TWI384991B - Treatment of heatstroke with products obtained from cultivation of paecilomyces farinosus g30801 - Google Patents

Description

Treatment of a hot stroke with a product obtained from the cultivation of Paecilomyces farinosus G30801

The present invention relates to the medical use of Paecilomyces farinosus G30801 for the treatment of heat strokes. In particular, the present invention relates to the treatment of a hot stroke using a water-extracted product made from a culture of Penicillium chrysogenum G30801, which has been deposited. The accession number BCRC 930108 is deposited at the Biosource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) (300 Food Road, Hsinchu City, 300). Taiwan).

In recent years, due to the impact of global warming, the world has suffered from heat waves. According to 2003 data, heat waves have deprived more than 36,600 people of their lives (S. Chatterjee et al. (2005), Shock, 24: 341-347). This shows that the extreme heat affects humans so much, and the heat-induced heatstroke is the most serious and urgent, and its prognosis is the worst.

Hot stroke is also known as sunstroke, which results from prolonged exposure to hot and humid conditions. Because human beings are warm-blooded animals, if they are in a hot and humid environment for a long time, the body temperature will rise, and the body can withstand the thermal pressure and temperature change threshold is quite limited, so when the body temperature exceeds the body can tolerate the maximum compensation At the limit, it will cause a variety of systemic pathological reactions (for example, inflammatory reactions, free radicals, etc.), which may cause multiple organ failure or injury, dysfunction of tissue or endocrine hormone system, and even life-threatening. Symptoms of heat stroke include, for example, hypothermia, loss of perspiration, weakness, headache, nausea, vomiting, weakness, elevated body temperature (normal body temperature will exceed 41 ° C), skin flushing and dryness, confusion, dizziness , photophobia, lower blood pressure, rapid and weak pulse, etc., severe cases may fall into a semi-conscious state.

Because the pathological conditions and symptoms of hot stroke are quite complex, the detailed pathological mechanism for causing a hot stroke has not been known so far. It has been reported that whole-body hyperthermia causes DNA fragmentation and increases the level of p53 and caspase-3 activity leading to cells. Apoptosis (S. Chatterjee et al. (2005), supra). In addition, hot stroke can cause tumor necrosis factor-alpha (TNF-α) and interleukin-1β (interleukin) in the central nervous system and peripheral blood stream. -1 beta, IL-1β) [known to be overproduction of proinflammatory cytokines], and excessive production of these cytokines and arterial hypotension, Cerebral ischemia and neuronal damage and high mortality rate are associated (SH Chen et al. (2007), Shock, 27: 663-71). It has also been reported that interleukin-10 (IL-10) [a known anti-inflammatory cytokine] inhibits the production of TNF-α and IL-1β. Therefore, arterial hypotension and cerebral ischemia and injury can be improved by increasing the production of IL-10 (SH Chen et al. (2007), supra; and CP Chang et al. (2007), Neuropharmacology, 52: 1024). -33).

In addition to changes in the expression of cytokines, thermal stroke can also cause cerebrovascular dysfunction, for example, increased intracranial pressure, decreased cerebral blood flows, and cerebral cerebral pressure (cerebral) Perfusion pressure), arterial hypotension, and cerebral ischemia. When the experimental animals are induced to have a hot stroke, there is a significant decrease in blood pressure and an increase in intracranial pressure, and diminished cerebral perfusion causes cerebral ischemia and hypoxia (MT Lin et Al. (1992), Experimentia, 48: 225-227; and CJ Shih et al. (1984), Journal of Neurosurgery, 60: 1246-1252).

It has also been reported in the literature that cerebral ischemia causes a large number of monoamine neurotransmitters to be released (MT Lin et al. (1995), American Journal of Physiology, 269: H487-H490; TY Kao and MT. Lin (1996), Journal of Applied Physiology, 80: 680-684; and YL Yang et al. (1998), Brain Research, 795: 121-127), nitric oxide metabolites and free radicals ( Free radicals are produced (SH Hsiao et al. (2007), Resuscitation, 73:437-45; and CY Yang and MT Lin (2002), Stroke, 33:790-794), causing brain neuronal damage (cerebral Neuronal damage). Therefore, symptoms such as central nervous abnormality and circulatory dysfunction similar to humans may occur in experimental animals that are induced to have a hot stroke, which may be the main cause of death of experimental animals caused by hot stroke (MT) Lin et al. (1992), supra; CH Hung et al. (2005), Shock, 23: 426-433; and CJ Shih et al. (1984), supra).

At present, there is no specific medicine for hot stroke in the clinic. Only the cooling treatment is given immediately to reduce the rectal temperature (<39.4 °C) and the skin temperature (30-33 °C), and maintain the airway patency. To maintain blood with sufficient oxygenation, and to maintain arterial blood pressure above 60mmHg to maintain adequate blood perfusion in the organ, and to prevent kidney failure and arrhythmia.

Supportive therapy followed by a cooling treatment can prevent problems with organ failure and help restore organ function, but patients who survive these treatments may still have permanent neurological damage sequelae. Therefore, there is a need in the medical community today to develop useful drugs for the treatment of hot strokes.

An entomopathogenic fungi [or entomogenous fungi] is an insect-infested fungus that has high host specificity for insects and does not infect insects. Animal or plant. The path of infection by entomopathogenic fungi is mainly attached to the external body surface of the insect by microscopic spores. Under appropriate temperature and moisture (usually high humidity), spores present on the surface of the outer shell of the insect germinate to form hyphae, which will penetrate the insect. The cuticle reaches the body cavity of the insect [ie, the hemocoel]. The invading hyphae utilize the nutrients in the insects for growth and multiplication, which in turn destroys the tissues and organs of the insects and causes insects to die.

The widely known use of entomopathogenic fungi is for use as agricultural biological insecticides. In addition, certain entomopathogenic fungi have been found to have great developmental value for medical use, and among the more common and/or important entomopathogenic fungi including Paecilomyces farinosus , which belong to the ascomycete Ascomycetes, Eurotiales, Trichocomaceae, Paecilomyces .

Paecilomyces fuliginea [another species called Isaria farinosa] has a high degree of insecticidal virulence, which is mainly parasitic on Lepidoptera, Coleoptera, and membranous wings. Hymenoptera insect larvae. It has been reported that polysaccharides or bioactive metabolites isolated from fermentation cultures of Penicillium chrysogenum have significant anti-tumor and anti-oxidant effects (G. Lang et al. (2005), J. Nat Prod., 68: 810-811; YH Jiang et al. (2005), Journal of Food Biochemistry, 29: 323-335; and YH Jiang et al. (2008), Microbiological Research, 163: 424-430).

Master's thesis by Zhang Yuren at the Institute of Biotechnology (Southern Taiwan University) of Nantai University of Science and Technology [Name: "Effect of the bioactive metabolites of Cordyceps sp. fungus In the cancer cell)"], Zhang Yuren uses a liquid medium containing different carbon sources and a nitrogen source to carry out fermentation culture of Paecilomyces pulveris, and investigates the obtained fermentation broth of Penicillium chrysogenum The ability to inhibit the growth of the following cancer cells: human lung adenocarcinoma cells A549, human cervical epithelioid carcinoma cells HeLa, human hepatoblastoma cells HepG2 (human Hepatoblastoma cells HepG2) and human breast adenocarcinoma cells MCF7.

In the Master's thesis by Huang Mei, from the Institute of Biotechnology, Nantai University of Science and Technology [name: "Investigation of the proteome maps and anti-cancer activities of isolated strain of Cordyceps sinensis "] Huang Meiren uses different liquid medium (glucose, sucrose, fructose or maltose as carbon sources, respectively) or different solid medium [soybean or black bean as matrix] The fermentation culture of Paecilomyces fuliginea G308-1 was carried out, and the harvested mycelia (mycelia) was subjected to protein profiling. The master's thesis also discusses the water extract liquid or alcohol extract liquid of the mycelium of the genus Paecilomyces gizae obtained by liquid or solid fermentation culture to inhibit the growth of the following cancer cells. The ability: human cervical cancer cells HeLa (human cervical cancer cells HeLa), human breast cancer cells MCF-7 (human breast cancer cells MCF-7), human prostate cancer cells DU-145 (human prostate cancer cells DU-145) Human hepatoma cell HepG2 (human hepatoma cells HepG2).

Master's thesis by Xiong Qihui, Institute of Biotechnology, Nantai University of Science and Technology [Name: "Studies on the molecular mechanism of the cell cycle arrest and apoptosis in the inhibition of the growth of human hepatoma cell line HepG2] Human HepG2 cell line by hot-water extracts from Paecilomyces farinosus mycelium)"], Xiong Qihui uses soybean as a substrate to carry out solid-state fermentation culture of mycelium of Penicillium chrysogenum, and to explore the harvested mycelium of Penicillium chrysogenum The molecular and physiological mechanism of Paecilomyces farinosus mycelia water extract (PFW) in inhibiting the growth of human hepatoma cells HepG2. The experimental results show that the water extract of the mycelium of Penicillium chrysogenum causes the cell cycle of HepG2 cells to be arrested in the G0/G1 phase (G0/G1 phase) and activated by HepG2 cells. Caspase-9 and caspase-3 perform a series of intracellular signal molecule transduction, which induces cells Apoptosis.

In a previous study, Li Songtai et al. cultured P. sphaeroides G30801 in solid medium containing rice and explored the hot-water extract (HW) of the harvested mycelium in activating RAW264.7 cells. The utility of the above. The results showed that HW significantly increased the mRNA level and protein expression of IL-1 and IL-6 in RAW264.7 cells, indicating that HW may be in the immune system by activating macrophage. There is an up-regulation activity (ST Lee et al. (2007), The 22th Joint Annual Conference of Biomedical Science: P503, page 107).

Upon investigation, the applicant unexpectedly discovered that in addition to the ability to inhibit tumor/cancer cell growth, the fermentation product of Penicillium chrysogenum G30801 can also have beneficial therapeutic effects on experimental animals that are induced to have a hot stroke. Therefore, the fermentation product of this strain is expected to be available for the treatment of hot stroke.

Summary of invention

Accordingly, the present invention provides a pharmaceutical composition for treating a hot stroke, comprising a product obtained by cultivating Paciilomyces farinosus G30801, wherein the powder is prepared Penicillium sp. G30801 is deposited with the Bioresource Conservation and Research Center of the Food Industry Development Institute under the accession number BCRC 930108.

The invention also provides a method of treating an individual having or suspected of having a hot stroke comprising administering to the individual a product obtained by culturing P. pseudomonas G30801.

The above and other objects, features and advantages of the present invention will become apparent from

Detailed description of the invention

For the purposes of this specification, it will be clearly understood that the words "comprising" means "including but not limited to" and the words "comprises" have a corresponding meaning.

It is to be understood that if any of the previous publications is quoted here, the prior publication does not constitute an acknowledgement that in Taiwan or any other country, the former publication forms a common general in the art. Part of the knowledge.

All technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains, unless otherwise defined.

In developing drugs that can be used to treat hot strokes, Applicants have discovered that Paecilomyces farinosus G30801 has industrial application potential in this regard. Thus, the present invention discloses the use of Paecilomyces fuliginea G30801 for the preparation of a medicament for the treatment of hot stroke.

Paecilomyces fuliginea G30801 was deposited at the Biosource Collection and Research Center of the Food Industry Research and Development Institute (FIRDI) on January 22, 2008 under the registration number BCRC 930108. , BCRC) (300 Food Road, Hsinchu City, Taiwan).

According to the present invention, the cultivation of Penicillium fuliginea G30801 is carried out by introducing the mycelial inoculum of one of the Penicillium sp. G30801 to a solid culture.

As used herein, the terms "cultivation," "culturing," and "fermentation" are used interchangeably. Further, the terms "solid cultures", "solid fermented cultures", and "fermented cultures" may be used interchangeably.

According to the present invention, when solid state culture is used to grow Penicillium chrysogenum G30801, it can be carried out using a solid medium containing at least one of the following: rice, soybean, black bean, wheat ( Wheat, barley, rye, buckwheat, oat, corn, job's tears, yam, potato, sweet potato , cassava, taro, arrowroot, lotus plants, elephant foot, edible canna, Canada potato, sorghum, silkworm cocoon (silkworm chrysalis), wheat bran, and rice bran.

In a preferred embodiment of the invention, the solid state culture is carried out using a solid medium comprising at least one of: rice, soybeans, and black beans. In a more preferred embodiment of the invention, the solid state culture is carried out using a solid medium containing rice.

According to the present invention, the solid medium may further comprise a nitrogen source selected from the group consisting of (NH ₄ ) ₂ SO ₄ , (NH ₄ ) ₃ PO ₄ , NH ₄ NO ₃ , NH ₄ Cl, Casein acid, urea, peptone, tryptone, meat extract, yeast extract, yeast powder, yeast powder, yeast powder, yeast powder, yeast powder, yeast powder, yeast powder, yeast powder, yeast powder, yeast powder, yeast powder, yeast powder, yeast powder, yeast powder, yeast powder And their combination. In a preferred embodiment of the invention, the solid medium further comprises peptone.

According to the present invention, the solid medium may further comprise an inorganic salt selected from the group consisting of K ₂ HPO ₄ , KH ₂ PO ₄ , MgSO ₄ , CaCl ₂ , NaCl, MnSO ₄ , FeCl ₃ , NaHCO. ₃ , MgCl ₂ , FeSO ₄ , and combinations thereof. In a preferred embodiment of the invention, the solid medium further comprises KH ₂ PO ₄ .

Further, according to the present invention, the solid medium can also be formulated into an agar medium containing nutrients required for the growth of Paecilomyces variabilis, and the preparation of the agar medium is based on the professional literacy of those skilled in the art. Within the scope of routine technology. For example, the agar medium can be prepared using a carbon source, a nitrogen source, and an inorganic salt which will be described below.

According to the present invention, carbon sources suitable for formulating the agar medium include, but are not limited to, glucose, fructose, lactose, sucrose, maltose, galactose, Mannose, trehalose, starch, molasses, potato starch, corn starch, malt extract, and the like. In a preferred embodiment of the invention, the agar medium contains potato starch.

According to the present invention, a nitrogen source suitable for use in formulating the agar medium includes, but is not limited to, (NH ₄ ) ₂ SO ₄ , (NH ₄ ) ₃ PO ₄ , NH ₄ NO ₃ , NH ₄ Cl, casein amino acid, Urea, peptone, tryptone, meat extract, yeast extract, yeast powder, and the like. In a preferred embodiment of the invention, the agar medium contains a yeast extract.

According to the present invention, the agar medium may further comprise an inorganic salt selected from the group consisting of K ₂ HPO ₄ , KH ₂ PO ₄ , MgSO ₄ , CaCl ₂ , NaCl, MnSO ₄ , FeCl ₃ , NaHCO ₃ , MgCl _{2 .} , FeSO ₄ , and combinations thereof. In a preferred embodiment of the invention, the agar medium further comprises MgSO ₄ and KH ₂ PO ₄ .

According to the present invention, the agar medium may further contain a natural substance including, but not limited to, soybean flour, black bean meal, wheat meal, barley meal, Rye flour, buckwheat flour, oat meal, corn flour, job's tears flour, yam flour, potato flour , sweet potato flour, cassava flour, taro flour, bean cake, bean meal, soy protein extract, peanut cake, corn flour (maize flour), rice bran, vinasse, silkworm pupa meal, fish meal, milk powder, yeast powder, and corn steep liquor and many more.

According to the present invention, the product obtained after the cultivation can be used to prepare a medicament for treating a hot stroke. In a preferred embodiment of the present invention, the culture formed after the cultivation of the P. solani G30801 in a solid state culture is subjected to a water-extraction treatment to obtain a water-extracted product.

According to the present invention, the water extraction treatment is such that the culture of the Paecilomyces fuliginea G30801 is mixed with water at an appropriate ratio for a period of time, followed by centrifugation to obtain an aqueous supernatant. Preferably, the ratio of the culture of the Paecilomyces fuliginea G30801 to water is 1:1 to 1:50, more preferably 1:10. The aqueous supernatant can be further dehydrated to form a dried powder.

Applicants have discovered that when an experimental animal in which a hot stroke is induced is administered a water-extracted product of a culture of Paecilomyces fuliginea G30801 according to the present invention, their symptoms of heat stroke include cerebrovascular dysfunction (eg, intracranial pressure) Increased, decreased cerebral blood flow, decreased cerebral perfusion pressure, arterial hypotension, and cerebral ischemia, systemic inflammatory response, oxidative stress, and multiple organ damage, etc., have all been significantly improved.

Accordingly, the present invention provides a pharmaceutical composition for treating a hot stroke comprising a product obtained by the cultivation of Penicillium chrysogenum G30801. In a preferred embodiment of the invention, the product comprises a culture formed after the solid state culture. In another preferred embodiment of the invention, the product comprises a water-extracted product obtained by extracting the culture formed after the solid state culture with water.

The pharmaceutical composition according to the present invention may be administered by a parenteral route selected from the group consisting of: intraperitoneal injection, subcutaneous injection, intramuscular injection. And intravenous injection.

In a preferred embodiment of the invention, the pharmaceutical composition is formulated for administration by intravenous injection.

In another preferred embodiment of the invention, the pharmaceutical composition is formulated to be administered by intraperitoneal injection.

In still another preferred embodiment of the invention, the pharmaceutical composition is formulated into a dosage form suitable for oral administration.

The pharmaceutical composition according to the present invention may further comprise a pharmaceutically acceptable carrier which is widely used in pharmaceutical manufacturing techniques. For example, the pharmaceutically acceptable carrier can comprise one or more agents selected from the group consisting of solvents, emulsifiers, suspending agents, decomposers, binders, excipients, stabilizers, chelating agents, diluents, gums Coagulants, preservatives, lubricants, absorption delaying agents, liposomes, and the like.

In a preferred embodiment of the present invention, the pharmaceutical composition comprises a pharmaceutically acceptable solvent, and the solvent is any one of the following: water, physiological saline, phosphate buffered physiological saline, sugar-containing solution, and An aqueous solution of alcohol. In a preferred embodiment of the invention, the solvent is water.

The invention also provides a method of treating an individual having or suspected of having a hot stroke comprising administering to the individual a product obtained by culturing P. pseudomonas G30801. In a preferred embodiment of the invention, the product comprises a culture formed after the solid state culture. In another preferred embodiment of the invention, the product comprises a water-extracted product obtained by extracting the culture formed after the solid state culture with water.

According to the present invention, the dosage and the number of administrations will vary depending on the severity of the disease to be treated, the route of administration, and the weight, age, physical condition and response of the individual to be treated. In general, the daily dose of the pharmaceutical composition according to the present invention is usually from 0.3 mg/kg body weight to 1.2 mg/kg body weight, in a single dose or divided into several doses, and can be parenterally Or oral administration.

Detailed description of the preferred embodiment

The invention is further described in the following examples, but it should be understood that these examples are for illustrative purposes only and are not to be construed as limiting.

Example Experimental Materials: A, experimental animals:

Male Sprague-Dawley (SD) rats (body weights approximately 300 to 350 g) and male ICR mice (body weights approximately 30 to 35 g) used in the following examples were obtained from the National Experimental Research Institute experimental animals. The center is the Experimental Animal Center of the National Taiwan University Medical College. All experimental animals were housed in an independent air-conditioned animal room with light and dark for 12 hours, room temperature maintained at 24 ± 1 ° C, and relative humidity maintained at 50-70%, and water and feed were adequately supplied. The breeding environment and experimental procedures of the experimental animals are in line with the international laboratory animal management standards.

General experimental method: A. Monitoring of physiological parameters of rats:

In the following examples, the applicant selected four physiological parameters [ie, blood pressure (BP), mean arterial pressure (MAP), heart rate (HR), and rectal temperature). The rats were monitored, wherein monitoring of blood pressure, mean arterial pressure, and heart rate of the rats was performed in accordance with the procedure described below.

First, rats were anesthetized by intraperitoneal injection (dose of 1.4 g/kg) of urethane (Sigma Chemical Co.) formulated in 0.9% physiological saline to the rats. Thereafter, a PE-50 polyethylene tube (PE prepared with 0.9% physiological saline and a heparin concentration of 30 U/mL) was inserted into the femoral artery and the femoral vein of the anesthetized rats, respectively. -50 polyethylene tube, Becton Dickinson), which was then fixed with a 4/0 surgical suture.

The other end of the PE-50 polyethylene tube inserted into the femoral artery was connected to a Capto SP844 Physiological Pressure Transducer (Memscap AS), which was further connected to a four-channel A physiological multi-function recorder (PowerLab/8 SP, ADInstruments) of a bridge amplifier (Quad Bridge Amp, AD Instruments) was used to monitor blood pressure, mean arterial pressure, and heart rate in rats. As for the PE-50 polyethylene tube inserted into the femoral vein, the other end thereof was connected with a syringe for the intravenous injection in the following examples.

In addition, the rectal temperature of the rat was monitored by inserting a copper-constantan thermocouple wire connected to a thermometer (HR1300, Yokogawa, Japan) from the anus of the rat into the rectum. .

B, Induction of heatstroke:

The induction of heat stroke in rats was carried out in accordance with the method described in JR Kuo et al. (2003), Intensive Care Medicine, 29(9): 1567-73, with minor modifications. Briefly, the body of the rat (physiological parameter is monitored) is coated in a water-fillable heating pad, and the gap between the front and rear ends of the heating pad is plugged with a toilet paper to expose only the head of the rat. unit. The heating pad stabilizes the body temperature of the rat (which is expressed as anal temperature) by communicating with a constant temperature water bath having a temperature of 24 °C. After the anus temperature of the rat was maintained at 36.5 ± 1 ° C for 30 minutes, the water in the constant temperature water bath was heated and maintained at 43 ° C, so that the rat was exposed to an ambient temperature of 43 ± 0.5 ° C. (ambient temperature, T _a ). The rats were allowed to observe the physiological parameters of the rats at this ambient temperature for a period of time. When the physiological parameters of the rats met the following conditions, it indicated that the hot stroke was induced:

(1) The mean arterial pressure is rapidly reduced and reduced by 25 mmHg;

(2) The anus temperature is higher than 42 °C.

In addition, the induction of hot stroke in mice was carried out in accordance with the method described in S. Chatterjee et al. (2005) (supra). Briefly, hot stroke was induced by placing the mouse in an oven set at 41.2 ° C and a relative humidity set at 50 to 55% for 1 hour.

C, statistical analysis (Statistical analysis):

The experimental data obtained in the following examples are all expressed as "standard error of mean (SEM)". All experimental data were first analyzed by repeated sampling of variance analysis (ANOVA) to confirm whether there was a difference in the experimental group, and then the Duncan's multiple range test was used to analyze the two experimental groups. The difference between the two. If the statistical analysis obtained is p < 0.05, this indicates statistical significance.

Example 1 Preparation of water-extracted products of rice fermented culture of rice fermentation culture of Penicillium chrysogenum G30801 (Preparation of water-extracted products of rice fermented culture of Paecilomyces farinosus G30801): A. Preparation of mycelium inoculum of Penicillium sp. G30801:

Penicillium chrysogenum G30801 was inoculated into a potato dextrose broth (PDB) (Himed, M096) and cultured in a constant temperature shaking incubator (17 ° C, 100 rpm) for 7 days. The resulting culture was used as a mycelial inoculum of Penicillium chrysogenum G30801.

B. Preparation of rice fermentation culture of Penicillium sp. G30801:

300 g of rice was taken and 300 mL of an aqueous solution containing 2% peptone and 0.1% KH ₂ PO ₄ was added, followed by cooking at 100 ° C to obtain a rice medium.

The obtained rice medium was placed in a 1000 mL wide mouth plastic bottle, and sterilized at 121 ° C and 1.5 atmospheres for 30 minutes. When the temperature of the wide-mouth plastic bottle was lowered to room temperature, 10 mL of the mycelium inoculum of Paecilomyces militaris G30801 was inoculated to the rice medium in the wide-mouth plastic bottle. After 20 days of static fermentation at 17 ° C, a rice fermented culture was obtained.

C. Preparation of water-extracted product of rice fermentation culture of Penicillium sp. G30801:

The resulting rice fermentation culture was freeze-dried and ground into a powder. 1 g of rice fermentation culture powder was added to 10 mL of sterile water having a temperature of 65 ° C, and stirred and mixed to form a mixture (in which the mixing ratio of the rice fermentation culture powder and the sterile water was 1:10), and then The mixture was maintained at a temperature of 65 ° C and shaken for 2 hours for water extraction. Thereafter, the mixture was centrifuged at 10,000 rpm (model CF16RX, Hitachi) at a temperature of about 25 ° C for 10 minutes to obtain a supernatant. The supernatant was vacuum dried at 45 ° C to obtain a water-extracted product of a rice fermentation culture of Penicillium sp. G30801 as a dried powder.

The thus obtained water-extracted product was dissolved in sterile water to form a solution A having a concentration of 0.6 mg/mL. In the animal experiments of the following examples, the experimental group of animals to which this solution A was induced to induce thermal stroke was referred to as "rice fermentation culture group".

Further, the rice medium was directly taken and subjected to the same water extraction treatment, and the thus obtained water-extracted product of the rice medium was formulated in the same manner as a solution B having a concentration of 0.6 mg/mL. In the animal experiments of the following examples, the experimental group of animals to which this solution B was administered was induced to be called "rice medium group".

In addition, in the animal experiments of the following examples, the animal experimental group at 24 ° C where no hot stroke was induced and which was not given any of the above solutions was taken as a "normal temperature control group".

Example 2. Effect of water-extracted product of rice fermentation culture of Penicillium chrysogenum G30801 on survival time of rats induced to have a heat stroke experimental method:

Male Sprague-Dawley rats were randomly divided into 2 groups (ie, rice fermentation culture group and rice medium group, each group n=8), and according to the "General Laboratory Method" above, "A, Monitoring of Physiological Parameters" Methods for anesthesia and monitoring of physiological parameters. Thereafter, heat stroke was induced in each group of rats in accordance with the method described in "B, Induction of Hot Stroke" of "General Experimental Method" above.

When hot stroke was induced, each group of rats was immediately moved to an environment at a temperature of 24 ° C, and then the rats in the rice fermentation culture group were intravenously injected with the solution A prepared in Example 1 (containing The water-extracted product of the rice fermentation culture of Penicillium chrysogenum G30801 was dosed at 0.6 mg/kg), and the rats of the rice culture group were intravenously injected with the solution B prepared in Example 1 (containing rice). The water-extracted product of the medium was dosed at 0.6 mg/kg), and then the changes in physiological parameters and the difference in survival time (ST) between the two groups of rats were observed. The survival time of the rats was calculated based on the time interval from the induction of heat stroke to death (respiratory arrest, mean arterial pressure, and heart rate was reduced to zero), and according to the "general experimental method" above. The method described in "C, Statistical Analysis" analyzes the experimental data obtained.

result:

When the rats were exposed to a temperature of 43 ° C, the anus temperature and mean arterial pressure (MAP) of the rats continued to increase with the increase of exposure time, they were from 36.5 ± 0.2 ° C and 90.5 ± 1.95 respectively. The mmHg is increased to approximately 43.4 ± 0.05 ° C and 121.5 ± 6.85 mm Hg. When the exposure time was about 70 minutes, the mean arterial pressure of the rats began to decrease rapidly, indicating that a hot stroke was induced. At the 15th minute after the hot stroke was induced, the mean arterial pressure had decreased to 48.0 ± 6.22 mmHg, but the anus temperature was still at a high temperature (greater than 42 ° C).

The difference in survival time between the rice medium group and the rice fermentation culture group was as shown in Fig. 1. The survival time of the rice medium group and the rice fermentation culture group was about 23.5 ± 1.52 minutes and about 221, respectively. ±37.5 minutes. This result shows that the water-extracted product of the rice fermentation culture of Penicillium chrysogenum G30801 according to the present invention can significantly prolong the survival time of the rats in which hot stroke is induced.

Example 3. Effect of water-extracted product of rice fermentation culture of Penicillium chrysogenum G30801 on cerebrovascular dysfunction in rats induced to have a heat stroke experimental method:

Male SD rats were randomly divided into 3 groups (ie, rice fermentation culture group, rice medium group, and normal temperature control group, each group n=8), and according to the "general experimental method" above, "A, physiological parameters Monitoring the methods described for anesthesia and monitoring of physiological parameters. After that, each group of rats was subjected to the following monitoring:

A, rat cerebral temperature (T _c ), cerebral blood flow (CBF) and cerebral oxygen pressure (PO ₂ ) monitoring:

The head and ear portions of the rat were fixed on a Stereotaxic frame (Kopf model 1406, David Kopf Instruments), and the scalp of the rat was incised with a scalpel to expose the skull. Next, use a fine electric drill (NE120, Nskvolvere Vmax) to drill a hole 0.8mm [that is, on the sagittal suture] and move it to the left side 4mm to the left side (bregma), then carefully with a tweezers Pick up the meninges to avoid massive bleeding. Then, with reference to Paxinos and Watson's map (G. Paxinos and C. Watson (1982), The rat brain in stereotaxic coordinates, New York, Academic Press), one for detecting brain temperature, cerebral blood flow, and brain oxygen A laser Doppler Probe (OxyFlo 2000, Oxford Optronix Ltd., Oxford, UK) was inserted into the striatum (Corpus striatum) of the rat brain (AP: -0.8 mm, L: +4mm, DV: -4mm). The probe was coupled to a cerebral blood flow recorder (OxyFlo monitor, Oxford Optronix Ltd.) by an optical fiber. When the cerebral blood flow of the rats is in a stable state [that is, the cerebral blood flow does not change more than 10 blood perfusion units (BPU)] for 1 hour, the brain temperature and cerebral blood are continuously recorded. Flow and cerebral oxygen partial pressure.

B. Monitoring of intracranial pressure (ICP) in rats:

Rats in each group were additionally monitored for intracranial pressure according to the following procedure: using a drill to drill a hole 0.8 mm posterior to the anterior iliac crest and 1.5 mm to the right, and then carefully picking up the meninges with forceps. Thereafter, a 24 gauge hypodermic needle (about 7.5 cm long) was attached to the stereotaxic instrument, and the hypodermic needle was inserted from the drilled hole into the lower 3.6 mm of the brain surface (ie, the lateral ventricle of the rat). AP: -0.8mm, L: +1.5mm, DV: -3.6mm), the end of the hypodermic needle is connected with a PE-50 polyethylene tube filled with physiological saline, by observing the PE-50 polyethylene tube The level of saline is used to determine if the insertion position is correct. When the liquid level of the physiological saline in the tube cannot be lowered, the position of the hypodermic needle is slightly adjusted up and down until the liquid level can be smoothly lowered. Next, the PE-50 polyethylene tube was attached to a physiological multifunction recorder (PowerLab/8SP, AD Instruments). After the rat's blood pressure was stable for 0.5 hours, the intracranial pressure of the rats was recorded.

C, the induction of hot stroke:

In addition to the rats in the normal temperature control group, after the brain temperature, cerebral blood flow, cerebral oxygen partial pressure, and intracranial pressure were recorded for 40 minutes, according to the "general experimental method" above, "B, induction of hot stroke" The method described herein induces a heat stroke to the remaining groups of rats. When hot stroke was induced, the rats were immediately moved to a temperature of 24 ° C, and then the rats in the rice fermentation culture group were intravenously injected with the solution A prepared in Example 1 (containing The water-extracted product of the rice fermentation culture of Penicillium chrysogenum G30801 was dosed at 0.6 mg/kg), and the rats of the rice culture group were intravenously injected with the solution B prepared in Example 1 (containing rice). The water-extracted product of the medium was dosed at 0.6 mg/kg.

During the above operation, brain temperature, cerebral blood flow, cerebral oxygen partial pressure, and intracranial pressure of each group of rats were continuously recorded. The cerebral blood flow is expressed as a percentage of the measured value relative to the reference value, where the reference value is the average of the values of the cerebral blood flow measured at the time of the steady state of the rat. Cerebral perfusion pressure is obtained by subtracting the intracranial pressure from the mean arterial pressure of the rat. The obtained experimental data was analyzed in accordance with the method described in "C, Statistical Analysis" of "General Experimental Method" above.

result:

2A to 2C show changes in cerebral blood flow, cerebral oxygen partial pressure, and brain temperature with time in each group of rats in the present example. As can be seen from Fig. 2A to Fig. 2C, the parameters of the rats in the normal temperature control group were maintained at a stable value throughout the experiment, and there was no statistical difference. The cerebral blood flow, cerebral oxygen partial pressure and brain temperature of the rats in the rice medium group gradually increased with the increase of the ambient temperature, and the cerebral blood flow, the cerebral oxygen partial pressure and the brain temperature were respectively from the steady state reference values (in terms of Increased by 100%, 14±1.4 mmHg, and 36.0±0.1°C to 139±22.4%, 19±3.6 mmHg, and 42.2±0.3°C of the reference value. However, at the 20th minute after the hot stroke was induced, the cerebral blood flow and cerebral oxygen partial pressure of the rats in the rice culture group were reduced to 7.4% of the reference value and 1.6 mmHg, respectively, while the brain temperature was maintained at 42 ± 0.5 °C. .

As for the rats in the rice fermentation culture group, their cerebral blood flow, cerebral oxygen partial pressure, and brain temperature were not significantly different from those in the rice culture group before the hot stroke was induced, but were induced in the hot stroke. After the 20th minute, in addition to the brain temperature, the cerebral blood flow (100% of the reference value) and the cerebral oxygen partial pressure (16.3 mmHg) of the rats in the rice fermentation culture group and the rats of the rice culture group were included. Both were statistically significant ( p < 0.05).

In addition, FIGS. 3A to 3C respectively show changes in mean arterial pressure, intracranial pressure, and cerebral perfusion pressure with time in each group of rats in the present example. As can be seen from Figs. 3A to 3C, the respective monitoring data of the rats of the normal temperature control group were maintained at a stable value throughout the experiment, and there was no statistical difference. The mean arterial pressure, intracranial pressure and cerebral perfusion pressure of the rats in the rice medium group gradually increased with the increase of the ambient temperature, and the mean arterial pressure, intracranial pressure and cerebral perfusion pressure were 90±2.5mmHg and 6.9±, respectively. 0.6 mmHg and 80±2.7 mmHg were increased to 149±7.4 mmHg, 11.9±0.8 mmHg, and 136±8.2 mmHg. However, when hot stroke was induced, the mean arterial pressure of the rats in the rice medium group rapidly decreased (from 149 ± 7.4 mmHg to 124 ± 7 mmHg). At the 20th minute after the hot stroke was induced, the mean arterial pressure and cerebral perfusion pressure of the rats in the rice medium group were reduced to 6.8 mmHg and 0 mmHg, respectively, but the intracranial pressure continued to increase to 17 mmHg, while the anus temperature remained at 42.5±. 0.1 ° C until the rat died.

As for the rats in the rice fermentation culture group, their respective monitoring data were not significantly different from those in the rice culture group before the hot stroke was induced, but in the 20th minute after the hot stroke was induced, The mean arterial pressure, intracranial pressure, and cerebral perfusion pressure were maintained at 43.8 mmHg, 9.6 mmHg, and 35.6 mmHg, respectively. These results were statistically significant compared with the rats in the rice medium group ( p <0.05).

The above results show that the water-extracted product of the rice fermentation culture of Paecilomyces fuliginea G30801 according to the present invention can effectively improve the phenomenon of cerebrovascular dysfunction in rats induced by hot stroke.

Example 4. Water-extracted product of rice fermentation culture of Penicillium chrysogenum G30801 Extracellular ischemia and damage and oxidative stress in the brain of rats induced to have a heat stroke )Impact

In this example, extracellular ischemia and injury are the concentration of glutamate and glycerol in the cerebral dialysate of the rat striatum and lactate versus pyruvate ( The concentration ratio of pyruvate is used as an evaluation index, and the oxidative pressure is used as an evaluation index of the content of nitric oxide (NO) and hydroxyl radical in the cerebral dialysate.

experimental method: A. Collection of cerebral dialysate in rats:

Male SD rats were randomly divided into 3 groups (ie, rice fermentation culture group, rice medium group, and normal temperature control group, each group n=8), and according to the "general experimental method" above, "A, physiological parameters Monitoring the methods described for anesthesia and monitoring of physiological parameters. In addition, microdialysis probes were placed on each group of rats according to the following procedure: the meninges of the rats were picked up in the manner described in section A of Example 3 above, and one was used for collection. A microdialysis probe (CMA/12, Carnegie Medicine, Stockholm, Sweden) of cerebral dialysate was inserted into the striatum of the rat brain (AP: -0.8 mm, L: +4 mm, DV: -5.2mm). Next, using a Microinjection pump (CMA100, Carnegie Medicine, Stockholm, Sweden), an artificial cerebrospinal fluid (aCSF) (containing 149 mM chlorine) at a flow rate of 1.2 μL/min. Sodium, 2.8 mM potassium hydride, 1.2 mM calcium chloride, 1.2 mM magnesium chloride, 0.25 mM ascorbic acid, and 5.4 mM glucose, pH 7.2-7.4) were injected into the inlet of the microdialysis probe. Thereafter, using a CMA 140 fraction collector (CMA140 fraction collector, Carnegie Medicine, Stockholm, Sweden) at a time interval of 20 minutes and a flow rate of 1.2 μL/min from the outlet of the microdialysis probe (outlet The rat brain dialysate was collected into Appendorff tubes (24 μL/tube).

B. Induction of hot stroke:

In addition to the rats in the normal temperature control group, after the cerebral dialysate was collected for 40 minutes, the rats in the other groups were induced according to the method described in "B, Hot Stroke Induction" of the "General Experimental Method" above. Hot stroke. When hot stroke was induced, the rats were immediately moved to a temperature of 24 ° C, and then the rats in the rice fermentation culture group were intravenously injected with the solution A prepared in Example 1 (containing The water-extracted product of the rice fermentation culture of Penicillium chrysogenum G30801 was dosed at 0.6 mg/kg), and the rats of the rice culture group were intravenously injected with the solution B prepared in Example 1 (containing rice). The water-extracted product of the medium was dosed at 0.6 mg/kg.

During the induction of hot stroke, the cerebral dialysate of each group of rats was continuously collected until the 70th minute after the hot stroke was induced.

C. Analysis of indicator substances in brain dialysate:

The cerebral dialysate collected above was subjected to the following analysis:

1. Determination of the concentration of glutamate, glycerol, pyruvate and lactate:

The concentrations of glutamine, glycerol, pyruvic acid, and lactic acid in rat brain dialysate were determined by using a CMA600 Microdialysis Analyser (CMA Microdialysis Inc.) and referring to the manufacturer's instructions. Data reading.

2. Determination of nitric oxide (NO) content:

For the determination of nitric oxide content, see CP Chang et al. (2007), Neuropharmacology, 52: 1024-1033. Briefly, the content of nitric oxide is provided by use of a high performance liquid chromatography with a system of NO _x in the analyzer (ENO-20, EICOM corporation) and the operating instructions provided by the manufacturers with reference to the measurement sample NO ₂ ^- content, then the measured NO ₂ ^{- -} and NO ₃ and NO ₃ ^- content is calculated by adding. The expression of one of the contents of nitrogen oxide in the cerebral dialysate is expressed as a percentage value of the calculated content of nitric oxide relative to the reference value, wherein the reference value is within 40 minutes of starting the collection of the cerebral dialysate. The average value of the measured content of nitric oxide.

3. Determination of hydroxyl radical content:

The determination of the content of hydroxyl radicals is as described in CY Yang and MT Lin 2002, Stroke, 33: 790-794, and is equipped with a two-channel electrochemical detector [LC- 4C, Bioanalytical system (BAS), West Lafayette, USA] High performance liquid chromatography (HPLC) [CC-5E, Bioanalytical system (BAS), West Lafayette] for 2,3-dihydroxybenzoic acid (2,3) -DHBA) and 2,5-DHBA determination, the column used and operating conditions are as follows: Analytical column is Alltima reverse phase C ₁₈ column (Alltima reverse-phase C ₁₈ column [BAS, 150X1MM ID, particle size (particle size) 5mm]; mobile phase: 0.1M chloroacetic acid, 26.87 nM disodium EDTA, 688.76 nM sodium octylsulfate and 10% acetamidine nitrate (acetonitrite) (pH 3.0); the flow rate was controlled to 1 mL/min; the retention times of 2,3-DHBA and 2,5-DHBA were 9.07 and 5.44 minutes, respectively. The content of hydroxyl radicals was measured. The content of 2,3-DHBA and 2,5-DHBA obtained is added up and calculated. Hydroxyl groups in brain dialysate The content of the base is expressed as a percentage of the calculated content of hydroxyl radicals relative to the reference value, wherein the reference value is the average of the DHBA content measured during the 40 minutes from the start of collection of the brain dialysate. value.

The experimental data obtained in the above analysis were analyzed in accordance with the method described in "C, Statistical Analysis" of the "General Experimental Method" above.

result:

4A to 4H show physiological parameters of each group of rats and the content of the indicator substance in their cerebral dialysate as a function of time, respectively.

As can be seen from FIGS. 4A to 4H, after the hot stroke was induced, the rats in the rice medium group were intravenously injected with the solution B, the glutamic acid concentration, the glycerol concentration in the cerebral dialysate, and the lactic acid relative to pyruvate. The concentration ratio of the rats in the control group increased with time, and the content of DHBA and NO also increased rapidly with time.

In contrast, after the rats in the rice fermentation culture group were intravenously injected with the solution A, the phenomenon that the index substances in their cerebral dialysate were increased was remarkably improved. This result shows that the water-extracted product of the rice fermentation culture of Penicillium chrysogenum G30801 according to the present invention can effectively improve the extracellular ischemia and damage of the brain of a rat induced to have a hot stroke, and can provide significant Antioxidant effect.

Example 5. Effect of water-extracted product of rice fermentation culture of Penicillium chrysogenum G30801 on the expression of proinflammatory cytokines and anti-inflammatory cytokines caused by hot stroke

In this example, the Applicant evaluated the water-extracted product of the rice fermentation culture of Paecilomyces fuliginea G30801 according to the present invention for measuring the TNF-α and IL-10 concentrations in the rat serum for the hot stroke. The resulting effects of pro-inflammatory cytokines and anti-inflammatory cytokines.

experimental method:

Male SD rats were randomly divided into 3 groups (ie, rice fermentation culture group, rice medium group, and normal temperature control group, each group n=8), and according to the "general experimental method" above, "A, physiological parameters Monitoring the methods described for anesthesia and monitoring of physiological parameters. Thereafter, in addition to the rats of the normal temperature control group, the hot stroke was induced according to the method described in "B, induction of hot stroke" of the "general experimental method" above.

When hot stroke was induced, the rats were immediately moved to a temperature of 24 ° C, and then the rats in the rice fermentation culture group were intravenously injected with the solution A prepared in Example 1 (containing The water-extracted product of the rice fermentation culture of Penicillium chrysogenum G30801 was dosed at 0.6 mg/kg), and the rats of the rice culture group were intravenously injected with the solution B prepared in Example 1 (containing rice). The water-extracted product of the medium was dosed at 0.6 mg/kg.

At the 15th minute after the hot stroke was induced, 5 mL of blood was taken from the femoral artery cannula by using a syringe, and centrifuged at 3,000 rpm for 10 minutes at 25 ° C to obtain a serum sample. . Rat TNF-α kit (Rat TNF-α kit, R&D systems, Cat. No. DY510) and rat IL-10 kit (Rat IL-10 kit, R&D systems, Cat. No. DY522) were used and The manufacturer's instructions are used to measure the concentration of TNF-α and IL-10 in the resulting serum samples.

The obtained experimental data was analyzed in accordance with the method described in "C, Statistical Analysis" of "General Experimental Method" above.

result:

5A and 5B show the concentrations of TNF-α (proinflammatory cytokines) and IL-10 (anti-inflammatory cytokines) in the serum of each group of rats at the 15th minute after the hot stroke was induced, respectively.

As can be seen from Figures 5A and 5B, the concentrations of TNF-α and IL-10 in the serum of the rats in the rice medium group were 621 pg/mL and 75 pg/mL, respectively, when compared with those of the rats in the normal temperature control group. The concentration of TNF-α was significantly increased while the concentration of IL-10 was only increased by a small amount.

In contrast, the concentrations of TNF-α and IL-10 in the serum of the rats in the rice fermentation culture group were 56 pg/mL and 622 pg/mL, respectively, when compared with those of the rats in the rice medium group, TNF- The concentration of α was significantly reduced while the concentration of IL-10 was significantly increased, and these values were statistically significant ( p < 0.01).

The above results show that the water-extracted product of the rice fermentation culture of Penicillium chrysogenum G30801 according to the present invention can inhibit the expression of proinflammatory cytokines (TNF-α) and increase anti-inflammatory in rats induced by hot stroke. The performance of cytokines (IL-10) has a significant anti-inflammatory effect.

Based on the above experimental results, the applicant speculates that treating the rats with induced hot stroke with the water-extracted product of the rice fermentation culture of P. pulcherii G30801 according to the present invention can improve arterial hypotension. And excessive production of cerebral ischemia and oxidative stress, which may be achieved by reducing the expression of pro-inflammatory cytokines and reducing the production of NO to maintain a moderately constant systemic and cerebral blood flow and Improve nerve damage caused by hot strokes.

In the following animal experiments, the applicant was treated with hot pressure to induce a hot stroke in the unanesthetized awake mice, and the solution A prepared in Example 1 (the rice fermentation culture containing Paecilomyces militaris G30801, respectively) The water-extracted product) and solution B (water-extracted product containing rice medium) were used for efficacy evaluation experiments.

Example 6. Effect of the dose of the water-extracted product of the rice fermentation culture of Penicillium chrysogenum G30801 on the survival rate and body temperature of mice induced to have a heat stroke experimental method:

Male ICR mice were randomly divided into 6 groups (n=8 for each group), and a thermal stroke was induced according to the method described in "B, Induction of Hot Stroke" of "General Experimental Method" above. Thereafter, each group of mice that were induced to have a hot stroke was moved to an environment at a temperature of 24 ° C, and each group of mice was intraperitoneally injected at different doses (0.3 mg/kg, 0.6 mg/). Kg and 1.2 mg/kg) of solution A prepared in Example 1 and (water-extracted product of rice fermentation culture containing P. stipitis G30801) and solution B (water-extracted product containing rice medium) . At the same time as the experiment, a copper-constantan thermocouple wire connected to a thermometer (HR1300, Yokogawa, Tokyo, Japan) was inserted into the rectum from the anus of the mouse, and 0, 0.5, 1 after the hot stroke was induced. Their anus temperature was measured at 2, 4, 7, and 24 hours.

Each group of mice was then housed at 24 ° C for 4 days, and the survival rate of each group of mice was calculated at 0, 12, 24, 48, 72 or 96 hours after the induction of hot stroke. The survival rate of each group of mice was calculated by referring to the following formula:

A=(B/8)×100%

Where: A = survival rate B = number of survival at 0, 12, 24, 48, 72 or 96 hours after induction of hot stroke in each group of mice

The obtained experimental data was analyzed in accordance with the method described in "C, Statistical Analysis" of "General Experimental Method" above.

result:

Figure 6 shows that mice that were induced to have a hot stroke were intraperitoneally injected with different doses (0.3 mg/kg, 0.6 mg/kg, and 1.2 mg/kg) of solution A (a rice fermentation culture containing P. pallidum G30801). When water-extracted product) or solution B (water-extracted product containing rice medium), they were kept at 24 ° C for a survival rate after 4 days.

As can be seen from Fig. 6, when mice induced to have a hot stroke were intraperitoneally injected with different doses of solution B, their survival rate after being cultured at 24 ° C for 4 days was zero. In contrast, when mice that were induced to have a hot stroke were intraperitoneally injected with different doses of solution A, at a dose of 0.3 mg/kg, the survival rate after they were reared for 4 days was 50%, while at the dose. In the case of 0.6 mg/kg and 1.2 mg/kg, the survival rate after they were reared for 4 days reached 100%. This result shows that the water-extracted product of the rice fermentation culture of Paecilomyces fuliginea G30801 according to the present invention can significantly improve the survival rate of mice which are induced to have a hot stroke.

Figure 7 shows that mice that were induced to have a hot stroke were intraperitoneally injected with solution A (water-extracted product of rice fermentation culture containing P. stipitis G30801 at a dose of 0.6 mg/kg) or solution B (containing rice). The water-extracted product of the medium, at a dose of 0.6 mg/kg, their survival rate as a function of time. As can be seen from Fig. 7, the mice in the rice medium group were reduced to zero after 24 hours of induction of hot stroke.

Cerebrovascular dysfunction in mice induced by hot stroke causes a decrease in body temperature in mice. Therefore, Applicants further measured changes in rectal temperature in mice that were induced to have a hot stroke. Referring to Fig. 8, when a mouse induced to have a hot stroke is intraperitoneally injected with solution A (a water-extracted product of a rice fermentation culture containing Paecilomyces militaris G30801 at a dose of 0.6 mg/kg), it is apparent that It improves the body temperature of mice because of the induction of hot stroke.

Example 7. Effect of water-extracted product of rice fermentation culture of Penicillium chrysogenum G30801 on extracellular ischemia and injury and oxidative stress in the brain of mice induced to have a heat stroke

In the present embodiment, extracellular ischemia and injury are used as an evaluation index for the concentration of glutamic acid and glycerol in the brain tissue fluid of hypothhalamus of mice and the ratio of lactic acid to pyruvic acid. The oxidative pressure is used as an indicator of the content of nitric oxide (NO) in the brain tissue fluid.

experimental method:

Male ICR mice were randomly divided into 3 groups (i.e., rice fermentation culture group, rice medium group, and normal temperature control group, each group n = 24). In addition to the mice of the normal temperature control group, the hot stroke was induced according to the method described in "B, Induction of Hot Stroke" of "General Experimental Method" above. Thereafter, each group of mice in which hot stroke was induced was moved to an environment at a temperature of 24 ° C, and then the mice in the rice fermentation culture group were intraperitoneally injected with the solution A (containing powder) prepared in Example 1. The water-extracted product of the rice fermentation culture of Penicillium sp. G30801 was dosed at 0.6 mg/kg), and the mice of the rice culture group were intraperitoneally injected with the solution B prepared in Example 1 (containing rice medium). The water-extracted product was dosed at 0.6 mg/kg.

Each group of mice was then subjected to brain tissue fluid collection according to the following procedure: intravenous injection of urethane (2.8 g/kg) at 0, 2, and 4 hours after the induction of hot stroke, respectively. To sacrifice the mice (n=8 in each group), and then cut the scalp and skull of the mouse to take out the brain tissue, and the person with a spherical structure at the bottom of the brain tissue is the hypothalamus. About 0.2 g of the hypothalamic tissue was added to 1 mL of homogenization buffer (Pefabloc SC) [50 mM Tris buffer containing 10% sucrose, pH 6.8], and homogenized by a homogenizer, followed by 4 Centrifuge (12,000 rpm) for 5 minutes at ° C, and then transfer the supernatant thus obtained to a microcentrifuge tube for use.

The obtained mouse brain tissue fluid is determined by the method described in "C. Analytical substance analysis of C. brain dialysate" of the experimental method of Example 4 above to determine the concentration of glutamic acid, glycerin, pyruvic acid, and lactic acid, and the content of NO. . The expression of glutamic acid and glycerol in brain tissue fluid is expressed as a percentage value of the concentration measured at each time point relative to the reference value, wherein the reference value is the concentration measured at the 0th hour. .

The obtained experimental data was analyzed in accordance with the method described in "C, Statistical Analysis" of "General Experimental Method" above.

result:

9A to 9D show the contents of the indicator substances in the brain tissue fluid of the hypothalamus of each group of mice at 0, 2, and 4 hours after the hot stroke was induced, respectively. As can be seen from Fig. 9A to Fig. 9D, at the 4th hour after the hot stroke was induced, the glutamic acid concentration, the glycerol concentration, and the lactic acid in the brain tissue fluid of the mice in the rice medium group were compared with the mice in the normal temperature control group. The concentration ratio of pyruvic acid and the content of NO are both increased. In contrast, in the brain tissue fluid of the mice in the rice fermentation culture group, the glutamic acid concentration, the glycerin concentration, the ratio of the concentration of lactic acid to pyruvic acid, and the content of NO were increased due to the induction of hot stroke. Significantly improved. This result shows that the water-extracted product of the rice fermentation culture of Paecilomyces fuliginea G30801 according to the present invention can effectively improve the extracellular ischemia and damage of the brain of the mouse induced to have a hot stroke, and has a remarkable Antioxidant effect.

Example 8. Effect of water-extracted product of rice fermentation culture of Penicillium chrysogenum G30801 on the expression of proinflammatory cytokines and anti-inflammatory cytokines caused by heat stroke and the effects of cell damage and organ dysfunction

In this example, in addition to using TNF-α, IL-1β and IL-10 as indicators for evaluating the amount of inflammatory cytokines and anti-inflammatory cytokines, the applicant further selected blood urinary nitrogen (BUN). ), creatinine, serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), and alkaline phosphatase ( Alkaline phosphatase (ALP) is used as an indicator of the function of the organ to be evaluated to evaluate the effect of the water-extracted product of the rice fermentation culture of the Penicillium chrysogenum G30801 according to the present invention on cell damage and organ dysfunction caused by hot stroke.

experimental method:

Male ICR mice were randomly divided into 3 groups (i.e., rice fermentation culture group, rice medium group, and normal temperature control group, each group n = 8). In addition to the mice of the normal temperature control group, the other groups of mice were induced to have a hot stroke according to the method described in "B, Induction of Hot Stroke" of the "General Experimental Method" above.

Thereafter, each group of mice in which hot stroke was induced was moved to an environment at a temperature of 24 ° C, and then the mice in the rice fermentation culture group were intraperitoneally injected with the solution A (containing powder) prepared in Example 1. The water-extracted product of the rice fermentation culture of Penicillium sp. G30801 was dosed at 0.6 mg/kg), and the mice of the rice culture group were intraperitoneally injected with the solution B prepared in Example 1 (containing rice medium). The water-extracted product was dosed at 0.6 mg/kg.

Next, at the 4th hour after the hot stroke was induced, 3 mL of blood was taken from the heart of each group of mice using a syringe, and centrifuged at 3,000 rpm for 10 minutes at 25 ° C, whereby the obtained serum was Used to perform the analysis below.

A. Determination of the concentration of TNF-α, IL-1β and IL-10 in serum:

The concentrations of TNF-α, IL-1β and IL-10 in the serum were respectively used in the mouse TNF-α kit (R&D systems, Cat. No. DY410), and the mouse IL-1β set. Group (Mouse IL-1β kit) (R&D systems, Cat. No. DY401) and mouse IL-10 kit (R&D systems, Cat. No. DY417) and according to the manufacturer's operation Guidelines for measurement.

B. Determination of the concentration of urea nitrogen (BUN), creatinine, SGOT, SGPT and ALP in serum:

The concentrations of urea nitrogen, creatinine, SGOT, SGPT, and ALP in the serum were determined by the Department of Pathology of the Chimei Hospital of the consortium according to the "standard test procedure" approved by the hospital.

result:

10A to 10C show the concentrations of TNF-α, IL-1β, and IL-10 in the serum of each group of mice at the 4th hour after the hot stroke was induced, respectively. As can be seen from Fig. 10A to Fig. 10C, at the 4th hour after the hot stroke was induced, the concentrations of TNF-α and IL-1β in the serum of the rice medium group were compared with those of the normal temperature control group, respectively. Increasing to 940 pg/mL and 320 pg/mL, but the concentration of IL-10 was only increased in small amounts (26.2 pg/mL). In contrast, the concentrations of TNF-α, IL-1β, and IL-10 in the serum of the rice fermentation culture group mice were 28 pg/mL, 53 pg/mL, and 286 pg/mL, respectively, which showed TNF- in their serum. The phenomenon that the concentration of α and IL-1β was increased due to the induction of hot stroke was significantly improved and the concentration of IL-10 was increased. These data were statistically significant compared with those of the mice in the rice medium group ( p <0.05). This result shows that the water-extracted product of the rice fermentation culture of Paecilomyces fuliginea G30801 according to the present invention has a remarkable anti-inflammatory effect.

In addition, FIGS. 11A to 11E respectively show the concentrations of urea nitrogen, creatinine, ALP, SGOT, and SGPT in the serum of each group of mice at the 4th hour after the hot stroke was induced. As can be seen from Fig. 11A to Fig. 11E, in the fourth hour after the hot stroke was induced, urea nitrogen, creatinine, ALP, SGOT, and SGPT in the serum of the rice medium group mice were compared with the mice of the normal temperature control group. The concentration is significantly increased. In contrast, at the 4th hour after the hot stroke was induced, the concentrations of urea nitrogen, creatinine, ALP, SGOT, and SGPT in the serum of the rice fermentation culture group were compared with those of the rice medium group. Significantly reduced and exhibited statistical significance ( p < 0.05). This result shows that the water-extracted product of the rice fermentation culture of Penicillium chrysogenum G30801 of the present invention can effectively improve cell damage and organ dysfunction caused by hot stroke.

Example 9. Water-extracted product of rice fermentation culture of Penicillium chrysogenum G30801 for inducible nitric oxide synthase (iNOS) in hypothalamus of mice induced to have a heat stroke And the effects of apoptosis on liver, kidney and spleen experimental method:

Male ICR mice were randomly divided into 3 groups (i.e., rice fermentation culture group, rice medium group, and normal temperature control group, each group n = 8). In addition to the mice of the normal temperature control group, the other groups of mice were induced to have a hot stroke according to the method described in "B, Induction of Hot Stroke" of the "General Experimental Method" above.

Thereafter, each group of mice in which hot stroke was induced was moved to an environment at a temperature of 24 ° C, and then the mice in the rice fermentation culture group were intraperitoneally injected with the solution A (containing powder) prepared in Example 1. The water-extracted product of the rice fermentation culture of Penicillium sp. G30801 was dosed at 0.6 mg/kg), and the mice of the rice culture group were intraperitoneally injected with the solution B prepared in Example 1 (containing rice medium). The water-extracted product was dosed at 0.6 mg/kg.

Then, at the 4th hour after the hot stroke was induced, each group of mice was sacrificed by intravenous injection of urethane (2.8 g/kg), followed by 0.05 mol/L anticoagulant phosphate buffer. The solution was perfused with cold 4% paraformaldehyde (Sigma Chemical Co.). Thereafter, the brain was quickly taken out and frozen with liquid nitrogen for the following immunohistochemistry stain. Further, the spleen, kidney, and liver of the mouse were taken out to perform the following terminal transferase-mediated nick end labeling assay.

A, immunohistochemical staining (immunohistochemistry stain):

The frozen brain was placed on a rotary microtome (CM1900, Leica) having a cryostat function to perform a coronal section (thickness of about 5 μm). The cut brain slices were thawed and placed on a glued glass slide. Next, the slide was immersed in a 10% MeOH/3% H ₂ O ₂ /sodium phosphate buffer mixture and allowed to react at room temperature for 30 minutes to block endogenous peroxidase. Thereafter, the slides were placed at room temperature and pre-incubated with 2% normal goat serum (NGS) and 0.2% Triton X-100 (Sigma Chemical Co.) for 0.5 hours to remove immunoglobulin (IgG). Non-specific binding reaction.

After pre-incubation, the sections were immersed in rabbit anti-NOS antiserum (rabbit anti-NOS antiserum, Sigma) diluted 50-fold with PBS containing 0.2% Triton X-100 and 1% azide at 4 °C. Overnight in Chemical Co., followed by PBS for 30 minutes, and then incubated with biotinylated goat anti-rabbit IgG (Sigma Chemical Co.) diluted 500-fold with PBS. After 2 hours, it was washed several times with PBS. Next, the sections were incubated with the AB mixture [avidin-biotin complex, 1:200, Vectastain] for 2 hours, followed by 3,3'-diaminobenzidine (3). 3'-diaminobenzidine, DAB) (Daceo Co.), nickel ammonium sulfate, and 0.003% hydrogen peroxide were colored, and the sections were finally sealed on a glass slide.

Immunohistochemical staining results were observed at a magnification of 400X by using a Zeiss upright microscope (Carl Zeiss up-right microscope, Carl Zeiss, Jena, Germany), which was shown to have a reddish-brown mark. It means that there is iNOS performance. The number of iNOS-positive cells was determined using Image Analysis Software Image-Pro Plus 6.3 (Media Cybernetics Co.).

B. End-transferase-mediated incision end labeling analysis:

The removed spleen, kidney, and liver were each immersed in 4% paraformadehyde for 24 hours, followed by dehydration, paraffin embedding, and sectioning (thickness: 4 μm/tablet). After dewaxing, sections were subjected to TUNEL staining (presenting green) using a commercial kit TdT-FragELTM DNA fragmentation Detection Kit (Oncogene) followed by the manufacturer's instructions, followed by 4,6-dimercapto -2-Phenylpurine (4',6-diamidino-2-phenylindole, DAPI) was counter-stained (blue). Fluorescence staining results were observed at a magnification of 400X by using a Carl Zeiss upright fluorescence microscope, which was shown to have a green fluorescent marker [ie TUNEL positive (TUNEL) Positive)] indicates that apoptosis has occurred. The number of TUNEL positive cells was determined using the image analysis software Axioscope version 4 (Carl Zeiss, Jena, Germany).

result: A, immunohistochemical staining:

12A to 12C show inducible nitric oxide synthase (iNOS) observed by immunohistochemical staining of the inferior colliculus of each group of mice obtained at 4 hours after the induction of hot stroke, respectively. Performance results. In addition, the number of iNOS-positive cells measured in the hypothalamus of each group of mice is shown in Table 1 below.

From Fig. 12A to Fig. 12C and Table 1, it can be seen that at the 4th hour after the hot stroke was induced, there was a large amount of iNOS expression in the hypothalamus of the rice culture group mice, and the lower hypothalamus of the rice fermentation culture group mice. The amount of iNOS expression in the case was significantly reduced. This result shows that when the mouse is induced to have a hot stroke, the iNOS in the hypothalamus is largely expressed and the brain nerve cells are attacked by free radicals. However, if the water-extracted product of the rice fermentation culture of the Penicillium chrysogenum G30801 according to the present invention is administered immediately after the hot stroke is induced, the expression amount of iNOS in the hypothalamus of the mouse is reduced, thereby reducing the brain Free radical damage to nerve cells.

B. End-transferase-mediated incision end labeling analysis:

Figures 13 to 15 show the results of double staining of TUNEL-DPAI in the spleen, kidney and liver sections of each group of mice obtained at 4 hours after the induction of hot stroke, and the number of TUNEL-positive cells measured. It is shown in Table 2 below.

As can be seen from Fig. 13 to Fig. 15 and Table 2, a large number of apoptotic cells appeared in the spleen, kidney and liver sections of the rice medium mice, and appeared in the spleen, kidney and liver sections of the rice fermentation culture group. The number of apoptotic cells is significantly reduced. This result indicates that the spleen, kidney and liver cells of the mouse cause DNA breakage in the cells due to the induction of hot stroke, which in turn triggers apoptosis. If the water-extracted product of the rice fermentation culture of Paecilomyces fuliginea G30801 according to the present invention is administered immediately after the hot stroke is induced, the phenomenon of apoptosis occurring in the spleen, kidney and liver of the mouse is clearly improve. Therefore, the water-extracted product of the rice fermentation culture of Paecilomyces fuliginea G30801 according to the present invention can reduce cell damage and avoid organ dysfunction.

Based on the above experimental results, the water-extracted product of the rice fermentation culture of Paecilomyces fuliginea G30801 according to the present invention can improve cerebrovascular dysfunction, acute inflammatory response, and multiple organ damage in mice induced to have a hot stroke. Symptoms are therefore available for the treatment of hot strokes.

All documents and patent documents cited in the present specification are incorporated herein by reference in their entirety. In the event of a conflict, the detailed description of the case (including definitions) will prevail.

While the invention has been described with respect to the specific embodiments of the invention described above, many modifications and changes can be made without departing from the scope and spirit of the invention. It is therefore intended that the invention be limited only by the scope of the appended claims.

Fig. 1 shows that the rice fermentation culture group and the rice culture medium group in the second embodiment of the present invention were intravenously injected with solution A after the induction of hot stroke (the water of the rice fermentation culture containing P. stipitis G30801). The difference between the survival time of the extracted product) and the solution B (the water-extracted product containing the rice medium), wherein "*" indicates p <0.01; and FIGS. 2A to 2C respectively show the respective examples in the present embodiment 3. Cerebral blood flow, cerebral oxygen partial pressure and brain temperature change with time in the group rats, wherein the intersection point of the dotted line indicated by the arrow and the X axis represents the time point at which the hot stroke is induced and the intravenous injection is performed; "*" It is indicated that when the rice medium group is compared with the normal temperature control group, p <0.05; and "+" means: when the rice fermentation culture group is compared with the rice medium group, p <0.05; FIGS. 3A to 3C respectively show the present embodiment 3 The mean arterial pressure, intracranial pressure, and cerebral perfusion pressure of each group of rats changed with time, and the intersection of the dotted line and the X-axis indicated by the arrow represents the induction of hot stroke and the intravenous injection. Intermediate point; "*" denotes: when m medium and normal temperature control group, p <0.05; and "+" means: Comparison with rice culture group when m fermentation culture group, p <0.05; Figs. 4A to 4H The physiological parameters of each group of rats in Example 4 of the present invention and the content of the indicator substance in their cerebral dialysate are changed with time, wherein the intersection of the straight line indicated by the arrow and the X-axis represents the induction of hot stroke and The time point at which intravenous injection was performed; DHBA and NO represent dihydroxybenzoic acid and nitric oxide, respectively; "*" means: when the rice medium group is compared with the normal temperature control group, p <0.05; and "+ "Expression: when the rice fermentation culture group was compared with the rice medium group, p <0.05; Figures 5A and 5B show that each group of rats in Example 5 of the present case was in the serum 15 minutes after the induction of hot stroke. the concentration of TNF-α and IL-10, wherein "*" denotes: m when the medium temperature and normal control group, p <0.01; and "+" means: when the object group and m m fermentation Comparative raising yl group, p <0.01; FIG. 6 shows that when the mice were induced heat stroke injected intraperitoneally with different doses (0.3mg / kg, 0.6mg / kg and 1.2mg / kg) of solution A (containing a powder intended When the water-extracted product of the rice fermentation culture of Penicillium sp. G30801 or the solution B (the water-extracted product containing rice medium), they were kept at 24 ° C for 4 days after survival; Figure 7 shows The mice induced to have a hot stroke were intraperitoneally injected with solution A (water-extracted product of rice fermentation culture containing Paecilomyces militaris G30801 at a dose of 0.6 mg/kg) or solution B (water containing rice medium) The extracted products, at a dose of 0.6 mg/kg, their survival rate with time; Figure 8 shows that mice that were induced to have a hot stroke were injected intraperitoneally with solution A (rice containing Paecilomyces militaris G30801) The water-extracted product of the fermentation culture, at a dose of 0.6 mg/kg) or solution B (water-extracted product containing rice medium at a dose of 0.6 mg/kg), their anal temperature changes with time. where "*" indicates p <0.01; Figs. 9A to 9D show the first stroke is 0 after the heat-induced 2 and the content of 4 hours, brain tissue fluid hypothalamus of mice in each group of indicator substance, where "*" denotes: When comparing the m medium temperature and normal control group, p <0.01; and "+" means: When the rice fermentation culture group was compared with the rice medium group, p <0.01; FIGS. 10A to 10C show the TNF-α, IL-1β in the serum of each group of mice at the 4th hour after the hot stroke was induced, respectively. The concentration of IL-10, where "*" means: when the rice medium group is compared with the normal temperature control group, p <0.05; and "+" means: when the rice fermentation culture group is compared with the rice medium group, p <0.05; 11A to 11E show the concentrations of urea nitrogen, creatinine, ALP, SGOT, and SGPT in the serum of each group of mice at the 4th hour after the hot stroke was induced, respectively, where "*" indicates: when the rice fermentation culture group Compared with the normal temperature control group, p <0.05; and "+" means that when the rice fermentation culture group was compared with the rice medium group, p <0.05; and Fig. 12A to Fig. 12C showed the fourth hour after the hot stroke was induced, respectively. Subgroup of mice obtained The results of inducible nitric oxide synthase (iNOS) were observed in the immunohistochemical staining of the chubby, where the reddish-brown mark indicates the presence of iNOS; Figure 13 shows the fourth hour after the hot stroke was induced. The TUNEL-DAPI double staining results of the spleen sections of each group of mice obtained showed that green fluorescent markers showed apoptosis, while DAPI stained blue; Figure 14 shows TUNEL-DAPI double staining of kidney sections of each group of mice obtained at 4 hours after the induction of hot stroke, where green fluorescent markers appear, indicating apoptosis, and being stained by DAPI Then, it is blue; and FIG. 15 shows the TUNEL-DAPI double staining result of liver slices of each group of mice obtained at the 4th hour after the hot stroke is induced, in which green fluorescent markers appear to indicate cells. Apoptosis occurs and is stained blue by DAPI.

Claims (18)

A pharmaceutical composition for treating a hot stroke, comprising a fermentation culture of Paecilomyces farinosus G30801, wherein the Paecilomyces genus G30801 is deposited with the Food Industry Development Institute under the registration number BCRC 930108 Bioresource Conservation and Research Center. The pharmaceutical composition of claim 1, wherein the fermentation culture is obtained by introducing the mycelium inoculum of one of the Paecilomyces genus G30801 to a solid culture. The pharmaceutical composition of claim 2, wherein the solid state culture is carried out using a solid medium comprising at least one of: rice, soybean, black bean, wheat, barley, rye, buckwheat, oat, corn, Coix seed, yam, potato, sweet potato, cassava, taro, Ge Yujin, lotus, mushroom, edible canna, Jerusalem artichoke, sorghum, silkworm cocoon, bran and rice bran. The pharmaceutical composition of claim 2, wherein the fermentation culture is a solid fermentation culture. A pharmaceutical composition according to claim 4, wherein the solid fermentation culture is further subjected to a water extraction treatment to obtain a water-extracted product. A pharmaceutical composition according to claim 1, which is in a form for parenteral administration. For example, the pharmaceutical composition of claim 1 is in a dosage form for intravenous injection. For example, the pharmaceutical composition of claim 1 is a peritoneum The dosage form for intrainjection. For example, the pharmaceutical composition of claim 1 is in a dosage form for oral administration. A fermented culture of Paecilomyces fuliginea G30801 for the preparation of a medicament for treating a hot stroke, the Paecilomyces sp. G30801 is deposited in the Bioresource Conservation Institute of the Food Industry Development Institute under the registration number BCRC 930108 Research center. The use of claim 10, wherein the fermentation culture is obtained by introducing the mycelium inoculum of one of the Paecilomyces genus G30801 to a solid culture. The use of the invention of claim 11, wherein the solid state culture is carried out using a solid medium comprising at least one of: rice, soybean, black bean, wheat, barley, rye, buckwheat, oats, corn, coix seed, Yam, potato, sweet potato, cassava, taro, Ge Yujin, lotus, mushroom, edible canna, Jerusalem artichoke, sorghum, silkworm cocoons, bran and rice bran. The use of claim 11, wherein the fermentation culture is a solid fermentation culture. The use of claim 13, wherein the solid fermentation culture is further subjected to a water extraction treatment to obtain a water-extracted product. The use of claim 10, wherein the pharmaceutical product is in a form for parenteral administration. The use of claim 10, wherein the pharmaceutical product is in a dosage form for intravenous injection. The use of claim 10, wherein the pharmaceutical product is in a dosage form for intraperitoneal injection. The use of claim 10, wherein the pharmaceutical product is in a dosage form for oral administration.