KR101567325B1 - A functional compositoion comprising Chrysanthemum morifolium extract - Google Patents

Abstract

The present invention relates to a method for producing a chrysanthemum extract, Preparing a first herbal medicine extract; Preparing a second herbal medicine extract; Mixing the chrysanthemum extract, the first herbal medicine extract and the second herbal medicine extract to produce a first composition comprising a chrysanthemum extract; Mixing a saccharide in the first composition to produce a second composition comprising a chrysanthemum extract; And fermenting the second composition to produce a third composition comprising an extract of Chrysanthemum chrysanthemum. The present invention relates to a method for preparing a food composition comprising a chrysanthemum extract and a food composition prepared thereby, And can also provide a food composition comprising a chrysanthemum extract capable of preventing nerve stress and diseases associated therewith.

Description

A food composition comprising chrysanthemum extract (Chrysanthemum morifolium extract)

The present invention relates to a food composition containing a chrysanthemum extract, and more particularly to a food composition comprising a chrysanthemum extract effective for improving liver function or activating neurons.

The efficacy of chrysanthemum has been widely used as a folk remedy for a long time, and it has been regarded as an important ingredient in the untouched life in China. It has also been used as a product of chrysanthemum tea and chrysanthemum in modern times.

According to the "medicinal plant dictionary" about the efficacy of chrysanthemums, it is said that "it is good to apply cold water headache, dizziness, juice with jujube to bite area and toothache etc." It is light, it does not get old, it lengthens, and it strengthens the eyebrows and brightens the eyes, it has the fever in the head or the bulge, it treats dizziness, dizziness, "And in the" herbal gangmok "," If you take a long time, it is good for the blood, light body, and does not grow old. It comforts the stomach, helps the five, and regulates the limbs. In addition, it is effective for cold, headache and dizziness ".

In addition, it is well known that it is good for fever, pneumonia, diphtheria, gastroenteritis, eye disease, nausea, hypertension, cough, headache, pneumonia, stomatitis, skin care, fever, calm, tinnitus, It clears up the mind and gets popular among examinees especially.

It also cleanses the blood and keeps the body from fever and makes it look better. It cleanses the skin, improves blood circulation, excretes waste products, and is rich in vitamin C, making it shiny and soft.

These effects include vitamin A, B1, B2, C, iron, choline, stachydrin, adenine, trimethylcyclohexanoic acid, carbosilicic acid, acacetin, corin stakadrine, Santamine (yellow pigment), and essential oils. However, studies on the efficacy and functional components of chrysanthemum are still very few.

On the other hand, reactive oxygen species (ROS), called free radicals, are oxidized compounds produced by metabolic processes in plants and animals, and are strongly influenced by the causes of atherosclerosis and cancer in modern humans and aging. Is emerging.

Free radicals are atoms, molecules, or parts of molecules that have orbits in which electrons are not paired to the outside. Free radicals can be divided into electrically neutral, anionic radicals, and cation radicals, but they are generally highly reactive, unstable, and readily react with other molecules to have stable electron configurations.

Active oxygen is a strong oxidizing oxygen that attacks the living tissue and damages the cells because it is generated during the metabolic processes as the oxygen that enters the body during the breathing process is used for the oxidation process.

In other words, according to the radical theory of toxicity of oxygen, oxygen in aerobic metabolism is indispensable for life maintenance, but in respiratory metabolism, active oxygen accepts energy and the rotation direction of unpaired electron changes singlet oxygen (O ₂ ) (O ₂ ^- ), hydroxyl radical (OH), hydrogen peroxide (H ₂ O ₂ ), alkoxy radical (RO ·), and the like. , peroxyl radical (ROO), and the like.

In addition, radical living body such as Fe ⁺³ in the presence of iron or copper in the molecule, O ₂ ^- by being to generate a lot of process H ₂ O ₂ with a strong reactivity, activity sansogi of OH from which is converted to Fe ⁺² such Radicals are known to undergo several steps to produce secondary radicals such as O ₂ ^- , H ₂ O ₂ , RO and ROO.

During the oxidation process for energy production in the body, a large amount of active oxygen is produced. The excess of active oxygen in the body causes oxidative damage to the cellular components lipids, proteins, nucleic acids, sugars and DNA, thereby inhibiting normal metabolism of cells.

It has been reported that when these active oxygen are generated in large amounts or chronically active oxygen is generated and the antioxidant defensive system is not balanced, it can cause various diseases.

Antioxidant systems have been developed by the biological system against oxidative damage by sustained exposure to reactive oxygen species. The antioxidant system of the biological system can be divided into three categories: (i) inhibition of the generation of free radicals, (ii) elimination of generated free radicals, and (iii) repair.

In order to inhibit the production of free radicals, the biological system has antioxidant systems such as catalase, glutathione peroxidase, phospholipid hydroperoxidase and glutathione-S-transferase.

Vitamin C, vitamin E, uric acid, bilirubin, albumin, carotenoids, and flavonoids effectively cleave the generated free radicals, and DNA repair enzymes, transferases,

Although the generation of free radicals and the elimination of free radicals are performed in a general biological system, when excessive free radicals are generated, the functions of the living body are impaired and the activity of the cells is lost, thereby inducing immune system damage and cell death. Therefore, it is required to search for natural products that suppress the generation of free radicals, eliminate the generated free radicals, inhibit cell death, and study the evaluation of physiological activity.

Therefore, the applicant of the present invention intends to develop a food composition containing a chrysanthemum extract containing an oxidative stress-inhibiting effect by preparing a fermentation broth mixed with chrysanthemum and various herbal medicines.

Korean Patent Publication No. 10-1990-0005901

It is an object of the present invention to provide a food composition comprising a chrysanthemum extract containing chrysanthemum and a variety of herbal medicines in a fermentation broth to improve liver function or oxidative stress.

The objects of the present invention are not limited to the above-mentioned objects, and other objects not mentioned can be clearly understood by those skilled in the art from the following description.

In order to solve the above-mentioned problems, the present invention provides a composition comprising 5 to 15 wt% of a chrysanthemum extract; 20 to 40% by weight of a first herbal medicine extract; 20 to 30% by weight of a second herbal medicine extract; 30 to 40% by weight of saccharides; And from 0.1 to 10% by weight of other substances.

The present invention also relates to a method for producing a chrysanthemum extract, Preparing a first herbal medicine extract; Preparing a second herbal medicine extract; Mixing the chrysanthemum extract, the first herbal medicine extract and the second herbal medicine extract to produce a first composition comprising a chrysanthemum extract; Mixing a saccharide in the first composition to produce a second composition comprising a chrysanthemum extract; And fermenting the second composition to produce a third composition comprising a chrysanthemum extract. The present invention also provides a method for producing a food composition comprising the chrysanthemum extract.

In addition, the present invention provides a method for producing a Chinese herbal medicine, wherein the chrysanthemum extract is 5 to 15 wt%, the first herb extract is 20 to 40 wt% of the first herb extract, the second herb extract is 20 to 30 wt% 30 to 40% by weight of the extract of Chrysanthemum chrysanthemum.

According to the present invention as described above, it is possible to provide a food composition containing an extract of chrysanthemum which has an effect of improving liver function and can prevent nerve stress and related diseases.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic flow chart for explaining a method for producing a food composition comprising a chrysanthemum extract according to the present invention. FIG.
FIG. 2 is a graph showing the results of measuring the DPPH scavenging ability of FCLA and FCLB. FIG.
3 is a graph showing the results of measurement of the oxygen radical absorbance capacity of FCLA and FCLB.
4 is a graph showing the results of measuring cytotoxicity of FCLA and FCLB on HepG2 cells.
5 is a graph showing antioxidative activities of FCLA and FCLB in a human hepatocyte HepG2 model.
6 is a graph showing the intracellular antioxidative activity of fermented products FCLA and FCLB against ethanol-induced oxidative damage.
7 is a graph showing the result of measurement of lipid accumulation.
FIG. 8 is a graph showing the results of measurement of PC12 neuron survival rate through MTT assay. FIG.
Figure 9 is a graph showing the PC12 neuronal protective effect of FCLA and FCLB on H ₂ O ₂ -induced oxidative damage.

BRIEF DESCRIPTION OF THE DRAWINGS The advantages and features of the present invention, and the manner of achieving them, will be apparent from and elucidated with reference to the embodiments described hereinafter in conjunction with the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Is provided to fully convey the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Reference will now be made in detail to the preferred embodiments of the present invention, examples of which are illustrated in the accompanying drawings. &Quot; and / or "include each and every combination of one or more of the mentioned items. ≪ RTI ID = 0.0 >

Although the first, second, etc. are used to describe various components, it goes without saying that these components are not limited by these terms. These terms are used only to distinguish one component from another. Therefore, it goes without saying that the first component mentioned below may be the second component within the technical scope of the present invention.

The terminology used herein is for the purpose of illustrating embodiments and is not intended to be limiting of the present invention. In the present specification, the singular form includes plural forms unless otherwise specified in the specification. The terms " comprises "and / or" comprising "used in the specification do not exclude the presence or addition of one or more other elements in addition to the stated element.

Unless defined otherwise, all terms (including technical and scientific terms) used herein may be used in a sense commonly understood by one of ordinary skill in the art to which this invention belongs. Also, commonly used predefined terms are not ideally or excessively interpreted unless explicitly defined otherwise.

The terms spatially relative, "below", "beneath", "lower", "above", "upper" And can be used to easily describe a correlation between an element and other elements. Spatially relative terms should be understood in terms of the directions shown in the drawings, including the different directions of components at the time of use or operation. For example, when inverting an element shown in the figures, an element described as "below" or "beneath" of another element may be placed "above" another element . Thus, the exemplary term "below" can include both downward and upward directions. The components can also be oriented in different directions, so that spatially relative terms can be interpreted according to orientation.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic flow chart for explaining a method for producing a food composition comprising a chrysanthemum extract according to the present invention. FIG.

Referring to FIG. 1, a method of preparing a food composition containing a chrysanthemum extract according to the present invention includes a step of producing a chrysanthemum extract (S111).

The "Chrysanthemum" is a Chrysanthemum species belonging to the family Asteraceae, originating in Asia and northern Europe. These chrysanthemums are widely distributed in the mountainous regions of southern central part of Korea, and they have been widely used as natural fragrance materials of traditional foods, mainly in the countries of origin and small countries, and the effects of these chrysanthemums on gastrointestinal diseases and headaches are described. Examples of the types of chrysanthemums include, for example, Chrysanthemum aphrodite, Chrysanthemum arcticum, Chrysanthemum argyrophyllum, Chrysanthemum arisanense, For example, Chrysanthemum boreale, Chrysanthemum coronarium, Chrysanthemum crassum, Chrysanthemum glabriusculum, Chrysanthemum hypargyrum, Chrysanthemum lavandulifolium, Chrysanthemum morifolium, Chrysanthemum morii, Chrysanthemum okiense, Chrysanthemum aureus room (Chrysanthemum lavandulifolium, Chrysanthemum orchestra, Chrysanthemum oreastrum, Chrysanthemum potentilloides, Chrysanthemum segetum, Chrysanth < RTI ID = 0.0 > emum shiwogiku, Chrysanthemum sinuatum, Chrysanthemum vestitum, Chrysanthemum weyrichii, Chrysanthemum zawadskii, and the like. In the above example, But is not limited to, preferably, Chrysanthemum morifolium.

In the present invention, Chrysanthemum morifolium was used to obtain a chrysanthemum extract.

The above-mentioned "chrysanthemum extract" means a substance obtained from the above-mentioned chrysanthemum, and the chrysanthemum extract can be obtained by using an extraction method commonly used in the art without limitation.

The chrysanthemum used in the production of the chrysanthemum extract may be natural or dried, but the chrysanthemum may be more preferable in terms of nutrition.

On the other hand, in the present invention, the chrysanthemum extract may include chrysanthemums in a bloom or a dry state without collecting them in a bloom or a dry state, ≪ / RTI >

For example, in the method of producing an extract in the form of an extract, the chrysanthemum is added in an amount of 1 to 10 parts by weight on the basis of 100 parts by weight of water, the water is heated to 50 to 90 캜, the temperature is maintained, Min to 60 min. ≪ / RTI >

Therefore, the meaning of the " extract " described below can be understood as meaning the concept including the state of collecting or extract as described above, and does not limit the meaning of the extract in the present invention.

Next, a method for preparing a food composition containing the chrysanthemum extract according to the present invention includes a step of preparing a first herbal medicine extract (S112).

In the present invention, the first herbal medicine may include Prunellae Spica, Portulaca oleracea, Acorus gramineus or Pycnostelma paniculatum. More specifically, the first herbal medicine extract may contain The extract of Prunellae spica and the extract of Portulaca oleracea may include the extract of Prunellae spica and the extract of Portulaca oleracea, Group 2 The first herbal medicine extract may contain the Acorus gramineus extract and the Pycnostelma paniculatum extract.

Reminiscent of the flowering ear of dried lentil. It means a herbal medicine which is used as a medicinal product for gynecological diseases, skin diseases, tuberculosis, etc.

In addition, the leggings are also known as pig grasses, thistle grasses and horse beetles, and are perennial weeds belonging to the genus Chrysanthemum. They are said to be long-lived if they eat leggings, and they are also called chrysanthemum do

In addition, the above-mentioned Seokchangpo is a plant that grows mainly in the crevices of the waterside, and it can be planted in the waterside of the mooring and seaweed below the forest due to its strong voice, and its leaves have a unique aroma (β-asarone) In the oriental medicine, it is mainly used for the dryness, vitality and the mountain wind, and it is prescribed and used as the main ingredient of the medicine which is called "

In addition, the above-mentioned mountain hake is a perennial herb that grows widely in Korea and can be seen in wild hills and grasses. It is a medicinal substance which is strong in the action to stabilize the mind and to stop the pain, .

The "first herbal medicine extract" means a substance obtained from the first herbal medicine, and the first herbal medicine extract can be obtained by using an extraction method commonly used in the art without limitation.

As described above, in the present invention, the first herbal medicine extract may be an extract of the first group first herbal medicine or a first herbal medicine extract of the second group, and the first herbal medicine extract of the first group may be an extract of Prunellae Spica ) Extract and the Portulaca oleracea extract, and the second group first herbal medicine extract may include the Acorus gramineus extract and the Pycnostelma paniculatum extract.

Next, a method of preparing a food composition containing the chrysanthemum extract according to the present invention includes a step of producing a second herbal medicine extract (S113).

In the present invention, the second herb medicine may include Ganoderma lucidum, Acanthopanax senticosus, Schisandra chinensis, Hovenia dulcis Thumb, Lycii Fructus and Corni Fructus .

The Ganji is a 1-year-old mushroom belonging to the genus Phalaenopsis mongolica and is classified as a commodity medicine (上品 药) with the wild ginseng in the main river gangmyeon. "When the ganji are buried, the body becomes lighter, Is expressed.

It grows in the northern part of the Chukwolryeong, and its base is gray to grayish brown. There are a lot of thorns on the whole, especially thorns on the petiole, with 3 ~ 5 small leaves and inverted long oval to long elliptical There are sharp cogs on the edge.

In addition, the omija is a deciduous vine plant with the buttercups of the buttercups, and the stem is sparse in the branches and the leaves with the leaves are out of order. Leaves are thin, ovate or wide elliptical, pointed end with small sawtooth at edge, leaf vein part is slightly ovoid. It is used for the treatment of asthma, diarrhea, nausea, hyperactivity and neurological diseases as Jinhae, tincture, excitement, diarrhea, and antipyretic agent in one room.

In addition, the humpback is a deciduous broad-leaved arboreous tree belonging to the family Seagrass, which is originated in East Asia and is also grown in other areas. It grows at 50-800 m above sea level in southern central part of Korea and is distributed in Japan and China. The fruit stem is irregularly branched, it is uneven and sweet, and it can be eaten. The wood is used variously for building materials, musical instruments, sculptures, etc. The fruit stem is sweet, so that the fruit stem is dipped. (酒 毒 毒) and Jinpo (鎭 吐) is used as medicinal.

It is also known that Goji is known to be effective in decomposing fatty liver.

In addition, it is a deciduous arboreous tree belonging to the stratum corneum (Cornaceae), and it is known that it is used as a tonic, astringent, and antipyretic in one room by extracting seed from fruit and drying it in the sun.

The "second herbal medicine extract" means a substance obtained from the second herbal medicine, and the second herbal medicine extract can be obtained by using an extraction method commonly used in the art without limitation.

1, a method for preparing a food composition comprising a chrysanthemum extract according to the present invention comprises mixing the chrysanthemum extract, the first herb extract, and the second herb extract to obtain a first composition comprising a chrysanthemum extract (S120).

At this time, in mixing the chrysanthemum extract, the first herbal medicine extract and the second herbal medicine extract, an appropriate amount may be mixed so as to be the final composition ratio of the composition including the chrysanthemum extract described later.

1, a method for preparing a food composition containing a chrysanthemum extract according to the present invention comprises mixing a saccharide in the first composition to prepare a second composition containing an extract of chrysanthemum (S130).

The saccharide may be a monosaccharide or a disaccharide and may be, for example, sucrose, phosgene, xylose, glucose, fructose, lactose, maltose, saccharose, etc. However, It does not.

At this time, in mixing the saccharide into the first composition, an appropriate amount may be mixed so as to be the final composition ratio of the composition including the chrysanthemum extract described later.

Meanwhile, although not shown in the drawing, the second composition containing the chrysanthemum extract may include other substances, for example, sodium chloride, yeast or lactic acid bacteria.

1, a method of preparing a food composition comprising the extract of chrysanthemum according to the present invention includes a step of fermenting the second composition to prepare a third food composition containing an extract of chrysanthemum (S 140 ).

At this time, the fermentation process may be performed by mixing sodium chloride, yeast or lactic acid bacteria as the other composition with the second composition.

The fermentation process may be performed for 30 to 120 days by a general fermentation process, and the fermentation process is not limited to the fermentation process of the present invention.

The food composition containing the chrysanthemum extract according to the present invention can be prepared by the above-described production methods.

The food composition comprising the chrysanthemum extract according to the present invention comprises 5 to 15% by weight of the chrysanthemum extract, 20 to 40% by weight of the first herbal medicine extract, 20 to 30% by weight of the second herbal medicine extract and 30 to 40% And may include from 0.1 to 10% by weight of other materials.

The first herbal medicine extract may be an extract of the first group first herb medicine or a first herb medicine extract of the second group, and the first herb medicine extract of the first group may contain extracts of Prunellae spica and Portulaca oleracea ) Extract, and the second group first herbal medicine extract may include the Acorus gramineus extract and the Pycnostelma paniculatum extract.

Meanwhile, although not shown in the figure, the food composition containing the chrysanthemum extract according to the present invention can be prepared as drinking water. In this case, 20 to 70 parts by weight of water is mixed with 100 parts by weight of the food composition containing the extract of chrysanthemum , A chrysanthemum extract, and the like.

In addition, the preparation of the drinking water can be performed not only by preparing a stock solution containing a food composition containing chrysanthemum extract, but also by mixing it with water, as described above. Alternatively, After the second composition containing the chrysanthemum extract is prepared by mixing the saccharides, the second composition mixed with water may be fermented to prepare the drinking water.

In this case, the third composition containing the chrysanthemum extract may be prepared by mixing 20 to 70 parts by weight of water with respect to 100 parts by weight of the second composition containing the extract of chrysanthemum, and fermenting the second composition mixed with water have.

Hereinafter, experimental examples and effects according to the present invention will be described. However, the present invention is not limited to the following experimental examples and effects.

Experiment and analysis contents

[Experimental Material]

Acetic acid and acetone, sodium hydroxide were obtained from Junsei Chemical (Tokyo, Japan) from Sigma-Aldrich (St. Louis, Mo., USA), and fluorescein and trolox, cupric sulfate, hydrogen peroxide, potassium phosphate and neocuproine were used. 2,2'-Azobis (2-amidinopropane) dihydrochloride (AAPH) was purchased from Wako Pure Chemical (Osaka, Japan). The enzyme-linked immunosorbent assay (ELISA) reader and the fluorescence reader were instrumented by Tecan (Salzburg, Austria).

[Preparation of sample]

The herbal medicines are dipped in water, washed with water and then soaked in 0.1% physiological saline overnight. After washing the herbal medicines immersed in physiological saline with primary distilled water, sterilized water of 50% by weight was separately added to FCLA and FCLB by varying the constituents of main medicinal materials as shown in Table 1, and then mixed with sugar, Fermented UF-10, Korean UCD). After a lapse of 100 days from the start of fermentation, the fermentation broth was filtered through a cotton pad and used for analysis.

[Total phenol content ( Total phenolic content )]

Total phenol content was measured by the Folins-Denis method, which is widely used as an analytical method. Add 1 mL of fermentation broth, 1 mL of distilled water, dilute it, add 2 mL of 1 N Folin-Ciocalteu reagent, leave at room temperature for 3 minutes, add 2 mL of 10% Na ₂ CO ₃ solution, The reaction was carried out at room temperature. 200 μL of this mixture was taken and absorbance was measured at 700 nm using a spectrophotometer (UV-1601, Shimadzu, Tokyo, Japan). Gallic acid was used as a standard and the total phenol content was expressed in units of mg / 100 g of gallic acid equivalents (GAE).

[ Oxygen radical absorbance capacity  ( ORAC ) assay ]

The scavenging activity of peroxy radicals was assessed by the oxygen radical absorbance capacity (ORAC) method, which was partially modified by Kurihara et al. For analysis, a 96-well black microplate (flat bottom type) was used to minimize fluorescence interference. 100 μL of 40 nM fluorescein dissolved in 75 mM potassium phosphate buffer (pH 7.4) was added to the plate and dissolved in the same buffer to prepare 0.1, 0.25, 0.5, 1 and 2.5 mM samples. 50 μL of sample was added to the plate, and then 50 μL of 20 mM 2, 2'-azobis (2-methylpropionamidine) dihydro-chloride (AAPH) was added to produce peroxyl radical. 6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl acid (trolox), a water-soluble vitamin E derivative used as a control, was newly prepared and used in each experiment. The fluorescence analyzer was set up to stabilize for 5 seconds after shaking the 96-well plate containing the reaction mixture for 10 seconds before the measurement. Samples were set up to measure fluorescence for a total of 200 minutes and fluorescein was measured at excitation wavelength 485 nm and emission wavelength 535 nm. The final result was calculated as the difference between the fluorescence value of the test sample and the fluorescence value of the blank, and all results were expressed in terms of trolox equivalents (TE, Trolox Equivalents, μM).

[Cell survival rate Cell Viability )]

MTT (3- [4,5-dimethyl-thiazol-2-yl] -2,5-diphenyl tetrazolium bromide) assay was performed to measure the effect of fermentation broths FCLA and FCLB on cell viability. HepG2 cells (1 × 10 ⁵ cells / mL) were dispensed into 96-well plates at a density of 1 × 10 ⁴ cells / well in a volume of 100 μL and incubated at 37 ° C and 5% CO ₂ for 24 hours , 10, 50, and 100 μg / mL of the fermentation broth, cultured for 30 minutes, and treated with 100 mM ethanol for 24 hours. After culturing for 24 hours, 10 μL of MTT solution (5 mg / mL) was added and incubated at 37 ° C. for 1 hour. After removing all the culture solution, 100 μL of DMSO was added and allowed to stand at room temperature for 15 minutes to dissolve the formazan crystal Absorbance was measured on a 540 nm absorbance filter of an ELISA microplate reader (Bio-Tek). When the number of control cells was taken as 100%, the relative inhibition rate of cell growth was obtained.

[ Intracellular reactive oxygen species  ( ROS ) Measure]

Using HepG2 cells, the level of intracellular ROS was measured using DCFH-DA. In a 96-well plate, 5 × 10 ⁴ cells / mL of HepG2 were incubated at 37 ° C, 5% CO ₂ were incubated in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. After 24 hours, the medium was removed and 200 μL of fluorescence-stable Hank's balanced salt solution (HBSS) was dispensed into each well. Each sample was treated with a final concentration of 100 μg / mL and incubated for 30 minutes. After incubation, the cells were washed with HBSS, and 2 μL of 80 mM AAPH was added to 200 μL of HBSS to induce peroxyl radical formation and cultured for 30 minutes. After incubation for 30 min with addition of 40 mM DCFH-DA with a fluorescent probe, the fluorescence was detected at 485 nm and 535 nm using a GENios fluorescence plate reader (Tecan Trading AG, Salzburg, Austria) The degree of occurrence was measured. Negative controls were treated with DCFH-DA only and positive controls with AAPH and DCFH-DA. The relative fluorescence values of each sample and AAPH were compared with the fluorescence value of the control group as 100%.

[ Lipid accumulation  ( Oil Red  O Staining )]

HepG2 cells treated with 100 mM ethanol for 24 hours were washed with phosphate buffered saline (PBS, pH 7.4), and 10% (v / v) ) formalin-PBS for 1 hour. The dried cells were stained with 0.6% (w / v) Oil Red O-60% isopropanol for 15 minutes and washed once with 70% ethanol and sterilized water. The cells were stained with 4% Nonidet P-40-isopropanol for 5 minutes and the absorbance was measured at 520 nm using a microplate reader (Bio-Tek).

[ MTT assay ]

MTT reduction assay was performed to measure the protective effect of each cell. Cells were plated in 96-well plates at a concentration of 5 × 10 ⁴ cells / mL in 100 mL portions and incubated for 24 h at 37 ° C in 5% CO ₂ incubator, and then treated with various concentrations of PSE-1. After incubation for 30 minutes, 20 mM glutamate was added and cultured for 24 hours. 10 mL of MTT (5 mg / mL) solution dissolved in PBS buffer was added to each well and reacted for 1 hour. After formazan formation was confirmed, the medium was completely removed and 100 mL of DMSO was added to dissolve the formazan formed on the well bottom, and the absorbance was measured at 540 nm using an ELISA reader (Model 680, BioRad, USA). The relative inhibition of cell growth was determined when control cells were 100%.

[ Microscopy 관측 of morphologic changes ]

Glutamate-induced cell lines were cultured for 24 h at 5 × 10 ⁴ cells / mL in a 6-well plate to observe the effect of fermentation on the morphological changes. Cells were treated at various concentrations and cultured for 30 min. The cells were exposed to 20 mM glutamate for 24 hr. The morphological characteristics of the PC12 cell line were photographed at 100-fold using a phase-contrast microscope (Nikon, Japan) .

[Statistical processing Statistical 분석 )]

Statistical analysis of all data was performed using SPSS for Windows according to each item. The mean and error were calculated and the significance was verified for the mean values at 95% confidence level ( p <0.05). F-values were obtained by one-way ANOVA and Duncan's multiple range test was used to verify the significance of each item.

Results and Discussion

[Total phenol content ( Total phenol content )]

The antioxidant activities of herbal medicines and natural products were highly correlated with phenolic compounds. Thus, in this study, the total phenolic content of FCLA and FCLB was analyzed to determine the phenol content of FCLA and FCLB in Table 1 above. As a result, as shown in Table 2, the total phenol content of FCLB was 962.76 ± 1.97 μg / mL in FCLA and 1,473.46 ± 6.77 μg / mL in FCLB, indicating that FCLB was about 1.5 times higher than that of FCLA. This result can be interpreted that it contains high phenol contents compared with chrysanthemum and black pepper.

[ DPPH Radical  Scavenging activity]

The DPPH radical elimination method is a widely used method for analyzing antioxidant functionality.

FIG. 2 is a graph showing the results of measuring the DPPH scavenging ability of FCLA and FCLB. FIG.

As shown in Fig. 2, the DPPH radical scavenging ability was increased in a concentration-dependent manner at 0.5-10 mg / mL for both FCLA and FCLB. IC ₅₀ represents the 50% scavenging is FCLA a 26.02 mg / mL, FCLB was found excellent DPPH radical scavenging activity of FCLA to 44.53 mg / mL. Except cell experiments in In vitro antioxidant assays are largely due to three radical scavenging mechanisms, which can infer the radical scavenging mechanism of antioxidants. A representative example is a mechanism of eliminating radicals through electron donating ability, and the DPPH radical erasing method is included here. The second is the method of eliminating radicals through hydrogen donating ability and the ORAC assay using AAPH. Finally, there is a mechanism for radical scavenging through both electron and hydrogen donating ability. These results indicate that the electron donating ability of FCLA and FCLB contributes to DPPH radical scavenging activity, and that the electron donating ability of various plant extracts and flavonoids may contribute to their antioxidant activity.

[ Oxygen radical absorbance capacity  ( ORAC ) assay ]

Next, we investigated the peroxyl radical scavenging activity of FCLA and FCLB. Various methods have been reported to analyze antioxidant activity by many researchers. Among them, ORAC analysis was first attempted by the Ghiselli and Glazer research teams, and the current ORAC analysis method is widely used by the Cao research team. The ORAC method can be applied to a variety of plant materials, tissues and blood of the organism as well as food components, and is highly utilized. EFSA and the European Food Safety Authority of the US Food and Drug Administration (USDA) and the United States Department of Agriculture The ORAC value is presented. The ORAC _{ROO ·} How to measure the current antioxidant activity using AAPH as a peroxyl radical generator is the most widely used. The production of peroxyl radicals is well known as ROS, which promotes fat oxidation in particular.

3 is a graph showing the results of measurement of the oxygen radical absorbance capacity of FCLA and FCLB.

As shown in FIG. 3, both FCLA and FCLB increased the concentration-dependent activity at 10-500 μg / mL and showed the highest activity at 500 μg / mL of FCLA. The ORAC _{ROO ·} method is an experimental method based on the hydrogen donating ability of the sample. This result shows that both fermentations FCLA and FCLB effectively remove peroxyl radicla based on the hydrogen donating ability. In particular, FCLA of 500 μg / mL showed antioxidant activity of trolox 14.3 TE (trolox equivalents, μM), well known as antioxidant vitamin, and FCLB showed antioxidant activity of 13.6 TE.

[ MTT assay Cell viability measurement by Cell viability assay )]

The cytotoxicity of FCLA and FCLB to HepG2 cells was evaluated by MTT assay. The MTT assay can be used to analyze the cytotoxicity of FCLA and FCLB by measuring the mitochondrial activity of reducing the yellow water soluble substrate MTT tetrazolium to a bluish purple water-insoluble MTT formazan by dehydrogenase.

FIG. 4 is a graph showing the results of measuring the cytotoxic characteristics of FCLA and FCLB on HepG2 cells. FIG.

Referring to FIG. 4, when FCLA and FCLB were treated for 30 minutes at a concentration of 10-100 μg / mL, there was no significant cytotoxicity compared with the control (NC). Several researchers have reported that natural products and polyphenols induce apoptosis in cancer cell models. Chen et al. Reported significant cell death of hepatocellular carcinoma cell line SK-HEP-1 when treated with 20-80 μM of fisetin for 24 h. Khan et al. Reported that when 48 h of 10-60 μM fisetin was treated, Cell death was observed in LNCaP.

However, HepG2 used in this experiment did not show any cytotoxicity. Ahmad et al. Reported that epigallocatechin-3-gallate (EGCG), a typical flavonoid of green tea, inhibited cancer cell-selective apoptosis by differently modulating the expression of nuclear factor κB, a transcription factor, in cancer cell lines and normal cell lines. These results suggest that phytochemicals derived from natural products can regulate transcription factors and gene and protein expression related to apoptosis in cancer cell lines and normal cell lines. Based on the cell survival results in this study, FCLA and FCLB up to 100 μg / mL were used in subsequent experiments.

[ AAPH Induced Oxidative  For damage Fermentation product FCLA  And FCLB of Intracellular  Antioxidant activity]

Antioxidative activities of FCLA and FCLB were evaluated in human hepatocyte HepG2 model. Recently, the DCFH-DA method has been widely used by many researchers as a method for measuring intracellular reactive oxygen species. When DCFH-DA, which has permeability to animal cells, is introduced into the cytoplasm, DCFH-DA is hydrolyzed by DCFH by the esterase present in the cytoplasm. When active oxygen is present, DCFH, which is not fluorescent, Oxidized and converted to a strong green-colored fluorescent DCF. Therefore, qualitative and quantitative analysis is possible by measuring fluorescence intensity of DCF through fluorescence microscope and fluorescence analyzer.

5 is a graph showing antioxidative activities of FCLA and FCLB in a human hepatocyte HepG2 model.

5, HepG2 cells were treated with FCLA and FCLB, respectively, and the inhibitory effects of AAPH-induced oxidative stress on ROS production were examined. As a result, both FCLA and FCLB were found to be 5-100 μg / mL inhibited oxidative stress induced by AAPH in a concentration dependent manner and showed a significantly higher inhibitory activity at high concentration (50, 100 μg / mL) of FCLA and FCLB. In particular, 100 μg / mL of FCLA inhibited oxidative stress by 61.3% compared to the control, and FCLB inhibited oxidative stress by 68.35%. AAPH is well known as an active oxygen that induces cell membrane damage and peroxidation of intracellular lipids. Therefore, this result shows that the fermentation products FCLA and FCLB can effectively inhibit lipid peroxidation in the cells.

[Ethanol-induced Oxidative  For damage Fermentation product FCLA  And FCLB of Intracellular  Antioxidant activity]

Effect of FCLA and FCLB fermentation broth on the survival of HepG2 hepatocyte cells after induction of oxidative stress by treatment of 10, 50, and 100 μg / mL of fermentation broth for 30 minutes with 100 mM ethanol for 24 hours Respectively.

6 is a graph showing the intracellular antioxidative activity of fermented products FCLA and FCLB against ethanol-induced oxidative damage.

Referring to FIG. 6, cell viability was reduced by 30% compared with the control by 100 mM ethanol treatment. Cell viability increased by 24.9-16.5% and 16.5-18.8% by FCLA and FCLB treatments, respectively. The results of this experiment show that the fermentations FCLA and FCLB effectively reduce ethanol-induced oxidative damage.

[ Lipid accumulation  Measure ( Oil Red  O staining )]

Oil Red O dye is combined with triglyceride to become red color and is widely used to evaluate the accumulation of intracellular fat. The effects of fermentation broth, FCLA and FCLB, on the accumulation of HepG2 hepatocyte were investigated. Oxidative stress was induced by 100 mM ethanol treatment for 30 min.

7 is a graph showing the result of measurement of lipid accumulation.

Referring to FIG. 7, in the ethanol-treated group, the fat content was significantly increased (8%) as compared with the control group, and the fat accumulation by ethanol was inhibited by 17.1-19.6% in both FCLA and FCLB. However, there was no significant difference between FCLA and FCLB. The results of this experiment show that the fermented product can be fully utilized as a food material that can prevent alcoholic fatty liver.

[ MTT assay Through PC12  Nerve cell survival rate]

To determine the effect of fermentation broth on cell viability, MTT (3- [4,5-dimethyl-thiazol-2-yl] -2,5-diphenyl tetrazolium bromide) assay was performed on PC12 neurons.

FIG. 8 is a graph showing the results of measurement of PC12 neuron survival rate through MTT assay. FIG.

Referring to FIG. 8, FCLA and FCLB were treated for 30 minutes at a concentration of 1-50 μg / mL. For comparison, no cytotoxicity was found in all treatments when treated with the same concentration of vitamin C. Based on the results of PC12 cell survival, FCLA and FCLB up to 50 μg / mL were used in the subsequent experiments.

[ H ₂ O ₂ Induced Oxidative  For damage FCLA  And FCLB of PC12  Nerve cell protection effect]

The effect of fermentation broth (FCLA) and FCLB (1, 10, and 50 μg / mL) for 30 minutes and 1 mM H ₂ O ₂ for 2 hours induce oxidative stress Respectively.

Figure 9 is a graph showing the PC12 neuronal protective effect of FCLA and FCLB on H ₂ O ₂ -induced oxidative damage.

Referring to Figure 9, it was by H ₂ O ₂ treatment decreased the cell viability of 78% than that of the control, and FCLA FCLB both concentration-dependent manner eotneunde shows the cytoprotective effect in FCLA 37.9- than H ₂ O ₂ treatment (PC) 51.%, and FCLB increased the cell viability by 33.7-128.5%. The cell survival rate was increased by 128.5% in 50 μg / mL FCLB compared to PC, and the oxidative damage protection level was the same as that of 50 μg / mL vitamin C (131.5%). Therefore, we confirmed the high cytoprotective effect of FCLB on oxidative stress induced by H ₂ O ₂ .

The results of the experiment are summarized as follows.

First, total phenol contents of FCLA and FCLB were analyzed and the total phenol contents of FCLB were significantly higher.

In addition, DPPH radical scavenging activity, ORAC value, and ROS production inhibitory activity showed a concentration-dependent effect, and there was no difference between FCLA and FCLB.

In addition, FCLA and FCLB for oxidative stress induced by ethanol in hepatocyte HepG2 cells did not show cytotoxicity at 10-100 μg / mL, and both FCLA and FCLB showed protective effects against ethanol-induced cytotoxicity Respectively. Both FCLA and FCLB inhibited increased fat accumulation due to ethanol - induced oxidative stress.

In addition, FCLA and FCLB analysis of oxidative stress induced H ₂ O ₂ -induced neuronal PC12 cells showed that neither FCLA nor FCLB showed cytotoxicity at 1-50 μg / mL, and both FCLA and FCLB Dependent protection against H ₂ O ₂ -induced cytotoxicity.

These results indicate that the fermentation broths FCLA and FCLB are useful as food or health functional food materials for improving liver function, nerve stress and related diseases.

Therefore, the food composition containing the chrysanthemum extract according to the present invention has an effect of improving liver function, and also can prevent nerve stress and related diseases.

While the present invention has been described in connection with what is presently considered to be practical exemplary embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, It will be understood. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (8)

5 to 15% by weight of chrysanthemum extract;
20 to 40% by weight of a first herbal medicine extract;
20 to 30% by weight of a second herbal medicine extract;
30 to 40% by weight of saccharides; And
0.1 to 10% by weight of other materials,
Wherein the first herbal medicine extract is a first group first herbal medicine extract or a second group first herbal medicine extract, the first group first herbal medicine extract comprises Prunellae Spica extract and Portulaca oleracea extract, The second group of first herbal medicine extracts include Acorus gramineus extract and Pycnostelma paniculatum extract,
The second herbal medicine extract includes Ganoderma lucidum, Acanthopanax senticosus, Schisandra chinensis, Hovenia dulcis Thumb, Lycii Fructus and Corni Fructus,
Wherein the other substance is sodium chloride, yeast or lactic acid bacteria. delete delete delete Producing a chrysanthemum extract;
Preparing a first herbal medicine extract;
Preparing a second herbal medicine extract;
Mixing the chrysanthemum extract, the first herbal medicine extract and the second herbal medicine extract to produce a first composition comprising a chrysanthemum extract;
Mixing a saccharide in the first composition to produce a second composition comprising a chrysanthemum extract; And
And fermenting the second composition to produce a third composition comprising a chrysanthemum extract,
Wherein the first herbal medicine extract is a first group first herbal medicine extract or a second group first herbal medicine extract, the first group first herbal medicine extract comprises Prunellae Spica extract and Portulaca oleracea extract, The second group of first herbal medicine extracts include Acorus gramineus extract and Pycnostelma paniculatum extract,
The second herbal medicine extract comprises a food composition comprising Ganoderma lucidum, Acanthopanax senticosus, Schisandra chinensis, Hovenia dulcis Thumb, Lycii Fructus and Corni Fructus. Way. delete delete 6. The method of claim 5,
Wherein the first herb extract is 20 to 40 wt%, the second herb extract is 20 to 30 wt%, the saccharide is 30 to 40 wt%, the second herb extract is 20 to 30 wt% &Lt; RTI ID = 0.0 &gt; 1, &lt; / RTI &gt;

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