CN114965763A - Traditional Chinese medicine composition for oropharyngeal inflammatory diseases and multi-index quantitative determination method - Google Patents

Traditional Chinese medicine composition for oropharyngeal inflammatory diseases and multi-index quantitative determination method Download PDF

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CN114965763A
CN114965763A CN202210549381.3A CN202210549381A CN114965763A CN 114965763 A CN114965763 A CN 114965763A CN 202210549381 A CN202210549381 A CN 202210549381A CN 114965763 A CN114965763 A CN 114965763A
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spray
ethanol
kaihoujian
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menthol
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CN114965763B (en
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张海
王珏犇
蒋露
龙银青
田成旺
陈金鹏
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Guizhou Sanli Pharmaceutical Co ltd
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Abstract

The invention relates to a traditional Chinese medicine composition for oropharyngeal inflammatory diseases and a multi-index quantitative determination method. In one aspect, the invention relates to a method for simultaneously and quantitatively measuring multi-index medicinal components of Kaihoujian spray, wherein the prescription of the Kaihoujian spray is as follows: radix Ardisiae Crenatae, radix Sophorae Tonkinensis, periostracum Cicadae, Mentholum and water; the active ingredients comprise matrine, bergenin and trifoliate red sandalwood glycoside; the method uses high performance liquid chromatography to simultaneously determine effective components therein, and has high detection efficiency. The invention also relates to a Chinese medicinal composition which can be called as Kaihoujian spray and is used for treating the oropharyngeal inflammatory diseases, and the Chinese medicinal composition has the effects of clearing away heat and toxic materials, and relieving swelling and pain; the traditional Chinese medicine composition can be clinically used for acute and chronic pharyngolaryngitis, tonsillitis, sore throat, stomatitis, swollen and painful gum, swollen and painful throat caused by lung and stomach intrinsic heat, dry mouth and bitter taste, swollen and painful gum, dental ulcer and recurrent aphtha with the symptoms.

Description

Traditional Chinese medicine composition for oropharyngeal inflammatory diseases and multi-index quantitative determination method
Technical Field
The invention belongs to the technical field of medicines, and relates to a traditional Chinese medicine composition for oropharyngeal inflammatory diseases, which is a traditional Chinese medicine preparation of Kaihoujian spray of Miao medicine and has the effects of clearing away heat and toxic materials, and relieving swelling and pain; can be clinically used for treating acute and chronic pharyngolaryngitis, tonsillitis, sore throat, stomatitis and gingival swelling and pain; for example, it can be used for treating swelling and pain of throat, dry mouth, bitter taste of mouth, swelling and pain of gum, oral ulcer, and recurrent aphtha caused by lung and stomach heat accumulation. The traditional Chinese medicine composition can be particularly used for treating infantile oropharyngeal inflammatory diseases.
Background
The infant is tender and delicate in viscera, is the body of 'young yin and young yang', is not full in shape and qi, is normally loose in striae, is easy to be infected by exogenous pathogenic factors, and is easy to grab articles beside the body into the mouth due to curiosity and exuberance of the children on external things. With the influence of external factors such as climate change, environmental pollution and the like, families are not paying attention to the environment, children are easy to enter the environment from the mouth, and the secret of infant care (Dayun) is recorded in the book: the infants have delicate viscera, frequent insufficiency of the spleen and dysfunction of transportation and transformation, and are very easy to get ill. Epidemic toxin is in the mouth, nose and lung, stagnates in lung and spleen, and is caused by hands and feet, smokes the mouth and throat, and penetrates the skin externally, and causes herpes. "paediatrics clinical oral ulcer belongs to the disease that the common incidence is higher, it means that oral mucosa surface takes place superficial ulcer, and the pathogenesis is mainly coxsackie virus, bacterial infection, hand-foot-and-mouth disease and recurrent aphtha etc. and the infant often shows symptoms such as salivation, fever and crying [ Zhang Bo, etc.. montmorillonite powder is united kaihoujian and is compared with Bingpeng san and is treated the curative effect analysis of infant oral ulcer [ J ]. Chinese woman child health research, 2017,28(S1):495], wherein the hand-foot-and-mouth disease is acute fever eruption infectious disease caused by enterovirus, is better developed for infants under 3 years old, is mainly manifested as red maculopapule and small vesicular rash of the skin of the hand and the hip, and the mucous membrane in the oral cavity is scattered in the small ulcer and the small vesicular rash, fever, reduced appetite, food refusal or vomiting after eating can occur, and the disease can be transmitted through close contact, respiratory tract and digestive tract. In addition, the infantile tonsillitis and the acute pharyngitis are common respiratory infectious diseases of children, and the children are taken as main pathogenic people, so that the children have the characteristics of acute onset of disease, fast disease progression and the like, and the symptoms such as angina, fever, cough, expectoration, hoarseness, dysphagia and the like are mainly clinically manifested; the acute pharyngolaryngitis of children is frequently generated in infants from 6 months to 3 years old, the infants can develop the acute pharyngolaryngitis all the year round, the early stage of the acute pharyngolaryngitis is manifested by burning and drying of the pharynx, pain is accompanied after the transition of the disease state, the pain is aggravated when being coughed and swallowed, and the chronic pharyngolaryngitis easily progress to the chronic pharyngolaryngitis [ Sunjing, and the like ] Kaihu spray (child type) for treating the acute pharyngitis and the acute tonsillitis of the children is discussed [ J ]. China journal of Experimental and formulary, 2019,25(10): 33-40; clinic analysis of Kaihoujian spray combined with Kegan Liyan oral liquid for treating acute tonsillitis in children [ J ] New Chinese medicine, 2016,48(03):158 and 160 ].
The Chinese medicine Kaihoujian spray is a Miao medicine compound preparation produced by Guizhou Sanli company, the content is light brown to brown liquid, the taste is sweet, slightly bitter and slightly numb, the spray has the cool feeling of mint, and the prescription is as follows: radix Ardisiae Crispae, radix Sophorae Tonkinensis, periostracum Cicadae, and Mentholum. The Kaihoujian spray has the functions of clearing away heat and toxic material, eliminating swelling, relieving pain, etc. and is used clinically in treating acute and chronic laryngopharyngitis, tonsillitis, sore throat, stomatitis, gingival swelling and pain, etc. The eight-claw golden dragon is collected to the quality standard of traditional Chinese medicinal materials and national medicinal materials in Guizhou province (2003 edition), and comprises three basic sources, namely: the ardisia crenata sims, the crispateleaf ardisia crenata and the red cool umbrella are collected and loaded into 2015 and 2020 edition of Chinese pharmacopoeia, so that the octopus dragon in 2019 edition of quality standard of traditional Chinese medicinal materials and national medicinal materials in Guizhou province only collects two basic sources of the crispateleaf ardisia crenata and the red cool umbrella, and the octopus dragon in the prescription of the Kaihoujian spray is named as the ardisia crenata sims.
The Kangjian spray directly acts on oral mucosa in a spray administration mode, the maximum medicine concentration is easily formed at a focus position, the Kangjian spray has the advantages of high bioavailability, quick response, strong effect and the like, a mint cooling feeling is generated after spraying, pain of an infant patient caused by methods such as smearing and the like is avoided, and compliance of the infant patient is improved [ Penjian, and the like ] the curative effect observation of the Kangjian anti-inflammatory oral liquid combined with the Kangjian spray on treating the infantile hand-foot-and-mouth disease [ J ] the traditional Chinese medicine guide report, 2012, 18(03):41-42 ].
Ardisia crenata is dried root of Ardisia crenata of Myrsinaceae. Collected in autumn and winter, cleaned and dried in the sun. Radix Ardisiae Crenatae is cool in nature, bitter and pungent in taste, enters lung and stomach channels, has the effects of clearing heat and removing toxicity, relieving swelling and stasis, promoting blood circulation to arrest pain, and dispelling pathogenic wind and removing dampness, is clinically used for treating acute pharyngitis, tonsillitis, sore throat, rheumatic arthralgia, traumatic injury and other symptoms, and is called as a good medicine for the throat by Miao nationality.
Radix sophorae tonkinensis is bitter and cold in property and toxic in property, enters lung and stomach channels, has the effects of clearing heat and removing toxicity, and reducing swelling and relieving sore throat, and is used for treating diseases such as fire toxin accumulation, sore throat, lung heat cough, tonsillitis pharyngitis, aphthous stomatitis and the like [ national pharmacopoeia committee, pharmacopoeia of the people' S republic of China, first part [ S ]. Beijing: the Chinese medicine science and technology publishing company, 2020, Kaibao Bencao records that the medicines mainly relieve toxicity, relieve pain and eliminate blister and swelling toxin, and the subprostrate sophora root in Bencao Jing Shu is the upper medicine for removing toxicity and clearing heat; the book Ben Cao Zhen she is the first essential herb for relieving swelling and pain in throat.
Cicada slough is sweet and cold in property, enters lung and liver channels, has the effects of dispelling wind and heat, improving eyesight, removing nebula, relieving sore throat, promoting eruption and the like, and is mainly used for treating wind and heat type cold, sore throat, hoarseness, febrile convulsion, itching and frequent cough and the like clinically [ Moxinlan, and the like. The compendium of materia Medica records: the cicada is mainly used for treating all wind-heat syndromes, ancient people use the cicada for treatment, later use the cicada for treatment of meridian and collateral in zang-fu organs, and the cicada is used for treatment; for sores and ulcers, wind-heat, it is used with Chan tui. Record in Ben Cao Yan Yi (augmented materia Medica): for blurred vision, nebula. Decoction of Hu Su is added to treat the infantile eczema. The book of the drug property treatise is also written: it is indicated for children's epilepsy due to intense heat and convulsions all over the body, and also for quenching thirst.
Menthol, also known as menthol, has pharmacological effects such as analgesic, anticancer, and anti-inflammatory effects [ Dedu-menthol has neuroprotective effect on LPS-induced Parkinson disease model and its mechanism [ D ]. Jilin university, 2021.DOI: 10.27162/d.cnki.gjilin.2021.005422 ].
Research shows that the fenugreek spray has better clinical curative effect on proteus, staphylococcus aureus, candida albicans and the like [ highly conserved and convenient analysis of the curative effect of the fenugreek spray on treating the acute pharyngolaryngitis of children [ J ]. Chinese medical abstracts (otorhinolaryngology), 2022,37(01):72-73], in addition, the fenugreek spray can be used together with other medicines or treatment methods, and can be used for treating the herpetic angina of children [ phyllocrytis, and the like besides the enhanced anti-inflammatory effect, the clinical curative effect and the safety of the oral liquid of the laryngopharynx cleaning and the fenugreek spray on treating the herpetic angina of children [ J ]. clinical reasonable medication journal, 2021,14(21):51-54] and the laryngeal cough [ Song Xiao, and the like ] acupoint application is matched with the fenugreek spray to treat the laryngeal cough of children [ J ]. practical traditional Chinese medicine journal, 2015,31(08): 775-776), and the like.
CN114200040A (application No. 202111415900.9) discloses a content determination method of Kaihoujian spray (children type), which adopts high performance liquid chromatography to establish a content determination method for determining the content of matrine, bergenin, trifolio red sandalwood glycoside and macaine in the Kaihoujian spray (children type), and uses bergenin as an internal standard substance for one-test multi-evaluation. The detection result is accurate and stable, and the method can be used for quality control of Kaihoujian spray (children type); meanwhile, the invention can reduce the detection cost and detection time, reduce the workload and improve the efficiency, and lays a foundation for improving the quality standard of the Kaihoujian spray (children type). However, the chromatographic run time of each measurement of this method is as long as 70min, different substances require the use of different detection wavelengths for this purpose requiring switching of the detection wavelengths during the run, and the calculation requires a cumbersome method of employing relative correction factors, the efficiency and applicability of which are to be improved.
However, there is still a need in the art for new pistachio sprays, e.g. new methods to control the quality of pistachio sprays.
Disclosure of Invention
The invention aims to provide a fenugreek spray or a method for controlling the quality of the fenugreek spray. It has been found that the method of the present invention can provide an effective quality test for Kanghou spray, and further can effectively test the chemical quality of Kanghou spray in the production process, storage and transportation process or use process of medicine. The present invention has been completed based on such findings.
Therefore, the invention provides a method for simultaneously and quantitatively measuring multi-index active ingredients of the Kaihoujian spray, wherein the active ingredients comprise matrine, bergenin and trifolium pterocarpus santaline.
The method according to the first aspect of the invention, comprising the operations of:
(1) providing a high performance liquid chromatograph, and providing a reference substance and a test substance of the drug effect component;
(2) chromatographic conditions are as follows:
uses octadecylsilane chemically bonded silica as a chromatographic column of a filler,
the column temperature was 25 c,
the measurement wavelength was 203nm,
mobile phase: acetonitrile (a) -0.1% formic acid solution (B) and mobile phase eluted with a gradient as follows:
t/min A/％
0 3
5 5
25 14
40 28
50 35
55 45
flow rate: 1 mL/min;
(3) preparation of control solutions: preparing control solutions of matrine, bergenin and trifoliolate pterocarpus santalinus control with 40% methanol as solvent, wherein the concentrations of the control solutions are 40-90 μ g/ml, such as about 65 μ g/ml, 70-130 μ g/ml, such as about 100 μ g/ml and 4-9 μ g/ml, such as about 6.5 μ g/ml;
(4) preparing a test solution: precisely sucking 2mL of Kaihoujian spray into a 10mL measuring flask, diluting with 40% methanol to scale, and filtering with 0.45 μm microporous membrane to obtain the final product;
(5) and (3) determination: respectively absorbing the test solution and various reference solutions, injecting the test solution and various reference solutions into a liquid chromatograph, recording a chromatogram, determining the retention time of each drug effect component in the chromatogram of the test solution according to the retention time of a main peak of the chromatogram of the reference, calculating the content of each drug effect component in the test solution according to the peak areas of each drug effect component in the chromatogram of the reference solution and the chromatogram of the test solution and the concentration of the reference solution, and calculating the content of each drug effect component in the kakko Swinhonis spray according to the dilution times.
The method according to the first aspect of the present invention, wherein the injection amount in the step (5) into the liquid chromatograph is 5 to 50. mu.L, for example, 10. mu.L.
The method according to the first aspect of the present invention, wherein the packing of the chromatography column has a particle size of 5 μm.
The method according to the first aspect of the invention, wherein the inner diameter of the chromatography column is 4.6.
The method according to the first aspect of the present invention, wherein the length of the chromatography column is 250 mm.
The method according to the first aspect of the invention, wherein the chromatography column is a 4.6X 250mm, 5 μm Welch Ultimate Plus C18 brand standard chromatography column.
According to the method of the first aspect of the invention, the peak areas and the concentrations of the matrine, the bergenin and the red sandalwood glycoside in the reference solution respectively satisfy R within the concentration ranges of 10-520 μ g/mL, 10-510 μ g/mL and 1-50 μ g/mL when measured by using the reference solution 2 >A linear relationship of 0.999.
According to the method of the first aspect of the present invention, the RSD of the chromatographic peak areas of the test solution measured by 6 times of continuous 6-sample injection measurement is less than 1%, especially less than 0.5%, for all of the 6 measurements of matrine, bergenin and pterocarpus santalinus glycoside.
According to the method of the first aspect of the present invention, the RSD of the chromatographic peak area of the test solution after 6 times of measurement of matrine, bergenin and trifolium indicum pterocarpus santalin is less than 1%, especially less than 0.5% after 0, 2, 4, 8, 12 and 24 hours of sample measurement after the test solution is placed at room temperature for 24 hours.
According to the method of the first aspect of the present invention, the RSDs of the chromatographic peak areas of 6 determinations of matrine, bergenin and trifolium pterocarpum glycoside are less than 1% when the test solution prepared from 6 samples of the same batch of test sample is subjected to sample injection determination.
The process according to the first aspect of the present invention, wherein 1.2% isopropanol and 0.2% ammonium chloride are also added to the acetonitrile in the mobile phase. In the present invention, as not otherwise specified, for the meaning of%,% is volume/volume% for a liquid/liquid mixture,% is mass/volume% for a solid/liquid mixture, and% is mass/mass% for a solid/solid mixture.
The process according to the first aspect of the present invention, wherein 1.2% isopropyl alcohol and 0.2% ammonium chloride are further added to the 0.1% formic acid solution in the mobile phase.
The method according to the first aspect of the invention, wherein the prescription of the pistachio spray is: 220-330 g of ardisia crenata, 220-330 g of subprostrate sophora, 180-270 g of cicada slough, 0.8-1.2 g of menthol, 5.5ml of essence, 1g of citric acid, 1-2.5 g of sodium benzoate, 20ml of ethanol and a proper amount of water, and the mixture is prepared into 1000 ml.
The method according to the first aspect of the invention, wherein the prescription of the pistachio spray is: 250g of ardisia crenata, 250g of subprostrate sophora, 200g of cicada slough, 1g of menthol, 5.5ml of pineapple essence, 1g of citric acid, 1g of sodium benzoate, 20ml of ethanol and a proper amount of water, and the mixture is prepared into 1000 ml.
The method according to the first aspect of the invention, wherein the prescription of the pistachio spray is: 313g of ardisia crenata, 313g of subprostrate sophora, 250g of cicada slough, 1g of menthol, 5.5ml of waxberry essence, 1g of citric acid, 2.5g of sodium benzoate, 20ml of ethanol and a proper amount of water, and the mixture is prepared into 1000 ml.
The method according to the first aspect of the invention, wherein the pistachio spray is prepared by: decocting the four medicinal materials except the menthol and the other three medicinal materials such as the ardisia crenata and the like in water twice, 2 hours for the first time and 1 hour for the second time, merging decoction, filtering, concentrating the filtrate to obtain clear paste with the relative density of 1.05-1.10 (50 ℃), adding ethanol to ensure that the ethanol content reaches 80%, standing for 24 hours, filtering, recovering the ethanol from the filtrate under reduced pressure, concentrating to obtain clear paste with the relative density of 1.10-1.20 (80 ℃), taking 20ml of menthol, sodium benzoate, citric acid, pineapple essence and ethanol, stirring and dissolving, adding the mixture into the extract, adding water to a specified amount, uniformly stirring, filtering and filling to obtain the composition.
Further, the invention provides a sword spray for throat, which is prepared by the following steps: 220-330 g of ardisia crenata, 220-330 g of subprostrate sophora, 180-270 g of cicada slough, 0.8-1.2 g of menthol, 5.5ml of essence, 1g of citric acid, 1-2.5 g of sodium benzoate, 20ml of ethanol and a proper amount of water, and the mixture is prepared into 1000 ml.
According to the second aspect of the invention, the prescription of the Kaihoujian spray is as follows: 250g of ardisia crenata, 250g of subprostrate sophora, 200g of cicada slough, 1g of menthol, 5.5ml of pineapple essence, 1g of citric acid, 1g of sodium benzoate, 20ml of ethanol and a proper amount of water, and the mixture is prepared into 1000 ml.
According to the second aspect of the invention, the prescription of the Kaihoujian spray is as follows: 313g of ardisia crenata, 313g of subprostrate sophora, 250g of cicada slough, 1g of menthol, 5.5ml of waxberry essence, 1g of citric acid, 2.5g of sodium benzoate, 20ml of ethanol and a proper amount of water, and the mixture is prepared into 1000 ml.
According to the second aspect of the invention, the Kaihoujian spray is prepared by the following steps: decocting the four medicinal materials except the menthol and the other three medicinal materials such as the ardisia crenata and the like in water twice, 2 hours for the first time and 1 hour for the second time, merging decoction, filtering, concentrating the filtrate to obtain clear paste with the relative density of 1.05-1.10 (50 ℃), adding ethanol to ensure that the ethanol content reaches 80%, standing for 24 hours, filtering, recovering the ethanol from the filtrate under reduced pressure, concentrating to obtain clear paste with the relative density of 1.10-1.20 (80 ℃), taking 20ml of menthol, sodium benzoate, citric acid, pineapple essence and ethanol, stirring and dissolving, adding the mixture into the extract, adding water to a specified amount, uniformly stirring, filtering and filling to obtain the composition.
In the above-described steps of the preparation method of the present invention, although the specific steps described therein are distinguished in some detail or in language specific to the steps described in the preparation examples of the following detailed description, those skilled in the art can fully summarize the above-described method steps in light of the detailed disclosure of the entire disclosure of the invention.
Any embodiment of any aspect of the invention may be combined with other embodiments, as long as they do not contradict. Furthermore, in any embodiment of any aspect of the invention, any feature may be applicable to that feature in other embodiments, so long as they do not contradict. The invention is further described below.
All documents cited herein are incorporated by reference in their entirety and to the extent such documents do not conform to the meaning of the present invention, the present invention shall control. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even though such terms and phrases are intended to be described or explained in greater detail herein, reference is made to the term and phrase as being inconsistent with the known meaning and meaning as is accorded to such meaning throughout this disclosure.
The Kaihoujian spray is a product of Guizhou Sanli pharmacy. The Miao medicine function of the children type Kaihoujian spray mainly comprises the following main functions: xuga-24 craba, the genus of Asaham, is critical to Ann-Fengmen; steepness: na, monning palace, mong gagong ang, jiangzhu, agagucy; the Chinese medicine indications are as follows: clear heat and remove toxicity, relieve swelling and alleviate pain. Can be used for treating acute and chronic pharyngolaryngitis, tonsillitis, sore throat, stomatitis, and gingival swelling and pain. The Miao medicine function of the adult Kaihoujian spray mainly comprises the following main functions: treating Song dynasty with Mongolia scales; mongolian, larola; the Chinese medicine indications are as follows: clear heat and remove toxicity, relieve swelling and alleviate pain. Can be used for treating swelling and pain of throat, dry mouth, bitter taste of mouth, gingival swelling and pain, oral ulcer, and recurrent aphtha caused by lung and stomach heat.
Ardisia crenata is dried root of Ardisia crenata Sims of Myrsinaceae. The ardisia crenata mainly contains triterpenoid saponins, coumarins and other types; the saponin structure type is mainly pentacyclic triterpenoid oleanane type derivatives, and aglycone thereof has 2 types: epoxy ethers and 12-alkenes; coumarins are mainly bergenin; also contains some other classes of ingredients: ardisia japonica quinone, friedelin, beta-sitosterol and daucosterol.
The radix Sophorae Tonkinensis is dried root and rhizome of Sophora tonkinensis Gagnep. The radix Sophorae Tonkinensis contains alkaloid, saponin, flavonoid, polysaccharide, etc., and its main medicinal components are alkaloid such as matrine and oxymatrine, and flavonoid such as trifolium pterocarpus santalin.
The invention simultaneously determines three effective components through a chromatographic condition, and can efficiently detect the quality of the Kaihoujian spray.
Drawings
FIG. 1: chromatogram of matrine (retention time t about 9.1min) control.
FIG. 2: bergenin (retention time t about 19.2min) control chromatogram.
FIG. 3: trifolium pterocarpus santalin (retention time t about 46.7min) control chromatogram.
FIG. 4: chromatogram of test sample of KAIHONGJIAN spray shows three chromatographic peaks of matrine (about 9.1min), bergenin (about 19.2min), and santalin (about 46.7 min).
FIG. 5: the chromatogram of the negative test solution shows no three chromatographic peaks of matrine, bergenin and trifolium pterocarpus santaline.
Detailed Description
The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible. In the invention, all the medicinal materials are dry medicinal materials and accord with the rules of pharmacopoeia if not specified. In the following examples of the present invention, the charged amount of each material was calculated in parts by weight, and when actually charged, the total weight of the whole materials per batch was not less than 5 kg.
In the following description of the present invention, when preparing the Chinese medicinal composition, the raw material herbs and the auxiliary materials are all in the same batch, unless otherwise specified.
Example 1: preparing Kaihoujian spray
Prescription: 250g of ardisia crenata, 250g of subprostrate sophora, 200g of cicada slough, 1g of menthol, 5.5ml of pineapple essence, 1g of citric acid, 1g of sodium benzoate, 20ml of ethanol and a proper amount of water, and the mixture is prepared into 1000ml (children type).
The preparation method comprises the following steps: decocting three materials of ardisia crenata, subprostrate sophora and cicada slough in water twice, the first time is 2 hours, the second time is 1 hour, merging decoction, filtering, concentrating filtrate to obtain clear paste with the relative density of 1.05-1.10 (50 ℃), adding ethanol to ensure that the ethanol content reaches 80%, standing for 24 hours, filtering, decompressing the filtrate, recovering the ethanol, concentrating to obtain clear paste with the relative density of 1.10-1.20 (80 ℃), taking menthol, sodium benzoate, citric acid, pineapple essence and 20ml of ethanol, stirring for dissolving, adding the mixture into the extract, adding water to a specified amount, stirring uniformly, filtering, and filling to obtain the finished product.
Example 2: preparing Kaihoujian spray
Prescription: 313g of ardisia crenata, 313g of subprostrate sophora, 250g of cicada slough, 1g of menthol, 5.5ml of waxberry essence, 1g of citric acid, 2.5g of sodium benzoate, 20ml of ethanol and a proper amount of water, and the mixture is prepared into 1000ml (adult type).
The preparation method comprises the following steps: decocting three materials of ardisia crenata, subprostrate sophora and cicada slough with water twice, wherein the first time is 2 hours, the second time is 1 hour, merging decoction, filtering, concentrating filtrate to obtain clear paste with the relative density of 1.05-1.10 (50 ℃), adding ethanol to ensure that the ethanol content reaches 80%, standing for 24 hours, filtering, decompressing filtrate, recovering ethanol and concentrating to obtain clear paste with the relative density of 1.10-1.20 (80 ℃), taking menthol, sodium benzoate, citric acid, pineapple essence and 20ml of ethanol, stirring for dissolving, adding the mixture into the extract, adding water to a specified amount, stirring uniformly, filtering and filling to obtain the traditional Chinese medicine.
Example 3: preparing Kaihoujian spray
Prescription: 280g of ardisia crenata, 280g of subprostrate sophora, 225g of cicada slough, 1g of menthol, 5.5ml of waxberry essence, 1g of citric acid, 1.8g of sodium benzoate, 20ml of ethanol and a proper amount of water, and the mixture is prepared into 1000 ml.
The preparation method comprises the following steps: decocting three materials of ardisia crenata, subprostrate sophora and cicada slough in water twice, the first time is 2 hours, the second time is 1 hour, merging decoction, filtering, concentrating filtrate to obtain clear paste with the relative density of 1.05-1.10 (50 ℃), adding ethanol to ensure that the ethanol content reaches 80%, standing for 24 hours, filtering, decompressing the filtrate, recovering the ethanol, concentrating to obtain clear paste with the relative density of 1.10-1.20 (80 ℃), taking menthol, sodium benzoate, citric acid, pineapple essence and 20ml of ethanol, stirring for dissolving, adding the mixture into the extract, adding water to a specified amount, stirring uniformly, filtering, and filling to obtain the finished product.
Example 4: preparing Kaihoujian spray
Prescription: 220g of ardisia crenata, 220g of subprostrate sophora, 180g of cicada slough, 0.8g of menthol, 5.5ml of waxberry essence, 1g of citric acid, 1.5g of sodium benzoate, 20ml of ethanol and a proper amount of water, and the mixture is prepared into 1000 ml.
The preparation method comprises the following steps: decocting three materials of ardisia crenata, subprostrate sophora and cicada slough in water twice, the first time is 2 hours, the second time is 1 hour, merging decoction, filtering, concentrating filtrate to obtain clear paste with the relative density of 1.05-1.10 (50 ℃), adding ethanol to ensure that the ethanol content reaches 80%, standing for 24 hours, filtering, decompressing the filtrate, recovering the ethanol, concentrating to obtain clear paste with the relative density of 1.10-1.20 (80 ℃), taking menthol, sodium benzoate, citric acid, pineapple essence and 20ml of ethanol, stirring for dissolving, adding the mixture into the extract, adding water to a specified amount, stirring uniformly, filtering, and filling to obtain the finished product.
Example 5: preparing Kaihoujian spray
Prescription: 330g of ardisia crenata, 330g of subprostrate sophora, 270g of cicada slough, 1.2g of menthol, 5.5ml of waxberry essence, 1g of citric acid, 2g of sodium benzoate, 20ml of ethanol and a proper amount of water, and the mixture is prepared into 1000 ml.
The preparation method comprises the following steps: decocting three materials of ardisia crenata, subprostrate sophora and cicada slough in water twice, the first time is 2 hours, the second time is 1 hour, merging decoction, filtering, concentrating filtrate to obtain clear paste with the relative density of 1.05-1.10 (50 ℃), adding ethanol to ensure that the ethanol content reaches 80%, standing for 24 hours, filtering, decompressing the filtrate, recovering the ethanol, concentrating to obtain clear paste with the relative density of 1.10-1.20 (80 ℃), taking menthol, sodium benzoate, citric acid, pineapple essence and 20ml of ethanol, stirring for dissolving, adding the mixture into the extract, adding water to a specified amount, stirring uniformly, filtering, and filling to obtain the finished product.
Test example 1: method for simultaneously and quantitatively measuring multi-index medicinal components of Kaihoujian spray
In another study, the inventor of the present application has found that the serum pharmaceutical chemistry basis of the xiu jian spray comprises index components such as matrine, bergenin, and pterocarpan glycoside, and the index components are typical pharmacodynamic substances of the xiu jian spray, and the experimental example provides a new method for rapidly and effectively performing quality detection on the xiu jian spray by trying to simultaneously measure the three index components by using an HPLC method.
1. Instruments and reagents
Waters e2695 high performance liquid chromatograph (Waters corporation), electronic balance SOP Secure 125-1CN (one hundred thousand, Sartorius corporation).
Acetonitrile, formic acid, methanol, deionized water and the like are all commercial products meeting the requirement for determination.
2. Reagent
Matrine (reference, lot No. T22M10F88874, purity no less than 98%), bergenin (reference, lot No. H14J10Z79791, purity no less than 98%), trifoliosid (reference, lot No. Y11J11H108216, purity no less than 98%), all of which were purchased from Shanghai-sourced leaf Biotechnology, Inc.
Kaihoujian spray is provided by Guizhou Sanli pharmaceutical products, Inc. (child type, lot numbers: KHJ20190127, KHJ20190610, KHJ20190816) or is made by embodiments of the present invention.
3. Chromatographic conditions
A chromatographic column: welch Ultimate Plus C18 (4.6X 250mm, 5 μm),
the column temperature is 25 ℃, the measuring wavelength is 203nm,
mobile phase: acetonitrile (a) -0.1% formic acid solution (B) and mobile phase eluted with a gradient as follows:
table: mobile phase gradient elution procedure
Figure BDA0003654044990000081
Figure BDA0003654044990000091
Flow rate: 1mL/min, and the sample size is 10. mu.L.
4. Preparation of control solutions
Weighing a proper amount of matrine, bergenin and red sandalwood glycosides reference substances, placing the reference substances into a measuring flask, fixing the volume with 40% methanol to obtain a stock solution with the concentration of 0.5230mg/mL, 1.0210mg/mL and 0.1034mg/mL, filtering the stock solution through a 0.45-micrometer microporous filter membrane to obtain a stock solution, and diluting the stock solution (for example, diluting the stock solution by 2-20 times, for example, diluting by 5-15 times) by using the same solvent according to the determination requirement (for example, estimating the concentration of the test solution and achieving the concentration equivalent to the concentration of the test solution) to prepare a reference substance solution. When preparing the solution for the stock solution of the reference substance, the concentration of the stock solution may be higher or lower than the above concentration, for example, the concentrations of the three substances may be 0.2 to 1mg/mL, 0.5 to 2mg/mL, and 0.02 to 0.5mg/mL, respectively.
5. Preparation of test solution
Precisely sucking 2mL of Kaihoujian spray (KHJ20190127) into a 10mL measuring flask, diluting with 40% methanol to scale, and filtering with 0.45 μm microporous membrane.
6. Preparation of negative test solution
Preparing a negative test solution without ardisia crenata and subprostrate sophora root by referring to a method of national drug standard (WS-10132(ZD-0132) -2002) of Kaihoujian spray (children type) issued by the State food and drug administration: adding 6 times of water into 20g of cicada slough, decocting for two times, decocting for 2 hours for the first time and 1 hour for the second time, combining decoction liquids, filtering, concentrating filtrate to obtain clear paste with the relative density of 1.05-1.10 (50 ℃), adding ethanol to ensure that the ethanol content reaches 80%, standing for 24 hours, filtering, decompressing the filtrate, recovering the ethanol, concentrating to obtain clear paste with the relative density of 1.10-1.20 (80 ℃), taking 0.1g of menthol, 0.55ml of pineapple essence, 0.1g of citric acid and 0.1g of sodium benzoate, dissolving the mixture by using 2ml of ethanol, adding the mixture into the extract after stirring, adding water to 100ml, stirring uniformly, and filtering to obtain a negative sample solution without ardisia crenata and subprostrate.
7. Specialization inspection
Detecting matrine reference substance solution, bergenin reference substance solution, red sandalwood glycoside reference substance solution, kakko Swinhonis spray test solution and negative test solution according to the above chromatogram conditions, wherein the chromatogram is shown in figures 1 to 5, the abscissa in the figure is time (min), the ordinate is AU value, and no scale is displayed due to excessive reduction of the chromatogram (scale proportion of 5 figures is different); however, the abscissa serves as a qualitative basis and the quantitative basis is the peak area otherwise provided from the HPLC instrument, and therefore the scale on the ordinate does not appear to affect the practice of the invention.
The result shows that the negative test solution has no corresponding peak at the chromatographic peak positions of the matrine, the bergenin and the red sandalwood glycoside, and the chromatographic condition has good specificity for the determination of the three components.
8. Methodology survey
8.1. Investigation of linear relationships
Taking the three reference substance stock solutions, diluting the stock solutions step by step to prepare aqueous solution of kuh-seng alkali 0.5230, 0.2615, 0.1046, 0.0523, 0.0262 and 0.0105mg/mL, solution of bergenin 0.5105, 0.2042, 0.1021, 0.0511, 0.0204 and 0.0102mg/mL, and solution of santalin 0.0517, 0.0259, 0.0103, 0.0052, 0.0026 and 0.0010 mg/mL. The measurement is carried out according to the chromatographic conditions of the test example, a linear curve is drawn and analyzed by taking the peak areas of the three components as ordinate (y) and the concentration as abscissa (x), and a linear regression equation and coefficients are shown in the following table.
Table: linear regression equation and range for three reference samples
Composition (A) Linear regression equation R 2 Linear Range (mg/mL)
Matrine y＝21261x-44.535 0.9999 0.0105-0.5230
Bergenin y＝27569x+86.733 0.9992 0.0102-0.5105
Trifolium Millettiae Spectabilis glycoside y＝93260x-34.107 0.9996 0.0010-0.0517
The results show that when the concentrations of the matrine, the bergenin and the trifoliate red sandalwood glycoside are respectively in the concentration ranges of 0.0105-0.5230mg/mL, 0.0102-0.5105mg/mL and 0.0010-0.0517mg/mL, the linear relation between the peak areas and the concentrations is good, and the content determination of the matrine, the bergenin and the trifoliate red sandalwood glycoside can be carried out by adopting an external standard one-point method. The content of the substance to be detected in the test sample can be calculated by using a linear regression equation, or the content of the substance to be detected in the test sample can be calculated by using a reference solution with a suitable concentration (for example, a concentration equivalent to that of the substance to be detected in the test sample) according to an external standard method.
8.2. Precision test
Taking the test sample solution prepared in the test example, carrying out continuous 6-time sample injection determination according to the chromatographic conditions of the test example, recording the chromatographic peak areas of the matrine, the bergenin and the trifolium red sandalwood glycoside and calculating RSD (%), and the results are shown in the following table.
Table: KHJ precision test results (n ═ 6)
Composition (I) Peak area Peak area mean RSD(％)
Matrine 1375815～1385910 1381317 0.28
Bergenin 2912891～2937736 2923715 0.33
Trifolium Millettiae Spectabilis glycoside 605800～609007 607096 0.21
The results showed that the RSD of all three components in the sample solution was 1% or less, indicating that the instrument precision was good under the chromatographic conditions. When the peak area in the above table is calculated by using a linear regression equation of 8.1. linear relation investigation, y is the peak area, x is the concentration taking mu g/ml as a unit, and the concentrations calculated by three components such as matrine and the like in the test solution are 65.0 mu g/ml, 106.1 mu g/ml and 6.5 mu g/ml respectively; performing HPLC determination according to test solution prepared by diluting spray 5 times, thereby calculating concentrations of three components such as matrine in KANGHONGJIAN spray (KHJ20190127) respectively at 325.0 μ g/ml, 530.5 μ g/ml, and 32.5 μ g/ml; the same applies below. According to the concentrations of the three components measured in the test solution, when preparing the control solution, preferably when the concentrations are calculated by an external standard method, the concentrations of the three components in the control solution are 40-90 μ g/ml, such as about 65 μ g/ml, 70-130 μ g/ml, such as about 100 μ g/ml, and 4-9 μ g/ml, such as about 6.5 μ g/ml, respectively. Of course, the concentration of the control solution can also be adjusted if the concentration of the test solution changes significantly.
8.3. Stability test
Taking the test sample solution prepared in the test example, standing at room temperature for 0, 2, 4, 8, 12 and 24 hours, measuring according to the chromatographic conditions of the test example, recording chromatographic peak areas of matrine, bergenin and trifolium indicum pterocarpus santaline and calculating RSD (%), and obtaining the results shown in the following table.
Table (b): KHJ results of stability test
Composition (A) Peak area (0 to 24h, n is 6) Peak area mean RSD(％)
Matrine 1375815～1385910 1380708 0.25
Bergenin 2912891～2947003 2926493 0.46
Trifolium Millettiae Spectabilis glycoside 605800～609007 606836 0.20
The results show that the RSD of the three components in the test solution is within 1 percent, and that KHJ is stable within 24 hours.
8.4. Repeatability test
2mL of the test solution is precisely absorbed from 6 bottles of KHJ (KHJ20190127), the test solution is prepared according to the method of the test example, the chromatographic conditions of the test are used for measurement, the chromatographic peak areas of the matrine, the bergenin and the trifoliolate pterocarpan glycoside in the test solution are respectively recorded, and RSD (%) is calculated, and the results are shown in the table below.
Table: sample repeatability test results (n ═ 6)
Composition (I) Peak area Peak area mean RSD(％)
Matrine 1367527～1380705 1372410 0.34
Bergenin 2863709～2929903 2909063 0.81
Trifolium Millettiae Spectabilis glycoside 598458～606406 602444 0.42
The result shows that the RSD of each component is within 1 percent, which indicates that the tested solution has good reproducibility under the chromatographic condition and meets the requirement of content determination.
8.5. Sample application recovery test
Precisely sucking 1mL of Kaihoujian spray (KHJ20190127) into a 5mL measuring flask, adding 0.0523mg/mL of the sophocarpidine solution, 0.1021mg/mL of the bergenin solution and 0.0052mg/mL of the trifolium pterocarpus santaline solution which are described in the test example into the measuring flask respectively by 0.5mL, fixing the volume of 40% methanol to a scale, filtering the solution through a 0.45-micron filter membrane to obtain a sample solution with the sample loading rate of 50%, and setting n to be 3. 1mL and 1.5mL of control solutions were added to prepare 100% and 150% sample solutions, respectively, according to the above procedures. The chromatographic conditions of the test example are used for measurement, the chromatographic peak areas of the matrine, the bergenin and the red sandalwood glycoside of the trifolium are recorded, the average recovery rate and the RSD (%) are calculated, and the results are shown in the table.
Table: sample recovery test results
Figure BDA0003654044990000121
The results show that the RSD of the matrine, the bergenin and the red sandalwood glycoside of the trifoliate beans under the condition is less than 2 percent, and the requirement of methodology verification is met.
9. Measurement results
The contents of matrine, bergenin and trifoliolate pterocarpan glycoside in three batches of kaihoujian sprays KHJ20190127, KHJ20190610 and KHJ20190816 and five batches of kaihoujian sprays prepared in the embodiments 1 to 5 of the present invention were measured, and the results are shown in the following table.
Table: index content of effective component in the composition (μ g/ml)
Matrine Bergenin Trifolium Millettiae Spectabilis glycoside
KHJ20190127 325.7 531.2 32.2
KHJ20190610 331.6 526.4 33.5
KHJ20190816 327.3 534.8 32.6
Example 1 328.6 530.5 33.3
Example 2 411.3 662.2 40.1
Example 3 368.7 596.8 37.3
Example 4 289.5 465.4 28.5
Example 5 431.8 694.5 42.7
The test example establishes a method for measuring the content of multiple index components in Kaihoujian spray by adopting an HPLC technology, comprises the content measurement of matrine, bergenin and trifoliolate pterocarpan glycoside, and the RSD of precision, repeatability, stability and sample adding recovery rate in a calculation methodology all meet the requirements in a certain linear range, thereby showing that the method is feasible for simultaneously measuring the three index pharmacodynamic components.
Test example 2: method for simultaneously and quantitatively measuring multi-index medicinal components of Kaihoujian spray
When various test solutions were measured in test example 1 above, it was found that after the test solutions were injected into the liquid chromatograph, the column pressure at the initial test was usually between 80 and 90bar, but after the test solutions were injected several times, the column pressure gradually increased, for example, after 5 test solutions were injected, the column pressure increased to between 120 and 130bar, while after 10 test solutions were injected, the column pressure increased to between 150 and 160bar, and after 15 test solutions were injected, the column pressure increased to between 210 and 220bar, and further increase of the column pressure was unacceptable; the column pressure can be returned to the initial state after 2 hours by backflushing the column with a mobile phase a/mobile phase B at a ratio of 10/90, but this operation is not desirable in large sample size analysis tests; it has been found that this increase in column pressure does not occur when testing the control solution, indicating that this increase in column pressure is due to the relevant components in the test solution. The inventor tries to use other brands of C18 columns (the column length, the column inner diameter and the packing granularity are the same), namely a Supelcoc C18 column, an Agilent C18 column and a Waters C18 column, tests are carried out according to test example 1, and the results also show that the column pressure is unacceptably increased along with the tests, for example, the column pressure of the three columns is increased to 202-210 bar, 185-192 bar and 205-212 bar respectively after 12 test sample solutions are injected into the three columns for testing. The inventor tries to simultaneously add 1.2% isopropanol and 0.2% ammonium chloride in mobile phase A, namely acetonitrile, and mobile phase B, namely 0.1% formic acid solution, so that both A and B contain two additives with the same concentration, and the elution is carried out according to the gradient elution procedure of experimental example 1, and other operation conditions are kept unchanged, and the results show that the initial column pressure is between 80 and 90bar when the test sample solution and other solutions are tested, the column pressure is still kept in the column pressure state after various solutions are tested for more than 30 times, for example, the column pressure is still between 85 and 95bar when the test sample solution is injected for 30 times, and the column pressure is still between 92 and 97bar when the test sample solution is injected for 50 times. Further, the inventors tried to add 1.2% isopropanol to both mobile phase a, i.e. acetonitrile, and mobile phase B, i.e. 0.1% formic acid solution, so that both a and B contain the same concentration of the additive, and still perform elution according to the gradient elution procedure of test example 1, and the other operation conditions are kept unchanged, and the results show that, when testing the sample solution and other solutions, the initial column pressure is between 80 and 90bar, the column pressure is still gradually increased when testing the sample solution, for example, the column pressure is increased to between 109 and 115bar after injecting 5 sample solutions, the column pressure is increased to between 133 and 140bar after injecting 10 sample solutions, and the column pressure is increased to between 160 and 168bar after injecting 15 sample solutions. Further, the inventor tries to add 0.2% ammonium chloride to both mobile phase a, i.e. acetonitrile, and mobile phase B, i.e. 0.1% formic acid solution, so that both a and B contain the same concentration of the additive, and still perform elution according to the gradient elution procedure of experimental example 1, and the other operation conditions are kept unchanged, and the results show that, when testing the sample solution and other solutions, the initial column pressure is between 80 and 90bar, the column pressure is still gradually increased when testing the sample solution, for example, the column pressure is increased to between 115 and 122bar after injecting 5 sample solutions, the column pressure is increased to between 140 and 150bar after injecting 10 sample solutions, and the column pressure is increased to between 180 and 190bar after injecting 15 sample solutions. From this, it is known that the simultaneous addition of both isopropyl alcohol and ammonium chloride to the mobile phase can avoid the problem of an increase in column pressure when testing the sample solution.
For this reason, in this test example 2, the same operation conditions as in test example 1 were used except that 1.2% isopropyl alcohol and 0.2% ammonium chloride were added to both of the mobile phase a, i.e., acetonitrile, and the mobile phase B, i.e., 0.1% formic acid solution, and various test solutions were prepared using test example 1, and the results of the various tests were substantially the same as in test example 1, and typical results were as follows: 1) retention time: matrine t is about 9.1min, bergenin t is about 19.1min, and trifolium red sandalwood glycoside t is about 46.5min, which is basically unchanged; 2) linear regression equation: matrine y ═ 21238x-44.457 and R 2 0.9999, bergenin y 27577x +86.624 and R 2 0.9997, pterocarpus santalin y 93253x-34.145 and R 2 0.9998; 3) KHJ20190127 precision of 20190127 test sample: the mean values of the areas of the matrine and the bergenin are 1380561 and 0.25 respectively, 2924216 and 0.22 respectively, 607144 and 0.35 respectively; 4) KHJ20190127 24h stability test of 20190127 test article: the mean values of the areas of the matrine and the bergenin are 1380268 and 0.33 respectively, 2923523 and 0.41 respectively, 604327 and 0.38 respectively; 5) KHJ20190127 repeatability test of 20190127 test article: the mean values of the areas of the matrine and the bergenin are 1372084, 0.51%, 2908473 and 0.43%, 602836 and 0.62%; 6) KHJ20190127 sample application recovery test of 20190127 test: the average recovery rate of three concentrations of matrine is 99.64% and RSD is 1.33%, the average recovery rate of three concentrations of bergenin is 101.46% and RSD is 1.26%, and the average recovery rate of three concentrations of trifolium pterocarpus santalin is 101.84% and RSD is 1.64%; 7) the content (mug/ml) of matrine, bergenin and red sandalwood glycoside of 5 batches of fenugreek sprays is simultaneously measured, and the result is that: KHJ20190127 the contents of the three components are 324.4 μ g/ml, 530.9 μ g/ml, 32.3 μ g/ml, KHJ2019The contents of three components of 0610 are respectively 332.2 mu g/ml, 525.6 mu g/ml and 32.6 mu g/ml, the contents of three components of KHJ20190816 are respectively 326.7 mu g/ml, 535.5 mu g/ml and 32.5 mu g/ml, the contents of three components of example 1 are respectively 329.4 mu g/ml, 531.4 mu g/ml and 32.4 mu g/ml, the contents of three components of example 2 are respectively 412.5 mu g/ml and 661.3 mu g/ml, 41.3 mu g/ml, 367.8 mu g/ml, 595.7 mu g/ml and 37.2 mu g/ml for the three components in example 3, 288.3 mu g/ml, 464.5 mu g/ml and 28.6 mu g/ml for the three components in example 4, and 430.5 mu g/ml, 695.3 mu g/ml and 43.4 mu g/ml for the three components in example 5.
The HPLC method of test example 2, in which 2 reagents are supplemented and added simultaneously in the mobile phase, can be used for measuring the content of multiple index components in Kanghou spray, including the content of matrine, bergenin and trifolio red sandalwood glycoside, and has excellent methodological performance.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims (10)

1. The method for simultaneously and quantitatively measuring the multi-index medicinal components of the Kaihoujian spray comprises the following steps: 220-330 g of ardisia crenata, 220-330 g of subprostrate sophora, 180-270 g of cicada slough, 0.8-1.2 g of menthol, 5.5ml of essence, 1g of citric acid, 1-2.5 g of sodium benzoate, 20ml of ethanol and a proper amount of water, and the mixture is prepared into 1000 ml; the effective components include matrine, bergenin and trifoliolate pterocarpan glycoside.
2. The method according to claim 1, comprising the operations of:
(1) providing a high performance liquid chromatograph, and providing a reference substance and a test substance of the drug effect component;
(2) chromatographic conditions are as follows:
uses octadecylsilane chemically bonded silica as a chromatographic column of a filler,
the column temperature was 25 c,
the measurement wavelength was 203nm,
mobile phase: acetonitrile (a) -0.1% formic acid solution (B) and mobile phase eluted with a gradient as follows:
t/min A/％ 0 3 5 5 25 14 40 28 50 35 55 45
flow rate: 1 mL/min;
(3) preparation of control solutions: preparing control solutions of matrine, bergenin and trifoliolate pterocarpus santalinus control with 40% methanol as solvent, wherein the concentrations of the control solutions are 40-90 μ g/ml, such as about 65 μ g/ml, 70-130 μ g/ml, such as about 100 μ g/ml and 4-9 μ g/ml, such as about 6.5 μ g/ml;
(4) preparing a test solution: precisely sucking 2mL of Kaihoujian spray into a 10mL measuring flask, diluting with 40% methanol to scale, and filtering with 0.45 μm microporous membrane to obtain the final product;
(5) and (3) determination: respectively absorbing the test solution and various reference solutions, injecting the test solution and various reference solutions into a liquid chromatograph, recording a chromatogram, determining the retention time of each drug effect component in the chromatogram of the test solution according to the retention time of a main peak of the chromatogram of the reference, calculating the content of each drug effect component in the test solution according to the peak areas of each drug effect component in the chromatogram of the reference solution and the chromatogram of the test solution and the concentration of the reference solution, and calculating the content of each drug effect component in the kakko Swinhonis spray according to the dilution times.
3. The method according to claim 1, wherein the amount of sample is 5 to 50 μ L, such as 10 μ L, when the injection into the liquid chromatograph in step (5) is performed.
4. The method of claim 1, wherein:
the grain diameter of the filler of the chromatographic column is 5 mu m;
the inner diameter of the chromatographic column is 4.6;
the length of the chromatographic column is 250 mm; and/or
The column was a Welch Ultimate Plus C18 brand standard 4.6X 250mm, 5 μm column.
5. The method according to claim 1, wherein the peak area and the concentration of the matrine, the bergenin and the red sandalwood glycoside in the reference solution satisfy R within the concentration ranges of 10-520 μ g/mL, 10-510 μ g/mL and 1-50 μ g/mL respectively, when the determination is carried out by using the reference solution 2 >A linear relationship of 0.999.
6. The method according to claim 1, wherein the sample solution is subjected to 6 consecutive sample injections, and the RSD of the chromatographic peak area of each of the 6 determinations of matrine, bergenin and trifolium pterocarpum glycoside is less than 1%, especially less than 0.5%.
7. The method of claim 1, wherein:
the sample solution is placed at room temperature for 24 hours, and after 0, 2, 4, 8, 12 and 24 hours, the RSD of the chromatographic peak area of 6 times of determination of the matrine, the bergenin and the trifolium red sandalwood glycoside is less than 1 percent, especially less than 0.5 percent;
respectively carrying out sample injection measurement on test solution prepared from 6 samples of the same batch of test products, wherein RSD of chromatographic peak areas of 6-time measurement of matrine, bergenin and trifoliolate pterocarpan glycoside is less than 1%;
in the mobile phase, 1.2% of isopropanol and 0.2% of ammonium chloride are also added into acetonitrile;
in the mobile phase, 1.2% of isopropanol and 0.2% of ammonium chloride are also added into 0.1% of formic acid solution;
the prescription of the Kaihoujian spray is as follows: 250g of ardisia crenata, 250g of subprostrate sophora, 200g of cicada slough, 1g of menthol, 5.5ml of pineapple essence, 1g of citric acid, 1g of sodium benzoate, 20ml of ethanol and a proper amount of water, and the mixture is prepared into 1000 ml.
8. The method of claim 1, wherein the pistachio spray is formulated as: 313g of ardisia crenata, 313g of subprostrate sophora, 250g of cicada slough, 1g of menthol, 5.5ml of waxberry essence, 1g of citric acid, 2.5g of sodium benzoate, 20ml of ethanol and a proper amount of water, and the mixture is prepared into 1000 ml.
9. The method of claim 1, wherein the kaihoujian spray is prepared by: decocting the four medicinal materials except menthol and the other three medicinal materials such as ardisia crenata in water twice, the first time is 2 hours, the second time is 1 hour, merging decoction, filtering, concentrating filtrate to obtain clear paste with the relative density of 1.05-1.10 (50 ℃), adding ethanol until the ethanol content reaches 80%, standing for 24 hours, filtering, recovering ethanol from the filtrate under reduced pressure, concentrating to obtain clear paste with the relative density of 1.10-1.20 (80 ℃), taking menthol, sodium benzoate, citric acid, pineapple essence and 20ml of ethanol, stirring for dissolving, adding the mixture into the extract, adding water to a specified amount, uniformly stirring, filtering and filling to obtain the traditional Chinese medicine composition.
10. A Kaihoujian spray is prepared by the following steps: 220-330 g of ardisia crenata, 220-330 g of subprostrate sophora, 180-270 g of cicada slough, 0.8-1.2 g of menthol, 5.5ml of essence, 1g of citric acid, 1-2.5 g of sodium benzoate, 20ml of ethanol and a proper amount of water, and the mixture is prepared into 1000 ml; for example, the prescription of the pistachio spray is: 250g of ardisia crenata, 250g of subprostrate sophora, 200g of cicada slough, 1g of menthol, 5.5ml of pineapple essence, 1g of citric acid, 1g of sodium benzoate, 20ml of ethanol and a proper amount of water, and the mixture is prepared into 1000 ml; for example, the prescription of the pistachio spray is: 313g of ardisia crenata, 313g of subprostrate sophora, 250g of cicada slough, 1g of menthol, 5.5ml of waxberry essence, 1g of citric acid, 2.5g of sodium benzoate, 20ml of ethanol and a proper amount of water, and the mixture is prepared into 1000 ml; for example, the kaihoujian spray is prepared by the following steps: decocting the four medicinal materials except the menthol and the other three medicinal materials such as the ardisia crenata and the like in water twice, 2 hours for the first time and 1 hour for the second time, merging decoction, filtering, concentrating the filtrate to obtain clear paste with the relative density of 1.05-1.10 (50 ℃), adding ethanol to ensure that the ethanol content reaches 80%, standing for 24 hours, filtering, recovering the ethanol from the filtrate under reduced pressure, concentrating to obtain clear paste with the relative density of 1.10-1.20 (80 ℃), taking 20ml of menthol, sodium benzoate, citric acid, pineapple essence and ethanol, stirring and dissolving, adding the mixture into the extract, adding water to a specified amount, uniformly stirring, filtering and filling to obtain the composition.
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