TWI541018B - Taiwan Cordyceps extract for the manufacture of anti-respiratory virus drugs - Google Patents

Description

Use of Taiwan Cordyceps extract for the preparation of anti-respiratory virus drugs

The invention relates to the use of Taiwan Cordyceps extract for preparing anti-respiratory virus medicine

Cordyceps formosana Kobayasi & Shimizu was first discovered in July 1976 by Japanese Kobayashi and other males on the fallen wood of Fenghuang Mountain in Xitou, Taiwan. In 1981, it was officially named Cordyceps formosana Kobayasi & Shimizu . There have also been reports in mainland China that in 2002, Anhui Agricultural University discovered that there were Taiwan Cordyceps in Huangshan, China. In July 2009, the research and development personnel of Taiwan Muqi Biotech Co., Ltd. acquired the Cordyceps sinensis in Xitou, Taiwan and separated it. It was identified as Taiwan Cordyceps by relevant experts. The applicant established the Taiwan Cordyceps Research Group to conduct an in-depth study on Cordyceps sinensis, and successfully carried out artificial fermentation of Cordyceps species in Taiwan, successfully growing fruiting bodies in artificial medium, and using live larvae of beetle-like beetles (for example) The larvae of barley worms have artificially cultivated Cordyceps sinensis.

It has been studied that Cordyceps sinensis contains more bioactive components, such as adenosine, mannitol, cordycepin, alizarin, oleic acid, ergosterol, stearic acid, palmitic acid and other chemical components. Studies have found that Cordyceps sinensis can significantly enhance the body's immune function; it has strong free radical scavenging active substances; it has strong anti-tumor activity, and also has antibacterial, anti-inflammatory, anti-oxidant, etc. effect.

Respiratory syncytial virus (RSV) is a common cause of bronchiolitis and pneumonia in infants worldwide. Almost all children have experienced one or more infections at the age of 2, and the peak age of infection is 2 Month to 8 months. It is the leading cause of lower respiratory tract infection in infants and young children, and the primary cause of hospitalization for young children due to respiratory diseases. Many studies have also shown that severe RSV infection in infants is a risk factor for asthma later, and its severity far exceeds other Microbial pathogens. It has also been found that RSV infection is also an important cause of immunosuppression in adults and the elderly.

Currently, there is no specific and effective treatment for RSV infection. The antiviral drug ribavirin (ribavirin) is currently the only chemotherapeutic drug approved by the FDA for the prevention and treatment of RSV infection, but it can cause headache, leukopenia, irreversible anemia, serum bile due to bone marrow cytotoxicity. Side effects such as elevated erythropoietin are limited in clinical application, and are only strictly used in high-risk and severely ill children, and their efficacy has been controversial in recent years. Human monoclonal antibodies palivizumab (Synagis ^® ) and intravenous immunoglobulin (RSV-IGIV) have been registered for use with good efficacy, but due to the cost of treatment and the safety of serum products, in the prevention and treatment of RSV infection, Careful consideration is needed, but not universally. To date, no vaccine has been used clinically to prevent RSV infection. Therefore, the development of drugs with reliable efficacy and small side effects to prevent and treat RSV infection is still one of the important issues to be solved in the world.

Applicant's research found that Taiwan Cordyceps extract has a good anti-RSV effect. At present, there is no anti-RSV effect of Taiwan Cordyceps extract. Research reports and patent documents.

The present invention relates to the use of a Cordyceps sinensis extract for the preparation of a medicament against respiratory viruses.

It is an object of the present invention to provide a use of a Cordyceps sinensis extract for the preparation of a medicament against respiratory viruses.

The object of the present invention is achieved by the following technical means: the extract of Taiwan Cordyceps sinensis is artificially fermented mycelium of Cordyceps sinensis or artificially growing fruit body in artificial medium or larvae of coleoptera by artificially cultivating Taiwan Cordyceps for ethanol extraction Rotate to obtain a paste. The above obtained extract is subjected to a treatment such as drying, and then a conventional excipient is added, and any conventional dosage form acceptable for pharmacy is prepared according to a conventional preparation process, including, but not limited to, a capsule, a tablet, a granule, and a gel. , sustained release and oral liquid.

The Taiwan Cordyceps extract is an artificial fermented mycelium of Cordyceps militaris, an artificial medium for growing fruit bodies or a larva of a coleoptera, and the larvae of the coleoptera are artificially cultivated by ethanol extraction, and the anti-respiratory syncytial virus activity test shows that Good inhibition of respiratory syncytial virus activity.

Experimental Example 1 Inhibition of Respiratory Syncytial Virus Activity by Artificial Fermentation Mycelium Extract of Cordyceps sinensis

Cytotoxicity test

HEp-2 cells (human laryngeal carcinoma epithelial cells) were inoculated into a 96-well cell culture plate, cultured at 37 ° C in a 5% CO ₂ incubator. After the cells were grown into a single layer, the culture solution was removed, washed twice with PBS, and added. 0.1 mL was used to maintain the liquid half-dilution test solution, and 0.1 mL of the maintenance solution was set as a blank control. Continue to incubate at 37 ° C, 5% CO ₂ incubator for 2 to 5 days, observe the cytopathic condition under the microscope every day, including the shedding, rounding, shrinkage, formation of granules and vacuoles in the cytoplasm, record After the results, the half toxicity concentration (TC ₅₀ ) was calculated.

2. Antiviral test

The HEp-2 cell culture method was the same as above, the culture medium was removed, washed with PBS, and 0.1 mL (1OOTCID ₅₀ /0.1 mL virus) virus suspension and 0.1 mL of the maximum cell non-toxic concentration were added (the cells detected under light microscope were not observed). The maximum concentration of the test product that produces toxicity is the highest concentration of the test solution after the half-dilution, and the maintenance solution is used as a blank control. The cells were cultured at 37 ° C for 2 to 5 days in a 5% CO ₂ incubator. The pathological changes caused by the virus were detected under the microscope and recorded. Ribavirin was used as a positive control drug.

Calculate the median inhibitory concentration (EC ₅₀₎ and a therapeutic index TI = TC ₅₀ / EC _50.

3. Anti-respiratory syncytial virus activity of artificial fermentation mycelium extract of Cordyceps sinensis

Artificial fermented mycelium extract of Cordyceps sinensis: The artificial fermented mycelium of Cordyceps sinensis was extracted by 20% ethanol and rotary evaporated to obtain a paste, which is an artificial fermentation mycelium extract of Cordyceps sinensis.

The specific results are shown in Table 1. Table 1 Inhibition of Respiratory Syncytial Virus by Extract of Mycelium of Cordyceps Artificial Fermentation

Experimental Example 2 Inhibition of respiratory syncytial virus activity by fruiting body extract of Cordyceps sinensis

Cytotoxicity test

HEp-2 cells (human laryngeal carcinoma epithelial cells) were inoculated into a 96-well cell culture plate, cultured at 37 ° C in a 5% CO ₂ incubator. After the cells were grown into a single layer, the culture solution was removed, washed twice with PBS, and added. 0.1 mL was used to maintain the liquid half-dilution test solution, and 0.1 mL of the maintenance solution was set as a blank control. Continue to incubate at 37 ° C, 5% CO ₂ incubator for 2 to 5 days, observe the cytopathic condition under the microscope every day, including the shedding, rounding, shrinkage, formation of granules and vacuoles in the cytoplasm, record After the results, the half toxicity concentration (TC ₅₀ ) was calculated.

2. Antiviral test

The HEp-2 cell culture method was the same as above, the culture medium was removed, washed with PBS, and 0.1 mL (1OOTCID ₅₀ /0.1 mL virus) virus suspension and 0.1 mL of the maximum cell non-toxic concentration were added (the cells detected under light microscope were not observed). The maximum concentration of the test product that produces toxicity is the highest concentration of the test solution after the half-dilution, and the maintenance solution is used as a blank control. The cells were cultured at 37 ° C for 2 to 5 days in a 5% CO ₂ incubator. The pathological changes caused by the virus were detected under the microscope and recorded. Ribavirin was used as a positive control drug.

Calculate the median inhibitory concentration (EC ₅₀₎ and a therapeutic index TI = TC ₅₀ / EC _50.

3. Anti-respiratory syncytial virus activity of fruit extracts of Cordyceps sinensis

Fruit body extract of Cordyceps sinensis: Take the fruiting body of Taiwan Cordyceps grown on artificial medium, extract it with 50% ethanol, and spin to obtain the paste, which is the fruiting body extract of Taiwan Cordyceps.

The specific results are shown in Table 2.

Experimental Example 3 Inhibition of Respiratory Syncytial Virus Activity by Extract of Taiwan Cordyceps (Insect, Grass Combination) Artificially Raised by Coleoptera of the Coleoptera (eg, Barley Larvae) 1. Cytotoxicity Test

HEp-2 cells (human laryngeal carcinoma epithelial cells) were inoculated into a 96-well cell culture plate, cultured at 37 ° C in a 5% CO ₂ incubator. After the cells were grown into a single layer, the culture solution was removed, washed twice with PBS, and added. 0.1 mL was used to maintain the liquid half-dilution test solution, and 0.1 mL of the maintenance solution was set as a blank control. Continue counter 37 ℃, 5% CO ₂ incubator for 2-5 days, daily cytopathic under a microscope, comprising exfoliated cells in monolayer, rounding, shrinkage, cytoplasmic vacuoles and granules are formed, the recording After the results, the half toxicity concentration (TC ₅₀ ) was calculated.

2. Antiviral test

The HEp-2 cell culture method was the same as above, the culture medium was removed, washed with PBS, and 0.1 mL (1OOTCID ₅₀ /0.1 mL virus) virus suspension and 0.1 mL of the maximum cell non-toxic concentration were added (the cells detected under light microscope were not observed). The maximum concentration of the test product that produces toxicity is the highest concentration of the test solution after the half-dilution, and the maintenance solution is used as a blank control. Cell counter 37 ℃, 5% CO ₂ incubator for 2-5 days. The pathological changes caused by the virus were detected under the microscope and recorded. Ribavirin was used as a positive control drug.

Calculate the median inhibitory concentration (EC ₅₀₎ and a therapeutic index TI = TC ₅₀ / EC _50.

3. Anti-respiratory syncytial virus activity of barley worm Cordyceps sinensis extract

Barley Cordyceps sinensis extract: Take the barley worm, Taiwan Cordyceps (worm, grass combination), extract it with 80% ethanol, and spin to obtain a paste, which is the barley worm and Taiwan Cordyceps extract.

The specific results are shown in Table 3.

It is the same from the above experimental results. From the above experimental results, it can be seen that the Cordyceps extract of Taiwan has a significant anti-respiratory syncytial virus (RSV) effect. Can be used in the development and application of pharmaceuticals.

Claims (8)

A use of a Cordyceps formosana Kobayasi & Shimizu extract for the preparation of a drug against respiratory syncytial virus (RSV). The use according to claim 1, wherein the Cordyceps sinensis is an artificially fermented mycelium of Cordyceps militaris, a larvae artificially grown by an artificial medium, or artificially bred by a larva of a coleoptera. The use according to claim 1, wherein the Cordyceps extract of Taiwan is extracted with ethanol in an amount of 10% to 90%. The use according to claim 1, wherein the Cordyceps extract of Taiwan is extracted with ethanol in an amount of 20% to 80%. The use according to claim 2, wherein the Cordyceps extract of Taiwan is extracted with ethanol in an amount of 10% to 90%. The use according to claim 2, wherein the Cordyceps extract of Taiwan is extracted with ethanol in an amount of 20% to 80%. The use according to claim 1, wherein the anti-respiratory syncytial virus drug is a capsule, a tablet, a granule, a gel, a sustained release or an oral solution. The use according to any one of claims 1 to 7, wherein the anti-respiratory syncytial drug is administered at a dose ranging from 1.5 μg/mL to 50.0 μg/mL.