CN1267110C - Elsholizia extract prepn and its quality control method and new use - Google Patents
Elsholizia extract prepn and its quality control method and new use Download PDFInfo
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Abstract
The present invention discloses a preparation of herba elsholtziae volatile oil, a quality control method, and a new purpose thereof. The preparation of herba elsholtziae volatile oil has the functions of resisting viruses, resisting bacteria, resisting inflammatory, stopping diarrhea, alleviating pain and removing heat, and can suppress stomach intestine hyperfunction.
Description
Technical field
The preparation, method of quality control that the present invention relates to a kind of Chinese medicine extract with and new purposes, particularly relate to a kind of preparation, method of quality control and new purposes of Herba Moslae extract.
Background technology
Diarrhoea (infectious, non-infectious) sickness rate height, also higher in the certain areas case fatality rate, be transworld serious problems.The traditional Chinese medical science is to the then determination of treatment based on pathogenesis obtained through differentiation of symptoms and signs of carrying out of diarrheal treatment more, cold-damp syndrome person huoxiang zhengqi powder master it, damp-heat syndrome person GEGEN QINLIAN TANG master it, dyspepsia card person lenitive pill master it, stagnation of liver-QI takes advantage of spleen person's tongxieyao formula master it, weakness of the spleen and stomach person SHENLING BAISHU SAN master it, kidney yang deficiency card person SISHEN WAN master it.So also there is the soil single-prescription of employing that gastroenteritis is carried out the treatment according to differentiation of diseases person.In the last few years, the report of Chinese traditional treatment viral diarrhea was many, but it is less to form the new drug person.The characteristics of Chinese medicine are to be good at recuperating gastrointestinal tract function, few side effects, but the action intensity of pathogen slightly is inferior to Western medicine.How modern medicine treats with the physical exercise therapy of antimicrobial drug fluid,matching infectious diarrhea, has obviously improved curative effect.The research of nearly microbial ecological agent has during the last ten years caused scholar's attention with application, has carried out vaccine research simultaneously, but owing to cause that the diarrheal pathogen is a lot, lacks cross immunity each other, so the development of vaccine is made slow progress always.Along with going deep into of research, also more and more clearer to the toxic and side effects of antimicrobial drug, as toxicity and gastrointestinal reaction, anaphylaxis and superinfection etc. to kidney, liver, neural spirit, blood.At present viral diarrhea is still lacked specific medicine, the report that external useful lactobacillus, interferon, virazole etc. are treated, domestic useful viral infection chicken, the preparation antibody preparation carries out therapist from ovum gallinaceum then, but still among research and development.In recent years developed the rotavirus live vaccine, among applying.According to the data of WHO, it is inferior that the whole world annual diarrhoea case can reach 30-50 hundred million examples, has 500-1000 ten thousand cases dead because of serious diarrhoea approximately.Inferior, non-, draw the area below 5 years old among the child, annual diarrhoea person is taken place more than 1,000,000,000 person-times, the dead about 5,000,000.According to reports, China suffers from diarrhoea for annual about 8.36 hundred million person-times, and wherein child below 5 years old accounts for 2.09 hundred million person-times, accounts for 1/4.According to Ministry of Public Health in 1993 report, China's acute gastroenteritis two all prevalences are that 11.74%, two all acute diseases constitute by the disease kind and account for 8.36%, come after acute nasopharyngitis and the influenza, occupy ill the 3rd of two weeks of the resident of urban and urual areas of whole country.Rotavirus is to be found by Bishop etc. in 1973.1.4 hundred million person-times of rotavirus diarrhea can take place every year in whole world child below 5 years old, and death can reach 1,000,000 people.Rotavirus is the important cause of disease of infantile diarrhea, has found to cause the one-tenth Human reoviruslike agent of adult diarrhea afterwards again.
Summary of the invention
The object of the invention is to provide a kind of treatment diarrheal Herba Moslae extract preparation.
The object of the invention also is to provide the method for quality control of Herba Moslae and extract thereof.
The object of the invention also is to provide the new pharmaceutical use of Herba Moslae and extract thereof.
The present invention seeks to be achieved through the following technical solutions:
The preparation of Herba Moslae volatile oil: the Herba Moslae herb, cutting adds 6-10 times of water gaging, and vapor distillation extracted 4-8 hour, isolated the volatilization oil reservoir, promptly.
Pharmaceutical preparation of the present invention contains the Herba Moslae volatile oil of 1%-99% and the excipient of 99%-1% (medicine that comprises other adapted), preferably contain the Herba Moslae volatile oil of 30%-80% and the pharmaceutical excipient of 70%-20% (comprising the medicine that other compatibility is used), preferably contain the Herba Moslae volatile oil of 60%-70% and the excipient of 40%-30% (comprising the medicine that other compatibility is used).
Press practice of pharmacy, Herba Moslae volatile oil of the present invention can be prepared into the various clinical pharmaceutical dosage form, comprise the dosage form of oral formulations or parenterai administration.Said oral formulations is selected from any in tablet, capsule, pill, granule, suspensoid, drop pill, the oral liquid; Said parenterai administration dosage form is selected from a kind of in the middle of injection, aerosol, suppository or the subcutaneous administration dosage form.
Adjuvant in the medicine of the present invention is meant conventional excipient, as solvent, disintegrating agent, correctives, antiseptic, coloring agent, binding agent etc.The medicine that other compatibility in the medicine of the present invention is used, the MOLA oil that refers to effective dose is certain medicine material, again compatibility other allowed the Chinese medicine or the chemical drugs that share.
Above-mentioned volatile oil (MOLA oil) is yellowish supernatant liquid; Special aroma is arranged, acrid in the mouth; Store with the passing of time, color deepens slightly.
Contain following discriminating and/or content assaying method in the method for quality control of MOLA oil:
Differentiate: by the MOLA oil content assaying method, high-efficient liquid phase chromatogram has corresponding absworption peak in the retention time identical with the carvacrol reference substance with thymol.
Assay: measure according to high performance liquid chromatography (2000 editions appendix VID of Chinese Pharmacopoeia).
Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; 50-70: 50-30: 1-3 methanol-water-glacial acetic acid is a mobile phase; The detection wavelength is 276nm.Number of theoretical plate calculates by the thymol peak should be not less than 10000, calculates by the carvacrol peak and should be not less than 15000.The preparation of reference substance solution, precision take by weighing thymol reference substance 8mg and thymol reference substance 4mg, put in the 100ml measuring bottle, add methanol to scale, shake up, and, contain thymol 80 μ g among every ml that is, contain carvacrol 40 μ g.The about 0.017g of this product is got in the preparation of need testing solution, and accurate the title decides, and puts in the 10ml measuring bottle, adds methanol to scale, shakes up, and precision is measured 0.9ml, to the 10ml measuring bottle, adds methanol and is diluted to scale, shakes up, promptly.Algoscopy, accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly.This product contains carvacrol (C
10H
14O) and thymol (C
10H
14O) total amount must not be less than 55%.
Differentiate: by the MOLA oil content assaying method, high-efficient liquid phase chromatogram has corresponding absworption peak in the retention time identical with the carvacrol reference substance with thymol.
Contain following content assaying method in the method for quality control of Herba Moslae of the present invention:
Assay: precision takes by weighing the about 0.2g of Herba Moslae fine powder, puts in the 100ml tool plug conical flask, accurate methanol 20ml, the close plug of adding, claim decide weight, supersound process 20-30 minute, put coldly, weight decided in title again, supply the weight that subtracts mistake with methanol, shake up, filter, get subsequent filtrate as need testing solution.Measure according to high performance liquid chromatography (2000 editions appendix VID of Chinese Pharmacopoeia).Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; 50-70: 50-30: 1-3 methanol-water-glacial acetic acid is a mobile phase; The detection wavelength is 276nm.Number of theoretical plate calculates by the thymol peak should be not less than 10000, calculates by the carvacrol peak and should be not less than 15000.The preparation of reference substance solution, precision take by weighing thymol reference substance 8mg and thymol reference substance 4mg, put in the 100ml measuring bottle, add methanol to scale, shake up, and, contain thymol 80 μ g among every ml that is, contain carvacrol 40 μ g.Algoscopy, accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly.Herba Moslae contains carvacrol (C
10H
14O) and thymol (C
10H
14O) total amount must not be less than 0.80%.
The pharmacodynamic study result of Herba Moslae of the present invention and volatile oil thereof shows that MOLA oil has following function:
1. the antivirus action of MOLA oil: 12.5～25nl/ml MOLA oil can suppress children's's rotavirus RDTA-I type (Wa strain) growth and breeding on African green monkey kidney cell.
2. the antibacterium of MOLA oil effect: MOLA oil can reduce Salmonella enteritidis abdominal cavity infection mortality of mice (P＜0.05); Can reduce the content (P＜0.05～0.01) of pathogenic bacterium in the mice stool of Salmonella enteritidis peroral infection; External, to common pathogens such as Escherichia coli, Shiga bacillus, proteus vulgaris, staphylococcus aureus, staphylococcus epidermidiss, stronger inhibition growth effect is arranged, and test is with in the 44 strain bacterial strains, and minimum inhibitory concentration≤1ul oil/ml accounts for 79.5%.
3. the anti-diarrhea effect of MOLA oil: loose stool rate, diarrhea rate, diarrhoea index and the diarrhoea degree (P＜0.05～0.001) that reduce the diarrhoea model mice due to the 100% Folium Sennae decoct significantly.
4. MOLA oil is to the influence of gastrointestinal function: this medicine can reduce hyperfunction mouse small intestine propulsion functions (P＜0.05～0.001) due to normal and the neostigmine, prolong defecation time, reduce defecation quantity (P＜0.05～0.001), slow down gastric emptying motion (P＜0.01～0.001), intestinal smooth muscle excitement due to the reduction stomachial secretion function (P＜0.001), stripped intestinal smooth muscle spontaneous vasomotion of inhibition normal guinea pig and acetylcholine.
5. the analgesic effect of MOLA oil: MOLA oil reduces very significantly by the mouse writhing number due to 0.7% acetic acid (all P＜0.001), and suppression ratio reaches 51.3～55.7%.
6. the antiinflammatory action of MOLA oil: MOLA oil suppresses significantly by dimethylbenzene induced mice ear swelling (P＜0.01～0.001), suppress the vascular permeability (P＜0.05～0.001) that histamine causes significantly, suppress abdominal cavity leukoplania (P＜0.05～0.001) due to the sodium carboxymethyl cellulose.
7. the antipyretic effect of MOLA oil: MOLA oil saccharomycetic rat temperature is raise and endotoxin due to the rabbit body temperature rising inhibitory action (P＜0.05～0.001) is significantly all arranged.
Following experimental example is used to further specify the present invention.
Experimental example:MOLA oil suppresses the effect of children's's rotavirus ROTA-I type (Wa strain)
Materials and methods:
1. medicine: tried the thing MOLA oil, faint yellow, proportion 0.95726g/ml, 16mg oil/1g crude drug, lot number: 20000601, Beijing big tree institute of Pharmaceutical Research provides.Getting the 0.5ml MOLA oil adds the 0.5ml tween 80 (Shanghai the 18 pharmaceutical factory produces, lot number: add the dilution of 4ml growth-promoting media after 960520) being mixed.It is standby to put 4 ℃ of refrigerators.
Contrast medicine GEGEN QINLIAN DIWAN, the Liuzhou city pharmaceutical factory of traditional Chinese medicine produces, and accurate word (1991) is defended No. 017004 in osmanthus, lot number: 990721.Get 10 milliliters of dissolvings of 1g medicine adding distil water, 2000 rev/mins, centrifugal 10 minutes, get the supernatant water-bath and boil sterilization in 10 minutes, put 40 ℃ of refrigerators and preserve.
The above-mentioned medicinal preceding pH=7.2 that transfers
2. cell: African green monkey kidney (Vero) passage cell, the cell that grows up to monolayer is individual cells through the digestion of trypsinization liquid, is made into 60,000 cell/ml with growth-promoting media, inserts the cell pipe, the 1ml/ pipe.
3. viral: children's's rotavirus ROTA-1 type (Wa strain) is provided TCID by Virology Inst., China Academy of Preventive Medicine Sciences
5010
-7, during test with 100 TCID
500.1ml.Before the Wa strain is used, add 370 ℃ of water-baths of 0.05ml pancreatin (200ug/ml) 30 minutes with 1ml virus liquid.
4. reagent: Eagles MEM dry powder, U.S. GIBCO product.Hyclone, the Tianjin Blood Research Institute is produced; Lot number: 9925, L-glutaminate, Sigma import packing, lot number: 911015.Penicillin, streptomycin.
The reagent preparation:
Growth-promoting media: Eagles adds 1% mycillin, 1% glutamine, 10% calf serum, with preceding accent PH=7.4.
Keep liquid: Eagles adds 1% mycillin, 1% glutamine, and 200ug/ml pancreatin (final concentration is 2ug/ml) is with preceding accent PH=7.4.
Cell dissociation buffer: 0.25% pancreatin, with no calcium magnesium phosphate buffer preparation.
5. test articles for use and instrument:
Culture bottle, test tube, measuring pipette, incubator, refrigerator, baking box, water bath, low temperature refrigerator, liquid nitrogen container etc. are homemade.
6. test method:
(1) MOLA oil, GEGEN QINLIAN DIWAN and tween 80 are to the toxic action of Vero cell: will dilute good medicine, tween 80 inserts and grows up in the test tube of cell monolayer, puts 37 ℃ of incubators and cultivates, every day, observation of cell changed.
(2) medicine is to the direct killing effect of virus: put 37 ℃ of incubators 1 hour after will diluting good medicine and viral mixed in equal amounts.Access grows up in the test tube of cell monolayer then, and every pipe 0.2ml is put 37 ℃ of absorption 1 hour.Keep liquid 1ml with keeping to add after liquid is washed twice.Put 37 ℃ of incubators and cultivate, observation of cell pathological changes every day (CPE), when virus control occurs ++ ++ the time, termination test.
(3) medicine inhibitory action that virus is bred in cell: the virus that will handle inserts in the cell pipe, every pipe 0.1ml is put 37 ℃ of absorption 1 hour, with keep liquid wash after twice add respectively contain different pharmaceutical concentration keep liquid 1ml, put 37 ℃ of cultivations, every day the observation of cell pathological changes, when the virus control pipe occurs ++ ++ the time, termination test.
Annotate: cytopathy :+25%, ++ 50%, +++75%, ++ ++ 100%.
More than medicine contrast, virus control, cell contrast are established in test simultaneously, and every test group 4 solencytes repeat to do experiment twice.
The result:
1. MOLA oil the results are shown in Table 1 to the toxicity test of Vero cell.
Table 1 MOLA oil is to the toxicity test result of Vero cell
| Group | Drug level/ml | Cell pipe number | Cytotoxicity pipe number |
| MOLA oil | 100nl 50nl 25nl 12.5nl | 4 4 4 4 | 4 0 0 0 |
| GEGEN QINLIAN DIWAN | 5mg 2.5mg 1.25mg | 4 4 4 | 4 0 0 |
| Tween 80 | 200nl 100nl | 4 4 | 2 0 |
| The Vero contrast | 4 | 0 |
4 concentration MOLA oil are inserted the cell pipe respectively, and final concentration is 100,50,25,12.5nl/ml.Each concentration 4 pipe was observed 7 days.Pair cell was toxic when MOLA oil concentration was 100nl/ml as a result, and concentration is 50,25, and pair cell does not have toxicity during 12.5nl/ml; Pair cell is toxic during GEGEN QINLIAN DIWAN 5mg/ml, and pair cell does not have toxicity when 2.5mg/ml and 1.25mg/ml; Pair cell does not have toxicity during tween 80 100nl/ml.
2. MOLA oil is to the direct killing effect result of the test of virus: the results are shown in Table 2.
Table 2 MOLA oil is to the direct killing effect result of the test of virus
| Group | Drug level/ml | The inoculated tube number | Positive pipe number | The negative tube number |
| The MOLA oil group | 50nl 25nl | 8 8 | 8 8 | 0 0 |
| The GEGEN QINLIAN DIWAN group | 10mg 5mg | 8 8 | 8 8 | 0 0 |
| The tween 80 group | 100nl 50nl | 8 8 | 8 8 | 0 0 |
| Wa strain cellular control unit matched group | 8 8 | 8 0 | 0 8 |
It is as shown in the table: each administration group of MOLA oil does not have direct deactivation to children's's rotavirus ROTA-1 type.
MOLA oil to virus in intracellular breeding inhibitory action result of the test: the results are shown in Table 3.
Table 3: MOLA oil to virus in intracellular breeding inhibitory action result of the test:
| Group | Drug level/ml | The pipe number | Positive pipe | Negative tube |
| The MOLA oil group | 25nl 12.5nl 6.25nl 3.12nl | 8 8 8 8 | 0 0 2 8 | 8 8 6 0 |
| The GEGEN QINLIAN DIWAN group | 2.5mg 1.25mg | 8 8 | 8 8 | 0 0 |
| The tween 80 group | 100nl 50nl | 8 8 | 8 8 | 0 0 |
| The contrast of Wa strain control cells | 8 8 | 8 0 | 0 8 |
It is as shown in the table, when MOLA oil concentration is 25nl/ml, 12.5nl/ml, can suppress virus fully in intracellular breeding, when concentration is 6.25nl/ml, virus is bred certain inhibitory action in cell, when concentration is 3.1nl/ml virus being bred in cell does not have inhibitory action.GEGEN QINLIAN DIWAN, each concentration group of tween 80 is bred in cell virus does not have inhibitory action.
Embodiment 1:Herba Moslae quality control:
Precision takes by weighing the about 0.2g of Herba Moslae fine powder, puts in the 100ml tool plug conical flask, accurate methanol 20ml, the close plug of adding, claim decide weight, supersound process 30 minutes is put coldly, and weight decided in title again, supply the weight that subtracts mistake with methanol, shake up, filter, get subsequent filtrate as need testing solution.Measure according to high performance liquid chromatography (2000 editions appendix VID of Chinese Pharmacopoeia).Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; 60: 40: 2 methanol-water-glacial acetic acid are mobile phase; The detection wavelength is 276nm.Number of theoretical plate calculates by the thymol peak should be not less than 10000, calculates by the carvacrol peak and should be not less than 15000.The preparation precision of reference substance solution takes by weighing thymol reference substance 8mg and thymol reference substance 4mg, puts in the 100ml measuring bottle, adds methanol to scale, shakes up, and, contains thymol 80 μ g among every ml that is, contains carvacrol 40 μ g.Algoscopy, accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing inject chromatograph of liquid, measure, that is and, Herba Moslae contains carvacrol (C
10H
14O) and thymol (C
10H
14O) total amount must not be less than 0.80%.
Embodiment 2:The method of quality control of MOLA oil
Assay: measure according to high performance liquid chromatography (2000 editions appendix VID of Chinese Pharmacopoeia).
Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; 60: 40: 2 methanol-water-glacial acetic acid are mobile phase; The detection wavelength is 276nm.Number of theoretical plate calculates by the thymol peak should be not less than 10000, calculates by the carvacrol peak and should be not less than 15000.The preparation precision of reference substance solution takes by weighing thymol reference substance 8mg and thymol reference substance 4mg, puts in the 100ml measuring bottle, adds methanol to scale, shakes up, and, contains thymol 80 μ g among every ml that is, contains carvacrol 40 μ g.The about 0.017g of this product is got in the preparation of need testing solution, and accurate the title decides, and puts in the 10ml measuring bottle, adds methanol to scale, shakes up, and precision is measured 0.9ml, to the 10ml measuring bottle, adds methanol and is diluted to scale, shakes up, promptly.Algoscopy, accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing inject chromatograph of liquid, measure, that is and, this product contains carvacrol (C
10H
14O) and thymol (C
10H
14O) total amount must not be less than 55%.
Differentiate: by the MOLA oil content assaying method, high-efficient liquid phase chromatogram has corresponding absworption peak in the retention time identical with the carvacrol reference substance with thymol.
Embodiment 3:Capsular preparation
MOLA oil 250 grams, starch 2500 grams carry out drying earlier with starch, cross 120 mesh sieves, then with the MOLA oil mix homogeneously, cross twice 120 mesh sieves, and fully mixing is made 1000 capsules altogether, and every intragranular contains 250 milligrams of MOLA oil.Oral, every day 2-3 time, each 1-2 grain.
Embodiment 4:The preparation of drop pill
Take by weighing the 300g Macrogol 4000, in water-bath, melt, add MOLA oil raw material 100g, stir, in the impouring insulating tube, regulate thermostat, make medicinal liquid under 80-90 ℃, splash in the liquid paraffin that cooled off (temperature ± 4 ℃), after dripping off, to blot paraffin oil on the pill impouring filter paper, add a small amount of Pulvis Talci again, mixing gets 1000 of MOLA oil drop pill.Oral, a 2-3 grain, three times on the one, one after each meal.
Embodiment 5:The preparation of capsule
Herba Moslae 1000g, medical starch 1000g, mix homogeneously, the 0# capsule of packing into, every 0.35g, treatment diarrhoea, each oral 1-2 grain, twice of every day.
Embodiment 6:The preparation of tablet
MOLA oil raw material 1000g, medical starch 500g, mix homogeneously is used an amount of alcohol granulation, through the pelletizing machine granulate, tabletting, every 0.25g, oral, each 1-2 sheet, twice of every day.
Embodiment 7:The preparation of granule
MOLA oil raw material 100g, starch 1000g, Icing Sugar 400g, mix homogeneously is used an amount of alcohol granulation, and drying, granulate, packing are promptly.Oral, a 5g, twice on the one.
Embodiment 8:The preparation of injection
MOLA oil 10g, propylene glycol 20ml, Polyethylene Glycol-40050ml, water for injection 300ml mixes heating in water bath 30 minutes, add benzyl alcohol 50ml, reuse water for injection adds to 1000ml, handles 10 minutes in ultrasound wave, heats 30 minutes in the water-bath again, transfer pH value 5.5-6.5, filter clear and bright, embedding, the sterilization promptly.Every 2ml, intramuscular injection, a 2ml, 1-2 time on the one.
Claims (2)
1, the application of Herba Moslae in diarrheal medicine due to the preparation treatment rotavirus.
2, the application of Herba Moslae volatile oil in diarrheal medicine due to the preparation treatment rotavirus.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02153995 CN1267110C (en) | 2002-12-09 | 2002-12-09 | Elsholizia extract prepn and its quality control method and new use |
| CNB2005100049926A CN100428925C (en) | 2002-12-09 | 2002-12-09 | Preparation of Herba Moslae extract and quality control thereof |
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| CNB2005100049930A Division CN100347539C (en) | 2002-12-09 | 2002-12-09 | Herba Moslae quality control |
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| CN100418514C (en) * | 2006-06-05 | 2008-09-17 | 谢华 | Soft capsule with elslholtzia and method for preparing same |
| CN102451228A (en) * | 2010-10-26 | 2012-05-16 | 中国医学科学院药用植物研究所 | Preparation method and application of antibacterial and antiviral elsholtzia densa benth volatile oil |
| CN109419846B (en) * | 2017-08-31 | 2021-07-16 | 安徽天康(集团)股份有限公司 | Application of herba Moslae volatile oil in treating norovirus infectious diarrhea |
| CN107843676A (en) * | 2017-11-15 | 2018-03-27 | 广州美瑞泰科生物工程技术有限公司 | The detection method of carvacrol and Thymol in a kind of plant extract feed additive |
| CN111135223B (en) * | 2020-02-13 | 2022-02-22 | 湖南省中医药研究院 | Application of flavored herba Moslae oral liquid in preparation of medicine for treating infantile rotavirus enteritis complicated with myocardial injury |
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