Application of Dendrobium officinale flower extract in preparing medicine or health care product for preventing cerebral ischemia

Abstract

The present invention discloses the application of a Chinese medicinal material Dendrobium officinale flower extract in the preparation of a drug or health product for preventing cerebral ischemia. The present invention finds that the Dendrobium officinale flower extract can significantly improve the neurobehavioral deficit symptoms of rats with cerebral ischemia-reperfusion, and significantly reduce the cerebral infarction volume of rats with cerebral ischemia-reperfusion. The Dendrobium officinale flower extract is prepared by extracting the flowers of the Chinese medicinal material Dendrobium officinale with alcohol and water, and the yield is 13.71-18.39%. The Dendrobium officinale flower extract can be used to prepare a drug or health product for preventing ischemic stroke, reduce the degree of brain tissue damage, and provide an effective solution for the prevention of the disease.

Description

Technical Field

[0001] The invention relates to a new application of a Dendrobium officinale flower extract in preparing medicines or health products, and mainly relates to the application of the Dendrobium officinale flower extract in preparing medicines or health products for preventing cerebral ischemia, and belongs to the field of medical technology. Background Art

[0002] Stroke is one of the more common cerebrovascular diseases in clinical practice. It is characterized by high morbidity, high disability rate and high mortality rate. It is a global disease that seriously threatens human health. The types of stroke are mainly divided into ischemic stroke, hemorrhagic stroke and transient ischemic attack. Ischemic stroke is mainly caused by insufficient blood supply to brain tissue due to vascular embolism or arteriosclerosis, resulting in brain tissue damage. Hemorrhagic stroke is mainly caused by rupture of cerebral blood vessels, blood seeping into the brain, resulting in interruption of normal brain tissue blood flow, thereby causing brain tissue damage. Ischemic stroke accounts for 70-80% of all stroke types. The pathogenesis and pathophysiological process of stroke are complex and have not yet been thoroughly elucidated. It is currently believed that it may be related to brain tissue energy metabolism disorders, free radical damage, calcium overload, excessive release of excitatory amino acids (EEA), brain cell apoptosis, and inflammatory response.

[0003] At present, there is a lack of clinically effective anti-stroke drugs. So far, tissue plasminogen activator (tPA) is the only drug approved by the US Food and Drug Administration for the treatment of focal cerebral ischemia. However, tPA has a narrow time window for thrombolytic therapy, a high risk of bleeding, and severe reperfusion injury, so it can only benefit a small number of clinical patients from thrombolytic therapy. Conventional drugs such as statins, antiplatelet aggregation drugs, and vasodilators can have a certain therapeutic effect, but their adverse reactions to patients cannot be underestimated. Overall, stroke-related therapeutic drugs and treatments are still in a very scarce state. Therefore, the development of stroke treatment drugs is particularly important and urgent.

[0004] Dendrobium officinale flower is the fresh or dried flower of Dendrobium officinale Kimura et Migo, an orchidaceae plant. Dendrobium officinale has the effects of benefiting the stomach and promoting the production of body fluid, nourishing yin and clearing away heat. It is used for damage to body fluid caused by febrile diseases, dry mouth and thirst, insufficient stomach yin, poor appetite and retching, persistent deficiency heat after illness, excessive yin and fire, bone steaming and fatigue fever, blurred vision, and weakness of tendons and bones. At present, the research on Dendrobium officinale mainly focuses on reducing blood sugar and enhancing immunity. There is no report on the effect of Dendrobium officinale flower and its extract in improving ulcerative colitis and its use in drugs for treating this disease.

[0005] Purpose of the Invention

[0006] The technical problem solved by the present invention is to provide an application of a Dendrobium officinale flower extract in the preparation of a drug or health product for preventing cerebral ischemia, thereby providing an effective solution for the treatment of cerebral ischemia.

[0007] Technical Solution

[0008] Application of Dendrobium officinale flower extract in preparing medicine or health care product for preventing cerebral ischemia.

[0009] The Dendrobium officinale flower extract is prepared by extracting with alcohol and water, and the yield of the Dendrobium officinale flower extract is 13.71-18.39%.

[0010] The Dendrobium officinale flower extract is prepared by the following method: the Dendrobium officinale flower is reflux-extracted with 95% ethanol 10 times the volume of the crude drug for 3 times, each time for 3 hours, and then reflux-extracted with 75% ethanol 10 times the volume of the crude drug for 2 hours, filtered, combined the extracts, and decompressed to remove the ethanol to obtain a total extract.

[0011] The Dendrobium officinale flower extract is characterized in that, by weight percentage, the Dendrobium officinale flower extract contains 1.10-2.31% of flavonoids, 10.78-13.82% of polysaccharides, and 0.70-1.04% of amino acids.

[0012] Furthermore, the prevention of cerebral ischemia refers to the prevention of acute ischemic stroke, transient ischemic attack and chronic cerebral ischemia caused by various factors;

[0013] Furthermore, the preventive effect of Dendrobium officinale flower extract on cerebral ischemia is characterized by targeting high-risk groups without acute symptoms, and patients who have already suffered cerebral ischemia and received treatment, with the purpose of avoiding recurrence;

[0014] Furthermore, the Dendrobium officinale flower extract can be prepared into various dosage forms of anti-stroke drugs with pharmaceutically acceptable excipients according to conventional preparation methods.

[0015] Furthermore, the Dendrobium officinale flower extract can be prepared into various anti-stroke health products with health products including health foods and functional foods with acceptable auxiliary materials according to conventional preparation methods.

[0016] Beneficial Effects

[0017] The present invention adopts a cerebral ischemia-reperfusion rat model to investigate the anti-cerebral ischemia effect of Dendrobium officinale flower extract. The results show that oral administration of Dendrobium officinale flower extract can significantly improve the neurobehavioral deficit symptoms of cerebral ischemia rats and reduce the cerebral infarction volume of cerebral ischemia rats, providing an effective solution for the prevention of stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1Effects of Dendrobium officinale flower extract on neurobehavioral deficits in rats with cerebral ischemia-reperfusion (). Sham: sham operation control group; MCAO / R: model group; MCAO / R+10mg / kg DOM: low-dose group of Dendrobium officinale flower extract (10mg / kg); MCAO / R+20mg / kg DOM: high-dose group of Dendrobium officinale flower extract (20mg / kg); MCAO / R+NBP: butylphthalide positive drug group; ## Compared with the sham-operated control group, ## P<0.01; **Compared with the model group, **P<0.01, *P<0.05.

[0019] Figure 2 Effect of Dendrobium officinale flower extract on cerebral infarction volume in rats with cerebral ischemia-reperfusion A: TTC-stained brain slices; B: Percentage of cerebral infarction volume; Sham: sham operation control group; MCAO / R: model group; MCAO / R+10mg / kg DOM: low-dose group of Dendrobium officinale flower extract (10mg / kg); MCAO / R+20mg / kg DOM: high-dose group of Dendrobium officinale flower extract (20mg / kg); MCAO / R+NBP: butylphthalide positive drug group; ## Compared with the sham-operated control group, ## P<0.01; **Compared with the model group, **P<0.01, *P<0.05. DETAILED DESCRIPTION

[0020] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0021] Example 1: Preparation of Dendrobium officinale flower extract

[0022] Dendrobium officinale flowers are fresh or dried flowers of the orchid Dendrobium officinale Kimura et Migo, purchased from Bozhou, Anhui. Ethanol and other reagents were all analytically pure.

[0023] The flower of Dendrobium candidum was extracted with 95% ethanol 10 times the volume of the crude drug for 3 times, each time for 3 hours, and then extracted with 75% ethanol 10 times the volume of the crude drug for 2 hours, filtered, combined, and the extracts were decompressed to remove the ethanol to obtain the flower extract of Dendrobium candidum. The flower extract of Dendrobium candidum was diluted with distilled water and prepared into a liquid for activity study.

[0024] Example 2: Confirmation of the chemical composition structure of Dendrobium officinale flower extract 1. The method for determining the content of flavonoids in Dendrobium officinale flower is as follows:

[0025] ① Preparation of standard solution and sample solution: Weigh 1 mg of rutin, place it in a 10 mL volumetric flask, add appropriate amount of distilled water to dissolve and dilute to scale, shake well for later use. Weigh 5 mg of Dendrobium officinale flower extract dried to constant weight at 105°C, place it in a 25 mL volumetric flask, add appropriate amount of distilled water to dissolve and dilute to scale, shake well for later use.

[0026] ② Drawing of standard curve: Accurately measure 0mL, 1.0mL, 2.0mL, 3.0mL, 4.0mL, and 5.0mL of 0.1mg / mL rutin standard solution in a 10.0mL volumetric flask, add 0.3mL of 5% NaNO2 solution by volume, shake well, let stand for 6 minutes, add 0.3mL of 10% Al(NO3)3 solution by volume, shake well, let stand for 6 minutes, add 4.0mL of 1mol / LNaOH solution, shake well, let stand for 1.5 minutes, and make up to 10.0mL with 30% ethanol solution by volume. Use tube No. 0 as blank control to measure the absorbance at a wavelength of 510nm. Draw the standard curve with solution concentration C as the ordinate and absorbance A as the ordinate, and the linear regression equation is C=0.0768A+0.000961, R 2 =0.9999.

[0027] ③ Determination of flavonoid content in the sample: Take 1 mL of the sample solution, operate according to the standard curve preparation steps, and determine the absorbance. The flavonoid content in the flower of Dendrobium candidum is calculated to be 1.10-2.31%.

[0028] 2. The method for determining the content of polysaccharides in Dendrobium officinale flowers is as follows:

[0029] ① Preparation of standard solution and sample solution: Weigh 10 mg of standard glucose dried to constant weight at 105°C, place in a 10 mL volumetric flask, add appropriate amount of distilled water to dissolve and dilute to scale, shake well for later use. Weigh 5 mg of Dendrobium candidum flower extract dried to constant weight at 105°C, place in a 25 mL volumetric flask, add appropriate amount of distilled water to dissolve and dilute to scale, shake well for later use.

[0030] ② Drawing of the standard curve: Accurately pipette 10mL of glucose stock solution into a 100mL volumetric flask, dilute to the mark with water, and obtain a 0.1mg / mL glucose standard solution. Pipet 0, 0.2, 0.4, 0.6, 0.8, and 1.0mL of the glucose standard solution into a 10mL stoppered graduated test tube, make up to 1.0mL with distilled water, add 1mL of 5% phenol, shake well, and then immediately add 5mL of concentrated sulfuric acid, shake well, let stand for 5min, heat in a 90℃ water bath for 20min, then cool in an ice bath for 5min, and measure the absorbance at a wavelength of 488nm. Plot a standard curve with solution concentration C as the ordinate and absorbance A as the ordinate, and the linear regression equation is C=0.112A+0.00136, R 2 =0.9993.

[0031] ③ Determination of polysaccharide content in the sample: Take 1 mL of sample solution, operate according to the standard curve preparation steps, and determine the absorbance. The polysaccharide content in the flower of Dendrobium candidum is calculated to be 10.78-13.82%.

[0032] 3. The method for determining the content of amino acids in Dendrobium officinale flowers is as follows:

[0033] The amino acid was determined by automatic amino acid analyzer, and the determination method was based on GB / T 5009.124-2003.

[0034] ① Preparation of standard solution and sample solution: Accurately pipette 0.20, 0.40, 0.60, 0.80, and 1.00 mL of Type B and Type AN-Ⅱ amino acid mixed standards into a 25 mL volumetric flask, dilute with 0.02 mol / L hydrochloric acid solution, and store at 0-4 ° C. Weigh 0.1 mg of Dendrobium officinale flower extract dried to constant weight at 105 ° C, place in a 10 mL volumetric flask, add appropriate amount of sample diluent to dissolve and dilute to scale, shake well and pass through a 0.22 μm membrane for testing.

[0035] ②Chromatographic conditions: Chromatographic column: LCAK07 / Li; injection volume: 50 μL; detection wavelength: 570 nm, 440 nm; ninhydrin flow rate: 0.25 mL / min, mobile phase flow rate: 0.45 mL / min; reactor temperature: 130 °C; mobile phase gradient: see Table 1.

[0036] Table 1 Mobile phase elution gradient

[0037]

[0038] Note: A is pH 2.90 lithium citrate buffer solution; B is pH 4.20 lithium citrate buffer solution; C is pH 8.0 lithium citrate buffer solution; D is pH 2.20 lithium citrate buffer solution.

[0039] ③ Determination of amino acid content in samples: The amino acid content in Dendrobium officinale flowers was measured to be 0.70-1.04%.

[0040] Example 3: Effect of Dendrobium officinale flower extract on neurobehavioral deficits in rats with cerebral ischemia-reperfusion

[0041] Experimental materials: Healthy male SD rats (weight 280-300g). They were kept in the laboratory of the SPF animal experiment center (temperature: 22℃±1℃, humidity: 60%±5%), fed with standard feed and drinking purified water. The experimental plan was designed in accordance with the Guidelines for the Use of Laboratory Animals in China and approved by the Animal Ethics Committee. The number of animals used and the harm to animals were reduced as much as possible during the experiment. The positive drug Butylphthalide was purchased from CSPC Pharmaceutical Group Enbi Pharmaceutical Co., Ltd.

[0042] Experimental groups: SD rats were randomly divided into sham operation control group, model group, low-dose group (10 mg / kg) of Dendrobium officinale flower extract, high-dose group (20 mg / kg), and butylphthalide group (20 mg / kg, positive control), with 12 rats in each group.

[0043] Establishment of the animal model of cerebral ischemia-reperfusion: The middle cerebral artery occlusion / reperfusion (MCAO / R) model was prepared by the suture method. Rats were anesthetized by intraperitoneal injection of ketamine (80 mg / kg) and xylazine (10 mg / kg), and the common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA) were isolated. The common carotid artery was clamped with a micro-artery clamp, and the trunk of the external carotid artery was freed and ligated 3-4 mm away from the bifurcation of the common carotid artery. The micro-artery clamp temporarily clamped the internal carotid artery, and a small incision was made in the external carotid artery with ophthalmic scissors. A 4-0 monofilament nylon line coated with methylpolysiloxane was inserted, and a loose knot was tied at the incision of the external carotid artery to block the blood reflux of the internal carotid artery. The micro-artery clamp of the internal carotid artery was removed, and the nylon line was gently pushed into the internal carotid artery to reach the anterior cerebral artery in the skull, blocking the opening of the middle cerebral artery (MCA). The insertion depth was about 18-22 mm. The micro-arterial clamp of the common carotid artery was removed, and the subcutaneous tissue and skin were sutured layer by layer after disinfection. The nylon thread was slowly withdrawn 2 hours after surgery for reperfusion. All other operations were the same as those in the sham operation group except that the nylon thread was not inserted. The rectal temperature of all rats was maintained at 37℃ throughout the experiment.

[0044] Neurobehavioral deficit scoring: After 24 hours of reperfusion, the neurobehavioral deficit scoring was performed before the animals were killed. Longa scoring criteria: 0, normal, no neurological deficit; 1, left forelimb extension disorder, weakened function; 2, circling to the left, left forelimb paralysis, severe weakened function, after the rat's tail was lifted, the rat kept twisting towards the damaged area; 3, leaning to the left side when walking; 4, limb paralysis, coma; 5, death.

[0045] Results: Figure 1As shown, the neurological function of animals in the Sham group was normal. Compared with the Sham group, the neurological injury score of animals in the MCAO / R model group was significantly increased; compared with animals in the MCAO / R model group, DOM (10, 20 mg / kg) showed a dose-dependent reduction in neurological deficit score (Longa score). In short, DOM showed an improvement effect on ischemic brain injury.

[0046] Example 4: Effect of Dendrobium officinale flower extract on cerebral infarction volume in rats with cerebral ischemia-reperfusion

[0047] Experimental materials: TTC (2,3,5-triphenyltetrazolium chloride) was purchased from Sigma Aldrich. Other relevant experimental materials were the same as those in Example 1.

[0048] Experimental groups: SD rats were randomly divided into sham operation control group, model group, low-dose and high-dose groups of Dendrobium officinale flower extract, and butylphthalide group (20 mg / kg, positive control), with 10 rats in each group. Other relevant experimental schemes were the same as in Example 1.

[0049] Determination of cerebral infarction volume: Rats (n = 10) were killed by cervical dislocation 24 hours after reperfusion. The whole brain was quickly removed and placed in a -20℃ refrigerator for a short period of freezing. The brain was placed on an ice tray to remove the olfactory bulb. The forebrain was cut into 5 slices with a thickness of 2 mm along the coronal plane. The brain slices were placed in 1.5 mL of 2% TTC solution and incubated in the dark for 30 minutes. The brain slices were then fixed in 10% paraformaldehyde overnight and photographed for analysis. The cerebral infarction volume was measured and calculated using Image-Pro plus 5.1 software. Cerebral infarction volume = total cerebral infarction area × brain slice thickness (2 mm). Because brain edema affects the cerebral infarction volume, the cerebral infarction dead area was corrected, and the cerebral infarction dead area = measured cerebral infarction area × {1-[(ipsilateral hemisphere area – contralateral hemisphere area) / contralateral hemisphere area]}.

[0050] Results: As shown in the figure, no infarction was found in the brain area of ​​the animals in the Sham group, but infarction appeared in the animals in the MCAO / R model group after 2h of ischemia and 24h of reperfusion, with obvious ischemic core area and semi-dark area; compared with the Sham group, the infarction volume of the MCAO / R model group increased significantly; compared with the animals in the model group, the brain infarction volume of the animals in the low-dose and high-dose groups (10 and 20 mg / kg) of DOM was significantly reduced. In addition, the positive control NBP can significantly reduce the infarction volume.

[0051] discuss

[0052] The present invention uses a cerebral ischemia-reperfusion rat model to investigate the anti-stroke effect of Dendrobium officinale flower extract, and the results show that oral administration of Dendrobium officinale flower extract can significantly improve the neurobehavioral deficit symptoms of stroke rats and significantly reduce the cerebral infarction volume of stroke rats. Therefore, Dendrobium officinale flower extract has an anti-stroke effect. Using Dendrobium officinale flower extract as an active substance, it is used alone or in combination with other pharmacologically active compounds or extracts to form a compound for use, and various dosage forms of anti-stroke drugs or health products are prepared according to conventional preparation methods in the pharmaceutical field, or it is prepared into a compound preparation with other drugs, etc., which is used to reduce adverse reactions in drug action while maintaining the therapeutic effect, which can provide an effective solution for the prevention of stroke.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described by referring to the preferred embodiments of the present invention, it should be understood by those skilled in the art that various changes can be made in practice and details without departing from the spirit and scope of the present invention as defined in the attached claims.

Claims

1. A use of a Dendrobium officinale flower extract in the preparation of a drug for treating ischemic stroke, characterized in that: The Dendrobium officinale flower extract is prepared by the following steps: refluxing and extracting the Dendrobium officinale flower with water 10 times the volume of the crude drug for 3 times, each time for 3 hours, then refluxing and extracting with 75% ethanol 10 times the volume of the crude drug for 2 hours, filtering, combining the extracts, and removing the ethanol under reduced pressure to obtain the Dendrobium officinale flower extract; by weight percentage, the Dendrobium officinale flower extract contains 1.10-2.31% flavonoids, 10.78-13.82% polysaccharides, and 0.70-1.04% amino acids.

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