KR102629866B1 - Biorenovation of Distylium racemosum gall extract - Google Patents

Abstract

The present invention relates to a method for bioconversion of an extract of Chrysanthemum chinensis, a bioconversion product bioconverted by this method, and an anti-inflammatory composition containing the bioconversion product. The bioconversion method of the present invention includes the step of mixing the extract of Chrysanthemum Chrysanthemum and the bacterial cells of the Bacillus genus JD3-7 strain (KACC 92346P) to produce a mixture, and the step of incubating the mixture.

Description

Biorenovation of Distylium racemosum gall extract}

The present invention relates to the bioconversion of an extract from the plant, and specifically, a bioconversion method that can bioconvert the extract from the plant, showing excellent anti-inflammatory activity and low toxicity, the excellent anti-inflammatory activity obtained through this, and It relates to a biotransformation product with low toxicity and an anti-inflammatory composition containing the biotransformation product.

Inflammation is a very complex innate defense system of the human body caused by exogenous pathogens or endogenous stimuli that invade the human body, and causes inflammation locally as a repair mechanism for the recovery of damaged tissues. This inflammatory response is induced by immune cells in the body expressing various mediators such as cytokines, PGE2 (prostaglandin E ₂ ), and free radicals to recognize and eliminate inflammatory factors. In particular, immune cells, known as a type of immune cell, Macrophages play an important role in the immune system by providing immediate defense against pathogens. LPS (lipopolysaccharide), one of the toxins derived from Gram-negative bacteria, is a representative inflammation-causing factor and is known to stimulate macrophages to produce various mediators involved in various inflammations. Among them, the representative pro-inflammatory cytokine (pro) -The production and secretion of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), which are inflammatory cytokines, activate NF-κB (nuclear factor-κB). Inflammation-inducing genes iNOS (inducible nitric oxide synthase) and COX-2 (cyclooxygenase-2) are expressed. When iNOS is expressed during the inflammatory response, highly reactive nitric oxide (NO) is produced, and overproduced NO is closely related to several inflammatory diseases, including tissue damage, genetic mutation, and serious autoimmune diseases. It has been reported that there is. In addition, COX-2, expressed at the site of inflammation, when excessively induced by various pro-inflammatory substances, causes various degenerative diseases or induces chronic inflammation by promoting the synthesis of PGE2, an inflammatory mediator. In this way, inflammation in the human body is part of the immune response, but when an abnormal inflammatory response occurs, inflammation-related cells produce excessive inflammatory mediators, resulting in the appearance of various diseases called chronic inflammatory diseases. Currently, as the inflammatory basis of various diseases such as arteriosclerosis, asthma, and rheumatoid arthritis has become clear, research is being actively conducted to discover anti-inflammatory materials for the purpose of preventing and treating inflammatory diseases, and many natural products such as NO and cytotoxic Anti-inflammatory activity through a kine inhibition mechanism has been reported.

Meanwhile, there are many biological resources around us that are not fully utilized due to lack of appropriate research. Accordingly, the need for research to utilize biological resources with low practical value in various ways and new utilization technologies based on this is emerging.

Algae tree is one of these biological resources, and although its whitening and antioxidant effects have been reported, it has not yet been actively utilized. In particular, fir trees have the characteristic of forming insect nests of various shapes on their leaves and branches by more than eight types of pests, including aphids, and these insect nests of fragrant trees are rarely utilized, and related research is also being conducted. It is almost non-existent. In addition, because the green foliage can be toxic, there are restrictions when trying to use it in products applied to the human body, especially food or medicine.

Accordingly, the present inventors attempted to develop a technology for utilizing the phyllodes of the ginseng as a new anti-inflammatory substance by focusing on the physiological activity of the foliage of the ginseng tree, especially the anti-inflammatory activity as mentioned above, while at the same time solving the toxicity problem. By solving this problem, we sought to develop technology that can be safely used as food or medicine.

Kim, H. R., G. N. Park, B. K. Jung, W. J. Yoon, Y. H Jung, and K. S. Chang (2016) Antioxidant Activity of Solvent Fractions from Distylium racemosum in Jeju. Korean journal of clinical laboratory science. 48(2): 62-67. Lee, M. J (2008) Isolation of Functional Cosmetic Ingredient from Distylium racemosum Leaves. master's thesis. University of jeju, jeju island, korea.

Therefore, the main purpose of the present invention is to provide a method to solve the toxicity problem so that the plant can be utilized usefully, especially as an anti-inflammatory substance, and can be safely used as a food or medicine.

Another object of the present invention is to provide a new anti-inflammatory substance using the Chrysanthemum ginseng tree based on the above method.

According to one aspect of the present invention, the present invention provides a method of producing a mixture by mixing an extract of the Chrysanthemum vulgaris and the bacterial cells of the Bacillus genus JD3-7 strain (KACC 92346P), and incubating the mixture at a temperature. A method for bioconversion of extracts is provided.

In the bioconversion method of the present invention, it is preferable that the extract of Chrysanthemum vulgaris is extracted with water, ethanol, or a mixed solvent thereof.

In the bioconversion method of the present invention, the step of producing the mixture is preferably a step of mixing the extract of Chrysanthemum chinensis and the bacteria in a buffer solution.

In the bioconversion method of the present invention, the step of generating the mixture includes mixing the extract and the bacteria in a buffer solution so that the concentration of the extract in the mixture is 1 g/l or less. It is desirable to be

In the bioconversion method of the present invention, the buffer solution is preferably a phosphate buffer solution containing 1 to 3% (v/v) glycerin and having a pH of 7 to 7.5.

In the bioconversion method of the present invention, the incubation is preferably at 25 to 35°C for 2 to 4 days.

According to another aspect of the present invention, the present invention provides a bioconversion product of the extract of Chrysanthemum chinensis bioconverted by the above bioconversion method.

According to another aspect of the present invention, the present invention provides an anti-inflammatory composition containing the biotransformation.

According to the present invention, the extract of Chrysanthemum vulgaris can be bioconverted into a substance with low toxicity and exhibits excellent anti-inflammatory activity, such as inhibiting the production of nitric oxide and PGE2 and inhibiting the expression of iNOS, COX-2, IL-1β and IL-6. You can. Accordingly, the underused Chrysanthemum sinensis can be safely used to manufacture anti-inflammatory products, such as anti-inflammatory functional cosmetics or foods, or medicines for the prevention or treatment of inflammatory diseases.

Figure 1 shows the results of HPLC analysis of D. arborvitae extract (DR), bioconversion of D. arborvitae extract (DRB), and control group (BAE).
Figure 2 shows the results of an experiment on the cytotoxicity of D. arborvitae extract (DR), bioconversion of D. rhododendron extract (DRB), and control group (BAE).
Figure 3 shows the results of an experiment on the inhibitory activity of nitric oxide (NO) production of R. vulgaris extract (DR), bioconversion of R. vulgaris extract (DRB), and control group (BAE).
Figure 4 shows the results of an experiment on the inhibitory activity of PGE2 production of the biotransformation of the extract of Chrysanthemum Chrysanthemum.
Figure 5 shows the results of an experiment on the inhibitory activity of iNOS and COX-2 expression of the biotransformation of the extract of Chrysanthemum Chrysanthemum.
Figure 6 shows the results of an experiment on the inhibitory activity of IL-1β, IL-6, and TNF-α expression of the biotransformation of the extract of Chrysanthemum chinensis.
Figure 7 shows the accession of Bacillus JD3-7 strain (KACC 92346P).

The bioconversion method of the present invention is characterized in that it includes the step of mixing the extract of Chrysanthemum Chrysanthemum and the bacterial cells of the Bacillus genus JD3-7 strain (KACC 92346P) to produce a mixture, and the step of incubating the mixture.

The plant is an evergreen shrub or tree classified into the genus of the plant and the plant family, and its scientific name is ' Distylium racemosum '. In the present invention, the galls produced in such green trees are used. Chrysanthemum refers to an abnormal lump-shaped bulge that can be seen in plants, and in the case of fir trees, it is mainly produced on leaves and branches. It is known that more than 8 types of organisms, including aphids, are the cause of the formation of fragrant aphids, and their shapes are diverse. In one embodiment of the present invention, fragrant inflorescence is a fibrous inflorescence produced on the leaves of fir tree.

In the present invention, the Chrysanthemum rhododendron extract is extracted using the Chrysanthemum rhododendron as an extraction raw material, and can be prepared by conventional extraction methods in the art, such as boiling water extraction, ultrasonic extraction, and reflux extraction. Preferably, the extract is extracted using a solvent selected from the group consisting of water, C1 to C4 lower alcohols, and mixtures thereof, and more preferably, it is extracted using water, ethanol, or a mixed solvent thereof. This extract is more suitable for bioconversion into a substance that exhibits excellent effects, i.e., excellent anti-inflammatory activity and low toxicity, through the bioconversion method of the present invention. For the same reason, the extract of Chrysanthemum Chrysanthemum is more preferably extracted with water or a mixed solvent of water and ethanol, and more preferably with a mixed solvent of water and ethanol. In addition, the mixed solvent of water and ethanol is preferably a 10 to 99% (v/v) ethanol solution, more preferably a 30 to 90% (v/v) ethanol solution, and more preferably 40% (v/v) ethanol solution. to 90% (v/v) ethanol solution, more preferably 50 to 90% (v/v) ethanol solution, more preferably 60 to 80% (v/v) ethanol solution, and more preferably 50 to 90% (v/v) ethanol solution. Preferably it is a 65 to 75% (v/v) ethanol solution.

In the present invention, the amount of solvent when producing the extract of the Chrysanthemum vulgaris is based on 1 g of the weight (e.g., dry weight) of the extraction raw material, that is, the weight (e.g., dry weight) of the sage, preferably 2 to 100 g, more preferably 5 to 50 g, more preferably. is used at a rate of 5 to 20 g. The extraction temperature is preferably 0 to 100°C, more preferably 5 to 90°C, and even more preferably 10 to 80°C. In particular, when using a mixed solvent of water and ethanol as a solvent, the extraction temperature is preferably 5 to 35°C, more preferably 10 to 30°C, and even more preferably 15 to 25°C. The extraction time is preferably 1 hour to 10 days, more preferably 12 hours to 5 days, and even more preferably 1 day to 3 days. According to this, a more suitable extract can be prepared for bioconversion into a substance showing excellent effects, that is, excellent anti-inflammatory activity and low toxicity through the bioconversion method of the present invention.

In the present invention, the extract of Chrysanthemum Chrysanthemum may have been subjected to additional processes such as filtration, concentration, reduced pressure drying, and freeze-drying after the extraction process as described above.

The Bacillus JD3-7 strain, which is the strain used in the biotransformation of the present invention, was isolated from soybean native soybeans in Jeju Island and has been deposited in the Microbial Bank (Korean Agricultural Culture Collection, KACC) of the National Institute of Agricultural Sciences under the accession number KACC 92346P.

Bacillus JD3-7 strain can be cultured according to normal culture conditions for cultivating Bacillus microorganisms. For example, it can be cultured in LB (Luria Bertani) medium or NB (nutrient broth) medium at a temperature of 25 to 35°C.

Cells of the Bacillus JD3-7 strain can be obtained through conventional methods such as filtration or centrifugation of the culture medium after culturing the strain.

In the present invention, the step of creating a mixture is a step of mixing the extract of the extract of the plant and the cells (or their enzymes) of the Bacillus genus JD3-7 strain so that bioconversion of the extract of the plant of the plant can be achieved. This can be achieved by adding and mixing tree extract and Bacillus JD3-7 strain. At this time, the medium may be a medium, for example, a medium in which the Bacillus JD3-7 strain can grow, and may be water or a water-based buffer. In one embodiment of the invention, the medium is a buffer solution.

The buffer preferably contains 1 to 3% (v/v) glycerin, more preferably 1.5 to 2.5% (v/v), more preferably 1.7 to 2.3% (v/v), even more preferably Contains 1.8 to 2.2% (v/v) glycerin. Additionally, the buffer solution is preferably a buffer solution of pH 6.5 to 8, and more preferably a buffer solution of pH 7 to 7.5. Additionally, the buffer solution is preferably a phosphate buffer solution. According to these buffer conditions, more effective bioconversion can be achieved.

Add and mix the Bacillus genus JD3-7 extract and the Bacillus genus JD3-7 strain to the medium, preferably so that the concentration of the Corona vulgaris extract in the mixture formed by this mixing is 1 g/l or less. This was in consideration of the possibility of death of the Bacillus JD3-7 strain due to the toxicity of the extract of the Chrysanthemum ginseng and the resulting lower bioconversion rate. Through experiments, efficient bioconversion was confirmed up to a concentration of 1g/l. At the same time, in order to bioconvert as much of the extract as possible through a single reaction, preferably 100 mg/l to 1 g/l, more preferably 200 mg/l to 1 g/l, more preferably 300 mg/l to 1 g/l, more preferably 400 mg/l to 1 g/l, more preferably 500 mg/l to 1 g/l, more preferably 600 mg/l to 1 g/l, more preferably Preferably, the concentration is 700 mg/l to 1 g/l, more preferably 800 mg/l to 1 g/l, and more preferably 900 mg/l to 1 g/l.

In the present invention, the incubation step is a step of incubating to induce biotransformation by the Bacillus JD3-7 strain (possibly by the action of an enzyme produced by this strain), and the extract of Chrysanthemum Chrysanthemum and Bacillus JD3- This can be achieved by putting a mixture of 7 strains of bacterial cells into a typical thermostat and maintaining it for a certain period of time. The incubation temperature at this time is preferably 20 to 40°C, more preferably 25 to 35°C, more preferably 27 to 33°C, and even more preferably 29 to 31°C, and the incubation time is preferably 1. hours to 10 days, more preferably 12 hours to 7 days, more preferably 1 to 5 days, more preferably 2 to 4 days. According to these incubation conditions, more efficient bioconversion can be achieved.

The biotransformation product of the present invention is characterized in that it is obtained by bioconverting an extract of Chrysanthemum Chrysanthemum using the bioconversion method described above. In one embodiment of the present invention, the biotransformation product may exhibit low cytotoxicity compared to the non-biotransformation extract of Chrysanthemum Chrysanthemum. For example, it may exhibit lower cytotoxicity at the same concentration, or it may exhibit the same level of cytotoxicity at a higher concentration. In addition, it can exhibit excellent anti-inflammatory activity, for example, inhibition of nitric oxide production, inhibition of PGE2 (Prostaglandin E2) production, inhibition of iNOS and COX-2 expression, and inhibition of IL-1β and IL-6 expression.

The anti-inflammatory composition of the present invention is characterized in that it contains the above-described biotransformation.

The anti-inflammatory composition of the present invention may be an anti-inflammatory functional cosmetic composition, an anti-inflammatory functional food composition, or a pharmaceutical composition for preventing or treating inflammatory diseases.

The anti-inflammatory composition of the present invention may be composed of only the biotransformation product, and may include only the biotransformation product as an active ingredient. In addition, the anti-inflammatory composition of the present invention may further include other substances as active ingredients along with the biotransformation product. At this time, other substances may be derived from biological resources other than the algae, may be processed by other methods, or may be compounds.

The anti-inflammatory composition of the present invention may further include additives, such as cosmetically, foodologically, or pharmaceutically acceptable additives, in addition to the active ingredient (e.g., the biotransformation of the present invention), and may be in an appropriate form. It can be formulated as: These additives may be appropriately selected depending on the intended use of the composition, mode of application (e.g., dosage form), etc.

The anti-inflammatory composition of the present invention may contain, for example, 0.01 to 100% by weight of the biotransformation product of the present invention.

If the anti-inflammatory composition of the present invention is a pharmaceutical composition, it can be administered orally or parenterally. When administered parenterally, it can be administered by various methods such as subcutaneous injection, and can be used in the form of a general pharmaceutical preparation. The dosage can be adjusted depending on the condition of the administration subject, the administration route, and the form of administration, and those skilled in the art will be able to adjust it within a variety of ranges depending on the symptoms. The anti-inflammatory composition of the present invention may be administered, for example, at 0.01 to 400 mg per 1 kg of body weight of the subject, based on the weight (eg, dry weight) of the biotransformant of the present invention contained in the composition.

Hereinafter, the present invention will be described in more detail through examples. Since these examples are merely for illustrating the present invention, the scope of the present invention is not to be construed as limited by these examples.

[Example]

Example 1. Preparation of Chrysanthemum Chrysanthemum Extract

In August 2020, samples of pigments produced on the leaves of Distylium racemosum were collected in Silrye-ri, Namwon-eup, Seogwipo-si, Jeju-do. 70% (v/v) ethanol at a 10-fold weight ratio was added to 1 g of the collected Chungyoung sample, and extraction was repeated twice for 48 hours at room temperature. The extract was then filtered through a filter (TOYO ROSHI KAISHA, Tokyo, Japan). The filtered extract was concentrated using a vacuum concentrator, then freeze-dried and used for the next bioconversion.

Example 2. Bioconversion of Chrysanthemum Chrysanthemum Extract

Bacillus sp. JD3-7 strain (KACC 92346P) was cultured in nutrient broth (Nutrient Broth, Difco, USA) for 18 hours, and then the culture was centrifuged at 4,000 rpm for 15 minutes to remove the bacteria. Obtained. The obtained cells were washed twice with PG buffer (50mM phosphate buffer, 2% (v/v) glycerin). The washed bacterial cells were suspended in PG buffer, and 100 mg of the D. vulgaris extract (DR) of Example 1 was added per 100 ml of suspension and incubated at 30°C for 72 hours to induce a biotransformation reaction. This reaction was named DRB, and the bacterial suspension without DR was used as a control (BAE). The reaction solution was centrifuged at 4000 rpm for 15 minutes, the supernatant was collected, concentrated using a vacuum concentrator, freeze-dried at -110°C, and used in the following experiment.

Example 3. Evaluation of the physiological activity of the extract biotransformation of Chrysanthemum chinensis

3-1. method

3-1-1. HPLC analysis

For HPLC analysis, the D. arborvitae extract (DR) of Example 1, the D. arborvitae extract bioconversion (DRB) of Example 2, and the control group BAE were dissolved in DMSO (dimethyl sulfoxide, Sigma-Aldrich, St. Louis, USA). did. A Shimadzu SpectroMonitor 3200 digital UV/Vis detector was used for the analysis, and water (Solvent A) with 0.1% TFA (Trifluoroacetic acid, SAMCHUN, Korea) and acetonitrile (Solvent B, Sigma-Aldrich, St. Louis, USA) were used as solvents. It was used as. The sample was analyzed for 30 minutes under gradient conditions by changing acetonitrile (Solvent B) from 0.1 min: 10% to 30 min: 100%, using a column (Phenomenex 4㎛ Hydro-RP 80Å, 250×4.6). ㎜) was conducted at a temperature of 40°C, a flow rate of 1.0 mL/min, and a wavelength of 254 nm.

3-1-2. Experimental materials and cell culture

LPS used in the experiment was purchased from Sigma-Aldrich (St. Louis, USA), and RAW 264.7 cells were purchased from the Korea Cell Line Bank. For culturing the cell line, a culture medium containing 10% FBS (fetal bovine serum), 100 U/ml penicillin, and 100 μg/ml streptomycin was used in DMEM (Dulbecco's Modified Eagle Medium, Gibco, New York, USA), and cultured in a CO ₂ incubator (37). Subculture was performed every 2 days at ℃, 5% CO ₂ ).

3-1-3. Cytotoxicity measurements

MTT analysis was used to confirm cytotoxicity of the sample in RAW 264.7 cells. RAW 264.7 cells were inoculated into _a ^24- well plate at a _{concentration} of 7.0 1㎍/㎖) were simultaneously treated and reacted for 24 hours. Afterwards, MTT reagent was added to RAW 264.7 cells, reacted in an incubator for 3 hours, the supernatant was removed, DMSO was added to each well to dissolve formazan blue, and then the cells were incubated at 570 nm using an ELISA reader. The absorbance was measured.

3-1-4. Measurement of NO production inhibition activity

RAW 264.7 cells were inoculated in _a ²⁴ -well plate at a _{concentration} of 7.0 The diluted samples were simultaneously processed and reacted for 24 hours. Afterwards, 100㎕ of culture medium and 100㎕ of Griess reagent [1% (w/v) sulfanilamide, 0.1% (w/v) naphylethylenediamine in 2.5% (v/v) phosphoric acid] were mixed 1:1 in a 96-well plate. After dark reaction for 10 minutes, absorbance was measured at 540 nm using an ELISA reader.

3-1-5. Measurement of PGE2 production inhibition activity

Prostaglandin E2 (PGE2) in cell culture was measured using a mouse enzyme-linked immnunosorbent assay (ELISA) kit (R&D Systems Inc., Minneapolis, Minnesota, USA). RAW _264.7 cells were inoculated in a ²⁴ -well plate at a concentration _of 7.0 100, 200, and 400㎍/㎖) were treated simultaneously and reacted under the same conditions for 24 hours. Afterwards, the recovered cell culture fluid was centrifuged at 10,000 rpm for 3 minutes to remove the precipitate, and the recovered supernatant was used for PGE2 measurement.

3-1-6. Western blot analysis

RAW 264.7 cells seeded in a 6-well plate at a concentration of 5 × 10 ⁵ cells/well were cultured in a CO ₂ incubator (37°C, 5% CO ₂ ) for 24 hours, and then incubated with DRB (100, 200) diluted in DMEM. , 400㎍/㎖) and LPS (1㎍/㎖) were treated simultaneously and reacted under the same conditions for 24 hours. Afterwards, _the medium _was removed and washed twice with PBS, and the proteins were lysed in lysis buffer [1 , 1㎍/㎖ aprotinin, 1㎍/㎖ pepstatin, and 1㎍/㎖ leupeptin] and then centrifuged at 12,000rpm for 30 minutes to separate the supernatant and pellet. After quantifying the protein in the supernatant using a BCA kit (Bio-Rad, USA), 20 μg of protein was separated by electrophoresis on 10% SDS-PAGE containing 10% polyacrylamide. Afterwards, the membrane was transferred to a PVDF (poly-vinylidene difluoride) membrane (Milipore, Burlington, Massachusetts, USA) at 200 mA for 2 hours, and then the membrane was placed in 5% skim milk (sol. TBST) and blocked for 2 hours at room temperature. Afterwards, it was washed three times for 10 minutes with TBST. Primary antibodies (iNOS antibody (1:1,000, Bio-Rad, USA), COX-2 antibody (1:1,000, Rockland Immunochemicals, Inc., USA), β-actin antibody clone AC-74 (1:10,000, Sigma) , USA) was used to react overnight at 4°C, and then washed three times with TBST solution and then reacted with secondary antibody (Jackson ImmunoResearch, USA) diluted 1:10,000 at room temperature for 90 minutes. With TBST. The membrane washed three times for 10 minutes was reacted with an ECL kit (Bio-Rad, USA) and developed using an imaging densitometer (model GS-700, Bio-rad, USA) using the ImageJ program (NIH, Bethesda, USA). Maryland, USA), the area of iNOS and COX-2 expression compared to β-actin was quantified and displayed in a graph.

3-1-7. Measurement of inhibition activity of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) production

Pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) in cell culture were analyzed using mouse TNF-α ELISA kit (Invitrogen, California, USA), mouse IL-6 ELISA kit (BD Biosciences, California, USA), It was measured using a mouse IL-1β ELISA kit (R&D Systems Inc., Minneapolis, Minnesota, USA). _RAW 264.7 cells were distributed in a ²⁴ - _well plate at a concentration of 7.0 ) and LPS (1㎍/㎖) were treated simultaneously and reacted under the same conditions for 24 hours. Afterwards, the culture medium was centrifuged (10,000 rpm, 3 minutes) to remove sediment, and the supernatant was recovered to measure the amount of proinflammatory cytokines (TNF-α, IL-6, IL-1β) produced in the culture medium.

3-1-8. Statistical processing

All experiments were repeated three times, and the results were expressed as mean value ± standard deviation. Statistical analysis was performed using analysis of variance to verify the significance of each treatment section (*p<0.05; **p<0.01). , ANOVA) followed by multiple comparisons using student's t-test.

3-2. result

3-2-1. HPLC analysis

As a result of HPLC analysis of the Diorchid extract (DR) of Example 1, the biotransformation product of the Diorchid extract (DRB) of Example 2, and BAE as a control, a new peak that did not exist in DR was confirmed in DRB. and the results are shown in Figure 1. This change in the chromatogram indicates that certain components present in the extract of Chrysanthemum Chrysanthemum have undergone biotransformation and changed into a different structure.

3-2-2. Comparative cytotoxicity measurements

Macrophages play an important role in regulating the body's innate and adaptive immunity by producing and secreting secondary mediators such as NO, PGE2, leukotriene, and pro-inflammatory cytokines. Therefore, the cytotoxicity caused by DR, DRB, and BAE was analyzed using the MTT assay using macrophages, which play an important role in inflammation, and the cell survival rate was measured by treating each sample at concentrations of 100, 200, and 400㎍/㎖. It is shown in 2. DR showed high cytotoxicity with cell survival rates of 79.8% at a concentration of 100 μg/ml, 50.1%, and 33.1% at concentrations of 200 μg/ml and 400 μg/ml, respectively. On the other hand, DRB extract showed a survival rate of more than 95% at all concentrations and showed improved cell survival rate compared to DR, and BAE treated at the same concentration also showed a survival rate of more than 90%. This is believed to be because the components in DR have been converted through bioconversion and the toxicity has disappeared. Accordingly, in the NO analysis, ELISA kit, and Western blot to be carried out in the future, experiments were conducted at concentrations of 100, 200, and 400㎍/㎖, which are the range in which DRB extract does not show cytotoxicity.

3-2-3. Comparison of NO production inhibition activity

NO (nitric oxide), one of the various inflammatory factors in the human body, is known to have important physiological functions such as human defense, signal transduction, and removal of tumors or bacteria. NO produced by iNOS during the inflammatory process causes inflammation. It has been reported to cause inflammatory diseases by promoting the biosynthesis of mediators and intensifying the inflammatory response. Therefore, to investigate the effect of DRB extract on NO production in RAW 264.7 cells stimulated with LPS, NO inhibitory activity was determined by treating LPS (1 μg/ml) with DR, DRB, and BAE at concentrations of 100, 200, and 400 μg/ml. was measured, and the results are shown in Figure 3. DR and DRB treated at concentrations of 100, 200, and 400㎍/㎖ suppressed the increased NO due to LPS in a similar trend, and at the highest concentration of 400㎍/㎖, they showed a similar level of inhibitory activity as the LPS untreated group. However, according to Figure 2, since a cell survival rate of less than 80% was observed in RAW 264.7 cells treated with DR, it is believed that the NO inhibitory activity shown in this result is due to toxicity rather than the inherent activity of DR. On the other hand, DRB showed significant inhibitory activity within a concentration that did not cause cytotoxicity, and since no significant NO inhibitory activity was observed in BAE, it is believed that the inhibitory activity of DRB is an intrinsic activity unrelated to cytotoxicity and BAE. These results suggest that the bioconversion technique can have a positive effect on enhancing the physiological activity of the extracts of the extracts of the extracts of the extracts of the extracts of the extracts of the extracts of the extracts of the extracts of the extracts of the extracts of the extracts, and the potential use of the extracts of the extracts of the extracts that have little practical value can be used as a preventative and therapeutic agent for inflammatory diseases.

3-2-4. PGE2 production inhibitory activity

PGE2 produced within the human body is a representative inflammatory mediator and is a measure of the degree of inflammatory response. Generally, in the early stages of inflammation, PGE2 induces the activity of inflammation by promoting local vasodilation and activation of neutrophils, macrophages, mast cells, and immune-related cells, and causes pain, swelling, and fever, leading to a continuous inflammatory response. It has been reported to play a key role in maintaining In this experiment, RAW 264.7 cells stimulated with LPS were treated with DRB extract at concentrations of 100, 200, and 400 μg/ml to measure the inhibitory activity of PGE2, and the results are shown in Figure 4. DRB was found to significantly inhibit the increased PGE2 in LPS-induced RAW 264.7 cells in a concentration-dependent manner, and at the highest concentration of 400㎍/ml, it was found to inhibit it to a level similar to that of the LPS-untreated group. These results suggest that DRB will have a positive effect on the deepening of the inflammatory response and the synthesis of excessive inflammatory cytokines by effectively inhibiting PGE2.

3-2-5. Inhibition of iNOS and COX-2 expression

When an inflammatory response occurs in the human body, macrophages and various immune-related cells are activated and induce the expression of iNOS and COX-2, known as pro-inflammatory proteins. During the inflammatory response, excessive amounts of NO are produced from L-arginine via NOS (nitric oxide synthase) and have been reported to be a cause of cancer or various inflammatory diseases. In addition, the expression of COX-2 is secondarily induced by inflammatory stimuli, cytokines, growth factors, and TGF-β, and prostaglandins induced by COX-2 cause inflammatory responses and cytokine synthesis by lymphocytes and macrophages. Adjust. In general, NO and PGE2 produced in inflammatory reactions are controlled by the expression of iNOS and COX-2, so anti-inflammatory activity can be evaluated by inhibiting the expression of iNOS and COX-2. Therefore, in this experiment, Western blot was performed to investigate how DRB, which had excellent NO and PGE2 production inhibitory activity, affected the expression of iNOS and COX-2 proteins, and the results are shown in Figure 5. DRB significantly suppressed the expression level of iNOS and COX-2 in LPS-induced RAW 264.7 cells compared to the LPS-only treatment group. In addition, considering that this reduction level shows a similar trend to that of NO and PGE2, it is believed that DRB is effective in suppressing gene expression of iNOS and COX-2 and that their expression is closely related to the production of NO and PGE2. Therefore, this suggests that DRB exhibits anti-inflammatory activity by reducing the production of NO and PGE2 through suppressing the expression of iNOS and COX-2 in the LPS-induced inflammatory response, and is effective in suppressing iNOS and COX-2 in various inflammatory processes. It is believed that significant effects can be expected in the treatment of diseases.

3-2-6. Inhibits production of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β)

Cytokines are proteins secreted by macrophages activated by inflammatory substances such as LPS and are factors that mediate inflammatory responses by regulating the activity, proliferation, and differentiation of immune cells. In order for an inflammatory response to proceed in the human body, the production of inflammatory cytokines, in addition to inflammatory mediators such as NO and PGE2, is inevitably accompanied. Among them, TNF-α, IL-1β, and IL-6 are pro-inflammatory cytokines that are produced in large quantities by stimulation. It has been reported that when produced, it catalyzes inflammatory reactions and worsens human diseases. In this experiment, the effect of DRB on the expression of IL-1β, IL-6, and TNF-α in LPS-induced RAW 264.7 cells was investigated using an ELISA kit, and the results are shown in Figure 6. DRB treated at 100, 200, and 400㎍/㎖ significantly suppressed the increased IL-1β and IL-6 caused by LPS in RAW 264.7 cells in a concentration-dependent manner, especially at the highest concentration of 400㎍/㎖ for IL-6. A level of inhibition similar to that of the LPS untreated group was observed at 28.5%. On the other hand, as the results of TNF-α measurement showed a slight inhibition level of 85.4%, 80.7%, and 74.4%, it is believed that DRB extract does not significantly affect the production mechanism of TNF-α.

Agricultural Biotechnology Research Institute KACC92346P 20210401

Claims (8)

Creating a mixture by mixing the extract of Chrysanthemum Chrysanthemum and the bacterial cells of the Bacillus JD3-7 strain (KACC 92346P);
A method for bioconversion of an extract of the ginseng tree, comprising the step of incubating the mixture. According to paragraph 1,
The bioconversion method wherein the extract of the Chrysanthemum vulgaris is extracted from the Chrysanthemum vulgaris with water, ethanol, or a mixed solvent thereof. According to paragraph 1,
The step of generating the mixture is a step of mixing the extract and the bacterial cells in a buffer solution. According to paragraph 3,
The step of generating the mixture is a step of mixing the extract of the Chrysanthemum ginseng in the mixture to a concentration of 1 g/l or less. According to paragraph 3,
The bioconversion method, wherein the buffer solution is a phosphate buffer solution containing 1 to 3% (v/v) glycerin and having a pH of 7 to 7.5. According to paragraph 3,
The bioconversion method wherein the incubation is incubation at 25 to 35°C for 2 to 4 days. A biotransformation product of an extract of Chrysanthemum vulgaris bioconverted by the bioconversion method of any one of claims 1 to 6. An anti-inflammatory composition comprising the biotransformation of claim 7.