CN111904970B - Application of pulsatilla chinensis saponin B4 in preparation of medicine for treating asthma - Google Patents

Application of pulsatilla chinensis saponin B4 in preparation of medicine for treating asthma Download PDF

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CN111904970B
CN111904970B CN202010836864.2A CN202010836864A CN111904970B CN 111904970 B CN111904970 B CN 111904970B CN 202010836864 A CN202010836864 A CN 202010836864A CN 111904970 B CN111904970 B CN 111904970B
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saponin
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CN111904970A (en
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杨世林
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Guangxi Xinhai Pharmaceutical Technology Co Ltd
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    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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Abstract

The invention discloses application of pulsatilla saponin B4 in preparing a medicament for treating asthma of mammals including human beings. The invention has high bioavailability, quick effect, direct action on focus and good curative effect on asthma.

Description

Application of pulsatilla chinensis saponin B4 in preparation of medicine for treating asthma
Technical Field
The invention relates to the technical field of medicines. More specifically, the invention relates to an application of pulsatilla saponin B4 in preparing a medicine for treating asthma.
Background
Pulsatillae radix is dried root of Pulsatilla chinensis (Bunge) Regel of Pulsatilla of Ranunculaceae, and has effects of clearing heat and detoxicating, cooling blood and relieving dysentery. Modern pharmacological studies show that the saponins in pulsatilla chinensis is the main active component of pulsatilla chinensis, and has the effects of enhancing immune function, resisting inflammation and the like. The pulsatilla saponin B4 is pentacyclic triterpenoid saponin extracted from pulsatilla and having pharmacological activity, and has pharmacological effects of resisting inflammation, resisting bacteria, regulating immunity, resisting tumor, resisting oxidation, resisting virus and the like, but the effect of the pulsatilla saponin B4 on treating asthma is not reported.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.
The invention also aims to provide the application of the pulsatilla chinensis saponin B4 in preparing the medicine for treating asthma, which has high bioavailability and quick response, can directly act on focus and has good curative effect on asthma.
To achieve these objects and other advantages in accordance with the present invention, there is provided a use of pulsatillae radix saponin B4 in the manufacture of a medicament for treating asthma in a mammal including a human.
Preferably, the medicine contains the pulsatilla saponin B4 with a therapeutically effective amount and a pharmaceutically acceptable carrier.
Preferably, the medicament is formulated into a pharmaceutically acceptable dosage form.
Preferably, the medicament is formulated as an aerosol.
Preferably, the medicament is formulated as a spray.
Preferably, the dosage of the pulsatilla saponin B4 is not less than 10 mg/kg-d.
The invention at least comprises the following beneficial effects:
first, white head Weng Zaogan B4 has an asthma treating effect, and its mechanism of action may be related to the reduction of blood, alveolar lavage fluid and lung tissue inflammatory factors such as IL-6, TNF- α, and the reduction of blood leukocyte (WBC), lymphocyte (LYMPH) and Neutrophil (NEUT) levels. The treatment effect of the pulsatilla saponin B4 aerosol and the spray on rat bronchial asthma induced by OVA induction is stronger than that of pulsatilla saponin B4 for injection, and the dosage of the pulsatilla saponin B4 aerosol and the spray is less than that of pulsatilla saponin B4 for injection, so that the asthma treatment effect of the pulsatilla saponin B4 aerosol and the spray is more remarkable than that of pulsatilla saponin B4 for injection;
secondly, as the aerosol and the spraying agent have the advantages of small dosage, uniform distribution, quick response, high bioavailability, convenient use and the like, are suitable for asthma, can directly reach the focus, and reduce the side effect of gastrointestinal tracts, the invention provides scientific basis for preventing and treating the upper respiratory tract infection diseases such as asthma and the like by the following Chinese pulsatilla saponin B4 aerosol and the spraying agent so as to better serve the clinic.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Detailed Description
The present invention is further described in detail below with reference to examples to enable those skilled in the art to practice the invention with reference to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
It is to be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified.
Use of pulsatilla saponin B4 in the manufacture of a medicament for the treatment of asthma in a mammal, including a human.
The pharmaceutical preparation contains the pulsatilla saponin B4 with effective treatment amount, and the balance of pharmaceutically acceptable carriers and excipients which are nontoxic and inert to human and animals.
The pharmaceutically acceptable carrier or excipient is one or more selected from solid, semi-solid and liquid diluents, fillers and pharmaceutical adjuvants. The pharmaceutical preparation of the present invention is used in the form of a dose per unit body weight. The extract of the present invention can be administered to a patient in need of treatment by oral administration or injection. For oral administration, it can be made into tablet, sustained release tablet, controlled release tablet, capsule, dripping pill, pellet, suspension, emulsion, powder or granule (nanometer preparation), oral liquid, etc.; for injection, the composition can be made into sterilized aqueous or oily solution, sterile powder for injection, liposome or emulsion.
For example, when the compound aerosol is prepared, besides the proper propellant, the proper additive is selected mainly according to the physicochemical properties of the medicine to prepare the aerosol of a certain type so as to meet the requirements of clinical medication. The aerosol excipient mainly comprises a latent solvent and a propellant, wherein the latent solvent is glycerol, propylene glycol, ethanol, water or an ethanol-water mixed system, and the propellant mainly comprises trichloromonofluoromethane (F11), dichlorodifluoromethane (F12) and dichlorotetrafluoroethane (F114), or a mixture of any two of the trichloromonofluoromethane, the dichlorodifluoromethane and the dichlorotetrafluoroethane. In addition, the aerosol can be added with surfactant, cosolvent, suspending agent, antioxidant, etc. to optimize the aerosol prescription. The pulsatilla saponin B4 aerosol is mainly used for inhalation therapy and is prepared into solution type aerosol, so clarity and mouthfeel comfort are mainly considered in prescription design.
For example, when the spray is prepared, ethanol, glycerol, water, propylene glycol or a mixture thereof is selected as a solvent; the fatty acid sorbitan substance, the polysorbate substance, the polyoxyethylene fatty alcohol ether substance, the polyoxyethylene-polyoxypropylene copolymer, the polyethylene glycol substance, the cyclodextrin substance, the sodium dodecyl sulfate, the cetyl trimethyl ammonium bromide or the mixture thereof is taken as a solubilizer; sodium dodecyl sulfate, laurocapram, lauric acid, sodium laurate, lecithin, oleic acid, poloxamer, choline phosphate, etc. or a mixture thereof as absorption enhancer. The pulsatilla saponin B4 spray is mainly used for inhalation or spraying treatment and is prepared into a solution type spray, so clarity and comfortable mouthfeel are mainly considered in prescription design.
< example 1>
The preparation method of the pulsatilla saponin B4 aerosol comprises the following steps: collecting Pulsatillae saponin B4, 100g,60% medicinal ethanol 1000g 11 1000g, dissolving the pulsatilla chinensis saponin B4 in 60 percent ethanol under reflux, standing for 48 hours in a cold place, filtering,adding 60% ethanol to the filtrate to 1000mL, packaging into 200 bottles with 5mL each, and pressing propellant F 11 And (6) sealing the cover.
The aerosol prepared in example 1 is colorless, clear and transparent, has a pH of 7.52, has a total number of puffs per bottle of 500 times, has an average value of 10 puffs of 0.092mL per puff, accounts for 92% of the marked puffs, contains 9.81mg of the main drug per puff, mostly has a particle size of 5 μm or less, and is subjected to stability test by a constant temperature acceleration test method, wherein the aerosol can stably exist after being placed at a high temperature (40 ℃) for 10 days (the pH is changed from 7.52 to 7.48, and the content is changed from 9.1% to 9.6%), the anemonin B4 aerosol can stably exist after being placed at a low temperature (-18 ℃) for 10 days (the pH is changed from 7.52 to 7.48, and the content is changed from 9.1% to 9.6%), and only the content is reduced after being placed at a low temperature (4500 ± 500 Lx) for 10 days (the pH is changed from 7.52 to 7.56, and the content is changed from 10% to 9.7%, so that a proper device and the aerosol should be kept in a dark place.
< example 2>
The preparation method of the pulsatilla saponin B4 aerosol comprises the following steps: collecting pulegone saponin B4, 50g, borneol 5mg, vitamin C1g, medicinal ethanol 8g,60% ethanol 990g, F 12 1000g. Dissolving Borneolum in medicinal ethanol, slowly adding 60% ethanol into Borneolum solution, adding radix Pulsatillae saponin B4 and vitamin C, heating in water bath, stirring to dissolve, standing at cold place for 48 hr, filtering, adding 60% ethanol to 1000mL, packaging into 200 bottles with 5mL each, and pressing F 12 And (6) sealing the cover.
The aerosol prepared in example 2 is colorless, clear and transparent, has a pH of 7.12, has a total number of puffs per bottle of 500 times, has a volume of 0.095mL per puff, accounting for 95% of the indicated puffs, contains 4.73mg per puff, has a particle size mostly below 5 μm, and is subjected to stability test by a constant temperature acceleration test method, and is stable after being placed at a high temperature (40 ℃) for 10 days (the pH is changed from 7.12 to 7.09, and the content is changed from 4.94% to 4.95%), and stable after being placed at a low temperature (-18 ℃) for 10 days (the pH is changed from 7.12 to 7.13, and the content is changed from 4.91% to 4.92%), and is reduced only after being placed at a low temperature (4500 ± 500 Lx) for 10 days (the pH is changed from 7.12 to 7.19, and the content is changed from 5.0% to 4.75%), so that a suitable device and a light-shielding device are adopted and should be stored.
< example 3>
The preparation method of the pulsatilla saponin B4 aerosol comprises the following steps: taking 4, 10g of pulsatilla saponin B, 1g of sodium metabisulfite, 999g of water 12 :F 11 =3:7, 1000g of mixed propellant. Dissolving Pulsatillae radix saponin B4 and sodium pyrosulfite in water, heating to dissolve, cooling at room temperature, standing at cold place for 48h, filtering, adding 1000mL of water to the filtrate, packaging into 200 bottles, each bottle containing 5mL, pressing propellant, and sealing.
The aerosol prepared in example 3 is colorless, clear and transparent, has a pH of 7.14, has a total number of puffs per bottle of 500 times, has a volume of 0.098mL per puff, accounts for 98% of the labeled puff, contains 0.98mg per puff, has a particle size of mostly less than 5 μm, and is stably stored at a high temperature (40 ℃) for 10 days (the pH is changed from 7.14 to 7.09, and the content is changed from 0.98% to 0.99%), and is stably stored at a low temperature (-18 ℃) for 10 days (the pH is changed from 7.14 to 7.15, and the content is changed from 0.98% to 0.99%), and is reduced only after being stored at a low temperature (4500 ± 500 Lx) for 10 days (the pH is changed from 7.14 to 7.19, and the content is changed from 1.00% to 0.92%), and is stored in a suitable dark device.
< example 4>
The preparation method of the pulsatilla saponin B4 aerosol comprises the following steps: collecting Pulsatillae saponin B4, 40g, borneolum Syntheticum 20mg,80% ethanol 1000g 114 1200g. Dissolving Borneolum Syntheticum with 80% ethanol, adding pulsatilla saponin B4, stirring to dissolve completely, standing at cold place for 48 hr, filtering, adding 80% ethanol to 1000mL of filtrate, bottling in 200 bottles, each bottle is 5mL, and pressing F 114 And (6) sealing the cover.
The aerosol prepared in example 4 is colorless, clear and transparent, has a pH of 7.48, has a total number of puffs per bottle of 500 times, has a volume of 0.090mL per puff, accounting for 90% of the indicated puffs, contains 3.56mg of the main drug per puff, mostly has a particle size of 5 μm or less, and is subjected to stability test by a constant temperature acceleration test method, and is stable after being placed at a high temperature (40 ℃) for 10 days (the pH is changed from 7.48 to 7.43, and the content is changed from 3.95% to 3.96%), stable after being placed at a low temperature (-18 ℃) for 10 days (the pH is changed from 7.48 to 7.48, and the content is changed from 3.92% to 3.95%), and is reduced only after being placed at a low temperature (4500 ± 500 Lx) for 10 days (the pH is changed from 7.48 to 7.57, and the content is changed from 4.00% to 3.82%), and thus should be stored in a suitable device and protected from light.
< example 5>
The preparation method of the pulsatilla saponin B4 aerosol comprises the following steps: collecting Pulsatillae saponin B4, 80g,30% ethanol 1000g 12 1000g. Dissolving anemonin B4 with 30% ethanol, standing in cold place for 48h, filtering, adding 30% ethanol to 1000mL of filtrate, packaging into 200 bottles with 5mL each, and pressing F 12 And (6) sealing the cover.
The aerosol prepared in example 5 is colorless, clear and transparent, has a pH of 7.36, has a total number of puffs per bottle of 500 times, has an average value of 10 puffs of 0.091mL per puff of 91% of the labeled puff, contains 7.12mg of the main drug per puff, mostly has a particle size of 5 μm or less, and is subjected to stability test by a constant temperature acceleration test method, and is stable after being placed at a high temperature (40 ℃) for 10 days (the pH is changed from 7.36 to 7.32, and the content is changed from 7.98% to 7.94%), stable after being placed at a low temperature (-18 ℃) for 10 days (the pH is changed from 7.36 to 7.35, and the content is changed from 7.93% to 7.91%), and is reduced only after being placed at a low temperature (4500 ± 500 Lx) for 10 days (the pH is changed from 7.36 to 7.41, and the content is changed from 8.00% to 7.82%), so that a suitable light-shielding device is adopted and is kept away from the light.
< example 6>
The preparation method of the pulsatilla chinensis saponin B4 spray comprises the following steps: taking 4,5g of pulsatilla saponin B, 10mL of propylene glycol, 80,0.75mL of polysorbate, and adding 20% ethanol to 100mL. Adding the pulsatilla chinensis saponin B4 into polysorbate 80, stirring uniformly, adding 20% ethanol to a sufficient amount, stirring uniformly by a homogenizer, and quantitatively filling into a spray container to obtain a finished product. When in use, the spray is directly sprayed into the oral cavity, and 1 to 3 sprays are carried out at a time.
The spray prepared in example 6 is colorless, clear and transparent, has a pH of 7.02, has a total number of puffs per bottle of 500 times, has a spraying amount of 0.095mL per puff, accounting for 95% of the marked spraying amount, contains 9.75mg of the main drug per puff, mostly has a particle size of 5 μm or less, and is subjected to stability test by a constant temperature acceleration test method, and is reduced in content (the pH is changed from 7.02 to 7.06, and the content is changed from 5.0% to 4.96%) after being placed under illumination (4500 ± 500 Lx) for 10 days, and is stable (the pH is changed from 7.02 to 7.03, and the content is changed from 4.92% to 4.98%) after being placed under low temperature (-18 ℃) for 10 days, and is stable (the pH is changed from 7.02 to 7.00, and the content is changed from 4.91% to 4.84%) only under high temperature (40 ℃) conditions, so that the spray should be preserved at a proper temperature to avoid high temperature.
< example 7>
The preparation method of the pulsatilla saponin B4 spray comprises the following steps: taking 4,5g of pulsatilla saponin B, 15mL of absolute ethyl alcohol and 400 65mL of polyethylene glycol, and adding water to 100mL. Weighing the pulsatilla chinensis saponin B4 according to the proportion, dissolving the pulsatilla chinensis saponin B4 in absolute ethyl alcohol and polyethylene glycol 400, uniformly stirring, adding water to a sufficient amount, uniformly stirring by using a homogenizer, and quantitatively filling into a spray container to obtain a finished product. When in use, the spray is directly sprayed into the oral cavity, and 1 to 3 sprays are carried out at a time.
The spray prepared in example 7 is colorless, clear and transparent, has a pH of 7.13, has a total number of puffs per bottle of 500 times, has a spraying amount of 0.095mL per puff, accounting for 95% of the indicated spraying amount, contains 9.5mg of the main drug per puff, mostly has a particle size below 5 particles, and is subjected to stability test by a constant temperature acceleration test method, wherein the content of the spray is reduced after the spray is placed under illumination (4500 ± 500 Lx) for 10 days (the pH is changed from 7.13 to 7.17, and the content is changed from 5.0% to 4.98%), and the spray can be stably stored under low temperature (-18 ℃) for 10 days (the pH is changed from 7.13 to 7.14, and the content is changed from 4.92% to 4.96%), and the spray can be stably stored under appropriate temperature only after the spray is placed under high temperature (40 ℃) for 10 days (the pH is changed from 7.13 to 7.08, and the content is changed from 4.95% to 4.84%).
< example 8>
The preparation method of the pulsatilla saponin B4 spray comprises the following steps: taking 1g of pulsatilla chinensis saponin B4, 188 g of poloxamer and 35% ethanol, and adding the total volume to 100mL. Weighing the pulsatilla chinensis saponin B4 and the poloxamer according to the proportion, dissolving in 35% ethanol, uniformly stirring by using a homogenizer, and quantitatively filling into a spray container to obtain a finished product. When in use, the spray is directly sprayed into the oral cavity, and 1 to 5 sprays are carried out at a time.
The spray prepared in example 8 is colorless, clear and transparent, has a pH of 7.05, has a total number of puffs per bottle of 500 times, has a spraying amount of 0.095mL per puff, accounting for 95% of the marked spraying amount, contains 1.9mg of the main drug per puff, has a particle size mostly below 5 drugs, and is subjected to stability test by a constant temperature acceleration test method, and has a reduced content (a pH of 7.05 to 7.13 and a content of 100% to 96%) after being placed under illumination (4500 ± 500 Lx) for 10 days, and a stable presence (a pH of 7.05 to 7.12 and a content of 98% to 98%) after being placed under low temperature (-18 ℃) for 10 days (a pH of 7.05 to 7.11 and a content of 98% to 89%) and therefore should be stored at a proper temperature to avoid high temperature.
< example 9>
The preparation method of the pulsatilla saponin B4 spray comprises the following steps: adding 1g of pulsatilla chinensis saponin B4, 2g of sodium dodecyl sulfate and 50% ethanol to 100mL. Weighing the pulsatilla chinensis saponin B4 and the sodium dodecyl sulfate according to the proportion, dissolving in 50% ethanol, stirring uniformly by using a homogenizer, and quantitatively filling into a spray container to obtain a finished product. When in use, the spray is directly sprayed into the oral cavity, and 1 to 3 sprays are carried out at a time.
The spray prepared in example 9 is colorless, clear and transparent, has a pH of 7.18, has a total number of puffs per bottle of 500 times, has a puff volume of 0.095mL, accounting for 95% of the marked puff volume, contains 1.96mg of the main drug per puff, mostly has a particle size below 5 particles, and is subjected to stability test by a constant temperature acceleration test method, wherein the content of the spray is reduced after the spray is placed under illumination (4500 using the constant temperature acceleration test) for 10 days (the pH is changed from 7.18 to 7.14, and the content is changed from 100% to 98%), the spray can be stably stored after the spray is placed under a low temperature (-18 ℃) for 10 days (the pH is changed from 7.18 to 7.18, and the content is changed from 97% to 96%, and the spray can be stably stored under a high temperature (40 ℃) for 10 days (the pH is changed from 7.18 to 7.14, and the content is changed from 98% to 90%), so that the spray should be stored at a proper temperature to avoid high temperature.
< example 10>
The preparation method of the pulsatilla chinensis saponin B4 spray comprises the following steps: taking 1g of pulsatilla chinensis saponin B4,1g of hydroxypropyl-beta-cyclodextrin, 1g of sorbitan monostearate (Span 60), and adding 60% ethanol to 100mL. Weighing the pulsatilla chinensis saponin B4 and the hardy bergamot in the proportion, dissolving in 60% ethanol, stirring uniformly by a high-speed homogenizer, and quantitatively filling into a spray container to obtain a finished product. When in use, the spray is directly sprayed into the oral cavity, and 1 to 5 sprays are carried out at a time.
The spray prepared in example 10 is colorless, clear and transparent, has a pH of 7.16, has a total number of puffs per bottle of 500 times, has a puff volume of 0.095mL as an average of 10 puffs, accounts for 95% of the indicated puff volume, has a primary drug content of 1.96mg per puff, mostly has a particle size of 5 or less, and is subjected to stability test by a constant temperature acceleration test method, wherein the spray has a reduced content (the pH is changed from 7.16 to 7.19, and the content is changed from 100% to 96%) after being placed under illumination (4500 using the constant temperature acceleration test) for 10 days, and can be stably stored (the pH is changed from 7.16 to 7.19, and the content is changed from 95% to 94%) after being placed under a low temperature (-18 ℃) for 10 days (the pH is changed from 7.16 to 7.11, and the content is changed from 98% to 92%) so that the spray should be stored at a proper temperature to avoid high temperature.
1. Material
1.1 Experimental animals
The species are as follows: brown Norway rat, grade: SPF grade, number of pre-purchased animals and sex: 104, males. Age: 6-7 weeks of age at the time of purchase and 7-8 weeks of age at the start of the test. Weight: the weight average number of the bought product is 180-200g, and the weight average number of the produced product is 220-240g. The source is as follows: beijing Weitonglihua laboratory animal technology Co., ltd, production license number: SCXK (Jing) 2016-0011. Animal welfare: this organization was certified by AAALAC (the international committee for laboratory animal evaluation and management). The test has been approved by the IACUC (committee for the management and use of laboratory animals), approval serial No.: IACUC-A2019175-P002-01. And (3) treating the rest animals: after the experiment is finished, if the residual animals exist, the experimental animal management part is handed over.
1.2 drugs and reagents
Pulsatillae radix saponin B4 (Jiangxi Bencao Tian Gong Tech Co., ltd., 2019030101); ovalbumin (Ovalbumin, OVA, sigma Aldrich, SLBV 6597); aluminum hydroxide gel (aluminum hydroxide gel, invivoGen, 5594); pertussis Toxin (Pertussis Toxin, PTX, list Biological laboratories. Inc).
1.3 animal groups
Group design: experimental design 13 groups: a blank control group, a model control group, a pulsatilla saponin B4 (10 mg/kg) caudal vein group, a pulsatilla saponin B4 aerosol group (examples 1-5 groups), and a pulsatilla saponin B4 spray group (examples 6-10 groups). Animal number and sex: each group had 8 males. The grouping method comprises the following steps: groups were randomized according to rat body weight using the PRISTIMA version 7.2.0 data system.
1.4 Molding method
According to the earlier research result of the mechanism, the experiment is supposed to adopt 1 mL/OVA subcutaneous injection for split injection, simultaneously 0.5mL/0.1 mu g/mL PTX for intraperitoneal injection for sensitization (D1), and after 7 days, the same operation is carried out for intensifying sensitization (D8); after 7 days of the last sensitization, 0.1mg/mL OVA was injected via trachea for 2 times (D15, D22) to perform molding.
1.4.1 sensitization
Sensitization route: subcutaneous injection (OVA emulsion) and intraperitoneal injection (PTX); sensitization frequency: 2 times, D1 and D8 sensitization method: each group of rats was administered with OVA emulsion or physiological saline at a concentration corresponding to the subcutaneous injection (about 200. Mu.L each in 5-point injection, about 0.2 mL/point, subcutaneous injection in both feet, subcutaneous injection in both inguinal regions, and intraperitoneal injection) in Table 2, and then 0.1. Mu.g/mL of PTX or DPBS was administered in intraperitoneal injection (i.p.) at 0.5mL.
1.4.2OVA excitation
Excitation pathway: intratracheal instillation excitation frequency: d15 and D22. The excitation method comprises the following steps: after anesthetizing the rats with 2% -5% isoflurane each time at a similar time point, the rats were vertically fixed and administered by slowly dropping 0.25ml of 1mg/mL OVA or DPBS into the trachea with a syringe (smooth and near spherical needle tip) under the guidance of an optical fiber. The supine position is maintained until consciousness is completely restored and then the supine position is returned to the cage.
1.4.3 dose design and basis
According to the previous research result, the dosage of the pulsatilla saponin B4 tail vein group administration medicament in the test is 10mg/kg. Pulsatillae saponin B4 aerosol group (example 1-5 groups), pulsatillae saponin aerosol obtained according to the above formula, was administered 1mL each time, once a day. The pulsatilla saponin B4 spray group (example 6-10 groups) was administered twice a day at 1mL per time. The blank and model controls were given an equal volume of 0.9% sodium chloride solution. Frequency and period of administration: administration was started on the day of the second sensitization (before sensitization), 1 time/day, for 14 consecutive days. The Day of first sensitization was defined as Day 1 of the experiment, day 1 (D1).
1.4.4 statistics of data
Firstly, LEVENE test is adopted to carry out the homogeneity test of the variance, and when the variance Ji Shi (P is more than 0.05), single-factor analysis of variance (ANOVA) is adopted to carry out the statistical test; when the variance is not uniform (P is less than 0.05), statistical analysis is carried out by using Kruskal-Wallis H rank sum test (K-W method). When the single-factor variance analysis shows that the difference has statistical significance (P is less than or equal to 0.05), the LSD method is adopted for statistical analysis; when one-way anova showed no statistical significance for the difference (P > 0.05), the statistical analysis was complete. When the Kruskal-Wallis H rank sum test shows that the difference is statistically significant (P is less than or equal to 0.05), the Mann-Whitney U test (M-W method) is adopted to compare the difference among the groups; statistical analysis was concluded when Kruskal-Wallis H rank sum test showed no statistical significance for the difference (P > 0.05).
2. Results and discussion
2.1 Effect of Pulsatillae saponin B4 on levels of IL-6, TNF-alpha in lung tissue homogenates
Compared with a blank control group, the levels of IL-6 and TNF-in the lung tissues of rats in the model control group are obviously increased (P < 0.001); compared with the model control group, the levels of IL-6 and TNF-tissues in the lung tissues of rats in the tail vein group, the aerosol group and the spray group are obviously reduced (P <0.001 or P < 0.05), and the effects of the aerosol group and the spray group are better than those of the tail vein injection group, which is detailed in table 1.
TABLE 1
Figure BDA0002640017730000091
Figure BDA0002640017730000101
Comparing with model group, P <0.05, P <0.01, P <0.001
2.2 Effect of Pulsatillae saponin B4 on IL-6, TNF-Wash content in alveolar lavage fluid (BALF)
Compared with a blank control group, the level of the alveolar lavage fluid IL-6 and TNF-fluid of the rats in the model control group is obviously increased (P < 0.001); compared with the model control group, the levels of IL-6 and TNF-tissue in the lung tissues of rats in the tail vein group, the aerosol group and the spray group are obviously reduced (P is less than 0.001 or less than 0.05), and the effects of the aerosol group and the spray group are better than those of the tail vein injection group, which is detailed in table 2.
TABLE 2
Figure BDA0002640017730000102
Comparing with model group, P <0.05, P <0.01, P <0.001
2.3 influence of white head Weng Zaogan B4 on IL-6, TNF-glycoside content in serum
Compared with a blank control group, the serum IL-6 and TNF-mouse level of the rat of the model control group is obviously increased (P < 0.01); compared with the model control group, the levels of IL-6 and TNF-tissues in the lung tissues of rats in the tail vein group, the aerosol group and the spray group are obviously reduced (P <0.001 or P < 0.05), and the effects of the aerosol group and the spray group are better than those of the tail vein injection group, which is detailed in table 3.
TABLE 3
Figure BDA0002640017730000111
Note: comparison with model group, P <0.05, P <0.01, P <0.001
2.4 influence of Pulsatillae saponin B4 on blood general index
Compared with a blank control group, the level of blood WBC, NEUT and LYMPH of the rats in the model control group is obviously increased (P < 0.01); compared with the model control group, the levels of blood WBC, NEUT and LYMPH of the rats in the tail vein group, the aerosol group and the spray group are obviously reduced (P is less than 0.05 or P is less than 0.01), and the effects of the aerosol group and the spray group are better than those of the tail vein injection group, which is detailed in table 4.
TABLE 4
Figure BDA0002640017730000112
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Figure BDA0002640017730000121
3. Conclusion
The root cause of asthma is bronchitis, which belongs to nonspecific inflammation, and the nonspecific inflammation is mostly caused by infection and turns into asthma after long-term effective treatment. Furthermore, asthma is a chronic airway inflammation involving a variety of cells, particularly mast cells, eosinophils and T lymphocytes, and thus detection of the inflammatory factors IL-6, TNF-6 and WBC, LYMPH, NEUT levels is directly linked to asthma, and reduction of these indices can reflect therapeutic effects on asthma. Experimental results show that the anemoside B4 aerosol and the spray have the effect of treating asthma, and the action mechanism of the anemoside B4 aerosol and the spray can be related to the reduction of the levels of blood, alveolar lavage fluid and lung tissue inflammatory factors such as IL-6 and TNF-asthma and the reduction of the levels of White Blood Cells (WBC), lymphocytes (LYMPH) and neutral granulocytes (NEUT) in the blood. The pulmonioside B4 aerosol and spray have stronger treatment effect on rat bronchial asthma induced by OVA than pulmonioside B4 for injection, and the dosage of the pulmonioside B4 for injection is less than that of pulmonioside B4 for injection, which indicates that the pulmonioside B4 aerosol and spray have more remarkable asthma treatment effect than pulmonioside B4 for injection.
The number of apparatuses and the scale of the process described herein are intended to simplify the description of the present invention. Applications, modifications and variations of the present invention will be apparent to those skilled in the art.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable to various fields of endeavor for which the invention may be embodied with additional modifications as would be readily apparent to those skilled in the art, and the invention is therefore not limited to the details given herein and to the examples shown and described without departing from the generic concept as defined by the claims and their equivalents.

Claims (2)

1. Use of pulsatilla saponin B4 in the manufacture of a medicament for the treatment of asthma in a mammal, including a human, characterized in that the medicament is an aerosol or spray.
2. The use according to claim 1, wherein the medicament comprises a therapeutically effective amount of pasqueflower saponin B4 and a pharmaceutically acceptable carrier.
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