CN101167822B - Tibetan medicine bongga extraction and its application in preparing antiviral medicine - Google Patents
Tibetan medicine bongga extraction and its application in preparing antiviral medicine Download PDFInfo
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- CN101167822B CN101167822B CN2007102020673A CN200710202067A CN101167822B CN 101167822 B CN101167822 B CN 101167822B CN 2007102020673 A CN2007102020673 A CN 2007102020673A CN 200710202067 A CN200710202067 A CN 200710202067A CN 101167822 B CN101167822 B CN 101167822B
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Abstract
The invention relates to a banga extract and the application in preparing antiviral medicine, which can not only inhibit various etiological agent bacteria, but also can play evident antiviral function through a plurality of connections such as inhibiting virus absorption, directly killing living virus, and inhibiting proliferation of virus, thereby the extract can be applied in preparing medicine which resists bacteria and virus, and simultaneously can be applied in food, health products and cosmetics. The banga extract of the invention can be made into medicine compound in preparing of the medicine which resists bacteria and virus in single-flavor or compound style, in particular to the application of the single-flavor or the compound style in preparing medicine compound for curing genital herpes. Simultaneously, the extract can be used incorporated in food, health products and cosmetics. The banga extract has the advantages of rich raw material, low cost, simple extraction technique, and can be conveniently shaped into various shapes, and has important developing and applying prospect.
Description
Technical field
The present invention is that Tibetan medicine bongga extraction and this extract thereof are antibiotic in preparation, the application in the antiviral drugs, belongs to national pharmaceutical field.
Background technology
At present a large amount of continuous risings of bacterial drug resistance of using antibiotic to cause have become one of common difficult problem of paying close attention in the whole world.Especially the resistant rate of staphylococcus aureus reaches more than 90%.Herpes simplex virus type II mainly causes the genital area mucocutaneous infections.Virus enters body through respiratory tract, oral cavity, genitals mucosa and damaged skin, dives to occupy in human normal mucosa, blood, saliva and the sensory nerve ganglion cell.When Abwehrkraft des Koepers descended, during such as heating, gastrointestinal dysfunction, menstruation, gestation, focal infection and mood change, the HSV-II that hides in the body was activated and falls ill
[11]Synthetic drug more sure antiviral drugs in the treatment of herpes simplex is ACV, but the persister of clinical continuous discovery ACV in recent years, and drug resistance has growth trend with the prolongation of courses of pharmaceuticals.
Therefore, antibiotic, the antiviral drugs of developing new high-efficiency low-toxicity have broad prospects, the multiple target effect of Chinese medicine mechanism, low toxic and side effects, be easy to metabolism, the advantage such as have no drug resistance makes it become the focus of antibiotic, the antiviral drugs of screening high-efficiency low-toxicity.
The Tibetan medicine material is grown in height above sea level more than 3000 meters, and the habitat is unique, and pharmacologically active is strong, is the treasure-house of new drug research.Tibetan medicine list loud, high-pitched sound is the herb of ranunculaceae plant Aconitum naviculare and aconitum tanguticumStapf.Be born in grass slope, high mountain mountain region or moistening meadow.Be distributed in Shaanxi, SOUTH OF GANSU, East of Qinghai Province, western Sichuan, northwestern Yunnan Province and Eastern Tibet.Bitter in the mouth, cool in nature, slightly poisonous.The function that heat-clearing and toxic substances removing is arranged.Be used for the infectious disease heating, liver, gallbladder-heat disease, lung-heat, intestinal heat, influenza, alimentary toxicosis.
Take a broad view of report both domestic and external, be showed no antibiotic, the antivirus action research of total ethanol extract, volatile oil and alkaloids extract of Tibetan medicine list loud, high-pitched sound.The invention provides Tibetan medicine list loud, high-pitched sound total ethanol extract, volatile oil and alkaloids extract and these extracts uses in antibiotic, the antiviral drugs of preparation.Compare with existing antibiotic, antiviral synthetic drug, the drug action of Tibetan medicine bongga extraction is strong, and toxicity is low, and is safe and reliable, and Tibetan medicine list loud, high-pitched sound abundant raw material, inexpensive, extraction process is simple, preparation cost is low, can make various dosage forms.
Goal of the invention:
The application in the preparation antibacterial and antiviral drug of Tibetan medicine list loud, high-pitched sound total ethanol extract, volatile oil and alkaloids extract and these extracts thereof is provided.
Summary of the invention:
Method for preparing extractive of the present invention is as follows
1, the preparation of Tibetan medicine list loud, high-pitched sound total ethanol extract
The herb of Tibetan medicine list loud, high-pitched sound is ground into coarse powder, extracts take 95% ethanol as solvent refluxing, and the sucking filtration slagging-off through being evaporated to without the alcohol flavor, obtains the total ethanol extract.
2, the preparation of Tibetan medicine list loud, high-pitched sound volatile oil
Get the dry herb of Tibetan medicine list loud, high-pitched sound, pulverize, with 95% ethanol extraction, Recycled ethanol, it is outstanding molten with water to extract extractum, uses petroleum ether extraction, reclaim petroleum ether, petroleum ether extract adds ether dissolution, adds distilled water and carries out steam distillation, collect distillate, use extracted with diethyl ether, merge ether extraction liquid, reclaim ether, use anhydrous sodium sulfate dehydration, get yellow green grease, peat-reek is arranged, for subsequent use.Being take vapor distillation as example in this technique, can also be CO
2Supercritical extraction, this is that those skilled in the art can understand.
3, Tibetan medicine list loud, high-pitched sound fat-soluble biological alkaline extraction
Tibetan medicine list loud, high-pitched sound total ethanol extract adds an amount of 5% hydrochloric acid stirring and dissolving, transfer to pH=3, hold over night, remove by filter insoluble impurity, filtrate extracts respectively 3 times with petroleum ether, ether and removes impurity, acid mother liquid is added strong aqua ammonia transfer the pH=10 chloroform extraction, merge each time chloroformic solution, decompression is removed chloroform and is got Fat-soluble alkaloids.
4, Tibetan medicine list loud, high-pitched sound water-soluble biological alkaline extraction
Tibetan medicine list loud, high-pitched sound total ethanol extract adds an amount of 5% hydrochloric acid stirring and dissolving, transfer to pH=3, hold over night, remove by filter insoluble impurity, filtrate extracts respectively 3 times with petroleum ether, ether and removes impurity, acid mother liquid is added mother solution n-butanol extraction after strong aqua ammonia transfers pH=10 with chloroform extraction, merge each time butanol solution, reduce pressure and remove n-butyl alcohol and get water-soluble alkaloid.
Above-mentioned Tibetan medicine bongga extraction provided by the present invention not only can suppress the Various Diseases indigenous bacteria, and can suppress viruses adsorption, directly the too many levels such as inactivation of viruses, virus replication be brought into play obvious antivirus action, so this extract can be antibiotic in preparation, use in the antiviral drugs.Tibetan medicine bongga extraction provided by the invention can be prepared into pharmaceutical composition with single or compound recipe mode in antibiotic, the antiviral drugs of preparation, especially single or compound recipe mode are used in the pharmaceutical composition of preparation treatment genital herpes.Simultaneously, also can in food, health product and cosmetics, use.Because the preparation of the pharmaceutical dosage forms such as soft capsule, hard capsule, drop pill, tablet, dispersible tablet, granule, injection, oral liquid, suppository is that the researcher in this field is to understand, can realize easily that Tibetan medicine bongga extraction and respective carrier make up and be prepared into various pharmaceutical dosage forms: soft capsule or hard capsule or drop pill or tablet or dispersible tablet or granule or injection or oral liquid or suppository.Also it can be mixed in food, health product and the cosmetics and use.
The specific embodiment:
Embodiment 1 Tibetan medicine bongga extraction antibacterial action
1, strain and culture medium
Experimental strain has Gansu Province Center for Disease Control (CDC) and Clinical Laboratory center, Gansu Province to provide.Be listed as follows:
Belong to strain
Staphylococcus staphylococcus aureus ATCC 25923 (Staphylococcus aureus)
Resistant Staphylococcus epidermidis (Staphylococcus epidermidis),
Escherichia coli belongs to bacillus ATCC 25922 (Escherichia coli),
Enterococcus faecalis (Enterococcus faecalis)
Bacillus bacillus subtilis (Bacillus subtilis)
Bacillus cereus (Bacillus cereus)
Candida Candida albicans (Candida albicans)
Culture medium is used in experiment: antibacterial MH culture medium, fungus PDA culture medium is all available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
2 fungistatic effect detection methods:
2.1 fungistatic effect is measured the inhibition zone size with cylinder plate method
Activation bacterium liquid is diluted to 10 with physiological saline solution
5～10
6Cfu/ml is for subsequent use.Add 0.2ml bacterium liquid to dry MH culture medium flat plate, smoothen.Place 5min, make culture medium fully absorb bacterium liquid.Each dull and stereotyped 6 in sterilized Oxford cup of placing is placed 5min, makes Oxford cup adsorbed close in media surface.Add 0.1g/ml water extract thing 100ul in the cup of Oxford.Positive control levofloxacin and fluconazol.The positive control antibacterial is 40 μ g/ml levofloxacin with concentration, and Candida albicans is 200 μ g/ml fluconazol with concentration, all adds 100ul.Negative control adds sterilized water.Then flat board is placed 37 ℃ of constant incubators to cultivate 24h (candida albicans 48h), go out with slide calliper rule to measure antibacterial circle diameter, calculating mean value, D represents bacteriostatic activity with antibacterial circle diameter.The fungistatic effect criterion is: D≤8mm is insensitive, and 8mm＜D≤13mm is low responsive, and 13mm＜D≤19mm is medium sensitivity, and D＞19mm is extremely sensitive.
2.2 the mensuration of minimal inhibitory concentration MIC adopts Microdilution plate method
The test bacterial concentration is 10
6Cfu/ml.Good bacterium liquid is added in the 96 porocyte culture plates to draw dilution, every hole 100ul, and every kind of medicine 100ul, 10 holes are diluted in contrast successively, and last hole does not add medicine (only adding culture medium and antibacterial) and adds dilution bacterium liquid 100ul, is the bacterial growth control wells.Stay the string hole not add antibacterial (only adding culture medium and medicine) and make the medicine control wells, positive control is done doubling dilution with levofloxacin and fluconazol by explanation.Place the 1min that vibrates on the agitator, make in the hole behind the abundant mixing of solution, microwell plate is added a cover and is placed in the square enamel tray that is covered with wet gauze, hatches 24h (candida albicans 48h) in 37 ℃ of incubators, observes without the contained lowest concentration of drug in bacterial growth hole and is minimal inhibitory concentration.
3 fungistatic effects
Table 1: different Tibetan medicine bongga extraction inhibition zones (mm)
| The total ethanol extract | Volatile oil | Fat-soluble alkaloids | Water-soluble alkaloid | Positive control | |
| Staphylococcus aureus | 11 | 10 | 16 | 16 | 26 |
| Resistant Staphylococcus epidermidis | 9 | 13 | 14 | 11 | 29 |
| Escherichia coli | 12 | 11 | 17 | 15 | 25 |
| Enterococcus faecalis | 16 | 15 | 15 | 13 | 25 |
| Bacillus subtilis | 17 | 14 | 16 | 11 | 26 |
| Bacillus cercus | 19 | 12 | 15 | 11 | 26 |
| Candida albicans | 18 | 12 | 16 | 14 | 10 |
Annotate: a Tibetan medicine list loud, high-pitched sound total ethanol extract, volatile oil and alkaloids addition are the 1mg/ cup, and b positive control antibacterial is 40 μ g/ml levofloxacin with concentration, and Candida albicans is 200 μ g/ml fluconazol with concentration, all adds 100ul.The negative contrast of 5%DMSO.
Table 2: different Tibetan medicine bongga extraction minimal inhibitory concentrations (MIC) (mg/ml of unit)
| The total ethanol extract | Volatile oil | Fat-soluble alkaloids | Water-soluble alkaloid | Positive control | |
| Staphylococcus aureus | 6.25 | 0.5 | 18.75 | 3.125 | 0.002 |
| Resistant Staphylococcus epidermidis | 12.5 | 0.5 | 18.75 | 3.125 | 0.002 |
| Escherichia coli | 25 | 1 | 9.375 | 6.25 | 0.004 |
| Enterococcus faecalis | 12.5 | 1 | 18.75 | 3.125 | 0.002 |
| Bacillus subtilis | 6.25 | 0.5 | 18.75 | 3.125 | 0.002 |
| Bacillus cercus | 25 | 1 | 18.75 | 6.25 | 0.004 |
| Candida albicans | 12.5 | 0.5 | 4.688 | 3.125 | 0.1 |
Annotate: positive control antibacterial levofloxacin, the Candida albicans fluconazol all adds 100ul.The negative contrast of 5%DMSO.
The result shows: Tibetan medicine list loud, high-pitched sound total ethanol extract, volatile oil and alkaloids extract have broad-spectrum antiseptic and bactericidal action.To gram positive bacteria: staphylococcus aureus, drug resistance staphylococcus epidermidis, bacillus cereus, bacillus subtilis; Gram negative bacteria: escherichia coli, enterococcus faecalis; The effect that deep infection fungus Candida albicans has killing and suppresses to grow has important practice significance and using value, can use in the food that is prepared with antibacterial and/or bactericidal action, health product, cosmetics and medicine.
Embodiment 2: Tibetan medicine bongga extraction antivirus action
1, experiment material
Virus: HSV-II type (3 generation) 20060913, Lanzhou Institute of Biological Products vaccine research chamber provides (herpessimplex virus, HSV-II)
Cell: African green monkey kidney cell (Vero cell) Lanzhou Institute of Biological Products provides.Cultivate with DMEM culture medium+7% Ox blood serum.
Animal: cleaning level kunming mice, male and female half and half, body weight 18～22g is purchased from Lanzhou Institute of Biological Products's Animal House.
Reagent: tetrazolium bromide (MTT, Fluka Biochemika company), dimethyl sulfoxide (Sigma company produces for dimethylsulfoxide, DMSO), DMEM (GIBCO company), standard new-born calf serum (sino-america joint-venture Lanzhou people's marine growth Engineering Co., Ltd), injection acyclovir (ACV, 9-(2-hydroxyl ethoxymethyl-) guanine, Wuhan Pusheng Pharmaceutical Co., Ltd., the accurate word H42020129 of product batch number 060811 traditional Chinese medicines).
2, experimental technique
2.1 experiment in vitro
2.1.1 drug cell toxicity test (mtt assay)
With cell suspension with 8 * 10
5/ ml density is inoculated in the 96 porocyte culture plates, every hole 200 μ l, 37 ℃, 5%CO
2, 100% relative humidity is cultivated 24h.Total extract, Fat-soluble alkaloids and the equal doubling dilution of water-soluble biological alkali liquor add in the cell suspension hole to following 6 concentration: 16,8,4,2,1,0.5mg/ml, and each drug level repeats 4 holes, and other establishes the normal cell contrast.Continue to cultivate 33h under the same terms, observation of cell pathological changes (CPE) situation, and with mtt assay detection cell survival rate.
2.1.2 virus virulence is measured (plaque method)
With cell suspension with 8 * 10
5/ ml density is inoculated in the 24 porocyte culture plates, every hole 1mL, 37 ℃, 5%CO
2, 100% relative humidity is cultivated 24h.Viral dilution is following 6 concentration extremely: 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, 10
-8, adding in the cell suspension hole, each drug level repeats 4 holes, and other establishes the normal cell contrast, and the methylcellulose covering covers, and continues to cultivate 72h under the same terms, violet staining, counting.Infectious virus amount (PFU/ml)=(speckle number in every hole/every hole virus inoculation amount ml) * viral dilution multiple
2.1.3 medicine is invaded the blocking effect (plaque method) of cell to HSV-II
Test sample liquid is mixed with the HSV-II equal-volume, with this virus drugs mixed liquor vero cells infection.37 ℃, 5%CO
2Absorption 90min, washing adds the cell maintenance medium and puts 37 ℃, 5%CO
2Cultivate.Observe CPE every day, detect viral suppression ratio with the plaque method behind the 72h.Adopting therapeutic index (TI) to weigh medicine as evaluation index renders a service the inhibition of virus.Therapeutic index (TI)=half toxic concentration (TC50)/half suppresses dense (IC50).Each drug level repeats 4 holes, and normal cell contrast, medicine contrast and virus control are set simultaneously.
2.1.4 medicine is to the direct deactivation (plaque method) of HSV-II
Test sample liquid is mixed with the HSV-II equal-volume, behind 37 ℃ of effect 90min, with this virus drugs mixed liquor vero cells infection.37 ℃, 5%CO
2Absorption 90min, washing, the same method is cultivated and is checked.
2.1.5 medicine is to the inhibitory action (plaque method) of virion propagation
With virus infected cell, wash behind the absorption 90min, add the test sample liquid of variable concentrations, the same method is cultivated and is checked.Virus suppression ratio (%)=(the virus control group on average goes out speckle number-medicine-feeding test group and on average goes out the speckle number)/virus control group on average goes out the speckle number
2.2 experiment in the body
2.2.1 medicine is to the determination of acute toxicity of mice
Cleaning level Kunming mouse random packet, 6 every group, male and female half and half.According to the experiment in vitro result, select to suppress preferably total extract of HSV-II biosynthesis activity, with 400,500,625,800,1000,1250mg/kg dosage gastric infusion, other establishes negative control group, gives isopyknic normal saline.Normal feed water inlet, Continuous Observation 10d, record dead mouse situation is also calculated LD50.
2.2.2 viral encephalocoele model toxicity test
10 every group of cleaning level Kunming mouse random packet, male and female half and half.Behind the infection site routine disinfection, through the right ventricle injecting virus, viral dilution is following 6 concentration extremely: 10
-1, 10
-2, 10
-3, 10
-4, 10
-6, 10
-7, infective dose is every 0.05mL.Normal feed water inlet, Continuous Observation 10d, record dead mouse situation is also calculated LD50 with the Reed-Muench method.
2.2.3 medicine infects the protective effect of HSV-II mice to encephalocoele
10 every group of cleaning level Kunming mouse random packet, male and female half and half.Behind the infection site routine disinfection, through the right ventricle injecting virus, 24h behind the viral infection, drug treatment.By medicine LD50 1/2～1/4 as the high test dose of testing in the medicine body, LD50 1/12～1/16 as low test dose, every fraction medicine is established 2 dosage groups of height.Without the matched group (virus control group, Normal group) of medicine, gastric infusion 5d observes animal morbidity and death condition, continuously 30d continuously.
3, experimental result
3.1 experiment in vitro
3.1.1. drug cell toxicity test
Each effective site of table 3 Tibetan medicine list loud, high-pitched sound is to the toxicity (unit: mg/ml) of Vero cell
| Total extractum | Fat-soluble alkaloids | Water-soluble alkaloid | |
| TC50 | 2.6?3 | 2.77 | 2.77 |
Test sample liquid shows as to the toxic action of Vero cell that cell proliferation is slow, granule is more, refractivity is poor, form changes, the part cell breakage comes off.Because cell metabolic activity reduces or be dead, the viable count that mtt assay detects reduces.
3.1.2 virus virulence is measured (plaque method)
The Vero cell is responsive to HSV-II, and the Vero cell pathological changes effect CPE due to HSV-II infects is characterized as circle contracting swelling, fusion forms thyrsiform or starlike mutually, even the part cell detachment, forms focus.It is 10 that plaque counting gets HSV-II type (3 generation) 20060913 infectious virus amounts (PFU/ml)
7.57PFU/ml, this HSV-II virus adopts 10
-5The time every hole go out the speckle number and be about 40, easy counting is so adopt 10
-5Viral dilution degree as subsequent experimental.
3.1.3 medicine is invaded the blocking effect of cell to HSV-II
Each effective site of table 4 Tibetan medicine list loud, high-pitched sound is invaded the blocking effect of Vero cell to HSV-II
3.1.4 medicine is to the direct killing effect of HSV-II
Each effective site of table 5. Tibetan medicine list loud, high-pitched sound is to the direct deactivation of HSV-II
3.1.5 medicine is to the inhibitory action (plaque method) of HSV-II virion propagation
Each effective site of table 6. Tibetan medicine list loud, high-pitched sound is to the inhibitory action of HSV-II virion propagation
3.2 experiment in the body
3.2.1 medicine is to the determination of acute toxicity of mice
Because drug toxicity is relatively strong, mice is at once dead behind the maximal dose group mouse stomach.Dosage reduces, and the mouse survival rate raises, and the time-to-live growth also obtains Tibetan medicine list loud, high-pitched sound total extract LD50=512mg/kg.
3.2.2 viral encephalocoele model toxicity test
Occurred symptom after the mouse infection HSV-II virus on the 7th～10 day, dietary amount minimizing, weight loss, alarm hair, roll up, dead in 3～7d after the infected mice morbidity.Calculate LD50=10 with the Reed-Muench method
-1.38, thus, adopt 10 during experiment
-1The dilution factor inoculation, this is to make all minimum dilution factors of death of animal.
3.2.3 medicine infects the protective effect of HSV-II mice to encephalocoele
Table 7. is respectively organized average survival rate and time-to-live
| Grouping | Survival rate (%) | Time-to-live (t/d) |
| Virus control | 0 | 7 |
| Low dose group | 0 | 8 |
| High dose group | 10％ | 30 |
Embodiment 2 shows Tibetan medicine list loud, high-pitched sound total ethanol extract and alkaloids extract by multiple effective site, and too many levels, multipath ground are brought into play synergism and shown antiviral efficacy.Medicine is the Main Function approach of Tibetan medicine list loud, high-pitched sound extracorporeal antivirus effect to blocking effect and the direct kill virus that HSV-II invades cell.HSV-II is tunicary virus, and outermost peplos is comprised of lipid and glycoprotein, and wherein any composition changes, and can make the peplos degeneration, thus inactivation of viruses.Experimental result shows that each composition of Tibetan medicine list loud, high-pitched sound is stronger to the direct deactivation of HSV-II, and especially the effect of the direct deactivation HSV-II of total extract and water-soluble alkaloid is more remarkable than ACV.Can make the peplos degeneration with inactivation of viruses so infer these chemical constituents.The blocking effect that HSV-II invades cell studies show that Tibetan medicine list loud, high-pitched sound not only has antiviral activity, also has the effect that Cell protection to a certain degree prevents Virus entry.Utilize HSV-II encephalitis model research Tibetan medicine list loud, high-pitched sound total extract to the protective effect of HSV-II infecting mouse, the result shows that total extract shows certain antiviral activity.Each dosage treatment group of total extract is compared with untreated virus infected mice, and survival rate increases, and the time-to-live prolongs to some extent.These results have important practice significance and using value, can use in the antiviral food of preparation, health product, cosmetics and medicine.
Claims (3)
1. the extracting method of a list loud, high-pitched sound volatile oil is characterized in that: get the dry herb of Tibetan medicine list loud, high-pitched sound, pulverize, with 95% ethanol extraction, Recycled ethanol, it is outstanding molten with water to extract extractum, use petroleum ether extraction, reclaim petroleum ether, petroleum ether extract adds ether dissolution, add distilled water and carry out steam distillation, collect distillate, use extracted with diethyl ether, merge ether extraction liquid, reclaim ether, use anhydrous sodium sulfate dehydration, get yellow green grease, peat-reek is arranged, for subsequent use.
2. a bongga extraction is the list loud, high-pitched sound volatile oil that is prepared gained by extracting method claimed in claim 1.
3. the application of bongga extraction according to claim 2 in antibiotic, the antiviral drugs of preparation.
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| 徐愚聪等.藏药十一味金色丸的鉴别和没食子酸的含量测定.《华西药学杂志》.2004,第19卷(第1期),60-62. * |
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