CN103127196A - Preparation method and application of total extractive of artemisia rupestris L - Google Patents
Preparation method and application of total extractive of artemisia rupestris L Download PDFInfo
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- CN103127196A CN103127196A CN2013100870611A CN201310087061A CN103127196A CN 103127196 A CN103127196 A CN 103127196A CN 2013100870611 A CN2013100870611 A CN 2013100870611A CN 201310087061 A CN201310087061 A CN 201310087061A CN 103127196 A CN103127196 A CN 103127196A
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Abstract
The invention relates to a preparation method and application of a total extractive of artemisia rupestris L. The method comprises the following steps of: crushing a dry artemisia rupestris L. medicinal material, extracting by using ethanol as an organic solvent, carrying out resin separation and purification to obtain the total extractive of the artemisia rupestris L., wherein the total extractive of the artemisia rupestris L. mainly contains sesquiterpenoids and flavonoid constitutes, a sesquiterpenoid and the flavonoid constitute compound is in the mass percentage of 25-90%, a sesquiterpenoid compound is in the mass percentage of 5-25%, a flavonoid constitute compound is in the mass percentage of 20-65%, and rupestonic acid of the sesquiterpenoid compound is in the mass percentage of 3-15%. Proved by a primary activity screening test, the total extractive of the artemisia rupestris L., obtained by using the method provided by the invention, has remarkable in vitro and vivo influenza virus resistance, bacteriostasis and sterilization effects and can also be used for effectively preventing influenza viruses. The method disclosed by the invention is simple in process, free of high-temperature and high-pressure equipment, low in production cost, stable in quality of effective components and easy to control.
Description
Technical field
The present invention relates to a kind of preparation method of Herba Achilleae total extract, and total extract belongs to the national medicine technical field in the purposes of the grippal medicine of preparation treatment.
Background technology
Influenza (Influenza) is called for short influenza, is a kind of Acute respiratory infectious disease that is caused by influenza virus, and infectiousness is strong, and sickness rate is high, easily causes outbreak of epidemic or is very popular.The chemicals amantadines, rimantadine, zanamivir, ribavirin etc. that at present are used for preventing and treating influenza all have side effect in various degree.And the antiviral unique curative effect of Chinese medicine has obtained globally generally acknowledging, and the treatment by Chinese herbs influenza is " multicomponent, multipath, many target spots " onset, and side effect is low, therefore, seeks from Chinese medicine and the medicine of exploitation treatment influenza has broad prospects.
Compositae (Compositae) plant Herba Achilleae (Artemisia rupestris L.), all herbal medicine.Uighur claims " a diligent Ai Manni ", and Kazak claims " one is diligent ", and medication among the people is with a long history in Xinjiang.Heat-clearing and toxic substances removing is arranged, promoting digestion and invigorating the stomach, function of gallbladder promoting, the effect that antidotes against snake bite.Be used for dyspepsia, abdomen flatulence pain, hepatitis, urticaria, venom, cat fever etc.Mainly be distributed in the ground such as Xinjiang of China, the Central Asia, Europe.This medical material is recorded in China Qing Dynasty ZHAO Xue-Min supplementary Amplifications of the Compendium of Materia Medica, cloud: " Barkol goes out a kind of medicine, and the name Herba Achilleae is given birth in the remote mountains, without branch and leaf, and the soil of growing sturdily, abnormal smells from the patient such as Artemisia, the soldier that herds horses between April drives horse and enters the mountain, the receipts grass is taken and is returned, and the dealer is arranged to Lanzhou goods seller ".The proverb among the people of " family Herba Achilleae is arranged, all kinds of diseases and ailments are all removed " is spreading in Xinjiang since ancient times.
Contain the multiple compounds such as sesquiterpenoids, flavonoid, amino acids, glycoside, polysaccharide, volatile oil, polypeptide and alkaloid in the Herba Achilleae herb.Separate the sesquiterpenoids that obtains and mainly contain rupestonic acid and rupestonic acid glycolipid compound from Herba Achilleae.Separate the flavone compound that obtains and mainly contain luteolin, casticin, luteolin-7-O-β-D-Glucose glycoside, linarin, Japanese tiliacin, golden waist element second, absinthin, robinin, Quercetin, tiliannin, luteolin-7-O-glucose, rutin, different kaempferide, Yue Hua element etc. from Herba Achilleae.
Up to now, there is not yet document record or research report in the Herba Achilleae total extract total sesquiterpene and total flavones as the application of active component in the treatment influenza.Herba Achilleae is with a long history in medication among the people, clinical practice steady in a long-term is arranged, so its development prospect is wide.
Summary of the invention
In order to solve the deficiencies in the prior art, primary and foremost purpose of the present invention is to provide a kind of preparation method of Herba Achilleae total extract, and wherein the main component of total extract is sesquiterpene and flavone compound.
Another object of the present invention is to provide the purposes of Herba Achilleae total extract in the treatment influenza.
The preparation method of Herba Achilleae total extract of the present invention follows these steps to carry out:
A. after the Herba Achilleae herb of drying being ground into 2cm, be 60-80 ℃ of heating extraction of 10-95% ethanol temperature 1-3 time with concentration, extraction time 1-3 hour, be cooled to room temperature, filter, merge extractive liquid, is evaporated to without the alcohol flavor, obtains ethanol extract;
B. the extract concentration that step a is obtained is after the 0-15% dissolve with ethanol disperses, centrifugal (4000 rev/mins), filter, be 3-7 with the dilute hydrochloric acid adjust pH, then use resin isolation, first with the water elution of 2-6 times of column volume, with the 30-95% ethanol elution of 3-8 times of column volume, collect ethanol elution again, concentrating under reduced pressure eliminates solvent, drying can obtain the Herba Achilleae total extract.
In step a, the volume ratio of Herba Achilleae and ethanol is 1:10-30.
In step b, resin used is DM-130, AB-8, HPD-450, NKA-9, HPD-300, D101, HPD-400, HPD-500, HP-600, SP-825, LD601, LS-303B, LX-28, LX-38 or polyamide.
Sesquiterpene in the Herba Achilleae total extract that obtains by the method for the invention and the mass percent of flavone compound are 25-90%.
The mass percent of the sesquiterpenoids in the Herba Achilleae total extract is 5-25%, and the mass percent of flavone compound is 20-65%.
In the Herba Achilleae total extract, the mass percent of sesquiterpenoid rupestonic acid is 3-15%.
The Herba Achilleae total extract that obtains by the method for the invention is used in the purposes for preparing the grippal medicine of control.
The Herba Achilleae total extract that obtains by the method for the invention, in pharmaceutical preparation for the preparation for the treatment of influenza class medicine, contain the Herba Achilleae total extract in this pharmaceutical preparation or contain the compound recipe of Herba Achilleae total extract, be i.e. Western medicine or the active ingredient of Chinese herbs of other treatment influenza class of compatibility.
The preparation method of Herba Achilleae total extract of the present invention has following advantage and effect with respect to prior art:
(1) the Herba Achilleae total extract that adopts the method for the invention to obtain is yellow powder, and the mass percent of sesquiterpene and flavone compound is 25-90%.Wherein the mass percent of sesquiterpene is that the mass percent of 5-25%, flavone is 20-65%, and adopts first sesquiterpene and flavones ingredient in resin Sync enrichment Herba Achilleae in the production method of this Herba Achilleae total extract.This production method is simple, is fit to suitability for industrialized production.
(2) prove through pharmacodynamics test, this Herba Achilleae total extract have significant inside and outside resisiting influenza virus, antibacterial and the sterilization effect.
The specific embodiment
Embodiment 1: the preparation of Herba Achilleae total extract:
The Herba Achilleae herb medical material 10kg of drying is ground into 2cm, and adding 10 times of volumetric concentrations is that 10% alcohol heating reflux extracts 3 times, 80 ℃ of temperature, and time 2 h is cooled to room temperature, filters, and merge extractive liquid, is evaporated to without the alcohol flavor, obtains ethanol extract;
Be that 5% ethanol is dispersed to and contains crude drug 0.3g/ml with ethanol extract concentration, centrifugal (4000 rev/mins), filter, supernatant is regulated pH value as 5, DM-130 type macroporous adsorptive resins take dilute hydrochloric acid, first use 6 times of column volume water elutions, be 30% ethanol elution with 6 times of column volume concentration again, collect ethanol elution, concentrating under reduced pressure, vacuum drying becomes dry powder, namely gets Herba Achilleae total extract 560g.
Embodiment 2: the preparation of Herba Achilleae total extract:
The Herba Achilleae herb medical material 10kg of drying is ground into 2cm, and adding 14 times of volumetric concentrations is that 30% alcohol heating reflux extracts 3 times, 75 ℃ of temperature, and 1.5 hours time, be cooled to room temperature, filter, merge extractive liquid, is evaporated to without the alcohol flavor, obtains ethanol extract;
Be that 5% ethanol is dispersed to and contains crude drug 0.2g/ml with ethanol extract concentration, centrifugal (4000 rev/mins), it is 3 that supernatant is regulated pH value with dilute hydrochloric acid, AB-8 type macroporous adsorptive resins is first used 4 times of column volume water elutions, then is 40% ethanol elution with 5 times of column volume concentration, collect ethanol elution, concentrating under reduced pressure, vacuum drying becomes dry powder, namely gets Herba Achilleae total extract 600g.
Embodiment 3: the preparation of Herba Achilleae total extract:
The Herba Achilleae herb medical material 10kg of drying is ground into 2cm, and adding 16 times of volumetric concentrations is that 50% alcohol heating reflux extracts 3 times, 75 ℃ of temperature, and time 2 h is cooled to room temperature, and merge extractive liquid, is evaporated to without the alcohol flavor, obtains ethanol extract;
It is that 10% ethanol is dispersed to and contains crude drug 0.3g/ml that extract adds concentration, centrifugal (4000 rev/mins), it is 6 that supernatant is regulated pH value with dilute hydrochloric acid, HPD-450 type macroporous adsorptive resins is first used 8 times of column volume water elutions, then is 50% ethanol elution with 6 times of column volume concentration, collect ethanol elution, concentrating under reduced pressure, vacuum drying becomes dry powder, namely gets Herba Achilleae total extract 510g.
Embodiment 4: the preparation of Herba Achilleae total extract:
The Herba Achilleae herb medical material 10kg of drying is ground into 2cm, and the concentration that adds 18 times of volumes is that 60% alcohol heating reflux extracts 2 times, temperature 70 C, and 3 hours time, be cooled to room temperature, merge extractive liquid, is evaporated to without the alcohol flavor, obtains ethanol extract;
Be that 15% ethanol is dispersed to and contains crude drug 0.4g/ml with extract concentration, centrifugal (4000 rev/mins), supernatant is regulated pH value as 6 take dilute hydrochloric acid, NKA-9 type macroporous adsorptive resins is first used 4 times of column volume water elutions, then is 60% ethanol elution with 6 times of column volume concentration, collect ethanol elution, concentrating under reduced pressure, vacuum drying becomes dry powder, namely gets Herba Achilleae total extract 540g.
Embodiment 5: the preparation of Herba Achilleae total extract:
The Herba Achilleae herb medical material 10kg of drying is ground into 2cm, and adding 20 times of volumetric concentrations is that 70% alcohol heating reflux extracts 1 time, temperature 70 C, and 3 hours time, be cooled to room temperature, be evaporated to without the alcohol flavor, obtain ethanol extract;
Be that 8% ethanol is dispersed to and contains crude drug 0.2g/ml with extract concentration, centrifugal (4000 rev/mins), supernatant is regulated pH value as 3 take dilute hydrochloric acid, HPD-300 type macroporous adsorptive resins is first used 4 times of column volume water elutions, then is 70% ethanol elution with 6 times of column volume concentration, collect ethanol elution, concentrating under reduced pressure, vacuum drying becomes dry powder, namely gets Herba Achilleae total extract 705g.
Embodiment 6: the preparation of Herba Achilleae total extract:
The Herba Achilleae herb medical material 10kg of drying is ground into 2cm, and adding 12 times of volumetric concentrations is that 80% alcohol heating reflux extracts 2 times, 65 ℃ of temperature, and time 2 h is cooled to room temperature, and merge extractive liquid, is evaporated to without the alcohol flavor, obtains ethanol extract;
Be that 15% ethanol is dispersed to and contains crude drug 0.3g/ml with extract concentration, centrifugal (4000 rev/mins), supernatant is regulated pH value as 4 take dilute hydrochloric acid, D101 type macroporous adsorptive resins is first used 6 times of column volume water elutions, then is 80% ethanol elution with 6 times of column volume concentration, collect ethanol elution, concentrating under reduced pressure, vacuum drying becomes dry powder, namely gets Herba Achilleae total extract 635g.
Embodiment 7: the preparation of Herba Achilleae total extract:
The Herba Achilleae herb medical material 10kg of drying is ground into 2cm, and adding 20 times of volumetric concentrations is that 95% alcohol heating reflux extracts 2 times, temperature 60 C, and time 2 h is cooled to room temperature, and merge extractive liquid, is evaporated to without the alcohol flavor, obtains ethanol extract;
Be that 10% ethanol is dispersed to and contains crude drug 0.2g/ml with extract concentration, centrifugal (4000 rev/mins), supernatant is regulated pH value as 5 take dilute hydrochloric acid, HPD400 type macroporous adsorptive resins is first used 6 times of column volume water elutions, then is 45% ethanol elution with 4 times of column volume concentration, collect ethanol elution, concentrating under reduced pressure, vacuum drying becomes dry powder, namely gets Herba Achilleae total extract 590g.
Embodiment 8: the preparation of Herba Achilleae total extract:
The Herba Achilleae herb medical material 10kg of drying is ground into 2cm, and adding 15 times of volumetric concentrations is that 95% alcohol heating reflux extracts 2 times, temperature 60 C, and time 2 h is cooled to room temperature, and merge extractive liquid, is evaporated to without the alcohol flavor, obtains ethanol extract;
Be that 10% ethanol is dispersed to and contains crude drug 0.2g/ml with extract concentration, centrifugal (4000 rev/mins), supernatant is regulated pH value as 5 take dilute hydrochloric acid, HPD500 type macroporous adsorptive resins is first used 6 times of column volume water elutions, then is 30% ethanol elution with 4 times of column volume concentration, collect ethanol elution, concentrating under reduced pressure, vacuum drying becomes dry powder, namely gets Herba Achilleae total extract 615g.
Embodiment 9: the preparation of Herba Achilleae total extract:
The Herba Achilleae medicine herb material 10kg of drying is ground into 2cm, and adding 20 times of volumetric concentrations is that 70% alcohol heating reflux extracts 2 times, 65 ℃ of temperature, and time 2 h is cooled to room temperature, and merge extractive liquid, is evaporated to without the alcohol flavor, obtains ethanol extract;
Be that 10% ethanol is dispersed to and contains crude drug 0.2g/ml with extract concentration, centrifugal (4000 rev/mins), supernatant is regulated pH value as 4 take dilute hydrochloric acid, HPD600 type macroporous adsorptive resins is first used 6 times of column volume water elutions, then is 95% ethanol elution with 4 times of column volume concentration, collect ethanol elution, concentrating under reduced pressure, vacuum drying becomes dry powder, namely gets Herba Achilleae total extract 624g.
Embodiment 10: the preparation of Herba Achilleae total extract:
The Herba Achilleae herb medical material 10kg of drying is ground into 2cm, and adding 25 times of volumetric concentrations is that 95% alcohol heating reflux extracts 3 times, temperature 60 C, and time 2 h is cooled to room temperature, and merge extractive liquid, is evaporated to without the alcohol flavor, obtains ethanol extract;
Be that 10% ethanol is dispersed to and contains crude drug 0.2g/ml with extract concentration, centrifugal (4000 rev/mins), supernatant is regulated pH value as 5 take dilute hydrochloric acid, SP-825 type macroporous adsorptive resins is first used 6 times of column volume water elutions, then is 45% ethanol elution with 4 times of column volume concentration, collect ethanol elution, concentrating under reduced pressure, vacuum drying becomes dry powder, namely gets Herba Achilleae total extract 700g.
Embodiment 11: the preparation of Herba Achilleae total extract:
The Herba Achilleae herb medical material 10kg of drying is ground into 2cm, and adding 30 times of volumetric concentrations is that 60% alcohol heating reflux extracts 2 times, temperature 70 C, and time 2 h is cooled to room temperature, and merge extractive liquid, is evaporated to without the alcohol flavor, obtains ethanol extract;
Be that 5% ethanol is dispersed to and contains crude drug 0.2g/ml with extract concentration, centrifugal (4000 rev/mins), supernatant is regulated pH value as 5 take dilute hydrochloric acid, LD-601 type macroporous adsorptive resins is first used 5 times of column volume water elutions, then is 85% ethanol elution with 8 times of column volume concentration, collect ethanol elution, concentrating under reduced pressure, vacuum drying becomes dry powder, namely gets Herba Achilleae total extract 720g.
Embodiment 12: the preparation of Herba Achilleae total extract:
The Herba Achilleae herb medical material 10kg of drying is ground into 2cm, and adding 15 times of volumetric concentrations is that 90% alcohol heating reflux extracts 2 times, temperature 60 C, and 3 hours time, be cooled to room temperature, merge extractive liquid, is evaporated to without the alcohol flavor, obtains ethanol extract;
The extract water is dispersed to contains crude drug 0.2g/ml, centrifugal (4000 rev/mins), supernatant is regulated pH value as 5 take dilute hydrochloric acid, LS-303B type macroporous adsorptive resins is first used 4 times of column volume water elutions, then is 95% ethanol elution with 6 times of column volume concentration, collect ethanol elution, concentrating under reduced pressure, vacuum drying becomes dry powder, namely gets Herba Achilleae total extract 704g.
Embodiment 13: the preparation of Herba Achilleae total extract:
The Herba Achilleae herb medical material 10kg of drying is ground into 2cm, and adding 25 times of volumetric concentrations is that 95% alcohol heating reflux extracts 2 times, temperature 60 C, and time 2 h is cooled to room temperature, and merge extractive liquid, is evaporated to without the alcohol flavor, obtains ethanol extract;
Be that 10% ethanol is dispersed to and contains crude drug 0.2g/ml with extract concentration, centrifugal (4000 rev/mins), supernatant is regulated pH value as 5 take dilute hydrochloric acid, LX-28 type macroporous adsorptive resins is first used 5 times of column volume water elutions, then is 75% ethanol elution with 8 times of column volume concentration, collect ethanol elution, concentrating under reduced pressure, vacuum drying becomes dry powder, namely gets Herba Achilleae total extract 750g.
Embodiment 14: the preparation of Herba Achilleae total extract:
The Herba Achilleae herb medical material 10kg of drying is ground into 2cm, and adding 15 times of volumetric concentrations is that 80% alcohol heating reflux extracts, temperature 60 C, and time 2 h extracts 2 times, is cooled to room temperature, and merge extractive liquid, is evaporated to without the alcohol flavor, obtains ethanol extract;
Be that 3% ethanol is dispersed to and contains crude drug 0.2g/ml with extract concentration, centrifugal (4000 rev/mins), supernatant is regulated pH value as 5 take dilute hydrochloric acid, LX-38 type macroporous adsorptive resins is first used 6 times of column volume water elutions, then uses 6 times of column volume concentration 50% ethanol elutions, collect ethanol elution, concentrating under reduced pressure, vacuum drying becomes dry powder, namely gets Herba Achilleae total extract 723g.
Embodiment 15: the preparation of Herba Achilleae total extract:
The Herba Achilleae herb medical material 10kg of drying is ground into 2cm, and adding 25 times of volumetric concentrations is that 85% alcohol heating reflux extracts 2 times, temperature 60 C, and time 2 h is cooled to room temperature, and merge extractive liquid, is evaporated to without the alcohol flavor, obtains ethanol extract;
Be that 15% ethanol is dispersed to and contains crude drug 0.2g/ml with extract concentration, centrifugal (4000 rev/mins), it is 6 that supernatant is regulated pH value with dilute hydrochloric acid, polyamide type macroporous adsorptive resins is first used the water elution of 6 times of column volumes, then uses 95% ethanol elution of 5 times of column volumes, collect ethanol elution, concentrating under reduced pressure, vacuum drying becomes dry powder, namely gets Herba Achilleae total extract 728g.
Embodiment 16: the assay method of general flavone content in the Herba Achilleae total extract:
The preparation of reference substance solution: precision takes the control substance of Rutin 25mg in 120 ℃ of dry constant weights of temperature, put in the 50ml measuring bottle, add appropriate amount of ethanol, supersound process makes dissolving, let cool, add ethanol to scale, shake up, precision measures 20ml, put in the 50ml measuring bottle, add water to scale, shake up, namely get (containing anhydrous rutin 0.2mg in every 1ml);
The preparation of standard curve: precision pipettes reference substance solution 1ml, 2ml, 3ml, 4ml, 5ml, 6ml puts respectively in the 25ml measuring bottle, respectively adds water to 6ml, add 5% sodium nitrite solution 1ml, shake up, placed 6 minutes, add 10% aluminum nitrate solution 1ml, shake up, placed 6 minutes, hydro-oxidation sodium test solution 10ml, then add water to scale, shake up, placed 15 minutes, take corresponding reagent as blank, measure absorbance at the wavelength place of 510nm, take absorbance as vertical coordinate, concentration is abscissa, the drawing standard curve;
sample determination: precision takes above-described embodiment gained Herba Achilleae total extract sample 0.5g respectively, put in 100ml tool plug conical flask, precision adds methanol 50ml, weighed weight, supersound process 15 minutes, be cooled to room temperature, weighed weight again, supply the weight of less loss with methanol, filter, precision measures subsequent filtrate 1ml and puts in the 10ml measuring bottle, be diluted to scale with methanol, shake up, precision measures 2ml, put in the 25ml measuring bottle, respectively add water to 6ml, add 5% sodium nitrite solution 1ml, shake up, placed 6 minutes, add 10% aluminum nitrate solution 1ml, shake up, placed 6 minutes, hydro-oxidation sodium test solution 10ml, add water to again scale, shake up, placed 15 minutes, take corresponding reagent as blank, wavelength place at 510nm measures absorbance, read from standard curve the amount that contains anhydrous rutin sample solution, calculate the content 25%-90% of total flavones in Herba Achilleae total extract sample.
Embodiment 17: the assay method of total sesquiterpene content in the Herba Achilleae total extract:
Chromatographic condition: take octadecylsilane chemically bonded silica as filler; Take methanol-0.2% aqueous formic acid (67:33) as mobile phase; The detection wavelength is 245nm;
The preparation of reference substance solution: precision takes keto acid reference substance 5.6mg drying under reduced pressure to constant weight, is placed in the 10ml volumetric flask, adds methanol and dissolves in right amount and be settled to scale, shake up, then precision measures this solution 1ml in the 10ml volumetric flask, methanol constant volume, shake up, namely get (every ml contains keto acid 56 μ g);
The preparation of need testing solution: Herba Achilleae total extract sample 0.5g, put in 100ml tool plug conical flask, precision added 2% sodium hydroxide methanol 50ml reflux, extract, 3 hours, weighed weight, be cooled to room temperature, weighed weight is again supplied the weight of less loss with methanol, filter, precision measures subsequent filtrate 1ml and puts in the 10ml measuring bottle, be diluted to scale with methanol, shake up, and get final product;
Assay: precision is drawn reference substance solution and need testing solution 10 μ l respectively, enters chromatograph of liquid, measures, and calculates total sesquiterpene content (in rupestonic acid) in the Herba Achilleae total extract, should be between 5%-30%.
Embodiment 18: the assay method of rupestonic acid content in the Herba Achilleae total extract:
Chromatographic condition: with embodiment 17;
The preparation of reference substance solution: with embodiment 17;
The preparation of need testing solution: Herba Achilleae total extract sample 0.5g, to put in 100ml tool plug conical flask, precision adds methanol 50ml, weighed weight, supersound process 15 minutes is cooled to room temperature, more weighed weight, supply the weight of less loss with methanol, filter, precision measures subsequent filtrate 1ml and puts in the 10ml measuring bottle, is diluted to scale with methanol, shake up, and get final product;
Assay: precision is drawn reference substance solution and need testing solution 10 μ l respectively, enters chromatograph of liquid, measures, and calculates the content of rupestonic acid in Herba Achilleae total extract sample, should be between 3%-15%.
Embodiment 19: resisiting influenza virus experiment in the body of Herba Achilleae total extract:
Strain: influenza A virus Mus lung adapted strain PR8, available from China prevention medical courses in general institute institute of viruses, go down to posterity 1 time in 9 age in days chick embryo allantoic cavity inoculations before using, 35 ℃ of cultivation 48h of temperature collect allantoic fluid, with 3000rmin
-1Centrifugal 10min gets supernatant and is sub-packed in-80 ℃ of cryogenic refrigerators and saves backup;
Impact on the infecting mouse mortality rate: healthy kunming mice is divided into 5 groups at random by the sex body weight, i.e. model group, ribavirin group, Herba Achilleae total extract 0.03,0.06,0.12gkg
-1The dosage group, every group 10, all medicinal liquids are made into suspension by 0.5% sodium carboxymethyl cellulose (CMC-Na), the continuous gastric infusion 5d of each administration group, every day 2 times, model group is to 0.5%CMC-Na, 2h after administration in the 2nd day, each is organized mice and infects with 50 μ L Flu virus PR8 collunariums respectively, observes day by day the animal incidence and records death toll, observed 14 days, and calculated dead protective rate;
Dead protective rate (%)=(model group mortality rate-experimental group mortality rate)/model group mortality rate * 100%;
Result: after the mouse infection influenza virus, model group symptom occurred on the 7th day after infecting, and showing as that feed reduces, becomes thin, lethargy, being slow in action, and alarmmed mao, rolled up, adnormal respiration is until death, occurred peak mortality on the 10th day.Result is as shown in table 1, and the mortality rate of model group is 80%, and the mortality rate of Herba Achilleae total extract small dose group is 50%, in the Herba Achilleae total extract, the mortality rate of heavy dose of group is respectively 40% and 30%, compares with model group, can significantly reduce the dead mouse number;
The impact of table 1 Herba Achilleae total extract on the infecting mouse mortality rate:
Annotate: compare with model group,
*P＜0.01,
*P＜0.05.
Embodiment 20: the In Vitro Anti influenza virus test of Herba Achilleae total extract:
The toxicity of mensuration medicine to the Hep-2 cell: sample Herba Achilleae total extract and positive control drug ribavirin are made into respectively 2mgmL with the RPMI-1640 culture fluid
-1And 50mgmL
-1Mother solution, be diluted to 1:3,1:9,1:27,1:81,1:243,1:729,1:2187 totally 8 concentration with culture fluid again, the medicinal liquid that dilution is good is added to inoculation Hep-2 cell and has grown up in 96 well culture plates of monolayer every hole 100 μ L, blank is established in 4 multiple holes of every dilution factor simultaneously; Culture plate is put 37 ℃ of temperature, 5%CO
2Cultivate in incubator, observation of cell pathological changes situation under the every day inverted microscope is observed 4d, determines that the minimum extension rate that obvious regression does not appear in cell is maximal non-toxic concentration (TC
0), and with SPSS11.5 computed in software 50% toxic concentration TC
50
The impact of mensuration medicine on pathological changes caused by virus (CPE):
Get the culture plate of inoculating the Hep-2 cell and growing up to monolayer, inhale and abandon culture fluid, inoculate respectively 100 times of TCID
50Virus liquid 100 μ L, put 35 ℃ of temperature, 5%CO
2After absorption 1h, inhale and abandon virus liquid in incubator, establish blank group, virus control group, positive control drug ribavirin TC
0And get the TC that 5 concentration amount to six concentration, Herba Achilleae total extract after 3 times of dilutions
0And get the administration group that 5 concentration amount to six concentration after 3 times of dilutions, and every group is repeated four holes, and the administration group adds different dilution medicinal liquids, and matched group adds maintenance medium, every hole 100 μ L, culture plate is put 35 ℃ of temperature, 5%CO
2Cultivate in incubator, observation of cell pathological changes situation under the every day inverted microscope records experimental result when virus control group cytopathy reaches 4 grades, and cytopathy is by six grade standards judgements.And with SPSS11.5 computed in software half-inhibition concentration IC
50, selection index SI=TC
50/ IC
50
–: Growth of Cells is normal, occurs without pathological changes;
±: cytopathy is less than 10% of whole cell monolayer;
1: cytopathy accounts for below 25% of whole cell monolayer;
2: cytopathy accounts for below 50% of whole cell monolayer;
3: cytopathy accounts for below 75% of whole cell monolayer;
4: cytopathy accounts for more than 75% of whole cell monolayer;
The toxicity of medicine to the Hep-2 cell: sample Herba Achilleae total extract to the toxic action of Hep-2 cell show as cell proliferation slowly, become justify, come off, the part cell breakage, the Herba Achilleae total extract alleviates along with the reduction of drug level within the specific limits to the toxic action of Hep-2 cell, the results are shown in Table 2;
Table 2Hep-2 cell TC
0And TC
50Mensuration
The impact of medicine on pathological changes caused by virus (CPE): according to the result of influenza virus to the Hep-2 cytotoxic assay, be chosen in the non-toxic concn scope, medicine is tested with different concentration, the results are shown in Table 3;
The impact of table 3 Herba Achilleae total extract on pathological changes caused by virus (CPE)
It can be seen from the table: the Herba Achilleae total extract has obvious inhibitory action to influenza A virus, its IC
50Be 2.62 ± 1.22 μ gmL
-1, SI is 303.25.
Embodiment 21: bacteriostatic test in the body of Herba Achilleae total extract:
protective effect to the infection of staphylococcus aureus mice: get 60 of kunming mices, body weight 18-22g, male and female half and half, be divided at random 5 groups, model group, positive control drug Radix Isatidis granule group (5.2g/kg), the Herba Achilleae total extract is little, in, heavy dose of group (30mg/kg, 60mg/kg, 120mg/kg), every group 12, all medicinal liquids are made into suspension by 0.5%CMC-Na, each administration group is by setting the dosage gastric infusion, every day 2 times, for three days on end, model group gives equivalent 0.5%CMC-Na, 2h after the 1st administration in the 3rd day, lumbar injection staphylococcus aureus liquid 0.5ml/ only, dead distribution situation of each treated animal in observed and recorded 7 days, calculate dead protective rate and estimate the curative effect of medicine.Data are carried out statistical analysis with SPSS11.5 software, relatively use X between group
2Check, there is statistical significance P＜0.05;
Dead protective rate (%)=(model group mortality rate-administration group mortality rate)/model group mortality rate * 100%;
Shown in result, in the Herba Achilleae total extract, heavy dose of all can reduce the mortality rate that the infection of staphylococcus aureus mice causes, compare with model group, has significant difference (P＜0.05, P＜0.01), under clinical equivalent dosage, the Herba Achilleae total extract is suitable to protective effect and the Radix Isatidis granule of infection of staphylococcus aureus mice;
The protective effect of table 4 Herba Achilleae total extract to the infection of staphylococcus aureus mice
Annotate: compare with model group,
*P＜0.05,
*P＜0.01;
Protective effect to the coli-infection mice: get 60 of kunming mices, body weight 18-22g, male and female half and half, 2h after the 1st administration in the 3rd day, lumbar injection Escherichia coli bacteria liquid 0.5ml/ only, dead distribution situation of each treated animal in observed and recorded 7 days is calculated dead protective rate, estimate the curative effect of medicine with the mortality rate of animal, the results are shown in Table 5;
Dead protective rate (%)=(model group mortality rate-administration group mortality rate)/model group mortality rate * 100%;
Result: in the Herba Achilleae total extract, heavy dose of all can reduce the mortality rate that the coli-infection mice causes, compare with model group, all has significant difference (P＜0.05), under clinical equivalent dosage, the Herba Achilleae total extract is better than Radix Isatidis granule to the protective effect of coli-infection mice;
The protective effect of table 5 Herba Achilleae total extract to the coli-infection mice
Annotate: compare with model group,
*P＜0.05,
*P＜0.01.
Embodiment 22: the extracorporeal bacteria inhibitor test of Herba Achilleae total extract:
Get 1 aseptic 96 well culture plate, adopt micro-dilution method to measure bacteriostasis.Add medicinal liquid 200 μ L in the B1 hole, B2 to B12, every hole adds the broth medium of 100 μ L sterilizations, draw 100 μ L medicinal liquids and add the B2 hole from the B1 hole, draw 100 μ L to B3 holes after the mixing of B2 hole, doubling dilution to the B10 hole, discards 100 μ l after the mixing of B10 hole continuously by that analogy; After doubling dilution, the B1-B10 hole, every hole adds 10 μ L Escherichia coli bacteria liquids, and the B11 hole does not add medicinal liquid, adds 0.1mL bacterium liquid, observes the bacterial growth situation, and the B12 hole does not add medicinal liquid and bacterium liquid as blank; Get C1-C11 and repeat 1 batch, the C12 hole adds medicinal liquid 100 μ L, do not add antibacterial, observe tested medicine whether pollution is arranged, above operation repeats 2 96 orifice plates, 96 orifice plates is positioned over 37 ℃ of constant incubators of temperature cultivates observed result after 18-24h, with perusal, by contrasting with medicinal liquid control wells, bacterium liquid control wells, the muddy the highest drug dilution multiple that changes does not occur be this medicine minimal inhibitory concentration value.Each hole culture fluid of all clear asepsis growths is inoculated in M-H agar plate culture 24h, the lowest drug concentration that is no more than 5 take clump count is the minimal bactericidal concentration value of this medicine, Radix Isatidis granule with the Herba Achilleae total extract, the results are shown in Table 6 to colibacillary bacteriostasis detection method;
Table 6 Herba Achilleae total extract is to colibacillary bacteriostasis
Annotate: "-" expression solution is limpid, asepsis growth; "+" expression has bacterial growth;
The B5-B11 pore fungi liquid of Herba Achilleae total extract is done 10
-6, 10
-7, 10
-8Doubly dilution, get 0.1ml and be inoculated in the MH agar plate, and each dilution factor repeats 3 flat boards, observes counting after 37 ℃ of cultivation 24h of temperature, determines that further the Herba Achilleae total extract is to colibacillary minimum inhibitory concentration, minimum bactericidal concentration; Select B5, B6, B7, B11 hole to do respectively 10
-8Doubly dilution, then get bacterium liquid 0.1mL and be added drop-wise to 9cm MH agar plate central authorities, use aseptic L spreading rod to be coated with in the same direction evenly, each dilution factor repeats 3 flat boards, flat-plate inverted is placed in observes counting after 37 ℃ of constant incubators of temperature are cultivated 24h, get its average, compare with the count of bacteria result of bacterium liquid control wells B11, find out the Herba Achilleae total extract to colibacillary 50% minimum inhibitory concentration, 50% minimum bactericidal concentration.In like manner, the D2-D11 pore fungi liquid of Radix Isatidis granule is done 10
-6, 10
-7, 10
-8Doubly dilution, be inoculated in the MH agar plate, observes counting after 37 ℃ of cultivation 24h of temperature, determines that further the Herba Achilleae total extract to colibacillary minimum inhibitory concentration, minimum bactericidal concentration, the results are shown in Table 7;
Table 7 Herba Achilleae total extract is to colibacillary bacteriostasis
The B8-B11 pore fungi liquid of Herba Achilleae total extract is done 10
-6, 10
-7, 10
-8Doubly dilution, get 0.1ml and be inoculated in the MH agar plate, each dilution factor repeats 3 flat boards, observes counting after 37 ℃ of cultivation 24h of temperature, further determine the Herba Achilleae total extract to minimum inhibitory concentration, the minimum bactericidal concentration of staphylococcus aureus, again the B8-B11 hole is done respectively 10
-8Doubly dilution, then get bacterium liquid 0.1ml and be added drop-wise to 9cm MH agar plate central authorities, use aseptic L rod to be coated with in the same direction evenly, each dilution factor repeats 3 flat boards, flat-plate inverted is placed in observes counting after 37 ℃ of constant incubators of temperature are cultivated 24h, get its average, compare with the count of bacteria result of bacterium liquid control wells B11, find out the Herba Achilleae total extract to 50% minimum inhibitory concentration, 50% minimum bactericidal concentration of staphylococcus aureus; In like manner, the D2-D11 pore fungi liquid of Radix Isatidis granule is done 10
-6, 10
-7, 10
-8Doubly dilution, be inoculated in the MH agar plate, observes counting after 37 ℃ of cultivation 24h of temperature, determines that further the Herba Achilleae total extract to minimum inhibitory concentration, the minimum bactericidal concentration of staphylococcus aureus, the results are shown in Table 9;
The bacteriostasis of table 9 Herba Achilleae total extract to staphylococcus aureus
Result shows: the Herba Achilleae total extract has stronger antibacterial and bactericidal action to staphylococcus aureus and escherichia coli, and it is antibacterial, bactericidal action is better than the positive control drug Radix Isatidis granule.
Claims (7)
1. the preparation method of a Herba Achilleae total extract is characterized in that following these steps to carrying out:
A. after the Herba Achilleae herb of drying being ground into 2cm, be 60-80 ℃ of heating extraction of 10-95% ethanol temperature 1-3 time with concentration, extraction time 1-3 hour, be cooled to room temperature, filter, merge extractive liquid, is evaporated to without the alcohol flavor, obtains ethanol extract;
B. the extract concentration that step a is obtained is after the 0-15% dissolve with ethanol disperses, centrifugal 4000 rev/mins, filter, be 3-7 with the dilute hydrochloric acid adjust pH, then use resin isolation, first with the water elution of 2-6 times of column volume, with the 30-95% ethanol elution of 3-8 times of column volume, collect ethanol elution again, concentrating under reduced pressure eliminates solvent, drying can obtain the Herba Achilleae total extract.
2. method according to claim 1 is characterized in that in step a, the volume ratio of Herba Achilleae and ethanol is 1:10-30.
3. method according to claim 1 is characterized in that in step b that in institute, resin is DM-130, AB-8, HPD-450, NKA-9, HPD-300, D101, HPD-400, HPD-500, HP-600, SP-825, LD601, LS-303B, LX-28, LX-38 or polyamide.
4. method according to claim 1, the sesquiterpene in the Herba Achilleae total extract that it is characterized in that obtaining by the method for the invention and the mass percent of flavone compound are 25-90%.
5. method according to claim 4, the mass percent that it is characterized in that sesquiterpenoids in the Herba Achilleae total extract is 5-25%, the mass percent of flavone compound is 20-65%.
6. method according to claim 5, the mass percent that it is characterized in that sesquiterpenoid rupestonic acid in the Herba Achilleae total extract is 3-15%.
7.. the purposes of Herba Achilleae total extract according to claim 1 is characterized in that the Herba Achilleae total extract that obtains by the method for the invention is used for the purposes at the grippal medicine of preparation control.
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| CN111317719A (en) * | 2018-12-13 | 2020-06-23 | 复旦大学 | Artemisia rupestris nasal spray with antiviral activity |
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| CN115197238B (en) * | 2022-02-26 | 2024-04-19 | 河南中医药大学 | Preparation method and application of germacrane sesquiterpenoids with hypoglycemic activity |
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