CN110507679A - A kind of preparation method and applications of SIJIQING extract - Google Patents

A kind of preparation method and applications of SIJIQING extract Download PDF

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Publication number
CN110507679A
CN110507679A CN201910943641.3A CN201910943641A CN110507679A CN 110507679 A CN110507679 A CN 110507679A CN 201910943641 A CN201910943641 A CN 201910943641A CN 110507679 A CN110507679 A CN 110507679A
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chinese holly
preparation
extract
sijiqing
acid
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钟英杰
李志明
李亮
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Qingdao Rongtai Biotechnology Co Ltd
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Qingdao Rongtai Biotechnology Co Ltd
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Priority to CN202010187175.3A priority patent/CN111053795A/en
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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)
    • 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)
    • A61K36/19—Acanthaceae (Acanthus family)
    • 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)
    • A61K36/56—Loganiaceae (Logania family), e.g. trumpetflower or pinkroot
    • 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)
    • A61K36/70—Polygonaceae (Buckwheat family), e.g. spineflower or dock
    • A61K36/704—Polygonum, e.g. knotweed
    • 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)
    • A61K36/80—Scrophulariaceae (Figwort family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • 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
    • A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • 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

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  • Health & Medical Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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Abstract

The present invention provides a kind of preparation method and applications of SIJIQING extract, belong to field of veterinary.Its technical solution are as follows: preparation method crosses 10 mesh~24 meshes, obtains Chinese holly powder the following steps are included: S1, take appropriate Chinese holly crushed after being dried, spare；S2, it takes Chinese holly powder obtained in step S1 to add acidic ethanol refluxing extraction 1.5~3.0 hours of appropriate concentration, and is filtered, collect the dregs of a decoction and filtrate, filtrate is spare；S3, filtrate decompression obtained in step S2 is concentrated into 60 DEG C and measure obtain relative density be 1.01~1.06 medicinal extract；S4, medicinal extract obtained in step S3 is spray-dried, obtains SIJIQING extract.The invention has the benefit that SIJIQING extract of the invention, it is inhibited to porcine pseudorabies virus infection development, simultaneously for polyphenol components contained in Chinese holly, and polyphenol components are usually to exist with its esters, refluxing extraction is carried out using acidic ethanol, greatly promotes recovery rate.

Description

A kind of preparation method and applications of SIJIQING extract
Technical field
The present invention relates to a kind of field of veterinary more particularly to the preparation method and applications of SIJIQING extract.
Background technique
Porcine pseudorabies (Pseudorabies PR) are caused by porcine pseudorabies virus (Pseudorabies PRV) A kind of acute infectious disease.The disease is in pig break out and spread.It can cause pregnant sow miscarriage, stillborn foetus, boar infertility, newborn piglet Mortality, growing and fattening pigs expiratory dyspnea, growth retardation etc. are one of the serious infectious diseases for endangering global pig breeding industry.Viral disease The therapeutic agent research and development of disease are slower, mainly since virus has a stringent parasitics, after virus infection virus, in host cell It is interior to utilize cellular enzymes system copies；Some viral nucleic acids are integrated in cell chromosome, it is difficult to remove；Virus parasite in the cell, So that also having damage to body cell while drug killing virus.Existing antiviral chemicals are because toxicity is too big or resistance to Pharmacological property, clinic are difficult to obtain satisfactory effect.Existing vaccine cannot provide swinery protection completely, bring to its anti-system Great difficulty is badly in need of finding new prevention and control scheme.Traditional Chinese medicine toxic side effect is small, and drug resistance is low, cheap, drug effect Target spot is more, and have immunoregulatory function, screen high-efficiency low-toxicity anti virus herb be current antiviral research hotspot it One.
Summary of the invention
The purpose of the present invention is to provide a kind of SIJIQING extracts, have to porcine pseudorabies virus infection development and inhibit Effect, at the same for polyphenol components contained in Chinese holly, and polyphenol components are usually to exist with its esters, using acidity Ethyl alcohol carries out refluxing extraction, greatly promotes recovery rate.
The present invention is realized by following measure:
A kind of preparation method of SIJIQING extract, which is characterized in that the preparation method comprises the following steps:
S1, appropriate Chinese holly crushed after being dried is taken, crosses 10 mesh~24 meshes, obtains Chinese holly powder, it is spare；
S2, it takes Chinese holly powder obtained in step S1 to add acidic ethanol refluxing extraction 1.5~3.0 hours of appropriate concentration, goes forward side by side The dregs of a decoction and filtrate are collected in row filtering, and filtrate is spare；
S3, filtrate decompression obtained in step S2 is concentrated into 60 DEG C and measure obtain relative density be 1.01~1.06 leaching Cream；
S4, medicinal extract obtained in step S3 is spray-dried, obtains SIJIQING extract.
Specific feature of the invention is also:
The preparation method comprises the following steps:
S1, appropriate Chinese holly crushed after being dried is taken, crosses 10 mesh~24 meshes, obtains Chinese holly powder, it is spare；
S2, it takes Chinese holly powder obtained in step S1 to add acidic ethanol refluxing extraction 2 hours of appropriate concentration, and is filtered, The dregs of a decoction and filtrate are collected, filtrate is spare；
S3, filtrate decompression obtained in step S2 is concentrated into 60 DEG C and measure obtain relative density be 1.03 medicinal extract；
S4, medicinal extract obtained in step S3 is spray-dried, obtains SIJIQING extract.
The 20%-70% ethyl alcohol for adding 7-10 times of weight of the Chinese holly powder acid containing 0.1%-0.5% for measuring volume in the S2 step Solution carries out refluxing extraction.
Acidic ethanol in the S2 step is 20%~70% ethyl alcohol containing 0.1%~0.5% acid, and the property acid ethyl alcohol is 0.1~0.5% acid is dissolved in the ethyl alcohol that 100ml concentration is 20~70%.
Chinese holly in the step S1 is the herb of Chinese holly or one or more of mixtures of leaf, in the S2 step Acid be one or more of concentrated hydrochloric acid, phosphoric acid, glacial acetic acid, citric acid.
The SIJIQING extract is used to prepare the application in the drug of resisting pstudorabies virus.
The invention has the benefit that SIJIQING extract of the invention, has porcine pseudorabies virus infection development Inhibiting effect, while for polyphenol components contained in Chinese holly, and polyphenol components are usually to exist with its esters, are added After appropriate amount of acid, polyphenol components are in free state, and lipophilicity enhancing is soluble in high concentration ethanol, than individually using water or certain dense Degree ethyl alcohol recovery rate wants high, carries out refluxing extraction using acidic ethanol, greatly promotes recovery rate.
Detailed description of the invention
Fig. 1 is that plaque-forming assay measures what 6 kinds of Chinese herbal medicine extracts were proliferated PRV in experiment of the embodiment of the present invention one Inhibitory effect figure.
Specific embodiment
In order to clarify the technical characteristics of the invention, being illustrated below by specific embodiment to this programme.
Embodiment 1:
The preparation method comprises the following steps:
S1, appropriate Chinese holly crushed after being dried is taken, crosses 10 mesh~24 meshes, obtains Chinese holly powder, it is spare；
S2, it takes Chinese holly powder obtained in step S1 to add acidic ethanol refluxing extraction 1.5 hours of appropriate concentration, and carried out The dregs of a decoction and filtrate are collected in filter, and filtrate is spare；
S3, filtrate decompression obtained in step S2 is concentrated into 60 DEG C and measure obtain relative density be 1.02 medicinal extract；
S4, medicinal extract obtained in step S3 is spray-dried, obtains SIJIQING extract.
In the S2 step plus 7 times of Chinese holly powder weight amount volumes 30% ethanol solutions containing 0.2% acid flow back It extracts.
Acidic ethanol in the S2 step is 30% ethyl alcohol containing 0.2% acid, and the property acid ethyl alcohol is that 0.2% acid is molten In the ethyl alcohol that 100ml concentration is 30%.
Chinese holly in the step S1 is the herb of Chinese holly or one or more of mixtures of leaf, in the S2 step Acid be one or more of concentrated hydrochloric acid, phosphoric acid, glacial acetic acid, citric acid.
The SIJIQING extract is used to prepare the application in the drug of resisting pstudorabies virus.
Embodiment 2:
The preparation method comprises the following steps:
S1, appropriate Chinese holly crushed after being dried is taken, crosses 10 mesh~24 meshes, obtains Chinese holly powder, it is spare；
S2, it takes Chinese holly powder obtained in step S1 to add acidic ethanol refluxing extraction 2 hours of appropriate concentration, and is filtered, The dregs of a decoction and filtrate are collected, filtrate is spare；
S3, filtrate decompression obtained in step S2 is concentrated into 60 DEG C and measure obtain relative density be 1.03 medicinal extract；
S4, medicinal extract obtained in step S3 is spray-dried, obtains SIJIQING extract.
In the S2 step plus 8 times of Chinese holly powder weight amount volumes 40% ethanol solutions containing 0.3% acid flow back It extracts.
Acidic ethanol in the S2 step is 40% ethyl alcohol containing 0.3% acid, and the property acid ethyl alcohol is that 0.3% acid is molten In the ethyl alcohol that 100ml concentration is 40%.
Chinese holly in the step S1 is the herb of Chinese holly or one or more of mixtures of leaf, in the S2 step Acid be one or more of concentrated hydrochloric acid, phosphoric acid, glacial acetic acid, citric acid.
The SIJIQING extract is used to prepare the application in the drug of resisting pstudorabies virus.
Embodiment 3:
The preparation method comprises the following steps:
S1, appropriate Chinese holly crushed after being dried is taken, crosses 10 mesh~24 meshes, obtains Chinese holly powder, it is spare；
S2, it takes Chinese holly powder obtained in step S1 to add acidic ethanol refluxing extraction 3 hours of appropriate concentration, and is filtered, The dregs of a decoction and filtrate are collected, filtrate is spare；
S3, filtrate decompression obtained in step S2 is concentrated into 60 DEG C and measure obtain relative density be 1.05 medicinal extract；
S4, medicinal extract obtained in step S3 is spray-dried, obtains SIJIQING extract.
In the S2 step plus 9 times of Chinese holly powder weight amount volumes 65% ethanol solutions containing 0.4% acid flow back It extracts.
Acidic ethanol in the S2 step is 65% ethyl alcohol containing 0.4% acid, and the property acid ethyl alcohol is that 0.4% acid is molten In the ethyl alcohol that 100ml concentration is 65%.
Chinese holly in the step S1 is the herb of Chinese holly or one or more of mixtures of leaf, in the S2 step Acid be one or more of concentrated hydrochloric acid, phosphoric acid, glacial acetic acid, citric acid.
The SIJIQING extract is used to prepare the application in the drug of resisting pstudorabies virus.
The inventor of the preparation method of this SIJIQING extract is being directed to the inhibited aspect of PRV infection development, into Series of experiment research is gone, it is determined that this SIJIQING extract is inhibited to PRV infection development.
Experiment one, plaque assays detect Chinese herbal medicine extract to the inhibiting effect of PRV infection development
1, measurement of the Chinese medicine to cytotoxic concentration
The Chinese holly for preparing completion in Herba Andrographitis, ganoderma lucidum, folium isatidis, dandelion and marine algae extract and the present invention is taken to extract Object adds water to be made into the solution of various concentration, it is incorporated DMEM cell culture medium respectively according to the solubility of different extracts, The filtering of 0.22um filter, -80 DEG C of preservations.With 6 kinds of Chinese herbal medicine extracts of mtt assay and micro- sem observation to the cell of Vero cell Toxicity.Obtain this 6 kinds of Chinese herbal medicine extracts is respectively to the safe concentration of Vero cell: 1.69g/L, 3.75g/L, 2.5g/L, 6.25g/L, 3.125g/L, 0.59g/L, the above safe concentration illustrate that 6 kinds of Chinese herbal medicine extracts are very low to Vero cytotoxicity, spirit Sesame extract can even promote cell to increase.
2, plaque assays
Under conditions of to cytotoxic, 6 kinds of Chinese herbal medicine extracts of detection are tested using plaque test, PRV is infected in vitro The inhibitory effect of proliferation.
Plaque assay of viruses is the method for a kind of inspection and Accurate Determining virus titer.Diluted viral suspension is added single In confluent monolayer cells culture bottle, after viruses adsorption, then the semisolid nutrient agar rouge of a melting layer is covered, makes virus in monolayer cell culture In limited diffusion.
The virus of each infectious can produce the focus of infection of a limitation in cell monolayer.With reactive dye bromine Change diphenyltetrazolium bromide (MTT) dyeing, living cells can dye navy blue by the MTT of yellow, be infected by the virus and the cell that destroys It is not colored, form macroscopic plaque/plaque (plaque).Be added Herba Andrographitis, Chinese holly, ganoderma lucidum, folium isatidis, dandelion and Seaweed solution can reduce the formation of plaque since Chinese medical extract solution has certain antiviral activity.
Cell is routinely cultivated, after growing up to fine and close cell monolayer, by maintaining liquid and porcine pseudorabies venom containing Chinese medicine Mixed in equal amounts, the cell monolayer that 25 DEG C of effect 2h postoperative infections are newly formed, addition is covered containing 1.5% Ago-Gel after discarding supernatant liquid Lid liquid, is frozen into gelatin to it, and culture plate is overturn, 35 DEG C, 5%CO are set23d is cultivated in incubator.Daily observation cytopathy Become situation, removes gelatin culture medium, fix 30min with 10% formaldehyde, discard formaldehyde, with four nitrogen of reactive dye brominated diphenyl base Azoles (MTT) dyeing records plaque number with tap water rinse.
3, plaque assays result
Plaque assays can accurately measure the gradient of infection of virus, and the reduction of virus plaque number means to infect in a certain virus liquid Venereal disease poison granule content is reduced, viral appeal decline, this test result as shown in Figure 1, Chinese holly group almost without plaque It is formed, prompts the drug to be suppressed to the duplication of virus, the formation of porcine pseudorabies virus plaque can be blocked.
In attached drawing 1 the result shows that: 6 kinds of Chinese herbal medicine extracts are inhibited to PRV infection development.Chinese holly, ganoderma lucidum Extract inhibitory effect is most obvious, this may be due in extract alcohol radical, ketone group and PRV adhesion protein interaction to Influence the absorption of virus.
Test two, assay
1, chromatographic condition and system suitability
Using octadecylsilane chemically bonded silica as filler；Rule using acetonitrile as mobile phase A, using water as Mobile phase B, according to the form below Surely gradient elution is carried out；Detection wavelength is 210nm.Number of theoretical plate is calculated by pedunculoside peak should be not less than 3000.
The preparation of reference substance solution: taking pedunculoside reference substance appropriate, accurately weighed, adds 50% methanol that every lml is made and contains O.3mg solution to get.
The preparation of test solution: taking SIJIQING extract powder about 1g prepared by the present invention, accurately weighed, sets tool plug cone In shape bottle, 50% methanol 50ml, weighed weight is added in precision, and ultrasonic treatment (power 300W, frequency 40kHz) 30 minutes is let cool, Weighed weight again is supplied the weight of less loss with 50% methanol, is shaken up, filtration, take subsequent filtrate to get.
Measuring method: accurate absorption reference substance solution and each 10ul of test solution respectively, injection liquid chromatograph, measurement, To obtain the final product.
This product is calculated by dry product, (the C containing pedunculoside36H58010) 1.35% must not be less than.

Claims (6)

1. a kind of preparation method of SIJIQING extract, which is characterized in that the preparation method comprises the following steps:
S1, appropriate Chinese holly crushed after being dried is taken, crosses 10 mesh~24 meshes, obtains Chinese holly powder, it is spare；
S2, it takes Chinese holly powder obtained in step S1 to add acidic ethanol refluxing extraction 1.5~3.0 hours of appropriate concentration, goes forward side by side The dregs of a decoction and filtrate are collected in row filtering, and filtrate is spare；
S3, filtrate decompression obtained in step S2 is concentrated into 60 DEG C and measure obtain relative density be 1.01~1.06 leaching Cream；
S4, medicinal extract obtained in step S3 is spray-dried, obtains SIJIQING extract.
2. the preparation method of SIJIQING extract according to claim 1, which is characterized in that the preparation method includes following Step:
S1, appropriate Chinese holly crushed after being dried is taken, crosses 10 mesh~24 meshes, obtains Chinese holly powder, it is spare；
S2, it takes Chinese holly powder obtained in step S1 to add acidic ethanol refluxing extraction 2 hours of appropriate concentration, and is filtered, The dregs of a decoction and filtrate are collected, filtrate is spare；
S3, filtrate decompression obtained in step S2 is concentrated into 60 DEG C and measure obtain relative density be 1.03 medicinal extract；
S4, medicinal extract obtained in step S3 is spray-dried, obtains SIJIQING extract.
3. the preparation method of SIJIQING extract according to claim 2, which is characterized in that in the S2 step plus four seasons The 20%-70% ethanol solution of green 7-10 times of the powder weight acid containing 0.1%-0.5% for measuring volume carries out refluxing extraction.
4. the preparation method of SIJIQING extract according to claim 3, which is characterized in that the acid second in the S2 step Alcohol is 20%~70% ethyl alcohol containing 0.1%~0.5% acid.
5. the preparation method of SIJIQING extract according to claim 4, which is characterized in that the Chinese holly in the step S1 For the herb of Chinese holly or one or more of mixtures of leaf, the acid in the S2 step is concentrated hydrochloric acid, phosphoric acid, glacial acetic acid, lemon One or more of lemon acid.
6. the preparation method of SIJIQING extract according to claim 1, which is characterized in that the SIJIQING extract is used In preparing the application in resisting pstudorabies virus drugs.
CN201910943641.3A 2019-09-30 2019-09-30 A kind of preparation method and applications of SIJIQING extract Withdrawn CN110507679A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114052144A (en) * 2021-12-07 2022-02-18 青岛大学附属医院 Traditional Chinese medicine feed additive for improving growth performance of animals and preparation method thereof

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* Cited by examiner, † Cited by third party
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CN102151256B (en) * 2011-02-23 2012-10-10 中国农业大学 Application of protocatechuic acid in preparation of drugs for preventing and controlling livestock and poultry virus infectious diseases

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114052144A (en) * 2021-12-07 2022-02-18 青岛大学附属医院 Traditional Chinese medicine feed additive for improving growth performance of animals and preparation method thereof

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