KR20150037774A - Preparation of clove having enhanced antioxidative effect - Google Patents

Abstract

The present invention relates to a method for producing a fermented clove having enhanced antioxidant activity, and the present invention can dramatically enhance the antioxidant capacity of cloves, and can prevent or treat degenerative diseases closely related to oxidative stress To increase the health functionalities of the cloves.

Description

Preparation of clove having enhanced antioxidative effect "

More particularly, the present invention relates to a method for producing an extract of cloves having an increased antioxidative action and an antioxidant active composition using the same, and more specifically, to a method for producing an extract of clove, lentil or ethanol extract from a microorganism or an enzyme solution obtained from microorganisms, And a composition comprising the fermented oriental liquid having increased antioxidative activity by treating an enzyme solution obtained from plants such as spinach or the like, or a fraction thereof.

Clove (Caryophylli Flos.) Refers to the bud of a clove belonging to the clove. Cloves have a slightly rounded rod shape, corolla is spherical and 0.3 to 0.5 cm in diameter. The clove has a distinctive smell strong and the taste is spicy and irritating, and after a little paralysis of the tongue.

It is known that the essential oil component contains 15 ~ 20% of the essential oil component and the eugenol component accounts for 70 ~ 85% of the essential oil component. Acetylugenol, eugenol salicylate and terpene compounds having a structure similar to yugenol are known. In addition, it has been known that the effect of clove is due to its structure and antimicrobial action, and it has been used for some time as an indigestion due to a placebo (Genetic Algebra Revision 2, 2013, Compendium of Herbal Medicine Textbook, Dongmyungsa, 635).

On the other hand, fermentation is a phenomenon in which microorganisms decompose or change organisms with their own enzymes in a broad sense and produce end products that are unique to each other. In a narrow sense, fermentation refers to a process consisting of a complex reaction sequence in which carbohydrates are decomposed anaoxidally. Or a microorganism secretes various enzymes to oxidize, reduce or decompose organic compounds.

Fermented foods include mungolli made by koji mold, distilled spirits (brandy, rum, whiskey, vodka, gin shochu), vinegar made from acetic acid bacteria, wine and beer made from yeast, bread made from yeast, Soy sauce, kochujang, kimchi made with lactic acid bacteria, and western cheese, butter, and yogurt. Koreans also adapted to the natural environment and developed chestnut, kimchi, salted fish, vinegar, sikhye, and liquor.

When fermenting food extracts and the like, fermentation products are produced, so that there may be more nutrients than the original food. In soybeans, isoflavones are contained in an amount of about 1.5 to 2.5% per dry weight. Of these, glycosides (daisin and genistin), which are the glycoside forms, account for 60 to 70% of total isoflavones . In contrast, the contents of the aglycone-type compound, ie, daidzein and genistein, are only about 10 to 30 times smaller than those of each glycoside [Wang, HJ and Murphy AP 1994]. Isoflavone composition of America and Japanese soybeans in Iowa: Effects of variety, crop years, and location. J. Agric. Food Chem. 42: 1674-1677]. In the antimutagenic test using various soybean processed food extracts, the fermented food extracts showed much higher antimutagenic activity than the nonfermented foods, and the degree of antimutagenicity was increased in proportion to the aglycone content of the isoflavones contained in the food, The antioxidant activity in conventional meju and doenjang also increases in proportion to fermentation period, suggesting that the production of antioxidant activity during fermentation is increased [Peterson, G. and Barnes, S. 1991. Genistein Inhibition of the Growth of Human Breast Cancer Cells: Independent From Estrogen Receptors and the Multi-Drug Resistance Gene. Biochem. Biophysic. Res. Comm. 179: 661-667; Brandi, M. L. (1997) Natural and synthetic isoflavones in the prevention and treatment of chronic diseases. Calcif. Tissue. Int. 61 (Suppl 1): S5-8; Park, Kun Young, Mun Sook Hee, Lee Sook Hee. (1994) Antimutagenic Effect of Doenjang - Mutagenic Induced Inhibition of Aflatoxin B1 in Miso Stew and Miso Soup. Environmental Mutagens & Carcinogens 14: 145-152; Lee, Jong - Ho, Kim, Mi - hye, Clinical Line. (1991) Studies on Antioxidative Substances in Traditional Meju and Doenjang, 1. Lipid Oxidation and Browning during Meju Fermentation and Soybean Fermentation. Journal of the Korean Nutrition Society. 20: 148-155). Indeed, a study by the Ministry of Agriculture has shown that glycosylation through fermentation converts glycosides to non-glycosylated compounds with increased functionality and bioavailability [Lee, et al. (2002) Development of functional soybean products with enhanced bioactivity by fermentation. Final Report of Agriculture and Rural Development Projects of the Ministry of Agriculture and Forestry].

Therefore, the inventors of the present invention have determined that it is possible to produce cloves with increased health functional properties through fermentation.

Regarding the physiological activity of the clove, there is a pharmaceutical composition for treating and preventing brain cell damage induced by beta-amyloid (Korean Patent Laid-open No. 10-2009-0112268), which uses a fragrance component of oriental herbal medicine Although reactive oxygen species can participate in brain cell damage, it is not specific to the effect of removing reactive oxygen species, which is different from the increase in antioxidative activity by the fermentation of cloves claimed in the present invention.

Meanwhile, according to the present invention published by the present inventors in 2013, the content of dehydrouroguanol and dihydrogenol B in the cloaca is increased by the enzyme, and thus the effect of protecting the brain cell is increased (Eugenia caryophyllata) via increasing amounts of dehdrodieugenol and dehydrodiugenol (Eugenia caryophyllata) in the in vitro neuroprotective effect of Clove (2013) Proceedings of the Spring International Conference of the Pharmaceutical Society of Korea ). However, in this case, as a fermentation method, Lacta azela is limited to a specific purified enzyme, and its antioxidant activity is also limited to specific active oxygen in the test tube using DPPH as described above, which is different from what is claimed in the present invention. The ethanol extract of clove has an anti-diabetic effect, but it is based on clove extract before fermentation and is not related to the method of producing cloves with increased antioxidative activity proposed in the present invention (Minepei Kuroda et al., Hypoglycemic effects of clove (Syzygium aromaticum flower buds, J. Nat Med., 2012. 66: 394-399). There is also a literature related to the production of bisguanol using plant cell cultures, but there has been no report of increased antioxidant activity (Liliana Hernandez-Vazquez et al.) Screening of plant cell cultures for their capacity to dimerize eugenol and isoeugenol : Preparation of dehydrodieugenol on genetically diabetic KK-Ay mice and identification of the active ingredients (2011) J. Mol Catalysis B: Enzymatic. 72: 102-106) It has also been reported that bisguanol (or called dihydrodiguanol) is formed from eugenol by DPPH and shows radical scavenging ability (Renzo Bortolomeazzi et al., Formation of dehydrodiisoeugenol and dehydrodiugenol from the reaction of isoeugenol and eugenol with DPPH (2010) Food Chemistry 118: 256-265), which is limited to the specific oxygen radical scavenging activity produced by DPPH in vitro and suggests that living cell lines (ROS) and lipid peroxidation inhibitory activity, which is different from the antioxidant activity of the active oxygen species (Reactive Oxygen Species, ROS)

The present inventors have demonstrated that fermentation increases the antioxidant activity of the orient. In other words, the present inventors not only observed the difference in the inhibitory activity against active oxygen species and lipid peroxidation of the cloves before and after fermentation using live neuronal cells, but also indirectly observed an increase in the protective effect of brain cells which is closely related to the antioxidant activity The inventors of the present invention have accomplished the present invention by demonstrating that fermentation can create an improved culm for improved health function against various diseases caused by active oxygen.

Conventional clove and eugenol are known to be actinic and antiinflammatory, cancer cell toxic, insecticidal, and anti-diabetic, and it is reported that the antioxidative activity is increased by the present invention.

Accordingly, the object of the present invention is to enhance the antioxidant activity of cloves using an enzyme capable of being digested, or an enzyme solution derived therefrom, or a plant-derived enzyme, A health functional food, a natural substance new drug, and the like.

The present invention uses fermentation as a method for enhancing antioxidant activity of cloves, and relates to a food and pharmaceutical composition containing an extract of fermented clove and fermented clove. The clove used in the present invention may be clove ethanol extract or water extract or clove itself. In addition, an enzyme solution obtained from cloud mushroom, oyster mushroom or lacquer can be used for the fermentation, or an enzyme solution derived from wasabi or radish can be used.

According to the present invention, it was confirmed that the treatment of the ethanol extract of clove with the enzyme solution derived from cloud mushroom or radish significantly reduced the content of eugenol and the content of other compound, that is, biseugenol, increased sharply . In addition, the antioxidative activity of the extracts before and after fermentation were compared with those of the extracts before the fermentation. As a result, , And the effect was similar to that of normal cells that did not induce toxicity. Therefore, the present invention can be used to produce an effective amount of the antioxidant activity-enhancing antioxidative activity, and to provide a food or a medicinal product that can prevent and treat diseases such as degenerative diseases and various inflammations, Composition can be made.

FIG. 1 is a graph comparing the effect of reducing active oxygen species (ROS) in cloves before and after fermentation;
2 is a graph showing the lipid peroxidation inhibition effect increased by fermentation;
Fig. 3 is a graph showing changes in brain cell protective activity against the clove extract before and after fermentation; Fig.
FIG. 4 is a graph comparing the results of high-performance liquid chromatography before and after fermentation;
FIG. 5 is a graph showing the relative change of a compound that is newly produced and a compound that decreases when the clove extract is fermented with an enzyme solution derived from cloud mushroom;
6 shows HPLC results showing that similar results can be obtained by using commercially available peroxidase as a result of fermentation of cloves and using an enzyme solution derived from cloud mushroom;
FIG. 7 shows the TLC results showing that the enzyme solution was obtained from commercially available turnips and fermented by using the juice to obtain similar results as in the case of using the enzyme solution derived from cloud mushroom;
Figure 8 is a graph showing that biseugenol, a newly generated compound by fermentation, exhibits antioxidative activity.

Hereinafter, the present invention will be described in detail.

In the present invention, the enzyme solution derived from cloud mushroom was applied to the extract of clove and the antioxidative activities before and after the treatment were compared.

Fermentation was carried out as follows. That is, commercially available clove water or an ethanol extract, preferably a 95% ethanol extract, is prepared, the extract is suspended in water, the pH of the reaction solution is adjusted to 2 to 10, preferably 4.5, ml: the enzyme solution is adjusted to 1 to 100 mg / ml, preferably 14 mg / ml, and the reaction solution is reacted at 10 to 50, preferably 25, for 0.1 to 72 hours, preferably 1 hour. The fermentation broth was purchased from commercial mushroom in the market, poured into a mixer, filtered using gauze, and filtered.

In order to compare the antioxidant activity before and after fermentation, the effect on glutamate-induced intracellular ROS (Reactive Oxygen Species) was examined by fermentation and showed the ability to erase ROS by about 25% (Fig. 1). In addition, the antioxidative capacity against lipid peroxidation was measured using BODIPY, which is used as an index of another active oxygen species scavenging ability, and it was observed that fermentation significantly inhibits lipid peroxidation (FIG. 2). This fact can be interpreted as a result of the fact that the antioxidant effect of cloves is enhanced by fermentation.

In order to observe the protective effect of cranial nerve, which is closely related to antioxidant capacity, the neuronal cell death inhibitory effect of glutamate, which is an excitatory amino acid, on HT22 cultured human brain cell line was evaluated by MTT (3-4,5-dimethylthiazol- -yl) -2,5-diphenyl tetrazolium bromide) method [Kim, Min-Young et al. Isolation and Identification of Antioxidants from Petasites japonicus . Korean Journal of Food Science and Technology . 37 (8): 979984]. As a result, it was confirmed that the effect of inhibiting neuronal cell death after fermentation was increased about 5 times as compared to before fermentation (Fig. 3).

In addition, the compounds were analyzed by HPLC on the extracts before and after fermentation. As a result, the peak (compound 1) size of about 16 minutes of retention time was significantly reduced after fermentation, and after fermentation It was found that the peak of 27.5 minutes (compound 2) was very large instead (FIG. 4). Compounds 1 and 2 were found to be eugenol and biseugenol, respectively.

As a result of observing changes in the amount of Compound 2 produced in accordance with the fermentation time, it was found to be maximized about 1 hour after fermentation, and gradually decreased thereafter (FIG. 5, right). Therefore, it is most preferable to conduct the fermentation under the conditions of 10 to 50, preferably 25 to 0.1 to 72 hours, preferably 1 hour. Therefore, when the clove or clove extract is fermented, a substance having an increased content of Compound 2 can be obtained, and a composition having an enhanced antioxidative effect can be obtained. In addition, since it is known that antioxidants are related to various degenerative diseases such as dementia, inflammation, cancer, and aging, the present invention can produce cloves with increased health functionalities.

In addition to the cloud mushroom enzyme solution used in the present invention, the use of peroxidase, which has a similar action, showed that a new compound was produced almost similarly (FIG. 6). Therefore, the fermentation of the present invention is not limited to the cloudy mushroom enzyme solution, but an oxidase such as peroxidase or phenolase, which exhibits similar activity, can be used. Also, unlike the examples of the present invention, commercially available enzymes can be used as well as materials containing oxidizing enzymes such as turnip, lacquer, horseradish, oyster mushroom and the like. For example, when water extract of turnip was obtained and applied to the clove extract in the same manner, it was confirmed that compound 2 was produced on thin layer chromatography (FIG. 7)

The use of the present invention as described above can rapidly increase the amount of Compound 2, which is hardly before fermentation. As a result, it is confirmed that Compound 2 has an antioxidative activity (FIG. 8) Therefore, the turnip extract can achieve the object of the present invention.

When the antioxidant or antioxidant with an increased antioxidative activity according to the present invention is formulated in the form of a pharmaceutical preparation for preventing or treating a degenerative disease, the active ingredient is mixed with a pharmaceutically acceptable carrier The pharmaceutical composition may be formulated in the form of tablets, hard or soft capsules, chewing tablets, powders, preparations for oral administration such as solutions or suspensions, or preparations for parenteral administration such as injectable solutions or suspensions, It can be changed.

When the active ingredient of the present invention is formulated into preparations such as tablets, capsules, chewing tablets, powders, solutions and suspensions for the purpose of oral administration, binders such as gum arabic, corn starch, microcrystalline cellulose or gelatin, Disintegrants such as dicalcium phosphate or lactose, disintegrants such as alginic acid, corn starch or potato starch, lubricants such as magnesium stearate, sweeteners such as sucrose or saccharin and flavors such as peppermint, methyl salicylate or fruit flavor . When the unit dosage form is a capsule, a liquid carrier such as polyethylene glycol or fatty oil may be included in addition to the above components.

Injections in the form of solutions or suspensions for parenteral administration may also be administered parenterally, for example, subcutaneously, intravenously, intramuscularly or intraperitoneally. In general, injectable solutions or suspensions may be formulated in pharmaceutically acceptable liquid carriers such as water, saline, aqueous dextrose and related sugar solutions, non-volatile oils, glycols such as ethanol, glycerin, polyethylene glycols and propylene glycol, Can be prepared by homogeneously mixing the active ingredients of the present invention. In addition, auxiliaries such as solubilizing agents, antibacterial agents, chelating agents, buffering agents and preservatives may be included if necessary.

As the pharmaceutically acceptable carrier, any adjuvant which is pharmaceutically pure, substantially non-toxic and does not inhibit the action of the active ingredient may be used.

The fermented clove extract according to the present invention is a natural extract. The prophylactic or therapeutic agent of the present invention is not limited to the above-mentioned pharmaceutical preparations, but also beverages such as conventional soft drinks, mineral water and alcoholic beverages, chewing gum or caramel products, candies, Confectionery, or the like, or may be formulated into food supplements or food supplements by incorporating into health supplements including vitamins and minerals, or food additives.

The daily dose of the prophylactic or therapeutic agent according to the present invention may vary depending on various factors such as severity, complication, weight, age, sex, etc. of the subject to be administered, , Preferably 10 to 500 /, more preferably 10 to 100 /, per 1 to 3 times per day.

Hereinafter, the present invention will be described in more detail by way of examples, but they are not intended to limit the scope of the present invention in any way.

Example 1: Preparation and fermentation of ethanol extract of clove

Cloves were purchased from the market and used. 5.5 kg of cloves were added to 35 L of 95% ethanol, heated for 2 hours in a water bath for 5 hours, filtered, and dried with a rotary evaporator to obtain 30.8 g of dry solid. This was dispersed in 500 g of acetic acid buffer (pH 4.5) and then treated with 1 g of an enzyme derived from cloud mushroom and then fermented at 25 for 1 hour.

Example 2: Preparation of hard capsule

A 10-fold concentrate of the mixed extract of Example 1 10

D-sorbitol 10

Lactose 5

Colloidal silicon dioxide 5

Magnesium stearate 3

Total 33

These ingredients were blended in the specified amounts according to the conventional method of preparing the capsules, and filled into hard gelatine capsules of appropriate size so as to be a blend of 33 per capsule, to prepare the desired capsules.

Example 3: Preparation of tablets

A 10-fold concentrate of the mixed extract of Example 1 10

D-sorbitol 10

Lactose 5

Colloidal silicon dioxide 5

Magnesium stearate 3

                           Total 33

After mixing the above ingredients in the specified amounts, tablets were prepared according to the usual preparation method of tablets.

Example 4: Preparation of soft capsule

A 10-fold concentrate of the mixed extract of Example 1 10

Soybean oil 100

Palm oil 76

                           Total 186

After combining the ingredients in the specified amounts, soft capsule preparations were prepared according to the usual methods for preparing soft capsules.

Example 5: Preparation of injection

A 10-fold concentrate of the mixed extract of Example 1 10

Tocopherol 172

Selenium 30

Distilled water for injection

PH adjuster

The above components were prepared according to the conventional method of injection preparation in the injection of one vial (10).

Experimental Example 1: Effect of removing reactive oxygen species before and after fermentation

In order to investigate the antioxidative activity of fermentation, we examined the effect of ROS elimination on intracellular reactive oxygen species (ROS) before and after fermentation. As a result, when the fermented clove was treated with glucamate alone, the activity of the intracellular reactive oxygen species was reduced by about 25% (FIG. 1).

A concrete experimental example is as follows.

Flow cytometry analysis was performed to determine the effect of glutamate on oxidative stress in HT22 cells. In this experiment, CM-H2DCFDA [5- (and-6) -chloromethyl-2,7-dichlorodihydrofluo-resceindiacetate, acetylester, Invitrogen, USA] was used. When non-fluorescent CM- esterase or oxidative hydrolysis to rapidly deacetylate and react with reactive oxygen species in the cell to convert it to 2,7-dichlorofluoresein, which produces strong fluorescence. This experiment refers to the method used by Ishige et al. HT22 cells were cultured on a culture dish (Falcon, USA) at a density of 1.2 106 cells / ml for 24 hours. The prepared samples were treated for 12 hours by concentration and treated with 10 mM glutamate for 4 hours to induce the generation of active oxygen species in the cells. After that, trypsinization was performed by treating with 1 ml of 0.25% trypsin-EDTA and centrifuged to obtain cell pellet. Cells were washed once with phenol-red DMEM and then cultured at 37 for 15 min with 5 M CM-H2DCFDA. Then, centrifuge again to obtain cell pellet and wash again. Washing cells were resuspended in phenol red-free DMEM for analysis. Ten thousand cells were analyzed by fluorescence-activated cell sorting (FACS) sysetem. Fluorescence intensity was measured and quercetin (Sigma, USA) was used as a positive control.

Experiments on lipid peroxidation, another method of measuring intracellular antioxidative effect, showed that lipid peroxidation was inhibited by about 80% or more as compared with the case of only treatment with glutamate (FIG. 2).

Specific experimental examples are as follows. HT22 cells were cultured on a culture dish (Falcon, USA) at a density of 1.2 106 cells / ml for 24 hours. The prepared samples were treated for 12 hours by concentration and treated with 10 mM glutamate for 4 hours to induce the generation of active oxygen species in the cells. After that, trypsinization was performed by treating with 1 ml of 0.25% trypsin-EDTA and centrifuged to obtain cell pellet. Cells were washed once with phenol-red DMEM and then incubated at 37 for 15 min with 5 M BODIPY. Then, centrifuge again to obtain cell pellet and wash again. Washing cells were resuspended in phenol red-free DMEM for analysis. Ten thousand cells were analyzed by fluorescence-activated cell sorting (FACS) sysetem. Fluorescence intensity was measured and quercetin (Sigma, USA) was used as a positive control.

In conclusion, it can be seen that the antioxidant activity was increased by fermentation.

Experimental Example 2: Comparative measurement of brain cell protective activity by MTT method

HT22 cells, a neuronal cell line derived from mouse hippocampus, were treated with glutamate to induce oxidative stress, and cell viability was examined to examine the inhibitory effect of clove and fermented oriental neuronal cell death. This experiment is based on the principle that water-soluble MTT (3-4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide is converted into a water-insoluble substance called purmazine formazan by the action of mitochondrial redox- Is known to be suitable for evaluating cell viability. The cells were inoculated into each well of a 24-well plate at a cell density of 310 ⁴ cells / well and cultured for 24 hours. Twelve hours later, 10 mM glutamate was treated to induce oxidative stress. After 12 hours, the MTT solution (0.5 mg / ml) was treated for 4 hours. The MTT solution in the well plate was removed and the formazan produced was dissolved using Dimethylsulfoxide (DMSO). After shaking for 1 hour or more, the optical density (OD) was measured at 575 nm and the cell viability was calculated as a% value for the control using the following equation.

Cell survival rate (%) =

100

FIG. 3 is a graph comparing the protective effect of the extract of Clove cells before and after fermentation, showing a cell survival rate of about 20% before fermentation (gray bar graph) at a concentration of 10 ppm, %, Indicating that about 80% of neuronal cell protection effect was increased by fermentation.

In conclusion, fermentation of cloves increased the protection effect of brain cells by increasing antioxidant effect.

EXPERIMENTAL EXAMPLE 3: Changes in Cationic Compounds Before and After Fermentation

To investigate the changes of compounds in the clove extract, HPLC analysis was carried out. The analysis was carried out on a reversed phase column of XTerra RP18 (4.6250 mm, 5 m; Waters, USA) and the flow rate at room temperature was set at 0.8 m / min and 10 at the injection volume. The solvent phase was prepared by using water (H2O; solvent A) and acetonitrile (MeCN; solvent B) containing 1% acetic acid (HOAc) The total wavelength was analyzed using a photodiode array detector, and the optimum wavelength was set at 254 nm.

The clove extract before and after fermentation is evaporated to dryness, then methanol is added and centrifuged. The supernatant, which is a soluble part of methanol, was evaporated again and evaporated. Methanol was added to the dry solid at a rate of 100,000 ppm, and then a membrane of 0.45 was passed through the column.

As a result, as shown in FIG. 4, as shown in FIG. 4, after the fermentation (B), compared to the fermentation (A), the number of peaks of 1 was decreased, but the peak of 2 was increased, Was biseugenol.

Experimental Example 4: Measurement of optimum fermentation time

Compounds were analyzed by HPLC to observe changes in the contents of the compounds in the clove extracts according to the reaction time during the treatment with the enzyme from the cloud mushroom. 2 g of clove ethanol extract was dispersed in 20 ml of distilled water and 2 ml of acetic acid buffer (pH 4.5) containing 40 mg of cloudy mushroom-derived enzyme solution was added to each well. 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, and 17 hours. The reaction mixture was extracted with 100 ml of ethyl acetate (EtOAc), and then dehydrated and concentrated under reduced pressure to obtain an EtOAc soluble fraction. The cryopreserved EtOAC soluble fraction treated with the enzyme solution was subjected to HPLC analysis. At this time, zero time setting was used for analysis of clove extract treated with enzyme solution inactivated by auto clave.

As a result, when the enzyme solution was treated for 1 hour, the content of bisguanol was maximized and then decreased (FIG. 5).

Experimental Example 5: Changes in the content of bisguanol when fermentation of cloves was carried out using an enzyme solution other than cloud mushroom enzyme solution

In addition to the cloudy mushroom-derived enzyme solution used in the examples of the present invention, the clove juice, which is known to contain a large amount of commercial peroxidase and peroxidase, was used to ferment the cloves (Figs. 6 and 7). As a result, the product was detected on the HPLC or in a location consistent with the bisgeneol as a result of thin layer chromatography (FIGS. 6 and 7). As shown in FIG. 8, this compound has a strong antioxidative effect, and therefore, the increase of the compound content is related to the increase of antioxidative activity.

In conclusion, in addition to the enzyme solution derived from cloud mushroom used as an example in the present invention, an enzyme solution obtained from cloud mushroom, oyster mushroom and Japanese lacquer tree having similar effects, as well as plants and microorganisms such as commercially available peroxidase and turnip Similar results can be obtained.

When the present invention is used, it is possible to prepare an extract of cloves with an increased antioxidant activity, so that it can be used as a health functional food, a natural substance, and the like, and as the population ages, there is a surge of interest in health and degenerative diseases caused by active oxygen Therefore, it can be said that industrial value is very high.

1: CNT used in Fig. 1; Normal control, vCNT; Solvent control (0.05% DMSO-treated group). None; 5 mM glutamate alone. Q25; 25 M Quercetin (positive control). ** p <0.01 (compared to normal control), * p <0.05, ** p <0.01 (compared to 5 mM glutamate treated group).
Code used in Fig. 2: Above: FACS result. Below: A bar graph of FACS results
CNT; Normal control, None; 5 mM glutamate alone. EU10; Before fermentation. DE; After fermentation. QC10; 10 M Quercetin (positive control). # p < 0.05 (compared to normal control), * p < 0.05, ** p < 0.01 (compared to 5 mM glutamate treated group).
3: CNT used in Fig. 3; Normal control, vCNT; Solvent control (0.05% DMSO-treated group). None; 5 mM glutamate alone. Q25; 25 M Quercetin (positive control). ** p <0.01 (compared to normal control), * p <0.05, ** p <0.01 (compared to 5 mM glutamate treated group).
4: A: Clove extract. B: Clove extract after treatment with cloudy mushroom-derived enzyme solution. Eugenol standards (1). Newly produced compounds by fermentation (2).
5: Left: Changes in the compound (eugenol) decreased when the enzyme solution derived from cloudy mushroom was treated with time
Right: Changes in the compound (dehydrodieugenol) when treated with the cloudy mushroom-derived enzyme solution over time
6: A: Clove extract. B: Clove extract after peroxidase treatment. Eugenol (1). Compound produced by fermentation (2).
1 used in Fig. 7: 1; Clove extract (control), 2; + Turnip water extract treatment (+ 35% H ₂ O _2, ) _. 3; Turnip water extract (+ 35% H ₂ O ₂ ). 4; dehydrodieugenol standard
(A) UV 254 nm detection
(B) 10% Sulfuric acid + heating detection
8: CNT; Normal control, None; 5 mM glutamate alone. QC; 10 M Quercetin (positive control). # p < 0.05 (compared to normal control), * p < 0.05, ** p < 0.01 (compared to 5 mM glutamate treated group).

Claims (2)

A method of producing an extract of clove or clove having increased antioxidative activity through fermentation The composition according to claim 1, wherein the antioxidant activity is increased, and a composition for protecting and treating antioxidative and related degenerative diseases, comprising a fermented clove and fractions.

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