TW201225963A - Pharmaceutical composition for preventing or treating stroke - Google Patents

Abstract

A pharmaceutical composition for preventing or treating stroke is provided, comprising Antrodia camphorata as an active ingredient. Use of Antrodia camphorata in the manufacture of a medicament for preventing or treating stroke is also provided.

Description

201225963 VI. [Technical Field] The present invention relates to a pharmaceutical composition for preventing or treating stroke, and more particularly to a pharmaceutical composition using Antrodia camphorata as an active ingredient. [Prior Art] Stroke is a brain The rapid development of brain function caused by abnormal blood supply is lost. Medically known as cerebrovascular accident (CVA), the blood supply to the brain is disrupted, making it impossible for local cells in the brain to obtain sufficient nutrients and oxygen to cause damage to the nerves. Stroke can be divided into hemorrhage stroke and ischemia stroke. Hemorrhagic stroke comes from intracerebral hemorrhage and usually has a high mortality rate. Ischemic stroke is caused by cerebral thrombosis and cerebral embolism, and the local mortality of the brain is usually low, but it is easy to cause damage in neurobehavioral ability. Risk factors for φ stroke include age, hypertension, history of stroke, transient ischemic heart disease, diabetes, high cholesterol, smoking, and atrial fibrillation, among which the southerly blood pressure is a risk factor. Therefore, current agents for preventing recurrent stroke and treatment are usually thrombolytic agents, antiplatelet agents (eg, aspirine, dipyridamole), anticoagulants, antihypertensive drugs, statins (statins). )Wait. The present inventors have succeeded in finding an alternative medicine having low biotoxicity, few side effects, and the potential to protect the nerve damage of the brain after a stroke in view of the side effects of the above-mentioned drugs, and have completed the present invention. 201225963 SUMMARY OF THE INVENTION The present invention provides a pharmaceutical composition for preventing or treating stroke, comprising Antrodia camphorata as an active ingredient. The present invention further provides the use of Antrodia camphorata for the preparation of a medicament for preventing or treating stroke. The specific embodiments of the present invention are described in detail below. However, the following embodiments are only used to further disclose the technical contents of the present invention, and should not limit the scope of the invention. [Embodiment] The active ingredient 'Antrodia camphorata' of the present invention, including a shellfish body, a mycelium body, or a combination thereof, may be subjected to steps such as drying and grinding to form the fruit body and/or mycelium. Suitable as a dosage form for pharmaceuticals. The source of Antrodia camphorata is not particularly limited and can be obtained by a cultivation method or a culture method well known in the art. For example, a wild-type Antrodia camphorata fruit body, an artificially cultivated Antrodia camphorata fruit body, or a mycelium fermented with a culture liquid. The wild-type Antrodia camphorata fruit body is derived from the natural environment of its own host, Cinnamomum Kanehirai Hay (Lauraceae). However, since the burdock is a conservation plant and the number is scarce, the wild type A. sinensis is relatively difficult to obtain. The artificially cultivated A. camphorata body includes a eucalyptus cultivation method, a space bag cultivation method, and a solid deep layer cultivation method. The eucalyptus cultivation method uses burdock or other species of eucalyptus as a nutrient source, space pack cultivation method uses wood chip culture, and solid deep culture method uses wood powder plus acacia gum as a nutrient source. The artificial cultivation system implants the artificial seedlings: After the appropriate temperature and humidity control, the A. sinensis fruiting body is cultivated. Alternatively, 201225963 may use a liquid medium, for example, US 6,355,475 B1, which describes the use of yeast extract, potassium phosphite, magnesium sulfate hydrate and fructose, glucose, sucrose, maltose or corn starch as a carbon source to culture the hyphae of the anthraquinone. body. In one embodiment of the invention, the dried A. camphorata fruit or mycelium is ground and dissolved in a polar solvent. From the viewpoint of cytotoxicity and simplification of the process, an embodiment of the present invention dissolves an anthraquinone fruit body or a mycelium powder in water. It is known that the Antrodia camphorata fruit body can separate fatty acids, rutheniums, benzene derivatives, sesquiter penes, triterpenoids, sterols and the like by different solvents (Chen CC, et Al., Neuroprotective diterpenes from the fruiting body of

Antrodia camphorate, J. Nat. Prod. 2006, 69, p. 689-691.). Moreover, several studies have indicated that the mycelium of Antrodia camphorata is cytotoxic to LLC tumor cells (Nakamura N. et al., Five New Maleic and Succinic Acid Derivatives from the Mycelium of Antrodia camphorata ® and Their Cytotoxic Effects on LLC Tumor). Cell Line. J. Nat. Prod. (2004) 67, p.46-48.) 'And the fruiting bodies of Antrodia sinensis have immunomodulatory, antioxidant, liver-protecting effects (Chen CC, et al., Neuroprotective diterpenes from *' the fruiting body of

Antrodia camphorate, J. Nat. Prod. 2006, 69, p. 689-691.). Wild and solid culture of Antrodia camphorata, through the extraction of water and sterol products, it is speculated that the main triterpenoids have the activity of inhibiting angiotensin converting enzyme (angiotensin 201225963 converting enzyme), but the concentration required to inhibit 50% is extremely high. 0.312mg/ml and 0.176mg/kg, respectively; the extract can reduce the blood pressure of natural hypertensive rats by oral administration, but it does not affect the blood pressure of normal rats (Der-Zen Liu et al., Antihypertensive activities of a Solid-state culture of Taiwanofungus camphoratus (Chang-chih) in spontaneously hypertensive rats. Biosci.

Biotechnol. Biochem. 2007, 71, p. 23-30). However, there has been no research on the prevention and treatment of Antrodia camphorata and stroke. Further, in the present invention, the whole body of the Antrodia camphorata fruit body and/or mycelium is used as an active ingredient, and is not limited to a single compound in Antrodia camphorata, so as to suppress nerve behavior damage and brain infarction volume after stroke. The stroke described in this case, including hemorrhagic stroke and ischemic stroke. In a preferred embodiment of the invention, the nerve damage and cerebral infarction volume caused by ischemic stroke have a good mitigating effect. In particular, before and after the onset of ischemic stroke (prevention) and after the onset (treatment), the pharmaceutical composition of the present invention can be significantly slowed down for both neurobehavioral ability (4) and cerebral infarction volume. The dosage of the active ingredient of the present invention can be appropriately formulated according to the patient's body weight, age, physiological condition, dosage form, administration mode, and administration procedure (IV). Because Odia camphoma is a well-known traditional Chinese medicine and health food, although the current dose specification is 'but in the animal safety test of health foods and related literature', there is no obvious oral toxicity and teratogenicity. . The preferred embodiment of the present invention is 6 201225963 100 mg, more preferably 5 〇〇 mg. Further, it is preferably administered orally, but is not limited to the administration method of the pharmaceutical composition of 100 mg per kg of the experimental animal. The pharmaceutical composition of the sputum can further include a medicinal carrier, such as an emulsifier, a suspending agent, a scalp, a female formula, a fragrance, and an excipient used in the funeral. The preparation of the present invention is carried out in the following manner. The present invention is further described by way of examples. However, the present invention is not limited to the scope of the following examples. [Example 1] The selection of the material of the test material includes: (1) a fruit body cultivated in a space package (Hua Chun Biotechnology) (hereinafter referred to as factory AI-01); (7) a mycelium fermented in a solid state ( Bairong Industrial Co., Ltd.) (hereinafter referred to as AI-02); (3) Mycelium fermented in liquid form (Good M Biotechnology) (hereinafter referred to as "ai_〇3"); and (4) The fruiting body of the forged wood (Asian Biotech) (hereinafter referred to as "^ milk"). 201225963 All of the above materials were sequenced and analyzed by the Internal Transcribed Spacer (ITS) and confirmed to be Antrodia camphorata. Experimental Animals Experimental animals were 7-week-old male SD rats purchased from Lesco Biotech Co., Ltd. The rats used in this test were domesticated and quarantined for 1 week. This product has a wealth of basic reference materials and data for experimental animals, which can be applied to the ischemic stroke assessment test. φ Feeding and management of experimental animals Feeding environment: The experimental animals are kept in the biomedical experimental animal station of the Industrial Research Institute. The illumination time of the feeding area is automatically controlled to 12 hours bright, 12 hours dark, room temperature 23±2 ° C, relative humidity 40- 70%. Animals are free to get enough food and water. Clinical symptoms were observed and recorded daily by veterinarians and testers in the hospital during quarantine and trial to ensure the health of the experimental animals. • Animal observation: Clinical observations were performed daily during the trial and the animals were recorded for other clinical symptoms or death. Clinical symptoms were also observed as usual on holidays. All deaths and all _ abnormal symptoms and degrees were recorded in the animal clinical observation record. Dead animals are also subject to necropsy and to identify possible causes of death. 8 201225963 Animal grouping and individual identification After 1 week of domestication of experimental animals, the veterinarians of the hospital judged that the experimental animals were in good health, and then weighed and grouped by S type. 3 For one feeding cage, the individual animals were distinguished by ear tag number, and the Cage card was used to indicate the cage number, strain, age, animal number, test number, test group, enthalpy and trial period. Ischemic Stroke Animal Model (MCAO) Test #An animal model of ischemic stroke according to Dittmar, MS., et al., The

Role of ECA transection in the development of masticatory lesions in the MCAO filament model, Experimental

Neurology, (2005) 195, p.372-38. Methodology described. Specifically, the test animals were anesthetized with a gas of 5% isoflurane in N20/〇2 (70%/30%), cut along the midline of the scalp, and the monofilament nylon thread (front end covered with silicone) was passed through the right outer neck. The exteriia carotid artery (ECA) is inserted, along the right internal carotid artery (ICA)—to the part of the circle 〇f Willis, causing obstruction of the right middle cerebral artery. After one hour of ischemia, the monofilament nylon thread is removed, and the ischemic site of the right brain reaches the reperfusion of blood (reperfusi〇n). Experimental design and grouping The experimental animals were grouped into 6 groups, and each group was subjected to the following experimental steps: "(1) Prevention of nuclear type: each group of test animals was subjected to ischemic stroke animal model (MCA〇) 1 hour before, the above listed 樟 材料 materials AI-01, 9 201225963 AI-02, AI-03, AI-05 each 500mg / kg (Figure 1). (ii) treatment mode: make each group subject The test animals were administered 500 mg/kg of the above-mentioned anthraquinone materials AI-02 and AI-03 0.5 h after the ischemic stroke animal model (MCAO). In the above prevention mode and treatment mode, In addition, a blank control group (sham) that does not undergo MCAO surgery, does not administer anthraquinone material, and a solvent control group that performs MCAO surgery and water instead of anthraquinone material is administered to the test animals. Neurobehavioral assessment analysis neurological behavior assessment Rats were tested at 1, 2, and 24 hours after MCAO surgery in each group of animals, and scored according to the following conditions. The purpose of this evaluation was to evaluate the severity of rat brain neurological deficits. Degree. Lift the rat from the tail to make it drift The ground is about 20 to 30 cm. The forelimb extension of the rats is observed, and the severity of the brain nerve defects is scored according to the behavior of the rats. The scores are divided into four levels: Level 0: The forelimbs of the rats can reach the ground evenly, without Other neurological deficits occurred in normal rats. Stage 1: Contralateral contraction of the forelimbs of the rat to the damaged area of the brain. Level 2: Place the rats on the ground and apply lateral thrust, rat pair The resistance of the ipsilateral thrust in the damaged area of the brain decreased. Level 3: The rats were free to move, and the rats continued to circle to the opposite side of the damaged brain region, unable to go straight. Level 4: Rats due to severe nerve damage, The limbs showed weakness or epilepsy. 201225963 The results of the prevention mode and treatment mode are shown in Table 丨, Table 2, Figure 3, and Figure 4, respectively. Table 2, Figure 3, and Figure 4 The data were expressed as mean ± standard deviation (SE), and there was a difference between the groups and the solvent control group by Mest. Ρ$〇.〇5 indicates that there is statistically between the test group and the solvent control group. Significant difference.

According to the data in Table 1 and Table 2 above, in the prevention mode, the AI_〇3 group and the AI-05 group, compared with the solvent-controlled control group, had a significant reduction in the 24 hours after the MCA〇 operation. Activity of rat nerve injury behavior (p< 201225963 0.01). The remaining test groups in the prevention mode were statistically insignificant compared to the solvent control control group (Table 1; Figure 3). In the treatment mode, the AI-02 group and the AI-03 group had a tendency to reduce the behavior of nerve injury in rats at the 24 hour time after MCAO surgery compared with the solvent-controlled control group, but there was no statistically significant difference. (Table 2; Figure 4). Analysis of brain infarction area 24 hours after MCAO surgery, the brain of the test animal was taken out and placed in a low-temperature oxygenated physiological saline solution (0.95% normal saline), and the brain was coronally sliced to a thickness of 2 mm to remove the brain. The tip of the tip is 1 mm, and a total of seven pieces are cut. Then, brain tissue sections were infiltrated with 10/〇 TTC (2,3,5-triphenyltetrazolium chloride), and the reaction was carried out in an incubator at 37 ° C for 30 minutes. The sections were fixed in a 4% Formalin solution, recorded by a photographing system (MarcoPATH Digital Image System), and the percentage of cerebral infarct volume was calculated using image analysis software (ImageJ 1.42q). The corrected percentage of cerebral infarction = (B-A) / B X 100% · A. The area of the left cerebral hemisphere is not damaged B. The area of the left hemisphere. The results of brain infarct area analysis are shown in Tables 3, 4, 5A, 5B, 6A and 6B. Similarly, the results shown in Tables 3, 4, 5A, 5B, 6A, and 6B are expressed as mean ± standard deviation (SE), and t-test is used to compare each group with the solvent control group. There is a difference between 12 201225963. 0.05 indicates a statistically significant difference between the test group and the solvent control group. [Table 3] Mean ± standard deviation (SE) of infarct size (%, 5, 7, 9, 11, 13, 15 mm from the anterior end of the brain) and infarct volume (%) in each group in the prevention mode Group name 3 mm 5 mm 7 mm 9 mm 11 mm 13 mm 15 mm Total infarct volume (%) Blank control group 1.57 ± 0.62 0.96 ± 0.23 1.41 ± 0.73 0.53 ± 0.53 0.20 ± 0.20 0.17 Soil 0.17 2.79 ± 0.62 1.09 ± 0_11 Solvent control group 16.75 ± 3.35 32.53 ± 5.16 57.85 ± 5.15 48.04 ± 8.15 24.45 ± 12.19 14.95 ± 5.01 8.69 ± 3.26 29.04 ± 4.51 ΑΙ-01 Group 7.13 ± 2.76 21.48 ± 7_13 31.76 ± 7.19 * 24.62 ± 3.90 * 7.94 ± 4.25 4.22 ± 2.95 3.30 ±1.76 14.35 ±3.77* ΑΙ-02 Group 2.71 ±1.08* 8.90 ±3.01** 12.91 ±4.32*** 11.67 ±6.66** 3.97 ±1.92 2.49 ±1.17 0.68 ±0.55 6.69 ±1.56** ΑΙ-03 Group 3.41 ±1_25* 7.04 Earth 2.90** 14.32 ±5.75*** 13.90 ±2.40* 1.53 ±0.68 1.23 ±0.72 1.02 ±0.51 6.06 ±1.11** ΑΙ-05 Group 1.15 ±0.48** 21.29 ±9.10 25.23 ±6.50** 14.23 ±3.44* 0.83 ±0.43 1.17 ±0.47 0.52 ±0.48 9.20 ±2.65**

[Table 4] Mean ± standard deviation (SE) of infarct size (%, 5, 7, 9, 11, 13, 15 mm from the anterior end of the brain) and infarct volume (%) in each group in the treatment mode Group name 3 mm 5 mm 7 mm 9 mm 11 mm 13 mm 15 mm Total infarct volume (%) Solvent control group 16.51 ± 6.34 32.02 ± 7.23 55.06 ± 5.51 59.07 ± 3.38 38.20 ± 6.73 22.83 ± 6.24 5.57 ± 3.48 32.75 ± 4.12 AI-02 Group 4.56 ±3.36 18.08 ±9.72 34.85 ±8.02 40.66 ±5.57* 24.38 ±7.75 5.64 ±3.36* 2.94 ±1.64 18.73 ±5.24 AI-05 Group 5.37 ±1_40 12.66 ±3.97 35.75 ±4.96* 42.28 ±5.61* 17.66 ± 5.00* 2.04 ±0.68* 0.56 ±0.37 16.62 ±2.49* *:ρ$0·05. 13 201225963 According to the data in Table 3 and Table 4 above, in the prevention mode, compared with the solvent control group, the ΑΙ-01 group was at a distance of 7 mm and 9 mm from the anterior end of the brain, and the distance between the AI-02 group and the AI-03 group was At the 3, 5, 7 and 9 mm fronts of the brain, and the AI-05 group at 3, 7 and 9 mm from the anterior end of the brain, there was a significant reduction in infarct size (Table 3; Figure 5A). In the proportion of total infarct size, as shown in Table 3 and Figure 5B, the AI-01, AI-02, AI-03, and AI-05 groups significantly reduced the total infarct size of the brain compared to the vehicle control group. Activity. Further, based on the ratio of the total infarct size shown in Table 3, based on the ratio of the total infarct area of the solvent control group, the reduction ratio of the total infarct size of each test group was calculated. For example, the total infarct size reduction ratio of the AI-01 group was 50.58% (from (14.35-29.04)/29.04), and the total infarct size reduction ratio of the AI-02, AI-03, and AI-05 groups was 70.97% and 79.12%, respectively. And 68.31%. In the treatment mode, the AI-02 group was significantly smaller than the solvent control group at 9 and 13 mm from the anterior end of the brain, and the AI-03 group had a significant reduction in infarct size at 7, 9 and 11 mm from the anterior end of the brain. Activity (Table 4; Figure 6A). As shown in Table 4 and Figure 6B, the AI-03 group had a significantly reduced activity in the proportion of total infarct size of the brain compared to the vehicle control group. Although the AI-02 group has a tendency to reduce the proportion of total infarct size in the brain, it has not reached statistical significance. Further, based on the ratio of the total infarct size shown in Table 4, the total infarct size reduction ratios of the AI-02 and AI-03 groups were calculated to be 42.81 and 49.26% based on the total infarct size of the solvent control group. While the present invention has been described in its preferred embodiments, the present invention is not intended to limit the invention, and it is to be understood that those skilled in the art can make some changes and refinements without departing from the spirit and scope of the invention. The scope of the invention is defined by the scope of the appended claims.

15 201225963 [Simplified Schematic] Fig. 1 shows a preventive mode of administration of Antrodia camphorata materials 1 hour before the ischemic stroke (MCAO surgery) in an embodiment of the present invention, and 1, 2 and 24 hours after the operation. Perform neurobehavioral assessment analysis. Fig. 2 is a view showing the treatment mode of administration of Ganoderma lucidum material after 5 hours of ischemic stroke (MCAO surgery) in an embodiment of the present invention, and neurobehavioral evaluation analysis was performed at 1, 2, and 24 hours after the operation. Fig. 3 is a view showing the prevention mode of an embodiment of the present invention, in which each control group and the test group performed neurobehavioral evaluation analysis at 2 and 24 hours after the operation. φ Fig. 4 shows the neurobehavioral evaluation analysis of the control group and the test group at 1, 2, and 24 hours after the operation in the treatment mode of one embodiment of the present invention. Fig. 5A is a graph showing the percentage of brain infarction sites and infarct volume in each of the control group and the test group in the preventive mode of the present embodiment, and the brain infarct analysis region is in the range from the front end to 15 mm in the brain. Fig. 5B is a view showing the total infarct volume (%) of the brain in each of the control group and the test group in the preventive mode of the present embodiment, and the brain infarct analysis region is in the range from the front end to the 15 mm. Spring Fig. 6A shows the percentage of brain infarction sites and infarct volume in each of the control group and the test group in the treatment mode of one example of the present case, and the brain infarction analysis area is in the range of the brain from the front end to 15 mm. _ Fig. 6B shows the total infarct volume (%) of the brain in each of the control group and the test group in the treatment mode of one example of the present case, and the brain infarct analysis area is in the range from the front end to 15 mm in the brain. [Main component symbol description] None 0 16

Claims (1)

201225963 VII. Scope of application: Compositions, including Anshidia 1. An antidiabetic medicine (Antrodia camphoma) as the active ingredient _ 所述 之 (4) composition, including wild type and artificially cultivated 樟Chiba. A pharmaceutical composition, wherein the person is in a solid state culture, or a pharmaceutical composition as described in the pharmaceutical composition of the liquid formulation. 4. As described in claim 3 Cultivation includes eucalyptus cultivation, space planting, and raising. 5. If you apply for any of the scope of patents 1-4, it also includes a pharmaceutical carrier. 6. For example, in claim 1-4, the stroke includes ischemic stroke. The pharmaceutical composition described in Item M of the patent scope is administered orally. The pharmaceutical composition described in the eighth aspect of the patent is the same as the medicinal composition described in claim 8, wherein the sputum is administered pre-stroke. In H, please refer to the (4) composition described in item 8 of the patent scope, wherein the treatment is administered after stroke. The use of Antrodia camphorata for the preparation or prevention of a windy wind. 17 201225963 12. As described in section u of the patent application, include wild type and artificially cultivated Antrodia camphorata. Use, wherein the oyster bag 13. As described in claim 12, the cultivation includes eucalyptus cultivation, space k,: the artificial cultivating 14 1 is cultured in a solid state, or liquid culture. The lieutenant stroke includes the use of any of the items 1M3 of the Leo Wai Wai Wai, wherein the stroke includes an ischemic stroke.