CN116173085B - Acanthopanax senticosus extract and preparation method and application thereof - Google Patents

Acanthopanax senticosus extract and preparation method and application thereof Download PDF

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CN116173085B
CN116173085B CN202310173066.XA CN202310173066A CN116173085B CN 116173085 B CN116173085 B CN 116173085B CN 202310173066 A CN202310173066 A CN 202310173066A CN 116173085 B CN116173085 B CN 116173085B
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acanthopanax
acid
extract
preparation
membrane
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CN116173085A (en
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王震
屈孟云
巴跃民
魏学春
姜文良
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Heilongjiang Quanle Pharmaceutical Co ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
    • A61K36/254—Acanthopanax or Eleutherococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/12—Drugs for genital or sexual disorders; Contraceptives for climacteric disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/20—Hypnotics; Sedatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/10—Preparation or pretreatment of starting material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention relates to an acanthopanax extract and a preparation method and application thereof, wherein the acanthopanax extract is obtained by crushing and wetting acanthopanax plants, and a special compound enzyme reagent consisting of cellulase, pectase, beta-glucanase and plant protease is added in the hydrolysis process, so that the content of active ingredients of the acanthopanax extract is obviously improved.

Description

Acanthopanax senticosus extract and preparation method and application thereof
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to an acanthopanax root extract and a preparation method and application thereof.
Background
Acanthopanax senticosus (Radix Acanthopanacis Senticosl) is root, rhizome and stem of Acanthopanax senticosus of Araliaceae, and is mainly distributed in northeast China, russian far east region, north sea way of Japan, korean, etc. Starting from Shennong Ben Cao Jing (Shennong's herbal), the name is Acanthopanax root, tiger Ruthenia, and spiny Hovenia dulcis (northeast). The "Ben Cao gang mu" records that acanthopanax root can tonify qi, replenish essence, improve eyesight, lower qi, tonify five strains, seven impairments, and is light and aging resistant after long-term administration, and has the effects of replenishing qi, strengthening spleen, tonifying kidney and tranquillizing.
The main functional components of the acanthopanax are phenolic glycosides and certain aglycone components thereof, and mainly comprise acanthopanax total glycosides, total flavonoids and the like; the acanthopanax root total glycoside has 7 types, and the main active ingredients of the acanthopanax root total glycoside are acanthopanaxoside B and acanthopanaxoside E; the acanthopanax glycoside B is also called syringin (Syringin), has the highest relative content, is one of main medicinal components in the acanthopanax, and has the functions of resisting fatigue, enhancing immunity, protecting liver, releasing acetylcholine, increasing insulin secretion, protecting against radiation and the like; the eleutheroside E has effects of relieving stress, resisting anxiety, resisting stress, resisting ulcer and relieving fatigue;
The acanthopanax total flavone is an important natural active ingredient in acanthopanax, has wide pharmacological activity, and can dilate blood vessels, increase coronary blood flow, reduce myocardial oxygen consumption, resist myocardial ischemia, improve electrocardiogram, resist ectopic heart rhythm, reduce blood pressure and calm and tranquilize. Because of various biological activities and good clinical effects of the acanthopanax total flavonoids, the acanthopanax total flavonoids have become one of focuses of traditional Chinese medicine extraction and modern research of Chinese herbal medicines at home and abroad for many years.
About 90% of the traditional Chinese medicinal materials are plant medicinal materials, plant cells consist of cell walls and protoplasts, and the cell walls are compact structures composed of cellulose, hemicellulose, pectic substances, lignin and other substances and are generally divided into 3 layers, namely, a cell layer, a primary wall and a secondary wall. The primary wall is mainly composed of cellulose, hemicellulose and pectic substance. The structure of the primary wall is quite complex, microfibrils composed of cellulose molecules form the basic skeleton thereof, and the interstices between the microfibrils are filled with colloidal substances of pectic substance and hemicellulose. Like the primary wall, the skeleton of the secondary wall is also composed of microfibrils composed of cellulose molecules. In the process of extracting the traditional Chinese medicine, when the effective components in the cell protoplast are diffused to an extraction medium, double resistance of cell walls and cell stroma is overcome, so that proper enzymes such as cellulase, hemicellulase, pectase and the like are selected to act on medicinal plant cells, so that substances such as cellulose, hemicellulose, pectin and the like in the cell walls and the cell stroma are degraded, the compact structure of the cell walls is destroyed, the local loosening, swelling, collapse and other changes of the cell walls and the cell stroma structures are caused, the mass transfer resistance of the mass transfer barriers such as the cell walls and the cell stroma on the diffusion of the effective components from the cells to the extraction medium is reduced, and the extraction rate of the effective components is truly promoted from the mass transfer angle.
At present, the extraction of main functional active ingredients of acanthopanax comprises water extraction, alcohol extraction, supercritical CO 2 extraction and other methods, wherein the supercritical CO 2 extraction has high selectivity, short operation time and high yield of effective ingredients, but is limited by treatment capacity, low in yield and high in industrial development difficulty, so that the water extraction and the organic solvent extraction are still mainly used at present, and most of the main functional active ingredients only take single ingredients as indexes for measuring the quality of the extraction process parameters, so that the full extraction, separation and utilization of all the effective ingredients of the acanthopanax are difficult to ensure. There are also few methods for extracting the effective components of acanthopanax by enzyme method, including cellulase, pectase, protease and other different enzymes, for example, patent CN105131142a discloses an industrial production method of acanthopanax polysaccharide and its application, the patent uses supercritical extraction, enzymolysis (protease: cellulase: pectase=2-4:1-2:0-0.5), resin adsorption, the extraction rate of acanthopanax glycoside E reaches more than 90%, the polysaccharide content is more than 95%, the acanthopanax polysaccharide prepared by the method has better effect on tuberculosis liver injury, but the transfer rate of the extracted whole effective components is low, the preparation method is complex, and the cost is high in industrial application; CN112089741a discloses an acanthopanax extract composition, an extraction method and an injection thereof, which are obtained by adopting water extraction and alcohol precipitation, then adsorbing by using macroporous adsorption resin, and eluting by using ethanol, wherein the extract components comprise acanthopanaxoside B, acanthopanaxoside E, fructose, glucose, sucrose, maltose, chlorogenic acid, cryptochlorogenic acid and neochlorogenic acid, but the acanthopanax extract obtained by the method has low content of active ingredients and low extraction rate.
In conclusion, the preparation of the special enzyme preparation of the acanthopanax extract has wide application prospect in the extraction of traditional Chinese medicines, even the structure of the target active substances of the traditional Chinese medicines can be changed through enzyme reaction, and the preparation of the medical product with high added value is expected to be prepared, so that the preparation has important significance in promoting the development of the medical industry.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides an acanthopanax root extract and a preparation method and application thereof.
In a first aspect, the present invention provides a method for preparing an acanthopanax extract, comprising the steps of:
S1, harvesting acanthopanax in the north in winter, cleaning roots, rhizomes and/or stems of acanthopanax plant, freezing and drying outdoors at a temperature below minus 10 ℃ in winter, fully crushing to 60-80 meshes by using a crusher, and then carrying out water wetting for 3-12 hours;
S2, placing the acanthopanax root moistened in the step S1 in water with the mass of 5-8 times, then adding a special compound enzyme reagent, uniformly stirring, and hydrolyzing;
S3, inactivating enzyme of the material liquid hydrolyzed in the step S2, filtering and concentrating to obtain the acanthopanax extract.
Further, the pH of the water bath in the step S2 is 4.1 to 5.0, preferably 4.8.
Further, the pH in step S2 is adjusted with oxalic acid, acetic acid or citric acid.
Further, the hydrolysis temperature in the step S2 is 15 to 62 ℃, preferably 50+ -5 ℃.
Further, the hydrolysis time in the step S2 is 6-24 hours.
Further, the special complex enzyme reagent in the step S2 is composed of cellulase, pectase, beta-glucanase and plant protease.
Furthermore, the mass ratio of the cellulase, pectase, beta-glucanase and plant protease in the special compound enzyme reagent is 1 (0.5-2.0) (0.1-1.0) (1.0-1.8).
Further, the addition amount of the special compound enzyme in the step S2 is 1-6% of the weight of the substrate, preferably 3-6%.
Further, the hydrolysis process in the step S3 is stirred, and the hydrolysis time is shortened by half by stirring fully every 5 minutes.
Further, in the step S3, the ceramic membrane filtration and concentration adopts a ceramic membrane separation experimental device (151101 ceramic membrane sample, BCM-200/500-19×31×1016, membrane filtration pore diameter 50nm, 100nm, 200 nm) and a set of 2540 organic membrane concentration experimental device (reverse osmosis membrane 2540), the membrane area of the device is 0.24x1=0.24m 2, the device is composed of a feed pump, a circulating pump, a membrane assembly and the like, the feed pump provides system pressure, the circulating pump provides system flow, steam or cooling water can be used as heating or cooling medium, the process temperature (5-80 ℃) of the system is ensured to be maintained, batch intermittent operation process is adopted, the feed liquid is continuously filtered, when the retentate is concentrated to a certain concentration multiple, the ceramic membrane filtrate is transferred to an organic membrane device for concentration, the yield of the effective components is improved, and after each batch of liquid filtration is completed, the cleaning agent such as alkali and acid is used for membrane regeneration for the next reuse.
In a second aspect, the present invention provides an acanthopanax extract prepared by the preparation method of the acanthopanax extract.
Further, the acanthopanax root extract contains 0.5 to 2.5 parts of syringin, 0.5 to 2.5 parts of acanthopanaxoside E, 0.1 to 2.0 parts of isofraxidin, 0.05 to 0.5 parts of protocatechuic acid, 0.25 to 1.05 parts of vanillic acid, 0.5 to 1.5 parts of chlorogenic acid, 0.3 to 1.5 parts of 4-caffeoylquinic acid, 0.5 to 1.5 parts of 5-caffeoylquinic acid, 0.1 to 1.0 parts of 1, 5-dicaffeoylquinic acid, 0.1 to 1.5 parts of glucose, 0.1 to 0.5 parts of maltose, 0.15 to 1.0 parts of xylose and 0.2 to 1.5 parts of galactose according to parts by weight.
In a third aspect, the invention also provides the application of the acanthopanax extract in preparing medicines for regulating nervous system, immune system, endocrine system, cardiovascular and cerebrovascular system and/or reproductive system and improving anorexia;
further, the medicament also comprises a pharmaceutically acceptable carrier or diluent of the acanthopanax extract;
further, the endocrine system agents include, but are not limited to, drugs for regulating climacteric syndrome, diabetes, improving sleep, regulating blood sugar, etc.
Further, the pharmaceutical dosage forms include, but are not limited to, injection, lyophilized powder for injection, tablet, granule, capsule, syrup or mixture.
Compared with the prior art, the invention has the following beneficial effects:
According to the invention, the special compound enzyme reagent consisting of the cellulase, the pectase, the beta-glucanase and the plant protease is adopted to hydrolyze the acanthopanax plant, so that the content of the active ingredients in the acanthopanax extract is remarkably improved; meanwhile, the acanthopanax extract composition prepared by the invention has remarkable effects of regulating sleep, regulating blood sugar level and improving anorexia.
Drawings
FIG. 1 shows the effect of Acanthopanax senticosus extract on food intake in anorexia rats;
FIG. 2 shows the effect of Acanthopanax senticosus extract on the body mass of anorexia rats.
Detailed Description
The experimental methods of the present invention, in which specific conditions are not specified in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The terms "comprising" and "having" and any variations thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to the elements or modules listed but may alternatively include additional steps not listed or inherent to such process, method, article, or device.
The present invention will be further described in detail with reference to the following embodiments, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the description is only illustrative and is not intended to limit the scope of the invention. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the present invention.
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.
Example 1 an extract of acanthopanax of the present invention and a process for preparing the same
S1, cleaning roots, rhizomes and/or stems of acanthopanax plants, freeze-drying the roots, the rhizomes and/or the stems of acanthopanax plants outdoors at a temperature below 10 ℃, fully crushing the acanthopanax plants to 80 meshes, and then carrying out water wetting for 12 hours;
S2, placing the acanthopanax which is moistened in the step S1 in water with the weight of 6 times, wherein the temperature is 50 ℃, the pH is regulated to 4.8 by acetic acid, then adding a special compound enzyme reagent (the mass ratio of cellulase, pectase, beta-glucanase and plant protease is 1:0.5:0.5:1.2), wherein the addition amount of the special compound enzyme reagent is 5% of the weight of a substrate, stirring uniformly, stirring in the hydrolysis process, fully stirring once every 5 minutes, and hydrolyzing for 15 hours;
S3, inactivating enzyme of the material liquid hydrolyzed in the step S2, and filtering and concentrating by using a ceramic membrane to obtain the acanthopanax extract.
The ceramic membrane filtration concentration adopts a ceramic membrane separation experimental device (151101 ceramic membrane sample, BCM-200/500-19 x 31 x 1016, membrane filtration pore diameter of 100 nm) and a set of 2540 organic membrane concentration experimental device (reverse osmosis membrane 2540), the membrane area of the device is 0.24 x 1=0.24 m 2, the device consists of a feed pump, a circulating pump, a membrane component and the like, the feed pump provides system pressure, the circulating pump provides system flow, a steam heating medium is adopted, the process temperature (55 ℃) of the system is ensured to be maintained, batch treatment intermittent operation process is adopted, the feed liquid is continuously filtered, when the feed liquid retentate is concentrated to 2 times of concentration multiple, ceramic membrane filtrate is transferred to organic membrane equipment for concentration, the yield of active ingredients is improved, and after each batch of liquid filtration is completed, cleaning agents such as alkali, acid and the like are used for membrane regeneration so as to be reused next time.
Example 2 Acanthopanax senticosus extract and its preparation method
S1, cleaning roots, rhizomes and/or stems of acanthopanax plants, drying the roots, the rhizomes and/or the stems of acanthopanax plants outdoors at a temperature below 10 ℃, fully crushing the roots, the rhizomes and/or the stems into 80 meshes, and then carrying out water wetting for 12 hours;
S2, placing the acanthopanax which is moistened in the step S1 in water with the weight of 6 times, wherein the temperature is 45 ℃, the pH is regulated to 4.5 by acetic acid, then adding a special compound enzyme reagent (the mass ratio of cellulase, pectase, beta-glucanase and plant protease is 1:0.8:0.1:1.0), wherein the addition amount of the special compound enzyme reagent is 3% of the weight of a substrate, stirring uniformly, stirring in the hydrolysis process, fully stirring once every 5 minutes, and hydrolyzing for 24 hours;
S3, inactivating enzyme of the material liquid hydrolyzed in the step S2, and filtering and concentrating by using a ceramic membrane to obtain the acanthopanax extract.
The ceramic membrane filtration concentration adopts a ceramic membrane separation experimental device (151101 ceramic membrane sample, BCM-200/500-19 x 31 x 1016, membrane filtration pore diameter of 100 nm) and a set of 2540 organic membrane concentration experimental device (reverse osmosis membrane 2540), the membrane area of the device is 0.24 x 1=0.24 m 2, the device consists of a feed pump, a circulating pump, a membrane component and the like, the feed pump provides system pressure, the circulating pump provides system flow, a steam heating medium is adopted, the process temperature (55 ℃) of the system is ensured to be maintained, batch treatment intermittent operation process is adopted, the feed liquid is continuously filtered, when the feed liquid retentate is concentrated to 2 times of concentration multiple, ceramic membrane filtrate is transferred to organic membrane equipment for concentration, the yield of active ingredients is improved, and after each batch of liquid filtration is completed, cleaning agents such as alkali, acid and the like are used for membrane regeneration so as to be reused next time.
Example 3 Acanthopanax senticosus extract and its preparation method
S1, cleaning roots, rhizomes and/or stems of acanthopanax plants, drying the roots, the rhizomes and/or the stems of acanthopanax plants outdoors at a temperature below 10 ℃, fully crushing the roots, the rhizomes and/or the stems into 60 meshes, and then carrying out water wetting for 12 hours;
s2, placing the acanthopanax which is moistened in the step S1 in water with the weight of 6 times, wherein the temperature is 55 ℃, the pH is regulated to 4.1 by acetic acid, then adding a special compound enzyme reagent (the mass ratio of cellulase, pectase, beta-glucanase and plant protease is 1:2.0:1.0:1.0), wherein the addition amount of the special compound enzyme reagent is 6% of the weight of a substrate, stirring uniformly, stirring in the hydrolysis process, fully stirring once every 5 minutes, and hydrolyzing for 10 hours;
S3, inactivating enzyme of the material liquid hydrolyzed in the step S2, and filtering and concentrating by using a ceramic membrane to obtain the acanthopanax extract.
The ceramic membrane filtration concentration adopts a ceramic membrane separation experimental device (151101 ceramic membrane sample, BCM-200/500-19 x 31 x 1016, membrane filtration pore diameter of 100 nm) and a set of 2540 organic membrane concentration experimental device (reverse osmosis membrane 2540), the membrane area of the device is 0.24 x 1=0.24 m 2, the device consists of a feed pump, a circulating pump, a membrane component and the like, the feed pump provides system pressure, the circulating pump provides system flow, a steam heating medium is adopted, the process temperature (55 ℃) of the system is ensured to be maintained, batch treatment intermittent operation process is adopted, the feed liquid is continuously filtered, when the feed liquid retentate is concentrated to 2 times of concentration multiple, ceramic membrane filtrate is transferred to organic membrane equipment for concentration, the yield of active ingredients is improved, and after each batch of liquid filtration is completed, cleaning agents such as alkali, acid and the like are used for membrane regeneration so as to be reused next time.
Example 4 Acanthopanax senticosus extract and its preparation method
S1, cleaning roots, rhizomes and/or stems of acanthopanax plants, drying the roots, the rhizomes and/or the stems of acanthopanax plants outdoors at a temperature below 10 ℃, fully crushing the roots, the rhizomes and/or the stems into 60 meshes, and then carrying out water wetting for 12 hours;
S2, placing the acanthopanax which is moistened in the step S1 in water with the weight of 6 times, wherein the temperature is 62 ℃, the pH value is regulated to 4.8 by acetic acid, then adding a special compound enzyme reagent (the mass ratio of cellulase, pectase, beta-glucanase and plant protease is 1:0.8:1.0:1.8), wherein the addition amount of the special compound enzyme reagent is 1% of the weight of a substrate, stirring uniformly, stirring in the hydrolysis process, fully stirring once every 5 minutes, and hydrolyzing for 6 hours;
S3, inactivating enzyme of the material liquid hydrolyzed in the step S2, and filtering and concentrating by using a ceramic membrane to obtain the acanthopanax extract.
The ceramic membrane filtration concentration adopts a ceramic membrane separation experimental device (151101 ceramic membrane sample, BCM-200/500-19 x 31 x 1016, membrane filtration pore diameter of 100 nm) and a set of 2540 organic membrane concentration experimental device (reverse osmosis membrane 2540), the membrane area of the device is 0.24 x 1=0.24 m 2, the device consists of a feed pump, a circulating pump, a membrane component and the like, the feed pump provides system pressure, the circulating pump provides system flow, a steam heating medium is adopted, the process temperature (55 ℃) of the system is ensured to be maintained, batch treatment intermittent operation process is adopted, the feed liquid is continuously filtered, when the feed liquid retentate is concentrated to 2 times of concentration multiple, ceramic membrane filtrate is transferred to organic membrane equipment for concentration, the yield of active ingredients is improved, and after each batch of liquid filtration is completed, cleaning agents such as alkali, acid and the like are used for membrane regeneration so as to be reused next time.
Determination of the content of active ingredient in the acanthopanax extract prepared in test example one and examples 1 to 4
Determining the content of effective components in the extract composition of the acanthopanax group in examples 1-4 by adopting high performance liquid chromatography (China Pharmacopeia 2020 edition, four-part rule 0512), and applying ACQUITY UPLC HSS T (2.1×100mm,1.8 μm) chromatographic columns in chromatographic conditions and system applicability test; elution was performed according to the elution procedure in table 1 with 0.5% formic acid-water solution (V/V) as mobile phase a and acetonitrile solution (V/V) as mobile phase B, wherein the column temperature was 35 ℃, the flow rate was 0.5ml/min, detection wavelength: syringin, eleutheroside E, protocatechuic acid, vanillic acid, glucose, maltose, xylose and galactose are 275nm, isozinidine is 344nm, and chlorogenic acid, 4-caffeoylquinic acid, 5-caffeoylquinic acid and 1, 5-dicaffeoylquinic acid are 321nm.
TABLE 1 Mobile phase elution procedure
The specific method for measuring the content of the effective components of the acanthopanax senticosus extracts prepared in examples 1 to 4 by using the high performance liquid chromatography is as follows:
(1) Preparation of control solution
Taking proper amounts of syringin, eleutheroside E, isozindine, protocatechuic acid, vanillic acid, chlorogenic acid, 4-caffeoyl quinic acid, 5-caffeoyl quinic acid, 1, 5-dicaffeoyl quinic acid, glucose, maltose, xylose and galactose as reference substances, precisely weighing, respectively adding 15% methanol solution, ultrasonically dissolving to constant volume, preparing mixed solution, filtering with a microporous filter membrane of 0.22 μm, and collecting the subsequent filtrate.
(2) Preparation of test solutions
Precisely measuring 5ml of the acanthopanax extract in examples 1-4, placing in a 10ml measuring flask, adding 15% methanol solution to dilute to scale, shaking uniformly, filtering with a microporous filter membrane of 0.22 μm, and collecting the subsequent filtrate.
(3) The measurement method comprises precisely sucking 2 μl of each of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement.
The acanthopanax senticosus extracts prepared in examples 1 to 4 and measured by the high performance liquid chromatography are shown in table 2;
TABLE 2
Comparative example 1
The difference from example 1 is that the mass ratio of cellulase, pectase, beta-glucanase and plant protease in the special complex enzyme is 1:0.5:0:1.2, and the rest steps are the same as in example 1.
Comparative example 2
The difference from example 1 is that the mass ratio of cellulase, pectase, beta-glucanase and plant protease in the special complex enzyme is 1:0.5:1.2:1.2, and the rest steps are the same as in example 1.
Comparative example 3
The difference from example 1 is that the mass ratio of cellulase, pectase, beta-glucanase and plant protease in the special complex enzyme is 1:2.2:0.5:0.5, and the rest steps are the same as in example 1.
Comparative example 4
The difference from example 1 is that the hydrolysis pH is 4.0 and the remaining steps are identical to example 1.
Comparative example 5
The difference from example 1 is that the addition amount of the specific complex enzyme was 6.5%, and the rest of the procedure was the same as in example 1.
Effective component content of acanthopanax extract prepared in test example II and comparative examples 1 to 5
The results of measuring the content of the effective components of the acanthopanax senticosus extracts prepared in comparative examples 1 to 5 according to a high performance liquid chromatography of test examples are shown in table 3;
TABLE 2
As can be seen from comparison of the results of the first test example and the second test example, the preparation method of the acanthopanax extract has remarkable effects on improving the contents of syringin, acanthopanaxoside E, isofraxidin, protocatechuic acid, vanillic acid, chlorogenic acid, 4-caffeoylquinic acid, 5-caffeoylquinic acid, 1, 5-dicaffeoylquinic acid, glucose, maltose, xylose and galactose, wherein the components and proportion of cellulase, pectase, beta-glucanase and plant protease in the special complex enzyme have more remarkable effects on the effective components of the acanthopanax extract.
Test example III, influence of acanthopanax extract on blood sugar
Experimental animals: kunming mice, 18-22g in weight, male and female halves.
Experimental reagent: tetraoxapyrimidine, available from Sigma.
Experimental grouping: blank, model, examples 1-4, and comparative examples 1-5.
The experimental method is as follows:
and (3) model preparation: the tetraoxypyrimidine was prepared as a 2% aqueous solution (as-prepared) with physiological saline.
Taking Kunming mice, and administering to the mice after 12 hr of fasted period at a dosage of 50mg/kg of tetraoxypyrimidine solution
Mice were injected tail vein. After 3 days of continuous feeding, the eyes were fasted for 12 hours, blood was collected from the eyes, serum was separated, fasting blood glucose was measured, and blood glucose was greater than 14mmol/l as a model mouse for diabetes.
Grouping drug administration: taking 80 diabetic model mice, randomly dividing the mice into 8 groups of which 10 are respectively an example 1-4 group, a comparative example 1-5 group and a model group; the mice 10 of the Kunming species, which had a weight similar to that of the normal Kunming species, were taken as blank groups.
The acanthopanax extract of examples 1 to 4 and the acanthopanax extract of comparative examples 1 to 5 were administered by gavage in each of examples 1 to 4 and the acanthopanax extract of comparative examples 1 to 5 at a dose of 1.0g crude drug per kg, respectively, and the model group and the blank group were administered with physiological saline in the same volume according to a weight of 15ml/kg by gavage for 15 days continuously, and changes in the appearance characteristics of mice were observed every day.
The test animals were fasted for 12 hours after the end of the test, blood was collected from the orbital venous plexus, and fasting blood glucose was measured.
And (3) statistical treatment: the statistical analysis of the experimental data was done using SPSS statistical software.
Experimental results:
The effect on the blood glucose elevation in mice caused by tetraoxypyrimidine is shown in Table 3:
TABLE 3 Table 3
Note that: #P＜0.05,## P < 0.01 in the model group, * P < 0.05 in the blank group.
The results in Table 3 show that the acanthopanax extract prepared by the preparation method has remarkable blood sugar reducing effect.
Test example IV, influence of Acanthopanax senticosus extract on sleep adjustment
(1) The test method comprises the following steps:
Taking Kunming mice, weighing 18-22 g, controlling the temperature at 24+/-2 ℃ and the humidity at 55+/-15%, and 12: the experimental animals are free to eat and drink for 12 hours, the experimental animals are fasted and watered in the laboratory for one week before the experiment, 60 mice are taken to adapt to the environment, the experimental animals are randomly divided into 5 groups, each group is divided into 12 groups, namely a blank group (0.2 mL/20 g), a positive control group (0.2 mL/20 g), prepared acanthopanax extract high, medium and low dose groups (0.2 mL/20 g) are administrated by a gastric lavage way, the blank group/positive control group is administrated with distilled water with the same volume, diazepam tablets (2.5 mg/kg) are administrated simultaneously, the prepared acanthopanax extract high, medium and low dose groups are administrated with 4mL/kg, 2 mL/kg respectively, the prepared acanthopanax extract of the comparative example is administrated with 4mL/kg for one time of gastric lavage, each group is subjected to intraperitoneal injection with pentobarbital sodium (55 mg/kg) for 30 days after the last administration for 30min, and sleep awakening time is recorded.
(2) Statistical analysis:
the statistical analysis of the experimental data is completed by using SPSS statistical software;
(3) Experimental results
The effect of acanthopanax extract on sleep of mice caused by prolonged threshold dose of pentobarbital sodium is shown in table 4;
TABLE 4 Table 4
Note that: #P＜0.05,## P < 0.01 in comparison with the positive control group, ** P < 0.01 in comparison with the blank group.
The results in Table 4 show that the acanthopanax extract prepared by the preparation method of the present invention has obvious effect on the sleep regulation of mice after 30d of continuous administration.
Test example five, influence of acanthopanax extract on anorexia treatment
(1) Test method
50 SPF-grade SD rats, male and female rats, are randomly divided into 6 groups after 3 days of adaptive feeding, 10 blank groups and 50 experiment groups are fed with conventional feed, the experiment groups are fed with anorexia model feed (milk powder, dried fish floss, bean powder, corn flour, white sugar, fresh eggs and fresh fat meat (1:1:2:1:1.8:2), the raw materials are uniformly mixed and kneaded into a biscuit shape, the mixture is placed in a baking tray at about 2cm multiplied by 3cm, the mixture is baked for 30 minutes at 160 ℃, the mixture is hermetically packaged after room temperature is restored, the mixture is stored in a refrigerator at 4 ℃, the food intake and the body mass of the model group after 10 days of molding are respectively lower than 53% and 12% of the blank group, the molding is successful, the experiment groups after the molding is successfully divided into a model control group, a positive control group, a high dose group, a medium dose group and a low dose group (example 1), 10 groups are fed with conventional feed, the blank group and the model control group are fed with 10g/kg of drinking water, the high dose, the medium dose group and the low dose group are respectively fed with 5mL/kg of acanthopanax extract, 2.25 mL/kg of acanthopanax extract is fed with the drinking water, the mixture is fed with the mixture at 1.25mL/kg of acanthopanax extract, the mixture is fed with the mixture for 5 days of acanthopanax extract, the mixture is fed with the mixture at 1.5 m/kg of acanthopanax, the mixture is fed with the mixture for 1 day, and the mixture is fed with the food intake and 5mL of acanthopanax, and the mixture is fed with the mixture at 1 day at the food intake and 5m day;
(2) Experimental results
The result of the acanthopanax extract for treating anorexia is shown in figures 1-2;
In the test example, the model group has a significant difference, p <0.05, the positive control group, the high-medium-low dose group, the comparative example 4 and the comparative example 5 have a significant difference, p <0.05, and the comparative examples 1 to 3 have no significant difference, p >0.05, compared to the model group; as can be seen from the results of the accompanying figure 1, the acanthopanax extract prepared by the invention obviously increases the food intake of anorexia rats, the high and medium dosage groups can basically achieve the recovery capacity of the positive control group, the food intake can be recovered to the normal level within 7-14 days, and the results of the accompanying figure 2 show that the acanthopanax extract prepared by the invention can improve the body quality of anorexia rats, and the recovery capacity of the high and medium dosage groups is equivalent to that of the positive control group.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (6)

1. A preparation method of an acanthopanax root extract is characterized by comprising the following steps:
S1, cleaning roots, rhizomes and/or stems of acanthopanax plants, drying, fully crushing, and then carrying out water wetting for 3-12 hours;
S2, placing the acanthopanax which is moistened in the step S1 in water with the weight of 5-8 times, then adding a special compound enzyme reagent, wherein the addition amount of the special compound enzyme reagent is 1-6% of the mass of a substrate, the special compound enzyme reagent consists of (0.5-2.0): (0.1-1.0): (1.0-1.8) cellulase, pectase, beta-glucanase and plant protease, stirring uniformly, and hydrolyzing, and the hydrolysis pH is 4.1-4.8;
S3, inactivating enzyme of the material liquid hydrolyzed in the step S2, filtering and concentrating to obtain the acanthopanax extract.
2. The method for preparing acanthopanax extract according to claim 1, wherein the hydrolysis pH in step S2 is adjusted by oxalic acid, acetic acid or citric acid.
3. The method for preparing acanthopanax extract according to claim 1, wherein the hydrolysis temperature in the step S2 is 15-62 ℃.
4. An extract of acanthopanax root produced by the production method according to any one of claims 1 to 3.
5. The acanthopanax extract according to claim 4, wherein the acanthopanax extract contains syringin, acanthopanaxoside E, isofraxidin, protocatechuic acid, chlorogenic acid, vanillic acid, 4-caffeoylquinic acid, 5-caffeoylquinic acid and 1, 5-dicaffeoylquinic acid, glucose, maltose, xylose and galactose.
6. The use of acanthopanax extract according to claim 4 in preparing medicine for regulating endocrine system and improving anorexia.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107595893A (en) * 2017-08-25 2018-01-19 广西中医药大学 A kind of enzyme treated prepared slices of Chinese crude drugs and preparation method thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107595893A (en) * 2017-08-25 2018-01-19 广西中医药大学 A kind of enzyme treated prepared slices of Chinese crude drugs and preparation method thereof

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* Cited by examiner, † Cited by third party
Title
刺五加临床应用拓展研究;缪茂军等;江西省中医药学会2011年学术年会论文集;第1页第1-2段 *

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