 28.74 ppm, respectively. Therefore, the new herbal distillate has a higher content of eugenol derivatives than the conventional herbal distillate, so it is considered to have better physiological activity (FIG. 6).

Comparison of Aroma Components and Contents of Existing and Inventive Novel Herbal Distillates

Antioxidant Activity: DPPH radical scavenging activity of the new herbal distillate added with sacin and lettuce was added 22.02% and 94.0%, respectively, and it was determined that the novel herbal distillate of the present invention had excellent antioxidant activity. 7).

The present invention is useful in the biomedical industry because it has the effect of providing a novel herbal distillate added sessin and baekryepi to the existing herbal distillate and provides an antioxidant, anti-aging and anti-inflammatory functional extract.

Claims (5)

Steam of Chinese herbal medicine characterized in that it is extracted by using steam distillation apparatus after adding distilled water by drying dried jujube, Mokhyang, Cheongung, Dongguk, Clove, Gobon, Mint, Apple Leaf, Sessin and Sangbaekpi. Distillation extract preparation method.
Antioxidant or anti-aging functional composition containing the herbal steam distillation extract of claim 1 as an active ingredient.
An anti-inflammatory functional composition comprising the herbal distillation extract of claim 1 as an active ingredient.
The method of claim 1, wherein the steam distillation extract is a steam distillation extract of the herbal medicine characterized in that it contains eugenol derivatives as an active ingredient.
The steam distillation extract of herbal medicine according to claim 4, wherein the eugenol derivative is one of methyleugenol and acetyleugenol.

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