Patent Application
20240261359
The invention belongs to the field of traditional Chinese medicines, and it relates to a traditional Chinese medicine composition for preventing or treating respiratory diseases in winter and a formulation thereof, in particular a tea formulation. The invention further relates to a method of using said pharmaceutical composition and use thereof in the manufacture of medicaments.
Winter is a season when respiratory diseases frequently occur, and pharyngitis is one common symptom of respiratory diseases. The pharyngitis has the characteristics of repeated attack, stubborn symptoms, prolonged disease course and the like, which produce great adverse impacts on daily life, study and work of patients and causes great trauma to physical and mental health of the patients. In clinic, the treatment of pharyngitis is mainly based on the use of antiviral medicaments and antibacterial medicaments, whereas sometimes, they cannot achieve satisfactory therapeutic effects and some serious side effects are also involved. For the treatment of the pharyngitis, traditional Chinese medicines in China more focus on starting from the overall of diseases and emphasizing dialectical treatments, and the achieved therapeutic effects are often more satisfactory whilst fewer side effects are produced. However, traditional Chinese medicine formulations currently applied in clinic are relatively limited, and no traditional Chinese medicine formulation for preventing or treating pharyngitis in winter, which can be conveniently used and achieve significantly effects, is reported.
Herb tea, i.e., a tea formulation, refers to an oral formulation prepared by mixing decoction pieces or extracts with tea leaves or other adjuvants, and it may be classified into a block tea formulation, a bagged tea formulation and a decocted tea formulation, being an important member of the traditional Chinese medicinal treasury. The bagged tea formulation refers to a tea formulation prepared by charging into bags tea leaves, coarse decoction piece powder or a part of coarse decoction piece powder which absorb extractive solutions and then dried, wherein those charged into tea bags for drinking are also referred as tea bag formulations. The tea bag formulation has the advantages of large doses, small volumes, convenient taking and quick dissolution, and it reserves the advantages and characteristics of traditional decoctions in a maximum level and more conform to public habits and customs. The formulation is increasingly developed into one of popular traditional Chinese medicinal dosages. In winter, taking traditional Chinese medicine tea formulations can achieve the beneficial effects of early intervention and early prevention.
Therefore, there is a need that is still not met for the means of preventing or treating respiratory diseases, e.g., pharyngitis, in winter, improving life qualities of patients, and/or preventing recurrences of the pharyngitis
It is one object of the invention is to provide a traditional Chinese medicine composition and a traditional Chinese medicine formulation for preventing and/or treating respiratory diseases in winter.
It is another object of the present invention is to provide use of traditional Chinese herbal medicines in the manufacture of a traditional Chinese medicine composition or a traditional Chinese medicine formulation for preventing and/or treating respiratory diseases in winter.
It is a further object of the present invention to provide a method for preventing and/or treating respiratory diseases in winter, comprising administering an effective amount of a traditional Chinese medicine composition or a traditional Chinese medicine formulation to a subject in need.
The inventors, through intensive research and creative work, obtain a pharmaceutical composition. The inventors surprisingly find that the pharmaceutical composition can nourish yin and relieve sore throat, and have the efficacies of nourishing Qi, moistening lungs, clearing throats, relieving sore throats, and decontaminating, and it can effectively treat and/or prevent respiratory diseases in winter such as pharyngitis and the like, and effectively eliminate phlegm and/or relieve cough. On this basis, the inventors have finished the invention.
The invention may be described in various aspects, and the inventions described in these aspects and any forms thereof are, independently but also interrelatedly, combined with each other to form the subject matter of the invention.
In one aspect, the present invention provides a traditional Chinese medicine composition, a traditional Chinese medicine formulation or a tea bag formulation for treating and/or preventing respiratory diseases in winter.
The traditional Chinese medicine composition of the invention is composed of or prepared with the following traditional Chinese medicinal materials in medicinal forms:
The traditional Chinese medicine composition of the invention may be composed of or prepared with a mixture of decoction piece powders of the following traditional Chinese medicinal materials or a powder of a decoction piece mixture of the following traditional Chinese medicinal materials:
The traditional Chinese medicine composition of the invention may be also composed of the individual recipe granules of the following traditional Chinese medicinal materials:
The traditional Chinese medicine composition of the invention may be further composed of decoction piece extracts of the following traditional Chinese medicinal materials:
The traditional Chinese medicine formulation of the invention comprises the traditional Chinese medicine composition of the invention, and it may further comprise pharmaceutically acceptable adjuvants. In other words, the traditional Chinese medicine formulation of the invention comprises the following traditional Chinese medicinal materials or extracts thereof in medicinal forms:
The traditional Chinese medicine formulation of the invention may be formulated into an oral liquid or a decoction, comprising water extracts of the following traditional Chinese medicinal materials:
The traditional Chinese medicine formulation of the invention can be formulated into a tea bag formulation, comprising decoction piece powders of the following traditional Chinese medicinal materials:
The traditional Chinese medicine formulation of the invention may be formulated into granules, comprising decoction piece extracts of the following traditional Chinese medicinal materials:
The traditional Chinese medicine formulation of the invention may be formulated into pills, comprising decoction piece extracts of the following traditional Chinese medicinal materials:
The traditional Chinese medicine formulation of the invention may be formulated into capsules, comprising decoction piece extracts of the following traditional Chinese medicinal materials:
The traditional Chinese medicine formulation of the invention may be formulated into syrups, comprising decoction piece extracts of the following traditional Chinese medicinal materials:
The traditional Chinese medicine formulation of the invention may be formulated into ointments, comprising decoction piece extracts of the following traditional Chinese medicinal materials:
The tea bag formulation of the invention comprises decoction piece powders of the following traditional Chinese medicinal materials:
In the other aspect, the application provides use of traditional Chinese herbs in the manufacture of a traditional Chinese medicine composition, a traditional Chinese medicine formulation or a tea bag formulation for preventing and treating respiratory diseases in winter, the preventing and treating respiratory diseases including one, more or all of (1) nourishing Qi, moistening lungs, clearing throats, relieving sore throats, and/or decontaminating, (2) treating and preventing pharyngitis; (3) eliminating phlegm and (4) relieving cough, wherein the traditional Chinese medicine composition, the raditional Chinese medicine formulation or the tea bag formulation has the above-defined composition.
In a further aspect, the present invention provides a method for preventing and/or treating respiratory diseases in winter, comprising administering an effective amount of the traditional Chinese medicine composition, the traditional Chinese medicine formulation or the tea bag formulation of the invention to a subject in need, the preventing and/or treating respiratory diseases including one, more or all of (1) nourishing Qi, moistening lungs, clearing throats, relieving sore throats, and/or decontaminating, (2) treating and preventing pharyngitis; (3) eliminating phlegm and (4) relieving cough, and wherein the traditional Chinese medicine composition, the traditional Chinese medicine formulation or the tea bag formulation has the above-defined composition.
The traditional Chinese medicine composition, the traditional Chinese medicine formulation or the tea bag formulation of the invention can be used to produce technical effects as described in one, more or all of the following items (1) to (5):
The previous text has summarized the invention in general aspects, and further, the invention will be described below in details by combining examples.
To accurately appreciate the terms used in the invention, the meanings of a part of the terms are specifically defined below. For the terms that are not specifically defined here, they have meanings that are commonly appreciated and accepted by those skilled in the art. If the meanings of some term defined here are not consistent with the meanings that are commonly appreciated and accepted by those skilled in the art, the meaning of the term should be based on the meanings as defined here.
The term “Arctii Fructus” used in the invention refers to dried ripe fruits of compositae, Arctium lappa L.
The term “Belamcandae Rhizoma” used in the invention refers to dried rootstalk of Iride, Belamcanda chinensis (L.) DC.
The term “Platycodonis Radix” used in the invention refers to dried roots of Campanulaceae, Platycodon grandiflorum (Jacq.) A. DC.
The term “Paeoniae Radix Rubra” used in the invention refers to dried roots of Ranunculaceae, Paeonia lactiflora Pall or Paeonia veitchii Lynch.
The term “Perillae folium” used in the invention refers to dried leaves (or with shoots) of Labiate, Perilla frutescens (L.) britt.
The term “Lonicerae Japonicae Flos” used in the invention refers to dried buds or buds with first bloomed flowers of Caprifoliaceae, Lonicera japonica Thunb.
The term “charred hawthorn” used in the invention refers to hawthorn products with scorched brown surfaces and yellow brown interiors prepared by processing hawthorns according to a single stir-drying method (General Rule 0213) recorded in Pharmacopoeia of the People's Republic of China. The “hawthorn” refers to dried ripe fruits of Rosaceae, Crataegus pinnattifolia Bge. var. major N.E.Br, or Crataegus pinnattifolia Bge.
The term “Astragali Radix” used in the invention refers to dried roots of Leguminosae, Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge.
The term “Polygoni Cuspidati Rhizoma et Radix” used in the invention refers to dried rootstalk and roots of Polygonum, cuspidatum Sieb. et Zucc.
The term “Glycyrrhizae Radix et Rhizoma” used in the invention refers to dried roots and rootstalk of Leguminosae, Glycyrrhiza uralensis Fisch, Glycyrrhiza inflata Bat, or Glycyrrhiza glabra L.
The term “fermented tea” used in the invention refers to tea articles obtained by fermenting tea leaves, such as red tea, dark tea and oolong tea, and the “tea leaves” refer to leaves of Camellia sinensis.
The term “decoction piece powder of traditional Chinese medicine materials” used in the invention refers to an ingredient-full powder of traditional Chinese medicinal materials free of additives, which is processed by physical methods, such as coarse pulverizing, fine pulverizing, micronizing, physical wall-breaking pulverizing and the like.
The term “recipe granules” used in the invention refers to a single traditional Chinese medicine product prepared by extracting, concentrating, drying and other processes of traditional Chinese medicine decoction pieces according to a traditional decocting method of traditional Chinese medicine decoctions. The product retains the nature, taste and efficacies of traditional Chinese medicinal decoction pieces and has a stable and reliable quality, it may be applied in the prescription deployment of clinical diagnoses in traditional Chinese medical science and suited to requirements of treatments based on syndrome differentiations and prescription changes, and it has the advantages of no decoction and convenient taking.
The term “extract” used in the invention refers to a substance obtained by extracting said traditional Chinese medicinal materials in any forms (including traditional Chinese medicinal decoction pieces, traditional Chinese medicinal powder, such as micronized traditional Chinese medicinal powder) with a suitable pharmaceutically acceptable solvent such as water or an alcoholic aqueous solution, including specific effective ingredients and mixtures containing effective ingredients. The substance is suitable for rescues of patients in the decubation of severe diseases, e.g., Coronary Pneumonia, effectively preventing, treating or relieving sequelae or complications of infectious diseases or promoting function recoveries of damaged tissues, organs or systems caused by infectious diseases. Forms of the extract include, but are not limited to, solids, semisolids, solutions, suspensions, concentrates, ointments, and powders. Suitable water used in the invention for extracting traditional Chinese medicinal materials include various water which can be used for preparing traditional Chinese medicinal active extracts, e.g., medicinal water such as distilled water and deionized water.
The term “alcoholic aqueous solution” used in the invention refers to alcohol-containing aqueous solutions with suitable concentrations (e.g., low concentrations, particularly 10-50% v/v). Suitable alcohols include various alcohols, preferably ethanol. Under some conditions, an alcoholic aqueous solution with a concentration higher than 50% v/v can be also used.
The terms “patient” and “subject” used in the invention, interchangeably with each other, refer to mammals, particularly human being, suffering from respiratory diseases, e.g., upper respiratory diseases such as pharyngitis, laryngitis or pharyngolaryngitis.
The term “pharmaceutically acceptable adjuvants” used in the invention refers to any adjuvants conventionally used in the field of pharmaceutical formulations, as long as the adjuvants will not produce adverse reactions or impacts on intended qualities and therapeutic effects of the traditional Chinese medicine composition of the invention.
The term “acceptable additives in foods or beverages” used in the invention refers to any additives that can be added to food or beverages according to national laws and regulations and departmental regulations, such as flavoring agents, sweetening agents, materials used in both medicines and food, and the like.
The term “effective amount” used in the invention refers to an amount of the pharmaceutical composition of the invention that can prevent or treat said respiratory diseases or alleviate and/or relieve one or more symptoms of said respiratory diseases in a subject.
All numerical ranges disclosed in the application include the endpoints thereof and any sub-ranges therein that are not expressly listed.
The invention provides a pharmaceutical composition for preventing and/or treating respiratory diseases in winter, composed of the following traditional Chinese medicinal materials or extracts thereof in medicinal forms by weight parts:
In some embodiments, the pharmaceutical composition is composed of the following traditional Chinese medicinal materials or extracts thereof in medicinal forms by weight parts:
In specific embodiments, the pharmaceutical composition is composed of the following traditional Chinese medicinal materials or extracts thereof in medicinal forms by weight parts:
In specific embodiments, the pharmaceutical composition is composed of the following traditional Chinese medicinal materials or extracts thereof in medicinal forms by weight parts:
In specific embodiments, the pharmaceutical composition is composed of the following traditional Chinese medicinal materials or extracts thereof in medicinal forms by weight parts:
In specific embodiments, the pharmaceutical composition is composed of the following traditional Chinese medicinal materials or extracts thereof in medicinal forms by weight parts:
In preferred embodiments, the pharmaceutical composition is in a single dose form composed of the following traditional Chinese medicinal materials or extracts thereof in medicinal forms by weight parts:
In preferred embodiments, the pharmaceutical composition is in a single dose form composed of the following traditional Chinese medicinal materials or extracts thereof in medicinal forms by weight parts:
In preferred embodiments, the pharmaceutical composition is in a single dose form composed of the following traditional Chinese medicinal materials or extracts thereof in medicinal forms by weight parts:
In preferred embodiments, the pharmaceutical composition is in a single dose form composed of the following traditional Chinese medicinal materials or extracts thereof in medicinal forms by weight parts:
In some embodiments, the traditional Chinese medicinal materials used in the pharmaceutical composition are processed traditional Chinese medicinal materials, and for example, the Arctii Fructus is stir-fried Arctii Fructus.
In some embodiments, the traditional Chinese medicinal materials used in the pharmaceutical composition are pulverized; preferably, the pulverized medicinal powder passes through Sieve No. 1; more preferably, the pulverized powder passes through the Sieve No. 1 but not pass through Sieve No. 5.
In some embodiments, the water content of the pharmaceutical composition is less than 12%; preferably, less than or equal to 10%, or less than or equal to 8%; more preferably, less than or equal to 6%, or less than or equal to 5%.
The invention also provides a traditional Chinese medicine formulation for preventing and/or treating respiratory diseases in winter, comprising, by weight parts, the following traditional Chinese medicinal materials or extracts thereof in medicinal forms and pharmaceutically acceptable adjuvants:
In some embodiments, the traditional Chinese medicine formulation comprises, by weight parts, the following traditional Chinese medicinal materials or extracts thereof in medicinal forms and pharmaceutically acceptable adjuvants:
In some embodiments, the traditional Chinese medicine formulation comprises, by weight parts, the following traditional Chinese medicinal materials or extracts thereof in medicinal forms and pharmaceutically acceptable adjuvants:
In some embodiments, the traditional Chinese medicine formulation comprises, by weight parts, the following traditional Chinese medicinal materials or extracts thereof in medicinal forms and pharmaceutically acceptable adjuvants:
In some embodiments, the traditional Chinese medicine formulation comprises, by weight parts, the following traditional Chinese medicinal materials or extracts thereof in medicinal forms and pharmaceutically acceptable adjuvants:
In some embodiments, the traditional Chinese medicine formulation comprises, by weight parts, the following traditional Chinese medicinal materials or extracts thereof in medicinal forms and pharmaceutically acceptable adjuvants:
In preferred embodiments, the pharmaceutical composition is in a single dose form comprising the following traditional Chinese medicinal materials or extracts thereof in medicinal forms by weight parts:
In preferred embodiments, the pharmaceutical composition is in a single dose form comprising the following traditional Chinese medicinal materials or extracts thereof in medicinal forms by weight parts:
In preferred embodiments, the pharmaceutical composition is in a single dose form comprising the following traditional Chinese medicinal materials or extracts thereof in medicinal forms by weight parts:
In preferred embodiments, the pharmaceutical composition is in a single dose form comprising the following traditional Chinese medicinal materials or extracts thereof in medicinal forms by weight parts:
In some embodiments, the pharmaceutical composition is formulated in a single dose form, comprising 2-10 g, preferably 3-6 g, 3-5 g, 3-4.5 g, 4-6 g, or 4-5 g, more preferably 3 g or 4.5 g of the pharmaceutical composition of any of the above embodiments.
In some embodiments, by per gram of the pharmaceutical composition, in the traditional Chinese medicine formulation:
In some embodiments, by 4.5 grams of the pharmaceutical composition, in the traditional Chinese medicine formulation:
In some embodiments, the content of arctiin is detected by high performance liquid chromatography, and the chromatographic conditions are shown below:
The invention also provides a traditional Chinese medicinal oral liquid for preventing and/or treating respiratory diseases in winter, comprising, by weight parts, extraction solutions, preferably water extraction solutions, of the following traditional Chinese medicinal decoction pieces, and pharmaceutically acceptable adjuvants:
In some embodiments, the traditional Chinese medicinal oral liquid comprises, by weight parts, extraction solutions, preferably water extraction solutions, of the following traditional Chinese medicinal decoction pieces, and pharmaceutically acceptable adjuvants:
In some embodiments, the traditional Chinese medicinal oral liquid comprises, by weight parts, extraction solutions, preferably water extraction solutions, of the following traditional Chinese medicinal decoction pieces, and pharmaceutically acceptable adjuvants:
In some embodiments, the traditional Chinese medicinal oral liquid comprises, by weight parts, extraction solutions, preferably water extraction solutions, of the following traditional Chinese medicinal decoction pieces, and pharmaceutically acceptable adjuvants:
In some embodiments, the traditional Chinese medicinal oral liquid comprises, by weight parts, extraction solutions, preferably water extraction solutions, of the following traditional Chinese medicinal decoction pieces, and pharmaceutically acceptable adjuvants:
In some embodiments, the traditional Chinese medicinal oral liquid comprises, by weight parts, extraction solutions, preferably water extraction solutions, of the following traditional Chinese medicinal decoction pieces, and pharmaceutically acceptable adjuvants:
In preferred embodiments, the traditional Chinese medicinal oral liquid is in a single dose form comprising, by weight parts, extraction solutions, preferably water extraction solutions, of the following traditional Chinese medicinal decoction pieces, and pharmaceutically acceptable adjuvants:
In preferred embodiments, the traditional Chinese medicinal oral liquid is in a single dose form comprising, by weight parts, extraction solutions, preferably water extraction solutions, of the following traditional Chinese medicinal decoction pieces, and pharmaceutically acceptable adjuvants:
In preferred embodiments, the traditional Chinese medicinal oral liquid is in a single dose form comprising, by weight parts, extraction solutions, preferably water extraction solutions, of the following traditional Chinese medicinal decoction pieces, and pharmaceutically acceptable adjuvants:
In preferred embodiments, the traditional Chinese medicinal oral liquid is in a single dose form comprising, by weight parts, extraction solutions, preferably water extraction solutions, of the following traditional Chinese medicinal decoction pieces, and pharmaceutically acceptable adjuvants:
The invention also provides a traditional Chinese medicinal tea bag formulation for preventing and/or treating respiratory diseases in winter, comprising decoction piece powders of the following traditional Chinese medicine materials and acceptable additives in food or beverage by weight parts:
In some embodiments, the traditional Chinese medicine tea bag formulation comprises decoction piece powders of the following traditional Chinese medicine materials and acceptable additives in food or beverage by weight parts:
In some embodiments, the traditional Chinese medicine tea bag formulation comprises decoction piece powders of the following traditional Chinese medicine materials and acceptable additives in food or beverage by weight parts:
In some embodiments, the traditional Chinese medicine tea bag formulation comprises decoction piece powders of the following traditional Chinese medicine materials and acceptable additives in food or beverage by weight parts:
In some embodiments, the traditional Chinese medicine tea bag formulation comprises decoction piece powders of the following traditional Chinese medicine materials and acceptable additives in food or beverage by weight parts:
In some embodiments, the traditional Chinese medicine tea bag formulation comprises decoction piece powders of the following traditional Chinese medicine materials and acceptable additives in food or beverage by weight parts:
As appreciated by those skilled in the art, the weight parts of the above traditional Chinese medicinal materials are relative, and based on traditional Chinese medicinal theories, the amounts of one or more traditional Chinese medicinal materials may be reasonably adjusted according to actual needs. All evident variable forms of prescriptions comprising component with such reasonably adjusted amounts are within the scope of the invention.
As above described, the traditional Chinese medicine formulation according to the invention, in addition to the traditional Chinese medicine composition of the invention, may further comprise pharmaceutically acceptable adjuvants. Pharmaceutically acceptable adjuvants that can be used in the traditional Chinese medicine composition of the invention include any adjuvants commonly used in traditional Chinese medicine formulations, as long as the adjuvants do not adversely affect the quality, performances and therapeutic effects of the traditional Chinese medicine composition of the invention. Adjuvants commonly used in traditional Chinese medicine formulations include diluents, carriers, fillers, binders, wetting agents, disintegrating agents, absorption promoters, surfactants, adsorption carriers and lubricants. Commonly-used diluents mainly include sucrose, dextrin, starch, lactose, mannitol, xylitol, bifidus sugar, and the like. Commonly-used wetting agents mainly comprise water, ethanol with different concentrations and the like; commonly-used binders include polymer binders in a wide variety such as ethylcellulose, polyvinylpyrrolidone, sodium carboxymethylcellulose, polyethylene glycol, sodium alginate, and the like. Commonly-used disintegrating agents include microcrystalline cellulose, sodium carboxymethyl starch and the like. Those skilled in the art can select and determine suitable adjuvants in the traditional Chinese medicine formulation of the invention according to the disclosures in the specification. The choice of specific adjuvants will depend on administration modes for treating specific patents or types and states of diseases.
If necessary, the traditional Chinese medicine formulation of the invention may further comprise suitable additives. These additives are known in the art, for example, emulsifiers, fragrances, solubilizers, anticaking agents, antifoaming agents, binders, buffering agents, pH adjusters, propellants, chelating agents, preservatives and the like.
The traditional Chinese medicine composition and traditional Chinese medicine formulation of the invention can be easily prepared by those skilled in the art according to the methods described in the specification and known techniques in the art.
For example, those skilled in the art can prepare the pharmaceutical composition of the invention by a method comprising the following steps:
On this basis, those skilled in the art can easily prepare the traditional Chinese medicine formulation of the invention in a desired dosage according to the disclosure of the specification and known techniques in the art.
For the steps of extracting, filtering, concentrating, drying and sub-packaging involved in the process for preparation of the traditional Chinese medicine formulation of the invention, those skilled in the art can implement them with commonly-used methods and apparatus in the art, e.g., to filter an extraction solution or a filtrate with, e.g., 100- to 300-mesh sieves.
As appreciated by those skilled in the art, based on the disclosure of the specification, corresponding traditional Chinese medicinal materials may be used alone or in a mixture thereof, to prepare traditional Chinese medicinal active extracts and traditional Chinese medicine formulations applicable in the invention by conventional pulverizing, immersion extracting, and separating methods in the art, such as immersion, percolation, liquid-liquid extraction, water-extraction and alcohol precipitation, alcohol extraction and water precipitation, and dialysis. One or several active extracts in the traditional Chinese medicines used in the invention may be purchased in commercial routes and then formulated with the extracts of the other traditional Chinese medicines, to obtain the traditional Chinese medicinal active extracts of the invention. Traditional Chinese medicine compositions and traditional Chinese medicine formulations in these variable forms are in the scope of the invention.
The traditional Chinese medicine composition and traditional Chinese medicine formulation of the invention may be in a unit dose form. The term “unit dose form” means a physically dispersed unit suitable as a unit dose that may be administrated on human subjects and other mammals, each unit comprising the pharmaceutical composition and pharmaceutical formulations of the invention in predetermined amounts that, by making calculations, can produce desired therapeutic effects, and suitable pharmaceutically acceptable adjuvants.
The traditional Chinese medicine composition and traditional Chinese medicine formulation of the invention may significantly inhibit the release of cytokines TNF-α, IL-6 and IL-1β of LPS-induced THP-1 cells, indicating that they have anti-inflammatory effects. The results obtained with acute pharyngitis rat models show that the pharmaceutical composition and traditional Chinese medicine formulation of the invention have evident effects of preventing and treating acute pharyngitis caused by pathogenic microorganisms, e.g., Staphylococcus aureus.
Therefore, the traditional Chinese medicine composition and the traditional Chinese medicine formulation of the invention may be applied in uses as described in one, more or all of the following items 1) to 5):
Alternatively, the traditional Chinese medicine composition of the invention may be used for the manufacture of medicaments as described in one, more or all of the following items 1) to 4):
The traditional Chinese medicine composition and traditional Chinese medicine formulation of the invention may be administrated in any suitable modes and any suitable forms that are commonly used in the art. Preferably, the traditional Chinese medicine composition and traditional Chinese medicine formulation of the invention may be orally administration. For example, the traditional Chinese medicine composition and traditional Chinese medicine formulation of the invention may be orally taken twice in one day, for each time, 5 to 30 g, for example 10 to 20 g, preferably 10 g of the traditional Chinese medicine composition and traditional Chinese medicine formulation of the invention. Specific administration doses depend on factors such as weights of subjects, properties and serenity of diseases, administration modes of medicaments and administration cycles or time interval. As for some patients in special situations, particular administration modes should follow doctor's advices.
The traditional Chinese medicine composition and traditional Chinese medicine formulation of the invention may be used in combination with other medicaments and techniques for preventing and treating respiratory diseases that are known in the art.
The traditional Chinese medicine composition and traditional Chinese medicine formulation are expected to produce the technical effects as described in in one, more or all of the following items (1) to (5):
Embodiments of the invention will be described in detail below by combining examples. However, it will be appreciated by those skilled in the art that the following examples are only illustrative for the invention and they should not be contemplated to limit the scope of the invention. In the examples, those that are not specified in terms of specific conditions are conducted according to conventional conditions or conditions recommended by manufacturers. The used reagents or apparatus that are not specified in terms of manufacturers each are conventional commercially available products.
Methods for formulating medicinal solutions of the inventive pharmaceutical compositions used in the preparation examples of the invention each are to formulate a medicinal solution at a desired concentration with distillated water at room temperature, which is fully mixed with ultrasonic treatments for 20 minutes, not in need of removing dregs of a decoction, and to take the supernatant for administration, further dilution or storage for use.
The above individual medicinal materials were pulverized separately and all sieved by passing through Sieve No. 1 (10 mesh) to form coarse powders between Sieve No. 1 (10 mesh) and Sieve No. 5 (80 meshes), which, by charging them in a quantity of 1000 prescriptions according to the formulation proportions in the above prescriptions, were weighed in a total quantity of 27.0 kg, and the resultant powders were charged in a mixing machine for total mixing (the total mixing is aimed to enabling all the powders of medicinal materials to be uniformly mixed) for 60 minutes, to obtain a medicinal powder in a quantity of 26.9 kg, which, after sterilization, was packaged with tea filter paper of heat-sealing type into 3 g/bag or 4.5 g/bag (the yield in inner packaging process: 98-99.5%), and then was externally packaged to obtain a finished product (batch number: SJD2020001).
The prescription is the same as that in Preparation Example 1.
The above medicinal materials, by charging them in a quantity of 1000 prescriptions according to the formulation proportions in the above prescriptions, were weighed in a total quantity of 27.0 kg, and after the decoction pieces were mixed, the mixture was pulverized so that it totally passed through Sieve No. 1 (10 mesh) to form a coarse powder between Sieve No. 1 (10 mesh) and Sieve No. 5 (80 meshes). The resultant powder was charged in a mixing machine for total mixing (the total mixing is aimed to enabling all the powders of medicinal materials to be uniformly mixed) for 30 minutes, to obtain a medicinal powder in a quantity of 26.8 kg, which, after sterilization, was packaged with tea filter paper of heat-sealing type into 3 g/bag or 4.5 g/bag (the yield in inner packaging process: 98-99.6%), and then was externally packaged to obtain a finished product (batch number: SJD2020001).
The prescription is the same as that in Preparation Example 1.
The above medicinal materials, by charging them in a quantity of 1000 prescriptions according to the formulation proportions in the above prescriptions, were weighed in a total quantity of 27.0 kg, and after the decoction pieces were mixed, the mixture was pulverized so that it totally passed through Sieve No. 1 to form a coarse powder between Sieve No. 1 and Sieve No. 5 (80 meshes). The resultant powder was charged in a mixing machine for total mixing (the total mixing is aimed to enabling all the powders of medicinal materials to be uniformly mixed) for 30 minutes, to obtain a medicinal powder in a quantity of 26.7 kg, which, after sterilization, was packaged with tea filter paper of heat-sealing type into 3 g/bag or 4.5 g/bag (the yield in inner packaging process: 99.7%), and then was externally packaged to obtain a finished product (batch number: SJD2020002).
Presently, Thin-Layer Chromatography (TLC) was utilized to conduct qualitative identifications to one main medicament in the sample, Paeoniae Radix Rubra, and the other medicament, Belamcandae Rhizoma, and High Performance Liquid Chromatography (HPLC) was utilized to measure the content of one main medicament, Arctii Fructus. According to the standards of tea formulations (Pharmacopoeia of the People's Republic of China (2015 version), Part IV, Tea Formulations, General Rules 0188), an overall study on quality standards was conducted to provide scientific and effective bases for further studying the formulations.
Control samples for content measurement: Arctiin (batch No.: 110819-201812, purity: 95.0%), commercially available from National Institutes for Food and Drug Control.
Control samples for thin-layer identification: Peoniflorin (batch No.: 110736-201943, putridity: 95.1%), Irisflorentin (batch No.: 11557-201703, purity: 99.9%) and Belamcandae Rhizoma control medicinal material (batch No.: 120994-201206), each commercially available from National Institutes for Food and Drug Control.
According to Preparation Examples 1-3, the pharmaceutical compositions (SJD2020001, SJD202001, SJD2020002, SJD2020003 and SJD2020004) of the invention were prepared, and meanwhile, according to the same method, batches of samples lack of Paeoniae Radix Rubra, Belamcandae Rhizoma and Polygoni Cuspidati Rhizoma et Radix were respectively prepared as negative controls.
In negative control 1, except for the lack of Paeoniae Radix Rubra, the other medicinal materials and amounts thereof, and preparation processes each are the same as those in Preparation Example 3. A negative sample was provided in order to prove that the method for identification of Paeoniae Radix Rubra in samples is reliable, and to show that the characteristic spot of the Peoniflorin in Paeoniae Radix Rubra is really present in the samples, and in addition to Paeoniae Radix Rubra, the other medicinal materials are free of the Peoniflorin.
In negative control 2, except for the lack of Belamcandae Rhizoma, the other medicinal materials and amounts thereof, and preparation processes each are the same as those in Preparation Example 3. A negative sample was provided in order to prove that the method for identification of Belamcandae Rhizoma in samples is reliable, and to show that the characteristic spots of an Irisflorentin control sample (111557-201703) in the Belamcandae Rhizoma, and in addition to Belamcandae Rhizoma, the other medicinal materials are free of the Irisflorentin.
In negative control 3, except for the lack of Polygoni Cuspidati Rhizoma et Radix, the other medicinal materials and amounts thereof, and preparation processes each are the same as those in Preparation Example 3. A negative sample was provided in order to prove that the method for identification of Polygoni Cuspidati Rhizoma et Radix in samples is reliable, and to show the characteristic spot of a Polydatin control sample in Polygoni Cuspidati Rhizoma et Radix is really present in the sample, and in addition to Polygoni Cuspidati Rhizoma et Radix, the other medicinal materials are free of the Polydatin.
2 g of a medicinal powder of the sample (prepared in Preparation Example 3) were taken and added with 40 mL of ethanol, and they were shaken and filtered. The filtrate was evaporated to dryness, and the residue was dissolved by adding 5 ml of ethanol as the test sample solution.
Negative control 1 was taken and prepared into a negative control solution according to the same method.
0.5 g of Paeoniae Radix Rubra control medicinal material were taken and prepared into a control medicinal material solution according to the same method as that described in item 1.2.1.1 (1).
2 mg of a Peoniflorin control sample were taken and added with ethanol to formulate a control sample solution (2 mg·mL−1).
2 g of medicinal powders of the samples (prepared in Preparation Examples 1-3) were taken and added with 40 ml of ethanol, and they were shaken and filtered. The filtrate was evaporated to dryness, and the residue was dissolved by adding 5 ml of ethanol as the test sample solution.
Negative control 2 was taken and prepared into a negative control solution according to the same method.
0.5 g of Belamcandae Rhizoma control medicinal material were taken and prepared into a control medicinal material solution according to the same method as that described in item 1.2.1.1 (1).
2 mg of an Irisflorentin control sample were taken and added with ethanol to formulate a control sample solution (2 mg·mL−1).
2 g of medicinal powders of the samples (prepared in Preparation Examples 1-3) were taken and added with 20 ml of ethanol, and they were ultrasonically treated from 20 minutes and filtered. The filtrate was evaporated to dryness, and the residue was dissolved by adding 4 ml of methanol as the test sample solutions.
Negative control 3 was taken and prepared into a negative control solution according to the same method.
0.5 g of P Belamcandae Rhizoma control medicinal material were taken and prepared into a control medicinal material solution according to the same method as that described in item 1.2.1.1 (1).
A Polygoni Cuspidati Rhizoma et Radix control sample were taken and added with ethanol to formulate a control sample solution (1 mg·mL−1).
Inspections were conducted according to Pharmacopoeia of the People's Republic of China (2015 version), Part IV, Tea Formulations, General Rules 0188.
Measurements were conducted according to Pharmacopoeia of the People's Republic of China (2015 version), Part IV, moisture measurements (General Rule 0832).
The samples prepared in Preparation Examples 1-3 were taken in one batch respectively, 10 bags for each batch, and by inspecting according to the method of “loading difference” prescribed under Pharmacopoeia of the People's Republic of China (2015 version), Part IV, Tea Formulations, General Rules 0188, the weight of the products was 3 g, and the loading difference should be ±12%.
The samples prepared in Preparation Examples 1-3 were taken in one batch respectively, 3 bags for each batch, and according to the method of “Microbial Limits” prescribed under Pharmacopoeia of the People's Republic of China (2015 version), Part IV, Tea Formulations, General Rules 0188, the inspections were conducted.
The samples prepared in Preparation Examples 1-3 were taken in one batch respectively, each batch including 3 samples, and 3 g for each sample.
Preparation of samples: 3 g of a sample was taken and placed in a flask, and it was added with 180 mL of 85° C. purified water and immersed for 30 min. The sample was taken and added with methanol, and it was ultrasonically treated for 30 min. After cooling, it was transferred into a centrifugal tube wherein it was centrifuged for 15 min (5000 rpm). The supernatant was transferred into a 10 mL volumetric flask, and it was kept at a constant volume with 90% methanol and shaken. By filtering with a 0.45 μm microporous filtration membrane, the sample was obtained for use.
Chromatographic conditions: chromatographic column: Syncronis-AQ C18 (250 mm×4.6 mm, 5 μm); mobile phase: acetonitrile (A) and water (B); flow rate: 0.8 mL·min−1; wavelength: 230 nm; sampling quantity: 10 μL; column temperature: 30° C. Gradient elution procedure: 0-10 min, 14%-18% A; 10-18 min, 18% A; 18-22 min, 18%-19% A; 22-42 min, 19%-31% A; 42-50 min, 31%-55% A. The theoretical plate number, calculated according Arctiin peaks, was not less than 5000.
Thin-layer identification results were shown in
As can be seen from
As can be seen from
As can be seen from
Results are shown in Table 1.
As can be seen from Table 1, the water contents each were lower than 12% and in line with relevant requirements of tea formulations.
The results were shown in Table 2.
As can be seen from Table 2, the loading differences each met the requirements of tea formulations.
Inspection results of the microorganisms in the three batches of samples met relevant provisions.
An external standard method was used to calculate contents and take their average values, and the results were shown in Table 3.
To more representatively determine the content limits of the index ingredients of the inventive pharmaceutical compositions, the inventor, based on the Arctiin content standards specified in Pharmacopoeia of the People's Republic of China, determined the content limits of the index ingredients of the inventive pharmaceutical compositions. The specific contents are shown below:
According to the provisions of Pharmacopoeia of the People's Republic of China (2015 version), the Arctiin in stir-fried Arctii Fructus decoction pieces is 5.0%, and according to preparation processes of tea formulations, the proportion of the Arctii Fructus in a prescription is 11.1%. By combining the average yield 99% of the inventive pharmaceutical compositions in the pilot magnification productions of three batches, the content was calculated to be 5.5 mg/g, by 20% downward floating, being 4.4 mg/g, and each bag (4.5 g) with 19.8 mg was used as the standard of the arctiin content in the inventive pharmaceutical compositions.
The content limit standard of the inventive pharmaceutical compositions: each bag of the inventive pharmaceutical compositions contained not less than 19.8 mg of Arctii Fructus, in Arctiin (C27H34O11).
This experiment used rats suffered from acute pharyngitis caused by Staphylococcus aureus, in the three aspects of appearance indexes, pathological indexes, and biochemical indexes, to evaluate the medicinal effects of the inventive pharmaceutical compositions, thereby providing scientific bases for clinical applications of the inventive pharmaceutical compositions.
The inventive pharmaceutical composition samples (prepared in Preparation Example 3)
Amoxicillin capsules, Shandong Ziboxinda pharmaceutical Ltd.; batch number: 191040, in the valid period; specification: 0.25 g/tablet, usage and dose: referring to clinical usages; starting administration at the first day of modelling in a dose that is equivalent to clinical doses, each rat ig 0.36 g/kg, 1 time per day for 5 consecutive days.
SPF-grade SD rats, not being limited to female or male, with a weight of 180-220 g; provided by provided by Beijing Huafukang Biotechnology Ltd; certificate number: 1103222011009608; license number: SCXK (Jing) 2019-0008; feeding conditions: ABSL-2 laboratory, −20 Pa, temperature 22±1° C. and humidity 40-50%.
Staphylococcus aureus standard cell lines 6538, commercially available from ATCC.
1.7.1 Extracts of the inventive pharmaceutical compositions: clinical doses for human were 9 g/70 kg/d, and the dose for test rates were 3.5 g/kg/time, equivalent to 16 times of the clinical dose.
Formulations of medicinal solutions: before the test, distilled water was used to formulate a medicinal solution with a concentration of 0.35 g/ml, which was intragastrically administrated in 1 ml/100 g body weight/time, 2 times/day, and on 7th day after pre-administrations, modelling was started and the intragastric administration was continued for 5 days.
1.7.2 Amoxicillin capsules: referring to clinical usages. On 1st day, at 1 h after Staphylococcus aureus infection, the positive medicinal group was intragastrically administrated at a dose of 0.36 g/kg (equivalently equal to the clinical dose of a human), 1 time/day for 5 consecutive days.
Formulations of medicinal solutions: before the test, the granules inside capsules were formulated with distilled water into a medicinal solution with a concentration of 36 mg/ml, and stored at 4° C. for use.
Two 250 ml conical flasks were taken, and to each of them, 1 g of yeast extract, 2 g of peptone, 2 g of sodium chloride and 200 ml of deionized water were added and mixed uniformly, to formulate LB culture media which were placed in a 4° C. chromatography cabinet at 4° C. and stored for use.
Staphylococcus aureus colonies were inoculated into a conical flask charged with the LB culture medium and cultured by shaking at 37° C. for 17 hours. The bacterial solution was centrifuged at 3220 rcf for 1 min, and by discarding the supernatant, it was counted under a microscope; the density of the bacterial solution was adjusted to 1.9×109/ml.
28 SD rats were randomly divided into a normal group, a model group, an inventive pharmaceutical composition administrated group and an amoxicillin group, each group with 7 SD rats.
The group administrated with the pharmaceutical composition of the invention was is pre-administered in an intragastric route for seven days, at a dose of 3.5 g/kg, and twice per day; after 7 days, the other individual groups were modelled by injecting a Staphylococcus aureus bacterial solution on the pharynx, 1×108/rat for 2 consecutive days, and meanwhile, the administration was continued; for the amoxicillin group, the intragastric administration was started on the first day of modelling, at an administration dose of 0.36 g/kg, once per day; after the modelling was finished, the inventive pharmaceutical composition administrated group and the amoxicillin group were continuously administered for three days, and on 4th day, they were weighed and dissected for observation and detection.
With scoring standards (from Standards for Preparation of Animal Models Suffered from Acute Pharyngitis, enacted by Traditional Chinese medicine Experimental Pharmacology Professional Committee of Traditional Chinese Medicine Institute, Traditional Chinese Medicine Pharmacology and Clinics 2018; 34 (1)), according to color, gloss, secreted substances of tissues, and degrees of pharyngeal congestions and swelling, the lesions were classified into 4 grades.
With scoring standard (from Standards for Preparation of Animal Models Suffered from Acute Pharyngitis, enacted by Traditional Chinese medicine Experimental Pharmacology Professional Committee of Traditional Chinese Medicine Institute, Traditional Chinese Medicine Pharmacology and Clinics 2018; 34 (1)), according to weights, mobility, fur color and gloss of rats, they were classified into 4 grades, as shown in the following Table 4.
Before each group of animals were scarified, blood was taken by cutting tails and smeared on plates to conduct routine blood examinations about number of white blood cells, lymphocyte percentages and neutrophils percentages.
Material taking: rats were anesthetized and pharyngeal tissues were taken and fixed in 4% paraformaldehyde for more than 24 h. The tissues were taken from the fixing solution and in a fume hood, the tissues at the target region were trimmed with a surgical knife. The trimmed tissues and the corresponding tags were placed in a dehydration box.
Dehydration: the dehydration was conducted by placing the dehydration box in a hanging basket, and in a dehydration machine, the dehydration was conducted with alcohols with gradient concentrations in order. 75% alcohol 1 h-85% alcohol 1 h-90% alcohol 1 h-95% alcohol 1 h-anhydrous ethanol I 30 min-anhydrous ethanol II 30 min-alcohol benzene 5-10 min-xylene I 5-10 min-xylene II 5-10 min-wax I 1 h-wax II 1 h-wax III 1 h.
Embedding: the tissues immersed with wax were embedded in an embedding machine. The molten wax was placed in an embedding frame first, and before it was solidified, the tissues were taken from the dehydration box and placed in the embedding frame according to requirements on embedded faces and corresponding tags were attached. They were cooled on a freezing table at −20° C., and after wax solidification, the wax block was taken from the embedding frame and trimmed.
Slicing: the trimmed wax block was sliced on a paraffin slicing machine, in a thickness of 4 μm. The slices were floated on a slide spreading machine at 40° C. to spread the tissues, and a slide glass were used to take tissues and baked at an oven at 60° C. After water was baked to dryness and the wax to melt, the slide glass was taken and stored at normal temperature for use.
The paraffin slides were dewaxed into water; the slides, by being placing in order in xylene I 30 min-xylene II 30 min-anhydrous ethanol I 10 min-anhydrous ethanol II 10 min-95% alcohol 5 min-90% alcohol 5 min-80% alcohol 5 min-70% alcohol 5 min-distilled water, were washed.
Staining nuclei with Hematoxylin: slides were placed in Harris hematoxylin and stained for 5-10 min, and they were washed with tap water. After differentiating with 1% hydrochloric acid and alcohol for several seconds, they were washed with tap water for 10 min, returned to blue color with PBS for 5 min, and washed with running water.
Staining cytoplasm with Eosin: slides were placed in an Eosin solution and stained for 1-3 min.
Dehydration and sealing of slides: slices, by being placing in 95% alcohol I 5 min-95% alcohol II 5 min-anhydrous ethanol 15 min-anhydrous ethanol II 5 min-xylene I 5 min-xylene II 5 min in order, were dehydrated to transparent, and the sides were taken out of xylene, slightly dried in air and sealed with neutral gum.
A microscope is used for detections and the images were collected for analysis.
Rates were anesthetized. Blood was taken from abdominal aorta and injected into a procoagulant tube, standing for 45 min at room temperature, and it was centrifuged at 3500 rpm for 10 min. The supernatant was suction taken into a sterile EP tube and stored at −80° C. Before measurements, the samples were re-thawed at room temperature, and the IL-1β, IL-6 and TNF-α levels in serum were measured by enzyme-linked immunosorbent assay (ELISA). The operations were carried out according to the instruction of the kit, and each index was detected by an enzyme-labelled meter at the absorbance 450 nm.
The results were shown in Tables 5 and 6.
#P < 0.05,
##P < 0.01.
As shown by the results in Table 5, in comparison to the normal group, the weights of the rats in the model group were evidently reduced (P<0.01); the weights of thymus gland significantly decreased (P<0.01); in the model group, a phenomenon that animals were crowed and still was exhibited, and they exhibited bad mental states and evidently reduced mobility (P<0.01); the animals in the model group has dry and dull fur, and the scores for fur gloss were significantly increased (P<0.01).
In comparison to the rates in the model group, the rats in the inventive pharmaceutical composition administrated group had increased weights and thymus gland weights, obviously increased mobility, which are consistent with the characterizations for the behaviors of the rats in the amoxicillin group.
As shown by the results in Table 6, the pharyngeal inflammatory responses of the rats in the normal group were all in a “−” grade, and no inflammatory features appear. The model group had the pharyngeal inflammatory response in the grade “++” and the grade “+++” and evident lesions in pharyngeal mucosa and tissues, and thus it was successfully modelled. A majority of the animals in the inventive pharmaceutical composition administrated group were accompanied with small quantities of secreted substances, and slight congestion and swelling, and inflammatory symptoms were evidently improved, exhibiting the grade “+”, occasionally the grades “−” and “++”. Animals administrated with amoxicillin were recovered consistently with recovery conditions of pharyngeal lesions of the rats in the inventive pharmaceutical composition administrated group, most exhibiting the grade “+” and occasionally the grade “++”.
The results were shown in the following Table 7.
19.5 ± 6.77##
As shown by the results in Table 7, in comparison to the normal group, the number of white cells of the rat acute pharyngitis model group was increased (P<0.05), the lymphocyte percentages were evidently reduced, and the neutrophil percentages were evidently increased (P<0.01); in comparison to the model group, the lymphocyte percentages of the rats in the inventive pharmaceutical composition group and the amoxicillin group were increased (P<0.01), and the neutrophil percentages were reduced (P<0.01).
As shown in
The results are shown in the following Table 8 and in
254.04 ± 59.15#
As shown in the results of Table 8, in comparison to the normal group, the contents of the inflammatory factors TNF-α and IL-1β in peripheral blood of the rats in acute pharyngitis model were significantly increased (P<0.01), and the content of the IL-6 was reduced; in comparison to the model group, the inventive pharmaceutical composition group and the amoxicillin group can inhibit the expressions of TNF-α and IL-1β (P<0.01) in peripheral blood of rats.
The inventive pharmaceutical compositions can significantly improve lesion conditions of the pharyngeal mucosa of rats suffered with acute pharyngitis, increase the weights and the thymus gland weights of rats, improve the mobility of the rats, improve the fur states of rats, inhibit the increases of the number of white blood cells of the rats suffered with acute pharyngitis, the increase of the lymphocyte percentages, and reductions of the neutrophil percentages, and relieve the pathological lesion degrees of the pharyngeal tissues of the rats after infections, and it can also significantly reduce the content of TNF-α in the serum of rats suffered with acute pharyngitis. This indicates that: the inventive pharmaceutical compositions have evident effects for preventing and treating a rat acute pharyngitis model by staphylococcus aureus, thereby providing laboratory bases for clinical medicament administrations of the acute pharyngitis.
The experiment studied the impacts of the inventive pharmaceutical compositions on the releases of LPS-induced cytokine TNF-α, IP-10, IkBα, IL-6 and IL-1β of human monocyte THP-1 and preliminarily discusses the anti-inflammatory effects of the inventive pharmaceutical compositions.
The inventive pharmaceutical compositions (prepared in Preparation Example 3).
2.5 mg of the inventive pharmaceutical compositions were precisely weighed into 1 ml of double distilled water to obtain 2.5 mg/ml of a standard stock solution of the inventive pharmaceutical compositions, and by the means of a gradient dilutions with double distilled water, the sample solutions at 2.5 mg/ml, 1 mg/ml and 0.5 mg/ml were formulated.
1 mg of LPS was quickly centrifuged at less than 21° C. in a centrifuge and sufficiently dissolved by adding 1 mL of ddH2O. After filtering the solution by using a filter, it was sub-packaged to facilitate use in experimental processes, and stored at the temperature −20° C.
THP-1 cells used in the experiment were purchased from ATCC, being suspended cells, and thus upon passages, they were not in need of centrifugation. 20 mL of a RPMI cell culture solution containing 10% heat-inactivated fetal bovine serum and 1% bispecific antibodies were added into a 75 cm2 cell culture flask, and further added with 10 ml of cell solution from the original 25 cm2 culture flask, and they were placed into a temperature-constant CO2 cell incubator at 37° C. for continuous culture.
THP-1 cells were centrifuged at 800 rmp for 5 minutes and the supernatant was discarded. An appreciate amount of a cell cryopreserved liquid was taken according to actual situations, to precipitate cells which were rapidly re-suspended, and injected into a cryopreservation tube according to 1 mL/tube. The cryopreservation tube was placed in an isopropanol cryopreservation box, and after a gradient temperature decrease at −80° C., the cells were transferred in a liquid nitrogen tank for storage.
The cryopreservation tube was taken from liquid nitrogen, rapidly placed in a 37° C. water bath, and shaken quickly to thaw the cells. The cells were removed from the cryopreservation tube and added with 4 mL of the culture solution, and after being mixed uniformly, they were centrifuged for 5 minutes at 800 rpm. The supernatant was discarded, and the cell precipitates were re-suspended with 4 mL of the culture solution and then added with the culture solution to get a volume of 10 mL, and they were added to a 25 cm2 culture flask which was placed in a temperature-constant CO2 cell incubator at 37° C. where cells were cultured for 3-4 days. When the number of the cells reaches 1×106 per milliliter, the passage culture may be conducted.
A clear cell counting plate was taken and covered with a cover glass, and 10 μL of a uniformly dispersed cell suspension were added from the edge between the cover glass and the counting plate so as to fill the space between the counting plate and the cover glass. The plate was observed under an inverted microscope, to calculate the number of the cells in the four grid areas. For cells on the lines, only the cells on the left lines and on the right lines were counted, and clustered cells are counted as a single cell.
The density of cells was calculated according to the equation:
Cell numbers/mL=(cell numbers in four grid areas)/4×104×dilution time
The experiment uses CCK-8 and LDH test kits to investigate impacts of the pharmaceutical compositions of the invention with different concentrations on the viability of THP-1 cells.
The THP-1 cells in the logarithmic growth phase were taken and inoculated into a 96-well cell culture plate at a density of 1×106 cell/mL, each well with 100 μL. 2.5-0.5 μg/mL of the inventive pharmaceutical compositions were respectively added into the cell culture wells, each well with 1 μL and each concentration with 6 compound wells. Meanwhile, a control group without any medicaments was configured and subjected to CCK8 and LDH respectively. After 24 h cultures, the supernatant was suction taken and placed in the other new 96-well plate, and the absorbance (OD) was measured at 490 nm according to the operations of the LDH test kit. 10 μL of the CCK-8 solution were added to each well of the other 96-well plate, and incubated at 37° C. for 1 hour, and the absorbance (OD value) was measured by setting an enzyme-labelled meter at 450 nm.
THP-1 cells were incubated in a 6-well plate at a density of 1×106 cells/mL and after 24 h, the cells were treated in groups. In a model group, the LPS with a final concentration of 100 ng/ml was added; in the control group, the LPS was replaced with double distilled water; in the medicament-administrated group, the culture medium, in addition to the LPS, was further added the extracts of the inventive pharmaceutical compositions with final concentrations 25 μg/ml, 10 μg/ml and 5 μg/ml respectively. Cells were collected for measurements after they were pre-stimulated with the inventive pharmaceutical compositions for 4 hours and then stimulated by adding the LPS for 16 hours.
The step (8) was repeated.
The following operations were conducted on ice, and according to Table 9, 20 μL of a reaction system was formulated and vortexed and shaken vigorously. After the reaction system was mixed uniformly, it was centrifuged briefly to collect the solution on the side wall to the bottom of the tube, and then incubated at 42° C. for 15 minutes and 85° C. for 5 seconds. Upon completion, the sample was cooled by placing on ice.
On ice, 20 μL of a PCR reaction system was formulated according to the prescriptions listed in Table 10 by using a real-time quantitative PCR test kit. After the formulation was completed, the reaction system was vortexed so that it was fully and uniformly mixed. After centrifugation, a real-time quantitative PCR experiment was conducted. In this experiment, the reaction and cycle conditions of the real-time quantitative PR-PCR were set below: 95° C. 10 s, 60° C. 30 s, totally cycling 40 times. A melting curve was added at temperatures between 50° C. and 99° C. The sequences of the primers involved in the experiment were shown in Table 11.
After detections, the Ct value was obtained, and a ΔCt was the difference the Ct value of genes to be tested and the Ct value of internal reference genes. By analyzing the data of the reaction plate, the corresponding Ct value of each sample, in each reaction, was obtained, and by making calculations, the ΔCt value was obtained. After making comparisons to the control group, the values of 2−ΔΔCt were taken for making statistics.
Experimental data was subjected to single-way ANOVA analyses by using SPSS 20.0 software to assess the differences between the means of the data. In the case of P<0.05, it was contemplated that the data had statistical differences, and the data was expressed as means±S.E.m.
CCK-8 and LDH methods were used for detecting impacts of the inventive pharmaceutical compositions on the viabilities of the THP-1 cells at 25 μg/ml, 10 μg/ml and 5 μg/ml. The results showed that the viabilities of the THP-1 cells in the inventive pharmaceutical composition group at 5 μg/ml and 10 μg/ml had no significant difference as compared with those in a normal control group. Safety concentrations of medicaments were utilized to investigate the anti-inflammatory effects of the inventive pharmaceutical compositions (
In comparison to the control group, the LPS stimulation evidently induced a significant increase in mRNA expressions of TNF-α, IP-10, IKBα, IL-6 and IL-1β of THP-1 cells; in comparison to the LPS-stimulated group, 10 μg/ml of the inventive pharmaceutical compositions could significantly inhibit mRNA expressions of TNF-α, IP-10, IKBα, IL-6 and IL-1β of inflammatory THP-1 cells, indicating that the inventive pharmaceutical compositions could inhibit the release of LPS-induced cytokine (
The inventive pharmaceutical compositions can significantly inhibit the releases of cytokines TNF-α, IL-6 and IL-1β of LPS-induced THP-1 cells, indicating anti-inflammatory effects.
This experiment, based on a luciferase reporter gene system of ARE and NF-kB, investigated impacts of the inventive pharmaceutical compositions on the activities of the ARE and NF-KB, and preliminarily discussed the autoxidation and anti-inflammatory effects of the inventive pharmaceutical compositions.
A inventive pharmaceutical composition according to prescription was soaked and extracted twice, for every time, soaking for 40 minutes with boiling water in an quantity of 10 times thereof, and it was filtered and concentrated at a temperature below 60° C. to a sugar degree of 15-17 Brix and spray dried to obtain a medicament.
Dexamethasone, MedChemExpress Co. Ltd; batch number: HY-14648, in a valid period; specification: 500 mg; usages and doses: referring to clinical usages, formulated into a 10 mM mother liquor.
tBHQ ((Tertiary butylhydroquinone), MedChemExpress Co. Ltd; batch number: HY-100489, in a valid period; specification: 500 mg; usages and doses: referring to clinical usages, formulated into a 100 mM mother liquor.
293T cells, commercially available from ATCC cell bank.
Competent cells DH5a, commercially available from Tiangen Biochemical technology Ltd.
The inventive pharmaceutical compositions: 0.01 μg/mL, 0.1 μg/mL, 1 μg/mL, 10 μg/mL, 100 μg/mL.
Medicament Formulations: prior to test, a medicinal powder was formulated into a medicinal solution with the concentration of 100 mg/ml by distilled water, which was diluted into a test concentration in use.
After a triangular flask was sterilized at high temperatures and high pressures, 2.5 g of a dry powder of LB liquid culture medium were added into the flask, and fully dissolved by adding 100 ml of sterilized ultra-pure water. After attaching a filtration membrane on the opening of the flask, it was sterilized at high temperatures and high pressures. After the solution was cooled to about 55° C., it was added with 100 μL of 100 mg/mL ampicillin, and they were uniformly mixed to obtain a culture medium. Cells were cultured in a DMEM culture medium containing 10% FBS, and the passage was started when the cells in the flask grown to 80%-90%.
0.4 g of the dry powder of LB solid culture medium were taken. 0.3 g of the dry powder of LB solid culture medium were placed in a triangular flask which was sterilized at high temperatures and high pressures and added with 20 ml of sterilized ultrapure water, and they were heated to boil, dissolved by stirring and sterilized at high temperatures and high pressures. After the solution was cooled to 50-60° C., 50 μL of ampicillin (50 μg/mL) were added and mixed uniformly to obtain the culture medium; the above solution was sub-packaged in culture dishes, and after it was solidified by cooling, the culture dishes were covered with covers, inverted and stored a refrigerator at 4° C.
After competent cells DH5a were gently mixed to be uniform, 100 μL of the cells were taken and placed in a 15 mL centrifuge tube which were added with 1 μL of plasmid pGL4.37 sample. The mixture was placed on ice for 30 minutes and immediately transferred into a water bath at 42° C. where it was incubated for 45 s, and then it was transferred on ice where it stayed for 1-2 min to finish the transformation of the competent cells. 100 μL of the above transformed competent cells were suction taken into an appropriate amount of the LB liquid culture medium, and mixed uniformly. By a sterile coater, an appropriate amount of the above bacterial solution was coated onto the LB solid culture medium which was placed at room temperature until the bacterial solution was fully absorbed. At 37° C., the culture medium was inverted at 37° C. and cultured for 12 h.
In a biological safety cabinet, six bacterial granules were selected and added to six conical flasks containing appropriate amounts of the LB liquid culture medium respectively, to obtain six bacterial solution samples labelled with numbers 1 to 6. The above samples were kept at the temperature 37° C., and after they were oscillated at 160 rpm for 12 hours, 1 ml of the samples with individual numbers were taken and placed in a 1 mL centrifuge tube, and they were sequenced in Huada Gene, to identify positive clones.
293T cell lines were taken out of a liquid nitrogen tank and immediately placed in a temperature-constant water bath kettle at 37° C. which is slightly shaken so as to rapidly thaw the cell lines. A cryopreservation tube, after being wiped with alcohol cotton, was transferred to a biological safety cabinet, and the cells were suction taken into a centrifuge tube loaded with a complete culture medium by a pipette, and centrifuged at 1000 rpm and 4° C. for 3 min. The supernatant was discarded, and 5 mL of the complete culture medium were added. The cells were uniformly dispersed by blowing with a pipette and then they were transferred to a 25 cm2 culture flask. The cells were cultured in a 5% CO2 cell incubator at 37° C. After 24 h, the culture solution in the culture flask was replaced. According to growing states of the cells, the passage culture was conducted.
The cells were observed under a DMIL inverted phase-contrast microscope, and when the cells in the culture flask grown to 80 to 90%, the passage was conducted. The culture flask was removed from the culture incubator. The culture solution in the flask was discarded. 2 mL of a PBS buffer was added to rinse twice, and 500 μL of 0.25% pancreatin (containing 0.25% EDTA) was added to digest the cells for about 15 s until most of the cells bulge and become round. 1 mL of the complete medium was added to stop the digestion. The cell suspension was transferred into a centrifuge tube and centrifuged at 1000 rpm and 4° C. for 3 min. The supernatant was discarded. 4 mL of the complete medium was added to the centrifuge tube and gently blown and tapped until the cells were evenly dispersed, and then the cells were transferred to a new flask at 1:4 ratio.
A clear cell counting plate was taken and covered with a cover glass, and 10 μL of a uniformly dispersed cell suspension were added from the edge between the cover glass and the counting plate so as to fill the space between the counting plate and the cover glass. The plate was observed under an inverted microscope, to calculate the number of the cells in the four grid areas. For cells on the lines, only the cells on the left lines and on the upper lines were counted, and clustered cells are counted as a single cell.
The density of cells was calculated according to the equation:
Cell numbers/mL=(cell numbers in four grid areas)/4×104×dilution time
2.5 mg of the inventive pharmaceutical compositions were precisely weighed into 2.5 ml of water to obtain 100 mg/ml of a standard stock solution of the inventive pharmaceutical compositions, and by the means of a gradient dilutions with DMEM culture medium, the sample solutions at 100 μg/ml, 10 μg/ml, 1 μg/ml, 0.1 μg/ml and 0.01 μg/ml were formulated.
The detection on the cytotoxicity was conducted by grouping rates into a blank control group, and inventive pharmaceutical composition groups with five concentrations of 0.01 μg/mL, 0.1 kg/mL, 1 μg/mL, 10 μg/mL, and 100 μg/mL.
The detection on the anti-oxidative activity was conducted by grouping rats into a blank control group, a positive tBHQ medicinal group and inventive pharmaceutical composition groups with five concentrations of 0.01 μg/mL, 0.1 μg/mL, 1 μg/mL, 10 μg/mL, and 100 μg/mL.
The detection on the anti-inflammatory effects activity was conducted by grouping rats into a blank control group, a model group, a positive dexamethasone medicinal group, and inventive pharmaceutical composition groups with five concentrations of 0.01 μg/mL, 0.1 μg/mL, 1 μg/mL, 10 μg/mL, and 100 μg/mL.
The experiment uses CCK-8 and LDH test kits to investigate impacts of the pharmaceutical compositions of the invention with different concentrations on the cytotoxicity of 293T cells.
The 293T cells in the logarithmic growth phase were taken and inoculated into a 96-well cell culture plate at a density of 2×105 cell/mL, and after growth along the walls to 70-80%, the inventive pharmaceutical compositions at 0.01-100 μg/mL were respectively added into the cell culture wells, each well with 100 μL and each concentration with 6 compound wells. Meanwhile, a control group without any medicaments was configured, with 3 repetitions. After 24 h cultures, the supernatant was suction taken and placed in the other new 96-well plate, and the LDH release amount was detected according to the operations of the LDH test kit. After the initial 96-well plate was washed by PBS, each well was added with 100 μL of a diluted 1×CCK-8 working solution. The cells were incubated at 37° C. for 1 h. The absorbance (OD value) was measured by setting an enzyme-labelled meter at 450 nm.
According to the following equation, the cell survival rate was calculated:
Cell survival rate (%)=(As−Ab)/(Ac−Ab)×100%
The 293T cells in the logarithmic growth phase were taken and inoculated into a 96-well cell culture plate at a density of 2×105 cell/mL, and after growth along the walls to 70-80%, a PEI (1 mg/ml) transfecting agent was used to simultaneously transfect them with ARE and NF-κB luciferase reporter plasmids pGL4.37, pGL4.32 (100 ng/well) and a renilla luciferase reported plasmid pGL4.75 (10 ng/well), and they were cultured for 24 h.
After 24-h culture, the cells transfected with the ARE luciferase reporter plasmid pGL4.37 are added with tBHQ (10 μM) and safety concentrations of the sample solutions of the individual inventive pharmaceutical composition groups, and by setting a control group, and they were cultured 6 h; the cells transfected with NF-kB luciferase reporter plasmid pGL4.32 were respectively added with dexamethasone (10 μM) diluted by a TNF-α complete culture medium, and safety concentrations of the sample solutions of the individual inventive pharmaceutical composition groups, and by setting a control group, and a model group, and they were cultured for 6 h. The supernatant was discarded, and the cells were rinsed with PBS. After splitting, they were detected by using a Dual-Luciferase detection system. Each set of experiments were configured with 6 compound wells, with 3 repetitions. A relative luciferase activity value was obtained by comparing firefly luciferase activities and renilla luciferase activities. L/S=Luciferase activity value/Renilla activity value.
In comparison to the normal group, the 293T cell activities of the inventive pharmaceutical composition groups at 0.01-100 μg/ml did not have any significant differences, being medicinal safety concentrations for the 293T cells. The medicinal safety concentrations were used to discuss the antioxidative and anti-inflammatory effects of the pharmaceutical compositions of the invention (see
293T cell transfections were used to verify the antioxidative activities of the sample solutions of the inventive pharmaceutical compositions. The results showed that 1 μg/ml of the inventive pharmaceutical compositions may significantly induce the ARE luciferase activity (P<0.05) (see Table 12,
293T cells-transfected NF-kB promoter luciferase reporter plasmid was used to detect the anti-inflammatory activities of the inventive pharmaceutical compositions. The results showed that 0.01 μg/ml of the inventive pharmaceutical compositions can significantly inhibit the activity of TNF-α-induced NF-kB promoter (P<0.05, P<0.01), indicating that: the inventive pharmaceutical compositions at concentrations of 0.01, 0.1 and 1 μg/mL each had better anti-inflammatory activities (see Table 13,
###P < 0.001 vs model,
#P < 0.05 vs model.
The inventive pharmaceutical compositions do not have evident antioxidative effects, and the pharmaceutical compositions of the invention at 0.01-10 μg/ml can evidently inhibit the activity of TNF-α induced NF-kB, having significantly anti-inflammatory activity.
The invention and its embodiments have been described above generally and in detail. It will be appreciated by those skilled in the art that according to the teachings in the present application, various modifications, alternatives and other changes may be made directed to the details in the invention, and the technical solutions obtained by these changes each are in the scope of the invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202010989020.1 | Sep 2020 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/102174 | 6/24/2021 | WO |
