KR101245191B1 - Extract of fermented wild dropwort for stimulating hepatic function and preparing method thereof - Google Patents

Abstract

The present invention relates to the production of a buttercup fermentation solution for protecting and improving liver function, and specifically, a method of preparing a buttercup fermentation solution for protecting and improving liver function by naturally fermenting and ripening buttercups using a leaching agent, and a buttercup produced by the same. It relates to a fermentation broth. According to the present invention, the step of preparing water by washing with buttercup, mixing and mixing the brown sugar or oligosaccharide as a leaching agent with the buttercup and leach in a ratio (weight ratio) of 1: 5 to 5: 1, Provided is a method for producing a fermentation broth for protecting and improving liver function, which comprises a fermentation step of fermenting at 15 to 25 ° C. for 10 to 14 months. The buttercup fermentation broth prepared by the production method is natural without using an organic solvent It is safe by fermentation, and the active active ingredients of chlorogenic acid and caffeic acid are strengthened by natural fermentation and aging, so it is excellent in protecting and improving liver function.

Description

Extract of fermented fermented liquor for protecting and improving liver function and preparation method thereof {{Extract of fermented wild dropwort for stimulating hepatic function and preparing method

The present invention relates to the production of a buttercup fermentation solution for protection and improvement of liver function, specifically, a method of preparing a buttercup fermentation solution to protect and improve liver function by fermenting and fermenting buttercups naturally using leaching agent and the buttercup produced by the same. It relates to a fermentation broth.

The buttercup (Oenanthe stolonifera DC, Dropwort) is a perennial plant belonging to the mountain family, grows in wetlands where water can be supplied sufficiently, and is grown as a special crop in farmhouses. Buttercup is a representative alkaline food containing flavonoids, vitamins, potassium, calcium and iron from a food nutritional perspective. Buttercup contains 95% moisture, 2% crude protein, 0.3% crude oil, 0.7% crude ash, and contains essential oils such as essential oil and myristicin. In oriental medicine, it is known to be used in the treatment of fever, diuresis, jaundice, hypertension, or in preparation for detoxification after drinking. According to the cultivation method, it is reported that the buttercups grown in the field have higher activity than the buttercups grown in the rice fields. Buttercup is mainly used for vegetables and green juice. In this case, it is used as a fermented processed product for long-term storage because of storage problem.

Although buttercup is widely used in the private sector for liver protection and detoxification of hangovers, studies on active ingredient changes according to specific fermentation methods or fermentation conditions are insufficient. Therefore, the present inventors studied the change of flavor of fermentation broth according to various methods of fermentation of parsley, and the change of functional active ingredient according to fermentation or aging conditions, and accordingly fermented parsley by mixing with brown sugar or oligosaccharide as a leaching agent. And when aging it was found that the flavor is excellent and the specific useful ingredient is strengthened, and confirmed that it can be usefully used as a health functional food for liver function protection and improvement, and completed the present invention.

It is an object of the present invention to provide a method for producing a fermentation broth for protecting and improving liver function by fermenting and aging natural parsley under specific conditions using a leaching agent of brown sugar or oligosaccharide.

It is another object of the present invention to provide a buttercup fermentation broth, prepared by the above method, which is useful for protecting and improving liver function.

According to the present invention in order to achieve the above object, the step of preparing to wash water; Mixing the buttercups with brown sugar or oligosaccharide as a leaching agent in a ratio (weight ratio) of 1: 5 to 5: 1 in the washed and sterilized pottery; And it provides a method of producing a fermentation broth for the protection and improvement of liver function comprising a fermentation step of natural fermentation for 10 to 14 months at 15 ~ 25 ℃.

According to another embodiment of the present invention there is provided a method for producing a fermentation broth for liver function protection and improvement, further comprising the step of aging the leaching solution obtained in the fermentation step at a temperature of 4 ~ 6 ℃ refrigerated for 10 to 26 months. .

According to the present invention in order to achieve the above another object, there is provided a buttercup fermentation solution for the protection and improvement of liver function is prepared by the above production method and is strengthened chlorogenic acid and caffeic acid as the main active ingredient.

Buttercup fermentation solution prepared according to the present invention is safe by natural fermentation without the use of organic solvents, and by the natural fermentation and ripening, functional active ingredients of chlorogenic acid and caffeic acid are further strengthened to protect and improve liver function A buttercup fermentation liquid excellent in activity is obtained. In addition, the buttercup fermentation solution prepared according to the present invention has the advantage of the above liver function activation and at the same time exhibits an excellent flavor has the advantage that development as a health functional food is advantageous.

1 is a photograph showing the appearance of the buttercup fermentation broth prepared according to an embodiment of the present invention.
Figure 2 is a graph showing the protective effect against hepatocellular oxidative damage of the buttercup fermentation broth according to an embodiment of the present invention.
Figure 3 is a graph measuring the activity of the LDH outflow into the culture medium when damaged cell membrane in order to determine whether the buttercup fermentation broth according to an embodiment of the present invention has a protective effect against oxidative stress of hepatocytes induced by t-BHP to be.
Figure 4 is a graph showing the growth inhibitory effect of hepatic cancer cells (HepG2 cells) and normal liver cells (Chang cells) of brown sugar buttercup fermentation broth according to an embodiment of the present invention.
5 is a graph showing the effect of inhibiting the growth of liver cancer cells (HepG2 cells) and normal liver cells (Chang cells) of the oligosaccharide buttercup fermentation broth according to an embodiment of the present invention.
Figure 6 is a photograph showing the proliferation and morphological changes of hepatic cancer cell line HepG2 cells when treated with a buttercup fermentation solution according to an embodiment of the present invention.
7 is a graph showing the results of HPLC analysis for identifying the active ingredient of the buttercup fermentation broth according to the embodiment of the present invention.
8 is a graph showing the results of MS analysis for identifying the active ingredient of the buttercup fermentation broth according to an embodiment of the present invention.
9 is a graph showing the results of HPLC analysis for identifying the concentration of the active ingredient of the buttercup fermentation broth according to the embodiment of the present invention.
10 is a graph showing the results of HPLC reanalysis after mixing Example 4 and the active ingredient in a 1: 1 ratio to identify the active ingredient of the buttercup fermentation solution of the present invention.
11 is a graph showing the results of HPLC analysis of Example 4 and the organic solvent extract to identify the difference in content of the active ingredient according to the extraction method of the present invention.

The present invention is excellent in flavor when natural fermentation and ripening under specific conditions using brown sugar or oligosaccharide as a leaching agent instead of the conventional extraction method by organic solvents and is an active ingredient useful for improving liver function and protection Chlorogenic acid and caffeic acid are characterized in that the fermentation broth for the protection and improvement of liver function can be prepared.

According to an embodiment of the present invention, the steps of preparing water by washing the buttercup; Mixing the parsley and the brown sugar or oligosaccharide as a leaching agent in a ratio (weight ratio) of 1: 5 to 5: 1, preferably in a ratio (weight ratio) of 1: 1, in the washed and sterilized pottery; And it provides a method for producing a buttercup fermentation broth comprising a fermentation step of natural fermentation for 10 to 14 months at room temperature of 15 ~ 25 ℃. The buttercup can be used regardless of the type, the activity was better when using the stone parsley. When fermented and aged using brown sugar or oligosaccharide as a leaching agent, it has excellent sensory characteristics such as color, flavor, taste, and viscosity of the buttercup fermentation solution, and elution of chlorogenic acid and caffeic acid, active ingredients related to liver function improvement. It was easy to strengthen. When fermentation is less than 10 months, there is a problem that the active ingredients of chlorogenic acid and caffeic acid are not sufficiently eluted and strengthened, and when fermented at room temperature for more than 14 months, the buttercups recede and the flavor is deteriorated. There is a problem that the yield is reduced.

According to another embodiment of the present invention further comprises the step of aging the leaching solution obtained in the fermentation step while refrigerated for 10 to 26 months at a temperature of 4 ~ 6 ℃. Refrigerating only the leachate in this way is a problem that the extract is again absorbed by the buttercup solid (bak) when it is continuously aged at room temperature together with the buttercup solid, the extraction yield is reduced, the buttercup gourd is reduced, taste, flavor, nutrition Because there may be. Through the aging process, the reducing sugar content increased and the sensory preference such as taste and viscosity increased. In addition, there is an advantage that the color and taste of the extract is softened and the viscosity decreases through the aging process to increase the preference as a commodity. If the fermentation is less than 10 months, there is a problem that the flavor of the buttercup fermentation solution is insufficient, and if it is aged over 26 months, there is a problem that the functional active ingredient is reduced to reduce the liver function protection and improvement effect.

The buttercup fermentation broth prepared by the above method contains a large amount of flavonoids, and it was confirmed that the DPPH radical scavenging ability, the protective effect against oxidative damage of hepatocytes, and the hepatocellular growth inhibitory effect were excellent. As a result of the component analysis, it was confirmed that the buttercup fermentation broth contained a large amount of chlorogenic acid and caffeic acid, and it was found that such functional active ingredients were strengthened through the fermentation and aging process of the present invention to show such liver function protection and improving activity. . In addition, it was confirmed that the strengthening effect of the active ingredient is superior to the case of treating the buttercups by the fermentation and aging method of the present invention as compared to the case of the treatment with the general organic solvent extraction method. In addition, the present invention, the buttercup fermentation broth has a high reducing sugar content and excellent flavor can be developed as a health functional beverage.

EXAMPLE

Hereinafter, the present invention will be described in detail with reference to the following examples and test examples, but the present invention is not limited to these examples.

Example 1 Preparation of Fermented Buttercup

After washing the clean parsley harvested from Jeongdae-ri, Daechang, Bisulsan, Gachang-myeon, washed and sterilized into pottery, and added brown sugar (leaching agent) as a leaching agent in a ratio (weight ratio) of 1: 1. The buttercup fermented in sugar was left at room temperature (15-25 ° C.) for one year while the useful ingredient in the raw material was eluted by osmotic pressure and natural fermentation occurred.

Example 2 Preparation of Fermented Buttercup

Only the buttercup fermentation broth prepared in Example 1 was transferred to a storage container, and aged for one year while being refrigerated in a 4 ° C. cold room to prepare a buttercup fermentation broth.

Example 3 Preparation of Fermented Buttercup

Only the buttercup fermentation broth prepared in Example 1 was transferred to a storage container and further aged for 2 years while being refrigerated in a 4 ° C. cold storage room to prepare a buttercup fermentation broth.

Example 4 Preparation of Fermented Buttercup

After washing the clean parsley harvested from Jeongdae-ri, Daechang, Bisulsan, Gachang-myeon, washed and sterilized into pottery and mixed oligosaccharide as a leaching agent in a ratio of 1: 1 (weight ratio). The buttercup fermented in sugar was left at room temperature (15-25 ° C.) for one year while the useful ingredient in the raw material was eluted by osmotic pressure and natural fermentation occurred.

Example 5 Preparation of Fermented Buttercup

Only the buttercup fermentation broth prepared in Example 4 was transferred to a storage container and aged for one year while being refrigerated in a 4 ° C. cold storage room to prepare a buttercup fermentation broth.

Example 6: Preparation of Buttercup Fermentation Solution

Only the buttercup fermentation solution prepared in Example 4 was transferred to a storage container, and aged for 2 years while being refrigerated in a 4 ° C. cold storage room to prepare a buttercup fermentation solution.

Test Example 1: Physicochemical Properties of Buttercup Fermentation Broth

In order to determine the physicochemical properties of the buttercup fermentation broth prepared in the above example, pH, reducing sugar content, sugar content, color and amino nitrogen content were measured in the following manner.

pH, titration acidity measurement

The pH of the buttercup fermentation extract was measured using a pH meter (Orion 720A, USA), and the titratable acidity was added to 10 mL of the buttercup fermentation extract by adding 20 mL of distilled water until the pH reached 8.3 using a pH meter. Proper consumption was converted into lactic acid content (%).

Sugar, reducing sugar analysis

Sugar content of parsley fermentation extract is by using a digital Saccharimeters (Atago PAL-1, Japan) was measured as ° Brix, a reducing sugar is to dilute the parsley extract 10 ^three times, a spectrophotometer (Spectrophotometer 650, Beckman, USA) using the DNS method The value measured at 580 nm is expressed in%.

Chromaticity measurement

For chromaticity, L (lightness), a (redness / greenness), and b (yellowness / blueness) values were measured using a color QUEST II (Hunter Lab, USA), where the standard white plate was L = 92.68, a = -0.81 and a value of b = 0.86 were used.

The results are shown in Table 1 below.

sample Sugar (° Bx) pH Reducing Sugar (%) Titratable acidity (%) Amino nitrogen (mg%) Example 1 36.8 3.77 23.2 2.17 1125.2 Example 2 54.6 3.91 39.2 0.82 370.9 Example 3 54.6 4.01 39.7 0.80 326.9 Example 4 23.1 3.83 15.8 1.41 558.9 Example 5 40.0 3.43 31.5 1.39 241.9 Example 6 41.4 3.67 33.0 0.78 122.5

As shown in Table 1, the sugar, pH and reducing sugar of the brown sugar fermentation extract were higher, and the optimum acidity showed 2.17% of the brown sugar fermentation extract (Example 1), but the fermented buttercup fermented thereafter. In the case of, it was lowered to 0.82 to 0.80%, and the oligosaccharide extract was also lowered from 1.41% to 1 year (Example 4) to 1.39 to 0.78% at 2 to 3 years. In the case of amino nitrogen, both brown sugar and oligosaccharides showed high values in the first year and decreased with aging in the second to third years, and the brown sugar extract showed higher amino nitrogen content than the oligosaccharide extract.

The results of examining the appearance of the buttercup fermentation solution prepared in the above Example are shown in Table 2 and FIG. 1. As shown in FIG. 1, the brown sugar fermentation broth of Examples 1 to 3 exhibits a dark brownish brown color, whereas the oligosaccharide extract of Examples 4 to 6 has a 1 year fermentation broth, which is bright crimson and aged for 2 to 3 years. Red color was found to decrease. As shown in Table 2, the L sugar and b value decreases as ripening proceeds in the brown sugar extract, whereas the L value increases in the oligosaccharide extract while the a and b values decrease significantly. .

sample Chromaticity L a b Example 1 26.33 25.94 41.09 Example 2 11.42 29.29 17.70 Example 3 12.15 27.19 19.38 Example 4 55.90 35.32 16.49 Example 5 65.86 4.30 3.67 Example 6 63.86 1.30 -1.13

Test Example 2: Sensory Characteristics of Buttercup Fermentation Solution

Ten sensory fermentation agents of the buttercup fermentation broth were analyzed for sensory characteristics through flavor description test and sensory evaluation on the intensity of sensory characteristics was performed. In order to measure the sensory preference of the extract, the sensory test was carried out on the color, aroma, taste, body feeling, and overall preference in a 9-point preference scale method.

All experiments were repeated three times, and the result value was calculated using the SAS program (SAS Institute Inc., USA, ver. 9.0) to calculate the mean and standard deviation of each test zone, and the difference between each test zone was ANOVA. In case of significant difference at p <0.05 after analysis, Duncan's multiple range test (v.10.0) was used for post-verification.

The results are shown in Tables 3 and 4 below.

sample color Sour scent Sweet incense Chinese herbal incense sweetness Sour taste Viscosity Example 1 6.7 ± 0.5 6.3 ± 1.3 5.6 ± 1.3 5.7 ± 2.0 5.4 ± 1.5 6.0 ± 1.2 5.4 ± 1.7 Example 2 8.0 ± 0.0 5.9 ± 1.8 5.6 ± 1.0 6.0 ± 2.0 7.4 ± 1.4 4.4 ± 1.0 5.9 ± 1.7 Example 3 7.7 ± 0.0 6.1 ± 1.8 5.6 ± 1.0 6.1 ± 2.0 7.0 ± 1.4 4.9 ± 1.0 6.0 ± 1.7 Example 4 5.3 ± 0.8 6.0 ± 1.2 5.0 ± 1.0 3.9 ± 0.7 3.7 ± 0.8 6.9 ± 1.4 3.3 ± 0.8 Example 5 4.3 ± 0.8 5.6 ± 1.5 4.3 ± 1.3 4.0 ± 1.0 6.0 ± 1.6 6.6 ± 1.4 3.1 ± 0.7 Example 6 3.6 ± 0.8 5.6 ± 1.5 4.4 ± 1.3 3.9 ± 1.1 6.4 ± 1.6 5.7 ± 1.7 3.4 ± 0.5

As confirmed in Table 3, a total of seven sensory characteristics such as flavor characteristics such as sour flavor, sweet flavor, herbal medicinal flavor, sweetness, sour taste, and physical properties such as color and viscosity were derived. In all sensory properties except brown sugar extract showed stronger characteristics than oligosaccharide extract.

The results of the sensory test on the color, flavor, taste, viscosity and overall acceptability of the buttercup fermentation extract are shown in Table 4 below.

sample color incense flavor Viscosity Overall likelihood Example 1 4.6 ± 1.8 5.1 ± 0.9 4.9 ± 2.5 5.7 ± 1.0 4.7 ± 2.5 Example 2 3.7 ± 0.8 5.4 ± 0.8 7.3 ± 1.1 5.9 ± 0.9 7.4 ± 0.8 Example 3 3.9 ± 0.8 4.9 ± 0.8 6.9 ± 1.1 5.9 ± 0.9 7.0 ± 0.8 Example 4 7.0 ± 0.8 5.9 ± 1.6 2.9 ± 1.4 4.9 ± 1.2 2.9 ± 1.5 Example 5 6.6 ± 1.0 5.9 ± 1.1 3.9 ± 1.4 5.1 ± 1.2 4.3 ± 1.3 Example 6 5.7 ± 1.0 5.6 ± 1.4 4.7 ± 1.4 5.4 ± 1.5 4.7 ± 1.0

In the case of color and flavor, oligosaccharide extract and sugar extract in taste and viscosity showed high acceptability, and brown sugar extract showed high acceptability in overall acceptability. In addition, as ripening proceeds in brown sugar extract or oligosaccharide extract, the overall acceptability was found to increase significantly.

Test Example 3: Antioxidative Effect of Buttercup Fermentation Solution

1. Total Flavonoid Content

Dilute 0.1 mL of the stock solution, not the concentrate of each sample, to 0.9 mL of 80% ethanol, and then take 0.1 mL, mix it with 4.3 mL of 80% ethanol containing 10% aluminum nitrate and 1 M potassium acetate, and leave at room temperature for 40 minutes. Absorbance was measured at nm. At this time, the total flavonoid content was determined from the standard curve prepared using quercetin. The total flavonoid content measurement results are shown in Table 5 below.

sample Total flavonoids (㎍ / mL) Example 1 1696.85 ± 8.26 Example 2 354.42 ± 0.28 Example 3 343.52 ± 0.56 Example 4 288.36 ± 0.29 Example 5 105.33 ± 0.11 Example 6 58.67 ± 0.06

As a result, the flavonoid content of the fermentation broths of Examples 1 and 4 was high, and the flavonoid content showed a tendency to decrease with aging.

2. Measurement of DPPH Free Radical Scavenging Activity

The free radical scavenging activity of the sample stock was measured by reducing power of 1,1-diphenyl-2-picrylhydrazyl (DPPH), which is a stable radical. 0.2 mL of the dissolved 0.15 mM DPPH solution was added thereto, and the mixture was left at room temperature for 30 minutes and absorbance was measured at 517 nm. The free radical scavenging activity of each sample extract was expressed as IC ₅₀ , the concentration of the sample required to reduce the absorbance of the control without addition of the sample to ½. At this time, butyl hydroxyanisole (BHA; Butylated Hydroxy Anisole) was used as a control for comparing the relative activity. The measurement results are shown in Table 6 below.

sample IC ₅₀ (mg / mL) Example 1 41.92 ± 12.26 Example 2 64.87 ± 16.69 Example 3 62.68 ± 5.99 Example 4 120.15 ± 12.18 Example 5 105.42 ± 10.69 Example 6 186.85 ± 18.25

As confirmed in Table 6, the brown sugar fermentation broths of Examples 1 to 3 exhibited lower IC ₅₀ values, and both the brown sugar fermentation broth and the oligosaccharide fermentation broth showed a tendency to decrease the free radical scavenging ability with the aging period. It is found that this coincides with the decrease. This may be due to the change of unstable flavonoid compounds during fermentation.

Test Example 4 Protective Effect of Buttercup Fermentation Solution against Oxidative Damage by t-BHP

1. Measurement of Hepatocyte Survival Rate for Cell Culture and Damage Induction

Cytotoxicity against HepG2 cells, a human-derived liver cancer cell line, was measured by MTT method. The cell line HepG2 cells used in this experiment were distributed from Korea Cell Line Bank (Seoul, Korea) and used for subculture. The medium used for cell culture was used after incubating for 2-3 days in a 5% CO ₂ incubator at 37 ° C using MEM medium containing 10% FBS (fetal bovine serum) and 1% antibiotics (penicillin / streptomycin). . A frequency divider for HepG2 cell cultured in 1 × 10 ⁵ cells / well and then CO ₂ After incubation for 16 to 24 hours in the incubator, the samples diluted by concentration were treated with cells and incubated for 24 hours. Then t-BHP (tert-butylhydroperoxide, The final concentration of 1 mM) was added and cultured again for 4 hours, and then cell viability was measured by MTT (3- (4,5-dimethyl-thiazol-2-yl) -2,5-diphenyltetrazoliumbromide) assay. Each sample was treated with 10 ul of 5 mg / mL MTT solution in each well and cultured for 24 hours in each well and incubated for 4 hours in an incubator. After the incubation, the supernatant was removed, and formazan crystals were dissolved by adding 100 ul of DMSO to each well, and the absorbance was measured at 550 nm using a microplate reader. Is expressed as a percentage of the absorbance of the control.

In hepatocytes, t-BHP is converted to toxic substances such as peroxyl and alkoxyl radicals by cytochrom P-450 in primary cultured hepatocytes, which causes early lipid peroxidation. Another metabolic process is the detoxification of GSH peroxidase by binding to t-BHP and converting it to tert-butyl alcohol and oxidized glutathione. Through this metabolic process, a significant amount of t-BHP in the hepatocytes generates free radicals, which are directly and indirectly reacted with the unsaturated fatty acids constituting the cell membrane, resulting in the formation of MDA. do. Through this series of processes, t-BHP causes natural necrosis or cell necrosis of hepatocytes. In this experiment, in order to investigate the hepatocyte protective effect of the buttercup fermentation broth, the fermentation broth was pretreated to HepG2 cells by concentration, and then treated with t-BHP to observe cell viability. The results are shown in FIG. 2. When the cell viability of the untreated control group was 100%, the cell viability was significantly lowered to 42.8% in the experimental group treated with 1 mM t-BHP. This indicates that the hepatocytes are oxidatively damaged and necrotic. On the other hand, the treatment of buttercup fermentation broth at a final concentration of 0.001 ~ 1 mg / mL showed a protective effect from damage in a concentration-dependent manner. In particular, at 1 mg / mL, the survival rate of all the buttercup fermentation solutions of Examples 1 to 6 significantly increased to about 100%. In Fig. 2, Su 1 yr. Represents the brown sugar buttercup fermentation solution of Example 1, Su 2 yr. Represents the brown sugar buttercup fermentation solution of Example 2, and Su 3 yr. Represents the brown sugar buttercup fermentation solution of Example 3. In addition, Gold 1 yr. Represents the oligosaccharide buttercup fermentation solution of Example 4, Gold 2 yr. Represents the oligosaccharide buttercup fermentation solution of Example 5, Gold 3 yr. Represents the oligosaccharide buttercup fermentation solution of Example 6.

2. LDH (Lactate dehydrogenase) leakage inhibitory effect

Hepatocyte damage can be confirmed by measuring the amount of LDH released into the culture medium during hepatocyte damage. LDH activity in the medium was measured to investigate the effect on the hepatocyte damage of the buttercup fermentation broth. A frequency divider for HepG2 cell cultured in 96 well with 1 × 10 ⁵ cells / well and then CO ₂ After incubation for 16 to 24 hours in the incubator, the samples diluted by concentration were treated with cells and incubated for 24 hours. Then, t-BHP (final concentration 1 mM) was added and cultured again for 4 hours, and then LDH activity was measured using an LDH assay kit (BioVision, USA) for each cell culture. LDH is an enzyme that converts lactate to pyruvate. When an abnormality occurs in a cell, it flows out of the cell and increases in the medium. Whether or not there is a protective effect against t-BHP induced oxidative stress in hepatocytes was measured by LDH activity flowing into the culture medium upon cell membrane damage and the results are shown in FIG. 3. As shown in FIG. 3, LDH in the cells rapidly leaked into the culture medium by t-BHP, and the LDH concentration was not significantly changed by the maturation period but decreased in a concentration-dependent manner by pretreatment with the buttercup fermentation solution. It can be seen that there is a significant inhibitory effect in the case of sample treatment compared to the effect of brown sugar or oligosaccharide only.

Test Example  5: Effect of Buttercup Fermentation Solution on Hepatocellular Carcinoma Cell Growth

1. Cell culture and survival rate measurement

Cell viability of Chang cells, a human-derived normal liver cell line, and HepG2 cells, a liver cancer cell line, was measured by MTT method. The cell lines Chang and HepG2 cells used in this experiment were distributed from Korea Cell Line Bank (Seoul, Korea) and used for subculture. The medium used for cell culture was used after culturing for 2-3 days in 5% CO ₂ incubator at 37 ° C using MEM medium containing 10% FBS (fetal bovine serum) and 1% antibiotics (penicillin / streptomycin). .

In order to test the effect of inhibiting the liver cancer cell growth of the buttercup fermentation extract, the sample was treated by concentration in normal liver cell line and liver cancer cell line, and the effect on the growth of each cell was measured and the results are shown in FIGS. 4 and 5. As can be seen in FIGS. 4 and 5, neither the brown sugar buttercup fermentation broth nor the oligosaccharide buttercup fermentation broth had any effect on the growth of normal hepatocytes, and the oligosaccharide buttercup fermentation broth showed greater inhibitory activity than the brown sugar buttercup fermentation broth, in particular, Example 4 Significant inhibitory effects were found in the buttercup fermentation broth.

2. Observation of Morphological Changes in HepG2 Cells

HepG2 cells were incubated in 6-well plates (2 × 10 ⁶ cells / well) for 24 hours, and the solution of the buttercup fermentation solution of Examples 1 to 6 was treated by concentration. After removing the medium with PBS, the effect on the cell morphology and the number of cells was observed under the magnification of 100, 200, 400 times with inverted microscope (DM IRE2, Leica, Wetzlar, Germany). The reproducibility was confirmed by three experiments for the group not treated with the sample and the group treated with 0.1 and 1 mg / mL. The results are shown in Fig. Treatment with only brown sugar and oligosaccharide used as a control had no effect on cell morphology and number, and in the group treated with the buttercup fermentation solution of Examples 1 to 6, the number of cells was reduced in a concentration-dependent manner and the size was reduced.

Test Example 6 Analysis of Active Ingredients of Buttercup Fermentation Solution

1. HPLC analysis

HPLC was performed using Shimdazu Prominence LC20A with YMC C ₁₈ column (4 um, 150 mm x 4.6 mm id) at 40 ° C, buffer A (distilled water with 0.1% trifluoroacetic acid) and buffer B (0.1 70% acetonitrile containing% trifluoroacetic acid) was used as a gradient for 50 minutes at a flow rate of 1.0 ml / min. The gradient composition for separation is shown in Table 7. Detection was done at 254 nm using a UV-VIS detector (SPD-20A). The results are shown in FIG.

Time (min) Buffer A (%) Buffer B (%) 0 100 0 30 50 50 35 0 100 40 100 0 50 100 0

Analysis of the buttercup extract by HPLC showed two peaks at 14.2 and 14.6 min, which showed several peaks but a large distribution in the extract.

2. MS Analysis

As a result of HPLC analysis, the fraction of the peak showing two high intensities was received and analyzed by mass spectrometry (MS). The MS instrument used a Thermo Finnigan LTQ mass spectrometer from Thermo Electron Corp. It was ionized by ESI method and averaged 40 scans in the mass range of m / z 50-500 using negative ion mode. Sample injection was performed at a flow rate of 5 ul / min using a microsyringe. The analysis results are shown in FIG. 8.

Since the negative mode was used, the molecular weight obtained is [MH] ⁻ . Since the molecular weight of chlorogenic acid is 354, it becomes 353 in negative mode, and since the caffeic acid molecular weight is 180, it becomes 179 in negative mode. In FIG. 8, a peak corresponding to 353 was shown, and MS / MS results showed that 353 and 191 were detected to be chlorogenic acid. In addition, peaks corresponding to 179 appeared, and MS / MS results showed that 179 and 135 were detected as caffeic acid.

3. HPLC reanalysis

The control was used by dissolving in buffer A containing 1% methanol. Caffeic acid was prepared at a concentration of 0.1, 0.5, 1, 2, 5 pmol, and chlorogenic acid at a concentration of 0.1, 1, 2, 5 nmol and a calibration curve was shown in FIG. 9. In order to confirm that the two main peaks detected in the buttercup extract are chlorogenic acid and caffeic acid, which were confirmed by MS experiment, the buttercup extract, 0.1 mM chlorogenic acid or buttercup extract, and 0.1 mM caffeic acid were mixed in a 1: 1 ratio. HPLC was performed again. The results are shown in FIG. As a result of HPLC re-examination, only the peak of caffeic acid detected at 14.6 minutes was increased in the sample injected with caffeic acid, and only the peak of chlorogenic acid detected at 14.2 minutes was increased in the case of coinjection of chlorogenic acid. . Therefore, it was confirmed that caffeic acid and chlorogenic acid identified by MS were the main compounds in the buttercup fermentation broth. The relative amounts of chlorogenic acid and caffeic acid detected by HPLC are shown in Table 8.

sample Peak Peak area (%) Example 1 CGA ¹ 0.9 CA ² 1.3 Example 2 CGA 0.9 CA 1.5 Example 3 CGA 0.8 CA 1.2 Example 4 CGA 2.4 CA 3.0 Example 5 CGA 1.5 CA 3.0 Example 6 CGA 0.6 CA 1.2

(CGA ¹ : Chlorogenic acid, CA ² : Caffeic acid)

Test Example 7: Analysis of active ingredient content according to extraction method

Different extraction methods were used to compare the contents according to the fermentation of the two substances identified as active ingredients of buttercup extract. The results of analyzing the buttercup sample extracted with the organic solvent (ethanol) without fermentation and the buttercup extract of Example 4 after the fermentation process by HPLC are shown in FIG. 11. As a result of analyzing the two samples, the contents of chlorogenic acid and caffeic acid increased by fermentation. Chlorogenic acid increased by about 15.6 times (peak area of chlorogenic acid / area of total peak) by fermentation according to the present invention, and about 12.1 times (peak area / caffeic acid by fermentation according to the present invention in case of caffeic acid). The area of the total peak) was further increased. Therefore, the extraction method through the fermentation process according to the present invention can be said to be a superior method for increasing the content of chlorogenic acid and caffeic acid, which are the main active ingredients of buttercup, than the organic solvent extraction method.

Claims (5)

Washing and preparing buttercups; Mixing the buttercups with brown sugar or oligosaccharide as a leaching agent in a weight ratio of 1: 1 by washing and sterilizing pottery; And a fermentation step of naturally fermenting at 15-25 ° C. for 10-14 months,
Method of producing a buttercup fermentation broth for protecting and improving liver function, characterized in that the chlorogenic acid and the caffeic acid as the active ingredient is strengthened. The method of claim 1, wherein the fermentation broth for the protection and improvement of liver function is characterized in that it further comprises the step of aging while leaching the leaching solution obtained in the fermentation at a temperature of 4 ~ 6 ℃ for 10 to 26 months. delete A buttercup fermentation solution for protecting and improving liver function, prepared by the method of claim 1 or 2, containing chlorogenic acid and caffeic acid as active ingredients. The functional health drink for liver function protection and improvement containing the fermented buttercup of claim 4.

Images