CN105030884A - Chinese elder herb extract for treating influenza virus A1 and preparation method thereof - Google Patents
Chinese elder herb extract for treating influenza virus A1 and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a Chinese elder herb extract for treating influenza virus A1 and a preparation method thereof. The preparation method comprises the steps that (a) every 100 parts by weight of medicinal materials including 80-90 parts by weight of dried Chinese elder herbs and 10-20 parts by weight of dried licorice are mixed and smashed; (b) 65%-75% of ethyl alcohol is adopted for heat reflux extraction, extracting solutions are mixed and concentrated to be free of alcohol smell, and a concentrated solution is obtained; (c) petroleum ether, ethyl acetate and water-saturated n-butyl alcohol are sequentially used for extracting the concentrated solution obtained in the step (b) to respectively obtain a petroleum ether extract, an ethyl acetate extract and a water-saturated n-butyl alcohol extract; (d) the n-butyl alcohol extract is enriched by using macroporous resin, namely 7-9 column volumes of 20%-30% ethyl alcohol is used for flushing to remove polysaccharide firstly, then 9-11 column volumes of 55%-65% ethyl alcohol is used for elution, 55%-65% ethyl alcohol eluent is collected, and reduced-pressure concentration and spray drying are performed. The Chinese elder herb extract can be used for killing influenza virus A1.
Description
Technical field
The present invention relates to the field of Chinese medicines, be specifically related to a kind of Herba Blumeae Laciniatae extract for the treatment of Influenza A1 virus and preparation method thereof.
Background technology
The Herba Blumeae Laciniatae is herb or the leaf of feverfew six ear bell.The whole year can adopt.
Herba Blumeae Laciniatae original shape state six ear bell is sturdy draft, high 50-150cm.Main root is loose, has most fibrous root.Stem multi-branched, has bar rib, and long pubescence and tool handle glandular hair are carried out in top, the Mao Gengmi on sprout and rachis, the long 3-6cm of internode.Inferior leads has and reaches 2-4cm, the handle of the narrow wing of tool; The blade shape of falling ovum Long Circle or obovate, long 10-30cm, wide 4-6cm, the short point of tip, base portion is gradually narrow, downward one-tenth wing, and Lower Half qin shape divides, and top sliver is large, side sliver 2-3 couple, and edge sawtooth or bastard, below by thin pubescence; Lateral vein 5-7 couple; Middle leaf stockless, long 6-10cm, wide 2-4cm, edge has irregular tooth to carve, and qin shape is shallow sometimes splits; Upper leaf is minimum, does not divide, full edge or have tooth to carve.Head inflorescence is most, is arranged in oblong large panicle; Peduncle is by tool handle glandular hair and long pubescence; Involucre is cylindrical or bell; Involucre is cylindrical or bell; Phyllary 5-6 layer, aubergine, spends rear normal opisthotonos, outer linear, the back side by close pubescence and willing handle glandular hair, the oval shape lanceolar in middle level, internal layer is linear; Holder is cellular; Brightly yellowish color; Female flower is most, corolla eaves portion 2-3 fissure; Hermaphrodite flower corolla eaves portion 5 is split, by thin hair.Achene is cylindrical, and 10, tool rib, by thin hair; Pappus white, rough hair shape, difficult drop-off.May in October at florescence to next year.Be grown on dark and damp place, gardens, or on spacious meadow.Distribution Southwestern China portion and the ground such as Guangdong, Guangxi.
Summary of the invention
The object of the present invention is to provide a kind of Herba Blumeae Laciniatae extract, the preparation method of this extract and liquid phase analysis method, the pharmaceutical preparation containing this extract and utilize this extract to prepare the purposes of resisting influenza virus A 1 type medicine.
Above-mentioned purpose of the present invention is achieved by technical scheme below:
A kind of Herba Blumeae Laciniatae extract, this extract is prepared by following methods:
A () by weight, every 100 parts of medical materials comprise the dry Herba Blumeae Laciniatae 80 ~ 90 parts and 10 ~ 20 parts dry, Radix Glycyrrhizae, co-grinding; B () extracts with 65 ~ 75% alcohol heat reflux, merge extractive liquid, is concentrated into and obtains concentrated solution without alcohol taste; C () uses petroleum ether, ethyl acetate and water saturated n-butanol extraction successively to step (b) concentrated solution, obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively; D () n-butyl alcohol extract macroporous resin enrichment, first with 20 ~ 30% alcohol flushing, 7 ~ 9 column volume removing polysaccharide, then uses 55 ~ 65% ethanol elution, 9 ~ 11 column volumes, collects 55 ~ 65% ethanol elution, concentrating under reduced pressure, spraying dry.
Further, macroporous resin described in step (d) is AB-8 type macroporous resin.
Further, extracting with alcohol heat reflux the concentration of alcohol adopted described in step (b) is 70%.
Further, step (d) is by n-butyl alcohol extract macroporous resin enrichment, first with 25% alcohol flushing, 8 column volume removing polysaccharide, then uses 60% ethanol elution, 10 column volumes, collects 60% ethanol elution, concentrating under reduced pressure, spraying dry.
In order to be controlled Herba Blumeae Laciniatae extract differences between batches prepared by Different sources, batch medical material by the method setting up HPLC finger printing, the liquid phase analysis method of described extract is:
Chromatographic column: AgilentZorbaxSBC18 post (4.6mm × 250mm, 5 μm);
Mobile phase: A is methanol, and B is 0.1% phosphate aqueous solution;
Gradient elution program: 0.01 ~ 15min, A25% ~ 35%; 15 ~ 25min, A35% ~ 50%; 25 ~ 32min, A50%; 32 ~ 35min, A50% ~ 25%; 35 ~ 40min, A25%;
Flow rate of mobile phase: 1.0mLmin
-1;
Determined wavelength: 255nm;
Column temperature: 30 DEG C;
Sample size: 10 μ L.
Pharmaceutical preparation, the described Herba Blumeae Laciniatae extract containing treatment effective dose and pharmaceutically acceptable carrier.
The application of described Herba Blumeae Laciniatae extract in the medicine of preparation treatment Influenza A1 virus.
The application of described pharmaceutical preparation in the medicine of preparation treatment Influenza A1 virus.
When extract of the present invention is used as medicine, directly can uses, or use in the form of a pharmaceutical preparation.
Described pharmaceutical preparation contains the Herba Blumeae Laciniatae extract of the present invention for the treatment of effective dose, and all the other are acceptable on materia medica, nontoxic to humans and animals and pharmaceutically suitable carrier of inertia and/or excipient.
Described pharmaceutically suitable carrier or excipient are that one or more are selected from solid, semisolid and liquid diluent, filler and pharmaceutical preparation adjuvant.Pharmaceutical preparation of the present invention is used with the form of per weight dose.Extract of the present invention is applied to by form that is oral or injection the patient needing treatment.For time oral, tablet, slow releasing tablet, controlled release tablet, capsule, drop pill, micropill, suspensoid, Emulsion, powder or granule, oral liquid etc. can be made into; During for injecting, can be made into aqueous or oily solution, aseptic powder injection, liposome or the Emulsion etc. of sterilizing.
Advantage of the present invention: the present invention by extracting Herba Blumeae Laciniatae medical material, remove impurity, enrichment process, the extract of the Influenza A1 virus that can obtain medical treatment; Liquid phase analysis method provided by the invention may be used for the finger printing setting up this extract, for controlling Different sources, the differences between batches of extract prepared by batch medical material.
Accompanying drawing explanation
Fig. 1 is Herba Blumeae Laciniatae extractive HPLC chromatograms.
Detailed description of the invention
Further illustrate essentiality content of the present invention below in conjunction with embodiment, but do not limit scope with this.Although be explained in detail the present invention with reference to preferred embodiment, those of ordinary skill in the art should be appreciated that and can modify to technical scheme of the present invention or equivalent replacement, and does not depart from essence and the scope of technical solution of the present invention.
Embodiment 1: prepared by Herba Blumeae Laciniatae extract
Crude drug source: the Herba Blumeae Laciniatae and Radix Glycyrrhizae are purchased from Hui nationality's Chinese Medicinal Materials Markets.
Main agents: food-grade ethanol is purchased from Shanghai Ling Feng chemical reagent company limited; Pharmaceutical grade AB-8 macroporous resin is purchased from sky tunami letter resin company limited; Methanol is HPLC level, is purchased from TEDIA; 85% phosphoric acid is HPLC level, is purchased from TEDIA; Chromatographic grade pure water is heartily pure water.
By the Herba Blumeae Laciniatae of 8.5kg drying and the dry Radix Glycyrrhizae co-grinding of 1.5kg, extract (25L × 3 time) with 70% alcohol heat reflux, merge extractive liquid, is concentrated into and obtains concentrated solution (6L) without alcohol taste, petroleum ether (6L × 3 time) is used successively to concentrated solution, ethyl acetate (6L × 3 time) and water saturated n-butyl alcohol (6L × 3 time) extraction, obtain petroleum ether extract respectively, acetic acid ethyl ester extract and n-butyl alcohol extract 285g, with AB-8 macroporous resin (2kg after n-butyl alcohol extract dissolves with 5L distilled water, column volume 1.5L) enrichment, first use 25% alcohol flushing, 8 column volumes (12L), use 60% ethanol elution, 10 column volumes (15L) again, collect 60% eluent, concentrating under reduced pressure, spraying dry obtains Herba Blumeae Laciniatae extract extract powder and is about 155g.
Embodiment 2: 2. Herba Blumeae Laciniatae extract is prepared (80 parts of Herba Blumeae Laciniataes, 20 portions of Radix Glycyrrhizaes)
Preparation method: by the Herba Blumeae Laciniatae of 8.0kg drying and the dry Radix Glycyrrhizae co-grinding of 2.0kg, extract (25L × 3 time) with 70% alcohol heat reflux, merge extractive liquid, is concentrated into and obtains concentrated solution (6L) without alcohol taste, petroleum ether (6L × 3 time) is used successively to concentrated solution, ethyl acetate (6L × 3 time) and water saturated n-butyl alcohol (6L × 3 time) extraction, obtain petroleum ether extract respectively, acetic acid ethyl ester extract and n-butyl alcohol extract 285g, with AB-8 macroporous resin (2kg after n-butyl alcohol extract dissolves with 5L distilled water, column volume 1.5L) enrichment, first use 25% alcohol flushing, 8 column volumes (12L), use 60% ethanol elution, 10 column volumes (15L) again, collect 60% eluent, concentrating under reduced pressure, spraying dry obtains Herba Blumeae Laciniatae extract extract powder and is about 155g.
Embodiment 3: 3. Herba Blumeae Laciniatae extract is prepared (90 parts of Herba Blumeae Laciniataes, 10 portions of Radix Glycyrrhizaes)
Preparation method: by the Herba Blumeae Laciniatae of 9.0kg drying and the dry Radix Glycyrrhizae co-grinding of 1.0kg, extract (25L × 3 time) with 70% alcohol heat reflux, merge extractive liquid, is concentrated into and obtains concentrated solution (6L) without alcohol taste, petroleum ether (6L × 3 time) is used successively to concentrated solution, ethyl acetate (6L × 3 time) and water saturated n-butyl alcohol (6L × 3 time) extraction, obtain petroleum ether extract respectively, acetic acid ethyl ester extract and n-butyl alcohol extract 285g, with AB-8 macroporous resin (2kg after n-butyl alcohol extract dissolves with 5L distilled water, column volume 1.5L) enrichment, first use 25% alcohol flushing, 8 column volumes (12L), use 60% ethanol elution, 10 column volumes (15L) again, collect 60% eluent, concentrating under reduced pressure, spraying dry obtains Herba Blumeae Laciniatae extract extract powder and is about 155g.
Embodiment 4: 4. Herba Blumeae Laciniatae extract is prepared (75 parts of Herba Blumeae Laciniataes, 25 portions of Radix Glycyrrhizaes)
Preparation method: by the Herba Blumeae Laciniatae of 7.5kg drying and the dry Radix Glycyrrhizae co-grinding of 2.5kg, extract (25L × 3 time) with 70% alcohol heat reflux, merge extractive liquid, is concentrated into and obtains concentrated solution (6L) without alcohol taste, petroleum ether (6L × 3 time) is used successively to concentrated solution, ethyl acetate (6L × 3 time) and water saturated n-butyl alcohol (6L × 3 time) extraction, obtain petroleum ether extract respectively, acetic acid ethyl ester extract and n-butyl alcohol extract 285g, with AB-8 macroporous resin (2kg after n-butyl alcohol extract dissolves with 5L distilled water, column volume 1.5L) enrichment, first use 25% alcohol flushing, 8 column volumes (12L), use 60% ethanol elution, 10 column volumes (15L) again, collect 60% eluent, concentrating under reduced pressure, spraying dry obtains Herba Blumeae Laciniatae extract extract powder and is about 155g.
Embodiment 5: 5. Herba Blumeae Laciniatae extract is prepared (95 parts of Herba Blumeae Laciniataes, 5 portions of Radix Glycyrrhizaes)
Preparation method: by the Herba Blumeae Laciniatae of 9.5kg drying and the dry Radix Glycyrrhizae co-grinding of 0.5kg, extract (25L × 3 time) with 70% alcohol heat reflux, merge extractive liquid, is concentrated into and obtains concentrated solution (6L) without alcohol taste, petroleum ether (6L × 3 time) is used successively to concentrated solution, ethyl acetate (6L × 3 time) and water saturated n-butyl alcohol (6L × 3 time) extraction, obtain petroleum ether extract respectively, acetic acid ethyl ester extract and n-butyl alcohol extract 285g, with AB-8 macroporous resin (2kg after n-butyl alcohol extract dissolves with 5L distilled water, column volume 1.5L) enrichment, first use 25% alcohol flushing, 8 column volumes (12L), use 60% ethanol elution, 10 column volumes (15L) again, collect 60% eluent, concentrating under reduced pressure, spraying dry obtains Herba Blumeae Laciniatae extract extract powder and is about 155g.
Embodiment 6: liquid-phase chromatographic analysis
Need testing solution is prepared: in the brown volumetric flask of extract extract powder 5mg to 50mL that Example 1 method is obtained, add 30mL25% methanol aqueous solution ultrasonic dissolution, after being cooled to room temperature, continue to add 25% methanol aqueous solution standardize solution.
Analytical method:
High performance liquid chromatograph: Agilent1100, binary pump;
Chromatographic column: AgilentZorbaxSBC18 post (4.6mm × 250mm, 5 μm);
Mobile phase: A is methanol, and B is 0.1% phosphate aqueous solution;
Gradient elution program: 0.01 ~ 15min, A25% ~ 35%; 15 ~ 25min, A35% ~ 50%; 25 ~ 32min, A50%; 32 ~ 35min, A50% ~ 25%; 35 ~ 40min, A25%;
Flow rate of mobile phase: 1.0mLmin
-1;
Determined wavelength: 255nm;
Column temperature: 30 DEG C;
Sample size: 10 μ L.
Analyze the extract prepared by 10 batches of Different sources, batch Herba Blumeae Laciniatae, set up finger printing and mate, 1 ~ No. 5 peak all occurs in 10 batch sample chromatograms as a result.Therefore demarcate 5 peaks for total fingerprint peaks, set up the HPLC finger printing of this extract accordingly, the results are shown in Figure 1.
Embodiment 7: Herba Blumeae Laciniatae extract pharmacological testing
One, materials and methods
1.1 medicines: Herba Blumeae Laciniatae extract 1. ~ 5. obtain according to embodiment 1 ~ 5 respectively.Be dissolved in the dimethyl sulfoxide of minute quantity, be diluted to desired concn for experiment in vitro with cell maintenance medium; Experiment in vivo drug level used is interpreted into distilled water after dissolving with a small amount of Tween-80.Positive drug oseltamivir phosphate capsule (Oseltamivir that) is provided by China Agricultural University, is 100 μm of ol/L to the maximal non-toxic concentration of Madin-Darby canine kidney(cell line) (MDCK) (MDCK).
1.2 cells and Strain: mdck cell, provided by disease prevention and control center of Gansu Province influenza virus laboratory.Cell growth medium is the DMEM culture medium containing 10% hyclone, and maintenance medium is the DMEM culture medium containing 2% hyclone, and virus purification liquid is for containing the tryptic MEM culture medium of 40 μ g/mL.Influenza virus FM1 strain (A/PR/8/1934, IAV), provided by CDC, go down to posterity through 10 age in days chick embryo allantoic cavity inoculated and cultured, Microhemagglutination test titration virus titer, it is that experiment in vitro is used that hemagglutinative titer measures 1:256; The Strain of 1:512 is experiment in vivo strain.Murine Virus median lethal dose(LD 50) (LD50) is measured before formal experiment.
1.3 laboratory animals: Kunming mouse, weight 18 ~ 22g, male and female half and half, are provided by Lanzhou University's animal experimental center.
1.4 instruments and reagent: DMEM culture medium, MTT (Sigma company), L-glutaminate (Hua Lvyuan biotechnology), dimethyl sulfoxide (DMSO) (Sigma company), hyclone (Hangzhou Sijiqing Biological Engineering Material Co., Ltd.), the green source of students biotechnology of trypsin); A seating automatic electric heating pressure steam sterilizer (Shen, Shanghai peace), centrifuge (AnkeTDL80-2B), CO
2incubator (SHEL-LAB), inverted microscope (Olympus), Biohazard Safety Equipment (Shanghai Ruiyang Purification Equipment Co., Ltd.), microplate reader (ThermoMulti-skanMK3).
The mensuration of 1.5 virus infection titers
Well-grown mdck cell being added 96 orifice plates, grows up to after fine and close monolayer until cell, is that 1:256 Influenza A1 virus is diluted to 9 kinds of variable concentrations (10 with 10 times of the dilution methods with not containing antibiotic maintenance medium by hemagglutinative titer
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, 10
-8, 10
-9), each dilution factor establishes 8 holes, the virus liquid of 9 kinds of variable concentrations is inoculated into respectively 96 orifice plates growing up to MDCK cell monolayers, and every hole 50 μ L, is placed in 5%CO
2in constant incubator, after 37 DEG C of absorption 1h, abandon supernatant, wash 3 times with Hank ' s liquid, add the every hole 0.1mL of maintenance medium, establish cell control well 8 hole simultaneously, put 37 DEG C, 5%CO
2incubator is cultivated, daily check viral growth situation, and the metamorphosis of observation of cell under inverted microscope the hole count of recording feature sexual cell pathological changes (CPE) record result after 5d.Median infective dose (the TCID of virus is calculated by Reed-Muench method
50).Cell occurs that pathological changes (CPE) degree is by 6 grade standard records :-: Growth of Cells is normal, occurs without pathological changes; ±: cytopathy is less than 10% of whole cell monolayer; +: cytopathy is less than 25% of whole cell monolayer; ++: cytopathy is less than 50% of whole cell monolayer; +++: cytopathy is less than 75% of whole cell monolayer; ++++: cytopathy accounts for more than 75% of whole cell monolayer.Lower same.
1.6 drug cytotoxicity tests
Get well-grown mdck cell, cell is added 96 orifice plates, every hole 100 μ L (about 1 × 104/ hole), grow up to after monolayer until cell, with maintenance medium by Herba Blumeae Laciniatae extract 1. ~ 5. respectively dilution be 1.0, 0.50, 0.25, 0.125, 0.063, 0.032, 0.016, 0.008g/L, each mass concentration medicine is added culture plate with every hole 200 μ L, each mass concentration establishes 2 holes, establish normal cell controls group 4 hole simultaneously, put 37 DEG C, 5%CO2 incubator is cultivated, daily check cell growth status under inverted microscope, the metamorphosis of observation of cell, with CPE method observed and recorded medicine to after the toxicity data of mdck cell, carry out cell MTT colorimetry: every hole adds MTT dye liquor 20 μ L (5g/L), put 5%CO
2in incubator, cultivate 4h for 37 DEG C, abandon dyeing liquor, every hole adds DMSO150 μ L, and vibration 10min, surveys absorbance at microplate reader 490nm wavelength place, calculates median toxic concentration (TC
50) and maximal non-toxic concentration (TC
0).Experiment repetition 3 times, gets 3 meansigma methodss as a result.
1.7 Herba Blumeae Laciniatae extract In vitro antibacterial test
Mdck cell is inoculated in 96 orifice plates according to 2 × 105/mL, 0.1mL/ hole, cultivates after 2d forms cell monolayer and carry out In vitro antibacterial test.Divide normal cell controls group (adding maintenance medium); Virus control group; Drug control group is the oseltamivir phosphate capsule (Oseltamivir that) of 100 μm of ol/L; Herba Blumeae Laciniatae extract 1. ~ 5. respectively establish 4 groups, mass concentration is respectively 0.016,0.032,0.063,0.125g/L.Test is carried out in 3 kinds of modes: 1. medicine is to the direct deactivation of IAV: viral suspension and pastille culture fluid are at 37 DEG C, 5%CO
2incubator cultivates 1h postoperative infection cell.2. medicine to IAV absorption, invade the blocking effect of cell: with pastille culture fluid in advance with cell at 37 DEG C, 5%CO
2incubator cultivates 1h postoperative infection IAV.3. medicine inhibitory action that IAV is bred: cell first infects IAV1h, then adds pastille culture fluid, 37 DEG C, 5%CO
2calorstat is cultivated.Adopt 96 porocyte culture plates, every 1 group adds 4 holes, treats that virus control wells pathological changes reaches +++, when cell controls group is normal, carry out MTT dyeing, survey absorbance (A) value at microplate reader 490nm wavelength place, calculating cell survival rate and medicine are to the suppression ratio of IAV.Repeat 3 times.
Cell survival rate=A medicine group/A cell controls group × 100%
Suppression ratio=(A medicine group-A virus control group)/A virus control group × 100%
1.8 infected by influenza strains cause the effect of mice pneumonia
The Kunming mouse of 130 weight 18 ~ 22g is divided into 13 groups at random: model control group, Normal group, oseltamivir phosphate capsule group (3mg/kg) and Herba Blumeae Laciniatae extract 1. ~ 5. high and low dose (100,50mg/kg) group, male and female half and half, often organize 10.Except Normal group, each group is infected 1/3LD50 Influenza virus FM1 strain 50 μ L under ether light anesthesia, and after infection, 4h starts ig administration, 0.2mL/ time, continuous 5d; Normal group gives same volume sterile distilled water.Eyeball sacrificed by exsanguination animal is plucked after claiming weight after treatment 5d, open thoracic cavity and extract full lung, with brine twice, surface moisture is blotted with filter paper, claim lung quality with electronic balance, calculate Lung Exponent [Lung Exponent=lung quality/weight × 100%], lung index [lung index=(the average Lung Exponent of the average Lung Exponent-experimental group of virus control group)/average Lung Exponent × 100% of virus control group].Under cryogenic conditions, put in homogenizer by each group of lung and add normal saline (1:9), homogenate is made in grinding, 4 DEG C, get supernatant after the centrifugal 30min of 2500r/min, does Microhemagglutination test according to a conventional method.
The death protection of 1.9 pairs of In vivo infection influenza virus mices and the effect of extending life
Mice is divided at random model control group, oseltamivir phosphate capsule group (3mg/kg), Herba Blumeae Laciniatae extract 1. ~ 5. high and low dose (100,50mg/kg) group, often organize 10, each group is infected 25LD50 Influenza virus FM1 strain 50 μ L under ether light anesthesia, after infecting, 4h starts ig administration, 0.2mL/ time, every day 2 times, continuous 5d, separately establishes Normal group to give co-content sterile distilled water.Observe, record animal morbidity and death toll; observe 14d altogether, calculate Death prevention rate [Death prevention rate=virus control group death toll-administration group death toll)/virus control group death toll × 100%], increase in life span [increase in life span=(administration group mean survival time-virus control group mean survival time)/virus control group mean survival time × 100%].
1.10 statistical analysis: application SPSS13.0 statistical software, carry out the multifactor analysis of variance to experimental data.
Two, result
The mensuration of 2.1 virus infection titers: the median infective dose TCID calculating virus by Reed-Muench method
50be 10
-4.59/ 0.1mL, virus attack amount is 100TCID
50.
2.2 Herba Blumeae Laciniatae extracts are to the toxicity of mdck cell: Herba Blumeae Laciniatae extract 1. ~ 5. when 0.125g/L, mdck cell is had no adverse effects, with normal cell controls group without obvious difference, show Herba Blumeae Laciniatae extract lower than 0.125g/L mass concentration to mdck cell free of toxic effects.
2.3 In Vitro Anti Influenza A1 virus tests
2.3.1 medicine is to the direct deactivation of IAV
Extract 1. ~ 3. each dosage group and virus control group (model group) ratio, each group A value slightly raises (P<0.01); With control drug oseltamivir phosphate capsule ratio, each group A value declines (P<0.01) to some extent; Extract 4. ~ 5. each dosage group and model group be than without significant difference.(compare with normal cell controls group: △ △ P<0.01 in table 1; Compare with virus control group: * P<0.05, * * P<0.01; Compare with oseltamivir phosphate capsule group: ▲ P<0.05, ▲ ▲ P<0.01).
2.3.2 medicine is to IAV absorption, the blocking effect invading cell
Extract 1. ~ 3. each dosage group compare with model group, the A value significantly rising (P<0.01) of each group; Compared with control drug oseltamivir phosphate capsule, A value is without significant difference (P>0.05); Extract 4. ~ 5. each dosage group and model group be than without significant difference.In table 1.
2.3.3 to the inhibitory action of IAV propagation
Extract 1. ~ 3. each dosage group compare with model group, the A value significantly rising (P<0.01) of each group; Compared with control drug oseltamivir phosphate capsule, each group A value slightly declines (P<0.05); Extract 4. ~ 5. each dosage group and model group be than without significant difference.
The results are shown in Table 1.
Table 1 Herba Blumeae Laciniatae extract antiviral effect in vitro (x ± s, n=8)
2.4 impacts on mouse influenza virus pneumonia
High and low dose Herba Blumeae Laciniatae extract 1. ~ 3. the Lung Exponent of influenza virus infected there is suppression ratio; Lung tissue suspension viral hemoagglutination titre and Lung Exponent all decrease, significant difference (P<0.01) is had compared with model control group, compared with control drug group oseltamivir phosphate capsule, not statistically significant (P>0.05).Herba Blumeae Laciniatae extract 4. ~ 5. high low dose group and model group be without significant difference.
The results are shown in Table for 2 (comparing with model control group: * * P<0.01).
Table 2 Herba Blumeae Laciniatae extract is on the impact (x ± s, n=10) of IAV Lung Index of mice infected by Influenza virus and Hemagglutination titer
| Group | Dosage | Hemagglutination titer (InX) | Lung Exponent | Lung index/% |
| Blank group | - | - | 0.72±0.23** | - |
| Model group | - | 2.78±0.17 | 1.26±0.25 | - |
| Oseltamivir phosphate capsule group | 3 | 1.52±0.91** | 0.85±0.11** | 26.7 |
| Extract is low dose group 1. | 50 | 1.59±0.65** | 0.94±0.46** | 19.0 |
| Extract is high dose group 1. | 100 | 1.71±0.22** | 0.89±0.31** | 23.3 |
| Extract is low dose group 2. | 50 | 1.58±0.63** | 0.92±0.42** | 18.9 |
| Extract is high dose group 2. | 100 | 1.70±0.20** | 0.87±0.30** | 23.2 |
| Extract is low dose group 3. | 50 | 1.57±0.64** | 0.91±0.38** | 18.8 |
| Extract is high dose group 3. | 100 | 1.69±0.20** | 0.86±0.32** | 23.1 |
| Extract is low dose group 4. | 50 | 2.72±0.19 | 1.22±0.25 | 3.1 |
| Extract is high dose group 4. | 100 | 2.74±0.18 | 1.19±0.23 | 2.9 |
| Extract is low dose group 5. | 50 | 2.68±0.21 | 1.20±0.21 | 3.0 |
| Extract is high dose group 5. | 100 | 2.70±0.19 | 1.18±0.24 | 2.8 |
The protective effect of 2.5 pairs of IAV infecting mouse life
High and low dose Herba Blumeae Laciniatae extract 1. ~ 3. the Death prevention rate of influenza virus infected have significant difference (P<0.01) with increase in life span compared with model control group, high dose Herba Blumeae Laciniatae extract group compared with control drug oseltamivir phosphate capsule, not statistically significant (P>0.05); High and low dose Herba Blumeae Laciniatae extract 4. ~ the 5. Death prevention rate of influenza virus infected and increase in life span there was no significant difference compared with model control group.
The results are shown in Table for 3 (comparing with model control group: * * P<0.01).
Table 3 Herba Blumeae Laciniatae extract is to the protective effect of IAV infecting mouse life
| Group | Dosage | Animal/only | Death toll/only | Mean survival time/d | Death prevention rate/% | Increase in life span/% |
| Model group | - | 10 | 8 | 8.8 | - | - |
| Oseltamivir phosphate capsule group | 3 | 10 | 2 | 12.6** | 75.0 | 43.2 |
| Extract is low dose group 1. | 50 | 10 | 4 | 11.3** | 50.0 | 28.4 |
| Extract is high dose group 1. | 100 | 10 | 3 | 12.1** | 62.5 | 37.5 |
| Extract is low dose group 2. | 50 | 10 | 4 | 11.2** | 50.0 | 28.4 |
| Extract is high dose group 2. | 100 | 10 | 3 | 12.1** | 62.5 | 37.5 |
| Extract is low dose group 3. | 50 | 10 | 4 | 11.2** | 50.0 | 28.4 |
| Extract is high dose group 3. | 100 | 10 | 3 | 12.0** | 62.5 | 37.5 |
| Extract is low dose group 4. | 50 | 10 | 7 | 9.0 | 12.5 | 2.3 |
| Extract is high dose group 4. | 100 | 10 | 7 | 9.2 | 12.5 | 4.5 |
| Extract is low dose group 5. | 50 | 10 | 7 | 9.0 | 12.5 | 2.3 |
| Extract is high dose group 5. | 100 | 10 | 7 | 9.2 | 12.5 | 4.5 |
Many reports all show that oseltamivir phosphate capsule obviously can improve the every clinical symptoms caused by influenza virus, and therefore it is positive control medicine ideal in this model, and the antivirus action of its excellence is confirmed in this experiment.This experimental result confirms: Herba Blumeae Laciniatae extract has the effect of significant anti-IAV activity in vitro; The protective effect of IAV infecting mouse is shown: effectively reduce mouse lung index, reduction lung tissue suspension viral hemoagglutination is tired; and extend infecting mouse mean survival time; reduce and infect viral mouse death rate; and present certain amount-result relation, the research further but actual efficacy of its clinical treatment influenza infection needs.But the above-mentioned curative effect of Radix Glycyrrhizae content on Herba Blumeae Laciniatae extract has vital impact in the present invention, too high or too lowly all above-mentioned effect can not be produced.
Embodiment 8: the preparation of tablet
By embodiment 1 method first obtained extract, add excipient, pelletizing press sheet in itself and excipient weight than the ratio for 1:8.
Embodiment 9: the preparation of oral liquid
By embodiment 1 method first obtained extract, oral liquid method for making makes oral liquid routinely.
Embodiment 10: the preparation of capsule or granule
By embodiment 1 method first obtained extract, add excipient in itself and excipient weight than the ratio for 1:9, make capsule or granule.
Embodiment 11: the preparation of injection
Obtain extract by embodiment 1 method, inject with water, fine straining, injection is made in embedding sterilizing.
Embodiment 12: the preparation of aseptic powder injection
By embodiment 1 method first obtained extract, be dissolved in sterile water for injection, stirring makes molten, filters with aseptic suction funnel, more aseptic fine straining, and be sub-packed in ampoule, after frozen drying, aseptic sealing by fusing obtains injectable powder.
The effect of above-described embodiment is essentiality content of the present invention is described, but does not limit protection scope of the present invention with this.Those of ordinary skill in the art should be appreciated that and can modify to technical scheme of the present invention or equivalent replacement, and does not depart from essence and the protection domain of technical solution of the present invention.
Claims (8)
1. a Herba Blumeae Laciniatae extract, is characterized in that described extract is prepared by following methods: (a) by weight, every 100 parts of medical materials comprise the dry Herba Blumeae Laciniatae 80 ~ 90 parts and 10 ~ 20 parts dry, Radix Glycyrrhizae, co-grinding; B () extracts with 65 ~ 75% alcohol heat reflux, merge extractive liquid, is concentrated into and obtains concentrated solution without alcohol taste; C () uses petroleum ether, ethyl acetate and water saturated n-butanol extraction successively to step (b) concentrated solution, obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively; D () n-butyl alcohol extract macroporous resin enrichment, first with 20 ~ 30% alcohol flushing, 7 ~ 9 column volume removing polysaccharide, then uses 55 ~ 65% ethanol elution, 9 ~ 11 column volumes, collects 55 ~ 65% ethanol elution, concentrating under reduced pressure, spraying dry.
2. extract according to claim 1, is characterized in that: macroporous resin described in step (d) is AB-8 type macroporous resin.
3. extract according to claim 1, is characterized in that: extracting with alcohol heat reflux the concentration of alcohol adopted described in step (b) is 70%.
4. extract according to claim 1, it is characterized in that: step (d) is by n-butyl alcohol extract macroporous resin enrichment, first with 25% alcohol flushing, 8 column volume removing polysaccharide, use 60% ethanol elution, 10 column volumes again, collect 60% ethanol elution, concentrating under reduced pressure, spraying dry.
5. extract according to claim 1, is characterized in that: the liquid phase analysis method of described extract is:
Chromatographic column: AgilentZorbaxSBC18 post (4.6mm × 250mm, 5 μm);
Mobile phase: A is methanol, and B is 0.1% phosphate aqueous solution;
Gradient elution program: 0.01 ~ 15min, A25% ~ 35%; 15 ~ 25min, A35% ~ 50%; 25 ~ 32min, A50%; 32 ~ 35min, A50% ~ 25%; 35 ~ 40min, A25%;
Flow rate of mobile phase: 1.0mLmin
-1;
Determined wavelength: 255nm;
Column temperature: 30 DEG C;
Sample size: 10 μ L.
6. pharmaceutical preparation, is characterized in that: the extract according to claim 1 containing treatment effective dose and pharmaceutically acceptable carrier.
7. the application of extract according to claim 1 in the medicine of preparation treatment Influenza A1 virus.
8. the application of pharmaceutical preparation according to claim 6 in the medicine of preparation treatment Influenza A1 virus.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105250360A (en) * | 2015-11-12 | 2016-01-20 | 庄立 | Evening primrose extract and preparation method and medical application of extract |
| CN107773578A (en) * | 2016-08-28 | 2018-03-09 | 成都宝科生物科技有限公司 | A kind of common cnidium fruit P.E of resisiting influenza virus |
| CN110108828A (en) * | 2019-06-03 | 2019-08-09 | 广西中医药大学 | The method for building up of thick boisiana HPLC finger-print and UPLC finger-print |
| CN114306404A (en) * | 2020-09-30 | 2022-04-12 | 贵州宏宇药业有限公司 | A herba Blumeae Balsamiferae extract with anti-influenza virus effect, and its preparation method |
| CN114306405A (en) * | 2020-09-30 | 2022-04-12 | 贵州宏宇药业有限公司 | New use of herba Blumeae Balsamiferae extract in preparing medicine for resisting influenza virus |
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2015
- 2015-09-13 CN CN201510579862.9A patent/CN105030884A/en not_active Withdrawn
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105250360A (en) * | 2015-11-12 | 2016-01-20 | 庄立 | Evening primrose extract and preparation method and medical application of extract |
| CN107773578A (en) * | 2016-08-28 | 2018-03-09 | 成都宝科生物科技有限公司 | A kind of common cnidium fruit P.E of resisiting influenza virus |
| CN110108828A (en) * | 2019-06-03 | 2019-08-09 | 广西中医药大学 | The method for building up of thick boisiana HPLC finger-print and UPLC finger-print |
| CN114306404A (en) * | 2020-09-30 | 2022-04-12 | 贵州宏宇药业有限公司 | A herba Blumeae Balsamiferae extract with anti-influenza virus effect, and its preparation method |
| CN114306405A (en) * | 2020-09-30 | 2022-04-12 | 贵州宏宇药业有限公司 | New use of herba Blumeae Balsamiferae extract in preparing medicine for resisting influenza virus |
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