Traditional Chinese medicine extract as well as preparation method and application thereof

Abstract

The invention relates to the technical field of traditional Chinese medicines, in particular to a traditional Chinese medicine extract as well as a preparation method and application thereof, and the traditional Chinese medicine extract is extracted from a prescription comprising ligusticum wallichii and gastrodia elata. The traditional Chinese medicine extract contains 60 wt.%-70 wt.% of neutral sugar, the optimal ratio of ligusticum wallichii to gastrodia elata is selected, and the extract obtained from the ligusticum wallichii and gastrodia elata can exert specific pharmacodynamic activity and has a good curative effect on treatment of migraine and / or middle cerebral artery occlusion syndrome.

Description

Technical Field

[0001] The present application relates to the technical field of traditional Chinese medicine, and in particular to a traditional Chinese medicine extract and a preparation method and application thereof. Background Art

[0002] Migraine, the most common disabling primary headache disorder, is a common neurovascular disease in neurology. With increasing stress, the incidence of migraine has been on the rise in recent years. The clinical manifestations of migraine are recurrent, moderate to severe headaches, often occurring unilaterally.

[0003] The Da Chuanxiong formula, originating from Volume 2 of Liu Wansu's Xuanming Lunfang (Xuanming Lunfang) in the Jin Dynasty, consists of two herbs: Chuanxiong and Gastrodia elata. Chuanxiong is known as the "blood qi medicine," promoting blood circulation and qi circulation, dispelling wind and relieving pain. Gastrodia elata, known as the "head wind cure," calms the liver and suppresses yang, calming wind and stopping spasms. This herbal pair, with Chuanxiong as the main herb and Gastrodia elata as the auxiliary herb, was first described in the Daxiong Pills section of Shengji Zonglu (Shengji Zonglu). This formula promotes blood circulation, removes blood stasis, calms the liver and extinguishes wind, unblocks meridians and relieves pain. After thousands of years of clinical validation, it has demonstrated significant efficacy in treating migraines. Existing research on the chemical composition and pharmacology of the Chuanxiong-Gastrodia elata pair has primarily focused on evaluating the overall efficacy of the traditional Chinese medicine compound or investigating its small molecule components, with no reports on the polysaccharide fraction. Summary of the Invention

[0004] In view of this, the present application provides a Chinese herbal extract and its preparation method and application, in order to treat or intervene in migraine at an early stage.

[0005] In a first aspect, the present application provides a traditional Chinese medicine extract.

[0006] In some embodiments, the Chinese herbal medicine extract is extracted from a prescription comprising Chuanxiong rhizome and Gastrodia elata; the Chinese herbal medicine extract contains 60 wt.%-70 wt.% of neutral sugar.

[0007] In some embodiments, the Chinese herbal medicine extract further contains 10 wt.%-20 wt.% of uronic acid, and / or 0.5 wt.%-1.2 wt.% of protein;

[0008] Preferably, the Chinese herbal medicine extract contains 65.10 wt.% of neutral sugar, 14.22 wt.% of uronic acid, and 0.62 wt.% of protein;

[0009] More preferably, the weight ratio of Chuanxiong and Gastrodia elata in the prescription is (40-120): (10-30);

[0010] More preferably, the weight ratio of Chuanxiong and Gastrodia elata is 4:1.

[0011] In a second aspect, the present application provides a method for preparing a Chinese medicine extract.

[0012] In some embodiments, the preparation method comprises the following steps:

[0013] Extracting the Chinese medicinal materials including Chuanxiong rhizome and Gastrodia elata with alcohol to obtain an alcohol extraction residue and an alcohol extraction filtrate;

[0014] The alcohol extraction residue is subjected to water extraction again, the water extraction filtrate and the alcohol extraction filtrate are mixed, the mixed solution is separated by macroporous resin, washed with water, and the water eluate is collected;

[0015] The water eluate is subjected to alcohol precipitation and solid-liquid separation to obtain a precipitate.

[0016] In some embodiments, the macroporous resin is HP-20;

[0017] Preferably, the ratio of the sample load to the macroporous resin in the macroporous resin separation step is preferably 1:(1.5-2.5);

[0018] Preferably, the volume of the water eluent used in the macroporous resin separation step is 1-6 times the column volume.

[0019] In some embodiments, before the water extraction filtrate and the alcohol extraction filtrate are mixed,

[0020] The water extract filtrate and the alcohol extract filtrate are first concentrated and / or dried separately, and then mixed to form a mixed extract; the mixed extract is then dissolved in water, solid-liquid separation is performed, and the supernatant is collected and then separated by a macroporous resin;

[0021] Preferably, the alcohol-extracted filtrate is concentrated and / or dried to 0.8 g / L-1.2 g / L to form an alcohol-extracted extract;

[0022] The aqueous extract filtrate is concentrated and / or dried to 0.8 g / L-1.2 g / L to form an aqueous extract;

[0023] More preferably, the weight ratio of the alcohol extract to the water extract is 2-10:1.

[0024] In some embodiments, the preparation method further satisfies any one or more of the following A to D:

[0025] A: The alcohol extraction uses 50vol%-90vol% ethanol aqueous solution;

[0026] B: In the alcohol extraction step, the ethanol solution is refluxed and extracted at least twice, each time for 0.5 hours to 3 hours; the material-liquid ratio of each alcohol extraction is 1:(10-20), and the ratio relationship is g / mL;

[0027] C: In the water extraction step, water is added and decocted at least twice, each time for 0.5 hours to 1.5 hours; the material-liquid ratio of each water extraction is 1:(10-20), and the ratio relationship is g / mL;

[0028] D: The temperature of the water extraction is 60°C-90°C.

[0029] In some embodiments, before the water eluate is subjected to alcohol precipitation,

[0030] First, the water eluate is concentrated to 20%-30% of the original volume, and then alcohol precipitation is performed;

[0031] Preferably, 2-10 times the volume of a 50 vol%-95 vol% ethanol aqueous solution is added during the alcohol precipitation process;

[0032] More preferably, the alcohol precipitation time is 12 hours to 24 hours.

[0033] In some embodiments, in the solid-liquid separation step, the obtained solid is washed with an ethanol solution at least once, dissolved in water, and freeze-dried to obtain a precipitate;

[0034] Preferably, the concentration of the ethanol solution is 50 vol%-95 vol%.

[0035] In a third aspect, the present application also provides a pharmaceutical preparation.

[0036] In some embodiments, the pharmaceutical preparation comprises a preventive and / or therapeutically effective amount of a Chinese herbal extract;

[0037] The Chinese medicine preparation includes the Chinese medicine extract, or the Chinese medicine extract prepared by the preparation method;

[0038] Preferably, the Chinese herbal medicine extract is the only active ingredient in the pharmaceutical preparation; or the pharmaceutical preparation also includes other active ingredients or drugs; other active ingredients include nutritional supplements.

[0039] The Chinese medicine preparation can be a capsule, pill, powder, syrup, tablet, granule, plaster, mixture, pill, suppository, aerosol, ointment or injection.

[0040] The usage amount of the traditional Chinese medicine extract is 0.02g / kg / day-0.12g / kg / day.

[0041] In a fourth aspect, the present application also provides a Chinese herbal extract, or the use of the Chinese herbal extract prepared by the preparation method or the pharmaceutical preparation in the preparation of drugs for preventing and / or treating migraine or middle cerebral artery occlusion syndrome.

[0042] Preferably, the migraine is a migraine caused by taking nitroglycerin;

[0043] Optionally, the migraine detection index includes at least one of CGRP, MAO-A, MAO-B, and 5-HT levels.

[0044] The symptoms of migraine include recurrent unilateral moderate to severe headaches, as well as nausea, vomiting, phonophobia, and photophobia.

[0045] Compared with the prior art, the present invention has the following advantages:

[0046] The Chinese herbal extract of the present application is extracted from a prescription including Chuanxiong and Gastrodia elata; the Chinese herbal extract contains 60wt.%-70wt.% of neutral sugar, and the optimal ratio of Chuanxiong and Gastrodia elata is selected, and the extract obtained therefrom can exert specific pharmacological activity and has good therapeutic effect in treating migraine and middle cerebral artery occlusion syndrome.

[0047] Compared with other extracts of the Chuanxiong-Gastrodia elata Chinese medicine formula, the Chinese medicine extract of the present application has a better therapeutic effect at a lower dose and has fewer side effects.

[0048] The traditional Chinese medicine extracts of the present application have diverse targets, are less likely to produce drug resistance, and have no adverse reactions or fewer adverse reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is the immunofluorescence staining of brain tissues treated differently in Experiment 4;

[0050] Figure 2 This is a bar graph of CGRP content in brain tissues of different treatments in Experiment 4;

[0051] Figure 3 This is a bar graph of MAO-A content in brain tissues of different treatments in Experimental Example 4;

[0052] Figure 4 This is a bar graph of MAO-B content in brain tissues of different treatments in Experimental Example 4;

[0053] Figure 5 This is the thermal stability analysis diagram of different treatments in Experimental Example 6. DETAILED DESCRIPTION

[0054] The following examples are provided to further better understand the present application, but are not limited to the best implementation mode described herein, and do not limit the content and protection scope of the present application. Any product identical or similar to the present application obtained by anyone under the inspiration of the present application or by combining the features of the present application with other prior arts shall fall within the scope of protection of the present application.

[0055] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0056] In this application, the selection scope involving "and / or" and "or / and" includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, and the said any and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items.

[0057] In this application, the terms "preferred", "better", "more preferred" and "suitable" are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of this application.

[0058] In this application, the terms “further”, “further”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0059] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0060] In this application, when referring to percentage content, unless otherwise specified, for solid-liquid mixing and solid-solid mixing, it refers to mass percentage, and for liquid-liquid mixing, it refers to volume percentage.

[0061] In this application, references to percentage concentrations, unless otherwise specified, refer to final concentrations, which are the percentage of an added component in the system after the addition of that component.

[0062] In this application, wt.% refers to weight percentage, and vol% refers to volume percentage.

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.

[0064] This application investigates the Chuanxiong-Gastrodia elata herbal medicine, hoping to identify an active ingredient that is effective in treating migraines. During the experiment, a component extracted from the Chuanxiong-Gastrodia elata herbal medicine was found to have superior therapeutic effects. Further research is underway to investigate this component's chemical composition and pharmacological effects on migraines and middle cerebral artery occlusion.

[0065] In view of this, the present application provides a Chinese herbal extract and its preparation method and application, in order to find active ingredients, use smaller doses to treat or intervene in the occurrence and development of migraine and middle cerebral artery occlusion at an early stage, and reduce side effects.

[0066] The Chinese herbal medicine extract is extracted from a prescription comprising Chuanxiong rhizome and Gastrodia elata; the Chinese herbal medicine extract contains 60 wt.% to 70 wt.% of neutral sugar.

[0067] In some embodiments, the Chinese herbal medicine extract further contains 10 wt.%-20 wt.% of uronic acid, and / or 0.5 wt.%-1.2 wt.% of protein;

[0068] More preferably, the Chinese herbal medicine extract contains 60-70 wt.% of neutral sugar, 10-20 wt.% of uronic acid, and 0.5-1.2 wt.% of protein.

[0069] More preferably, the Chinese herbal medicine extract contains 65.10 wt.% of neutral sugar, 14.22 wt.% of uronic acid, and 0.62 wt.% of protein;

[0070] Neutral sugars are a type of monosaccharide, meaning they don't contain acidic or basic functional groups. Examples include glucose, fructose, and galactose. They are the building blocks of carbohydrates and typically consist of three to seven carbon atoms.

[0071] Uronic acids are compounds and their derivatives formed when the primary hydroxyl groups in sugars are oxidized to carboxyl groups. Naturally occurring uronic acids include D-glucuronic acid, D-galacturonic acid, and D-mannuronic acid. In the form of uronic acid glycosides bound to aglycones (uronides) or polyuronic acids, they are the main components of cell walls or mucus substances such as gum, pectin, hemicellulose, alginic acid, and bacterial polysaccharides. D-glucuronic acid binds to toxins in animals (glucuronic acid binding) and then excretes them. It is also a component of highly active substances such as heparin, chondroitin sulfate, and hyaluronic acid.

[0072] More preferably, the weight ratio of Chuanxiong and Gastrodia elata in the prescription is (40-120): (10-30);

[0073] More preferably, the weight ratio of Chuanxiong and Gastrodia elata is 4:1.

[0074] The optimal ratio of Chuanxiong and Gastrodia elata is selected, and polysaccharides are extracted from them in order to obtain a component with better pharmacological activity. Experiments have shown that the polysaccharide obtained when the weight ratio of Chuanxiong to Gastrodia elata is (40-120): (10-30), preferably 4:1, is more effective in treating migraines.

[0075] In a second aspect, the present application provides a method for preparing a Chinese medicine extract.

[0076] In some embodiments, the preparation method comprises the following steps:

[0077] Extracting the Chinese medicinal materials including Chuanxiong rhizome and Gastrodia elata with alcohol to obtain an alcohol extraction residue and an alcohol extraction filtrate;

[0078] The alcohol extraction residue is subjected to water extraction again, the water extraction filtrate and the alcohol extraction filtrate are mixed, the mixed solution is separated by macroporous resin, washed with water, and the water eluate is collected;

[0079] The water eluate is subjected to alcohol precipitation and solid-liquid separation to obtain a precipitate.

[0080] In some embodiments, the macroporous resin is HP-20;

[0081] Preferably, the ratio of the sample load to the macroporous resin in the macroporous resin separation step is preferably 1:(1.5-2.5);

[0082] Preferably, the volume of the water eluent used in the macroporous resin separation step is 1-6 times the column volume.

[0083] In some embodiments, before the water extraction filtrate and the alcohol extraction filtrate are mixed,

[0084] The water extract filtrate and the alcohol extract filtrate are first concentrated and / or dried separately, and then mixed to form a mixed extract; the mixed extract is then dissolved in water, solid-liquid separation is performed, and the supernatant is collected and then separated by a macroporous resin;

[0085] Preferably, the alcohol-extracted filtrate is concentrated and / or dried to 0.8 g / L-1.2 g / L to form an alcohol-extracted extract;

[0086] The aqueous extract filtrate is concentrated and / or dried to 0.8 g / L-1.2 g / L to form an aqueous extract;

[0087] More preferably, the weight ratio of the alcohol extract to the water extract is 2-10:1.

[0088] In some embodiments, the preparation method further satisfies any one or more of the following A to D:

[0089] A: The alcohol extraction uses 50vol%-90vol% ethanol aqueous solution;

[0090] B: In the alcohol extraction step, the ethanol solution is refluxed and extracted at least twice, each time for 0.5 hours to 3 hours; the material-liquid ratio of each alcohol extraction is 1:(10-20), and the ratio relationship is g / mL;

[0091] C: In the water extraction step, water is added and decocted at least twice, each time for 0.5 hours to 1.5 hours; the material-liquid ratio of each water extraction is 1:(10-20), and the ratio relationship is g / mL;

[0092] D: The temperature of the water extraction is 60°C-90°C.

[0093] In some embodiments, before the water eluate is subjected to alcohol precipitation,

[0094] First, the water eluate is concentrated to 20%-30% of the original volume, and then alcohol precipitation is performed;

[0095] Preferably, 2-10 times the volume of a 50 vol%-95 vol% ethanol aqueous solution is added during the alcohol precipitation process;

[0096] More preferably, the alcohol precipitation time is 12 hours to 24 hours.

[0097] After the concentration, the present application performs alcohol precipitation, and the alcohol precipitation is preferably carried out using ethanol, and the volume concentration of the ethanol is preferably 94%-96%, and more preferably 95%; the volume ratio of the ethanol to the water eluent after concentration is preferably (3.5-4.5):1, more preferably (3.8-4.2):1, and even more preferably 4:1.

[0098] In some embodiments, in the solid-liquid separation step, the obtained solid is washed with an ethanol solution at least once, dissolved in water, and freeze-dried to obtain a precipitate;

[0099] Preferably, the concentration of the ethanol solution is 50 vol%-95 vol%.

[0100] The present application adopts a method of first extracting with alcohol and then with water, and then adsorbing with a macroporous resin and then extracting the eluent with alcohol to separate the active ingredients in the mixture of Chuanxiong and Gastrodia elata.

[0101] HP20 macroporous adsorption resin, based on a unique rigid polystyrene / divinylbenzene matrix, features a controlled pore size distribution and a large specific surface area. It is commonly used for the purification of small peptides, oligonucleotides, and proteins. Experimental studies have shown that the HP20 macroporous adsorption resin used in this application can adsorb more polysaccharides, particularly active ingredients for treating migraines.

[0102] During the extraction of the polysaccharide components of the medicinal material, the first step is ethanol extraction, preferably with an ethanol concentration of 85-95% (illustratively, 85%, 90%, or 95% can be selected), more preferably 90%. The number of extractions is preferably 1-3, more preferably 2. To enrich the water-soluble fraction, the alcohol-extracted residue is decocted with water, the number of decoctions being 1-3, more preferably 2.

[0103] In the present application, deionized water is used as the elution solvent, and the elution volume is preferably 4 column volumes, so as to obtain the water elution fraction of Chuanxiong-Gastrodia elata.

[0104] In the present application, after obtaining the water-eluted fraction of Chuanxiong-Gastrodia elata, the water-eluted fraction of Chuanxiong-Gastrodia elata is subjected to alcohol precipitation to obtain Chuanxiong-Gastrodia elata fraction 2 (LG P). In the present application, the water eluate is preferably concentrated to 20%-30% of the volume of the water-eluted fraction of Chuanxiong-Gastrodia elata, more preferably 22%-28%, and even more preferably 25%. In the present application, the concentration can be performed by vacuum concentration at a temperature not exceeding 45°C.

[0105] In the present application, after the alcohol precipitation, the precipitate is collected; the collected precipitate is repeatedly washed with 95% ethanol 2-4 times, more preferably 3 times. In the present application, the precipitate is preferably collected by filtration, preferably by Buchner funnel filtration; in the present application, the precipitate is preferably freeze-dried after collection to obtain component 2 (LG P).

[0106] In a third aspect, the present application also provides a pharmaceutical preparation.

[0107] In some embodiments, the pharmaceutical preparation comprises a preventive and / or therapeutically effective amount of a Chinese herbal extract;

[0108] The pharmaceutical preparation comprises the Chinese herbal medicine extract, or the Chinese herbal medicine extract prepared by the preparation method;

[0109] Preferably, the Chinese herbal medicine extract is the only active ingredient in the pharmaceutical preparation; or the pharmaceutical preparation also includes other active ingredients or drugs; other active ingredients include nutritional supplements.

[0110] The Chinese medicine preparation can be a capsule, pill, powder, syrup, tablet, granule, plaster, mixture, pill, suppository, aerosol, ointment or injection.

[0111] The usage amount of the traditional Chinese medicine extract is 0.02g / kg / day-0.12g / kg / day.

[0112] In a fourth aspect, the present application also provides a Chinese herbal extract, or the use of the Chinese herbal extract prepared by the preparation method or the pharmaceutical preparation in the preparation of drugs for preventing and / or treating migraine or middle cerebral artery occlusion syndrome.

[0113] Preferably, the migraine is a migraine caused by taking nitroglycerin;

[0114] Optionally, the migraine detection index includes at least one of CGRP, MAO-A, MAO-B, and 5-HT levels.

[0115] The symptoms of migraine include recurrent unilateral moderate to severe headaches, as well as nausea, vomiting, phonophobia, and photophobia.

[0116] The middle cerebral artery occlusive syndrome includes cerebral stroke or cerebral infarction caused by middle cerebral artery occlusion.

[0117] The present application embodiment provides a preparation method of component 2 (LG P), which is simple to operate and has high implementation efficiency. The raw materials used in the operation process are safe, low-cost, and environmentally friendly, and have wide application potential and good development prospects. The component 2 (LG P) described in the present application is analyzed for thermal stability by thermogravimetry. The present application also proposes the application of component 2 in the preparation of migraine drugs or middle cerebral artery occlusion syndrome drugs. The constructed model is a nitroglycerin-induced migraine model in rats, and the migraine therapeutic drugs specifically refer to the callback migraine gold indicator calcitonin gene-related peptide (CGRP), pain-related indicators (β-EP), nitric oxide synthase (iNOS), 5-HT content and its related metabolic enzymes (MAO-A\MAO-B). By examining neurobehavioral and cerebral infarction area, it is proved that the extract component 2 (L-GP) of the present application has a good therapeutic effect on middle cerebral artery occlusion syndrome.

[0118] The present application is further described in detail below with reference to specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in this application.

[0119] The Rhizoma Chuanxiong and Rhizoma Gastrodiae used in the examples were obtained from Jiangsu Kangyuan Pharmaceutical Co., Ltd. (Batch No.: Z220402). All reagents or instruments used without manufacturer indicated were commercially available conventional products.

[0120] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0121] Example 1 Preparation of active fraction of Chinese medicine composition

[0122] This embodiment provides a Chinese herbal compound extract and a preparation method thereof, wherein the preparation method comprises:

[0123] 40g of Chuanxiong rhizome and 10g of Gastrodia elata were weighed and extracted twice with 90% ethanol for one hour each time. The solid-liquid separation was performed, and the resulting ethanol extracts were combined and concentrated to 1.0g / L to obtain an ethanol extract. The remaining residue was decocted twice with water for one hour each time. The solid-liquid separation was performed, and the resulting aqueous extracts were combined and concentrated to 1.0g / L to obtain an aqueous extract. The aqueous and ethanol extracts were mixed in a weight ratio of 4:1 to obtain a Chuanxiong rhizome-Gastrodia elata mixed extract. This was dissolved in an equal volume of deionized water and centrifuged at 5000 rpm for 10 minutes to separate the insoluble matter and supernatant. The supernatant was loaded onto a 10L column volume of macroporous resin (HP-20). After dynamic adsorption reached equilibrium, the supernatant was eluted with 40L of deionized water, and 40L of aqueous eluate solution A was collected. Four column volumes of 95% ethanol were added to the macroporous resin and concentrated to dryness under reduced pressure on a rotary evaporator to obtain fraction 1 (LG SM). The water-eluted solution A was concentrated to one-quarter of its original volume using a rotary evaporator under reduced pressure. Four volumes of 95% ethanol were then added for alcohol precipitation, removing most small impurities such as pigments. The polysaccharide precipitate was collected by filtration and washed three times with an equal volume of 95% ethanol to remove pigments and other impurities. The solution was then dissolved in water, concentrated to remove excess ethanol using a rotary evaporator under reduced pressure, and completely dissolved in water. The solution was then freeze-dried to obtain Fraction 2 (LGP).

[0124] Example 2 Preparation of active fraction of Chinese medicine composition

[0125] This embodiment provides a traditional Chinese medicine extract and a preparation method thereof, wherein the preparation method comprises:

[0126] 40g of Chuanxiong rhizome and 30g of Gastrodia elata were weighed and extracted twice with 90% ethanol for one hour each time. The resulting alcohol extract was concentrated to 1.2g / L to obtain an alcohol extract. The remaining residue was decocted twice with water for one hour each time. The resulting aqueous extract was concentrated to 1.0g / L to obtain an aqueous extract. The aqueous and alcohol extracts were mixed in a ratio of 6:1 to obtain a Chuanxiong rhizome-Gastrodia elata mixed extract. This was dissolved in an equal volume of deionized water and centrifuged at 5000 rpm for 10 minutes to separate the insoluble matter and supernatant. The supernatant was loaded onto a 10L column of macroporous resin. After dynamic adsorption reached equilibrium, the solution was eluted with 40L of deionized water, and 40L of aqueous eluate solution A was collected. Four column volumes of 95% ethanol were added for elution, and the solution was concentrated to dryness under reduced pressure on a rotary evaporator to obtain fraction 1 (LG SM). The water elution solution A was concentrated to one-quarter of its original volume using a rotary evaporator. Four times the volume of 95% ethanol was then added for alcohol precipitation, removing most small impurities such as pigments. The polysaccharide precipitate was collected by filtration and washed with an equal volume of 95% ethanol three times to remove pigments and other impurities. The solution was then dissolved in water, concentrated to remove excess ethanol using a rotary evaporator, and completely dissolved in water. The solution was then freeze-dried to obtain Fraction 2 (LGP).

[0127] Example 3 Preparation of active fraction of Chinese medicine composition

[0128] Weigh 120g of Chuanxiong rhizome and 10g of Gastrodia elata and extract twice with 90% ethanol for one hour each time. The resulting alcohol extract is concentrated to 0.8g / L to obtain an alcohol extract. The remaining residue is decocted twice with water for one hour each time. The resulting aqueous extract is concentrated to 1.2g / L to obtain an aqueous extract. The aqueous and alcohol extracts are mixed in a ratio of 10:1 to obtain a Chuanxiong rhizome-Gastrodia elata mixed extract. This extract is dissolved in an equal volume of deionized water and centrifuged at 5000 rpm for 10 minutes to separate the insoluble matter and supernatant. The supernatant is loaded onto a 10L column of macroporous resin. After dynamic adsorption reaches equilibrium, the extract is eluted with 40L of deionized water, and 40L of the aqueous eluate is collected. Four column volumes of 95% ethanol are added for elution and concentrated to dryness on a rotary evaporator under reduced pressure to obtain fraction 1 (LG SM). The water-eluted fraction was concentrated to one-quarter of its original volume using a rotary evaporator. Four times the volume of 95% ethanol was added for alcohol precipitation, removing most small impurities such as pigments. The polysaccharide precipitate was collected by filtration and washed with an equal volume of 95% ethanol three times to remove pigments and other impurities. Water was then added for dissolution, and excess ethanol was concentrated using a rotary evaporator. After complete dissolution with water, fraction 2 (LGP) was obtained by freeze-drying.

[0129] Experimental Example 4: Therapeutic Effects of Different Components of Traditional Chinese Medicine on Nitroglycerin-Induced Migraine in Rats

[0130] 1. Experimental Materials

[0131] 1.1 Animals

[0132] For the experiment, SPF-grade male Sprague-Dawley (SD) rats, 6-8 weeks old and weighing 200 ± 20 g, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (production license number: SCXK2019-0001). Rats were housed in a clean environment with a controlled temperature of 23 ± 2°C, a humidity of 50%-60%, and a 12-hour daylight cycle. Food was available ad libitum, and drinking water was filtered and autoclaved.

[0133] 1.2 Drugs and Reagents

[0134] Nitroglycerin injection (Guangzhou Baiyunshan Mingxing Pharmaceutical Co., Ltd., batch number 1210201); Flunarizine hydrochloride capsules (Xi'an Janssen Pharmaceutical Co., Ltd., batch number MDJ4985)

[0135] The total component (LG), component 1 (LG SM), and component 2 (LG P) of the Chuanxiong-Gastrodia elata mixed extract were prepared according to the method of Example 1.

[0136] 1.3 Instruments

[0137] Refrigerated centrifuge (ThermoFisher Scientific, USA); vortex mixer (Qilin Bell Instrument Manufacturing Co., Ltd.); electronic balance (Startorius, USA); pathology slicer (Leica Instrument Co., Ltd., Shanghai); upright optical microscope (Nikon Corporation, Japan)

[0138] 2. Dosage design

[0139] The rat dosage of the Chinese medicine composition (mixed extract) is 1.12 g crude drug / kg / day. Based on the extraction rate and yield of the medicinal materials, the rat dosage of component 1 is 150 mg / kg / day, and the equivalent rat dosage of component 2 is 200 mg / kg / day.

[0140] The daily dosage of flunarizine hydrochloride capsules is 10 mg / day. According to the body surface area conversion method, the equivalent dose for rats is 1 mg crude drug / kg / day.

[0141] 3. Experimental Methods

[0142] After the rats were adapted to feeding, they were randomly divided into 6 groups (8 rats in each group) according to their body weight, namely, a blank control group (Control), a model group (Model), a drug group 1 (Ligusticum chuanxiong-Gastrodia elata group) (LG, 1.12g crude drug / kg / day), a drug group 2 (component 2L-GP, 200mg / kg / day), a drug group 3 (component 1L-G SM, 150mg / kg / day), and a positive drug group (flunarizine hydrochloride capsule, 1mg / kg / day). Except for the blank control group, nitroglycerin injection (10mg / kg) was subcutaneously injected once a week for 5 consecutive weeks. The blank control group was subcutaneously injected with an equal volume of solvent. Two weeks after modeling (after 14 days), the treatment group began to receive medication once a day for 15 consecutive days. The blank control group and the model group were given equal volumes of solvent. After the final modeling, brain tissues were collected to detect nitric oxide synthase (iNOS), β-endorphin (β-EP), calcitonin gene-related peptide (CGRP), MAO-A, MAO-B, and 5-HT levels in the brain tissues. GraphPad Prism software was used to generate graphs for analysis of intergroup differences.

[0143] Brain tissue pathology was performed on rats induced with nitroglycerin. Modeling and drug administration were performed as described above. After the last modeling, the rat brain tissue was collected and fixed with 10% neutral formalin solution. After 48 hours, the tissue was embedded in paraffin and sliced at a thickness of 4 μm. The sections were then subjected to immunofluorescence staining according to the following procedures: ① Dewaxing of sections; ② Antigen retrieval; ③ Circle serum blocking; ④ Primary antibody (CGRP) incubation; ⑤ Secondary antibody incubation; ⑥ DAPI counterstaining; ⑦ Quenching of tissue autofluorescence; ⑧ Sealing; ⑨ Image acquisition.

[0144] Brain tissue pretreatment method: Modeling and drug administration were performed as described above. After the final modeling, brain tissue samples were collected from each group of rats, and the trigeminal ganglion was isolated using a rat brain stereotaxic instrument. The trigeminal ganglion was washed with saline and weighed. Then, 9 times the volume of saline was added for homogenization. The homogenate was centrifuged at 3000 rpm for ten minutes, and the supernatant was collected for biochemical analysis. Biochemical indicators included calcitonin gene-related peptide (CGRP), monoamine oxidase A (MAO-A), and monoamine oxidase B (MAO-B).

[0145] 4. Experimental Results

[0146] (1) Effects on biochemical parameters of rats with migraine induced by nitroglycerin

[0147] The CGRP fluorescence intensity in the migraine model group was higher than that in the control group. In addition, both LG and component 2 treatments attenuated the increased immunoreactivity of CGRP. Figure 1 and as shown in Table 1.

[0148] Table 1 Immunofluorescence quantitative analysis

[0149]

[0150] # Indicates that there is a statistical difference compared with the Control group. ## Indicates significant difference compared with the Control group. ### Indicates that the difference compared with the Control group is extremely significant; * Indicates that there is a statistical difference compared with the Model group. ** Indicates significant difference compared with the Model group. *** Indicates that the difference compared with the Model group is extremely significant.

[0151] The effect of the composition of the present application on the secretion of biochemical indicators in the brain tissue of rats with migraine induced by nitroglycerin. Figure 2-4 and as shown in Table 2. Figure 2 Histogram of CGRP content levels in midbrain tissue; Figure 3 Histogram of MAO-A content levels in brain tissue; Figure 4 Histogram of MAO-B levels in brain tissue; Data are expressed as mean ± SD, n = 3. **P < 0.001, **P < 0.01, *P < 0.05 compared with the control group.

[0152] In this study, experimental data are expressed as mean ± SEM. Data statistics and graphs were performed using GraphPad Prism 9.3. Significant differences between the control and treatment groups were analyzed using one-way ANOVA combined with Dunnett's post-hoc test. P values less than 0.05 were considered statistically significant. Component 2 (LG P) significantly reversed changes in biochemical parameters in the migraine model, and its efficacy was superior to that of component 1 (LG SM) and the active agent flunarizine hydrochloride capsules.

[0153] Table 2 Analysis of biochemical indexes

[0154]

[0155]

[0156] # Indicates that there is a statistical difference compared with the Control group. ## Indicates significant difference compared with the Control group. ### Indicates that the difference compared with the Control group is extremely significant; * Indicates that there is a statistical difference compared with the Model group. ** Indicates significant difference compared with the Model group. *** Indicates that the difference compared with the Model group is extremely significant

[0157] Test Example 5: Determination of the content of Chinese medicinal components

[0158] 1. Experimental Methods

[0159] 1. Purpose of the experiment

[0160] The contents of neutral sugar, uronic acid and protein in the component 2 sample prepared in Example 1 were determined by phenol-sulfuric acid method, m-hydroxybiphenyl method and Coomassie brilliant blue method, respectively.

[0161] 2. Experimental Reagents

[0162] D-glucose standard (Shanghai Maclean Biochemical Technology Co., Ltd.), concentrated sulfuric acid (Guangdong Guangshi Reagent Technology Co., Ltd.), phenol (Shanghai Maclean Biochemical Technology Co., Ltd.), 95% ethanol (Tianjin Damao Chemical Reagent Factory), Coomassie Brilliant Blue (Shanghai Maclean Biochemical Technology Co., Ltd.).

[0163] 3. Experimental instruments: Synergy HTX microplate reader (Bio-Tek, USA), Scilogex vortex shaker (Beijing Kebosail Technology Co., Ltd.), TOMY CAX-371 low-temperature high-speed centrifuge (TOMY KOGYO, Japan).

[0164] 4. Experimental Methods

[0165] (1) Glucose standard curve

[0166] The OD values of glucose standard solutions of different concentrations measured by the phenol-sulfuric acid method at 490 nm were used as the standard curve for neutral sugar content. The fitting equation of the standard curve was y = 0.0062x + 0.0542, and the correlation coefficient (R2) was 0.9994, indicating that the linear regression equation fitted well.

[0167] (2) Uric acid standard curve

[0168] The OD values of galacturonic acid standard solutions of different concentrations measured by the m-hydroxybiphenyl method at 520 nm were used as the standard curve of uronic acid content. The fitting equation of the standard curve was y=0.004x+0.0581, and the correlation coefficient (R2) was 0.9920, indicating that the linear regression equation fitted well.

[0169] (3) Protein standard curve

[0170] The OD values of bovine serum albumin standard solutions of different concentrations measured by the Coomassie Brilliant Blue method at 595 nm were used as a standard curve of protein concentration. The fitting equation of the standard curve was y = 0.0019x + 0.3448, and the correlation coefficient (R2) was 0.9965, indicating that the linear regression equation fitted well.

[0171] 5. Experimental Results

[0172] Table 3 shows the results of the determination of the contents of the main chemical components. The results show that Component 2 prepared in Example 1 of the present application contains a relatively high level of neutral sugars, a small amount of uronic acid, and a polysaccharide extract with almost no protein impurities.

[0173] Table 3 Chemical composition analysis of component 2

[0174]

[0175] Experimental Example 6 Analysis of Thermal Stability of Component 2 Sample Prepared in Example 1

[0176] 1. Experimental Procedure

[0177] 5 mg of the component 2 sample prepared in Example 1 was accurately weighed and placed in a crucible, and differential scanning calorimetry (DSC) and thermogravimetry (TG) analysis was performed using a simultaneous thermal analyzer.

[0178] Thermodynamic parameters: inert gas is nitrogen; nitrogen flow rate is 40 mL / min; heating range is 35°C-600°C; heating rate is 10°C / min.

[0179] 2. Experimental Results

[0180] The thermal stability of component 2 is Figure 5 The TG and DTG graphs shown are used to illustrate. The first stage (30°C-120°C) of the TG curve of component 2 shows a slight decrease with a weight loss rate of 4.15%, which is attributed to the removal of free water and bound water. The main weight loss of component 2 occurs in the second stage (230°C-380°C), which may be due to the decomposition of the polysaccharide structure. The weight loss rate in the second stage is 48.40%. The maximum weight loss rate is obtained at 295.83°C. Overall, the results indicate that the structure of component 2 (the traditional Chinese medicine extract of the present application) is thermally stable below 230°C.

[0181] In summary, the present application proposes the use of the Chinese herbal extract of the present application in the treatment of migraine, including reducing CGRP, iNOS, MAO-A, MAO-B and 5-HT in migraine models, and increasing β-EP levels. In addition, for migraine patients, the Chinese herbal extract of the present application has a lower dosage than the whole compound extract, and the polysaccharide component has better efficacy than the small molecule chemical component, is more easily accepted by patients, and has fewer toxic side effects when taken long-term.

[0182] Example 7 Therapeutic Effects of Different Components of Traditional Chinese Medicine on Middle Cerebral Artery Occlusion (MCAO) Model

[0183] 1. Experimental Materials

[0184] 1.1 Animals

[0185] Male Sprague-Dawley rats, SPF grade, weighing 240–260 g, were provided by Sibeifu (Beijing) Laboratory Animal Co., Ltd., license number: SCXK(Beijing)2019-0010. Housing conditions: room temperature 20–26°C, humidity 50–70%, free access to food and water, bedding changed twice weekly, and disinfected drinking water.

[0186] 1.2 Drugs and Reagents

[0187] TTC, Sigma, batch number BCBP3272Y, size 100g; 0.9% sodium chloride injection, Huaren Pharmaceutical (Rizhao) Co., Ltd., batch number 15121262, size 100ml; Edaravone, Yangtze River Pharmaceutical Group Nanjing Hailing Pharmaceutical Co., Ltd.; Isoflurane, Shenzhen Ruiwode Life Science Technology Co., Ltd. The total component (LG), component 1 (LG SM), and component 2 (LG P) of the Chuanxiong-Gastrodia mixed extract were prepared according to the method in Example 1.

[0188] 1.3 Instruments

[0189] Cannon camera, EOS 550D (Canon, Japan); BS224S electronic balance (Sartorius Scientific Instrument Co., Ltd.); DK-S26 constant temperature water bath (Shanghai Jinghong Experimental Equipment Co., Ltd.); small animal gas anesthesia machine (Shenzhen Ruiwode Life Science Technology Co., Ltd., model R500).

[0190] 2-dose design

[0191] The daily dosage of the Chinese medicine composition is 12g of crude drug per day. Based on the body surface area conversion method, the equivalent dose for rats is 12g / 60kg*(36.88 / 6.59)≈1.12g crude drug / kg / day. Based on the extraction rate (57%) and yield (component 1 = 20%, component 2 = 28%), the equivalent dose for component 1 in rats is 150mg / kg / day, and the equivalent dose for component 2 in rats is 200mg / kg / day. The equivalent dose of edaravone is 3mg / kg.

[0192] 3. Experimental Methods

[0193] After one week of adaptive feeding, rats were randomly divided into six groups: a sham group, a model group, drug group 1 (Chuanxiong-Gastrodia elata mixed extract LG, 1.12 g crude drug / kg / day), drug group 2 (component 2L-GP, 200 mg / kg / day), drug group 3 (component 1L-G SM, 150 mg / kg / day), and an edaravone positive control group (3 mg / kg). Except for the sham group, which consisted of eight rats per group, all other groups had 20 rats per group. Before the experiment, rats in each group were fasted for 12 hours and anesthesia was induced with 4% isoflurane, maintained with 2.5% isoflurane. Anesthetized rats were fixed in the supine position on the operating table. A midline incision was made in the neck, and the right common carotid artery was isolated and occluded for middle cerebral artery occlusion. The suture was inserted to a depth of approximately 18 mm. Two hours later, the suture was withdrawn to the bifurcation of the internal and external carotid arteries, resulting in reperfusion (24 hours). The sham-operated group underwent the same surgical procedure as the model group, except that the middle cerebral artery was not occluded. The corresponding drug was administered orally at the beginning of cerebral ischemia-reperfusion and 12 hours after reperfusion (the sham-operated and model groups were simultaneously given an equal volume of normal saline solution). Behavioral assessments were performed 24 hours after reperfusion, and the rats were sacrificed. Brain tissue was then collected for TTC staining.

[0194] 4. Detection indicators

[0195] 4.1 Neurobehavioral scoring

[0196] After 2 hours of ischemia and 24 hours of reperfusion, the rats were suspended approximately 35 cm above the ground by their tails, and the condition of their forelimbs was observed. The rats were then placed on a level surface and their shoulders were pushed to observe the difference in resistance. The rats were then placed on the ground and their walking was observed. A score of 0 was assigned if the rat's behavior was completely normal. A score of 1 was assigned if the rat's contralateral forelimb, while suspended by its tail, showed wrist and elbow flexion, shoulder internal rotation, elbow abduction, and adherence to the chest wall. A score of 2 was assigned if the rat's shoulder, when pushed against the surgical side, showed reduced resistance to movement. A score of 3 was assigned if the rat, while on the ground, circled the contralateral side. A score of 4 was assigned if the rat was severely injured and unable to move independently. A score of 5 was assigned if the rat died. Higher scores indicated more severe neurobehavioral impairment.

[0197] 4.2 Cerebral infarction area (TTC staining method)

[0198] Rats were anesthetized by intraperitoneal injection, and the brains were removed. The olfactory bulbs, cerebellum, and lower brainstem were removed and weighed wet. The brains were refrigerated at -20°C for 20 minutes and then cut coronally into five slices approximately 2 mm thick. The slices were quickly incubated in 5 ml of phosphate-buffered saline (PBS) containing 2% TTC at 37°C in the dark for 20 minutes, with the slices stirred every 5-6 minutes. After staining, normal tissue appears pink, while ischemic tissue appears white. After incubation, the brains were photographed, and Image-Pro Plus software was used to calculate the infarct area, normal area, and total brain area to calculate the cerebral infarction rate. The cerebral infarction rate = total infarct area / total brain area × 100%.

[0199] 5. Statistical methods

[0200] Experimental data are expressed as mean ± standard deviation and analyzed using GraphPad 9.3 statistical software. One-way analysis of variance was used to compare means between multiple groups, and the t-test was used to compare means between two groups. P < 0.05 was considered statistically significant.

[0201] 6. Experimental Results

[0202] As shown in Table 4, 24 hours after reperfusion, the rats were evaluated for neurobehavioral scores. Compared with the sham group, the neurobehavioral scores in the model group were significantly reduced (P < 0.001). Compared with the MCAO group, the LG, LG P, LG SM, and edaravone groups all improved the neurobehavioral scores of the rats, with significant differences (P < 0.001). The degree of improvement in the neurobehavioral scores of the rats in the LG P group was greater than that in the LG, LG SM, and edaravone groups.

[0203] Table 4 Neurobehavioral scores

[0204]

[0205] Note: Compared with the sham operation group, ###P<0.001; compared with the model group, *P<0.05, **P<0.01, ***P<0.001; data are expressed as Mean±SD.

[0206] Twenty-four hours after reperfusion, the rats were sacrificed, and the brains were frozen, sectioned, and stained with TTC. As shown in Table 5, the mean infarct size in the model group was 35.69±2.99%. Compared with the MCAO group, the LG, LG P, LG SM, and edaravone groups all reduced cerebral infarct size, with statistically significant differences (LG vs. model group: 29.90±6.04% vs. 35.69±2.99%, P < 0.05; LG P vs. model group: 21.25±7.56% vs. 35.69±2.99%, P < 0.001; LG SM vs. model group: 28.10±7.86% vs. 35.69±2.99%, P < 0.01; edaravone vs. model group: 23.27±8.83% vs. 35.69±2.99%, P < 0.001). The efficacy of LG P was better than that of LG group, LG SM group and edaravone group.

[0207] Table 5 Statistics of cerebral infarction area (%)

[0208]

[0209]

[0210] Note: Compared with the sham operation group, ### P<0.001; compared with the model group, *P<0.05, **P<0.01, ***P<0.001; data are expressed as Mean±SD.

[0211] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of this application.

Claims

1. A Chinese herbal medicine extract, characterized in that The Chinese herbal medicine extract is extracted from a prescription comprising Chuanxiong rhizome and Gastrodia elata; the Chinese herbal medicine extract contains 60 wt.% to 70 wt.% of neutral sugar.

2. The Chinese herbal extract according to claim 1, characterized in that The Chinese herbal medicine extract further contains 10wt.%-20wt.% of uronic acid and / or 0.5wt.%-1.2wt.% of protein; Preferably, the Chinese herbal medicine extract contains 65.10 wt.% of neutral sugar, 14.22 wt.% of uronic acid, and 0.62 wt.% of protein; More preferably, the weight ratio of Chuanxiong and Gastrodia elata in the prescription is (40-120): (10-30); More preferably, the weight ratio of Chuanxiong and Gastrodia elata is 4:

1.

3. The method for preparing the Chinese medicine extract according to claim 1 or 2, characterized in that: The preparation method comprises the following steps: Extracting the Chinese medicinal materials including Chuanxiong rhizome and Gastrodia elata with alcohol to obtain an alcohol extraction residue and an alcohol extraction filtrate; The alcohol extraction residue is subjected to water extraction again, the water extraction filtrate and the alcohol extraction filtrate are mixed, the mixed solution is separated by macroporous resin, washed with water, and the water eluate is collected; The water eluate is subjected to alcohol precipitation and solid-liquid separation to obtain a precipitate.

4. The preparation method according to claim 3, characterized in that: The model of the macroporous resin is HP-20; Preferably, the ratio of the sample load to the macroporous resin in the macroporous resin separation step is preferably 1:(1.5-2.5); Preferably, the volume of the water eluent used in the macroporous resin separation step is 1-6 times the column volume.

5. The preparation method according to claim 3 or 4, characterized in that Before the water extraction filtrate and the alcohol extraction filtrate are mixed, The water extract filtrate and the alcohol extract filtrate are first concentrated and / or dried separately, and then mixed to form a mixed extract; the mixed extract is then dissolved in water, solid-liquid separation is performed, and the supernatant is collected and then separated by a macroporous resin; Preferably, the alcohol-extracted filtrate is concentrated and / or dried to 0.8 g / L-1.2 g / L to form an alcohol-extracted extract; The aqueous extract filtrate is concentrated and / or dried to 0.8 g / L-1.2 g / L to form an aqueous extract; More preferably, the weight ratio of the alcohol extract to the water extract is 2-10:

1.

6. The preparation method according to any one of claims 3 to 5, characterized in that The preparation method also satisfies any one or more of the following A to D: A: The alcohol extraction uses 50vol%-90vol% ethanol aqueous solution; B: In the alcohol extraction step, the ethanol solution is refluxed and extracted at least twice, each time for 0.5 hours to 3 hours; the material-liquid ratio of each alcohol extraction is 1:(10-20), and the ratio relationship is g / mL; C: In the water extraction step, water is added and boiled at least twice, each time for 0.5 hours to 1.5 hours; the material-liquid ratio of each water extraction is 1:(10-20), and the ratio relationship is g / mL; D: The temperature of the water extraction is 60°C-90°C.

7. The preparation method according to any one of claims 3 to 6, characterized in that: Before the water eluent is subjected to alcohol precipitation, First, the water eluate is concentrated to 20%-30% of the original volume, and then alcohol precipitation is performed; Preferably, 2-10 times the volume of a 50 vol%-95 vol% ethanol aqueous solution is added during the alcohol precipitation process; More preferably, the alcohol precipitation time is 12 hours to 24 hours.

8. The preparation method according to any one of claims 3 to 7, characterized in that: In the solid-liquid separation step, the obtained solid is washed with an ethanol solution at least once, dissolved in water, and freeze-dried to obtain a precipitate; Preferably, the concentration of the ethanol solution is 50 vol%-95 vol%.

9. A pharmaceutical preparation, characterized in that The pharmaceutical preparation comprises a preventive and / or therapeutically effective amount of a Chinese herbal extract; The pharmaceutical preparation comprises the Chinese herbal extract according to claim 1 or 2, or the Chinese herbal extract prepared by the preparation method according to any one of claims 3 to 8; Preferably, the Chinese herbal medicine extract is the only active ingredient in the pharmaceutical preparation; or the pharmaceutical preparation also includes other active ingredients or drugs; other active ingredients include nutritional supplements.

10. Use of the Chinese medicinal extract according to claim 1 or 2, or the Chinese medicinal extract prepared by the preparation method according to any one of claims 3 to 8, or the pharmaceutical preparation according to claim 9 in the preparation of a drug for preventing and / or treating migraine or middle cerebral artery occlusive syndrome.

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