CN102379931A - Novel application of Chinese medicinal composition - Google Patents
Novel application of Chinese medicinal composition Download PDFInfo
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- CN102379931A CN102379931A CN2011103651053A CN201110365105A CN102379931A CN 102379931 A CN102379931 A CN 102379931A CN 2011103651053 A CN2011103651053 A CN 2011103651053A CN 201110365105 A CN201110365105 A CN 201110365105A CN 102379931 A CN102379931 A CN 102379931A
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Abstract
The invention discloses application of a Chinese medicinal composition to preparation of a medicine for resisting infection and/or treating inflammation and/or improving immunity. The Chinese medicinal composition is prepared from Chinese herbal medicines such as raw astragalus, Chinese angelica, honeysuckle flower, sweet wormwood herb, giant knotweed rhizome and the like. The composition has obvious effects of resisting the infection and the inflammation and regulating immune disorder.
Description
Technical field
The present invention relates to a kind of new purposes of Chinese medicine composition, particularly in the new purposes of treating infectious disease.
Background technology
The disorder of inflammatory reaction and immunologic function plays a part crucial in the generation of infectious disease, evolution, and multi-drug resistant bacteria infects no exception.The inflammatory reaction that it occurs after infection with non-drug-fast bacteria infection and the disorder of immunologic function are consistent; For no other reason than that it is effective not as non-fastbacteria to the treatment of multi-drug resistant bacteria; Can not remove this infective agent of multi-drug resistant bacteria timely and effectively; Also just mean and can not adjust the inflammatory reaction of its initiation and the disorder of immunologic function at short notice, thereby it is difficult to cause multi-drug resistant bacteria to infect delay, prognosis is relatively poor.
The polypeptide array technique can combine with autoantibody with polypeptide simulated albumin antigen, is considered to the novel protein site detection technique that genome times afterwards comprehensively and gene chip, protein chip compare favourably.Be widely used in fields such as antibody research, gene expression analysis, the new gene of discovery, medical diagnosis on disease and drug screening at present, had a extensive future.At present domestic should the technology more cancer research that is used for, like the early monitoring of tumor patient autoantibody, the research of antibody recognition epitope etc. are as using the EGFR autoantibody among the polypeptide chip research nonsmall-cell lung cancer patients serum; Other has the antibody chip of application to identify the unconventionality expression albumen in the nephridial tissue, carries out the early diagnosis and therapy monitoring of chronic nephropathy etc.In the basic research that the Chinese medicine of bacterial infection is intervened, do not retrieve related application as yet at present.
At present prior art openly the present composition be used to treat multi-drug resistant bacteria infect due to inflammatory reaction and immunologic function disorder.
Summary of the invention
The object of the present invention is to provide the application of a kind of Chinese medicine composition in preparation anti-infective, raising immunologic function medicine.
The objective of the invention is to realize through following technical scheme:
The present invention provides a kind of Chinese medicine composition in the preparation anti-infectives, to use;
The present invention provides a kind of Chinese medicine composition in preparation infection and/or treatment inflammation and/or raising immune drug, to use; Said Chinese medicine composition is processed by following crude drug:
Radix Astragali 30-120 weight portion Radix Angelicae Sinensis 5-20 weight portion
Flos Lonicerae 10-25 weight portion Herba Artemisiae Annuae 10-30 weight portion
Rhizoma Polygoni Cuspidati 5-20 weight portion.
Above-mentioned Chinese medicine composition is preferably processed by following crude drug:
Radix Astragali 40-110 weight portion Radix Angelicae Sinensis 6-19 weight portion
Flos Lonicerae 12-23 weight portion Herba Artemisiae Annuae 12-28 weight portion
Rhizoma Polygoni Cuspidati 5-20 weight portion.
Above-mentioned Chinese medicine composition is preferably processed by following crude drug:
Radix Astragali 55-95 weight portion Radix Angelicae Sinensis 8-15 weight portion
Flos Lonicerae 12-22 weight portion Herba Artemisiae Annuae 13-25 weight portion
Rhizoma Polygoni Cuspidati 5-20 weight portion.
Above-mentioned Chinese medicine composition is preferably processed by following crude drug:
Radix Astragali 85 weight portion Radix Angelicae Sinensis 15 weight portions
Flos Lonicerae 18 weight portion Herba Artemisiae Annuaes 22 weight portions
Rhizoma Polygoni Cuspidati 10 weight portions.
Above-mentioned Chinese medicine composition is preferably processed by following crude drug:
Radix Astragali 55 weight portion Radix Angelicae Sinensis 19 weight portions
Flos Lonicerae 24 weight portion Herba Artemisiae Annuaes 15 weight portions
Rhizoma Polygoni Cuspidati 6 weight portions.
Above-mentioned Chinese medicine composition is preferably processed by following crude drug:
Radix Astragali 110 weight portion Radix Angelicae Sinensis 8 weight portions
Flos Lonicerae 15 weight portion Herba Artemisiae Annuaes 28 weight portions
Rhizoma Polygoni Cuspidati 18 weight portions.
Above-mentioned application, infection wherein are due to the Pseudomonas aeruginosa;
Said Pseudomonas aeruginosa is CGMCC No.5317.
Above-mentioned Chinese medicine composition adds conventional adjuvant, according to common process, processes various dosage forms such as acceptable clinically water decoction, capsule, tablet, pill, oral liquid, injection, powder.
Said common process method comprises water extraction or alcohol extraction or first water extraction water extraction again after alcohol extraction or the first alcohol extraction again; It is further active constituent-enriched after above-mentioned conventional the extraction, can also to cross macroporous resin column.
For above-mentioned dosage form can be realized, need when these dosage forms of preparation, to add the pharmacy acceptable auxiliary, for example: filler, disintegrating agent, lubricant, suspending agent, binding agent, sweeting agent, correctives, antiseptic, substrate etc.Filler comprises: starch, pregelatinized Starch, lactose, mannitol, chitin, microcrystalline Cellulose, sucrose etc.; Disintegrating agent comprises: starch, pregelatinized Starch, microcrystalline Cellulose, carboxymethyl starch sodium, crospolyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, cross-linking sodium carboxymethyl cellulose etc.; Lubricant comprises: magnesium stearate, sodium lauryl sulphate, Pulvis Talci, silicon dioxide etc.; Suspending agent comprises: polyvinylpyrrolidone, microcrystalline Cellulose, sucrose, agar, hydroxypropyl emthylcellulose etc.; Binding agent comprises, starch slurry, polyvinylpyrrolidone, hydroxypropyl emthylcellulose etc.; Sweeting agent comprises: saccharin sodium, Aspartane, sucrose, cyclamate, enoxolone etc.; Correctives comprises: sweeting agent and various essence; Antiseptic comprises: parabens, benzoic acid, sodium benzoate, sorbic acid and its esters, benzalkonium bromide, fixed, the Folium eucalypti globueli (Eucalyptus globulus Labill.) wet goods of acetic acid chloroethene; Substrate comprises: PEG6000, PEG4000, insect wax etc.For making above-mentioned dosage form can realize pharmacy of Chinese materia medica, need when these dosage forms of preparation, to add acceptable other adjuvant of pharmacy (adjuvant of each dosage form record among the Fan Biting " pharmacy of Chinese materia medica ", Shanghai Science Press December in 1997 the 1st edition).
The present composition is set upright mortality rate that expelling pathogenic factors from the exterior can reduce the abdominal cavity infection rat, is regulated the effect of infecting back rat inflammation and immunologic derangement.
Bacterial strain preservation information:
The depositary institution that No. 1643, multidrug resistant Pseudomonas aeruginosa clinical separation strain according to the invention: China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Preservation date: on October 10th, 2011; Numbering: CGMCC No.5317; Classification name: Pseudomonas aeruginosa Pseudomonas aeruginosa.
The specific embodiment
Experimental example:
1. material and instrument
1.1 intervention medicine
1.1.1 set upright the expelling pathogenic factors from the exterior formula extraction: according to the method preparation of embodiment 1.Low concentration 0.5g/ml, middle concentration 0.99g/ml, high concentration 1.98g/ml.This is tested set rat and sets upright the low dose of dose in expelling pathogenic factors from the exterior side and be equivalent to 2.15 times of 70kg adult consumption, and middle dosage is equivalent to 6.3 times of 70kg adult consumption, and heavy dose of dose is equivalent to 12.6 times of 70kg adult consumption.
1.1.2 ceftazidime for inj: the batch number E20100102 of Shenzhen Zhijun Pharmaceutical Co., Ltd.
1.1.3 cefoperazone for inj sulbactam sodium: pfizer inc batch number 1039098.
1.1.4 imipenem for injection Cilastatin Sodium: Merck&Co., Inc.USA batch number 100231.
1.2 laboratory animal: the SD rat, male and female half and half, the cleaning level, body weight 200～220g, available from Test Animal Centre, Academy of Military Medical Sciences, P.L.A, quality certification SCXK-(army) 2007-004.
Experimental strain: No. 1643, multidrug resistant Pseudomonas aeruginosa clinical separation strain, numbering: CGMCC No.5317; Classification name: Pseudomonas aeruginosa Pseudomonas aeruginosa.
1.3 main agents and instrument
Silent generation that biochemistry goods (Beijing) company limited that flies of 1.3.1M-H agar plate: XWO1100301 match.
1.3.2RPMI-1640 culture medium: Sigma.
1.3.3 concanavalin A, Con A: Sigma.
1.3.4 lipopolysaccharide: Sigma.
1.3.5MTT：Sigma。
1.3.6 calf serum: people's marine growth company.
1.3.7 phosphate buffer (PBS) powder: ancient cooking vessel state Bioisystech Co., Ltd.
1.3.8Tris: Shanghai chemical reagent factory.
1.3.9NH4Cl: Red Star chemical plant, Beijing.
1.3.10 0.01NH4Cl-10％SDS：GIBCO。
1.3.11 Rat IL-1 β ELISA test kit: 96t ZABFBZAA01 Shanghai Excell Biology Product Co., Ltd..
1.3.12 Rat IL-2ELISA test kit: 96t ZJBFBZAZ03 Shanghai Excell Biology Product Co., Ltd..
1.3.13 Rat IL-4ELISA test kit: 96t ZCAABZAA03 Shanghai Excell Biology Product Co., Ltd..
1.3.14 Rat IL-10ELISA test kit: 96t ZLBGBZAZ02 Shanghai Excell Biology Product Co., Ltd..
1.3.15 Rat IFN-γ ELISA test kit: 96t ZCCZBZAA02 Shanghai Excell Biology Product Co., Ltd..
1.3.16 Rat TNF-α ELISA test kit: 96t ZDBZBZAA02 Shanghai Excell Biology Product Co., Ltd..
1.3.17 opacity tube.
1.3.18 micropipettor: GILSON P 0.1-1.0ml.
1.3.19 electronic scale: SCOUT SC6010 prunus mume (sieb.) sieb.et zucc. Teller. holder benefit Changzhou weighing apparatus company limited.
1.3.20 electronic micro balance: the flat more scientific instrument company limited in JA3003 Shanghai.
1.3.21 clinical thermometer: MC140 OMRON.
1.3.22 vertical automatic electric heating pressure steam sterilizer: AMA440N ATELL.
1.3.23 electro-heating standing-temperature cultivator: DNP9162 JINGHONG.
1.3.24 whirlpool blender: VORTEX-2 GENIE.
1.3.25 micro-stirrer: 75-2 type Shanghai medical analytical appearance factory.
1.3.26 low-temperature and high-speed centrifuge: Labofuge 400R Kendro.
1.3.27 superclean bench: Beijing is all green.
1.3.28 enzyme micro-plate reader: DNM-9602 PROLONG.
1.3.29 the long multi-functional ELIASA of all-wave: SepctraMR MRW DYNEX
1.3.30 Automatic Blood Cell Analyzer: LH 750 ANALYZER BECKMANCOULTER.
1.3.31 special proteins analyser: BN ProSpec SIEMENS.
1.3.32 AutoSpot peptide synthesizer; FX-7 digital image analyser,
1.3.33 PEG modified cellulose film, Fmoc-aminoacid, DCM, aminefreeDMF; Ethanol, Acetic anhydride, Acetic anhydride, DIPEA (Diisopropylamine); DIC (Diisopropylcarbodiimide), Bromophenolblue, Piperidine etc.
1.4 reagent preparation
1.5.1 the preparation of 10% chloral hydrate solution
Chloral hydrate 10g
Add distilled water 100ml
1.5.2 Tris-NH
4The preparation of Cl solution
Tris solution (0.17mol/L) 10ml
NH
4Cl solution (0.16mol/L) 90ml
1.6 the preparation of bacteria suspension
Experimental strain is inoculated in the activation of M-H agar culture medium, behind 37 ℃ of constant temperature culture 18～24h,, centrifugal with normal saline eluting bacterium colony; 3000rpm, 20min, supernatant discarded adds normal saline; Sucking-off 0.5ml adds in the new sterile test tube behind the mixing, adds an amount of normal saline, carries out than turbid bacterium liquid being diluted to 24 * 108CFU/ml with nephelometer; Using ELIASA to detect the OD value is 0.675 ± 0.005, again it is diluted to 0.48 * 108CFU/ml, as the experimental bacteria suspension.
2 experimental techniques
2.1 animal model
The SD rat is conventional to be raised a week, and room temperature is controlled between 18 ℃～22 ℃, freely obtains food, water, overnight fasting before the blood sampling.
According to Yang Jun, Zhang Shuwen, cloudy Cheng is grand, etc. the foundation [J] of fastbacteria abdominal cavity infection rabbit animal model. Chinese hospital infection magazine, 2008,18 (9): the 1129-1222 reported method is set up multidrug resistant Pseudomonas aeruginosa abdominal cavity infection rat model.Concrete grammar is following: the conventional raising, and after the mixing in 1: 1 of multidrug resistant Pseudomonas aeruginosa bacterium liquid (hereinafter to be referred as bacterium liquid) 2.0% tragacanth together, lumbar injection.Injection back ad lib, drinking-water.Matched group: isopyknic 2.0% tragacanth of lumbar injection.
2.2 animal divides into groups and handles
2.2.1 mortality rate protection experiment
88 of SD rats, male and female half and half are divided matched group, model group at random, set upright expelling pathogenic factors from the exterior side's small dose group, set upright dose groups in the expelling pathogenic factors from the exterior side, are set upright the heavy dose of group in expelling pathogenic factors from the exterior side (being B0, B1, B2, B3, B4), and totally 5 groups, 8 of B0 groups, all the other 4 groups every group each 20.Behind the B0 group lumbar injection tragacanth (8ml/kg), promptly give 0.9% normal saline and irritate stomach 2ml, once a day.Behind the B1 group lumbar injection bacterium liquid (8ml/kg), promptly give 0.9% normal saline and irritate stomach 2ml, once a day.Behind B2, B3, the B4 group lumbar injection bacterium liquid (8ml/kg), set upright expelling pathogenic factors from the exterior formula extraction 0.5g/ml, 0.99g/ml, 1.98g/ml respectively and irritates stomach, 2ml at every turn, once a day.
More than each group respectively at mortality rate of 6 hours statistics behind the lumbar injection, mortality rate of per thereafter 24 hours statistics the 5th day be whole last point of observation.Filter out and set upright the best dosage in expelling pathogenic factors from the exterior side and be: 9.9g/kg/ days.
96 of SD rats, male and female half and half, at random sub-model group, western medicine group, set upright expelling pathogenic factors from the exterior side's optimal dose group, therapy of combining Chinese and Western medicine group (being C1, C2, C3, C4), totally 4 groups, every group each 24.Behind the C1 group lumbar injection bacterium liquid (8ml/kg), promptly give 0.9% normal saline and irritate stomach 2ml, once a day.Behind the C2 group lumbar injection bacterium liquid (8ml/kg), promptly give ceftazidime, intramuscular injection, 0.45g/kg/ time, twice of every day.Behind the C3 group lumbar injection bacterium liquid (8ml/kg), promptly set upright expelling pathogenic factors from the exterior formula extraction (0.99g/ml) and irritate stomach, each 2ml, once a day.Behind the C4 group lumbar injection bacterium liquid (8ml/kg), promptly set upright expelling pathogenic factors from the exterior formula extraction (0.99g/ml) and irritate stomach, each 2ml once a day, gives ceftazidime, intramuscular injection, 0.45g/kg/ time, twice of every day simultaneously.
More than each group respectively at mortality rate of 6 hours statistics behind the lumbar injection, mortality rate of per thereafter 24 hours statistics the 5th day be whole last point of observation.
2.2.2 index test experience
200 of SD rats, male and female half and half are divided into blank control group, model group, western medicine group at random, set upright expelling pathogenic factors from the exterior side's optimal dose group, therapy of combining Chinese and Western medicine group (being D1, D2, D3, D4), and totally 5 groups, every group each 40.Behind isopyknic 2.0% tragacanth of matched group lumbar injection (5ml/kg), promptly give 0.9% normal saline and irritate stomach, each 2ml, once a day.D1, D2, D3, D4 group lumbar injection bacterium liquid are 5ml/kg, and all the other processing methods are identical with the mortality rate observation experiment.More than each group be divided into 3h behind the lumbar injection, 8h, 1d, 3d and 5d group again by time point, every group each 8, each group is left and taken blood and tissue specimen by corresponding time point shown in dividing into groups.1d, 3d and 5d organize and before leaving and taking blood and tissue specimen, measure the anus temperature.
36 of male SD rats are divided into blank control group, model group at random, set upright expelling pathogenic factors from the exterior side's optimal dose group, and totally 3 groups, every group each 12.Behind the model group lumbar injection lumbar injection bacterium liquid (5ml/kg), promptly give 0.9% normal saline and irritate stomach, each 2ml, once a day.Set upright (5ml/kg) behind expelling pathogenic factors from the exterior side's optimal dose group lumbar injection bacterium liquid, promptly set upright expelling pathogenic factors from the exterior formula extraction (0.99g/ml) and irritate stomach, each 2ml, once a day.Leave and take the above rat blood serum BIAO and BEN of respectively organizing behind the 7d ,-20 ℃ of preservations supply the polypeptide array detection to use.
2.3 the collection of BIAO and BEN and processing
2.3.1 blood specimen
Give lumbar injection 10% chloral hydrate solution, 4g/kg, after the anesthesia, routine disinfection rat abdomen skin is opened the abdominal cavity, separates ventral aorta, gives disposable sterilized blood taking needle and connects 2ml EDTA-K3 blood taking tube and the blank blood taking tube of 5ml respectively, gets blood.After blood sampling finished, EDTA-K3 blood taking tube censorship routine blood test after blank blood taking tube room temperature is placed 1h, is placed 1h for 4 ℃ again, and was centrifugal, 2500rpm, 20min, separation of serum ,-70 ℃ of preservations after the packing.
2.3.2 tissue specimen
Asepticly leave and take lung, small intestine puts into 4% formaldehyde fixed liquid, cut into slices, tectology observes.
2.4 detect index and method
2.4.1 the ELISA of rat blood serum IL-1 β detects
1. preparation:
(1) from refrigerator, took out test kit in 20 minutes in advance, with balance to room temperature.(2) concentrated cleaning solution is diluted (1: 20) with distilled water.(3) standard substance: add reagent diluent 0.5ml to the lyophilizing standard substance, left standstill 15 minutes, treat that it fully dissolves after, mixing (concentration is 2000pg/ml) gently.Then according to 2000,1000,500,250,125,62.5,31.25,0pg/ml concentration dilutes.(4) biotinylated antibody working solution: use to be configured to the biotinylated antibody working solution with reagent diluted concentrated biological elementization antibody (1: 100) in preceding 30 minutes.(5) enzyme conjugates working solution: use to concentrate enzyme conjugates (1: 100) with the reagent diluted in preceding 30 minutes and be configured to the enzyme conjugates working solution, the room temperature lucifuge is placed.
2. washing methods: use automatic washer, inject 350 μ l cleaning mixture, inject and sucking-off 20-30 second at interval.Wash plate 4 times.
3. operating procedure:
(1) from the sealing bag of balance to room temperature, takes out the required lath of test.(2) hole that blanks.(3) standard substance (100 μ l/ hole) have been added by the standard substance dilution process earlier, 0pg/ml hole additional examination dilution agent liquid (Assay Diluent) 100 μ l; In each sample aperture, add 50 μ l reagent diluents (Assay Diluent) and 50 μ l samples (this moment, sample was done 1: 2 times of dilution, and sample content will multiply by 2 during result of calculation); In sample and gauge orifice, add 50 μ l biotinylated antibody working solutions subsequently, seal reacting hole with the shrouding gummed paper.(4) room temperature (20-25 ℃), (low-limit frequency 100rpm), was hatched 120 minutes to use micro-shaker.(5) wash plate 5 times.(6) except that blank well, add enzyme conjugates working solution (100 μ l/ hole), seal reacting hole with shrouding glue.(7) room temperature (20-25 ℃), (low-limit frequency 100rpm), was hatched 60 minutes to use micro-shaker.(8) wash plate 5 times.(9) add chromogenic substrate (comprising blank well) 100 μ l/ holes, room temperature (22-25 ℃), lucifuge was hatched 10 minutes.(10) add stop buffer (comprising blank well) 100 μ l/ holes, measure OD450 value (in 10 minutes) behind the mixing at once.
4. the result judges:
(1) the OD value of each standard substance and BIAO and BEN deducts the OD value in zero hole.(2) manual drawing standard curve.Make abscissa with standard substance concentration, the OD value is made vertical coordinate, connects the coordinate points of each standard substance with sweep.OD value through BIAO and BEN is found its concentration on standard curve.(3) if BIAO and BEN OD value is higher than the standard curve upper limit, resurvey after should diluting, should multiply by extension rate during calculating concentration.
2.4.2 the ELISA of rat blood serum IL-10 detects
1. preparation:
(1) from refrigerator, took out test kit in 20 minutes in advance, with balance to room temperature.(2) concentrated cleaning solution is diluted (1: 20) with distilled water.(3) standard substance: add reagent diluent 0.5ml to the lyophilizing standard substance, left standstill 15 minutes, treat that it fully dissolves after, mixing (concentration is 4000pg/ml) gently.Then according to 4000,2000,1000,500,250,125,62.5,0pg/ml concentration dilutes.(4) biotinylated antibody working solution: use to be configured to the biotinylated antibody working solution with reagent diluted concentrated biological elementization antibody (1: 100) in preceding 30 minutes.(5) enzyme conjugates working solution: use to concentrate enzyme conjugates (1: 100) with the reagent diluted in preceding 30 minutes and be configured to the enzyme conjugates working solution, the room temperature lucifuge is placed.
2. washing methods: use automatic washer, inject 350 μ l cleaning mixture, inject and sucking-off 20-30 second at interval.Wash plate 4 times.
3. operating procedure:
(1) from the sealing bag of balance to room temperature, takes out the required lath of test.(2) hole that blanks.(3) respectively BIAO and BEN or variable concentrations standard substance (0pg/ml hole additional examination dilution agent liquid) are added in the respective aperture (100 μ l/ hole), seal reacting hole with shrouding glue, 37 ℃ of incubators were hatched 90 minutes.(4) wash plate 5 times.(5) except that blank well, add biotinylated antibody working solution (100 μ l/ hole).Seal reacting hole with shrouding glue, 37 ℃ of incubators were hatched 60 minutes.(6) wash plate 5 times.(7) except that blank well, add enzyme conjugates working solution (100 μ l/ hole).Seal reacting hole with shrouding glue, 37 ℃ of incubators, lucifuge was hatched 30 minutes.(8) wash plate 5 times.(9) add chromogenic substrate (comprising blank well) 100 μ l/ holes, 37 ℃ of incubators, lucifuge was hatched 15 minutes.(10) add stop buffer (comprising blank well) 100 μ l/ holes, measure OD450 value (in 10 minutes) behind the mixing at once.
4. the result judges: the ELISA with rat blood serum IL-1 β detects.
2.4.3ConA inductive spleen t-cell multiplication capacity is measured: adopt the MH method.
The aseptic Rats Spleen (operate in the ice bath and carry out) of winning, conventional preparation splenocyte suspension, counting splenocyte, reuse contain RPMI1640 culture medium adjustment cell concentration to the 5 * 106/mL of 10% calf serum, add in 96 plates 100 μ L/ holes.Add respectively and contain the complete RPMI1640 solution of ConA 100 μ L/ holes (the ConA final concentration is 5 μ g/mL), establish 5 multiple holes for every group, and establish no ConA cell contrast.96 orifice plates are put 37 ℃, and the 5%CO2 incubator is hatched 68h, takes out; Supernatant 100 μ L are abandoned in every hole, add the MTT10 μ L (final concentration 0.5mg/mL) of 5mg/mL, continue to hatch 4h; Add 0.01N HCl-10%SDS 100 μ L/ holes; Vibration shakes up, and 37 ℃ are spent the night, and ELIASA 570nm measures each hole A value down.
2.4.4LPS the mensuration of inductive spleen bone-marrow-derived lymphocyte multiplication capacity: adopt mtt assay.
The aseptic Rats Spleen (operate in the ice bath and carry out) of winning, conventional preparation splenocyte suspension, counting splenocyte, reuse contain RPMI1640 culture medium adjustment cell concentration to the 5 * 106/mL of 10% calf serum, add in 96 plates 100 μ L/ holes.Add respectively and contain the complete RPMI1640 solution of LPS 100 μ L/ holes (the LPS final concentration is 5 μ g/mL), establish 5 multiple holes for every group, and establish no LPS cell contrast.96 orifice plates are put 37 ℃, and the 5%CO2 incubator is hatched 68h, takes out; Supernatant 100 μ L are abandoned in every hole, add the MTT10 μ L (final concentration 0.5mg/mL) of 5mg/mL, continue to hatch 4h; Add 0.01N HCl-10%SDS100 μ L/ hole; Vibration shakes up, and 37 ℃ are spent the night, and ELIASA 570nm measures each hole A value down.
2.4.5 the ELISA of rat blood serum IFN-γ detects
1. preparation:
(1) from refrigerator, took out test kit in 20 minutes in advance, with balance to room temperature.(2) concentrated cleaning solution is diluted (1: 20) with distilled water.(3) standard substance: add reagent diluent 1.0ml to the lyophilizing standard substance, left standstill 15 minutes, treat that it fully dissolves after, mixing (concentration is 2000pg/ml) gently.Then according to 2000,1000,500,250,125,62.5,31.25,0pg/ml concentration dilutes.(4) biotinylated antibody working solution: use to be configured to the biotinylated antibody working solution with reagent diluted concentrated biological elementization antibody (1: 100) in preceding 30 minutes.(5) enzyme conjugates working solution: use to concentrate enzyme conjugates (1: 100) with the reagent diluted in preceding 30 minutes and be configured to the enzyme conjugates working solution, the room temperature lucifuge is placed.
2. washing methods: use automatic washer, inject 350 μ l cleaning mixture, inject and sucking-off 20-30 second at interval.Wash plate 4 times.
3. operating procedure:
(1) from the sealing bag of balance to room temperature, takes out the required lath of test.(2) hole that blanks.(3) standard substance (100 μ l/ hole) have been added by the standard substance dilution process earlier, 0pg/ml hole additional examination dilution agent liquid (Assay Diluent) 100 μ l; In each sample aperture, add 50 μ l reagent diluents (Assay Diluent) and 50 μ l samples (this moment, sample was done 1: 2 times of dilution, and sample content will multiply by 2 during result of calculation); In sample and gauge orifice, add 50 μ l biotinylated antibody working solutions subsequently, seal reacting hole with the shrouding gummed paper.(4) room temperature (20-25 ℃), (low-limit frequency 100rpm), was hatched 120 minutes to use micro-shaker.(5) wash plate 5 times.(6) except that blank well, add enzyme conjugates working solution (100 μ l/ hole), seal reacting hole with shrouding glue.(7) room temperature (20-25 ℃), (low-limit frequency 100rpm), was hatched 60 minutes to use micro-shaker.(8) wash plate 5 times.(9) add chromogenic substrate (comprising blank well) 100 μ l/ holes, room temperature (22-25 ℃), lucifuge was hatched 10 minutes.(10) add stop buffer (comprising blank well) 100 μ l/ holes, measure OD450 value (in 10 minutes) behind the mixing at once.
4. the result judges: the ELISA with rat blood serum IL-1 β detects.
2.4.6 the ELISA of rat blood serum IL-4 detects
1. preparation:
(1) from refrigerator, took out test kit in 20 minutes in advance, with balance to room temperature.(2) concentrated cleaning solution is diluted (1: 20) with distilled water.(3) standard substance: add reagent diluent 1.0ml to the lyophilizing standard substance, left standstill 15 minutes, treat that it fully dissolves after, mixing (concentration is 2000pg/ml) gently.Then according to 2000,1000,500,250,125,62.5,31.25,0pg/ml concentration dilutes.(4) biotinylated antibody working solution: use to be configured to the biotinylated antibody working solution with reagent diluted concentrated biological elementization antibody (1: 100) in preceding 30 minutes.(5) enzyme conjugates working solution: use to concentrate enzyme conjugates (1: 100) with the reagent diluted in preceding 30 minutes and be configured to the enzyme conjugates working solution, the room temperature lucifuge is placed.
2. washing methods: use automatic washer, inject 350 μ l cleaning mixture, inject and sucking-off 20-30 second at interval.Wash plate 4 times.
3. operating procedure:
(1) from the sealing bag of balance to room temperature, takes out the required lath of test.(2) hole that blanks.(3) respectively BIAO and BEN or variable concentrations standard substance (0pg/ml hole additional examination dilution agent liquid) are added in the respective aperture (100 μ l/ hole), seal reacting hole with shrouding glue, 37 ℃ of incubators were hatched 90 minutes.(4) wash plate 5 times.(5) except that blank well, add biotinylated antibody working solution (100 μ l/ hole).Seal reacting hole with shrouding glue, 37 ℃ of incubators were hatched 60 minutes.(6) wash plate 5 times.(7) except that blank well, add enzyme conjugates working solution (100 μ l/ hole).Seal reacting hole with shrouding glue, 37 ℃ of incubators, lucifuge was hatched 30 minutes.(8) wash plate 5 times.(9) add chromogenic substrate (comprising blank well) 100 μ l/ holes, 37 ℃ of incubators, lucifuge was hatched 15 minutes.(10) add stop buffer (comprising blank well) 100 μ l/ holes, measure OD450 value (in 10 minutes) behind the mixing at once.
4. the result judges: the ELISA with rat blood serum IL-1 β detects.
2.4.7 polypeptide array detection drug-resistant protein antibody
1. according to the white sequence of Pseudomonas Aeruginosa medicated egg, select ultra wide spectrum lactamase (TEMs/ESBLs) TEM (ABB76656), made 9 polypeptide arrays with the polypeptide array technique.Every kind of albumen has arranges 93 polypeptide on 3 every TEM of repetition polypeptide array (ABB76656) protein polypeptide array; Configuration 0.3M Fmoc-Freamine; Confining liquid I:2% (v/v) aceticanhydride in DMF; Confining liquid II:2% (v/v) acetic anhydride+2% (v/v) DIPEAin DMF removes to protect solution (20% (v/v) piperidine in DMF).Be placed on activatory PEG-cellulose membrane on the Autospotter; The Fmoc-Freamine that moves liquid 30 μ l according to Automatic Program reacted 2x20 minute to ad-hoc location and film on the activatory PEG-cellulose membrane; Then the PEG-cellulose membrane immerse according to the order of sequence (reacted 5 minutes) among the confining liquid I and confining liquid II in (reacting 20 minutes); Carry out the side chain sealing, clean 4 times each 30 seconds then with DMF.Add then and go to protect (2 times, each 5 minutes) in the solution, be used to remove N-terminal Fmoc blocking group; Go to clean 3 times with DMF after the protection, in each 30 seconds, then reuse ethanol is dry.If the aminoacid that is used for next time is synthetic, repeat above step, all synthetic until each polypeptide.After all synthetic, remove the side chain protected group with TFAcocktail earlier, reuse CH2Cl2 cleans 4 times, then uses ethanol dry, and-20 ℃ of preservations are used for next step immunoreation experiment.According to experimental design, the polypeptide automatic synthesizer synthesizes the polypeptide array, and every polypeptide array laterally is 12 polypeptide points, vertically is 8 polypeptide points.(seeing accompanying drawing 1)
2. sealing: 4%skim milk+5%sucrose in TBST buffer, room temperature 4 hours.
3. anti-hatching: rat blood serum dilution in 1: 200; With polypeptide array reaction, spend the night under 4 ℃.
4. wash film: with TBST buffer rinsing, 10 minutes * 3 times.
5. two anti-hatching: two anti-(sheep anti mouse) IgG-HRP, diluted following 2 hours of room temperature in 1: 1000 or 1: 2000 with confining liquid.
6. wash film: with TBST buffer rinsing, 10 minutes * 3 times.
7. colour developing: add the ECL luminescence reagent, digital image.
3 statistical procedures methods
Adopt SPSS 17.0 statistical packages, the parameter measurement data is carried out t check, variance analysis, and enumeration data uses chi-square criterion to carry out statistical procedures.Each measurement data representes that with average ± standard deviation
inspection level is α=0.05.
4 results
4.1 various dose is set upright the influence of expelling pathogenic factors from the exterior side's treatment to the protection of abdominal cavity infection rats death rate
Behind the lumbar injection bacterium liquid, 6h adds up a mortality rate, mortality rate of every thereafter 24h statistics.All survivals in the matched group 5 days, model group with set upright each dose groups rat of expelling pathogenic factors from the exterior side and behind 6h, begin to occur dead, along with the prolongation of time, mortality rate raises gradually.The mortality rate that each time point is set upright each dose groups rat of expelling pathogenic factors from the exterior side behind the 6h all has reduction in various degree than model group.Chi-square criterion shows, compares no significant difference (seeing table 1) between the corresponding time point group of each dose groups.
Table 1 various dose is set upright the influence of expelling pathogenic factors from the exterior side's treatment to the protection of abdominal cavity infection rats death rate
Annotate: compare with identical time point model group,
*P＜0.05,
*P＜0.01
4.2 different treatment methods is to the influence of abdominal cavity infection rats death rate protection
Behind the lumbar injection bacterium liquid, 6h adds up a mortality rate, mortality rate of every thereafter 24h statistics.Each is organized rat and behind 6h, begins appearance death, and along with the prolongation of time, mortality rate raises gradually.The mortality rate of each treatment group rat of each time point all has reduction in various degree than model group behind the 6h.X2 checks demonstration, 48h, 72h, 120h, and the mortality rate of therapy of combining Chinese and Western medicine group rat reduces than model group, and significant difference (P＜0.05) compares no significant difference (seeing table 2) between each treatment group.
Table 2 different treatment methods is to the influence (n=24) of abdominal cavity infection rats death rate protection
Annotate: compare with identical time point model group,
*P＜0.05,
*P＜0.01
4.3 different treatment methods is to the influence of abdominal cavity infection rat blood serum IL-1 β level
Model group has begun to raise with each treatment group rat blood serum IL-1 β level 3h after infection.Each treatment group rat blood serum IL-1 β peak postpones to occur than model group, and model group rat blood serum IL-1 β maintains and is higher than normal level behind the 72h, and each treatment group rat rat blood serum IL-1 β is then near normal level.
Model group rat blood serum IL-1 β level all obviously raises significant difference than blank control group at each time point; Each treatment group rat blood serum IL-1 β level obviously raises significant difference than blank control group at 3h, 8h, 24h; Each treatment group rat blood serum IL-1 β level does not have significant difference at 72h, 5d than blank control group; Western medicine group rat blood serum IL-1 β level reduces significant difference than model group at 72h, 5d; Set upright expelling pathogenic factors from the exterior side's group rat blood serum IL-1 β level and raise than model group at 24h, reduce than model group at 5d, difference is all remarkable; Combination of Chinese and Western medicine group rat blood serum IL-1 β level raises than model group at 24h, reduces than model group at 72h, 5d, and difference is remarkable (seeing table 3) all.
Table 3 is respectively organized the variation (
n=8) of each time point serum il-1 β level of rat
Annotate: compare with model group,
*P＜0.05,
*P＜0.01
Compare △ P＜0.05, △ △ P＜0.01 with blank control group
4.4 different treatment methods is to the influence of abdominal cavity infection rat lymphocyte propagation level
Infect back 3h, T lymphopoiesis level raises in the model group Rats Spleen, than blank control group difference highly significant (P＜0.01); T lymphopoiesis level reduces in the western medicine group Rats Spleen, than blank control group significant difference (P＜0.05); Set upright in expelling pathogenic factors from the exterior side's optimal dose group and the therapy of combining Chinese and Western medicine group Rats Spleen T lymphopoiesis level and do not have significant difference than blank control group.Western medicine group, set upright in expelling pathogenic factors from the exterior side's optimal dose group and the therapy of combining Chinese and Western medicine group Rats Spleen T lymphopoiesis level and all reduce difference highly significant (P＜0.01) (seeing table 4) than model group.
Table 4 infects back 3h and respectively organizes the lymphopoietic variation of rat T (
n=6)
Annotate: compare with model group,
*P＜0.05,
*P＜0.01
Compare △ P＜0.05, △ △ P＜0.01 with blank control group
Infect back 3h, bone-marrow-derived lymphocyte propagation level raises in the model group Rats Spleen, than blank control group significant difference (P＜0.05); Bone-marrow-derived lymphocyte propagation level reduces in the western medicine group Rats Spleen, than blank control group difference highly significant (P＜0.01); Set upright in expelling pathogenic factors from the exterior side's optimal dose group and the therapy of combining Chinese and Western medicine group Rats Spleen bone-marrow-derived lymphocyte propagation level and do not have significant difference than blank control group.Bone-marrow-derived lymphocyte propagation level all reduces difference highly significant (P＜0.01) in western medicine group and the therapy of combining Chinese and Western medicine group Rats Spleen than model group; Set upright in expelling pathogenic factors from the exterior side's optimal dose group Rats Spleen bone-marrow-derived lymphocyte propagation level and all reduce significant difference (P＜0.05) (seeing table 5) than model group.
Table 5 infects the variation (
n=6) that back 3h respectively organizes rat bone-marrow-derived lymphocyte propagation
Annotate: compare with model group,
*P＜0.05,
*P＜0.01
Compare △ P＜0.05, △ △ P＜0.01 with blank control group
4.5 different treatment methods is to the influence of abdominal cavity infection rat blood serum Th1/Th2 level
Model group begins to occur raising with each treatment group rat blood serum Th1/Th2 level 3h after infection; 8h peaks; Begin subsequently to descend, model group rat blood serum Th1/Th2 maintains and is higher than normal level behind the 72h, and each treatment group rat blood serum Th1/Th2 is then near normal level.
Model group rat blood serum Th1/Th2 level raises than blank control group at 8h, 24h, 72h, 5d, and difference is all remarkable; Western medicine group rat blood serum Th1/Th2 level raises than blank control group at 8h, 24h, and difference is all remarkable; Set upright expelling pathogenic factors from the exterior side's group and combination of Chinese and Western medicine group rat blood serum Th1/Th2 level raises than blank control group at 8h, difference is all remarkable; Western medicine group and combination of Chinese and Western medicine group rat blood serum Th1/Th2 level descend than model group at 72h, 5d, and difference is all remarkable; Set upright expelling pathogenic factors from the exterior side's group rat blood serum Th1/Th2 level and descend than model group at 24h, 72h, 5d, difference is all remarkable; In addition, set upright expelling pathogenic factors from the exterior side's rat blood serum Th1/Th2 level and descend significant difference (P=0.040) at 24h than western medicine group; Combination of Chinese and Western medicine group rat blood serum Th1/Th2 level 5d than western medicine group with set upright expelling pathogenic factors from the exterior side group and descend, all significantly (P=0.003 P=0.005) (sees table 6) to difference.
Table 6 is respectively organized the variation (
n=8) of each time point serum T h1/Th2 level of rat
Annotate: compare with model group,
*P＜0.05,
*P＜0.01
Compare △ P＜0.05, △ △ P＜0.01 with blank control group
4.6 set upright expelling pathogenic factors from the exterior side's treatment pneumoretroperitoneum infected rats serum to the proteic influence of TEM-1
From the polypeptide array of TEM-1 drug-resistant protein and the response spectrum of rat blood serum, find that in three groups, there is not evident difference in the positive epi-position number that antibody is directed against.But, set upright in expelling pathogenic factors from the exterior side's group, be better than infected group and negative control group to the antibody response of 19-21 and 52-55 epi-position, treatment by Chinese herbs might increase the immunoreation of lymphocyte to these epitopes.
4.7 abdominal cavity infection rat organ tectology changes and sets upright the influence of expelling pathogenic factors from the exterior side's treatment
Abdominal cavity infection 8h, lung and small intestinal pathologic damage in various degree all appear in the most rats of model group, and telangiectasis hyperemia in the alveolar septum has more serosity to ooze out and a small amount of erythrocyte, neutrophilic granulocyte and macrophage in the alveolar space; Intestinal mucosa hyperemia, edema and cell infiltration are obvious, and body of gland partly disappears.Set upright expelling pathogenic factors from the exterior side's treatment group and compare with model group, the slight dilatation and congestion of blood capillary in the alveolar septum, cell infiltration is lighter; Intestinal mucosa mild hyperaemia, edema, cell infiltration is lighter, and body of gland does not have obvious disappearance.
Abdominal cavity infection 24h, the pathologic damage of lung and small intestinal all appears in the model group rat, and all change basic features are similar with 8h, and the scope of lung and injury of small intestine does not have obvious expansion.Set upright all change of expelling pathogenic factors from the exterior side's group basic feature and slightly increase the weight of, but do not see that lung and injury of small intestine scope further enlarge than 8h.
Abdominal cavity infection 72h, the telangiectasis of model group part rat alveolar wall is congested, visible red cell in the alveolar space, and the neutrophilic granulocyte and a small amount of macrophage of some are arranged; The intestinal mucosal injury characteristic is similar with 8h, but scope further enlarges.Set upright all of expelling pathogenic factors from the exterior side's group and change basic feature and alleviate to some extent than 24h, similar with 8h.
Abdominal cavity infection 5d, the further dilatation and congestion of model group part rat alveolar wall blood capillary, the visible connection is netted cellulose and erythrocyte in the alveolar space, and the neutrophilic granulocyte and a small amount of macrophage of some are arranged; Intestinal mucosa is further congested, edema, body of gland arrangement disorder, accidental cellulose.Setting upright expelling pathogenic factors from the exterior side's all change basic feature further alleviates than 8h.
The specific embodiment
Embodiment 1: water decoction
Radix Astragali 85g Radix Angelicae Sinensis 15g
Flos Lonicerae 25g Herba Artemisiae Annuae 20g
Rhizoma Polygoni Cuspidati 10g
Get the above-mentioned raw materials medicine, decocte with water 2 times, amount of water is a conventional amount used, merges aqueous extract, processes water decoction.
Embodiment 2: tablet
Radix Astragali 50g Radix Angelicae Sinensis 18g
Flos Lonicerae 24g Herba Artemisiae Annuae 15g
Rhizoma Polygoni Cuspidati 6g
Get the above-mentioned raw materials medicine, add alcohol at normal temperature and flooded 24 hours down, merge extractive liquid, reclaims ethanol, and concentrate drying adds conventional adjuvant, according to common process, processes tablet.
Embodiment 3: capsule
Radix Astragali 110g Radix Angelicae Sinensis 8g
Flos Lonicerae 15g Herba Artemisiae Annuae 28g
Rhizoma Polygoni Cuspidati 18g
Get the above-mentioned raw materials medicine, 10 times that add ethanol to medical material amount are carried out percolation, reclaim ethanol, and concentrate drying adds conventional adjuvant, according to common process, processes capsule.
Embodiment 4: oral liquid
Radix Astragali 40g Radix Angelicae Sinensis 20g
Flos Lonicerae 22g Herba Artemisiae Annuae 12g
Rhizoma Polygoni Cuspidati 8g
Get the above-mentioned raw materials medicine,, process oral liquid according to common process.
Embodiment 5: injection
Radix Astragali 100g Radix Angelicae Sinensis 10g
Flos Lonicerae 12g Herba Artemisiae Annuae 25g
Rhizoma Polygoni Cuspidati 15g
Get the above-mentioned raw materials medicine, add water extraction 3 times, each 1 hour, merging filtrate, concentrate drying adds conventional adjuvant, according to common process, processes injection.
Embodiment 6: granule
Radix Astragali 90g Radix Angelicae Sinensis 15g
Flos Lonicerae 15g Herba Artemisiae Annuae 25g
Rhizoma Polygoni Cuspidati 15g
Get the above-mentioned raw materials medicine, add water extraction 3 times, each 1 hour, merging filtrate, concentrate drying adds conventional adjuvant, according to common process, processes granule.
Description of drawings
Accompanying drawing 1: (sketch map has carried out the Position Number mark to each polypeptide point on the polypeptide chip to polypeptide array pattern sketch map, and each numbers the peptide sequence that corresponding adnexa provides.Below epi-position numeral in all polypeptide array interpretations number corresponding one by one with above-mentioned reference numbers and peptide sequence.)
Accompanying drawing 2 medicines influence picture to the abdominal cavity infection rat blood serum to TEM-1 albumen, and (a is TEM-1 and the reaction of blank control group rat blood serum; B is that TEM-1 and model group rat blood serum reaction c are TEM-1 and set upright the group rat blood serum reaction of expelling pathogenic factors from the exterior side)
Following embodiment all can realize the effect of above-mentioned experimental example.
Claims (9)
1. a Chinese medicine composition is used in preparation infection and/or treatment inflammation and/or raising immune drug, and said Chinese medicine composition is processed by following crude drug:
Radix Astragali 30-120 weight portion Radix Angelicae Sinensis 5-20 weight portion
Flos Lonicerae 10-25 weight portion Herba Artemisiae Annuae 10-30 weight portion
Rhizoma Polygoni Cuspidati 5-20 weight portion.
2. application as claimed in claim 1, said Chinese medicine composition is processed by following crude drug:
Radix Astragali 40-110 weight portion Radix Angelicae Sinensis 6-19 weight portion
Flos Lonicerae 12-23 weight portion Herba Artemisiae Annuae 12-28 weight portion
Rhizoma Polygoni Cuspidati 5-20 weight portion.
3. application as claimed in claim 1, said Chinese medicine composition is processed by following crude drug:
Radix Astragali 55-95 weight portion Radix Angelicae Sinensis 8-15 weight portion
Flos Lonicerae 12-22 weight portion Herba Artemisiae Annuae 13-25 weight portion
Rhizoma Polygoni Cuspidati 5-20 weight portion.
4. application as claimed in claim 1, said Chinese medicine composition is processed by following crude drug:
Radix Astragali 85 weight portion Radix Angelicae Sinensis 15 weight portions
Flos Lonicerae 18 weight portion Herba Artemisiae Annuaes 22 weight portions
Rhizoma Polygoni Cuspidati 10 weight portions.
5. application as claimed in claim 1, said Chinese medicine composition is processed by following crude drug:
Radix Astragali 55 weight portion Radix Angelicae Sinensis 19 weight portions
Flos Lonicerae 24 weight portion Herba Artemisiae Annuaes 15 weight portions
Rhizoma Polygoni Cuspidati 6 weight portions.
6. application as claimed in claim 1, said Chinese medicine composition is processed by following crude drug:
Radix Astragali 110 weight portion Radix Angelicae Sinensis 8 weight portions
Flos Lonicerae 15 weight portion Herba Artemisiae Annuaes 28 weight portions
Rhizoma Polygoni Cuspidati 18 weight portions.
7. application as claimed in claim 1, infection wherein are due to the Pseudomonas aeruginosa.
8. application as claimed in claim 7, wherein said Pseudomonas aeruginosa are CGMCC No.5317.
9. application as claimed in claim 1, wherein this Chinese medicine composition adds conventional adjuvant, according to common process, processes acceptable forms clinically.
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| CN102894234A (en) * | 2012-10-29 | 2013-01-30 | 浙江省淡水水产研究所 | Plant immunopotentiator capable of improving immunity of Chinese soft shell turtle |
| CN105288299A (en) * | 2015-11-30 | 2016-02-03 | 谢松芬 | Medicine for treating infection caused by wearing of contact lenses |
| CN105535094A (en) * | 2016-01-11 | 2016-05-04 | 中悦民安（北京）科技发展有限公司 | Traditional Chinese medicine composition for facilitating beast, bird and fish production performance |
| CN105769831A (en) * | 2016-04-26 | 2016-07-20 | 暨南大学 | Application of polyacetylene compounds to preparation of medicines for resisting drug-resistance bacteria |
| CN109393451A (en) * | 2017-08-18 | 2019-03-01 | 青岛瑞思德生物科技有限公司 | A kind of health food that immunity can be improved containing Enteromorpha |
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| CN102091134A (en) * | 2011-01-12 | 2011-06-15 | 刘清泉 | Traditional Chinese medicinal composition for resisting multi-medicine tolerance of antibiotics |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102894234A (en) * | 2012-10-29 | 2013-01-30 | 浙江省淡水水产研究所 | Plant immunopotentiator capable of improving immunity of Chinese soft shell turtle |
| CN105288299A (en) * | 2015-11-30 | 2016-02-03 | 谢松芬 | Medicine for treating infection caused by wearing of contact lenses |
| CN105535094A (en) * | 2016-01-11 | 2016-05-04 | 中悦民安（北京）科技发展有限公司 | Traditional Chinese medicine composition for facilitating beast, bird and fish production performance |
| CN105769831A (en) * | 2016-04-26 | 2016-07-20 | 暨南大学 | Application of polyacetylene compounds to preparation of medicines for resisting drug-resistance bacteria |
| CN109393451A (en) * | 2017-08-18 | 2019-03-01 | 青岛瑞思德生物科技有限公司 | A kind of health food that immunity can be improved containing Enteromorpha |
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