CN114942288B - Medicinal chemical composition of plasma in vivo in Kaihoujian spray and its confirmation method - Google Patents
Medicinal chemical composition of plasma in vivo in Kaihoujian spray and its confirmation method Download PDFInfo
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- CN114942288B CN114942288B CN202210549723.1A CN202210549723A CN114942288B CN 114942288 B CN114942288 B CN 114942288B CN 202210549723 A CN202210549723 A CN 202210549723A CN 114942288 B CN114942288 B CN 114942288B
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Abstract
The invention relates to a medicinal chemical composition of plasma in vivo in a throat-opening sword spray and a confirmation method thereof. In one aspect, the present invention relates to a method of identifying an active pharmaceutical chemical in a throat spray, the method comprising the acts of: providing a high resolution mass spectrometer and a liquid chromatograph, chromatographic conditions and mass spectrometric conditions, administering and taking blood from experimental animals, processing plasma samples, analyzing the plasma samples, determining absorbed proto-drug components and metabolic components from various plasmas, confirming that the common absorbed proto-drug is a plasma active drug chemical component according to the absorbed proto-drug common in various plasmas, and confirming that the plasma active drug chemical component is pterocarpan-side. Further comprising using a cell assay method to confirm that the active pharmaceutical chemical entering the cell is pterocarside. The spray has effects of clearing heat, detoxicating, detumescence and relieving pain, and can be used for treating acute and chronic pharyngolaryngitis, tonsillitis, laryngopharynx swelling and pain, stomatitis, gingival swelling and pain, etc.
Description
Technical Field
The invention belongs to the technical field of medicines, and relates to a traditional Chinese medicine composition for treating stomatitis, which is a traditional Chinese medicine preparation of a Miao medicine's Kaihoujian spray and has the effects of clearing heat, detoxicating, reducing swelling and relieving pain; can be used for treating acute and chronic pharyngolaryngitis, tonsillitis, laryngopharynx swelling and pain, stomatitis, and gingival swelling and pain in clinic; for example, it can be used for treating throat swelling and pain, dry mouth, bitter taste, gum swelling and pain, oral ulcer, recurrent aphtha caused by lung and stomach heat. The traditional Chinese medicine composition provided by the invention can be especially used for treating children's oral pharyngitis.
Background
The children are young, the viscera are tender and tender, the body is of the type of 'young yin and young yang', the skin striae and sweat pores are not filled, the skin striae are often sparse, the children are susceptible to exogenous evil, in addition, the children are curious and vigorous to the outside, and the children can easily grasp the articles at hand and put the articles into the mouth. Along with the influence of external factors such as climate change, environmental pollution and the like, families are easy to make children 'enter from the mouth' without paying attention to the influence of the external factors, and the 'baby raising' is described as: the infant viscera are tender, spleen is often deficient, and the infant viscera are easy to attack due to dysfunction of transportation and transformation. The epidemic toxin is caused by the invasion of the lung and spleen through the mouth and nose, and is sent to hands and feet, fumigated up the mouth and throat, and externally penetrated through the skin to form herpes. The clinical pediatric oral ulcer belongs to a common disease with higher incidence rate, and is characterized in that superficial ulcers occur on the surface of oral mucosa, the incidence causes are Coxsackie virus, bacterial infection, hand-foot-mouth disease, recurrent aphtha and the like, the infants often show symptoms such as salivation, fever, crying and the like [ Zhang Fubo, and the like ], the analysis of the curative effect of the combination of montmorillonite powder, open throat sword and borneol boron powder for treating the pediatric oral ulcer [ J ]. Chinese women and young health research, 2017,28 (S1): 495], wherein the hand-foot-mouth disease is an acute fever eruptive infectious disease caused by enterovirus and is mainly manifested by red maculopapules and small herpes on hands and feet, buttocks skin, the oral mucosa is scattered on the small ulcers and the small herpes, fever, hypo accompanying and even vomiting after refusing eating or eating, and the infant can be transmitted through the approaches such as close contact, respiratory tract and digestive tract. In addition, the tonsillitis and acute pharyngitis of the children are common respiratory tract infectious diseases, the infants are taken as main morbidity groups, the children have the characteristics of urgent onset, rapid disease progress and the like, and the clinical symptoms such as pharyngalgia, fever, cough, expectoration, hoarseness and dysphagia are mainly presented; the infant acute pharyngolaryngitis is frequently seen in infants from 6 months to 3 years old, the infant can be ill all year round, the early stage is manifested by glowing and dryness of the throat, the illness state is prolonged with pain, the pain is aggravated when cough and swallowing are caused, and the infant acute pharyngolaryngitis is easy to progress to chronic pharyngolaryngitis [ Sun Jing ], etc., the usage amount of the open throat sword spray (children) for treating the infant acute pharyngitis and acute tonsillitis is discussed [ J ]. Chinese experimental journal of prescriptions, 2019,25 (10): 33-40; guan Xiaojuan A.C. throat opening and relieving spray and a clinical analysis of children acute tonsillitis [ J ]. New Chinese medicine 2016,48 (03): 158-160] by combining the oral liquid for treating children acute tonsillitis.
The traditional Chinese medicine throat opening sword spray is a Miao medicine compound preparation produced by Guizhou Sanli company, the content of the Miao medicine compound preparation is light brown to brown liquid, the taste is sweet, slightly bitter and slightly tingling, the cool feeling of mint is achieved, and the prescription is as follows: radix Ardisiae Crispae, radix Sophorae Tonkinensis, periostracum Cicadae, and Mentholum. The spray for opening throat has the effects of clearing heat, detoxicating, detumescence and relieving pain, and is clinically used for treating acute and chronic pharyngolaryngitis, tonsillitis, sore throat, stomatitis, gingival swelling and pain, etc. Wherein, the radix Ardisiae Crispae is collected in quality standard of Chinese medicinal materials and national medicinal materials of Guizhou province (2003 edition), and comprises three basic sources: the cinnabar root, the bailiangjin and the red parachute are loaded into 2015 and 2020 edition of Chinese pharmacopoeia, so that the octopus macrophylla in 2019 edition of Guizhou Chinese herbal medicine quality standard is only loaded with the bailiangjin and the red parachute, and the octopus macrophylla in the throat-opening sword spray formula is named as the cinnabar root.
The oral cavity mucosa spray is directly acted on the oral cavity mucosa in a spray administration mode, the maximum medicine concentration is easy to form at the focus part, the oral cavity mucosa spray has the advantages of high bioavailability, quick response, strong action and the like, and the spray has the cool feeling of mint after spraying, thereby avoiding the pain of children patients caused by methods such as smearing and the like, improving the compliance of children patients [ Peng, and the like.
The radix Ardisiae Crenatae is dry root of radix Ardisiae Crenatae of Philippinidae. Collected in autumn and winter, washed and dried in the sun. Cinnabar root is cool in nature, bitter and pungent in taste, enters lung and stomach meridians, has the effects of clearing heat and detoxicating, relieving swelling and removing stasis, activating blood and relieving pain, dispelling wind and removing dampness, is clinically used for treating symptoms such as acute pharyngitis, tonsillitis, sore throat, rheumatalgia and traumatic injury, and is called as a good medicine of the laryngeal family by Miao nationality.
The radix sophorae tonkinensis has bitter and cold property and toxic property, enters lung and stomach channels, has the effects of clearing heat and detoxicating, and relieving swelling and sore throat, is used for treating symptoms such as fire toxin accumulation, sore throat, cough due to lung heat, throat obstruction of the tonsillitis, sore mouth and tongue and the like [ national formulary committee, pharmacopoeia of people' S republic of China, one part of [ S ]. Beijing: chinese medical science and technology Press, 2020], "Kaibao Bencao" describes that "main drugs are detoxified, analgesic, anti-vesicular and anti-tumor", "Bencao Jingshu" is a drug for detoxification and heat clearing; the "first key medicine for relieving sore throat" is carried in Ben Cao Qizhen ".
Cicada slough has sweet and cold nature, enters lung and liver channels, has the effects of dispelling wind-heat, improving eyesight, removing nebula, relieving sore throat, promoting eruption and the like, and is clinically used for treating wind-heat type common cold, sore throat, hoarseness, febrile convulsion, itching throat, frequent cough and other symptoms [ Meng Xianlan ], and the like. The description of Ben Cao gang mu is: cicada is mainly used for treating all wind-heat syndromes, old people use the cicada body, later people use the slough to treat the channels and collaterals of the zang-fu organs, and when the cicada body is used; for skin sores and ulcers due to wind-heat, it is indicated for periostracum Cicadae. Record in "materia medica derivative: "treating dizziness and nebula". But also decocting the shell juice in water can treat infantile sore and rash. The drug theory is also written: it is indicated for infantile convulsive epilepsy due to heat-strengthening of whole body and combined with thirst quenching.
Menthol, also known as menthol, has pharmacological actions such as pain relieving, anticancer, anti-inflammatory etc. [ Du Jian. Neuroprotection of menthol on LPS-induced parkinsonism model and its mechanism [ D ]. Jilin university, 2021.DOI:10.27162/d.cnki.gjlin.2021.005422].
The research shows that the spray has better clinical curative effects on bacillus proteus, staphylococcus aureus, candida albicans and the like [ high storage jiao ], the analysis of the curative effects of the spray for treating children acute sphagitis [ J ]. Chinese medical abstract (ear, nose and throat science), 2022,37 (01): 72-73], the spray for treating children herpetic angina [ leaf ice and the like ] can be combined with other medicaments or treatment methods, can also be used for treating children herpetic angina besides enhancing anti-inflammatory effect, and can also be used for treating the clinical curative effects of the spray for treating children herpetic angina and the safety thereof [ J ]. Clinical rational medicine journal, 2021,14 (21): 51-54], laryngeal cough [ Song Xiao and the like [ J ]. Acupoint application is matched with the spray for treating children laryngeal cough, 2015,31 (08): 775-776] and the like.
CN114200040a (application No. 202111415900.9) discloses a content determination method of a throat-opening sword spray (children), which adopts a high performance liquid chromatography to establish a content determination method for determining the content of matrine, bergenin, pterocarpine and bergenin in the throat-opening sword spray (children), and uses bergenin as an internal standard substance. The detection result is accurate and stable, and can be used for quality control of a throat opening sword spray (children type); meanwhile, the invention can reduce the detection cost and the detection time, reduce the workload, improve the efficiency and lay a foundation for improving the quality standard of the throat opening sword spray (children type). However, the chromatographic run time of each measurement of this method is up to 70min, different substances need to use different detection wavelengths for which detection wavelengths need to be switched during the run, and calculation needs to be carried out with a troublesome method of a relative correction factor, and the efficiency and applicability of the method of this document are to be improved.
However, there remains a need in the art for new open throat sword sprays, such as new methods to control the quality of open throat sword sprays.
Disclosure of Invention
The invention aims to provide a throat-opening sword spray, or a method for controlling the quality of the throat-opening sword spray. It has been unexpectedly found that the method of the present invention can provide an effective quality detection for a throat-opening sword spray, and thus can effectively detect the chemical quality of the throat-opening sword spray during the production process, the storage process or the use process of a pharmaceutical product. The present invention has been completed based on such findings.
Therefore, the first aspect of the invention provides a method for simultaneously and quantitatively determining multi-index medicinal components of the open throat sword spray, wherein the medicinal components comprise matrine, bergenin and pterocarpan Heterophylla.
The method according to the first aspect of the invention comprises the following operations:
(1) Providing a high performance liquid chromatograph, and providing a reference substance and a test sample of the medicinal components;
(2) Chromatographic conditions:
a chromatographic column using octadecylsilane chemically bonded silica as a filler,
the column temperature is 25 ℃,
the measurement wavelength was 203nm,
Mobile phase: acetonitrile (a) -0.1% formic acid solution (B), mobile phase was eluted with a gradient, the gradient being as follows:
| t/min | A/％ |
| 0 | 3 |
| 5 | 5 |
| 25 | 14 |
| 40 | 28 |
| 50 | 35 |
| 55 | 45 |
flow rate: 1mL/min;
(3) Preparing a reference substance solution: respectively preparing reference substance solutions of matrine, bergenin and pterocarpan henryi reference substance with 40% methanol as solvent, wherein the concentrations are 40-90 μg/ml, such as about 65 μg/ml, 70-130 μg/ml, such as about 100 μg/ml, 4-9 μg/ml, such as about 6.5 μg/ml;
(4) Preparing a test solution: precisely sucking 2mL of the throat-opening sword spray in a 10mL measuring flask, diluting 40% methanol to scale, and passing through a 0.45 μm microporous filter membrane to obtain the final product;
(5) And (3) measuring: respectively sucking the test solution and various reference solutions, injecting into a liquid chromatograph, recording the chromatogram, determining the retention time of each medicinal component in the test solution chromatogram according to the retention time of the main peak of the reference chromatogram, calculating the content of each medicinal component in the test solution according to the peak areas of each medicinal component in the reference solution chromatogram and the test solution chromatogram and the concentration of the reference solution, and calculating the content of each medicinal component in the throat-opening sword spray according to the dilution multiple.
The method according to the first aspect of the present invention, wherein the injection amount of the liquid chromatograph in the step (5) is 5 to 50. Mu.L, for example, 10. Mu.L.
The method according to the first aspect of the invention, wherein the packing particle size of the chromatographic column is 5 μm.
The method according to the first aspect of the invention, wherein the column inner diameter is 4.6.
The method according to the first aspect of the invention, wherein the chromatographic column is 250mm long.
The method according to the first aspect of the invention, wherein the chromatography column is a Welch Ultimate Plus C brand chromatography column of specification 4.6X250 mm,5 μm.
The method according to the first aspect of the invention uses a reference solution, wherein the peak area and the concentration of matrine, bergenin and sansholin in the reference solution respectively satisfy R within the concentration ranges of 10-520 mug/mL, 10-510 mug/mL and 1-50 mug/mL 2 >Linear relationship of 0.999.
The method according to the first aspect of the invention, wherein the sample solution is subjected to 6 consecutive sample injection assays, and the RSD of the chromatographic peak areas of the matrine, bergenin, and pterocarpan in the three 6 assays are all less than 1%, especially less than 0.5%.
The method according to the first aspect of the invention, wherein the sample solution is left at room temperature for 24 hours, and the RSD of the chromatographic peak areas measured 6 times of matrine, bergenin and pterocarpine are all less than 1%, especially less than 0.5%, measured by sample injection after 0, 2, 4, 8, 12 and 24 hours.
According to the method of the first aspect of the invention, the sample solutions prepared from 6 samples of the same batch of samples are respectively subjected to sample injection measurement, and the RSD of chromatographic peak areas measured 6 times by matrine, bergenin and pterocarpan trilobate is less than 1%.
The process according to the first aspect of the present invention, wherein in the mobile phase, acetonitrile is further added with 1.2% isopropyl alcohol and 0.2% ammonium chloride. In the present invention, as for the meaning of% it is% by volume/volume% in the case of a liquid/liquid mixture,% by mass/volume% in the case of a solid/liquid mixture, and% by mass/mass% in the case of a solid/solid mixture, unless otherwise specified.
The method according to the first aspect of the invention, wherein 1.2% isopropyl alcohol and 0.2% ammonium chloride are also added to the 0.1% formic acid solution in the mobile phase.
The method according to the first aspect of the present invention, wherein the throat opening sword spray is prescribed as follows: 220 to 330g of cinnabar root, 220 to 330g of subprostrate sophora, 180 to 270g of cicada slough, 0.8 to 1.2g of menthol, 5.5ml of essence, 1g of citric acid, 1 to 2.5g of sodium benzoate, 20ml of ethanol and a proper amount of water are prepared into 1000ml.
The method according to the first aspect of the present invention, wherein the throat opening sword spray is prescribed as follows: 250g of cinnabar root, 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 to prepare 1000ml.
The method according to the first aspect of the present invention, wherein the throat opening sword spray is prescribed as follows: 313g of cinnabar root, 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 is prepared into 1000ml.
The method according to the first aspect of the invention, wherein the throat-opening sword spray is prepared by the following steps: decocting the four medicinal materials except menthol in water for two times, wherein the first time is 2 hours, the second time is 1 hour, merging decoction, filtering, concentrating filtrate to fluid extract with the relative density of 1.05-1.10 (50 ℃), adding ethanol to ensure that the alcohol content reaches 80%, standing for 24 hours, filtering, recovering ethanol from the filtrate under reduced pressure, concentrating the filtrate to fluid extract with the relative density of 1.10-1.20 (80 ℃), taking menthol, sodium benzoate, citric acid, pineapple essence and 20ml of ethanol, stirring and dissolving, adding water to a specified amount, stirring uniformly, filtering, and filling to obtain the traditional Chinese medicine.
Further, the second aspect of the invention provides a throat-opening sword spray, which is prepared by the following steps: 220 to 330g of cinnabar root, 220 to 330g of subprostrate sophora, 180 to 270g of cicada slough, 0.8 to 1.2g of menthol, 5.5ml of essence, 1g of citric acid, 1 to 2.5g of sodium benzoate, 20ml of ethanol and a proper amount of water are prepared into 1000ml.
According to the second aspect of the invention, the throat-opening sword spray comprises the following components: 250g of cinnabar root, 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 to prepare 1000ml.
According to the second aspect of the invention, the throat-opening sword spray comprises the following components: 313g of cinnabar root, 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 is prepared into 1000ml.
According to the second aspect of the invention, the throat-opening sword spray is prepared by the following steps: decocting the four medicinal materials except menthol in water for two times, wherein the first time is 2 hours, the second time is 1 hour, merging decoction, filtering, concentrating filtrate to fluid extract with the relative density of 1.05-1.10 (50 ℃), adding ethanol to ensure that the alcohol content reaches 80%, standing for 24 hours, filtering, recovering ethanol from the filtrate under reduced pressure, concentrating the filtrate to fluid extract with the relative density of 1.10-1.20 (80 ℃), taking menthol, sodium benzoate, citric acid, pineapple essence and 20ml of ethanol, stirring and dissolving, adding water to a specified amount, stirring uniformly, filtering, and filling to obtain the traditional Chinese medicine.
Further, a third aspect of the present invention provides a method of identifying an active pharmaceutical chemical in a throat spray, the method comprising the acts of:
(1) Providing a high-resolution mass spectrometer and a liquid chromatograph, providing a reference substance and a test substance of the medicinal components, and providing SD rats and beagle dogs;
(2) Chromatographic conditions:
a chromatographic column using octadecylsilane chemically bonded silica as a filler,
the column temperature is 30 ℃,
the measurement wavelength was 203nm,
mobile phase: acetonitrile (a) -0.1% formic acid solution (B), mobile phase was eluted with a gradient, the gradient being as follows:
| time (min) | Mobile phase a (%) |
| 0 | 3 |
| 15 | 8 |
| 25 | 15 |
| 35 | 22 |
| 45 | 24 |
| 75 | 50 |
Sample injection amount 2 μl, flow rate: 1mL/min;
(3) Mass spectrometry conditions
Adopting a soft ionization electrospray ion source (ESI), wherein the scanning mode is positive/negative ions, atomizing gas is nitrogen, auxiliary gas 1 is 60PSI, auxiliary gas 2 is 60PSI, gas curtain gas is 35PSI, atomizing temperature is set to 600 ℃ (in specific operation, for example, in the positive/negative ion mode, setting the scanning range of primary mass spectrum parent ions to be 100-1800Da, selecting 4 highest peaks with IDA setting response values exceeding 100cps for secondary mass spectrum scanning, setting the scanning range of sub-ions to be 100-1800Da, starting dynamic background subtraction (Dynamic background subtraction, DBS), and the software used for data acquisition is SCIEX OS 1.4 version);
(4) Administration and blood sampling for experimental animals
Four healthy SD rats and two beagle dogs (for example, the beagle dogs are placed in a room with the humidity of 50% and the temperature of 25 ℃ and fed with free food and drinking water for a week), animals are fasted for 12 hours before being fed without water, randomly grouped and weighed, 0.545g of liquid medicine per kg of body weight is fed according to the conversion of human/animal (the oral gavage administration of the rats, the oral gavage administration of one dog and the oral spray administration of one dog) are fed, the same volume of physiological saline is fed to a blank group, the same volume of physiological saline is respectively used for anesthesia and blood sampling at 0h and 0.5h, 1h and 2h after the administration, the blood of the rats is sampled for 2mL, the beagle dogs are sampled for 5mL, a plasma sampler is shaken at a uniform speed after the blood sampling and mixed with heparin sodium in a tube to prevent coagulation, the upper plasma is collected by centrifugation for 10min at 3500r/min under the condition of 4 ℃, the plasma is packaged into 1mL of each EP tube and stored in a refrigerator at the temperature of-80 ℃ for standby;
(5) Treatment of plasma samples
Taking blank or drug-administered (frozen or fresh) rat and beagle plasma samples (frozen plasma is melted to room temperature in advance), taking 250 mul of the sample into a 2.5ml EP tube after vortex for 1min, adding 750 mul of 0.1% formic acid-acetonitrile (50:50) mixed solution (5% methyl ethyl ketone and 1.2% calcium silicate (120-mesh fine powder can be added into the mixed solution in advance), vortex for 5min, centrifuging for 5min at 4 ℃ under 13000r/min, transferring 500 mul of supernatant into a new EP tube after centrifugation, blow-drying under nitrogen flow, vortex for 1min after 25 mul of redissolution residues with an initial proportion of 0.1% formic acid aqueous solution=3:97), and then ultrasonic for 5min, centrifuging for 10min at 13000r/min at 4 ℃, taking supernatant, and placing the supernatant into a sample-taking bottle for HPLC-Q-TOF MS measurement;
(6) Plasma sample analysis
Comparing the chromatographic data and the mass spectrum data of the rat blank plasma, the stomach-lavage administration plasma, the beagle blank plasma, the oral cavity spray administration plasma and the stomach-lavage administration plasma, analyzing the prototype components absorbed into blood and the ion fragments of the metabolites according to the chromatographic data and the mass spectrum data, and carrying out structural identification on the prototype components and the ion fragments;
(7) The method comprises the steps of determining absorbed proto-drug components and metabolic components from rat plasma, determining absorbed proto-drug components and metabolic components from beagle oral spray plasma, determining absorbed proto-drug components and metabolic components from beagle lavage plasma, and confirming that the shared absorbed proto-drug is a plasma active pharmaceutical chemical component according to shared absorbed proto-drug in three plasmas.
The method according to the third aspect of the present invention, wherein the identified plasma active pharmaceutical chemical is pterocarpan-side.
The method according to the third aspect of the present invention, further comprising the following cell assay procedure:
(8.1) CO provision 2 Incubator, microscope, enzyme-labeled instrument, providing RAW246.7 cells, culture medium, PBS buffer solution, fetal bovine serum, penicillin-streptomycin solution;
(8.2) preparation of solution
Preparation of the liquid medicine: sucking 20 mu L of the open throat sword spray into a 15mL centrifuge tube, adding 5% FBS complete culture medium solution to 10mL, shaking uniformly, filtering with a 0.22 mu m filter membrane, and sterilizing to prepare 1402 mu g/mL mother liquor; sucking 5mL from mother solution into 15mL centrifuge tube, adding 5% FBS complete culture medium solution to 10mL, shaking, gradually diluting with 2 times of the total culture medium solution to obtain 1402, 701, 350.5 μg/mL medicinal liquid,
preparation of 10% fbs complete medium solution: taking a 50mL centrifuge tube, adding 5mL FBS, adding DMEM to the scale, adding 500 mu L penicillin-streptomycin solution to prepare the medicine, refrigerating and preserving the medicine in a refrigerator at 4 ℃,
preparation of 5% fbs complete medium solution: taking 50mL centrifuge tube, adding 2.5mL FBS, adding DMEM to scale, adding 500 μl penicillin-streptomycin solution, refrigerating at 4deg.C,
preparation of intracellular and extracellular fluid samples: taking RAW264.7 cells in logarithmic growth phase, and adjusting cell concentration to about 2×10 5 cells/mL, three 6-well plates were removed,adding 2mL of cell suspension and 1mL of 10% culture medium into each well, shaking uniformly, standing for 10min, and adding 5% CO 2 Incubating in an incubator; taking out three 6-hole plates incubated for 24 hours, sucking the old culture medium, adding 1mL of 5% culture medium, respectively adding 1mL of liquid medicine with different concentrations, and setting 3 compound holes to ensure that the final medicine-containing concentration is 701, 350.5 and 175.25 mug/mL; taking out after continuous incubation for 24 hours, sucking old culture of each concentration dosing hole into a 15mL centrifuge tube, centrifuging at 1000rpm/min for 5min, and taking supernatant as extracellular fluid; gently washing cells in holes with concentration of 2mL PBS solution, blowing down the cells according to the region, collecting cell suspension, adopting a repeated freeze thawing method to lyse the cells, centrifuging at 1000rpm/min for 5min, taking supernatant as intracellular fluid,
(8.3) chromatography and Mass Spectrometry
Using the chromatographic conditions of step (2) and the mass spectrometry conditions of step (3), the intracellular and extracellular fluids of the obtained open throat sword spray were subjected to chromatographic and mass spectrometry, and the chromatographic data and mass spectrometry data of the inner and outer fluid components analyzed in the positive and negative ion modes were used to confirm the active pharmaceutical chemical components capable of penetrating the cell membrane.
The method according to the third aspect of the present invention, wherein the throat opening sword spray is prescribed as follows: 220 to 330g of cinnabar root, 220 to 330g of subprostrate sophora, 180 to 270g of cicada slough, 0.8 to 1.2g of menthol, 5.5ml of essence, 1g of citric acid, 1 to 2.5g of sodium benzoate, 20ml of ethanol and a proper amount of water are prepared into 1000ml.
The method according to the third aspect of the present invention, wherein the throat opening sword spray is prescribed as follows: 250g of cinnabar root, 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 to prepare 1000ml.
The method according to the third aspect of the present invention, wherein the throat opening sword spray is prescribed as follows: 313g of cinnabar root, 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 is prepared into 1000ml.
According to the third unilateral method of the invention, the preparation method of the throat-opening sword spray comprises the following steps: decocting the four medicinal materials except menthol in water for two times, wherein the first time is 2 hours, the second time is 1 hour, merging decoction, filtering, concentrating filtrate to fluid extract with the relative density of 1.05-1.10 (50 ℃), adding ethanol to ensure that the alcohol content reaches 80%, standing for 24 hours, filtering, recovering ethanol from the filtrate under reduced pressure, concentrating the filtrate to fluid extract with the relative density of 1.10-1.20 (80 ℃), taking menthol, sodium benzoate, citric acid, pineapple essence and 20ml of ethanol, stirring and dissolving, adding water to a specified amount, stirring uniformly, filtering, and filling to obtain the traditional Chinese medicine.
Among the steps of the above-described preparation method of the present invention, although the specific steps described therein are distinguished in some details or language description from the steps described in the preparation examples of the following detailed description, the above-described method steps can be fully summarized by one skilled in the art based on the detailed disclosure of the present invention as a whole.
Any of the embodiments of any of the aspects of the invention may be combined with other embodiments, provided that they do not contradict. Furthermore, in any of the embodiments of any of the aspects of the present invention, any technical feature may be applied to the technical feature in other embodiments as long as they do not contradict. The present invention is further described below.
All documents cited herein are incorporated by reference in their entirety and are incorporated by reference herein to the extent they are not inconsistent with this invention. Furthermore, various terms and phrases used herein have a common meaning known to those skilled in the art, and even though they are still intended to be described and explained in greater detail herein, the terms and phrases used herein should not be construed to be inconsistent with the ordinary meaning in the sense of the present invention.
The open throat sword spray is a product of Guizhou Sanli pharmacy. The Miao medicine function main indications of the children type throat opening sword spray are as follows: xuegakahi scab, an Dangmeng; steep: nano, meng Ninggong, meng Gagong on, jiang Gangfang, shui Gaguo west; the indications of the traditional Chinese medicine are as follows: clearing away heat and toxic materials, and relieving swelling and pain. Can be used for treating acute and chronic pharyngolaryngitis, tonsillitis, laryngopharynx swelling and pain, stomatitis, and gingival swelling and pain. The seedling medicine function main indications of the adult throat opening sword spray are as follows: raising a mask Song Gongzheng; meng Gagong, luo Lami; the indications of the traditional Chinese medicine are as follows: clearing away heat and toxic materials, and relieving swelling and pain. Can be used for treating sore throat, dry mouth, bitter taste, gingival swelling and pain, oral ulcer, and recurrent aphtha caused by lung and stomach heat.
The radix Ardisiae Crenatae is dry root of Ardisiae Crenatae Ardisia crenata Sims of Philippine. The radix Ardisiae Crenatae mainly contains triterpene saponins, coumarin and other substances; wherein the structural type of the saponin is mainly pentacyclic triterpene oleanane derivatives, and the aglycone comprises 2 types: epoxy ethers and 12-ene; coumarin is mainly bergenin; also contains some other components: viol, friedelane, beta-sitosterol, and daucosterol.
The Sophora tonkinensis root is the dried root and rhizome of Sophora tonkinensis Sophora tonkinensis Gagnep. The radix Sophorae Tonkinensis contains chemical components such as alkaloid, saponin, flavonoids and polysaccharide, etc., and its main medicinal components are alkaloid such as matrine, oxymatrine, and flavonoid component such as pterocarpan-side.
The invention can detect three medicinal components simultaneously by using one chromatographic condition, and can detect the quality of the throat-opening sword spray with high efficiency.
Drawings
Fig. 1: matrine (retention time t about 9.1 min) control chromatogram.
Fig. 2: bergenin (retention time t about 19.2 min) control chromatogram.
Fig. 3: control chromatogram of pterocarpan trilobate (retention time t about 46.7 min).
Fig. 4: the spray comprises a chromatogram of a sample of a Kaihoujian spray, wherein three chromatographic peaks of matrine (about 9.1 min), bergenin (about 19.2 min) and pterocarpan Heterophylla (about 46.7 min) are shown.
Fig. 5: the negative test solution chromatogram shows no three chromatographic peaks of matrine, bergenin and pterocarpan side.
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. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope thereof. The present invention generally and/or specifically describes the materials used in the test as well as the test methods. Although many materials and methods of operation are known in the art for accomplishing the objectives of the present invention, the present invention will be described in as much detail herein. In the invention, all medicinal materials are dry medicinal materials and meet pharmacopoeia regulations unless specified otherwise. In the following examples of the present invention, the amounts of the respective materials to be charged are all in parts by weight, and at the time of actual charging, the total weight of all materials in each batch is not less than 5 kg.
When the traditional Chinese medicine composition is prepared in the following way, the raw medicinal materials and the auxiliary materials are all in the same batch unless specified.
Example 1: preparation of spray for opening throat and sword
Prescription: 250g of cinnabar root, 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 is prepared into 1000ml (children type).
The preparation method comprises the following steps: decocting radix Ardisiae Crenatae, radix Sophorae Tonkinensis and periostracum Cicadae in water twice for 2 hr for 1 hr, mixing decoctions, filtering, concentrating the filtrate to obtain fluid extract with relative density of 1.05-1.10 (50deg.C), adding ethanol to 80%, standing for 24 hr, filtering, recovering ethanol from the filtrate under reduced pressure, concentrating to obtain fluid extract with relative density of 1.10-1.20 (80deg.C), mixing menthol, sodium benzoate, citric acid, pineapple essence and ethanol 20ml, stirring for dissolving, adding water to prescribed amount, stirring, filtering, and packaging.
Example 2: preparation of spray for opening throat and sword
Prescription: 313g of cinnabar root, 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 is prepared into 1000ml (adult type).
The preparation method comprises the following steps: decocting radix Ardisiae Crenatae, radix Sophorae Tonkinensis and periostracum Cicadae in water twice for 2 hr for 1 hr, mixing decoctions, filtering, concentrating the filtrate to obtain fluid extract with relative density of 1.05-1.10 (50deg.C), adding ethanol to 80%, standing for 24 hr, filtering, recovering ethanol from the filtrate under reduced pressure, concentrating to obtain fluid extract with relative density of 1.10-1.20 (80deg.C), mixing menthol, sodium benzoate, citric acid, pineapple essence and ethanol 20ml, stirring for dissolving, adding water to prescribed amount, stirring, filtering, and packaging.
Example 3: preparation of spray for opening throat and sword
Prescription: 280g of cinnabar root, 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 is prepared into 1000ml.
The preparation method comprises the following steps: decocting radix Ardisiae Crenatae, radix Sophorae Tonkinensis and periostracum Cicadae in water twice for 2 hr for 1 hr, mixing decoctions, filtering, concentrating the filtrate to obtain fluid extract with relative density of 1.05-1.10 (50deg.C), adding ethanol to 80%, standing for 24 hr, filtering, recovering ethanol from the filtrate under reduced pressure, concentrating to obtain fluid extract with relative density of 1.10-1.20 (80deg.C), mixing menthol, sodium benzoate, citric acid, pineapple essence and ethanol 20ml, stirring for dissolving, adding water to prescribed amount, stirring, filtering, and packaging.
Example 4: preparation of spray for opening throat and sword
Prescription: 220g of cinnabar root, 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 is prepared into 1000ml.
The preparation method comprises the following steps: decocting radix Ardisiae Crenatae, radix Sophorae Tonkinensis and periostracum Cicadae in water twice for 2 hr for 1 hr, mixing decoctions, filtering, concentrating the filtrate to obtain fluid extract with relative density of 1.05-1.10 (50deg.C), adding ethanol to 80%, standing for 24 hr, filtering, recovering ethanol from the filtrate under reduced pressure, concentrating to obtain fluid extract with relative density of 1.10-1.20 (80deg.C), mixing menthol, sodium benzoate, citric acid, pineapple essence and ethanol 20ml, stirring for dissolving, adding water to prescribed amount, stirring, filtering, and packaging.
Example 5: preparation of spray for opening throat and sword
Prescription: 330g of cinnabar root, 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 is prepared into 1000ml.
The preparation method comprises the following steps: decocting radix Ardisiae Crenatae, radix Sophorae Tonkinensis and periostracum Cicadae in water twice for 2 hr for 1 hr, mixing decoctions, filtering, concentrating the filtrate to obtain fluid extract with relative density of 1.05-1.10 (50deg.C), adding ethanol to 80%, standing for 24 hr, filtering, recovering ethanol from the filtrate under reduced pressure, concentrating to obtain fluid extract with relative density of 1.10-1.20 (80deg.C), mixing menthol, sodium benzoate, citric acid, pineapple essence and ethanol 20ml, stirring for dissolving, adding water to prescribed amount, stirring, filtering, and packaging.
Test example 1: method for simultaneously quantitatively determining multi-index medicinal components of open throat sword spray
In another study, the inventor of the present application has found that the plasma medicinal chemical foundation of the open-throat sword spray comprises index components such as matrine, bergenin, pterocarpine, and the like, and the index components are typical pharmacodynamic substances of the open-throat sword spray, and the test example provides a new method for rapidly and effectively carrying out quality detection on the open-throat sword spray by using an HPLC method to try to simultaneously determine the three index components.
1. Instrument and reagent
Waters e2695 high Performance liquid chromatograph (Waters Inc.), electronic balance SOP Secura 125-1CN (one ten thousandth, sarlor Inc.).
Acetonitrile, formic acid, methanol, deionized water and the like are all commercial products meeting the requirements for measurement.
2. Reagent
Matrine (control, lot number T22M10F88874, purity not less than 98%), bergenin (control, lot number H14J10Z79791, purity not less than 98%), trifolium pratense pterocarpine (control, lot number Y11J11H108216, purity not less than 98%), all of which were purchased from Shanghai source leaf Biotechnology Co.
The open throat sword spray is provided by Guizhou Sanli pharmaceutical Co., ltd (children, lot numbers KHJ20190127, KHJ20190610, KHJ 20190816) or made by the present invention examples.
3. Chromatographic conditions
Chromatographic column: welch Ultimate Plus C18 (4.6X250 mm,5 μm),
the column temperature is 25 ℃, the measurement wavelength is 203nm,
mobile phase: acetonitrile (a) -0.1% formic acid solution (B), mobile phase was eluted with a gradient, the gradient being as follows:
| t/min | A/％ |
| 0 | 3 |
| 5 | 5 |
| 25 | 14 |
| 40 | 28 |
| 50 | 35 |
| 55 | 45 |
flow rate: 1mL/min, and the sample injection amount is 10 mu L.
4. Preparation of control solution
Respectively weighing a proper amount of matrine, bergenin and pterocarpan trilobate glycoside reference substances, putting the reference substances into a measuring flask, using 40% methanol to fix the volume, preparing stock solutions with the concentrations of 0.5230mg/mL, 1.0210mg/mL and 0.1034mg/mL, passing through a 0.45 mu m microporous filter membrane to obtain the stock solution, and diluting the stock solution (for example, diluting by 2-20 times, for example, diluting by 5-15 times) by using the same solvent according to the measurement requirement (for example, predicting the concentration of the sample solution and reaching the concentration equivalent to the concentration). In the case of preparing a stock solution for a control, a higher or lower concentration than the above concentration may be prepared, and for example, the stock solution concentrations of the above 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 solutions
Precisely sucking 2mL of the open throat sword spray (KHJ 20190127) in a 10mL measuring flask, diluting 40% methanol to the scale, and passing through a 0.45 μm microporous filter membrane.
6. Preparation of negative test sample solution
A negative test sample solution without cinnabar root and without subprostrate sophora was prepared by referring to the method of open throat sword spray (children) national pharmaceutical standard (WS-10132 (ZD-0132) -2002) issued by the national food and drug administration: decocting periostracum Cicadae 20g with 6 times of water twice for 2 hours for the first time and 1 hour for the second time, mixing decoctions, filtering, concentrating the filtrate to obtain fluid extract 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 ethanol from the filtrate under reduced pressure, concentrating to obtain fluid extract with the relative density of 1.10-1.20 (80 ℃), taking menthol 0.1g, pineapple essence 0.55ml, citric acid 0.1g and sodium benzoate 0.1g, dissolving with ethanol 2ml, stirring, adding water to 100ml, stirring uniformly, and filtering to obtain a negative test sample solution without radix Ardisiae Crenatae and radix Sophorae Tonkinensis.
7. Investigation of specificity
Measuring matrine reference substance solution, bergenin reference substance solution, trifolium Prague pteroside reference substance solution, kaihoujian spray test substance solution, and negative test substance solution according to the above chromatographic conditions, wherein the chromatograms are shown in figures 1-5, the abscissa is time (min), and the ordinate is AU value, and the scale is not shown (the scale ratio of 5 figures is different) due to excessive reduction of the chromatogram; however, the abscissa serves as a qualitative basis and the quantitative basis is the peak area additionally provided from the HPLC instrument, so that the scale of the ordinate is not shown and does not affect the practice of the invention.
The result shows that the negative sample solution has no corresponding peak at the chromatographic peak positions of matrine, bergenin and pterocarpan henryi, and the chromatographic condition has better determination specificity for three components.
8. Methodology investigation
8.1. Linear relationship investigation
Taking the three reference substance stock solutions, gradually diluting the stock solutions to prepare matrine solution 0.5230, 0.2615, 0.1046, 0.0523, 0.0262, 0.0105mg/mL, bergenin solution 0.5105, 0.2042, 0.1021, 0.0511, 0.0204, 0.0102mg/mL, and Trifolium pratense pterosin solution 0.0517, 0.0259, 0.0103, 0.0052, 0.0026, 0.0010mg/mL. The measurement was carried out under the chromatographic conditions of the test example, and a linear curve was drawn and analyzed by taking the peak areas of the three components as ordinate (y) and the concentrations as abscissa (x), and the linear regression equation and coefficients are shown in the following table.
Table: three reference linear regression equations and ranges
The results show that when the concentration ranges of matrine, bergenin and pterocarpine are respectively 0.0105-0.5230mg/mL, 0.0102-0.5105mg/mL and 0.0010-0.0517mg/mL, the peak area and the concentration have good linear relation, and the content of matrine, bergenin and pterocarpine can be measured by adopting an external standard one-point method. The content of the substance to be measured in the sample can be calculated by using a linear regression equation, or by using a reference solution with a suitable concentration (for example, a concentration equivalent to that of the substance to be measured in the sample) according to an external standard method.
8.2. Precision test
Taking the test example to prepare a test sample solution, carrying out continuous 6 sample injection measurement according to the chromatographic conditions of the test example, recording the chromatographic peak areas of matrine, bergenin and pterocarpan henryi and calculating RSD (%), and the results are shown in the table below.
Table: KHJ precision test results (n=6)
| Composition of the components | Peak area | Peak area mean value | RSD(％) |
| Matrine | 1375815～1385910 | 1381317 | 0.28 |
| Bergenin | 2912891～2937736 | 2923715 | 0.33 |
| Trifolium pratense pterocarpine glycoside | 605800～609007 | 607096 | 0.21 |
The results show that the RSD of the three components in the test sample solution is below 1%, which indicates that the instrument precision is good under the chromatographic condition. When the peak areas in the above table are calculated by using a linear regression equation of '8.1. Linear relation investigation', y is the peak area, x is the concentration in μg/ml, and the calculated concentrations of three components such as matrine in the sample solution are 65.0 μg/ml, 106.1 μg/ml and 6.5 μg/ml respectively; the solution is diluted 5 times according to the spray to prepare a test solution for HPLC measurement, thereby calculating the calculated concentration of three components such as matrine in the open throat sword spray (KHJ 20190127) to be 325.0 mug/ml, 530.5 mug/ml and 32.5 mug/ml respectively; the following is the same. According to the concentrations of the three components measured in the sample solution, when the control solution is prepared, preferably, the concentrations of the three components in the control solution are 40 to 90. Mu.g/ml, for example, about 65. Mu.g/ml, 70 to 130. Mu.g/ml, for example, about 100. Mu.g/ml, and 4 to 9. Mu.g/ml, for example, about 6.5. Mu.g/ml, respectively, when the contents are calculated by the external standard method. Of course, the concentration of the control solution can also be adjusted if the concentration of the test solution is significantly changed.
8.3. Stability test
The test sample solution is prepared in this test example, and after being left at room temperature for 0, 2, 4, 8, 12 and 24 hours, the measurement is carried out according to the chromatographic conditions of this test example, the chromatographic peak areas of matrine, bergenin and pterocarpan henryi are recorded, and the RSD (%) is calculated, and the results are shown in the following table.
Table: KHJ stability test results
The results showed that the RSD of these three components in the test solution were within 1%, indicating that KHJ was stable over 24 hours.
8.4. Repeatability test
2mL of each sample solution was precisely sucked from 6 bottles KHJ (KHJ 20190127), the sample solutions were prepared according to the method of the test example, the chromatographic conditions of the test were used for measurement, the chromatographic peak areas of matrine, bergenin and pterocarpine in each sample solution were recorded and RSD (%) was calculated, and the results are shown in the following Table.
Table: sample repeatability test results (n=6)
| Composition of the components | Peak area | Peak area mean value | RSD(％) |
| Matrine | 1367527～1380705 | 1372410 | 0.34 |
| Bergenin | 2863709～2929903 | 2909063 | 0.81 |
| Trifolium pratense pterocarpine glycoside | 598458～606406 | 602444 | 0.42 |
The result shows that the RSD of each component is within 1%, which means that the sample solution has good reproducibility under the chromatographic condition and meets the content measurement requirement.
8.5. Sample recovery test
Precisely sucking 1mL of the open throat sword spray (KHJ 20190127) into a 5mL measuring flask, adding 0.0523mg/mL of matrine solution, 0.1021mg/mL of bergenin solution and 0.0052mg/mL of three-leaf bean pterocarpin solution which are described in the test example into each 0.5mL, fixing the volume to a scale by 40% methanol, and filtering with a 0.45 μm filter membrane to obtain a sample-adding 50% sample solution, wherein n=3. 1mL of a control solution and 1.5mL of a control solution are respectively added according to the operation, and 100% of a sample and 150% of a sample solution are prepared. The measurement was carried out under the chromatographic conditions of the test example, and the chromatographic peak areas of matrine, bergenin and pterocarpan henryi were recorded and the average recovery rate and RSD (%) were calculated, and the results are shown in the following table.
Table: sample recovery test results
The result shows that the RSD of matrine, bergenin and pterocarpus santalinus under the condition is less than 2%, which accords with the methodological verification requirement.
9. Measurement results
The contents of matrine, bergenin and pterocarpine were measured for three batches of open-throat sword sprays KHJ20190127, KHJ20190610 and KHJ20190816 and five batches of open-throat sword sprays prepared in examples 1 to 5 according to the present invention, and the results are shown in the following table.
Table: content of index pharmacodynamic ingredient in composition (μg/ml)
| Matrine | Bergenin | Trifolium pratense pterocarpine 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 multi-index components in the open throat sword spray by adopting an HPLC technology, and comprises the steps of measuring the content of matrine, bergenin and pterocarpan in three-leaf beans, wherein the precision, the repeatability, the stability and the RSD of the sample adding recovery rate in a calculation methodology meet the requirements within a certain linear range, and the method is proved to be feasible for simultaneously measuring the three index medicinal components.
Test example 2: method for simultaneously quantitatively determining multi-index medicinal components of open throat sword spray
When various sample solutions are measured in the above test example 1, it was found that after the sample solutions are injected into the liquid chromatograph, the column pressure at the initial test is usually between 80 and 90bar, however, the column pressure gradually increases after the test sample solutions are injected a plurality of times, for example, the column pressure increases between 120 and 130bar after the test sample solutions are injected 5 times, the column pressure increases between 150 and 160bar after the test sample solutions are injected 10 times, the column pressure increases between 210 and 220bar after the test sample solutions are injected 15 times, and the column pressure further increases are unacceptable; the column pressure can be returned to the original state by backflushing the column with mobile phase a/mobile phase b=10/90 ratio mobile phase for 2 hours, but this operation is not preferable in large sample size analytical 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 caused by the relevant components in the test solution. The inventors tried to use other brands of C18 columns (column length, column inner diameter and packing particle size are the same), namely three of Supelcoc C18, agilent C18, waters C18 columns, and as a result of the test according to test example 1, it was also found that there was an unacceptable increase in column pressure as the test proceeded, for example, three columns were increased in column pressure between 202 to 210bar, 185 to 192bar, 205 to 212bar, respectively, after injection of 12 test sample solutions. The inventors tried to add 1.2% isopropyl alcohol and 0.2% ammonium chloride simultaneously to both mobile phase a, acetonitrile and mobile phase B, 0.1% formic acid solution, so that both additives were contained in a and B at the same concentration, and still eluted according to the gradient elution procedure of test example 1, keeping the other operating conditions unchanged, and as a result, it was revealed that the initial column pressure was maintained between 80 and 90bar when the test sample solution and other solutions were tested, the column pressure was maintained between 85 and 95bar after 30 times or more of testing each solution, for example, the column pressure was maintained between 92 and 97bar when the test sample solution was injected for 30 times. Further, the present inventors tried to add 1.2% isopropyl alcohol to both mobile phase a, acetonitrile and mobile phase B, 0.1% formic acid solution, so that both a and B contained the same concentration of the additive, and still eluted according to the gradient elution procedure of test example 1, keeping other operating conditions unchanged, and as a result, it was revealed that the initial column pressure was still gradually increased between 80 and 90bar when the test sample solution and other solutions were tested, for example, the column pressure was increased between 109 and 115bar after the injection of 5 test sample solutions, and between 133 and 140bar after the injection of 10 test sample solutions, and between 160 and 168bar after the injection of 15 test sample solutions. Further, the present inventors tried to add 0.2% ammonium chloride to both mobile phase a, acetonitrile and mobile phase B, 0.1% formic acid solution, so that both a and B contained the same concentration of the additive, and still eluted according to the gradient elution procedure of test example 1, keeping other operating conditions unchanged, and as a result, it was revealed that the initial column pressure was still gradually increased between 80 and 90bar when the test sample solution and other solutions were tested, for example, the column pressure was increased between 115 and 122bar after the injection of 5 test sample solutions, and between 140 and 150bar after the injection of 10 test sample solutions, and between 180 and 190bar after the injection of 15 test sample solutions. From this, it is known that the simultaneous addition of both isopropanol and ammonium chloride in the mobile phase can avoid the problem of increased column pressure when testing the sample solution.
For this reason, in this test example 2, except that 1.2% isopropyl alcohol and 0.2% ammonium chloride were simultaneously added to both the mobile phase a, acetonitrile and the mobile phase B, i.e., 0.1% formic acid solution, the other operating conditions were all carried out with reference to test example 1, various test solutions were still prepared using test example 1, the results of various tests were substantially the same as those of 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 pterocarpin t is about 46.5min, substantially unchanged; 2) Linear regression equation: matrine y=21238x-44.457 and R 2 Bergenin y=27577x+86.624 and r=0.9999 2 =0.9997, pterosiny= 93253x-34.145 and R 2 =0.9998; 3) KHJ20190127 precision of test article: matrine peak area mean= 1380561 and rsd=0.25%, bergenin peak area mean= 2924216 and rsd=0.22%, sanguinarine peak area mean= 607144 and rsd=0.35%; 4) 24h stability test of KHJ20190127 test article: matrine peak area mean= 1380268 and rsd=0.33%, bergenin peak area mean= 2923523 and rsd=0.41%, sanguinarine peak area mean= 604327 and rsd=0.38%; 5) Repeatability test of KHJ20190127 test article: matrine peak area mean= 1372084 and rsd=0.51%, bergenin peak area mean= 2908473 and rsd=0.43%, sanguinarine peak area mean= 602836 and rsd=0.62%; 6) Sample recovery rate test of KHJ20190127 test article: the average recovery of three concentrations of matrine was 99.64% and rsd=1.33%, and the average recovery of three concentrations of bergenin was 101.46% and rsd=1.26%, the average recovery of three concentrations of pterosin from trefoil is 101.84% and rsd=1.64%; 7) The contents (mug/ml) of matrine, bergenin and pterocarpin in 5 batches of open throat sword spray are measured simultaneously, and the result is that: three component contents of KHJ20190127 were 324.4. Mu.g/ml, 530.9. Mu.g/ml, 32.3. Mu.g/ml, KHJ20190610 were 332.2. Mu.g/ml, 525.6. Mu.g/ml, 32.6. Mu.g/ml, KHJ20190816 were 326.7. Mu.g/ml, 535.5. Mu.g/ml, 32.5. Mu.g/ml, 329.4. Mu.g/ml, 531.4. Mu.g/ml, 32.4. Mu.g/ml, 412.5. Mu.g/ml, 661.3. Mu.g/ml, 41.3. Mu.g/ml, 367.8. Mu.g/ml, 595.7. Mu.g/ml, 37.2. Mu.g/ml, 288.3. Mu.g/ml, 464.5. Mu.g/ml, 28.6. Mu.g/ml, 430.5. Mu.g/ml, 9243.4. Mu.g/ml, and 3.3. Mu.g/ml, respectively.
The HPLC method of test example 2 with 2 reagents added in the mobile phase can be used for measuring the content of multi-index components in the open throat sword spray, including the content measurement of matrine, bergenin and pterocarpan with three beans, and has excellent methodological performance.
Test example 3: method for researching and confirming chemical components of plasma active medicine in Kaihoujian spray
Method of
The effective substances in the traditional Chinese medicine and the compound are required to be transported to a target point through blood to generate pharmacological action, so that the components contained in the blood plasma after administration are the material basis for generating the drug effect. The composition includes prototype component and metabolite contained in the Chinese medicine compound, and the substances directly acting in the body can be determined by means of analysis of transitional component in blood after administration [ Yang Fangliang, zhangjing, sun Guoxiang, etc. ] the Chinese medicine composition fingerprint research method and thinking [ J ]. Chromatograph, 2016,34 (07): 715-725].
Most of traditional experimental methods are gastric lavage and administration, the open throat sword spray is oral spray and directly acts on focus, so that oral spray administration of beagle dogs is added on the basis of gastric lavage and administration, and prototype and metabolic components in medicated plasma are analyzed by HPLC-Q-TOF MS technology in the experimental example.
3.1 materials and instruments
Sciex X500R QTOF (high resolution Mass Spectrometry, SCIEX), waters e2695 liquid chromatograph (Agilent), welch Ulitimate Plus C (Yuehu Xu), SQP Secura 125-1CN (parts per million, sarlarius), BT25S electronic balance (parts per million, sarlarius), ST8R high speed cryocentrifuge (thermoelectric Thermo), AS3120 ultrasonic extractor (Ottsaint), G & G JJ600 electronic balance (G & G Co.).
Other instruments and reagents are readily available from commercial sources.
3.2 preparation of liquid medicine
Open throat sword spray (child type, lot 20200415) is available from three force pharmaceutical company, guizhou.
3.3 laboratory animals and RAW264.7 cells
Four SPF-grade SD male rats, 200+ -20 g in weight, were purchased from St Bei Fu (Beijing) laboratory animal technologies Co., ltd. The male animals of beagle dogs, with weights of 9.8kg and 11.4kg, were purchased from Shanghai Xingang laboratory animal farm.
RAW264.7 cells (mouse megakaryocyte leukemia cells) were purchased from the marsupenario life technologies company, cat No.: CL-0190.
3.4 pharmacological test methods
3.4.1 chromatographic conditions
Chromatographic column: welch Ulitimate Plus C18 (4.6X105 mm,5 μm)
Mobile phase: acetonitrile (A) -0.1% formic acid aqueous solution (B)
Sample injection amount 2 μl, column temperature 30 ℃, flow rate: gradient elution at 1mL/min is shown in the following Table
| Time (min) | Mobile phase a (%) |
| 0 | 3 |
| 15 | 8 |
| 25 | 15 |
| 35 | 22 |
| 45 | 24 |
| 75 | 50 |
3.4.2 Mass Spectrometry Condition
A soft ionization electrospray ion source (ESI) is adopted, the scanning mode is positive/negative ions, the atomizing gas is nitrogen, the auxiliary gas 1 is 60PSI, the auxiliary gas 2 is 60PSI, the gas curtain gas is 35PSI, and the atomizing temperature is set to 600 ℃. In the positive/negative ion mode, setting the scanning range of primary mass spectrum parent ion as 100-1800Da, selecting 4 highest peaks with IDA setting response value exceeding 100cps for secondary mass spectrum scanning, setting the scanning range of child ion as 100-1800Da, starting dynamic background subtraction (Dynamic background subtraction, DBS), and using software for data acquisition as SCIEX OS version 1.4.
3.4.3 administration and blood sampling in laboratory animals
Four healthy SD rats and two beagle dogs were kept in a room with 50% humidity and 25℃and fed with free food and water for one week. Animals were fasted for 12 hours before administration without water withdrawal, randomly grouped and weighed, 0.545g of the drug solution per kg of body weight (oral gavage administration of rats, oral gavage administration of dogs, oral spray administration of dogs) was given in terms of human/animal, the same volume of physiological saline was given to the blank group, blood was collected by anesthesia with 3% pentobarbital solution (1 mL/kg) for 0.5h, 1h, and 2h after administration, about 2mL of blood was collected from rats, about 5mL of blood was collected from beagle dogs, and after blood collection, the plasma sampler was shaken at a constant speed to mix with heparin sodium in a tube to prevent coagulation, and centrifuged for 10min at 3500r/min to collect upper plasma at 4℃and package into 1mL plasma per EP tube for storage in a refrigerator at-80 ℃.
3.4.4 treatment of plasma samples
Plasma samples were processed by the precipitated protein method, blank or post-administration (frozen or fresh) rat, beagle plasma samples (frozen plasma was thawed to room temperature in advance), 250 μl was taken after vortexing for 1min in a 2.5ml EP tube, 750 μl of a 0.1% formic acid-acetonitrile (50:50) mixed solution (to which 5% methyl ethyl ketone and 1.2% calcium silicate (which may be 120 mesh fine powder, pre-suspended in the mixed solution) were added), vortexing for 5min, then centrifugation for 5min at 4 ℃ for 13000r/min, 500 μl supernatant was transferred to a new EP tube after centrifugation, blow-dried under nitrogen, vortexing for 1min after 25 μl of re-dissolved residues with an initial proportion of mobile phase (acetonitrile: 0.1% formic acid aqueous solution=3:97), vortexing for 5min, centrifugation for 10min at 4 ℃ for 10 r/min, and taking supernatant and placing in a vial for HPLC-Q-TOF MS measurement.
3.5 experimental results
3.5.1 analysis of plasma samples
The plasma samples of rats and beagle dogs are treated by adopting a method of '3.4.4', and are sampled under the conditions of '3.4.1' and '3.4.2', and the plasma sample is subjected to structural identification by comparing chromatographic data and mass spectrum data of the plasma blank of the rats, the plasma blank of the beagle dogs, the plasma of the oral cavity spray administration and the plasma of the plasma blank of the beagle dogs, analyzing ion fragments of prototype components and metabolites absorbed into blood according to the chromatographic data and the mass spectrum data, and the positive and negative ion mode results of the plasma blank of the rats and the beagle dogs are shown in the following table.
Table: LC-MS data of absorption blood-entering prototype and metabolic component of rat
Table: beagle oral spray absorption blood prototype and metabolic component LC-MS data
Table: stomach lavage absorption blood prototype and metabolic component LC-MS data of beagle
3.5.2 examination of plasma sample treatment method
Experiment 3.5.2a: referring to the method of the treatment of the 3.4.4 plasma sample above, taking 5ml of beagle blank plasma in an EP tube, adding 100 μl of three control stock solutions (the addition amount of three substances is 52.3 μg, 102.1 μg and 10.34 μg respectively) prepared in test example 1, respectively, mixing uniformly by vortexing for 2min, adding 15ml of 0.1% formic acid-acetonitrile (50:50) mixed solution (5% methyl ethyl ketone and 1.2% calcium silicate (which can pass through 120-mesh fine powder and is pre-suspended in the mixed solution), vortexing for 5min, centrifuging for 5min at 4 ℃, transferring 2ml of supernatant to a new EP tube, blow-drying under nitrogen flow, centrifuging for 1min at the initial ratio of mobile phase (acetonitrile: 0.1% formic acid aqueous solution=3:97), centrifuging for 00r/min under 4 ℃ C. To 10min, taking supernatant, and placing in a HPLC bottle under the condition of '3.130 min' for measurement; in addition, the stock solution of three reference substances of matrine, bergenin and pterocarpan side is properly diluted by an initial proportion mobile phase and then HPLC is measured by the same method; the recovery of three chemicals from this blank plasma treatment was calculated, measured 5 times in parallel, and averaged, resulting in a recovery (n=5) of 94.6% and rsd=2.27%. This indicates that the method of processing plasma samples has a relatively high recovery rate. Referring to the method of "test 3.5.2a", except that no calcium silicate was added to the mixed solution, the recovery (n=5) was 63.6% and rsd=3.23% by the same method. Referring to the method of "test 3.5.2a", except that methyl ethyl ketone was not added to the mixed solution, the recovery (n=5) was 70.4% and rsd=2.06% by the same method. Referring to the method of "test 3.5.2a", except that methyl ethyl ketone was not added and calcium silicate was not added to the mixed solution, the recovery (n=5) was 64.7% and rsd=2.53% by the same method. These results indicate that the simultaneous addition of methylethylketone and calcium silicate to the solvent used to process the plasma sample has been unexpectedly found to significantly improve the recovery of the process.
3.6 cell assay
3.6.1 materials and reagents
RAW246.7 cells (marpranopsis), DMEM medium (Gibco), PBS buffer (hyclone), fetal bovine serum (Gibco), penicillin-streptomycin solution (marpranopsis).
3.6.2 laboratory apparatus
SW-CJ-2FD super clean bench (Antai air), CO 2 Incubator (model MCO-18AIC, sanyo Co.), BDS200-PH inverted microscope (Chongqing Ort optics), specramax M5 microplate reader (Molecular Devices Co.), TC20 fully automatic cell counter (Bio-Rad Co.).
3.6.3 chromatographic conditions: the settings were according to "3.4.1 chromatographic conditions".
3.6.4 mass spectrometry conditions: set according to "3.4.2 mass spectrometry conditions".
3.7 preparation of solutions
3.7.1 arrangement of liquid medicine
20 mu L of the throat-opening sword spray is sucked into a 15mL centrifuge tube, 5% FBS complete culture medium solution is added to 10mL, the mixture is shaken uniformly, and a 0.22 mu m filter membrane is filtered and sterilized to prepare 1402 mu g/mL mother liquor. 5mL of the mother solution was taken out of a 15mL centrifuge tube, and a 5% FBS complete medium solution was added to 10mL, shaken well, and diluted stepwise in 2 times to prepare 1402, 701 and 350.5. Mu.g/mL of the liquid medicine.
3.7.2 Preparation of 10% FBS complete Medium solution
Taking a 50mL centrifuge tube, adding 5mL FBS, adding DMEM to the scale, adding 500 mu L penicillin-streptomycin solution, and refrigerating at 4deg.C.
3.7.3 Preparation of 5% FBS complete Medium solution
Taking a 50mL centrifuge tube, adding 2.5mL FBS, adding DMEM to the scale, finally adding 500 mu L penicillin-streptomycin solution to prepare the finished product, and putting the finished product in a refrigerator at 4 ℃ for refrigeration and preservation.
3.8 preparation of intracellular and extracellular fluid samples
Taking RAW264.7 cells in logarithmic growth phase, and adjusting cell concentration to about 2×10 5 Taking out three 6-hole plates, adding 2mL of cell suspension and 1mL of 10% culture medium into each hole, shaking uniformly in cross, standing for 10min, and adding 5% CO 2 And (5) incubating in an incubator. Three 6-hole plates incubated for 24 hours are taken out, old culture medium is sucked off, 1mL of 5% culture medium is added, 1mL of liquid medicine with different concentrations is respectively added, 3 compound holes are arranged, and the final medicine-containing concentration is 701, 350.5 and 175.25 mug/mL. After further incubation for 24 hours, the old culture was removed, and the wells were aspirated into 15mL centrifuge tubes, and centrifuged at 1000rpm/min for 5min to obtain the supernatant as extracellular fluid. The cells in the holes with various concentrations are gently washed by 2mL of PBS solution, the cells are blown down according to the area to collect cell suspension, the cells are lysed by a repeated freeze thawing method, and the supernatant is taken as intracellular fluid after centrifugation at 1000rpm/min for 5 min.
3.9 experimental results
Obtaining an intracellular and extracellular fluid chromatogram and a mass spectrogram of the throat-opening sword spray under the chromatographic conditions, according to the obtained chromatograms and mass spectrograms, correcting the resolved extracellular and intracellular fluid components in positive and negative ion modes, and correcting the components in blank extracellular and intracellular fluid to finally obtain the pterocarcinoside component, wherein the results are shown in the table below.
Table: liquid LC-MS data of inside and outside of open throat sword spray cell
| Chemical formula | Time | Theoretical value | Actual measurement value | ppm | Ion mode | Fragment ions | Name of the name | Source |
| C 22 H 22 O 10 | 53.82 | 469.1107 | 469.1107 | 0 | [M+Na] + | 915.5112,151.0390,123.0783 | Trifolium pratense pterocarpine glycoside | Radix Sophorae Tonkinensis |
3.10 summary and discussion
In this test example 3, the positive and negative ion chromatograms of the transitional components in the plasma of the administration of rats and beagle dogs are further analyzed by the HPLC-Q-TOF MS technology, the possible transitional components of the spray for opening throat and sword in blood are analyzed by comparing the chromatographic/mass spectrum data results of the plasma of administration and blank plasma, the cleavage rule of the mass spectrum is analyzed, and finally 10 absorbed proto-drug components and 9 metabolic components are identified in the plasma of rats, wherein the proto-drug components comprise 7, 8-dihydroxy-4-methylcoumarin, three-bean pterocarpine, N-acetyldopamine and the like, and the metabolic components comprise bergenin, esculenton and the like. Identifying 2 absorbed proto-drug components and 9 metabolism components in the beagle oral spray plasma, wherein the proto-drug components comprise 7, 8-dihydroxyl-4-methylcoumarin and three-leaf bean pterocarpin, and the metabolism components comprise vitexin, sophocarpine oxide and the like. 5 absorbed proto-drug components and 9 metabolic components are identified in the beagle stomach-filling plasma, wherein the proto-drug components comprise 9 alpha/14 alpha-hydroxy matrine, three-leaf bean pterocarpine and the like, and the metabolic components comprise oxymatrine, sophocarpine oxide and the like. Meanwhile, the intracellular and extracellular fluid chromatograms of the throat-opening sword spray are analyzed, and are corrected by blank intracellular and extracellular fluids, so that the components which possibly penetrate cell membranes to generate the drug effect are presumed, and finally, the component of the pharmacodynamics basic substance analyzed and determined in the intracellular fluid is the pterocarcinoside.
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 characteristics 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 (8)
1. A method for identifying an active pharmaceutical chemical in a throat spray, the method comprising the steps of:
(1) Providing a high resolution mass spectrometer and a liquid chromatograph, providing a reference substance and a test substance of the chemical components of the active medicaments, and providing SD rats and beagle dogs;
(2) Chromatographic conditions:
a chromatographic column using octadecylsilane chemically bonded silica as a filler,
the column temperature is 30 ℃ and the temperature is not higher than the column temperature,
the measurement wavelength was 203nm,
mobile phase: acetonitrile is taken as a mobile phase A, 0.1% formic acid solution is taken as a mobile phase B, and the mobile phase adopts gradient elution, wherein the gradient is as follows:
at 0min, the mobile phase A is 3 percent,
At 15min, the mobile phase A is 8 percent,
The mobile phase A is 15% at 25min,
At 35min, mobile phase A was 22%,
At 45min, mobile phase A was 24%,
The mobile phase A was 50% at 75min,
sample injection amount 2 μl, flow rate: 1mL/min;
(3) Mass spectrometry conditions
Adopting a soft ionization electrospray ion source, wherein the scanning mode is positive/negative ions, the atomizing gas is nitrogen, the auxiliary gas 1 is 60PSI, the auxiliary gas 2 is 60PSI, the gas curtain gas is 35PSI, and the atomizing temperature is set to 600 ℃;
(4) Administration and blood sampling for experimental animals
Four healthy SD rats and two beagle dogs are placed in a room with humidity of 50% and temperature of 25 ℃, fed freely and fed with drinking water for adaptation for one week, animals are fasted for 12 hours before administration and not forbidden, randomly grouped and weighed, 0.545g of liquid medicine per kg of body weight is given according to the conversion of human/animal, the rats are orally and gastrically administrated, one dog is orally and subjected to oral spray administration, a blank group is given with the same volume of physiological saline, blood is respectively taken by anesthesia of 0h and 0.5h, 1h and 2h of administration with 3% pentobarbital solution at a dose of 1mL/kg, the rats are taken for 2mL of blood, the beagle is taken for 5mL of blood, a plasma sampler is shaken at a uniform speed after blood taking, the beagle is mixed with heparin sodium in a tube for preventing blood coagulation, upper plasma is collected by centrifugation for 10min at 3500r/min under the condition of 4 ℃, and 1mL of plasma is prepared for each EP tube according to the components, and stored in a refrigerator at-80 ℃;
(5) Treatment of plasma samples
Taking blank or post-administration rat and beagle plasma samples, vortex for 1min, take 250 μl into 2.5ml EP tube, add 0.1% formic acid-acetonitrile to volume ratio 50:50, vortexing for 5min, centrifuging for 5min at 13000r/min at 4deg.C, transferring 500 μl of supernatant to a new EP tube, blow drying under nitrogen flow, and mixing with acetonitrile as mobile phase at initial ratio: 0.1% formic acid aqueous solution = 3:97 re-dissolving the residue with 25 μl, vortexing for 1min, then ultrasound for 5min, centrifuging for 10min at 13000r/min at 4deg.C, collecting supernatant, and placing in sample injection vial for HPLC-Q-TOF MS measurement; the mixed solution is also added with 5 percent of methyl ethyl ketone and 1.2 percent of calcium silicate, wherein the calcium silicate is fine powder with 120 meshes, and is pre-suspended in the mixed solution;
(6) Plasma sample analysis
Comparing the chromatographic data and the mass spectrum data of the rat blank plasma, the stomach-lavage administration plasma, the beagle blank plasma, the oral cavity spray administration plasma and the stomach-lavage administration plasma, analyzing the prototype components absorbed into blood and the ion fragments of the metabolites according to the chromatographic data and the mass spectrum data, and carrying out structural identification on the prototype components and the ion fragments;
(7) The method comprises the steps of determining absorbed proto-drug components and metabolic components from rat plasma, determining absorbed proto-drug components and metabolic components from beagle oral spray plasma, determining absorbed proto-drug components and metabolic components from beagle lavage plasma, and confirming that the shared absorbed proto-drug is a plasma active pharmaceutical chemical component according to shared absorbed proto-drug in three plasmas.
2. The method of claim 1, wherein the identified plasma active pharmaceutical chemical is pterocarpine.
3. The method according to claim 1, characterized in that it further comprises the following cell experiment procedure:
(8.1) CO provision 2 Incubator, microscope, enzyme-labeled instrument, providing RAW246.7 cells, culture medium, PBS buffer solution, fetal bovine serum, penicillin-streptomycin solution;
(8.2) preparation of solution
Preparation of the liquid medicine: sucking 20 mu L of the open throat sword spray into a 15mL centrifuge tube, adding 5% FBS complete culture medium solution to 10mL, shaking uniformly, filtering with a 0.22 mu m filter membrane, and sterilizing to prepare 1402 mu g/mL mother liquor; sucking 5mL from mother solution into 15mL centrifuge tube, adding 5% FBS complete culture medium solution to 10mL, shaking, gradually diluting with 2 times of the total culture medium solution to obtain 1402, 701, 350.5 μg/mL medicinal liquid,
preparation of 10% FBS complete medium solution: taking a 50mL centrifuge tube, adding 5mL FBS, adding DMEM to the scale, adding 500 mu L penicillin-streptomycin solution to prepare the medicine, putting the medicine in a refrigerator at 4 ℃ for cold storage,
preparation of 5% FBS complete medium solution: taking 50mL centrifuge tube, adding 2.5mL FBS, adding DMEM to scale, adding 500 μl penicillin-streptomycin solution, refrigerating at 4deg.C,
Preparation of intracellular and extracellular fluid samples: taking RAW264.7 cells in logarithmic growth phase, and adjusting cell concentration to about 2×10 5 Taking out three 6-hole plates, adding 2mL of cell suspension and 1mL of 10% culture medium into each hole, shaking uniformly in cross, standing for 10min, and adding 5% CO 2 Incubating in an incubator; taking out three 6-hole plates incubated for 24 hours, sucking the old culture medium, adding 1mL of 5% culture medium, respectively adding 1mL of liquid medicine with different concentrations, and setting 3 compound holes to ensure that the final medicine-containing concentration is 701, 350.5 and 175.25 mug/mL; taking out after continuous incubation for 24 hours, sucking old culture of each concentration dosing hole into a 15mL centrifuge tube, centrifuging at 1000rpm/min for 5min, and taking supernatant as extracellular fluid; gently washing cells in holes with concentration of 2mL PBS solution, blowing down the cells according to the region, collecting cell suspension, lysing the cells by repeated freeze thawing method, centrifuging at 1000rpm/min for 5min, collecting supernatant as intracellular fluid,
(8.3) chromatography and Mass Spectrometry
Using the chromatographic conditions of step (2) and the mass spectrometry conditions of step (3), the intracellular and extracellular fluids of the obtained open throat sword spray were subjected to chromatographic and mass spectrometry, and the chromatographic data and mass spectrometry data of the inner and outer fluid components analyzed in the positive and negative ion modes were used to confirm the active pharmaceutical chemical components capable of penetrating the cell membrane.
4. The method of claim 1, wherein the throat opening sword spray is formulated as follows: 220-330 g of cinnabar root, 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 to prepare 1000ml.
5. The method of claim 1, wherein the throat opening sword spray is formulated as follows: 250g of cinnabar root, 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 to prepare 1000ml.
6. The method of claim 1, wherein the throat opening sword spray is formulated as follows: 313g of cinnabar root, 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 is prepared into 1000ml.
7. The method according to claim 4, wherein the throat-opening sword spray is prepared by the following steps: decocting radix Ardisiae Crenatae, radix Sophorae Tonkinensis and periostracum Cicadae in water twice for 2 hr for 1 hr, mixing decoctions, filtering, concentrating the filtrate to obtain extract with relative density of 1.05-1.10 at 50deg.C, adding ethanol to make ethanol content reach 80%, standing for 24 hr, filtering, recovering ethanol from the filtrate under reduced pressure, concentrating to obtain extract with relative density of 1.10-1.20 at 80deg.C, mixing menthol, sodium benzoate, citric acid, pineapple essence and ethanol 20ml, stirring for dissolving, adding water to prescribed amount, stirring, filtering, and packaging.
8. The method of claim 1, wherein the chromatographic conditions further comprise: the particle size of the packing of the chromatographic column is 5 mu m; the inner diameter of the chromatographic column is 4.6mm; the chromatographic column length was 250mm.
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