KR20230040170A - Biorenovation of Distylium racemosum leaf extract - Google Patents

Abstract

The present invention relates to a bioconversion of a Distylium racemosum leaf extract, a bioconversion product bioconverted by the method, and an anti-inflammatory composition comprising the bioconversion product. The bioconversion method of the present invention includes a step of mixing the Distylium racemosum leaf extract and cells of a strain JD3-7 of the genus Bacillus (KACC 92346P) to create a mixture, and a step for isothermal treatment of the mixture.

Description

Biorenovation of Distylium racemosum leaf extract}

The present invention relates to the bioconversion of a leaf extract of Coriander tree, and specifically, a bioconversion method capable of bioconverting the leaf extract of Coriander tree into a substance exhibiting superior anti-inflammatory activity, and a organism having excellent anti-inflammatory activity obtained thereby. It relates to a conversion product and an anti-inflammatory composition comprising the bioconversion product.

Inflammation is an immune response of biological tissues to protect the body from trauma or tissue damage caused by physical and chemical stimuli, and is a series of biological processes progressed by various immune cells. In general, the inflammatory response is caused by nitric oxide (NO), prostaglandin (PG), TNF-α (tumor necrosis factor-α), IL-6 (interleukin-6), and IL-6 secreted by various immune-related cells in damaged tissues. It is induced by various pro-inflammatory mediators, including pro-inflammatory cytokines such as 1β (interleukin-1β), which express inflammatory symptoms such as pain, edema, and fever and play an important role as mediators of various diseases. play a role Macrophages, known as the main inflammatory cells in the body, recognize LPS (lipopoly-saccharide), a gram-negative bacterium's external cell membrane toxin, through TLR (toll-like receptor) expressed on the cell surface, and activate the intracellular transcription factor NF. It activates the signaling pathway of -κB (nuclear factor-κB). NF-κB, which migrated into the nucleus of macrophages, induces the expression of inflammation-related genes iNOS (inducible nitric oxide synthase) and COX-2 (cyclooxygenase-2) This increases the production of inflammatory mediators such as NO and PGE2. Among them, NO is a kind of highly reactive radical. At low concentrations, it performs important physiological roles in the human body, such as signal transduction and immune action through the death of bacteria. However, when it is overexpressed, it causes inappropriate inflammation in the body. It is known to cause mutations in genes and damage to tissues and nerves. It has also been reported to induce various intractable diseases by increasing the catalytic activity of COX-2, triggering the signal transduction cascade, and inducing the acute expression of COX-2 to increase the production of PGE2. Considering that the production of excessive NO and PGE2, which causes these inflammatory diseases, is caused by inducible iNOS and COX-2, it can be seen that substances that inhibit iNOS and COX-2 gene expression are highly likely to be used as materials for regulating inflammatory responses. there is. Recently, as the inflammatory basis of various diseases has become clear, interest in inhibiting the activity of iNOS, COX-2, and various inflammatory mediators for the purpose of preventing and treating inflammatory diseases is increasing, and the search for safer anti-inflammatory materials is increasing. Research on the development of natural anti-inflammatory drugs centered on natural materials is being actively conducted.

On the other hand, many biological resources exist around us that are not fully utilized due to lack of proper research. Therefore, the need for research to utilize biological resources with low practical value in various ways and new utilization technologies based on this has emerged.

Cedar trees are also one of these biological resources. Distylium racemosum is an evergreen arboreal tree of the family Dicorrhiaceae, which grows wild on Jeju Island and islands in the South Sea, and is distributed in the south of Honshu in Japan, southeast China, and Taiwan. It is known to contain physiologically active ingredients such as flavonoid compounds such as catechin, quercetin, and quercitrin. Although the inhibitory effect of elastase has been studied, the anti-inflammatory effect through inhibition of NO production at the cellular level has been reported to be insignificant.

Accordingly, the inventors of the present invention tried to increase the utilization of the morocco tree by developing a method for increasing the anti-inflammatory activity of the morocco tree material (particularly, the leaf).

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. Ko, R. K., G. O. Kim, C. G. Hyun, D. S. Jung, N. H. Lee (2011) Compounds with Tyrosinase Inhibition, Elastase Inhibition and DPPH Radical Scavenging Activities from the Branches of Distylium racemosum Sieb. et Zucc. Phytother. Res. 25: 1451-1456. Kim, H. H., J. H. Kwon, K. H. Park, M. H. Kim, M. H. Oh, K. I. Choe, S. H. Park, H. Y. Jin, S. S. Kim, M. W. Lee (2012) Screening of Antioxidative Activities and Anti-inflammatory Activities in Local Native Plants. Kor. J. Pharmacogn. 43(1): 85-93.

Therefore, the main object of the present invention is to provide a method capable of increasing the anti-inflammatory activity of the leaves of Cyrax japonica.

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

According to one aspect of the present invention, the present invention is a step of producing a mixture by mixing the extract of the leaf extract of Bacillus genus JD3-7 (KACC 92346P), and the step of incubating the mixture. A method for bioconversion of an extract is provided.

In the bioconversion method of the present invention, it is preferable that the extract of the Coriander tree leaf is extracted with water, ethanol or a mixed solvent thereof from the Coriander tree leaf.

In the bioconversion method of the present invention, it is preferable that the step of generating the mixture is a step of mixing the leaf extract and the microbial cells in a buffer solution.

In the bioconversion method of the present invention, the step of generating the mixture is mixing the Coriander leaf extract and the cells in a buffer solution, so that the concentration of the Coriander leaf 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 is preferably a phosphate buffer 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 incubation at 25 to 35° C. for 2 to 4 days.

According to another aspect of the present invention, the present invention provides a bioconverted product of the extract of the leaf extract of Corrhiza sinensis bioconverted by the above bioconversion method.

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

According to the present invention, the extract of Zorrhox sinensis has more excellent anti-inflammatory activity, that is, more excellent nitric oxide and PGE2 production inhibitory activity, and more excellent iNOS, COX-2, TNF-α, IL-1β and IL-6 expression inhibitory activity. can be biotransformed into substances that exhibit Accordingly, it can be used to manufacture anti-inflammatory products, such as anti-inflammatory functional cosmetics or foods, or pharmaceuticals for preventing or treating inflammatory diseases.

Figure 1 shows the results of HPLC analysis of the Coriander leaf extract (DL), the bioconverted product of the Coriander leaf extract (DLB), and the control (BA).
Figure 2 shows the experimental results for the cytotoxicity of the Coriander leaf extract (DL), the bioconverted product of the Coriander leaf extract (DLB) and the control group (BA).
Figure 3 shows the experimental results for the nitric oxide (NO) production inhibitory activity of the Coriander leaf extract (DL), the bioconverted product of the Coriander leaf extract (DLB), and the control group (BA).
Figure 4 shows the experimental results for the PGE2 production inhibitory activity of the leaf extract (DL) and the bioconverted product (DLB) of the leaf extract of the algae tree.
Figure 5 shows the experimental results for the inhibitory activity of iNOS and COX-2 expression of the leaf extract (DL) and the bioconverted product (DLB) of the leaf extract of the leaf extract of japonica.
Figure 6 shows the experimental results for the inhibitory activity of IL-1β, IL-6 and TNF-α expression of the leaf extract (DL) and the bioconversion product (DLB) of the leaf extract of the leaf extract.
Figure 7 shows the accession of Bacillus genus JD3-7 strain (KACC 92346P).

The biotransformation method of the present invention is characterized in that it comprises the step of mixing the extract of the leaf extract of Bacillus genus JD3-7 (KACC 92346P) to produce a mixture, and the step of incubating the mixture.

Cedar tree is an evergreen shrub or tree classified in the genus Cedaraceae, and its scientific name is ' Distylium racemosum '. In the present invention, the leaf parts of the Cerrophylla tree are used.

In the present invention, the Coriander leaf extract is extracted using the Coriander tree leaf as an extraction raw material, and can be prepared by conventional extraction methods in the art, such as hot water extraction, ultrasonic extraction, and reflux extraction. It is preferably extracted using a solvent selected from the group consisting of water, C1 to C4 lower alcohol, and mixtures thereof, and more preferably extracted with water, ethanol, or a mixed solvent thereof. Such an extract is more suitable for bioconversion into a substance exhibiting excellent effects, that is, excellent anti-inflammatory activity, through the bioconversion method of the present invention. For the same reason, the extract of the leaf of Zorrhox is more preferably extracted with water or a mixed solvent of water and ethanol, and more preferably extracted 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 a 40% (v/v) ethanol solution. to 90% (v/v) ethanol solution, more preferably a 50 to 90% (v/v) ethanol solution, more preferably a 60 to 80% (v/v) ethanol solution, more It is preferably a 65 to 75% (v/v) ethanol solution.

In the present invention, the amount of the solvent when preparing the leaf extract of Jocorus is based on 1 g of the weight (eg, dry weight) of the extract raw material, that is, the leaves of Jocorus, preferably 100 to 1000 ml, more preferably 300 to 800 ml, and more It is preferably in a proportion of 400 to 600 ml. The extraction temperature is preferably 0 to 100°C, more preferably 5 to 90°C, and more preferably 10 to 80°C. In particular, when a mixed solvent of water and ethanol is used as a solvent, the extraction temperature is preferably 5 to 35°C, more preferably 10 to 30°C, and more preferably 15 to 25°C. The extraction time is preferably 1 hour to 10 days, more preferably 12 hours to 5 days, more preferably 1 day to 3 days. According to this, it is possible to prepare an extract more suitable for bioconversion into a substance exhibiting excellent effects, that is, excellent anti-inflammatory activity, through the bioconversion method of the present invention.

In the present invention, the extract of the Coriander tree leaf may be further subjected to a process such as filtration, concentration, vacuum drying, and freeze-drying after the extraction process as described above.

The Bacillus genus JD3-7 strain, which is a strain used in the bioconversion of the present invention, was isolated from soybean conventional soy sauce in Jeju Island and deposited with the Korean Agricultural Culture Collection (KACC) under the accession number KACC 92346P.

The JD3-7 strain of the genus Bacillus can be cultured according to conventional culture conditions for culturing microorganisms of the genus Bacillus. For example, it may be cultured in a temperature condition of 25 to 35° C. in LB (Luria Bertani) medium or NB (nutrient broth) medium.

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

In the present invention, the step of generating a mixture is a step of mixing them so that the bioconversion of the Jocorus leaf extract can be achieved by contacting the Bacillus genus JD3-7 strain (or its enzyme), It can be achieved by adding and mixing the tree leaf extract and Bacillus JD3-7 strain. At this time, the medium may be a medium, for example, a medium in which the strain JD3-7 of the genus Bacillus can grow, and may be water or a water-based buffer. In one embodiment of the present invention, the medium is a buffer.

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), still more preferably contains 1.8 to 2.2% (v/v) glycerin. In addition, the buffer solution is preferably a buffer solution of pH 6.5 to 8, more preferably a buffer solution of pH 7 to 7.5. In addition, the buffer is preferably a phosphate buffer. According to these buffer conditions, more effective bioconversion can be made possible.

Zorrhox leaf extract and Bacillus genus JD3-7 strain are added to the medium and mixed, preferably so that the concentration of the Zorrhox leaf extract in the mixture formed by this mixing is 1 g/L or less. This is in consideration of the possibility of inhibiting the activity of the JD3-7 strain of Bacillus genus due to an excessively high concentration of the extract of the leaf extract of Bacillus sp. At the same time, in order to bioconvert as much of the leaf extract as possible through one 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, even 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 incubation to induce bioconversion by the Bacillus sp. JD3-7 strain (probably by the action of the enzyme produced by this strain), It can be achieved by putting a mixture of cells of 7 strains into a conventional 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, more preferably 29 to 31 ° C, and the incubation time is preferably 1 hours to 10 days, more preferably 12 hours to 7 days, still more preferably 1 to 5 days, still more preferably 2 to 4 days. According to these incubation conditions, more efficient bioconversion can be made possible.

The bioconversion product of the present invention is characterized in that it is obtained by bioconversion of the Corrhoid leaf extract by the bioconversion method as described above. In one embodiment of the present invention, the bioconverted product has higher anti-inflammatory activity compared to the non-bioconverted Cyrax japonica leaf extract, for example, inhibition of nitric oxide production, inhibition of PGE2 (Prostaglandin E2) production, iNOS and COX- 2 expression inhibition, TNF-α, IL-1β and IL-6 expression inhibition activity can be shown. For example, it may exhibit higher anti-inflammatory activity at the same concentration or the same level of anti-inflammatory activity at a lower concentration.

The anti-inflammatory composition of the present invention is characterized in that it comprises the bioconvertant as described above.

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 consist only of the bioconvertant, and may include only the bioconvertant as an active ingredient. In addition, the anti-inflammatory composition of the present invention may further include other substances as active ingredients together with the bioconverter. Other substances at this time may be derived from other biological resources other than the leaves of the cedar tree, may be processed from the leaves of the cedar tree by other methods, or may be compounds.

The anti-inflammatory composition of the present invention may further include additives, for example, cosmetic, food, or pharmaceutically acceptable additives, in addition to the active ingredient (eg, the bioconversion of the present invention), in an appropriate form. It can be formulated as These additives may be appropriately selected depending on the use of the composition, application method (eg dosage form), and the like.

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

When the anti-inflammatory composition of the present invention is a pharmaceutical composition, it can be administered orally or parenterally, and can be administered in various ways such as subcutaneous injection during parenteral administration, and can be used in the form of a general pharmaceutical preparation. The dosage may be adjusted according to the condition of the subject to be administered, the route of administration and the form of administration, and those skilled in the art will be able to adjust it within various ranges, depending on the symptoms. The anti-inflammatory composition of the present invention may be administered, for example, in an amount of 0.01 to 25 mg per 1 kg of the subject's body weight based on the weight (eg, dry weight) of the bioconverter of the present invention contained in the composition.

Hereinafter, the present invention will be described in more detail through examples. Since these examples are intended to illustrate the present invention only, the scope of the present invention is not to be construed as being limited by these examples.

[Example]

Example 1. Preparation of Zorrhox leaf extract

In August 2020, the leaves of Distylium racemosum were collected in Sinrye-ri, Namwon-eup, Seogwipo-si, Jeju-do. After pulverizing the dried Morocco leaves, 500 ml of 70% (v/v) ethanol was added to 1 g of the resulting powder, followed by repeated extraction twice for 48 hours at room temperature. Afterwards, the extract was filtered through a paper filter (TOYO ROSHI KAISHA, Tokyo, Japan). After concentrating the filtered extract using a vacuum concentrator, it was freeze-dried at -110 ° C and used for the next bioconversion.

Example 2. Biotransformation of Coriander leaf extract

Bacillus sp. JD3-7 strain (KACC 92346P) was cultured in a nutrient medium (Peptone 5.0g/ℓ, Beef extract 3.0g/ℓ) (Nutrient Broth, Difco, USA) at 30℃ for 18 days. After culturing for 15 minutes, the culture solution was centrifuged at 4,000 rpm to obtain cells. After washing the obtained cells twice, suspending them in 100 ml of PG buffer (50 mM phosphate buffer, 2% (v/v) glycerin), and adding 100 mg of the extract (DL) of Example 1 to the A bioconversion reaction was induced by incubation at 30° C. for 72 hours. This reaction was named DLB, and a cell suspension to which DL was not added was used as a control (BA). After completion of the reaction, the pellet and the supernatant were separated by centrifugation, and the supernatant from which the pellet was removed was freeze-dried at -110 ° C and used in the next experiment.

Example 3. Evaluation of the physiological activity of the bioconverted product of the leaf extract of C.

3-1. method

3-1-1. HPLC analysis

For HPLC analysis, a Shimadzu SpectroMonitor 3200 digital UV/Vis detector was used, and water (Solvent A) and acetonitrile (Solvent B, Sigma-Aldrich, St. Louis, USA) added with 0.1% TFA (Trifluoroacetic acid, SAMCHUN, Korea) were used. was used as a solvent. The analysis conditions were carried out at a wavelength of 254 nm with a column (Phenomenex 4㎛ Hydro-RP 80Å, 250 × 4.6 mm), temperature 40 ° C, flow rate 1.0 ml / min, and the extract of C. japonica leaf (DL) of Example 1 and Example 1 The bioconversion product (DLB) and the control (BA) of C. elegans leaf extract of 2 were dissolved in DMSO (dimethyl sulfoxide, Sigma-Aldrich, St. Louis, USA). The sample was analyzed under gradient conditions for 30 minutes, and the gradient of the solvent was acetonitrile (Solvent B) from 0.1 minutes: 10% to 30 minutes: 100%.

3-1-2. Experiment 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 Korea Cell Line Bank. For the culture of cell lines, DMEM (Dulbecco's Modified Eagle Medium, Gibco, New York, USA) containing 10% FBS (fetal bovine serum), 100 U/ml penicillin, and 100 μg/ml streptomycin was used. It was cultured in an incubator under CO ₂ conditions and subcultured every 2 days.

3-1-3. Cytotoxicity measurement

Cytotoxicity of the sample to RAW 264.7 cells was measured by cell viability analysis using MTT assay. RAW 264.7 cells were seeded in a 24-well plate at a concentration of 7.0×10 ⁴ cells/well, pre-cultured for 24 hours in a 37°C, 5% CO ₂ incubator, and samples diluted with culture medium (6.25, 12.5, 25 [mu]g/ml) and LPS (1 [mu]g/ml) were co-treated to induce inflammation for 24 hours under the same culture conditions. Thereafter, MTT reagent was added and reacted in an incubator for 3 hours. After dissolving formazan blue formed in each well with DMSO, absorbance was measured at a wavelength of 570 nm using an ELISA reader.

3-1-4. NO production inhibition activity measurement

RAW 264.7 cells were seeded in a 24-well plate at a concentration of 7.0 × 10 ⁴ cells/well, pre-incubated for 24 hours in a 37°C, 5% CO ₂ incubator, and then diluted with LPS (1 μg/ml) (6.25 , 12.5, 25 μg/ml) were simultaneously treated to induce inflammation for 24 hours under the same culture conditions. Thereafter, 100 μl of Griess reagent [1% (w/v) sulfanilamide, 0.1% (w/v) naphylethylenediamine in 2.5% (v/v) phosphoric acid] was mixed with 100 μl of the culture medium in a 96-well plate and reacted in the dark for 15 minutes. Then, absorbance was measured at a wavelength of 570 nm using an ELISA reader.

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

RAW 264.7 cells were seeded in a 24-well plate at a concentration of 7.0 × 10 ⁴ cells/well and pre-incubated for 24 hours in a 37°C, 5% CO ₂ incubator, followed by LPS (1 μg/ml) and diluted sample ( 6.25, 12.5, and 25 μg/ml), and inflammation was induced for 24 hours under the same culture conditions. Thereafter, the collected culture medium was centrifuged at 10,000 rpm for 3 minutes to remove the precipitate, and the supernatant was collected and used in the experiment. ., Minneapolis, Minnesota, USA).

3-1-6. Western blot analysis

RAW 264.7 cells were inoculated in a 6-well plate at a concentration of 5×10 ⁵ cells/well, pre-incubated for 24 hours in a 37°C, 5% CO ₂ incubator, and diluted samples (6.25, 12.5, 25 μg/ml) ) and LPS (1 μg/ml) were simultaneously treated to induce inflammation for 24 hours under the same conditions. After washing the cells twice with PBS, lysis buffer [1 × RIPA (Upstate Cell Signaling Solution, New York, USA), 1 mM PMSF (phenylmethylsulfonyl fluoride), 1 mM Na ₃ VO ₄ , 1 mM NaF, 1 μg /mL aprotinin, 1 μg/mL pepstatin, and 1 μg/mL leupeptin], followed by dissolution for 1 hour, and then centrifugation (12,000 rpm, 30 minutes) to separate the supernatant and pellet. The protein concentration of the supernatant was quantified using a BCA kit (Bio-Rad, USA), and 20 μg of protein was electrophoresed on 10% SDS-PAGE containing 10% polyacrylamide, followed by PVDF (polyacrylamide). -vinylidene difluoride) membrane (Milipore, Burlington, MA, USA). After the transfer was completed, the membrane was put in 5% skim milk (sol. TBST), blocked at room temperature for 2 hours, washed three times for 10 minutes with TBST, and the primary antibody diluted with TBST (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) Bake and react overnight at 4°C. After the reaction, the membrane was washed 4 times with TBST solution, diluted with HRP (horseradish peroxidase)-conjugated secondary antibody (Jackson ImmunoResearch, USA) at a ratio of 1:10,000, and reacted for 90 minutes. After washing three times for 10 minutes with , it was reacted with an ECL kit (Bio-Rad, USA) and developed through an imaging densitometer (model GS-700, Bio-rad, USA). The expression levels of developed iNOS and COX-2 were graphed by quantifying the area of iNOS and COX-2 relative to β-actin using the ImageJ program (NIH, Bethesda, Maryland, USA).

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

RAW 264.7 cells were inoculated in a 24-well plate at a concentration of 7.0×10 ⁴ cells/well and cultured in a CO ₂ incubator (37°C, 5% CO ₂ ) for 24 hours, and diluted samples (6.25, 12.5, 25 μg) /ml) and LPS (1 μg/ml) were simultaneously treated to induce inflammation for 24 hours under the same conditions. Thereafter, the recovered culture medium was centrifuged (10,000 rpm, 3 minutes) to remove the precipitate, and the content of pro-inflammatory cytokines present in the culture medium was measured using the supernatant from which the precipitate was removed. For measurement, mouse TNF-α ELISA kit (Invitrogen, California, USA), mouse IL-6 ELISA kit (BD Biosciences, California, USA), and mouse IL-1β ELISA kit (R&D Systems Inc., Minneapolis, Minnesota, USA) were used. used

3-1-8. statistical processing

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

3-2. result

3-2-1. HPLC analysis

As a result of HPLC analysis of the Coriander leaf extract (DL) of Example 1, the bioconverted product of the Coriander leaf extract (DLB) and the control (BA) of Example 2, the existing DL existed at 2.5 to 7.5 minutes of DLB. A new peak that does not appear was confirmed (Fig. 1). This suggests the possibility that changes in various components present in the leaf extract of Zorrhox sinensis were induced by biochemical reactions during the bioconversion process.

3-2-2. Comparative measurement of cytotoxicity

MTT assay was performed to investigate the cell viability of DL, DLB and BA in RAW 264.7 cells induced by LPS. As a result of confirming cell viability by treating each sample at concentrations of 6.25, 12.5, and 25 μg/ml, DL and DLB showed cell viability of 85% or more within 25 μg/ml, and BA also showed cell viability of 80% or more. was (FIG. 2). Therefore, based on the results of this experiment, further experiments were conducted at the maximum concentration of 25 μg/ml, which does not show cytotoxicity.

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

In the inflammatory response induced by LPS, secondary mediators such as NO, PGE2, leukotriene, and pro-inflammatory cytokines produced and secreted from macrophages are known to play an important role in regulating innate and acquired immunity. Among them, NO produced by iNOS has a pathologically important meaning. A high concentration of NO not only promotes the biosynthesis of inflammatory mediators and promotes the inflammatory response, but also damages DNA, amplifies inflammation, septic shock and cell damage. It has been reported as a cause of various immune diseases such as inducing necrosis. Therefore, a substance that specifically inhibits NO means that various immune diseases can be effectively prevented and treated. Therefore, this experiment was conducted to investigate the effect of DLB on the production of NO increased by LPS stimulation in RAW 264.7 cells. After simultaneous treatment at a concentration of ug/ml, the amount of NO ^2- present in the cell culture medium was measured using Griess reagent. As a result of the measurement, DLB inhibited NO production by 22.9±2.8% and 43.1±2.5% compared to the LPS-only treatment group, and at the highest concentration of 25 μg/ml, it was 61.1±2.2%, showing significant inhibitory activity at all concentrations. Observed. On the other hand, no significant inhibitory activity was observed in DL, and no significant inhibitory activity was observed in BA either (FIG. 3). This suggests that the activity of DLB is an inherent effect independent of BA, and that the NO inhibitory effect of DLB compared to DL was improved. Therefore, DLB is thought to be effective as an anti-inflammatory material through NO inhibition mechanism.

3-2-4. PGE2 production inhibitory activity

PGE2 suppresses the production of inflammatory cytokines such as TNF-α, IL-1β, IL-8, and IL-12 in macrophages as a regulator of the body's immune response, and inhibits the production of anti-inflammatory cytokines such as IL-10. It plays an important role in the immune response, such as stimulating However, PGE2, which is overproduced by COX-2 during the inflammatory process in the body, has been reported to be involved in the deepening of inflammation by mediating vasodilation, edema, fever, pain, etc. It is also known to be involved. Therefore, this experiment was conducted to investigate the effect of DLB on the production of PGE2 in RAW 264.7 cells induced by LPS stimulation. As a result of the experiment, DLB suppressed the production of PGE2 by 27.1±3.7%, 43.4±0.9%, and 55.3±0.7% compared to the LPS-only treatment group, and no significant decrease was observed in RAW 264.7 treated with DL (FIG. 4). . Therefore, DLB has improved the efficacy of PGE2 production inhibition compared to conventional extracts through the biotransformation process, and it is thought that it can contribute to the improvement of the inflammatory response through the inhibition of PGE2 production.

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

Inflammation-inducing genes iNOS and COX-2 are known to be causative substances that induce an inflammatory response by inducing the activity of immune-related cells such as macrophages in a pathological environment such as inflammation. Among them, NO is a kind of highly reactive biogenic radical and is known as a representative indicator of inflammatory response. Most of NO produced in inflammatory response is synthesized from L-arginine and O ₂ by the action of iNOS. reported that iNOS expression is closely related to NO production. In addition, COX-2 is an inducible isoform of COX, one of the important factors involved in the inflammatory response. It is acutely expressed in pathological inflammatory reactions and synthesizes PGE2, an inflammatory mediator involved in pain and fever. It is known to be involved in inflammatory reactions and cause various intractable diseases. Thus, iNOS and COX-2 gene expression is an important mechanism by which inflammatory mediators are overproduced by NO and PGE2, and must be precisely controlled to prevent chronic inflammatory responses. Therefore, in this experiment, Western blot analysis was performed to investigate the effect of DLB on the expression of iNOS and COX-2 proteins involved in the production of NO and PGE2 and their correlation. RAW 264.7 cells stimulated with LPS (1μg/ml) were treated with DL and DLB at concentrations of 6.25, 12.5, and 25μg/ml for 24 hours to induce inflammation, and expression of iNOS and COX-2 was confirmed. In RAW 264.7 cells treated with LPS, no significant reduction in activity was observed compared to the group treated with LPS alone, whereas in RAW 264.7 cells treated with DLB, iNOS expression was significantly reduced. In addition, the decrease in iNOS expression by DLB and the tendency of NO suppression were similar, confirming that the NO reduction activity was closely related to the expression of iNOS (FIG. 5a). Likewise, COX-2 expression was significantly suppressed in RAW 264.7 cells treated with DLB, and it was confirmed that the trend was similar to that of PGE2 (Fig. 5b).

Based on these results, it is believed that the NO and PGE2 inhibitory activity of DLB is due to the reduction of iNOS and COX-2 gene expression, and it is considered to be effective as an anti-inflammatory agent targeting their mechanism of action.

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

Proinflammatory cytokines produced and secreted by macrophages activated by inflammatory mediators play an important role in the initial inflammatory response by inducing the production of other cytokines. Among them, TNF-α, IL-6, and IL-1β are representative pro-inflammatory cytokines. They increase during systemic inflammatory response to activate NF-κB, a transcription factor, and consequently increase the production of other cytokines, including NO and PGE2. It is known to prolong the inflammatory response. Therefore, in this experiment, the effect of DLB on the increased production of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) in RAW 264.7 cells induced by LPS stimulation was investigated. As a result of treating RAW 264.7 cells induced with LPS (1μg/ml) with DL and DLB at concentrations of 6.25, 12.5, and 25μg/ml, DL showed significant effects on TNF-α, IL-6, and IL-1β. While there was no inhibitory activity, DLB increased TNF-α by 18.3±6.2%, 27.0±2.6%, and 37.9±3.1%, IL-6 by 37.8±1.2%, 50.2±2.4%, 63.8±1.3%, and IL-1β showed significant inhibitory activity at 15.5±2.2%, 32.2±2.6%, and 47.4±1.5% (FIG. 6). It was confirmed that DLB showed improved inhibitory activity in contrast to DL, and exhibited anti-inflammatory activity by most effectively inhibiting IL-6.

Agricultural Biotechnology Research Institute KACC92346P 20210401

Claims (8)

Generating a mixture by mixing the extract of Corphyra tree leaf and the cells of the Bacillus genus JD3-7 strain (KACC 92346P), and
A method for bioconversion of a leaf extract of C. alfalfa comprising the step of incubating the mixture. According to claim 1,
The bioconversion method of claim 1, wherein the leaf extract of the Coriander tree is extracted with water, ethanol or a mixed solvent thereof from the leaves of the Coriander tree. According to claim 1,
The step of generating the mixture is a step of mixing the Corphyra leaf extract and the cells in a buffer, a bioconversion method. According to claim 3,
The step of producing the mixture is a step of mixing so that the concentration of the Corphyra leaf extract in the mixture is 1 g / ℓ or less, bioconversion method. According to claim 3,
The buffer is a phosphate buffer containing 1 to 3% (v / v) glycerin, pH 7 to 7.5, bioconversion method. According to claim 3,
The incubation is an incubation at 25 to 35 ° C. for 2 to 4 days, a bioconversion method. A bioconverted product of a leaf extract of C. alfalfa bioconverted by the bioconversion method according to any one of claims 1 to 6. An anti-inflammatory composition comprising the bioconversion of claim 7.

Images