CN103933350A - Use of rhizoma bletillae ethyl acetate extract - Google Patents
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Abstract
The invention provides use of a rhizoma bletillae ethyl acetate extract in preparing anti-clinical drug-resistance bacteria drugs. The rhizoma bletillae ethyl acetate extract has a good inhibiting effect to various Gram negative bacteria, Gram positive bacteria and drug-resistance bacteria and can effectively protect bacteria infected individuals. The pharmacodynamics activity of the extract is equivalent to that of antibiotics. In particular, the extract can further effectively resist methicillin-resistant staphylococcus aureus, thereby providing a better choice for clinical medication.
Description
The application is application number: the divisional application of 201210590325.0 patent application.The applying date of original application: on December 31st, 2012, denomination of invention: the purposes of Pseudobulbus Bletillae (Rhizoma Bletillae) ethyl acetate extract.
Technical field
The present invention relates to the new purposes of Pseudobulbus Bletillae (Rhizoma Bletillae) ethyl acetate extract.
Background technology
Infect class disease, be regarded as extremely dangerous disease always, until Alexander Fleming chances on penicillin and for after clinical, the struggle of the mankind and infectious disease has just had an important breakthrough in nineteen twenty-eight.But be widely used along with antibiotic, the harm of abuse of antibiotics is far beyond people's the imagination: the Jevons of Britain has just found methicillin-resistant staphylococcus aureus (MRSA) first, MRSA, from finding to infect so far almost extend over the entire globe, has become one of important pathogen of nosocomial infection.Research is found, MRSA is except to methicillin resistance, beta-lactam and the equal drug resistance of cephalosporins of and methicillin same structure all to other, MRSA also can be by changing antibiotic target site, produce modification enzyme, reduce membrane permeability and produce the different mechanisms such as a large amount of PABA, aminoglycoside, Macrolide, Tetracyclines, fluorine quinolione class, sulfonamides, rifampicin are all produced to drug resistance in various degree, there is wide spectrum drug resistance, and vancomycin becomes the last line of defense of capturing MRSA infection.As can be seen here, the speed that bacterial drug resistance produces, far beyond the speed of new drug development, if continue development, can cause because of drug resistance existing all antibiotic invalid some day, and people are sick may " yielding to no remedy " enter the rear antibiotic epoch thus.
The stem reality that the proper mankind develop rapidly in the face of fastbacteria and body drug resistance and in badly battered, Chinese medicine is shown one's talent as new antibacterials is strong with its antibiotic property, toxic and side effects is little and be subject to the favor of extensive patients.
Pseudobulbus Bletillae (Rhizoma Bletillae), has another name called sweet, the Pseudobulbus Bletillae (Rhizoma Bletillae), Bletilla striata (Thunb.ex A.Murray)Rchb.f. etc., is orchid Pseudobulbus Bletillae (Rhizoma Bletillae) Bletilla striata(Thunb.) dry tuber of Reichb.f., main product in Zhejiang, the ground such as Guizhou, Hubei, Sichuan, Hunan, Henan, Shaanxi.Contain bibenzyl, the luxuriant and rich with fragrance class of dihydro, join the compound such as luxuriant and rich with fragrance class, pyrene class.The traditional Chinese medical science thinks, its property is puckery and receive, and can enter lung hemostasis, and granulation promoting is controlled skin ulcer.Research shows, Pseudobulbus Bletillae (Rhizoma Bletillae) have hemostasis, anticancer, protection gastric mucosa, plasma substitute, the migration of promotion horn cell formation, promote vascular endothelial cell tactophily, antiulcer, prevention of intestinal adhesion, promote the pharmacological actions such as wound healing, immunological enhancement.Clinically can be used for that upper respiratory tract etc. is hemorrhage, the treatment of decubital ulcer, burn and scald, tumor, pulmonary tuberculosis, acne, pertussis etc.
Find by literature search, existing pharmacological research mainly concentrates on Pseudobulbus Bletillae (Rhizoma Bletillae) hemostasis equivalence and uses, and the research of bacteriostatic activity is domestic rarely has report.
Summary of the invention
The object of the present invention is to provide the new purposes of Pseudobulbus Bletillae (Rhizoma Bletillae) ethyl acetate extract.
The invention provides the purposes of Pseudobulbus Bletillae (Rhizoma Bletillae) ethyl acetate extract in preparation antibacterials.
Further, described medicine is the medicine of anti-clinical drug-resistant bacterium.
Further, described clinical drug-resistant bacterium is methicillin-resistant staphylococcus aureus.
Further, described medicine is the medicine of anti-gram negative bacteria, gram positive bacteria.
Further, described gram negative bacteria is bronchus literary composition blood Bao Te bacterium, moraxelle catarrhalis, Pseudomonas aeruginosa, Klebsiella Pneumoniae or escherichia coli; Described gram positive bacteria is staphylococcus aureus, megacoccus, staphylococcus saprophyticus, enterococcus faecalis or staphylococcus epidermidis.
Wherein, described medicine is the medicine for the treatment of bacterial infection disease.
Wherein, described Pseudobulbus Bletillae (Rhizoma Bletillae) ethyl acetate extract is prepared by the following method: get Pseudobulbus Bletillae (Rhizoma Bletillae), ethanol extraction; Alcohol extract reclaims the extractum after ethanol, then extracts by systematic solvent extraction, gets ethyl acetate part, obtains Pseudobulbus Bletillae (Rhizoma Bletillae) ethyl acetate extract.
Further, described concentration of alcohol is 80～95%V/V.
Further, in described systematic solvent extraction, extract by petroleum ether, ethyl acetate successively.
Wherein, described medicine is oral agents, injection or the external preparation forming taking Pseudobulbus Bletillae (Rhizoma Bletillae) ethyl acetate extract as active fraction preparation.
Pseudobulbus Bletillae (Rhizoma Bletillae) ethyl acetate extract all has good inhibitory action to various gram negative bacterias, positive bacteria and fastbacteria; can effectively protect antibacterial infected individuals; its drug activity and antibiotic are suitable; especially this extract can also be effectively to methicillin-resistant staphylococcus aureus resistance, for clinical application provides better selection.
Detailed description of the invention
Pseudobulbus Bletillae (Rhizoma Bletillae) ethyl acetate extract of the present invention, extracts according to systematic solvent extraction conventional in Chemistry for Chinese Traditional Medicine field, and concrete operations can be referring to following embodiment preparation method.In addition, also can directly extract according to the method for existing bibliographical information, as Lu Bo etc., the impact of Pseudobulbus Bletillae (Rhizoma Bletillae) the different extracted parts on Platelet Aggregation in Rabbits, PLA's Acta Pharmaceutica Sinica, 2005 years 5 phases.
The preparation method of embodiment 1 Pseudobulbus Bletillae (Rhizoma Bletillae) ethyl acetate extract
Take Bletilla striata medicinal materials 250g, pulverize, add 95%v/v alcohol heating reflux and extract twice, 10 times of amounts for the first time, 8 times of amounts for the second time, each each 1 hour.Extracted twice liquid is merged, filter, filtrate decompression reclaims solvent, obtains crude extract 31g.Get crude extract, the about 250ml that adds water, ultrasonic suspendible, is transferred in 1000ml separatory funnel, adds petroleum ether extraction 8 times, each 300ml, remaining water layer is extracted with ethyl acetate 10 times again, each 250ml, reclaim under reduced pressure ethyl acetate, obtains ethyl acetate extract 8g.
The preparation method of embodiment 2 Pseudobulbus Bletillae (Rhizoma Bletillae) ethyl acetate extracts
Take Bletilla striata medicinal materials 500g, pulverize, add 80%v/v alcohol heating reflux and extract twice, 10 times of amounts for the first time, 8 times of amounts for the second time, each each 1 hour.Extracted twice liquid is merged, filter, filtrate decompression reclaims solvent, obtains crude extract.Get crude extract, after being dried, adding petroleum ether jolting and extract 5 times, remaining water layer extracts 5 times with ethyl acetate jolting again, combined ethyl acetate position, and reclaim under reduced pressure ethyl acetate, obtains ethyl acetate extract.
The preparation method of embodiment 3 Pseudobulbus Bletillae (Rhizoma Bletillae) ethyl acetate extracts
Take Bletilla striata medicinal materials 100g, pulverize, add 90%v/v alcohol heating reflux and extract twice, 12 times of amounts for the first time, 10 times of amounts for the second time, each each 1 hour.Extracted twice liquid is merged, filter, filtrate decompression reclaims solvent, obtains crude extract.Get crude extract, after being dried, add petroleum ether warm macerating 3 times, remaining water layer is used ethyl acetate warm macerating 5 times again, combined ethyl acetate position, and reclaim under reduced pressure ethyl acetate, obtains ethyl acetate extract.
By test example, beneficial effect of the present invention is described below.
Test example 1 antibacterial tests
1 material and instrument
1.1 medicines and preparation
For reagent thing, Pseudobulbus Bletillae (Rhizoma Bletillae), purchased from branch, Beijing Tongrentang Chengdu, is accredited as the dry tuber of orchid Pseudobulbus Bletillae (Rhizoma Bletillae) Bletilla striata (Thund.) Reichb.f. through qualification teacher Ma Yuntong of teaching and research room of Chengdu University of Traditional Chinese Medicine.Four active sites of Pseudobulbus Bletillae (Rhizoma Bletillae) are taked respectively following methods preparation.
Positive drug, vancomycin hydrochloride for injection, specification: 500,000 units, Zhejiang Medicine Co, lot number: 111204; Cefotaxime sodium for injection, specification: 1.0g, NCPC Hebei Huamin Pharmaceutical Co., Ltd., lot number: XF3120707; Cefixime tablets, specification 0.2g/ sheet, Zhejiang Haizheng Pharmaceutical Co company limited, lot number 120501.
Modeling medicine: Mucin form Porcine Stomach (Type II), specification: 100g, SIGMA-ALDRICH, lot number: 018K0079.
1.1.1 the extracting method of ethyl acetate extract takes Bletilla striata medicinal materials 250g, pulverizes, and adds 95%v/v alcohol heating reflux and extracts twice, 10 times of amounts for the first time, 8 times of amounts for the second time, each each 1 hour.Extracted twice liquid is merged, filter, filtrate decompression reclaims solvent, obtains crude extract 31g.Residue medicinal residues are for subsequent use.Get crude extract, the about 250ml that adds water, ultrasonic suspendible, is transferred in 1000ml separatory funnel, adds petroleum ether extraction 8 times, each 300ml, reclaim under reduced pressure petroleum ether.Water layer is extracted with ethyl acetate 10 times again, each 250ml, and reclaim under reduced pressure ethyl acetate, obtaining ethyl acetate extract 8g(is Pseudobulbus Bletillae (Rhizoma Bletillae) ethyl acetate extract of the present invention).Residue water layer is for subsequent use.
1.1.2 after alcohol extraction, remaining medicinal residues in " 1.1.1 " are got at decocting liquid position, and the water (about 2000ml) that adds 8 times of amounts decocts 1 hour, and by filtered through gauze, filtrate is heated to 60 DEG C, and concentrating under reduced pressure obtains decocting liquid position 95g after alcohol extraction.
1.1.3 the extracting method of n-butanol portion is got remaining water layer in " 1.1.1 ", adds n-butanol extraction 10 times, each 250ml, and reclaim under reduced pressure n-butyl alcohol, obtains n-butanol portion extract 7g.Residue water layer is for subsequent use.
1.1.4 extract Hou Shui position and get remaining water layer liquid in " 1.1.3 ", be heated to 60 DEG C, concentrating under reduced pressure, water extractive part 12g after must extracting.
1.2 bacterial strain
1.2.1 reference culture escherichia coli ATCC25922, staphylococcus aureus ATCC25923(Sichuan Industrial Institute of Antibiotics give).
1.2.2 strain subject is identified through VITEK-32, VITEK-60 automatic microbe identification and analysis instrument, then 22 strain pathogen isolated from clinical specimens of 10 kinds again identifying through Biolog Bacteria analyzer (U.S.) and API20E, 20NE, Staph series and conventional method.
1.3 instruments and reagent Mueller-Hinton agar (OXOID, lot number: 729683), nutrient agar (Hangzhou microorganism reagent company limited, lot number: 20100831-02), disposable sterilized culture dish (the healthy biological company limited in Jiangsu), Mcfarland Standard(bioMeri é ux, Inc. lot number: 821772701) etc.
2 methods
2.1 In Vitro Bacteriostatics tests adopt agar plate doubling dilutions to measure respectively decocting liquid position after Pseudobulbus Bletillae (Rhizoma Bletillae) ethyl acetate extract, alcohol extraction, extraction Hou Shui position and the In Vitro Bacteriostatic of n-butanol portion to 11 kinds, 26 pathogen strain bacterium and reference culture.
2.1.1 4 different parts of the dull and stereotyped preparation of pastille carry out doubling dilution with distilled water or DMSO diluent respectively, are diluted to respectively 1:1,1 ︰ 2,1 ︰ 4,1 ︰ 8,1 ︰ 16,1 ︰ 32,1 ︰ 64,1 ︰ 128,1 ︰ 256 totally 9 gradients.In disposable sterilized culture dish, add respectively the dilute liquid medicine 1ml of above-mentioned variable concentrations gradient and the MH culture medium of 14ml sterilizing, fully mix, dry for subsequent use.
2.1.2 bacterium liquid configuration: strain subject and susceptibility Quality Control bacterial strain are adjusted to bacterial concentration 1.5 × 10 with physiological saline solution
6cFU/ml.
2.1.3 lowest bacteria fogging-resistant concentration determining adopts multiple spot inoculation instrument the bacterium liquid of experimental strain to be added to pastille flat board and the positive control flat board of variable concentrations gradient, and makes negative control with physiologic saline for substitute bacterium liquid.37 DEG C of constant temperature culture 18～24h, observe each bacterial strain at the growing state containing in variable concentrations gradient medicine flat board.In the situation that positive and negative contrasts is all set up, taking in culture dish without the minimum mass concentration of bacterial growth as the minimal inhibitory concentration (MIC) of test position to this bacterium.
2.2 antibacterial activity in vivo tests
2.2.1 trial test
The mensuration of experimental strain virulence: 37 DEG C of tested methicillin-resistant staphylococcus aureus of constant temperature culture, escherichia coli, to exponential phase, become the bacterium liquid of variable concentrations gradient with normal saline dilution.Bacterium liquid and the 10% gastric Mucin mixed in equal amounts of getting variable concentrations, by the bacterium liquid measure intraperitoneal injection of mice of 0.5ml/20g, and contrast with 5% gastric Mucin.Under the not dead prerequisite of control mice, record the mortality rate in mice 14d, measure all dead minimum amount of bacteria of mice, i.e. the minimum lethal dose of this bacterium liquid (MLD).
The prerun of effective dose in body: multiple various dose groups are set methicillin-resistant staphylococcus aureus, the caused infection model mice of escherichia coli are carried out to lumbar injection, every group 4,1 time/d, and administration in 2 days in advance, 0.5h counteracting toxic substances after administration in the 3rd day, observe the death condition in animal 14d, determine formal test dosage.
2.2.2 antibacterial activity in vivo
Get 18～22g SPF level KM mice, male and female half and half,, are divided into vancomycin hydrochloride for injection group by 10 every group, cefotaxime sodium for injection group, Cefixime tablets group, Pseudobulbus Bletillae (Rhizoma Bletillae) 1,0.5,0.25,0.125g/kg group, the blank group of normal saline, model group, gastric Mucin matched group.All administrations in 2 days in advance, 1 time/d, 0.5h counteracting toxic substances after administration in the 3rd day, chooses methicillin-resistant staphylococcus aureus, escherichia coli bacterium liquid lumbar injection containing 5% gastric Mucin.Wherein taking physiologic saline for substitute medicine as model group; Taking administration neither also not counteracting toxic substances as blank group; Only inject 5% gastric Mucin that does not contain antibacterial as gastric Mucin matched group taking not administration.Survival condition after observation mice counteracting toxic substances in 14d, adds up its survival rate.
3 results
The antibacterial activity in vitro at 3.1 Pseudobulbus Bletillae (Rhizoma Bletillae) different activities positions adopts agar plate doubling dilution to measure the MIC of 4 positions of Pseudobulbus Bletillae (Rhizoma Bletillae) to 26 strain pathogen isolated from clinical specimens and reference culture, the results are shown in Table 1.
Table 1: the In Vitro Bacteriostatic of 4 positions of Pseudobulbus Bletillae (Rhizoma Bletillae) to strain subject
Note: " >=" represents that Pseudobulbus Bletillae (Rhizoma Bletillae) extract part, in the time of test Cmax, does not show bacteriostatic activity.
As shown in Table 1, Pseudobulbus Bletillae (Rhizoma Bletillae) has bacteriostatic activity to tested pathogen.Wherein, after alcohol extraction, decocting liquid and extraction Hou Shui position do not show the bacteriostatic activity to strain subject in the time of test Cmax, and n-butanol portion and ethyl acetate extract all show stronger bacteriostatic activity, wherein especially obvious with the bacteriostatic activity of ethyl acetate extract, be 65 μ g/ml to the minimum MIC of strain subject.N-butanol portion, except 1 strain bronchus literary composition blood Bao Te bacterium is not shown bacteriostatic activity, all shows stronger bacteriostatic activity to other strain subjects, is 16.667mg/ml to the minimum MIC of strain subject.Ethyl acetate extract all shows very strong bacteriostatic activity to all strain subjects, has broad-spectrum antibacterial activity, and to " super fastbacteria "---methicillin-resistant staphylococcus aureus also has good bacteriostatic activity.
The antibacterial activity in vivo of 3.2 Pseudobulbus Bletillae (Rhizoma Bletillae) ethyl acetate extracts
The endogenous protective of table 2 Pseudobulbus Bletillae (Rhizoma Bletillae) ethyl acetate extract to methicillin-resistant staphylococcus aureus infection model mice
Ip: drug administration by injection; Ig: oral administration.
As shown in Table 2, Pseudobulbus Bletillae (Rhizoma Bletillae) ethyl acetate extract has very strong protective effect to methicillin-resistant staphylococcus aureus infecting mouse, wherein especially strong with the protection of lumbar injection 0.5g/kg, protection to mice has reached 100%, and lumbar injection 0.25,0.125g/kg also reach 90%, 40% to the protection of mice; Gavage 1,0.5g/kg reach 50%, 40% to the protection of mice.
The endogenous protective of table 3 Pseudobulbus Bletillae (Rhizoma Bletillae) ethyl acetate extract to infection due to Escherichia coli model mice
As shown in Table 3, Pseudobulbus Bletillae (Rhizoma Bletillae) ethyl acetate extract has very strong protective effect to infection due to Escherichia coli mice, and wherein especially strong with the protection of lumbar injection 0.5,0.25g/kg, protection reaches 80%; Gavage 1g/kg reaches 50% to the protection of infecting mouse.
4 conclusions
Pseudobulbus Bletillae (Rhizoma Bletillae) has broad-spectrum antibacterial activity in vitro, and Grain-negative, positive bacteria are all had to good bacteriostatic activity, and to " super fastbacteria "---methicillin-resistant staphylococcus aureus also has very strong bacteriostatic activity; The bacteriostatic activity of Pseudobulbus Bletillae (Rhizoma Bletillae) different parts is compared to research and find, wherein especially obvious with the bacteriostatic activity of ethyl acetate extract, show that ethyl acetate extract is its main active site.
Pseudobulbus Bletillae (Rhizoma Bletillae) also has very strong bacteriostatic activity in vivo, and this experimental selection gram positive bacteria methicillin-resistant staphylococcus aureus, gram-negative bacteria escherichia coli are test strain, and experimental result shows that Pseudobulbus Bletillae (Rhizoma Bletillae) also has very strong bacteriostatic activity in vivo.
Claims (9)
1. the purposes of Pseudobulbus Bletillae (Rhizoma Bletillae) ethyl acetate extract in the anti-clinical drug-resistant bacterium medicine of preparation.
2. purposes according to claim 1, is characterized in that: described medicine is the medicine of resisting gram-positive bacteria.
3. purposes according to claim 2, is characterized in that: described gram positive bacteria is staphylococcus aureus, megacoccus, staphylococcus saprophyticus, enterococcus faecalis or staphylococcus epidermidis.
4. purposes according to claim 1, is characterized in that: described clinical drug-resistant bacterium is methicillin-resistant staphylococcus aureus.
5. according to the purposes described in claim 1-4 any one, it is characterized in that: described medicine is the medicine for the treatment of bacterial infection disease.
6. purposes according to claim 1, is characterized in that: described Pseudobulbus Bletillae (Rhizoma Bletillae) ethyl acetate extract is prepared by the following method:
Get Pseudobulbus Bletillae (Rhizoma Bletillae), ethanol extraction; Alcohol extract reclaims the extractum after ethanol, then extracts by systematic solvent extraction, gets ethyl acetate part, obtains Pseudobulbus Bletillae (Rhizoma Bletillae) ethyl acetate extract.
7. purposes according to claim 6, is characterized in that: described concentration of alcohol is 80～95%V/V.
8. purposes according to claim 6, is characterized in that: in described systematic solvent extraction, extract successively by petroleum ether, ethyl acetate.
9. according to the purposes described in claim 1-8 any one, it is characterized in that: described medicine is oral agents, injection or the external preparation forming taking Pseudobulbus Bletillae (Rhizoma Bletillae) ethyl acetate extract as active fraction preparation.
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| CN201210590325.0A CN102988765B (en) | 2012-12-31 | 2012-12-31 | Usage of rhizoma bletillae ethyl acetate extractive |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106361731A (en) * | 2016-08-24 | 2017-02-01 | 成都中医药大学 | Bibenzyl compound, preparation method thereof, and application thereof in preparation of antitumor medicines |
| CN106397140A (en) * | 2016-08-30 | 2017-02-15 | 成都中医药大学 | Poly-bibenzyl type compound and preparation method and purpose thereof |
| CN106431852A (en) * | 2016-08-24 | 2017-02-22 | 成都中医药大学 | Bibenzyl compound, preparation method thereof and application of compound in preparation of antitumor drug |
| CN106495998A (en) * | 2016-08-30 | 2017-03-15 | 成都中医药大学 | A kind of bibenzyl ether compound and preparation method thereof and purposes |
-
2012
- 2012-12-31 CN CN201410151905.9A patent/CN103933350B/en not_active Expired - Fee Related
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| MASAE YAMAKI, ET AL: "Biphenanthrenes from Bletilla striata", 《PHYTOCHEMISTRY》 * |
| 任华忠，等: "白及化学成分其药理活性研究进展", 《亚太传统医药》 * |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106361731A (en) * | 2016-08-24 | 2017-02-01 | 成都中医药大学 | Bibenzyl compound, preparation method thereof, and application thereof in preparation of antitumor medicines |
| CN106431852A (en) * | 2016-08-24 | 2017-02-22 | 成都中医药大学 | Bibenzyl compound, preparation method thereof and application of compound in preparation of antitumor drug |
| CN106431852B (en) * | 2016-08-24 | 2019-04-23 | 成都中医药大学 | Bibenzyl compound and preparation method thereof and purposes in the preparation of antitumor drugs |
| CN106361731B (en) * | 2016-08-24 | 2020-02-04 | 成都中医药大学 | Bibenzyl compound, preparation method thereof and application thereof in preparing antitumor drugs |
| CN106397140A (en) * | 2016-08-30 | 2017-02-15 | 成都中医药大学 | Poly-bibenzyl type compound and preparation method and purpose thereof |
| CN106495998A (en) * | 2016-08-30 | 2017-03-15 | 成都中医药大学 | A kind of bibenzyl ether compound and preparation method thereof and purposes |
| CN106397140B (en) * | 2016-08-30 | 2019-03-29 | 成都中医药大学 | A kind of poly bibenzyl derivative and preparation method thereof and purposes |
| CN106495998B (en) * | 2016-08-30 | 2019-08-06 | 成都中医药大学 | A kind of bibenzyl ether compound and preparation method thereof and purposes |
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