CN113499326A - Pulsatillae saponin B4 atomization inhalation preparation and application - Google Patents
Pulsatillae saponin B4 atomization inhalation preparation and application Download PDFInfo
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- CN113499326A CN113499326A CN202110882261.0A CN202110882261A CN113499326A CN 113499326 A CN113499326 A CN 113499326A CN 202110882261 A CN202110882261 A CN 202110882261A CN 113499326 A CN113499326 A CN 113499326A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/007—Pulmonary tract; Aromatherapy
- A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- 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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/71—Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/02—Inorganic compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
The invention discloses an atomized inhalation preparation of pulsatilla saponin B4, which comprises a B4 solution or a B4 nano preparation. B4 solution dissolving Pulsatillae saponin B4 in physiological saline, and B4 nanometer preparation comprises: preparing solution with concentration of 2.5-10mg/mL with ethanol of Pulsatillae saponin B4, placing in normal temperature environment to obtain B4 ethanol solution, preparing solution with mass fraction of 0.02% with Tween 80 with water, and refrigerating at 0-4 deg.C to obtain water phase solution; under the condition of ice salt bath, rapidly adding the B4 ethanol solution into the aqueous phase solution with the volume of 20-30 times of the ethanol solution, keeping the stirring speed of the aqueous phase solution at 900-2000r/min, and keeping the stirring time not shorter than 4min to separate out the drug nanocrystals. The invention can inhibit TNFR2, Foxp3 and Treg cells from playing a role by combining with TNFR2 protein and Foxp3 protein.
Description
Technical Field
The invention relates to the technical field of medicines. More specifically, the invention relates to an atomized inhalation preparation of pulsatilla saponin B4 and application thereof.
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. Pulsatillae saponin B4 is pentacyclic triterpenoid saponin extracted from radix Pulsatillae, and has antiinflammatory, antibacterial, immunity regulating, antioxidant, and antiviral effects. The determination item of the Chinese pulsatilla content in section one of 'Chinese pharmacopoeia' of 2015 edition is specified as follows: the content of Pulsatillae saponin B4 should not be less than 4.6%.
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.
Still another object of the present invention is to provide an aerosolized inhalation formulation of pasqueflower saponin B4 and its use, which can inhibit TNFR2, Foxp3 and Treg cells from acting by binding to TNFR2 protein and Foxp3 protein.
To achieve these objects and other advantages in accordance with the present invention, there is provided an aerosolized inhalation formulation of pasqueoside B4, comprising a B4 solution or a B4 nano-formulation.
Preferably, the preparation method of the B4 solution comprises the following steps: dissolving pulsatilla saponin B4 in physiological saline to reach the concentration of 5-25 mg/mL.
Preferably, the preparation method of the B4 nano preparation comprises the following steps:
preparing solution of Pulsatillae saponin B4 with ethanol at concentration of 2.5-10mg/mL, placing in normal temperature environment to obtain B4 ethanol solution, preparing solution of stabilizer with water at mass fraction of 0.01-0.03%, and refrigerating at 0-4 deg.C to obtain water phase solution;
under the condition of ice salt bath, rapidly adding the B4 ethanol solution into the aqueous phase solution with the volume of 20-30 times of the ethanol solution, keeping the stirring speed of the aqueous phase solution at 900-2000r/min, and keeping the stirring time not shorter than 4min to separate out the drug nanocrystals.
Preferably, the resulting drug nanocrystals have a particle size of no greater than 1000 nm.
Preferably, the ice salt bath conditions are-15 ℃ and the crushed ice is mixed with NaCl in a 4:1 weight ratio.
Preferably, the initial temperature of the aqueous phase solution is 3 ℃.
Preferably, the initial temperature of the ethanol solution of B4 is 20 ℃.
Preferably, the stirring speed of the aqueous phase solution is kept at 1200 r/min.
Preferably, the volume ratio of the B4 ethanol solution to the aqueous phase solution is 1: 30.
The pulsatilla chinensis saponin B4 atomized inhalation preparation is applied as TNFR2 and Foxp3 protein inhibitors.
The invention at least comprises the following beneficial effects:
firstly, the invention carries out in vitro experiments: firstly, a computer simulates the binding force of anemonin B4(B4) and TNFR 2; detecting the protective effect of B4 on cell death by using actinomycin D and TNF to induce WEHI-13var cells; ③ transferring the TNFR2 reporter gene plasmid into Jurkat cells, and detecting the influence of B4 on TNFR 2; separating T lymphocytes from C57BL/6 mouse lymph nodes, screening CD4+ Foxp3+ double positive cells and Treg cells by using a sorting flow cytometer, and detecting the influence of B4 on TGF-beta and IL-10 after treatment; detecting the killing effect of B4 on A549 cells; sixthly, the TNFR2 is transfected into A549 cells, and the influence of B4 on the TNFR2 is detected.
Second, in vivo experiments of the invention: the effect of B4 on treating C57BL/6 inoculated Liews Lung Cancer (LLC): LLC cells were inoculated subcutaneously into the right forelimb of C57BL/6 mice at a concentration of 2X 106 cells/mL, and treated with different concentrations of B4 in groups until the tumor volume reached around 100mm3, once a day for 2 weeks with tumor volume measured every 2 days. Spleen and tumor tissues were removed from the mice, and Treg cells were detected. Tumor tissue detection of Treg and TNFR2 protein expression; ② B4 treatment C57BL/6 inoculation B16F10 effect: B16F10 cells were injected into C57BL/6 mice via tail vein at a concentration of 2X 106 cells/mL and 100. mu.L cells were administered by nebulization at different concentrations of B4 2 times a day for 2 weeks, and then lung metastasis of B16F10 was observed. B16F10-luc cells were injected into the tail vein of C57BL/6 cells as described above, and the fluorescence intensity of mouse lung tissue was measured every three days using a mouse in vivo imager as described above.
Thirdly, the pulsatilla chinensis saponin B4 solution for aerosol inhalation and the nano preparation are selected as research objects, the preparation process and the preparation formula design of the pulsatilla chinensis saponin B4 nano preparation for aerosol inhalation are researched, the in vivo and in vitro anti-lung cancer effect evaluation is carried out on the pulsatilla chinensis saponin B4 nano preparation, and the pulsatilla chinensis saponin B4 nano preparation is combined with TNFR2 for aerosol inhalation administration, so that TNFR2 and Treg cells are inhibited to play a role, and the immunity is adjusted. Provides basis for clinical application of B4 nanometer preparation in aerosol inhalation for treating lung cancer. Compared with other dosage forms, the aerosol can directly and rapidly reach lung tissues and has specific treatment effect on the lung, wherein the B4 nanometer preparation has small diameter, so that the medicine particle size is smaller (the conventional aerosol medicine particle size is not more than 10 mu M), and the aerosol is better in absorption.
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.
Drawings
Fig. 1 shows the effect of the crystallization temperature on the results (n ═ 3);
FIG. 2 is a graph showing the effect of stirring time on test results;
figure 3 is the effect of aqueous phase starting solution temperature on the results (n-3);
fig. 4 shows the effect of the initial temperature of the ethanol solution of B4 (n ═ 3);
figure 5 is the effect of aqueous phase stirring speed on the results (n-3);
fig. 6 shows the effect of the rate of addition of B4 in ethanol (n-3);
FIG. 7 is a graph of the effect of B4 ethanol solution concentration;
FIG. 8 is a graph of the effect of B4 on the volume ratio of ethanol solution to aqueous solution;
FIG. 9 is a computer simulated Docking of B4 and TNFR2 receptor proteins;
FIG. 10 is a graph of the effect of B4 on WEHI-13var cells;
FIG. 11 is the effect of B4 on TNFR2 reporter plasmid;
figure 12 is an assessment of B4 effect on Treg cells;
FIG. 13 is a direct killing effect of B4 on A549 and H1299 cells;
FIG. 14 is a graph of the effect of B4 on A549 cells stably expressing TNFR 2-GFP;
FIG. 15 shows the effect of B4 NANO-formulation aerosol inhalation on the growth of LLC tumors in mice;
FIG. 16 shows the effect of B4 Nanometallant nebulization and inhalation on TNFR2 and Foxp3 protein expression in LLC tumor tissues of mice;
FIG. 17 is a graph of the effect of nebulized delivery of B4 nano formulation on melanoma B16F10 lung cancer metastasis;
FIG. 18 is a small animal imaging test of the effect of B4 aerosolized drug delivery on melanoma B16F10 lung cancer metastasis;
FIG. 19 shows the effect of B4 Nanometallant nebulization inhalation administration on mouse blood;
FIG. 20 is a HE stain showing the effect of B4 NanoTagent aerosol inhalation administration on mouse lung tissue (100X).
Detailed Description
The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.
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.
< example 1>
A method for preparing solution of Pulsatillae saponin B4 for aerosol inhalation comprises dissolving Pulsatillae saponin B4 in physiological saline to reach concentration of 5 mg/mL. The obtained medicine has particle size not greater than 10 μ M.
< example 2>
A method for preparing solution of Pulsatillae saponin B4 for aerosol inhalation comprises dissolving Pulsatillae saponin B4 in physiological saline to reach concentration of 25 mg/mL. The obtained medicine has particle size not greater than 10 μ M.
< example 3>
A method for preparing solution of Pulsatillae saponin B4 for aerosol inhalation comprises dissolving Pulsatillae saponin B4 in physiological saline to reach concentration of 15 mg/mL. The obtained medicine has particle size not greater than 10 μ M.
< example 4>
The preparation method of the pulsatilla chinensis saponin B4 nano preparation for atomization and inhalation comprises the following steps:
preparing solution with concentration of 2.5mg/mL of pulsatilla chinensis saponin B4 with ethanol, placing at normal temperature (20 ℃) to obtain B4 ethanol solution, preparing solution with mass fraction of 0.02% of Tween 80 with water, and placing at 3 ℃ for refrigeration to obtain water phase solution;
under the condition of ice salt bath (-15 ℃, crushed ice and NaCl are mixed in a weight ratio of 4: 1), quickly adding the B4 ethanol solution into 30 times of the volume of the aqueous phase solution, keeping the stirring speed of the aqueous phase solution at 1200r/min, and stirring for 4min to precipitate drug nanocrystals with the particle size of not more than 1000 nm.
< example 5>
The preparation method of the pulsatilla chinensis saponin B4 nano preparation for atomization and inhalation comprises the following steps:
preparing solution with concentration of 5mg/mL of pulsatilla chinensis saponin B4 with ethanol, placing at normal temperature (20 ℃) to obtain B4 ethanol solution, preparing solution with mass fraction of 0.02% of Tween 80 with water, and placing at 3 ℃ for refrigeration to obtain water phase solution;
under the condition of ice salt bath (-15 ℃, crushed ice and NaCl are mixed in a weight ratio of 4: 1), quickly adding the B4 ethanol solution into 30 times of the volume of the aqueous phase solution, keeping the stirring speed of the aqueous phase solution at 1200r/min, and stirring for 4min to precipitate drug nanocrystals with the particle size of not more than 1000 nm.
< example 6>
The preparation method of the pulsatilla chinensis saponin B4 nano preparation for atomization and inhalation comprises the following steps:
preparing 10mg/mL solution of Pulsatillae saponin B4 with ethanol, placing at normal temperature (20 deg.C) to obtain B4 ethanol solution, preparing 0.02% Tween 80 with water, and refrigerating at 3 deg.C to obtain water phase solution;
under the condition of ice salt bath (-15 ℃, crushed ice and NaCl are mixed in a weight ratio of 4: 1), quickly adding the B4 ethanol solution into 30 times of the volume of the aqueous phase solution, keeping the stirring speed of the aqueous phase solution at 1200r/min, and stirring for 4min to precipitate drug nanocrystals with the particle size of not more than 1000 nm.
< preparation Process and formulation design >
The average particle size of the drug particles is used as an index to carry out the research of the preparation process of the drug nanocrystals.
1.1 reagents and instruments
1.1.1 instruments
A Thermo sicentistic magnetic heating stirrer; BSA2245-CW type electronic analytical balance; LA-920Horiba laser particle size one.
1.1.2 reagents
Pulsatillae saponin B4 crude drug (purity > 98%, prepared in laboratory).
1.1.3 Experimental methods and results
The particle size of the drug crystal in the prepared liquid is measured by adopting an LA-920Horiba laser particle sizer, and the influence of crystallization temperature, stirring time, initial temperature of aqueous phase solution, initial temperature of B4 ethanol solution, stirring speed of aqueous phase, adding speed of liquid medicine, concentration of B4 ethanol solution, volume ratio of B4 ethanol solution to aqueous solution on test results is respectively considered under the condition that other conditions are ensured to be certain by taking the particle size as an index.
1.1.3.1 influence of crystallization temperature
The crystallization temperature is crucial to the formation of the nano-crystal, so the influence of the crystallization temperature on the test result is firstly investigated in the process parameter research. The crystallization temperatures were set to room temperature (20 ℃ C.), an ice-water bath (0 ℃ C.) and an ice-salt bath (-15 ℃ C.; crushed ice 80g was mixed with NaCl 20 g), and other test conditions were set as in examples 4 to 6. The experimental results are shown in FIG. 1. The average particle size of the drug is gradually reduced with the reduction of crystallization temperature, probably because the solubility of the drug is reduced with the reduction of temperature, the drug is easy to reach a saturated state, so that the nucleation rate of the drug is increased, and the growth of crystal nuclei can be inhibited at low temperature, so that smaller nano crystals are formed.
1.1.3.2 Effect of stirring time on test results
In order to determine the stirring time of the mixed ethanol solution and water phase solution of B4, the influence of the stirring time at three crystallization temperatures of room temperature, ice water bath and ice salt bath on the experimental result is explored, and other experimental conditions are set as in examples 4-6. The test results of four time points of 5min, 10 min, 15min and 20min are examined under the condition of room temperature and water bath, and the test results of three time points of 1min, 3 min and 4min are examined because the liquid medicine can be greatly frozen about 4min under the condition of ice salt bath. The results are shown in fig. 2, under the conditions of normal temperature and ice-water bath, when the stirring time is increased from 5min to 15min, the average particle size of the drug crystals is gradually reduced, and when the stirring time is increased again, the average particle size of the drug crystals is increased; under the condition of ice salt bath, when the stirring time is increased from 1min to 4min, the average grain diameter of the medicine crystal is gradually reduced. Therefore, under the ice salt bath condition, the ethanol solution of B4 was added to the aqueous solution and stirred for 4 min.
1.1.3.3 Effect of initial temperature of aqueous solution
The initial temperatures of the aqueous solutions were set to room temperature (20 ℃ C.) and 3 ℃ respectively, and the effects thereof on the experimental results were examined, and the other experimental conditions were set as in examples 4 to 6. The crystallization temperature was set to ice salt bath. As a result, as shown in FIG. 3, the initial temperature of the aqueous solution was 3 ℃ and the average particle size of the drug crystals was small.
1.1.3.4 Effect of the initial temperature of the ethanol solution of B4
The initial temperature of the ethanol solution of B4 was set to room temperature (20 ℃ C.) and 3 ℃ C, respectively, and the influence thereof on the experimental results was examined, and other experimental conditions were set as in examples 4 to 6. The crystallization temperature was set to ice salt bath. As a result, as shown in FIG. 4, when the initial temperature of the ethanol solution of B4 was 20 ℃, the average particle size of the drug crystals was small. Probably because when the normal-temperature B4 ethanol solution is added into the aqueous phase solution which is placed in the ice salt bath and has the temperature of 3 ℃, the temperature suddenly drops, the solubility of the drug is reduced, the drug is easy to form a saturated state to form a large number of crystal nuclei, and the low temperature and the stabilizing agent inhibit the growth of the crystal nuclei, thereby forming smaller nano crystals.
1.1.3.5 Effect of aqueous stirring speed
The water phase stirring speed was set at 600, 900, 1200, 2000r/min, and the effect thereof on the test results was examined, and other test conditions were set as in examples 4 to 6. The crystallization temperature in the test was set to be an ice salt bath. As shown in FIG. 5, when the stirring speed is increased from 600r/min to 1200r/min, the particle size of the drug is gradually decreased, and when the particle size is increased to 2000r/min, the particle size of the drug is not significantly decreased, the stirring speed may be increased, the mixing speed of the drug solution and the water phase is increased, the drug is more easily saturated to form a large number of crystal nuclei, and the influence is not obvious after the rotating speed is increased to a certain value.
1.1.3.6 Effect of the rate of addition of B4 ethanol solution
The addition rate of the ethanol solution of B4 was set to rapid addition and dropwise addition, and the effect thereof on the test results was examined, and other test conditions were set as in examples 4 to 6. The crystallization temperature in this experiment was ice salt bath. The experimental results are shown in FIG. 6, and the average particle size of the drug at the time of rapid addition is smaller than that at the time of dropwise addition. The reason may be that, when added rapidly, the local drug concentration is higher and the drug is more likely to reach a saturation state to form a large number of crystal nuclei.
1.1.3.7B 4 Effect of ethanol solution concentration
The concentrations of B4 in ethanol were set to 2.5, 10 and 20mg/mL, and the influence thereof on the test results was examined, and other test conditions were set as in examples 4 to 6. The crystallization temperature in this experiment was ice salt bath. As shown in FIG. 7, the reason why the average particle size of the drug is the smallest at a concentration of 10mg/mL in the ethanol solution of B4 is probably that the drug concentration is increased to make it easier to form saturated state and to form crystal nuclei, and that the drug concentration is too high to make the local crystal nuclei have too high concentration and to easily collide and aggregate to cause crystal growth. Therefore, the test explores that the nebulized administration concentration of the B4 nano preparation is determined to be 2.5, 5 and 10 mg/mL.
1.1.3.8B 4 Effect of the volume ratio of ethanol solution to aqueous solution
VB4 ethanol solution: aqueous phase solutionThe test conditions were set to 1:10, 1:20 and 1:30, and the effects thereof on the test results were examined, and the other test conditions were set as in examples 4 to 6. The crystallization temperature in this experiment was ice salt bath. As shown in fig. 8, the average particle size of the drug gradually decreased with increasing volume ratio of the ethanol solution to the aqueous solution, probably because the formed crystal nuclei are less likely to aggregate with each other with increasing volume of the aqueous phase.
1.1.4 nodules
According to the test result, the finally selected process parameters are that the ice salt bath is at the crystallization temperature, the concentration of the B4 ethanol solution is 2.5, 5 and 10mg/mL, the stirring speed is 1200r/min, the volume ratio of the B4 ethanol solution to the water solution is 1:30, and the ethanol solution is rapidly added. B4 ethanol solution is at normal temperature, and the water phase solution is refrigerated to 3 ℃. The stirring time was 4 min.
< evaluation of in vitro Effect >
2.1 Experimental methods
(ii) affinity assay of B4 with TNFR2 receptor: the binding mechanical parameters of B4 and TNFR2 were calculated in a previous autodock software simulation.
(iii) B4 action on mouse fibrosarcoma cells WEHI-13 var: WEHI-13var cells are cultured together with actinomycin D (1 mu g/mL), after B4(5, 10, 20 mu M) is preincubated for 2h, TNF-alpha (5IU/mL) is added to induce 24h, and the protection effect of B4 on TNF-induced WEHI-13var cell death is detected by an MTS method.
Effect of B4 on TNFR2 reporter plasmid: the TNFR2 gene fragment is introduced into a Vector by using a Promoter drive Control Firefoy and Renilla Luciferase Vector plasmid Vector pGL4.54[ luc2/TK ] Vector to form a reporter gene Vector plasmid. After introducing the plasmid into Jurkat cells, the cells were treated with B4(5, 10, 20. mu.M) for 24h, and the fluorescence intensity in the well plate was measured using the Dual Glo luciferase assay system kit.
Fourthly, evaluating the effect of B4 on the Treg cells: t lymphocytes are separated from C57BL/6 mouse lymph nodes, CD4+ Foxp3+ double positive cells and Treg cells are screened out by a sorting flow cytometer, and after the Treg cells are pretreated by adding B4(20 mu M) for 2 hours, TNF (10ng/mL) is added for induction for 24 hours. Collecting supernatant to detect IL-10 and TGF-beta.
Direct killing effect of B4 on A549 and H1299 cells: after non-small cell lung cancer cells A549 and H1299, B4(5, 10, 20 mu M) expressing TNFR2 receptor are selected to treat the cells for 24 hours, MTS detects the killing effect of B4 on the cells.
Sixthly, the effect of B4 on A549 cells stably expressing TNFR 2-GFP: A549-TNFR2-GFP cell is planted in a 96-hole blackboard, after B4(5, 10 and 20 mu M) is processed for 24 hours, a PE automatic work station JANUS G3 full-automatic processing Workstation and a PE Operetta CLS high-content cell imaging system are adopted to detect the fluorescence intensity.
2.2 results of the experiment
(ii) binding of B4 to TNFR2 receptor
Binding of B4 to TNFR2 was detected using Autodock software, and B4 was found to bind to TNFR2 receptor protein at-9.6 kcal/mol. As shown in FIG. 9, the specific binding amino acids are SER65, ASN171, GLY24, ARG77, and GLU 23.
② B4 action on WEHI-13var cells
WEHI-13var cells and actinomycin D (1 mu g/mL) are cultured together, after B4(5, 10, 20 mu M) is preincubated for 2h, TNF-alpha (5IU/mL) is added for induction for 24h, the result of MTS cell viability detection is shown in figure 10, and # P < 0.001 is compared with a blank control group; p < 0.001, protective effect of B4 on TNF-alpha induced WEHI-13var cell death compared to TNF-alpha (5IU/mL) group.
Effect of B4 on TNFR2 reporter gene plasmid
After TNFR2 plasmid was introduced into Jurkat cells, the cells were treated with B4(5, 10, 20. mu.M) for 24h, and the fluorescence intensity in the well plate was measured using the Dual Glo luciferase assay system kit, and as a result, B4 was able to suppress the fluorescence intensity of TNFR2 and had an inhibitory effect on TNFR2, as shown in FIG. 11.
(iv) evaluation of Treg cell Effect by B4
T lymphocytes are separated from C57BL/6 mouse lymph nodes, CD4+ Foxp3+ double positive cells and Treg cells are screened out by a sorting flow cytometer, and after the Treg cells are pretreated by adding B4(20 mu M) for 2 hours, TNF (10ng/mL) is added for induction for 24 hours. Collecting supernatant to detect IL-10 and TGF-beta levels, the results are shown in FIGS. 12A and 12B, FIG. 12A is the effect of B4 on IL-10 levels in the supernatant of Treg cells; FIG. 12B is a graph of the effect of B4 on TGF- β levels in the supernatant of Treg cells, ## P < 0.001 compared to the blank control; b4 was able to suppress IL-10 and TGF- β levels in the supernatant of Treg cells compared to the TNF- α (5IU/mL) group.
Direct killing effect of B4 on A549 and H1299 cells
After non-small cell lung cancer cells A549 and H1299 expressing TNFR2 receptor are selected and treated by B4(5, 10, 20 mu M) for 24 hours, MTS detects that B4 has no killing effect on the cells, and the results are shown in FIGS. 13A and 13B, wherein FIG. 13A shows that B4 has the killing effect on the A549 cells, FIG. 13B shows that B4 has the killing effect on the H1299 cells, and B4 possibly plays a role in treating the non-small cell lung cancer by regulating immunity and tumor microenvironment.
Sixthly, the effect of B4 on A549 cells stably expressing TNFR2-GFP
A549-TNFR2-GFP cell species was treated for 24h in a 96-well blackboard with B4(5, 10, 20. mu.M), and the TNFR2 fluorescence intensity was measured. As shown in figure 14, B4 was able to inhibit TNFR2 fluorescence intensity in a549 cells compared to the blank control.
< evaluation of in vivo Effect of preparation >
Aerosol inhalation administration aerosol administration was accomplished by exposing mice to an aerosol nebulizer (Shanghai Yuyan scientific instruments, Inc.) through the nose and mouth. The B4 nano-formulation was added to an aerosol nebulizer and the mice were treated. The method specifically comprises the following steps: placing B4 nanometer preparation solution in a beaker connected with an atomizer, placing the mouse in a fixer, exposing mouth and nose, placing the fixer on the atomizer, atomizing for administration for 15min, taking down the fixer, taking out the mouse, and finishing administration.
3.1 Experimental methods
The B4 nanometer preparation has the functions of treating Lewis Lung Cancer (LLC) inoculated by C57BL/6 through atomization and inhalation: LLC cells were cultured at 2X 106The mice are inoculated with 100 mu L of the suspension per mL to the subcutaneous right forelimb of the C57BL/6 mouse until the tumor volume is 100mm3On the left and right, the mice were divided into model groups, B42.5mg/kg, B45 mg/kg, B410 mg/kg and gefitinib-positive drug group (40mg/kg), and 10 mice were treated. The B4 nanometer preparation is inhaled by atomization 2 times per day for 2 weeks, the gefitinib group is administrated by gastric gavage once per day for 2 weeks, the model group is inhaled by normal saline with the same dose by atomization once per day for 2 weeks. Tumor volumes were measured every 2 days. Spleen is taken from the mouse body to detect Treg cells. Tumor tissues were taken to examine the expression of TNFR2 and Foxp3 proteins in tumor tissues.
② B4 nanometer preparation atomizing inhalation administration treatment C57BL/6 inoculation B16F10 lung cancer metastasis: B16F10 cells at 5X 106Cells at a concentration of 100. mu.L/mL were injected via tail vein into C57BL/6 miceEstablishing a melanoma lung cancer metastasis model in vivo. The mice are divided into model groups, B42.5mg/kg, B45 mg/kg, B410 mg/kg and gefitinib positive drug groups (40mg/kg) 4 days after model building, and 10 mice are treated in each group. The model group and each dose group of B4 nano-formulation were administered by nebulization 2 times a day for 2 weeks. The gefitinib group was administered by gavage once daily for 2 weeks. Observing the lung metastasis of the mice, detecting the levels of neutrophils, leukocytes and lymphocytes in the blood of the mice and HE staining of lung tissues. B16F10-luc cells were injected into C57BL/6 cells via tail vein as described above, and fluorescence intensity of mouse lung tissue was measured using a mouse in vivo imager 14 days after administration as described above.
3.2 results of the experiment
3.2.1B 4 nanometer medicinal preparation for treating Lewis Lung Cancer (LLC) inoculated with C57BL/6 by aerosol inhalation
The nanometer B4 preparation has the growth promoting effect on LLC tumor in mouse
14 days after administration of the mice, the tumors were weighed and the volume was measured after sacrifice of the mice. The results are shown in fig. 15A and 15B, where fig. 15A shows the effect of B4 on mouse tumor weight, and fig. 15B shows the effect of B4 on mouse tumor volume,. P < 0.05,. P < 0.01,. P < 0.001, compared to the model group, and B4 nano-formulation was administered by aerosol inhalation for 14 days, it was able to inhibit tumor weight and tumor volume of LLC, and its inhibitory effect was better than that of the positive control gefitinib.
② the influence of B4 nanometer preparation atomizing inhalation on TNFR2 and Foxp3 protein expression in mouse LLC tumor tissue
Mouse tumor tissues are ground, supernatants are taken for quantification, and Western blot is used for detecting the expression condition of TNFR2 and Foxp3 proteins in the tumor tissues. The results are shown in fig. 16, B4 was able to inhibit the expression of TNFR2 and Foxp3 proteins.
The results indicate that B4 can play a role in treating non-small cell lung cancer by inhibiting TNFR2 and regulating Treg cells.
3.2.2B 4 nanometer medicinal preparation for treating lung cancer metastasis by C57BL/6 inoculation of B16F10
Influence of B4 nanometer preparation atomization administration on melanoma B16F10 lung cancer metastasis
2.5mg/kg, B45 mg/kg and B410 mg/kg of B4 nano preparation are subjected to atomization administration for 14 days, and lung tissues of mice are taken, as shown in figure 17, compared with a blank control group, melanoma lung metastasis of a model group is obvious, B4 atomization administration can inhibit melanoma lung cancer metastasis conditions of B16F10, and the effect of B4 on lung cancer metastasis inhibition is better than that of a positive control medicament gefitinib group.
② small animal imaging detection of influence of B4 nanometer preparation atomizing administration on melanoma B16F10 lung cancer metastasis
B16F10-luc cells at 5X 106100 mu L of cells with the concentration of 100 mu L/mL are injected into a C57BL/6 mouse through tail vein to establish a melanoma lung cancer metastasis model. After the atomization administration of the mouse, the fluorescence of the lung metastatic cells is detected by using a small animal imager. As shown in fig. 18, B4 nano-formulations inhibited melanoma lung metastasis after nebulization.
③ B4 the effect of the nanometer preparation atomizing inhalation administration on the routine blood of mice
Day15, mice were bled from their eyes and blood routine instrumentation was used to measure the levels of White Blood Cells (WBC), Neutrophils (NEU) and Lymphocytes (LYMPH) in the plasma of the mice. Experimental results as shown in fig. 19, a. WBC levels in plasma; B. NEU levels in plasma; C. plasma LYMPH levels P < 0.05, P < 0.01, P < 0.001vs model group, in fig. 19A-19C, from left to right: in fig. 19A, WBC levels in the model group were significantly elevated compared to the blank group, and the low and medium doses administered by B4 aerosol inhalation significantly inhibited WBC levels; in fig. 19B, NEU was significantly elevated in plasma of model mice compared to the blank group, and low, medium, and high doses of B4 aerosolized for inhalation and gefitinib positive drug were able to significantly inhibit NEU levels; in fig. 19C, the model group exhibited a decrease in LYMPH levels compared to the blank group, and the B4 nebulized inhalation administered high dose group exhibited a significant increase in LYMPH levels. The above results suggest that nebulized inhalation administration of B4 nano-formulations may be used to inhibit lung cancer metastasis by modulating mouse WBC, NEU, and LYMPH levels.
(B4) influence of aerosol inhalation administration of nano preparation on mouse lung tissue
HE staining further detects the effect of B4 nano-formulation aerosol inhalation administration on melanoma lung metastasis, and the results are shown in fig. 20, wherein the lung tissue nodules are significant in the model group, while the lung tissue nodules can be significantly inhibited by the B4 nano-formulation low, medium and high dose groups and the gefitinib group. The B4 nano preparation is proved to have obvious inhibition effect on melanoma lung metastasis by atomization and inhalation, and the effect is more obvious than that of gefitinib positive medicine.
The experimental results show that the B4 nanometer preparation can regulate the WBC, NEU and LYMPH levels in the blood of a B16-F10 melanoma mouse and inhibit the lung metastasis of the melanoma. And the low-dose effect of B4 is more obvious than that of gefitinib positive drug in preventing and treating melanoma lung metastasis.
The above results suggest that B4 can inhibit TNFR2 levels by binding TNFR2, reduce Treg cell numbers, and act as a TNFR2 small molecule inhibitor to treat lung cancer with TNFR2 and Foxp3 as targets. In addition, the B4 nano preparation can inhibit the lung cancer metastasis of mouse melanoma by regulating the immune cell level through atomization administration, and the B4 nano preparation has more remarkable effect on inhibiting the lung cancer metastasis of non-small cell lung cancer than a gefitinib-positive medicament.
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 in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
Claims (10)
1. The pulsatilla saponin B4 atomization inhalation preparation is characterized by comprising a B4 solution or a B4 nano preparation.
2. The pulsatillae radix saponin B4 aerosol inhalation formulation of claim 1, wherein the B4 solution is prepared by: dissolving pulsatilla saponin B4 in physiological saline to reach the concentration of 5-25 mg/mL.
3. The pulsatillae radix saponin B4 aerosol inhalation preparation of claim 1, wherein the B4 nano preparation is prepared by the following steps:
preparing solution of Pulsatillae saponin B4 with ethanol at concentration of 2.5-10mg/mL, placing in normal temperature environment to obtain B4 ethanol solution, preparing solution of stabilizer with water at mass fraction of 0.01-0.03%, and refrigerating at 0-4 deg.C to obtain water phase solution;
under the condition of ice salt bath, rapidly adding the B4 ethanol solution into the aqueous phase solution with the volume of 20-30 times of the ethanol solution, keeping the stirring speed of the aqueous phase solution at 900-2000r/min, and keeping the stirring time not shorter than 4min to separate out the drug nanocrystals.
4. The pulsatillae radix saponin B4 aerosol inhalation formulation of claim 3, wherein the obtained drug nanocrystals have a particle size of no more than 1000 nm.
5. The pulsatillae radix saponin B4 aerosol inhalation formulation of claim 3, wherein the ice salt bath condition is-15 ℃ and the crushed ice is mixed with NaCl in a weight ratio of 4: 1.
6. Pulsatillae saponin B4 aerosol inhalation formulation according to claim 3, wherein the initial temperature of the aqueous phase solution is 3 ℃.
7. The pulsatillae radix saponin B4 aerosol inhalation formulation of claim 3, wherein the initial temperature of the ethanol solution of B4 is 20 ℃.
8. The pulmonioside B4 aerosol inhalation formulation of claim 3, wherein the stirring speed of the aqueous solution is maintained at 1200 r/min.
9. The pulsatilla saponin B4 aerosol inhalation formulation according to claim 3, wherein the volume ratio of the ethanol solution B4 to the aqueous solution is 1: 30.
10. The use of pulsatillae radix saponin B4 aerosolized inhalation formulation according to any one of claims 3 to 9 as TNFR2 and Foxp3 protein inhibitor.
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| CN108938654A (en) * | 2017-07-07 | 2018-12-07 | 刘琦 | A kind of injection preparation of anemoside B4 |
| CN111249291A (en) * | 2020-03-19 | 2020-06-09 | 广西馨海药业科技有限公司 | Application of pulsatilla saponin B4 in preparation of medicine for treating/preventing bacterial pneumonia |
| CN111265535A (en) * | 2020-03-19 | 2020-06-12 | 广西馨海药业科技有限公司 | Application of pulsatilla chinensis saponin B4 in preparation of medicine for treating/preventing pulmonary fibrosis |
| CN111904970A (en) * | 2020-08-19 | 2020-11-10 | 广西馨海药业科技有限公司 | Application of pulsatilla chinensis saponin B4 in preparation of medicine for treating asthma |
| CN112315934A (en) * | 2020-10-27 | 2021-02-05 | 广西馨海药业科技有限公司 | Preparation process of pulsatilla chinensis saponin B4 enteric-coated tablet |
-
2021
- 2021-08-02 CN CN202110882261.0A patent/CN113499326B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108938654A (en) * | 2017-07-07 | 2018-12-07 | 刘琦 | A kind of injection preparation of anemoside B4 |
| CN111249291A (en) * | 2020-03-19 | 2020-06-09 | 广西馨海药业科技有限公司 | Application of pulsatilla saponin B4 in preparation of medicine for treating/preventing bacterial pneumonia |
| CN111265535A (en) * | 2020-03-19 | 2020-06-12 | 广西馨海药业科技有限公司 | Application of pulsatilla chinensis saponin B4 in preparation of medicine for treating/preventing pulmonary fibrosis |
| CN111904970A (en) * | 2020-08-19 | 2020-11-10 | 广西馨海药业科技有限公司 | Application of pulsatilla chinensis saponin B4 in preparation of medicine for treating asthma |
| CN112315934A (en) * | 2020-10-27 | 2021-02-05 | 广西馨海药业科技有限公司 | Preparation process of pulsatilla chinensis saponin B4 enteric-coated tablet |
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| Title |
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| HE LUAN等: "Anemoside B4 attenuates nephrotoxicity of cisplatin without reducing anti-tumor activity of cisplatin", 《PHYTOMEDICINE》 * |
| 狄孟华: "白头翁皂苷B4雾化吸入制剂的研究", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 * |
| 袁进等: "白头翁皂苷B4药理作用研究进展", 《中成药》 * |
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| CN113499326B (en) | 2022-07-01 |
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