KR20100100489A - A pharmaceutical composition containing cordyceps bassiana extracts having anti-inflamatory activity - Google Patents

Abstract

The present invention relates to an anti-inflammatory active pharmaceutical composition containing yellow dongchunghyeoncho extract, in particular, yellow dongchoongchocho butanol fraction as an active ingredient, the yellow dongchoongchocho butanol fraction (CBBF) according to the present invention is known as an important mediator for the inflammatory response It inhibits NO production through iNOS and COX-2, inhibits NF-kB activation, IKb, and inflammatory mediators, and regulates ROS production and NF-kB activation. There is an excellent effect that can suppress the production of Akt, PI-3K.

Description

A pharmaceutical composition containing Cordyceps bassiana extracts having anti-inflamatory activity}

The present invention relates to an extract of yellow Cordyceps sinensis having anti-inflammatory activity, and more particularly, to an anti-inflammatory active pharmaceutical composition containing an extract of Yellow Cordyceps sinensis, in particular a fraction of Yellow Cordyceps sinensis, as an active ingredient.

Cordyceps sinensis is a name given to the fact that it is in insects in winter and grasses in summer, and its name refers to all fungi parasitic to the seeds of insects, arthropods, fungi or higher plants. Scholarly, it belongs to the asymptomatic bacterium, incomplete bacterium, and splicing fungus. About 800 species of fungus invading insects have been reported, and 76 species have been collected and identified in Korea. Cordyceps Sinensis is a parasitic insect, and even the same insect may have different types of Cordyceps invading larvae and adults.

Cordyceps sinensis is a kind of insect parasitic bacteria that invade insect larvae, pupa and adult stages and form fruiting bodies based on it, and about 1200 species are distributed all over the world, and the cordyceps is distributed in 100 genera and about 750 species. In addition, about 70 species have been reported in Korea.

Cordyceps bassiana , a type of Cordyceps sinensis, is a type of Entomopathogenic Fungi that invades insects and forms fruiting bodies on the basis of it, and its taxonomic location is Ascomycota, Pyrenomycetes, and Bacillus fungi. It belongs to the genus Clavicipitales, Cordyceps, and Cordyceps incomplete genus. And the same name is Baekganggyun ( Beauveria bassiana ) and in our country is called Baekganggyun.

In general, Cordyceps sinensis is composed of about 10.8% moisture, about 8.4% fat, about 25.3% crude protein, about 28.9% carbohydrate, about 4.1% ash, and about 13% saturated fatty acid and unsaturated fatty acid. It contains about 82.2%, and vitamin B ₁₂ contains about 0.0029mg / g, and protein contains 17 kinds of essential amino acids which are indispensable to our body. Fats contain 13% saturated fatty acids and 82.2% unsaturated fatty acids. The higher the content of these unsaturated fatty acids, the greater the effect of reducing cholesterol. In addition, vitamin B12 is found only in animal foods, not in vegetable foods, and cannot be synthesized in the human body. It is necessarily consumed through foods, and the only thing in the plant is Cordyceps sinensis.

Cordyceps sinensis has been used as an antidote for tuberculosis and asthma jaundice and as an antidote for opioid poisoning, or as a tonic tonic or immune enhancer. In addition, herbal life has been reported to be effective in treating kidney and lung diseases. Herbal wood is said to have the effect of strengthening weakness and increasing immunity. In addition, Chinese metabolism Cordyceps sinensis is sweet, mild and strong lungs, so the record is good for respiratory diseases. Recently, health supplement products are made and marketed using Cordyceps sinensis in Korea, and many researches are underway to develop therapeutics and medicines by searching for new substances and discovering pharmacological components in Cordyceps sinensis. However, Cordyceps sinensis is very difficult to collect in the natural state, and even if collected, very small amount is collected, so it is practically difficult to develop products using only collected individuals and to search for pharmacological ingredients and new substances that are beneficial to human body.

Cordyceps sinensis has been shown to have antibacterial, antitumor and anticancer effects from cordycepin, a bioactive substance of Cordyceps militaris . The physiologically active effects in the materials extracted from culture mycelia of Cordyceps red (C. cicadae), reported that the anti-tumor therapy in a polysaccharide extracted from sclerotia fungus (C. ophioglossoides).

Since ancient times, Cordyceps sinensis is known as a elixir of bully life. It also serves as a nutritional tonic by protecting the lungs and strengthening the kidneys. Cordyceps sinensis has an immune function, and if this immune function is lost, it will be attacked by a virus or bacteria and will be exposed to all diseases. Cordyceps sinensis improves stamina and has a good therapeutic effect on colds, chronic cough, asthma, seizures, anemia, weakness, sexual dysfunction in men and hypertension. Cordyceps sinensis has a variety of effects, but the effect of the respiratory system is particularly excellent. Cordyceps was found to have a very high anticancer component of 83%. This ingredient has few side effects, enhances resistance, and is known to be excellent for bacterial and viral infections. Cordyceps sinensis is also effective in drug detoxification and inflammation control.

The importance of Cordyceps Sinensis can be considered in terms of its potential as a medicinal aid to humans and the development of microbial agents for pest control. The herbal medicine is derived from fruiting bodies formed from mummy larvae by C. sinensis, which has been used since ancient China. Cordyceps sinensis is composed of 10.84% moisture, 8.4% fat, 25.32% crude protein, 28.9% carbohydrate, and 4.1% ash. The fat component contains 13% saturated fatty acids and 82.2% unsaturated fatty acids. Vitamin B12 contains 0.29 mg per 100 g.

According to the record on the efficacy of Cordyceps sinensis, Cordyceps sinensis is a nutritional tonic that protects the lungs and strengthens the kidneys, enhancing immune function. Increasing immunity will increase resistance, making it less likely to cause any illness, as well as speeding up recovery. Cordyceps sinensis has a variety of effects, but the effect is particularly excellent in diseases of the respiratory system.

In relation to the conventional production method of yellow bunched fungus, the "mass production method of yellow bunched fungus fruit" (Korean Patent Application No. 10-2000-0035753) has been developed.

In the present invention, using the organic solvent extract of ethanol of yellow bundle Cordyceps, a natural substance, the yellow bundle Cordyceps butanol fraction was dried as a material to examine the anti-inflammatory effect of the Yellow Cordyceps. In the anti-inflammatory effect experiment, cell density according to the concentration of drug was measured by NO detection, RT-PCR, Western blot and reactive oxygen species (ROS) generation.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition having anti-inflammatory activity by containing a yellow cordyceps butanol fraction (CBBF).

The purpose of the present invention as described above is to extract the ethanol extract from the fruiting body of yellow bunched fungus, after obtaining a butanol fraction from it, IkB, p-Akt, p-P38, Erk and JNK related to the inflammation of the yellow bunched fungus sheath This was achieved by identifying the inhibitory effect and the inhibitory effect of ROS formation.

The present invention provides an anti-inflammatory active pharmaceutical composition containing the yellow hawthorn extract as an active ingredient.

In another aspect, the present invention provides an anti-inflammatory active health functional food composition containing the yellow bunched seaweed extract as an active ingredient.

The yellow bunched extract according to the present invention is preferably an ethanol extract or butanol fraction.

In the present invention, the term "active ingredient" refers to a substance or a group of substances (including medicinal herbs whose pharmacologically active ingredients are not known) which are expected to directly or indirectly express the efficacy effect of the medicine by inherent pharmacological action. It means containing a main component as.

Pharmaceutical compositions comprising the extract of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

The pharmaceutical dosage forms of the extracts of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection.

As defined herein, "health functional food" means a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to Act No. 6767 of the Health Functional Food Act, and "functional" means It means ingestion for the purpose of obtaining useful effects on health use such as nutrient control or physiological action on structure and function.

Anti-inflammatory active health functional food composition according to the present invention for the purpose of anti-inflammatory activity can be prepared and processed into a health functional food in the form of tablets, capsules, powders, granules, liquid, pills and the like.

In the present invention, the mechanism of inhibiting the production of inflammation-induced factors by the butanol fraction of the yellow bundling ethanol extract was examined. Inflammation is an immune response originating from damaged cells or tissues. It is a complex mechanism in which local vestibules and various immune cells in body fluids interact to induce enzyme activation, release of mediators, fluid infiltration, cell migration, and tissue destruction. It is a series of biological defense reactions.

Cells activated by lipopolysaccharide (LPS) are known to form a complex network by interacting many mediators such as COX-2, iNOS, NO and ROS with inflammatory cytokines. Therefore, the molecular approach for the regulation of inflammatory response is being studied in recent years, and many studies on the anti-inflammatory action through the substance extracted and purified from natural products have been made. In this regard, in the present invention, NO production is generated through iNOS and COX-2, which are known as important mediators for the inflammatory response after inducing macrophage activation by endotoxin LPS through a natural product called yellow bunched fungus that grows insects as a host. In addition, it was found that yellow butadiene subcutaneous butanol fraction inhibits the activation of NF-kB, a molecule known as transcription factor, IKb, an inflammatory mediator. It was also found that it inhibits the production of Akt and PI-3K, the signaling molecules involved in the regulation of ROS and NF-kB activation.

Therefore, in the present invention, the yellow bunched cordyceps butanol fraction (CBBF) was obtained as a bioactive substance, which can be used as an anti-inflammatory agent.

Yellow Bundo Cordyceps butanol fraction (CBBF) according to the present invention inhibits NO production through iNOS and COX-2, which are known as important mediators for the inflammatory response, and inhibits NF-kB activation as a molecule of IKb, an inflammatory mediator. CBBF according to the present invention has an excellent effect of inhibiting activation and inhibiting the production of Akt and PI-3K, which are signal molecules related to the regulation of ROS production and NF-kB activation. It is a very useful invention for pharmaceutical powder.

Hereinafter, preferred embodiments of the present invention will be described in more detail with reference to Examples. However, the scope of the present invention is not limited to these examples.

Example 1 Extraction of Biologically Active Substances from Yellow Bunny Cordyceps

Cordyceps bassiana fruiting body used in this example was re-dried in a laboratory oven at 50 ° C. for about a week, powdered, and used as an extract sample while being stored in a freezer.

After extracting the yellow bunched cordyceps fruiting body powder with 100% ethanol, the obtained ethanol extract was fractionated again with butanol. In the following experimental example, butanol fraction was used.

Experimental Example

In this Experimental Example, the anti-inflammatory effect of the Yellow Bundo Cordyceps butanol fraction prepared in Example 1 was tested using Raw 264.7 cell line. Cell lines were treated with 10% inactivated fetal bovine serum (FBS) and 100 u / mL penicillin using DMEM medium. Cell culture was performed in a 5% CO ₂ and 95% air environment in an incubator at 37 ℃.

Add Solution A (0.5 g of 1% sulfanilamide, 2.5 ml of 5% phosphoric acid, 50 ml of tertiary distilled water and Solution B (0.05 g of 0.1% naphthylenediamine, 2.5 ml of 5% phosphoric acid, 50 ml of tertiary distilled water) After mixing at a ratio of 1: 1 and injecting the cells, the NO concentration of the cells was detected.

Western blot

RAW 264.7 cells (macrophage, murine macrophage) were incubated in 10% FBS DMEM medium in 6-well plates and NO was measured with supernatant. Mix 96-well plate Griess reagents A and B in 1: 1 with Griess reagent (A: 1% sulfanilanide, B: .1% naphthylenediamine in 5% phosphoric acid) and mix the cell culture medium in the same amount (100ul) Put it. After incubation for 5 minutes at 37 ℃, it was measured with a 550nm microplate reader. After removing the prepared culture medium and the supernatant, washed with PBS (phospho buffer saline) and then put the phosphorus sample buffer (phospho sample buffer) (200ul) in each well to elute the cells and scraped to obtain a sample. The sample was boiled at 100 ° C. for 10 minutes, then separated by SDS gel, and then transferred (PVDF membrane). After blocking with 5% skim milk, antibodies were attached, and after washing, immunoreaction bands were confirmed by an ECL detection system.

ROS measurement (confocal microscope)

Raw 264.7 cells were incubated for one day at 37 ° C., 5% CO ₂ on an 18 mm coverslip. It was starvated for 16 hours, washed with phenol-red free medium, and then treated with yellow bunched cordyceps for 30 minutes. Subsequently, after 20 minutes of LPS treatment, DCF-DA was treated in the dark for 9 minutes and H ₂ O ₂ for 1 minute. ROS were measured after washing with phenol-free medium.

Experimental Example 1 Anti-inflammatory Effect of Ethanol Extracts and Various Fractions of Yellow Bundo Cordyceps

As a result of confirming the inhibitory ability of iNOS and COX-2 by treating the yellow bundle Cordyceps ethanol extract by concentration, as shown in Figure 2, iNOS and COX-2 both markedly reduced iNOS when treated with Yellow Cordyceps ethanol extract It was confirmed that COX-2 was also markedly reduced . By measuring NO with various fractions of the extract, the anti-inflammatory effect was confirmed indirectly, and the butanol fraction having the best effect was selected and used in subsequent experiments.

Experimental Example 2 Anti-inflammatory Effect of Yellow Bundo Cordyceps Butanol Fraction (CBBF)

Yellow Bunchongchocho butanol fraction (CBBF) increases NO, the final product of the signal, when inflammation is promoted by LPS. As shown in FIG. 3, it was confirmed that when treated with CBBF concentration, the inflammtory mediator decreased with concentration. In addition, INOS protein and mRNA levels of the inflammatory signaling protein was confirmed that the decrease in CBBF concentration increases.

Experimental Example 3: Inhibitory Effect of IkB, p-Akt, p-P38, Erk and JNK on Inflammation by CBBF

Based on the above results, IBBB, p-Akt, p-P38, Erk, and JNK, which are representative protein molecules of the NF-kB signal, a transcription factor related to inflammation, were identified. As shown in FIGS. 3 and 4, CBBF When treated by concentration, it was confirmed that IkB, p-Akt, p-P38, Erk and JNK related to inflammation decrease in a dose-dependent manner.

Experimental Example 4: Confirmation of the signal of CBBF by various inhibitors

In order to confirm the inflammatory signal pathway, to determine which signal is involved in CBBF, various inhibitors were treated with CBBF to confirm NO production, iNOS protein production, and expression level of iNOS at the mRNA level.

As a result, as shown in Figures 5 and 6, it was confirmed that CBBF is involved in the NF-kB, PI-3K, p38 production inhibition signal. In addition, it was found that some degree of involvement in ROS formation and NADPH oxidase inhibitory signal.

Experimental Example 5 Inhibitory Effect of CBBF on ROS Formation

As it was confirmed that CBBF is a signal that inhibits iNOS generation by ROS in FIG. 5, ROS inhibition of CBBF was confirmed using confocal microscopy to determine whether ROS could be inhibited. As a result, as shown in Figure 7, it was confirmed that the dose-dependent decrease in ROS according to the concentration of CBBF.

Experimental Example 6: Anti-inflammatory mechanism of CBBF

The anti-inflammatory mechanism of CBBF is as shown in Figure 8, it can be described as follows. Macrophages activated by LPS are known to cause inflammation by the interaction of many mediators such as COX-2, iNOS, NO and ROS with inflammatory cytokines. This patent, when the LPS-activated macrophages treated with yellow bunched fungus secant butanol fraction (CBBF), has been shown to inhibit the production of NO produced by iNOS, COX-2, which is known as an important parameter for the inflammatory response, In addition, it can be seen that it inhibits the activation of IKb, an inflammatory response mediator, a molecule that activates NF-kB, a transcription factor. In addition, IBB, p-Akt, p-P38, Erk, and JNK related to inflammation were reduced by treating the yellow bunched fungus butanol fraction (CBBF), and it was confirmed that ROS decreases with the concentration of CBBF. . Therefore, the present patent has obtained the result that the CBBF can be used as an anti-inflammatory agent and as a functional food or pharmaceutical agent as a bioactive substance.

1 is a diagram schematically showing the process of fractionation of yellow bunched fungus.

Figure 2 is a graph showing the anti-inflammatory effect of the yellow bunched Cordyceps ethanol extract.

Figure 3 is a graph showing the anti-inflammatory effect of the yellow bunched Cordyceps butanol fraction.

4 and 5 is a diagram showing the inhibitory effect of IkB, pAkt, p-P38, Erk and JNK related to inflammation caused by the Yellow Bundo Cordyceps butanol fraction.

6 and 7 is a diagram confirming the signal of the yellow bunching Chungchocho butanol fraction by various inhibitors.

Figure 8 is a diagram showing the effect of inhibiting the formation of ROS of the yellow bunched Cordyceps butanol fraction.

Figure 9 is a diagram showing the anti-base mechanism of the yellow bunched Cordyceps butanol fraction.

Claims (4)

Anti-inflammatory active pharmaceutical composition containing yellow bunched perilla extract as an active ingredient. The anti-inflammatory active pharmaceutical composition according to claim 1, wherein the yellow herb extract is a butanol fraction. Anti-inflammatory active dietary supplement composition containing the yellow bunched perilla extract as an active ingredient. The dietary supplement composition according to claim 3, wherein the yellow herb extract is a butanol fraction.

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