An acanthopanax senticosus injection for improving microcirculation and its preparation process

Abstract

The present invention provides an acanthopanax injection for improving microcirculation and its preparation process. The preparation method includes: (1) extracting acanthopanax and performing solid-liquid separation to obtain a filtrate; (2) enriching and purifying the filtrate through an adsorption column to obtain the acanthopanax extract; the filler of the adsorption column includes silica gel and a ligand; the ligand is selected from straight-chain or branched-chain alkyl groups with C3-C18; mixing the acanthopanax extract with sodium chloride and activated carbon for injection, boiling to obtain a mixture, and filtering the mixture to obtain the acanthopanax injection. The present invention uses a reverse-phase adsorption column to enrich and purify the acanthopanax filtrate, which can effectively remove sensitizing substances such as tannins and polysaccharide macromolecules, and can retain the active ingredients to the greatest extent; the acanthopanax injection provided by the present invention can effectively improve microcirculation. Experiments show that it has a therapeutic effect on mice with an acute renal ischemic reperfusion model.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and particularly relates to an acanthopanax injection for improving microcirculation and its preparation process. Background Art

[0002] Microcirculation refers to the blood circulation between arterioles and venules. The most fundamental function of blood circulation is to carry out material exchange between blood and tissues, and this function is achieved in the microcirculation part.

[0003] The microcirculation blood flow is adapted to the metabolic levels of human tissues and organs, enabling the normal operation of the physiological functions of various organs in the human body. When microcirculation dysfunction or microcirculation blood perfusion decreases, nutrients and oxygen cannot meet the needs of tissue metabolism, and at the same time, waste in tissues and organs cannot be discharged in time, which can lead to tissue and organ dysfunction or failure, and this becomes an important cause for the occurrence and development of many diseases.

[0004] Microcirculation refers to the blood circulation between arterioles and venules. The basic function of microcirculation is to carry out material exchange between blood and tissue fluid. Under normal circumstances, the blood flow of microcirculation is adapted to the metabolic levels of tissues and organs, ensuring the blood perfusion of each tissue and organ and regulating the volume of blood returning to the heart. If microcirculation disorders occur, it will directly affect the physiological functions of various organs. Therefore, improving human microcirculation plays an extremely important role in regulating human health.

[0005] Acanthopanax is the dry root, rhizome or stem of the acanthopanax senticosus (Rupr. et Maxim.) Harms of the Araliaceae family, which is mainly distributed in Heilongjiang, Jilin, Liaoning, Hebei and Shanxi. It has a pungent and slightly bitter taste, and is warm in nature. It belongs to the spleen, kidney and heart meridians, and has the effects of replenishing qi and strengthening the spleen, tonifying the kidney and calming the mind.

[0006] Modern pharmacological studies have shown that acanthopanax has the effects of protecting the central nervous system and cardiovascular system, anti-tumor, anti-aging, anti-radiation, enhancing the body's immunity, etc., and has significant curative effects especially in the treatment of ischemic stroke. Acanthopanax contains many active ingredients, mainly including triterpenoids, polysaccharides, lignans, coumarins, and various trace elements. Acanthopanax glycosides are the main active substances of acanthopanax. Its phenolic glycoside compounds mainly include acanthopanax glycoside A, acanthopanax glycoside B (syringin), acanthopanax glycoside B1, acanthopanax glycoside C, acanthopanax glycoside D and acanthopanax glycoside E, and its sugar components include glucose, fructose and arabinose, etc. Acanthopanax glycoside B (syringin) has the effects of anti-fatigue, enhancing immunity, protecting the liver, releasing acetylcholine, increasing insulin secretion, etc.; acanthopanax glycoside E has the effects of relieving stress, anti-anxiety, anti-stress, anti-ulcer and anti-fatigue, etc.; isofraxidin is the aglycone of acanthopanax glycoside B1, and has obvious anti-inflammatory and antibacterial effects.

[0007] Traditional Chinese medicine is composed of many different chemical components, and the organic combination of these different components is different from the simple addition of each individual component. Therefore, it is particularly important to study the extraction and preparation processes of multiple active components of traditional Chinese medicine.

[0008] The traditional process of the draft quality standard of Acanthopanax senticosus injection publicized by the National Pharmacopoeia Commission is as follows: Take Acanthopanax senticosus, crush it, decoct it with water, filter it, adjust the pH value of the filtrate to 10 - 12 with lime milk, stir it, adjust the pH value to 5.0 - 6.0 with 20% sulfuric acid, stir it, let it stand, concentrate the supernatant to a certain relative density, add ethanol to make the alcohol content 83 - 85%, let it stand, filter it, recover the ethanol from the filtrate, add injection water to adjust the total flavonoids to a certain content, adjust the pH value, heat it, refrigerate it, filter it, add sodium chloride to the filtrate and stir well or add activated carbon to the solution and boil it, cool it, filter it, adjust the pH value of the filtrate with sodium hydroxide, and filter it. Then fill and seal it, sterilize it, and it is ready. This method uses the traditional lime-sulfur method to extract the active components of Acanthopanax senticosus, with more steps, a more cumbersome process, and a long extraction cycle.

[0009] CN109010398A discloses an Acanthopanax senticosus injection and its preparation process. This process improves the process based on the key steps that are likely to cause product clarity and safety during the production of Acanthopanax senticosus injection, and adds sedimentation and separation treatment steps; effectively removes macromolecular substances rapidly when macromolecules in the refined liquid polymerize, significantly reduces impurity components, and effectively improves the clarity of the injection, but does not investigate the safety and medicinal effects of the injection.

[0010] CN104306417A discloses a low-toxic Acanthopanax senticosus injection and its preparation method. The Acanthopanax senticosus injection contains the following active components: containing 1.8 - 5.5 mg / mL of total flavonoids, 0.13 - 0.70 mg / mL of syringin, and 0.06 - 0.34 mg / mL of acanthoside E, and the amount of potassium ions ≤ 800 μg / mL. The method provides an Acanthopanax senticosus injection with a low potassium ion content. The potassium ion content control of this preparation is reduced from ≤ 1000 μg / mL in the national standard to below 800 μg / mL (or calculated as 800 μg / 5 mg of total flavonoids); it has low abnormal toxicity and high safety.

[0011] Therefore, developing an Acanthopanax senticosus injection that can effectively improve microcirculation is the research focus in this field. Summary of the Invention

[0012] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide an Acanthopanax senticosus injection for improving microcirculation and its preparation process, which can effectively improve the microcirculation of the body.

[0013] To achieve the purpose of this invention, the present invention adopts the following technical solutions:

[0014] In a first aspect, the present invention provides a method for preparing an acanthopanax senticosus extract, and the preparation method includes the following steps:

[0015] (1) Extract acanthopanax senticosus and perform solid-liquid separation to obtain a filtrate;

[0016] (2) Enrich and purify the filtrate through an adsorption column to obtain the acanthopanax senticosus extract;

[0017] The packing material of the adsorption column includes silica gel and a ligand; the ligand is selected from straight-chain or branched-chain alkyl groups with C3-C18.

[0018] The present invention uses an adsorption column to enrich and purify the filtrate obtained by extraction and solid-liquid separation, which can effectively remove sensitizing substances such as tannins and polysaccharide macromolecules in the filtrate, and can retain the active ingredients to the greatest extent. The entire experimental operation process is simple, and the reagents used in the enrichment and purification process can be reused.

[0019] The ligand is selected from straight-chain or branched-chain alkyl groups with C3-C18, and can be, for example, C3, C4, C8, C18, etc.

[0020] Preferably, a pretreatment step is also included before the extraction.

[0021] Preferably, the pretreatment is to crush the roots and stems of acanthopanax senticosus.

[0022] Preferably, the material-liquid ratio for extraction is 1:(6-10), and can be, for example, 1:7, 1:8, or 1:9, etc.

[0023] Preferably, the extraction temperature is 90-100 °C, and can be, for example, 92 °C, 95 °C, or 97 °C, etc., and the time is 2-3 h, and can be, for example, 2.3 h, 2.5 h, or 2.8 h, etc.

[0024] Preferably, the extraction is repeated 1-2 times.

[0025] Preferably, the solid-liquid separation includes filtration and ultrafiltration.

[0026] Preferably, the filtration uses a ceramic membrane.

[0027] Preferably, the pore size of the ceramic membrane is 30-750 nm, and can be, for example, 50 nm, 100 nm, 200 nm, 500 nm, 600 nm, etc.

[0028] Preferably, the material of the ceramic membrane includes any one of alumina, zirconia, or titanium oxide.

[0029] Preferably, the ultrafiltration uses a hollow fiber membrane.

[0030] Preferably, the molecular weight cut-off of the hollow fiber membrane is 5 - 800 kD, and can be, for example, 10 kD, 50 kD, 100 kD, 200 kD, 500 kD, 600 kD, 700 kD, 750 kD, etc.

[0031] Other specific point values within the above numerical ranges can be selected and will not be elaborated one by one here.

[0032] Preferably, the adsorption column is a reverse-phase chromatography column.

[0033] Preferably, the particle size of the packing is 5 - 60 μm, and can be, for example, 6 μm, 7 μm, 10 μm, 15 μm, 30 μm, 40 μm, or 60 μm, etc.

[0034] Preferably, the enrichment and purification include the steps of loading, first elution, second elution, and regeneration.

[0035] Preferably, the flow rate of the filtrate during loading is 10 - 14 BV / h, and can be, for example, 11 BV / h, 12 BV / h, or 13 BV / h, etc.

[0036] Preferably, the flow rate of the eluent for the first elution and the second elution is 10 - 14 BV / h, and can be, for example, 11 BV / h, 12 BV / h, or 13 BV / h, etc.

[0037] Preferably, the flow rate of the regeneration solution for regeneration is 10 - 14 BV / h, and can be, for example, 11 BV / h, 12 BV / h, or 13 BV / h, etc.

[0038] BV / h refers to the average liquid volume flowing through a unit volume of resin in the column per unit time.

[0039] Preferably, after the enrichment and purification, a step of vacuum concentration is further included.

[0040] Other specific point values within the above numerical ranges can be selected and will not be elaborated one by one here.

[0041] Preferably, the eluent for the first elution is ethanol aqueous solution a.

[0042] Preferably, the mass percentage of ethanol in the ethanol aqueous solution a is 25 - 35%, and can be, for example, 27%, 30%, or 32%, etc.

[0043] Preferably, the eluent for the second elution is ethanol aqueous solution b.

[0044] Preferably, the mass percentage of ethanol in the ethanol aqueous solution b is 55 - 65%, and can be, for example, 58%, 60%, or 63%, etc.

[0045] Preferably, the regenerating solution is an ethanol aqueous solution c.

[0046] Preferably, the mass percentage of ethanol in the ethanol aqueous solution c is 85-95%, for example, it can be 88%, 90% or 93%, etc.

[0047] Preferably, the preparation method includes the following steps:

[0048] (1) Extract Acanthopanax senticosus at 90-100 °C for 2-3 h, repeat the extraction 1-2 times, then filter through a 30-750 nm ceramic membrane, and then ultrafilter through a hollow fiber membrane with a molecular weight cut-off of 5-800 kd to obtain a filtrate;

[0049] (2) Enrich and purify the filtrate through an adsorption column. The enrichment and purification includes the steps of loading, first elution, second elution, and regeneration; the flow rate of the filtrate during loading is 10-14 BV / h, first elute with an ethanol aqueous solution a at a flow rate of 10-14 BV / h, second elute with an ethanol aqueous solution b at a flow rate of 10-14 BV / h, and regenerate with an ethanol aqueous solution c at a flow rate of 10-14 BV / h;

[0050] The mass percentage of ethanol in the ethanol aqueous solution a is 25-35%; the mass percentage of ethanol in the ethanol aqueous solution b is 55-65%; the mass percentage of ethanol in the ethanol aqueous solution c is 85-95%;

[0051] The adsorption column is a reversed-phase chromatography column. The packing of the adsorption column includes silica gel and a ligand, and the ligand is selected from straight-chain or branched-chain alkyl groups of C3-C18.

[0052] Other specific point values within the above numerical ranges can be selected and will not be elaborated one by one here.

[0053] In a second aspect, the present invention provides an Acanthopanax senticosus extract, which is prepared by the preparation method provided in the first aspect.

[0054] Preferably, the mass percentage content of syringin in the Acanthopanax senticosus extract is 2-7.46%, for example, it can be 2.3%, 3%, 4%, 5%, 6% or 7%, etc.

[0055] Preferably, the mass percentage content of eleutheroside E in the Acanthopanax senticosus extract is 2-4.82%, for example, it can be 2.5%, 3%, 4%, 4.5%, etc.

[0056] Preferably, the mass percentage content of isofraxidin in the Acanthopanax senticosus extract is 0.3-2.7%, for example, it can be 0.5%, 1%, 1.5%, 2% or 2.5%, etc.

[0057] Other specific point values within the above numerical ranges can be selected and will not be elaborated one by one here.

[0058] In a third aspect, the present invention provides an application of the acanthopanax senticosus extract as described in the second aspect in an injection.

[0059] In a fourth aspect, the present invention provides an acanthopanax senticosus injection, and the acanthopanax senticosus injection is prepared by the following method, and the preparation method includes: mixing the acanthopanax senticosus extract as described in the second aspect with sodium chloride and activated carbon for injection, boiling to obtain a mixture, and filtering the mixture to obtain the acanthopanax senticosus injection;

[0060] Preferably, the mass-volume ratio of the activated carbon for injection to the acanthopanax senticosus extract is (0.1 - 0.4) g:1 mL, and for example, it can be 0.15 g:1 mL, 0.2 g:1 mL, or 0.3 g:1 mL, etc.

[0061] Preferably, the boiling temperature is 98 - 105 °C, and for example, it can be 100 °C, 102 °C, or 104 °C, etc., and the time is 25 - 35 min, and for example, it can be 28 min, 30 min, or 33 min, etc.

[0062] Preferably, the filtration includes primary filtration and secondary filtration.

[0063] Preferably, the pore size of the primary filtration is 0.2 - 1.2 μm, and for example, it can be 0.4 μm, 0.6 μm, 0.8 μm, or 1 μm, etc.

[0064] Preferably, the cut-off molecular weight of the secondary filtration is 5 - 800 kD, and for example, it can be 10 kD, 50 kD, 100 kD, 200 kD, 500 kD, 600 kD, 700 kD, 750 kD, etc.

[0065] Preferably, the mixture is cooled to 30 - 40 °C before filtration, and for example, it can be 33 °C, 35 °C, or 38 °C, etc.

[0066] Preferably, after filtration, it further includes steps of dilution, filling, and sterilization.

[0067] Preferably, the diluent used for dilution is water for injection.

[0068] Preferably, the sterilization temperature is 114 - 118 °C, and for example, it can be 115 °C, 116 °C, or 117 °C, etc., and the time is 35 - 45 min, and for example, it can be 38 min, 40 min, or 43 min, etc.

[0069] Other specific point values within the above numerical ranges can be selected and will not be elaborated one by one here.

[0070] Compared with the prior art, the present invention has the following beneficial effects:

[0071] The present invention uses a reverse adsorption column to enrich and purify the filtrate obtained by extraction and solid-liquid separation, which can effectively remove sensitizing substances such as tannins and polysaccharide macromolecules in the filtrate, and can retain the active ingredients to the greatest extent. For example, syringin, eleutheroside E and isofraxidin. The highest mass percentage content of syringin in the acanthopanax extract provided by the present invention is 7.46%, eleutheroside E is 4.82%, and isofraxidin is 2.7%;

[0072] The acanthopanax injection prepared with the acanthopanax extract provided by the present invention can effectively improve microcirculation. Experiments show that it has a therapeutic effect on mice with acute renal ischemic reperfusion model;

[0073] The preparation process provided by the present invention is simple, and the reagents used can be recycled and reused, saving costs. Description of the Drawings

[0074] Figure 1 It is the mixed standard characteristic spectrum of the acanthopanax injection in Application Example 1;

[0075] Figure 2 It is the characteristic spectrum of the acanthopanax injection in Application Example 1;

[0076] Figure 3 It is the characteristic spectrum of the acanthopanax injection in Application Example 2;

[0077] Figure 4 It is the characteristic spectrum of the acanthopanax injection in Application Example 3;

[0078] Figure 5 It is the characteristic spectrum of the acanthopanax injection in Application Example 4;

[0079] Figure 6 It is the characteristic spectrum of the acanthopanax injection in Application Example 5;

[0080] Figure 7 It is the characteristic spectrum of the acanthopanax injection in Application Example 6;

[0081] Figure 8 It is the characteristic spectrum of the acanthopanax injection in the comparative example;

[0082] Figure 9 It is the kidney diagram of the sham operation group, positive drug group, model group and comparative example in Test Example 2;

[0083] Figure 10 It is the kidney diagram of Application Examples 1-4 in Test Example 2;

[0084] Figure 11 It is the kidney diagram of Application Examples 5-6 in Test Example 2;

[0085] Figure 12 For the blood flow diagrams on the surfaces of the left and right kidneys of the positive control group in Test Example 2;

[0086] Figure 13 For the blood flow diagrams on the surfaces of the left and right kidneys of the sham operation group in Test Example 2;

[0087] Figure 14 For the blood flow diagrams on the surfaces of the left and right kidneys of the model group in Test Example 2;

[0088] Figure 15 For the blood flow diagrams on the surfaces of the left and right kidneys of Application Example 1 in Test Example 2;

[0089] Figure 16 For the blood flow diagrams on the surfaces of the left and right kidneys of Application Example 2 in Test Example 2

[0090] Figure 17 For the blood flow diagrams on the surfaces of the left and right kidneys of Application Example 3 in Test Example 2

[0091] Figure 18 For the blood flow diagrams on the surfaces of the left and right kidneys of Application Example 4 in Test Example 2

[0092] Figure 19 For the blood flow diagrams on the surfaces of the left and right kidneys of Application Example 5 in Test Example 2

[0093] Figure 20 For the blood flow diagrams on the surfaces of the left and right kidneys of Application Example 6 in Test Example 2

[0094] Figure 21 For the blood flow diagrams on the surfaces of the left and right kidneys of the comparative example in Test Example 2. Detailed implementation manners

[0095] The technical solutions of the present invention will be further described below through specific implementation manners. Those skilled in the art should understand that the described embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0096] As used herein, the terms "comprising", "including", "having", "containing" or any other variation thereof are intended to cover non-exclusive inclusion. For example, a composition, step, method, article or device containing the listed elements is not necessarily limited to those elements, but may also include other elements not explicitly listed or elements inherent to such composition, step, method, article or device.

[0097] "Optional" or "any one" means that the matters or events described thereafter may or may not occur, and this description includes the cases where the events occur and the cases where the events do not occur.

[0098] The indefinite articles "a" and "an" before the elements or components of the present invention do not limit the quantity requirements (i.e., the number of occurrences) of the elements or components. Therefore, "a" or "an" should be interpreted as including one or at least one, and the singular form of the elements or components also includes the plural form, unless the quantity clearly refers only to the singular form.

[0099] The description of terms such as "one embodiment", "some embodiments", "exemplarily", "specific example" or "some examples" described in the present invention means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this article, the schematic expressions of the above terms are not necessarily directed to the same embodiment or example.

[0100] The sources of reagents, raw materials or equipment in the following examples are as follows:

[0101] C1 adsorption column: provided by Wusuli River Pharmaceutical Co., Ltd. The packing of the adsorption column includes a matrix and a ligand. The matrix is silica gel, and the ligand is a self-made straight-chain or branched-chain alkyl group of C18;

[0102] C2 adsorption column: provided by Wusuli River Pharmaceutical Co., Ltd. The packing of the adsorption column includes a matrix and a ligand. The matrix is silica gel, and the ligand is a self-made straight-chain or branched-chain alkyl group of C18.

[0103] Example 1

[0104] This example provides an acanthopanax extract, and the preparation method of the acanthopanax extract is as follows:

[0105] (1) Crushing: Crushing the roots and stems of acanthopanax, sieving, and making the crushed material into acanthopanax coarse powder;

[0106] (2) Water extraction: Taking 1.0 kg of acanthopanax coarse powder, adding 12 times the amount of purified water, heating and decocting for 2 hours, and collecting the decoction; adding 10 times the amount of purified water to the medicinal residues, heating and decocting for 1 hour, collecting the decoction, and combining the two decoctions;

[0107] (3) Clarification and filtration: Clarifying and filtering the acanthopanax decoction with a 500 nm ceramic membrane to obtain a clarified filtrate;

[0108] (4) Ultrafiltration: Ultrafiltering the clarified acanthopanax filtrate with a 750 kd hollow fiber membrane to obtain an ultrafiltrate;

[0109] (5) Enrichment and purification: When adsorbing the ultrafiltrate of Acanthopanax senticosus with a C1 adsorption column, the filler particle size of the adsorption column is 40 μm, the filler weight is 1.5 kg, the upper liquid volume is 10 BV, the upper liquid flow rate is 8 BV / h, and then elute with 30% and 60% ethanol respectively. The eluent dosage is 4 BV of 30% ethanol and 1 BV of 60% ethanol, and the elution flow rate is 12 BV / h. Finally, regenerate with 2 BV of 90% ethanol, and the regeneration flow rate is 12 BV / h. Collect the eluent and concentrate it under reduced pressure to obtain the Acanthopanax senticosus extract.

[0110] Example 2

[0111] This example provides an Acanthopanax senticosus extract, and the preparation method of the Acanthopanax senticosus extract is as follows:

[0112] (1) Crushing: Crush the roots and stems of Acanthopanax senticosus, sieve them, and make them into the crushed material of Acanthopanax senticosus coarse powder;

[0113] (2) Water extraction: Take 1.0 kg of Acanthopanax senticosus coarse powder, add 10 times the amount of purified water, heat and decoct for 2.5 hours, and collect the decoction; add 8 times the amount of purified water to the medicinal residue, heat and decoct for 1.5 hours, collect the decoction, and combine the two decoctions;

[0114] (3) Clarification and filtration: Clarify and filter the Acanthopanax senticosus decoction with a 500 nm ceramic membrane to obtain a clarified filtrate;

[0115] (4) Ultrafiltration: Ultrafilter the clarified filtrate of Acanthopanax senticosus with a 50 kd hollow fiber membrane to obtain an ultrafiltrate;

[0116] (5) Enrichment and purification: When adsorbing the ultrafiltrate of Acanthopanax senticosus with a C1 adsorption column, the filler particle size of the adsorption column is 40 μm, the filler weight is 1.5 kg, the upper liquid volume is 12 BV, the upper liquid flow rate is 12 BV / h, and then elute with 30% and 60% ethanol respectively. The eluent dosage is 4 BV of 30% ethanol and 1 BV of 60% ethanol, and the elution flow rate is 12 BV / h. Finally, regenerate with 2 BV of 90% ethanol, and the regeneration flow rate is 12 BV / h. Collect the eluent and concentrate it under reduced pressure to obtain the Acanthopanax senticosus extract.

[0117] Example 3

[0118] This example provides an Acanthopanax senticosus extract, and the preparation method of the Acanthopanax senticosus extract is as follows:

[0119] (1) Crushing: Crush the roots and stems of Acanthopanax senticosus, sieve them, and make them into the crushed material of Acanthopanax senticosus coarse powder;

[0120] (2) Water extraction: Take 1.0 kg of Acanthopanax senticosus coarse powder, add 8 times the amount of purified water, heat and decoct for 3 hours, and collect the decoction; add 6 times the amount of purified water to the medicinal residue, heat and decoct for 2 hours, collect the decoction, and combine the two decoctions;

[0121] (3) Clarification and filtration: The Acanthopanax senticosus decoction is clarified and filtered with a 500 nm ceramic membrane to obtain a clarified and filtered solution.

[0122] (4) Ultrafiltration: The clarified and filtered Acanthopanax senticosus solution is ultrafiltered with a 5 kd hollow fiber membrane to obtain an ultrafiltrate.

[0123] (5) Enrichment and purification: When the Acanthopanax senticosus ultrafiltrate is adsorbed by a C1 adsorption column, the filler particle size of the adsorption column is 40 μm, the filler weight is 1.5 kg, the upper liquid volume is 12 BV, the upper liquid flow rate is 12 BV / h, and then it is eluted with 30% and 60% ethanol respectively. The eluent dosage is 4 BV of 30% ethanol and 1 BV of 60% ethanol, and the elution flow rate is 12 BV / h. Finally, it is regenerated with 2 BV of 90% ethanol, and the regeneration flow rate is 12 BV / h. The eluate is collected and concentrated under reduced pressure to obtain the Acanthopanax senticosus extract.

[0124] Example 4

[0125] This example provides an Acanthopanax senticosus extract, and the preparation method of the Acanthopanax senticosus extract is as follows:

[0126] The difference from Example 1 is only that the C1 adsorption column is replaced with a C2 adsorption column, and the other raw materials, dosages and preparation methods are the same as those in Example 1.

[0127] Example 5

[0128] This example provides an Acanthopanax senticosus extract, and the preparation method of the Acanthopanax senticosus extract is as follows:

[0129] The difference from Example 2 is only that the C1 adsorption column is replaced with a C2 adsorption column, and the other raw materials, dosages and preparation methods are the same as those in Example 2.

[0130] Example 6

[0131] This example provides an Acanthopanax senticosus extract, and the preparation method of the Acanthopanax senticosus extract is as follows:

[0132] The difference from Example 3 is only that the C1 adsorption column is replaced with a C2 adsorption column, and the other raw materials, dosages and preparation methods are the same as those in Example 3.

[0133] Application Example 1

[0134] This application example provides an Acanthopanax senticosus injection, and the preparation method of the Acanthopanax senticosus injection is as follows:

[0135] The acanthopanax senticosus extract prepared in Example 1 was added to 0.3% sodium chloride and boiled for 30 minutes. Activated carbon for injection with a mass-to-volume ratio of 0.2% g / mL to the acanthopanax senticosus extract was added, and boiling was continued at 100 °C for 30 minutes. The liquid medicine was cooled to 30 °C, filtered through a 0.45 μm plate-and-frame filter, diluted with injection water to a crude drug ratio of 1:2, i.e., diluted with injection water to 2 L, filled, and sterilized at 116 °C for 40 minutes to obtain the acanthopanax senticosus injection.

[0136] Application Example 2

[0137] This application example provides an acanthopanax senticosus injection, and the preparation method of the acanthopanax senticosus injection is as follows:

[0138] The acanthopanax senticosus extract prepared in Example 2 was added to 0.4% sodium chloride and boiled for 30 minutes. Activated carbon for injection with a mass-to-volume ratio of 0.3% g / mL to the acanthopanax senticosus extract was added, and boiling was continued at 100 °C for 30 minutes. The liquid medicine was cooled to 40 °C, filtered through a 0.65 μm plate-and-frame filter, diluted with injection water to a crude drug ratio of 1:2, i.e., diluted with injection water to 2 L, filled, and sterilized at 116 °C for 40 minutes to obtain the acanthopanax senticosus injection.

[0139] Application Example 3

[0140] This application example provides an acanthopanax senticosus injection, and the preparation method of the acanthopanax senticosus injection is as follows:

[0141] The acanthopanax senticosus extract prepared in Example 3 was added to 0.3% sodium chloride and boiled for 30 minutes. Activated carbon for injection with a mass-to-volume ratio of 0.4% g / mL to the acanthopanax senticosus extract was added, and boiling was continued at 100 °C for 30 minutes. The liquid medicine was cooled to 35 °C, filtered through a 0.22 μm plate-and-frame filter, diluted with injection water to a crude drug ratio of 1:2, i.e., diluted with injection water to 2 L, filled, and sterilized at 116 °C for 40 minutes to obtain the acanthopanax senticosus injection.

[0142] Application Example 4

[0143] This application example provides an acanthopanax senticosus injection, and the preparation method of the acanthopanax senticosus injection is as follows:

[0144] The difference from Application Example 1 is only that the acanthopanax senticosus extract solution prepared in Example 1 is replaced with an equal amount of the acanthopanax senticosus extract solution prepared in Example 4, and the other raw materials, dosages, and preparation methods are the same as those in Example 1.

[0145] Application Example 5

[0146] This application example provides an acanthopanax senticosus injection, and the preparation method of the acanthopanax senticosus injection is as follows:

[0147] The difference between this and Application Example 2 is only that the Acanthopanax senticosus extract prepared in Example 2 is replaced with an equal amount of the Acanthopanax senticosus extract prepared in Example 5, and the remaining raw materials, dosages, and preparation methods are the same as those in Example 2.

[0148] Application Example 6

[0149] This application example provides an Acanthopanax senticosus injection, and the preparation method of the Acanthopanax senticosus injection is as follows:

[0150] The difference between this and Application Example 3 is only that the Acanthopanax senticosus extract prepared in Example 3 is replaced with an equal amount of the Acanthopanax senticosus extract prepared in Example 6, and the remaining raw materials, dosages, and preparation methods are the same as those in Example 3.

[0151] Comparative Example

[0152] This comparative example provides an Acanthopanax senticosus injection prepared by a traditional process, and the preparation method of the Acanthopanax senticosus injection is as follows:

[0153] Take Acanthopanax senticosus, crush it, decoct it with water for 3 h, filter it, adjust the pH value of the filtrate to 11 with lime milk, stir for 10 min, adjust the pH value to 5.0 with 20% sulfuric acid, continue to stir for 10 min, let it stand for more than 4 h, take the supernatant and concentrate it, add ethanol to make the alcohol content 85%, add it while stirring, control the system temperature not to exceed 30 °C, let it stand for more than 12 h, filter it, recover the ethanol from the filtrate to obtain an Acanthopanax senticosus extract;

[0154] Adjust the Acanthopanax senticosus extract to contain 12 mg of total flavonoids per milliliter with water for injection, adjust the pH value to 5.0, heat it at 115 °C for 40 minutes, cool it to room temperature, filter it, stir the filtrate with 0.3% sodium chloride, filter it, adjust the pH value of the filtrate to 6.0 with 20% sodium hydroxide, and filter it. Seal it and sterilize it at 116 °C for 40 minutes to obtain the Acanthopanax senticosus injection.

[0155] Test Example 1

[0156] The content of the active ingredients in the Acanthopanax senticosus extracts prepared in Examples 1-6, the Acanthopanax senticosus injections prepared in Application Examples 1-6, and the Acanthopanax senticosus injection prepared in the comparative example was determined by high performance liquid chromatography (General Rules 0512, Volume IV, Chinese Pharmacopoeia 2020 Edition).

[0157] Chromatographic conditions and system suitability test: Use octadecylsilane chemically bonded silica gel as the filler; use acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution according to the program in the following table; the detection wavelength is 220 nm; the column temperature is 30 °C. The number of theoretical plates calculated by the syringin peak should be not less than 10000; the resolution between the isofraxidin peak and the adjacent impurity peak should be greater than 1.5.

[0158] Time (min) Mobile Phase A (%) Mobile Phase B (%) 0-20 10-20 90-80 20-30 20-25 80-75 30-40 40 60 40-50 10 90

[0159] Preparation of reference substance solution: Weigh appropriate amounts of syringin reference substance, eleutheroside E reference substance, and isofraxidin reference substance accurately. Dissolve them in methanol (dissolve eleutheroside E reference substance in 50% methanol first) to prepare a mixed solution containing 40 μg of syringin and eleutheroside E each and 10 μg of isofraxidin per 1 mL. That is how you get it.

[0160] Preparation of test solution: Take about 0.2 g of this product, weigh it accurately, place it in a small beaker, dissolve it in 20 mL of 50% methanol in portions, transfer it to a 25-mL volumetric flask, sonicate it (power 250 W, frequency 50 kHz) for 10 minutes, take it out, let it cool, add 50% methanol to the mark, shake well, filter, and take the subsequent filtrate. That is how you get it. Herein: This product represents the acanthopanax extract prepared in Examples 1 - 6, the acanthopanax injection prepared in Application Examples 1 - 6, and the acanthopanax injection prepared in the comparative example respectively.

[0161] Determination method: Accurately pipette 10 μL of the reference substance solution and 10 μL of the test solution respectively, inject them into the liquid chromatograph, and conduct the determination according to the aforementioned detection parameters to obtain the characteristic chromatogram. The detection results of the main components are shown in Table 1.

[0162] Detection results: The mixed standard characteristic chromatogram of the acanthopanax injection in Application Example 1 is as Figure 1 shown; the characteristic chromatogram of the acanthopanax injection in Application Example 1 is as Figure 2 shown; the characteristic chromatogram of the acanthopanax injection in Application Example 2 is as Figure 3 shown; the characteristic chromatogram of the acanthopanax injection in Application Example 3 is as Figure 4 shown; the characteristic chromatogram of the acanthopanax injection in Application Example 4 is as Figure 5 shown; the characteristic chromatogram of the acanthopanax injection in Application Example 5 is as Figure 6 shown; the characteristic chromatogram of the acanthopanax injection in Application Example 6 is as Figure 7 shown; the characteristic chromatogram of the acanthopanax injection in the comparative example is as Figure 8 shown.

[0163] Table 1

[0164]

[0165] According to the tabular data, the highest mass percentage content of syringin in the acanthopanax senticosus extract obtained by the preparation method provided by the present invention is 7.46%, that of acanthoside E is 4.82%, and that of isofraxidin is 2.7%. Acanthoside is the main active substance of acanthopanax senticosus. Among them, acanthoside B (syringin) has the effects of anti-fatigue, enhancing immunity, protecting the liver, releasing acetylcholine, increasing insulin secretion, etc.; acanthoside E has the effects of relieving stress, anti-anxiety, anti-stress, anti-ulcer and anti-fatigue, etc.; isofraxidin is the aglycone of acanthoside B1 and has obvious anti-inflammatory and antibacterial effects. By enriching and purifying the main active substances in acanthopanax senticosus injection, the prepared acanthopanax senticosus injection has stronger advantages in effectiveness and quality controllability.

[0166] Test Example 2

[0167] Improvement effect of acanthopanax senticosus injection on microcirculation in mice with acute renal ischemia-reperfusion injury model

[0168] Experimental animals: C57BL / 6J mice, 6 - 8 weeks old, 20 - 22 g.

[0169] Experimental method:

[0170] (1) Acute renal ischemia-reperfusion injury model: Inject 200 μL of 5% chloral hydrate into the mice. After 3 min, the mice enter the anesthetized state. Using the double-clamp method, clamp the bilateral renal blood vessels for 40 min. If the kidneys turn from rosy to black, it indicates that the ischemia model is successful.

[0171] (2) Administration method: Inject the acanthopanax senticosus injections prepared in Application Examples 1 - 6 and Comparative Examples into the tail vein of the mice once every two days, 250 μL each time, for a total of 14 days of administration, and observe the growth status of the mice.

[0172] Positive drug (shuxuening injection): Inject it into the tail vein of the mice once every two days. Each time, inject 52 μL of the stock solution and dilute it to 250 μL with normal saline for a total of 14 days of administration, and observe the growth status of the mice.

[0173] Divided into sham operation group, model group, positive drug group, comparative example group, and example group. Administer the drug and normal saline by tail vein injection once every two days for a total of 14 days of administration.

[0174] (3) Detection indexes:

[0175] Observation of kidney morphology: Sacrifice the mice, take out the left and right kidneys, place them neatly according to the grouping, and observe the morphological differences of the kidneys. The mice injected with the acanthopanax senticosus injections prepared in Application Examples 1 - 6 and Comparative Examples are respectively called Application Examples 1 - 6 and Comparative Examples. Detection results: The kidneys of the sham operation group, positive drug group, model group and comparative example are as Figure 9 shown; the kidneys of Application Examples 1 - 4 are asFigure 10 as shown; the kidneys in Application Example 5 and Application Example 6 are as Figure 11 shown.

[0176] Renal blood flow index: Open the abdominal cavity of the mouse from the abdomen, expose the kidneys, and use a laser speckle blood flowmeter to observe the blood flow index and blood flow changes on the surfaces of the left and right kidneys; the test results are shown in Table 2. Test results: The blood flow diagrams on the surfaces of the left and right kidneys in the positive control group are as Figure 12 shown; the blood flow diagrams on the surfaces of the left and right kidneys in the sham operation group are as Figure 13 shown; the blood flow diagrams on the surfaces of the left and right kidneys in the model group are as Figure 14 shown; the blood flow diagrams on the surfaces of the left and right kidneys in Application Example 1 are as Figure 15 shown; the blood flow diagrams on the surfaces of the left and right kidneys in Application Example 2 are as Figure 16 shown; the blood flow diagrams on the surfaces of the left and right kidneys in Application Example 3 are as Figure 17 shown; the blood flow diagrams on the surfaces of the left and right kidneys in Application Example 4 are as Figure 18 shown; the blood flow diagrams on the surfaces of the left and right kidneys in Application Example 5 are as Figure 19 shown; the blood flow diagrams on the surfaces of the left and right kidneys in Application Example 6 are as Figure 20 shown; the blood flow diagrams on the surfaces of the left and right kidneys in the comparative example are as Figure 21 shown.

[0177] Table 2

[0178]

[0179] According to the table data and blood flow diagrams, it can be seen that the kidney conditions in the sham operation group and the positive drug group (Shuxuening injection group) are comparable. The kidneys of the mice are intact in shape, with a rosy color, presenting a healthy state, indicating that Shuxuening injection has a good effect on improving microcirculation; the kidney condition in the model group is the worst, presenting a necrotic state and gradually shrinking; the comparative example group also has a certain effect on improving microcirculation. The kidneys in Application Example Groups 1-6 are rosy in color and uniform in shape, indicating that Application Example Groups 1-6 all have good effects on improving microcirculation, and among them, the effects of Examples 4 and 5 on improving microcirculation are better.

[0180] The applicant declares that the present invention uses the above-mentioned embodiments to illustrate the process method of the present invention, but the present invention is not limited to the above-mentioned process steps, that is, it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement to the present invention, the equivalent substitution of the raw materials selected for the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A method for preparing an extract of Eleutherococcus senticosus, characterized in that, The preparation method comprises the following steps: (1) Extract Acanthopanax senticosus and perform solid-liquid separation to obtain a filtrate; the solid-liquid separation includes filtration and ultrafiltration; the filtration uses a ceramic membrane with a pore size of 30 - 750 nm; the ultrafiltration uses a hollow fiber membrane with a molecular weight cut-off of 5 - 800 kd; The temperature of the extraction is 90 - 100 °C and the time is 2 - 3 h; (2) Enrich and purify the filtrate through an adsorption column, and the enrichment and purification includes the steps of loading, primary elution, secondary elution, and regeneration to obtain the Acanthopanax senticosus extract; The adsorption column is a reversed-phase chromatography column; The eluent for the primary elution is an ethanol aqueous solution a with an ethanol mass percentage of 25 - 35%; The eluent for the secondary elution is an ethanol aqueous solution b with an ethanol mass percentage of 55 - 65%; The regeneration solution is an ethanol aqueous solution c with an ethanol mass percentage of 85 - 95%; The packing of the adsorption column includes silica gel and a ligand; the ligand is selected from straight-chain or branched-chain alkyl groups of C3 - C18; 2. The preparation method according to claim 1, characterized in that, The material-liquid ratio of the extraction is 1:(6 - 10); 3. The preparation method according to claim 1, characterized in that, The extraction is repeated 1 - 2 times; 4. The preparation method according to claim 1, wherein The particle size of the packing is 5 - 60 μm; 5. The preparation method according to claim 1, characterized in that The flow rate of the filtrate during loading is 10 - 14 BV / h; 6. The preparation method according to claim 1, wherein, The flow rates of the eluents for the primary elution and the secondary elution are 10 - 14 BV / h; 7. The preparation method according to claim 1, characterized in that, The flow rate of the regeneration solution for regeneration is 10 - 14 BV / h; 8. The preparation method according to claim 1, characterized in that, After the enrichment and purification, a step of vacuum concentration is further included; 9. The preparation method according to claim 1, characterized in that, The preparation method comprises the following steps: (1) Extract Acanthopanax senticosus at 90 - 100 °C for 2 - 3 h, repeat the extraction 1 - 2 times, then filter through a 30 - 750 nm ceramic membrane, and then perform ultrafiltration through a hollow fiber membrane with a molecular weight cut-off of 5 - 800 kd to obtain a filtrate; (2) Enrich and purify the filtrate through an adsorption column, and the enrichment and purification includes the steps of loading, primary elution, secondary elution, and regeneration; the flow rate of the filtrate during loading is 10 - 14 BV / h, perform primary elution with ethanol aqueous solution a at a flow rate of 10 - 14 BV / h, perform secondary elution with ethanol aqueous solution b at a flow rate of 10 - 14 BV / h, and perform regeneration with ethanol aqueous solution c at a flow rate of 10 - 14 BV / h; The ethanol aqueous solution a contains ethanol with a mass percentage of 25 - 35%; the ethanol aqueous solution b contains ethanol with a mass percentage of 55 - 65%; the ethanol aqueous solution c contains ethanol with a mass percentage of 85 - 95%; The adsorption column is a reversed-phase chromatography column, and the packing of the adsorption column includes silica gel and a ligand, and the ligand is selected from straight-chain or branched-chain alkyl groups of C3 - C18; 10. An acanthopanax senticosus extract, characterized in that, The Acanthopanax senticosus extract is prepared by the preparation method according to any one of claims 1 - 9; 11. The acanthopanax senticosus extract according to claim 10, characterized in that, The mass percentage content of syringin in the Acanthopanax senticosus extract is 2 - 7.46%; 12. The acanthopanax senticosus extract according to claim 10, wherein, The mass percentage content of acanthoside E in the Acanthopanax senticosus extract is 2 - 4.82%; 13. The acanthopanax senticosus extract according to claim 10, characterized in that, The mass percentage content of isofraxidin in the Acanthopanax senticosus extract is 0.3 - 2.7%; 14. Use of the acanthopanax senticosus extract according to any one of claims 10-13 in the preparation of an injection.

15. An acanthopanax senticosus injection, characterized in that, The acanthopanax senticosus injection is prepared by the following method, and the preparation method includes: Mix the acanthopanax senticosus extract according to any one of claims 10-13 with sodium chloride and activated carbon for injection, boil to obtain a mixture, and filter the mixture to obtain the acanthopanax senticosus injection.

16. The acanthopanax senticosus injection according to claim 15, wherein, The mass-volume ratio of the activated carbon for injection to the acanthopanax senticosus extract is (0.1-0.4) g:1 mL.

17. The acanthopanax senticosus injection according to claim 15, characterized in that, The filtration includes primary filtration and secondary filtration.

18. The acanthopanax senticosus injection according to claim 17, characterized in that, The pore size of the primary filtration is 0.2-1.2 μm.

19. The acanthopanax senticosus injection according to claim 17, wherein, The molecular weight cut-off of the secondary filtration is 5-800 kd.

20. The acanthopanax senticosus injection according to claim 15, characterized in that, After the filtration, there are also steps of dilution, filling, and sterilization.

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