CN111735896A - Method for constructing characteristic spectrum of dampness-resolving and toxin-vanquishing composition - Google Patents

Abstract

The invention discloses a method for constructing a characteristic spectrum of a dampness-resolving and toxin-vanquishing composition, wherein the dampness-resolving and toxin-vanquishing composition mainly comprises the following components: ephedra, fried bitter almond, gypsum, liquorice, patchouli, mangnolia officinalis, bran-fried rhizoma atractylodis, fried grass nut, rhizoma pinellinae praeparata, poria cocos, rheum officinale, astragalus membranaceus, semen lepidii and red paeony root; the construction method of the dampness-eliminating and toxin-vanquishing composition characteristic spectrum comprises the following steps: preparing paeoniflorin, liquiritigenin, chrysophanol-8-O-glucoside, glycyrrhizic acid, honokiol and magnolol mixed reference substance solution; preparing a test solution; then injecting the mixture into a liquid chromatograph for testing, and establishing a characteristic spectrum of the dampness-resolving and toxin-vanquishing composition. The method has good reproducibility, accuracy and reliability, can provide a data basis for controlling the quality of the dampness-resolving and toxin-vanquishing composition, and effectively ensures the stability and controllability of the product quality of the dampness-resolving and toxin-vanquishing composition.

Description

Method for constructing characteristic spectrum of dampness-resolving and toxin-vanquishing composition

Technical Field

The invention relates to the technical field of traditional Chinese medicine quality analysis and detection, in particular to a method for constructing a characteristic spectrum of a dampness-resolving and toxin-vanquishing composition.

Background

2019 the epidemic situation of pneumonia caused by infection of novel coronavirus (COVID-19) is a global overweight public health emergent event because of strong infectivity, rapid spread, common susceptibility of people and lack of specific drugs, and has already formed a pandemic in the global scope. The traditional Chinese medicine plays a unique and important role in the process of resisting the epidemic situation of the new coronary pneumonia. The Chinese medicine administration indicates that the golden flower cold-clearing granules, the honeysuckle plague-clearing granules, the Xuebijing injection, the lung-clearing toxin-expelling decoction, the dampness-resolving toxin-expelling prescription and the lung-ventilating toxin-expelling prescription in the three-medicine three-party play good roles in resisting the epidemic situation through research and screening.

The dampness-resolving and toxin-vanquishing formula consists of 14 traditional Chinese medicines, including raw ephedra herb, almond, raw gypsum, liquorice, agastache, mangnolia officinalis, rhizoma atractylodis, amomum tsao-ko, rhizoma pinellinae praeparata, poria cocos, raw rhubarb, raw astragalus, semen lepidii and red paeony root. Clinical experiments show that the dampness-resolving and toxin-vanquishing formula has outstanding effects of improving the symptoms of patients and increasing the negative conversion rate of nucleic acid. However, the research on dampness-resolving and toxin-vanquishing formulas at present mostly focuses on the research on pharmacology and curative effect, and no research on quality standards is available. And the existing production is only carried out in a small range, large-scale industrial production is not carried out, and the requirement on quality monitoring is relatively low.

The material basis of each medicine is researched in the literature 'research on material basis of dampness-resolving and toxin-vanquishing granule medicine taste for resisting novel coronavirus pneumonia (COVID-19)' (Chinese modern traditional medicine, No. 3 of 2020). However, no specific quality control method of the drug has been studied.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for constructing a characteristic spectrum of the dampness-resolving and toxin-vanquishing composition, which has good reproducibility, is accurate and reliable, can provide a data base for the large-scale production quality control of the dampness-resolving and toxin-vanquishing composition, and ensures the stability and controllability of the product quality of the dampness-resolving and toxin-vanquishing composition.

In order to solve the technical problems, the invention provides a method for constructing a characteristic spectrum of a dampness-resolving toxin-vanquishing composition, wherein the dampness-resolving toxin-vanquishing composition mainly comprises the following components: ephedra, fried bitter almond, gypsum, liquorice, patchouli, mangnolia officinalis, bran-fried rhizoma atractylodis, fried grass nut, rhizoma pinellinae praeparata, poria cocos, rheum officinale, astragalus membranaceus, semen lepidii and red paeony root;

the construction method of the dampness-eliminating and toxin-vanquishing composition characteristic spectrum comprises the following steps:

(1) respectively taking appropriate amount of penoniflorin reference substance, glycyrrhizin reference substance, chrysophanol-8-O-glucoside reference substance, glycyrrhizic acid reference substance, honokiol reference substance, and magnolol reference substance, adding solvent, and dissolving or extracting to obtain reference substance solution;

(2) extracting the dampness-resolving and toxin-vanquishing composition with an extraction solvent to obtain a test solution;

(3) injecting a preset amount of reference substance solution and test solution into a liquid chromatograph, and performing gradient elution by using octadecylsilane chemically bonded silica as a filler, acetonitrile as a mobile phase A and formic acid solution as a mobile phase B in the liquid chromatograph to establish a characteristic spectrum of the dampness-resolving and toxin-vanquishing composition.

As an improvement of the technical scheme, in the step (2), the extraction solvent is 50-100% of methanol or 50-70% of ethanol; the extraction method is ultrasonic extraction or reflux extraction.

As an improvement of the technical scheme, the step (2) comprises the following steps:

taking the dampness-resolving and toxin-vanquishing composition, grinding, taking 0.5-1.5 g of the composition, precisely weighing, placing the composition into a conical flask with a plug, adding 20-30 mL of 50-70% methanol, weighing, heating and refluxing for 30-90 minutes, taking out, cooling, weighing again, complementing the weight loss by using 50-70% methanol, shaking uniformly, filtering, taking the subsequent filtrate, and preparing the test solution.

As an improvement of the technical scheme, the step (2) comprises the following steps:

taking the dampness-resolving and toxin-vanquishing composition, grinding, taking 0.5-1.5 g of the composition, precisely weighing, placing the composition into a conical flask with a plug, adding 20-30 mL of 50-70% methanol, weighing, carrying out ultrasonic treatment with the power of 200-300 kW and the frequency of 35-45 kHz for 30-90 minutes, taking out, cooling, weighing again, complementing the loss weight with 50-70% methanol, shaking uniformly, filtering, and taking a subsequent filtrate to prepare a sample solution.

In the improvement of the technical scheme, in the step (3), the volume concentration of the formic acid solution is 0.05-0.2%.

As an improvement of the technical scheme, the volume concentration of the formic acid solution is 0.05%.

As an improvement of the above technical scheme, the gradient elution is carried out according to the following procedures:

0-1 min, wherein the content of mobile phase A is from 3% → 6%, and the content of mobile phase B is from 97% → 94%;

1-3 min, the mobile phase A is 6% → 11%, and the mobile phase B is 94% → 89%;

3-5 min, wherein the mobile phase A is 11% → 13%, and the mobile phase B is 89% → 87%;

5-15 min, wherein the mobile phase A is from 13% → 16%, and the mobile phase B is from 87% → 84%;

15-22 min, wherein the mobile phase A is 16% → 19%, and the mobile phase B is 84% → 81%;

22-30 min, wherein the mobile phase A is 19% → 23%, and the mobile phase B is 81% → 77%;

30-35 min, wherein the mobile phase A is 23% → 27%, and the mobile phase B is 77% → 73%;

35-40 min, wherein the mobile phase A is from 27% → 34%, and the mobile phase B is from 73% → 66%;

40-45 min, wherein the mobile phase A is 34% → 36%, and the mobile phase B is 66% → 64%;

45-50 min, wherein the mobile phase A is 36% → 50%, and the mobile phase B is 64% → 50%;

50-65 min, the mobile phase A is 50% → 60%, and the mobile phase B is 50% → 40%.

As an improvement of the technical scheme, in the step (3), respectively sucking 1-3 μ L of each of a reference solution and a sample solution, taking octadecylsilane chemically bonded silica as a filler, wherein the column length is 150mm, the inner diameter is 2.1mm, and the particle size is 1.7 μm; acetonitrile is used as a mobile phase A, and 0.05% -0.2% formic acid solution is used as a mobile phase B; the flow rate is 0.28-0.32 mL/min; the column temperature is 28-32 ℃, and the detection wavelength is 235-250 nm.

As an improvement of the technical scheme, in the step (3), respectively sucking 1 μ L of each of a reference solution and a sample solution, taking octadecylsilane chemically bonded silica as a filler, wherein the column length is 150mm, the inner diameter is 2.1mm, and the particle size is 1.7 μm; acetonitrile is taken as a mobile phase A, and a 0.05% formic acid solution is taken as a mobile phase B; the flow rate is 0.3 mL/min; the column temperature is 30 ℃, and the detection wavelength is 235-250 nm.

As an improvement of the technical scheme, in the step (3), when the detection time is 0-18 minutes, the detection wavelength is 235 nm; when the detection time is 18-65 minutes, the detection wavelength is 250nm.

As an improvement of the technical scheme, the characteristic spectrum of the dampness-eliminating and toxin-vanquishing composition comprises 11 characteristic peaks; wherein, the peak corresponding to the chrysophanol-8-O-glucoside reference peak is an S peak, and the relative retention time of each characteristic peak and the S peak is within +/-10% of a specified value; the prescribed values include: peak 2 was 0.43, Peak 3 was 0.60, Peak 5 was 0.82, Peak 6 was 0.96, Peak 8 was 1.25.

The implementation of the invention has the following beneficial effects:

the invention establishes the characteristic spectrum of the dampness-resolving and toxin-vanquishing composition for the first time, the characteristic spectrum can fully display the chemical component characteristics of the dampness-resolving and toxin-vanquishing composition, the information content of characteristic peaks is rich, and the quality information of the dampness-resolving and toxin-vanquishing composition can be comprehensively reflected, thereby achieving the aim of comprehensively and effectively controlling the product quality of the dampness-resolving and toxin-vanquishing composition. Meanwhile, the characteristic map construction method provided by the invention has the advantages of good reproducibility, accuracy, reliability and good stability.

Drawings

FIG. 1 is a characteristic diagram of the dampness-resolving and toxin-vanquishing composition of the present invention measured by different elution gradients;

FIG. 2 is a characteristic diagram of the dampness-resolving and toxin-vanquishing composition of the present invention measured by different chromatographic columns;

FIG. 3 is a characteristic diagram of the dampness-eliminating and toxin-vanquishing composition of the present invention measured by different absorption wavelengths;

FIG. 4 is a characteristic diagram of the dampness-resolving and toxin-vanquishing composition of the present invention measured by different mobile phases;

FIG. 5 is a characteristic diagram of the dampness-resolving and toxin-vanquishing composition of the present invention measured by using mobile phases with different concentrations;

FIG. 6 is a characteristic diagram of the dampness-resolving and toxin-vanquishing composition of the present invention extracted by different extraction solvents;

FIG. 7 is a characteristic spectrum of the dampness-resolving and toxin-vanquishing composition of the present invention extracted by different extraction methods;

FIG. 8 is a characteristic spectrum of the dampness-resolving and toxin-vanquishing composition of the present invention extracted at different extraction times;

FIG. 9 is a characteristic diagram of a dampness-resolving and toxin-vanquishing composition, a semen lepidii reference medicinal material and a semen lepidii-lacking negative sample in the characteristic diagram special study of the dampness-resolving and toxin-vanquishing composition;

FIG. 10 is a characteristic diagram of a dampness-resolving and toxin-vanquishing composition, a licorice control drug and a licorice-deficient negative sample in the invention;

FIG. 11 is a characteristic diagram of a dampness-resolving and toxin-vanquishing composition, a red peony root control medicinal material and a red peony root-lacking negative sample in the invention;

FIG. 12 is a characteristic diagram of a dampness-resolving and toxin-vanquishing composition, a Magnolia officinalis control medicinal material and a Magnolia officinalis-absent negative sample in the characteristic diagram special investigation of the dampness-resolving and toxin-vanquishing composition of the present invention;

FIG. 13 is a characteristic diagram of the dampness-resolving and toxin-vanquishing composition, a gypsum work control medicinal material and a poria cocos control medicinal material in the invention;

FIG. 14 is a characteristic diagram of the dampness-resolving and toxin-vanquishing composition, rhubarb reference medicinal material and rhubarb-lacking negative sample in the invention;

FIG. 15 is a characteristic diagram of a dampness-resolving and toxin-vanquishing composition, a herba Ephedrae control material, and a herba Ephedrae deficiency negative sample, which are examined according to the characteristic diagram specificity of the dampness-resolving and toxin-vanquishing composition of the present invention;

FIG. 16 is a characteristic map of a specific attribute of the dampness-resolving and toxin-vanquishing composition of the present invention, wherein the characteristic maps of a negative sample of a dampness-resolving and toxin-vanquishing composition, a parched grass nut working control drug and a parched grass nut lack are examined;

FIG. 17 is a characteristic map of a dampness-resolving and toxin-vanquishing composition, a herba pogostemonis control medicinal material and a herba pogostemonis-lacking negative sample in the characteristic map special investigation of the dampness-resolving and toxin-vanquishing composition of the invention;

FIG. 18 is a feature map of a dampness-resolving and toxin-vanquishing composition, a bran-fried rhizoma Atractylodis working control material, and a bran-fried rhizoma Atractylodis negative sample, which are examined according to the feature map specificity of the dampness-resolving and toxin-vanquishing composition of the present invention;

FIG. 19 is a feature map of the dampness-resolving and toxin-vanquishing composition, rhizoma pinellinae praeparata working control medicinal material, rhizoma pinellinae praeparata negative sample, rhizoma pinellinae praeparata and licorice root double negative sample according to the feature map specialization of the dampness-resolving and toxin-vanquishing composition of the present invention;

FIG. 20 is a feature map of a dampness-resolving and toxin-vanquishing composition, a radix astragali control medicinal material and a radix astragali-deficient negative sample in the feature map special investigation of the dampness-resolving and toxin-vanquishing composition of the invention;

FIG. 21 is a characteristic map of the dampness-resolving and toxin-vanquishing composition of the present invention, wherein the characteristic maps of the dampness-resolving and toxin-vanquishing composition, the fried bitter apricot kernel working control medicinal material and the non-fried bitter apricot kernel negative sample are examined;

FIG. 22 is a feature map of each control, negative sample, dampness-resolving and toxin-vanquishing composition in the feature map specificity study of the dampness-resolving and toxin-vanquishing composition of the present invention;

FIG. 23 is a characteristic diagram of the dampness-resolving and toxin-vanquishing composition measured by different instruments in the middle precision investigation of the characteristic diagram of the dampness-resolving and toxin-vanquishing composition according to the present invention;

FIG. 24 is a characteristic diagram of a dampness-resolving and toxin-vanquishing composition when different column temperatures are used for durability examination of the characteristic diagram of the dampness-resolving and toxin-vanquishing composition according to the present invention;

FIG. 25 is a graph of the durability of the dampness-resolving and toxin-vanquishing composition of the present invention using different flow rates;

FIG. 26 is a graph of the durability of the wet removing and toxin counteracting composition measured using different chromatographic columns according to the present invention;

FIG. 27 is an overlay of the characteristic spectra of a small sample of 15 batches of the dampness-eliminating and toxin-vanquishing composition of the present invention; wherein peak 1 is a paeoniflorin peak, peak 4 is a glycyrrhizin peak, peak 7 is a chrysophanol-8-O-glucoside peak, peak 9 is a glycyrrhizic acid peak, peak 10 is a honokiol peak, and peak 11 is a magnolol peak;

FIG. 28 is an overlay of the profiles of the 6 batches of damp-clearing and detoxifying composition production samples of the present invention; wherein peak 1 is a paeoniflorin peak, peak 4 is a glycyrrhizin peak, peak 7 is a chrysophanol-8-O-glucoside peak, peak 9 is a glycyrrhizic acid peak, peak 10 is a honokiol peak, and peak 11 is a magnolol peak;

FIG. 29 is a control profile of a small sample of a dampness-resolving and toxin-vanquishing composition of the present invention; wherein peak 1 is a paeoniflorin peak, peak 4 is a glycyrrhizin peak, peak 7 is a chrysophanol-8-O-glucoside peak, peak 9 is a glycyrrhizic acid peak, peak 10 is a honokiol peak, and peak 11 is a magnolol peak;

fig. 30 is a comparison characteristic spectrum of a sample produced from the dampness-resolving and toxin-vanquishing composition of the present invention, wherein peak 1 is a paeoniflorin peak, peak 4 is a glycyrrhizin peak, peak 7 is a chrysophanol-8-O-glucoside peak, peak 9 is a glycyrrhizic acid peak, peak 10 is a honokiol peak, and peak 11 is a magnolol peak.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings and detailed description.

The dampness-eliminating and toxin-vanquishing composition mainly comprises the following components: ephedra herb, fried bitter almond, gypsum, liquorice, cablin potchouli herb, officinal magnolia bark, bran-fried rhizoma atractylodis, fried grass nut, rhizoma pinellinae praeparata, Indian buead, rhubarb, astragalus, pepperweed seed and red paeony root.

The invention relates to a damp-resolving toxin-vanquishing composition, which takes ephedra, patchouli and gypsum as monarch drugs, wherein the ephedra and the patchouli have pungent, bitter and warm odor, and can relieve exterior syndrome, relieve asthma, resolve dampness and harmonize the middle warmer; gypsum, gypsum, pungent, sweet and cold in flavor, can clear and purge stagnated heat of lung and stomach and promote the production of body fluid, and the three herbs are combined to achieve the effects of relieving exterior syndrome, dispelling cold, eliminating dampness with aromatics, clearing heat and relieving asthma. The fried bitter almond, the rhizoma pinellinae praeparata, the magnolia officinalis, the rhizoma atractylodis fried with bran, the fried grass nut and the poria cocos are used as ministerial drugs, and the fried bitter almond, the rhizoma pinellinae praeparata and the magnolia officinalis are pungent, bitter and warm, promote qi circulation, descend adverse qi, resolve masses and relieve asthma; stir-frying rhizoma atractylodis and parched tsaoko nut with bran, and the rhizoma atractylodis and the tsaoko nut are pungent, bitter and warm, enter spleen and stomach meridians, dry dampness and invigorate spleen and are knotted by grumpy; poria, with the effects of removing dampness and invigorating spleen; the six herbs are used together to achieve the actions of drying dampness and strengthening spleen, moving qi and unblocking orifices, dredging striae and striae, and helping pathogen go out. Radix astragali, radix Paeoniae Rubra, semen Lepidii, and radix et rhizoma Rhei as adjuvant drugs, radix astragali, radix Et rhizoma Rhei, radix Paeoniae Rubra, radix Et rhizoma Rhei, bitter taste, slight cold, blood cooling, and blood stasis dispelling effects, and can be used for treating diseases such as impairment of vital qi in late stage of epidemic diseases, and blood stasis due to stagnation of qi; ting Li Zi is pungent and cold, and assists the principal drug Gypsum Fibrosum in clearing lung heat, and also has the effect of inducing diuresis to prevent or treat "damp lung (pulmonary edema) lesion"; the rhubarb, radix et rhizoma Rhei, bitter and cold in property, enters the large intestine channel to purge the fu-organs, the lung and the large intestine are exterior and interior, the monarch drug gypsum is used for assisting in clearing lung heat, and the red peony root is used for cooling blood and activating blood, the four drugs are used together as adjuvant drugs to achieve the effects of treating and protecting healthy qi, purging heat and cooling blood, and activating blood and dissolving stasis. The liquorice is used as a guiding drug, the liquorice is sweet and mild, the liquorice is used for harmonizing the effects of the drugs in the recipe, and the radix paeoniae rubra and the liquorice are used for decoction of slow and urgent medicines. The whole formula has the effects of relieving exterior syndrome, eliminating dampness, clearing heat, relieving asthma, tonifying qi and dissipating blood stasis.

The clinical findings show that the patients with severe coronary pneumonia have the following characteristics: firstly, fever is mainly manifested as lingering fever and difficult healing, but can be moderate or low fever, even no fever; ② the asthma suffocation and fatigue are obvious and also the main manifestations; ③ the patients with symptoms of poor appetite, loose stool, diarrhea and other digestive systems; fourthly, most of the tongue has thick and greasy coating. From the characteristics, the medicine accords with the pathogenic characteristics of damp pathogen: heavy turbidity obstructing qi and impairing yang, and sticky food descending. Dampness can cause diseases independently, and can be accompanied by cold and heat manifested as cold-dampness and damp-heat, wherein the heat can be caused by latent dryness or transformation by long-term stagnation of dampness. Pathogenic dampness, cold-dampness and damp-heat can combine with epidemic toxicity to cause disease, which is manifested by mild cold-dampness stagnation in lung and damp-heat accumulation in lung, common type of damp-toxicity stagnation in lung and cold-dampness obstruction in lung, and severe coronary pneumonia due to invasion of ying-blood and reverse transmission of pericardium if no treatment is given or disease development. Therefore, the new coronary pneumonia is considered to be marked as 'damp-toxin plague', the disease is located in the lung and closely related to the spleen, the pathological properties are that the cold and heat are mixed and deficiency and excess are seen, the pathological factors are toxin, dampness, heat, cold, stasis and deficiency, wherein the epidemic toxin is the root, the core pathogenesis is epidemic toxin and damp pathogen stagnation, and the new coronary pneumonia can block the chest and the lung due to invasion of cold and heat, the qi movement is abnormal in ascending and descending, the blood vessel is blocked, and the qi and yin are consumed. The pathological nature of the new coronary pneumonia is complex, and multiple pathological factors are involved.

The main disease location is in the lung, and the secondary disease location is in the spleen and stomach, and the damp toxin stagnation is the core pathogenesis of the disease, and can be divided into an initial stage, a middle stage, a critical stage and a recovery stage to carry out syndrome differentiation treatment, and the treatment methods comprise methods of eliminating dampness and promoting qi circulation, removing dirt and detoxifying, clearing lung and eliminating phlegm, promoting blood circulation and removing blood stasis, clearing hollow viscera and purgating, tonifying healthy qi and the like. Therefore, the compatibility of the dampness-resolving and toxin-vanquishing composition of the invention is based on the core pathogenesis, and the compatibility of the dampness-resolving and toxin-vanquishing composition is taken as a core treatment method for relieving exterior syndrome and resolving dampness, clearing heat and relieving asthma and dispelling toxin, and also has the functions of removing blood stasis and dredging collaterals, and tonifying qi and nourishing yin. Epidemic toxin is combined with cold-dampness, aversion to cold and fever, and it is suitable for relieving exterior syndrome, eliminating dampness and dispelling toxin; epidemic toxin is combined with damp-heat, loose stool is not comfortable, and fatigue and weakness are caused, so that the traditional Chinese medicine composition is suitable for clearing heat, eliminating dampness and removing toxicity, and also has the functions of tonifying qi and nourishing yin; block the chest and lung, dyspnea, oppression in the chest and shortness of breath, dyspnea should be treated with dyspnea, and blood stasis removing and collaterals dredging are also used.

The composition for eliminating dampness and detoxifying disclosed by the invention integrates the core pathogenesis of traditional Chinese medicine treatment in a novel coronavirus infection pneumonia diagnosis and treatment scheme (trial for the fifth edition), belongs to the problems of lung qi stagnation and lung qi obstruction caused by warm and damp mixed with each other, and has the effects of eliminating dampness and promoting qi circulation, dispersing lung qi and relieving asthma, clearing heat and eliminating phlegm, and tonifying qi and activating blood. The early-stage clinical observation shows that the traditional Chinese medicine composition can improve the clinical symptoms of severe novel coronavirus infection pneumonia, can obviously relieve the main symptoms of cough, hypodynamia, dry mouth or vomiting and the like for severe patients, and shortens the curing time after the traditional Chinese medicine and western medicine are combined for treatment. Obviously improves the respiratory function of the patient and shortens the time of oxygen inhalation. For common patients, the traditional Chinese medicine composition can obviously relieve fever symptoms and also can improve anorexia and chest distress symptoms. The medicine has obvious improvement on clinical symptoms of cough, hypodynamia, xerostomia or vomit and the like of the severe and common novel coronavirus infection pneumonia, and supplements the medicine for treating the severe and common novel coronavirus infection pneumonia which is urgently needed by the current epidemic situation.

Preferably, the dampness-eliminating and toxin-removing composition comprises the following components:

3-60 parts of ephedra, 4.5-90 parts of fried bitter almond, 7.5-150 parts of gypsum, 1.5-30 parts of liquorice, 5-100 parts of pogostemon cablin, 5-100 parts of mangnolia officinalis, 7.5-150 parts of bran-fried rhizoma atractylodis, 5-100 parts of fried grass nut, 4.5-90 parts of rhizoma pinellinae praeparata, 7.5-150 parts of poria cocos, 2.5-50 parts of rheum officinale, 5-100 parts of astragalus membranaceus, 5-100 parts of semen lepidii, 5-100 parts of red paeony root and a proper amount of auxiliary materials.

The dampness-resolving and toxin-vanquishing composition is prepared into a traditional Chinese medicine preparation, and the traditional Chinese medicine preparation is granules, decoction, powder, capsules, oral liquid, tablets or pills, but is not limited to the preparation.

In order to comprehensively reflect the quality information of the dampness-resolving and toxin-vanquishing composition and realize comprehensive and effective control of the product quality of the dampness-resolving and toxin-vanquishing composition, the invention provides a method for establishing a characteristic spectrum of the dampness-resolving and toxin-vanquishing composition, which is explained in detail as follows:

1 Instrument and reagent

The instrument comprises the following steps: waters ultra high performance liquid chromatograph (H-Class, Watts Ltd.), Thermo ultra high performance liquid chromatograph (Vanqish, Sammer fly technologies (China) Ltd.), Agilent SB C18 column (2.1 × 150mm, 1.8 μm), Phenomenex Luna Omega C18 column (2.1 × 150mm, 1.6 μm), Waters BEH C18 column (2.1 mm × 150mm, 1.7 μm), ten thousandth scale (ME 204E, Mettler Torlington Co.), millionth (XP 26, Mettler Torlo Co.), numerical control ultrasonic cleaner (KQ 500D, Kunzshan ultrasonic instruments Ltd.), constant temperature water bath (HWS 28 type, Shanghai-Hengzhen technology Ltd.), ultra pure water system (Dili-Q rect, Mileke Merge).

Reagent: ethanol (west longa science, ltd) and methanol (west longa science, ltd) were both analytically pure; the liquid phase is prepared by using formic acid (Tianjin Corp Mimi European chemical reagent Co., Ltd.), glacial acetic acid (Tianjin Corp Mimi European chemical reagent Co., Ltd.), 36% acetic acid (Tianjin Corp Mimi European chemical reagent Co., Ltd.), acetonitrile (Merck Co., Ltd.) and methanol (Merck Co., Ltd.) as HPLC chromatographic grade, and water is ultrapure water (self-made in a laboratory).

Reagent testing: liquiritigenin reference (batch No. wkq18030505, content: 98.0%, Dopperphy Biotech, Inc.); glycyrrhizic acid reference (batch number 110731 and 201720, content: 97.7%, China institute for food and drug identification); paeoniflorin reference (batch No. 110736-201943, content: 95.1%, China institute for food and drug identification); chrysophanol-8-O-glucoside reference substance (batch number: CFN99410, content: 98.0%, ChemFaces); magnolol reference substance (batch No. 110729-; honokiol reference substance (batch number: 110730-; herba Ephedrae reference drug (batch number: 121051) -200704, China institute for food and drug assay); radix Paeoniae Rubra reference material (batch No. 121093-; cortex Magnolia officinalis control drug (batch No. 121285 and 201303, China institute for testing food and drug); glycyrrhrizae radix control (batch No. 120904-201620, China food and drug testing research institute); the rhubarb reference drug (batch number: 120984-; poria cocos reference drug (batch number: 121117-; radix astragali control (batch No. 120974-201612, China food and drug testing research institute); herba Agastaches control medicinal material (batch No. 121135 201606, China food and drug testing research institute); the parched fructus Tsaoko working control medicinal material, Gypsum Fibrosum working control medicinal material, rhizoma Pinelliae Preparata working control medicinal material, bran parched rhizoma Atractylodis working control medicinal material, parched semen Armeniacae amarum working control medicinal material, and semen Lepidii working control medicinal material are all from Guangdong party pharmaceutical company Limited (the working control medicinal material of a certain medicinal material is medicinal material obtained by processing standard medicinal material according to processing technology recorded in pharmacopoeia of the people's republic of China (2015 edition)); the production samples of dampness-resolving and toxin-vanquishing particles (batch numbers: P1-P6) and the small samples of dampness-resolving and toxin-vanquishing particles (batch numbers: S1-S15) are all from Guangdong pharmaceutical Co., Ltd.), dextrin (batch number: FC1901004, carumururi pharmaceutic adjuvant limited), steviosin (batch no: TJ2004011, major carlo ltd).

Chromatographic condition and preparation of reference substance solution and sample solution

2.1 chromatographic conditions

Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filler (the column length is 150mm, the inner diameter is 2.1mm, and the particle size is 1.7 mu m); acetonitrile is taken as a mobile phase A, 0.05 percent formic acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the table 1; the flow rate is 0.30mL per minute; the column temperature is 30 ℃; the detection wavelength is 235nm in 0-18 minutes and 250nm in 18-65 minutes. The number of theoretical plates is not less than 50000 calculated according to chrysophanol-8-O-glucoside peak.

2.2 preparation of reference solutions

Preparation of control solutions: accurately weighing appropriate amount of penoniflorin reference substance, liquiritigenin reference substance, chrysophanol-8-O-glucoside reference substance, glycyrrhizic acid reference substance, honokiol reference substance, and magnolol reference substance, and adding methanol to obtain mixed reference substance solution containing penoniflorin 160 μ g, liquiritigenin 25 μ g, chrysophanol-8-O-glucoside 20 μ g, glycyrrhizic acid 40 μ g, honokiol 3 μ g, and magnolol 4 μ g per 1 mL.

Preparation of reference solution of reference drug: respectively taking ephedra, red paeony root, rhubarb, officinal magnolia bark, semen lepidii, tuckahoe, astragalus, liquorice, patchouli contrast medicinal materials, fried grass nuts, gypsum, rhizoma pinellinae praeparata, bran-fried rhizoma atractylodis and fried bitter apricot kernel work contrast medicinal materials which are about 0.5g, precisely weighing, putting into a conical flask with a plug, adding 25mL of water, heating and refluxing for 60 minutes, taking out, cooling, filtering, evaporating to dryness, dissolving with 25mL of 70% methanol, transferring into the conical flask with the plug, weighing, heating and refluxing for 60 minutes, taking out, cooling, weighing again, complementing the loss weight with 70% methanol, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the traditional Chinese medicine.

2.3 preparation of test solutions

Taking a proper amount of the dampness-resolving and toxin-vanquishing composition, grinding, taking about 1.0g, precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of 70% methanol, weighing, heating and refluxing for 60 minutes, taking out, cooling, weighing again, supplementing the loss weight with 70% methanol, shaking up, filtering, and taking the subsequent filtrate.

2.4 assay

Precisely sucking 1 μ L of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and measuring to obtain characteristic spectrum. The sample characteristic chromatogram should present 11 characteristic peaks, wherein 6 peaks should correspond to the retention time of the corresponding control peak, specifically, peak 1 is a paeoniflorin peak, peak 4 is a liquiritigenin peak, peak 7 is a chrysophanol-8-O-glucoside peak, peak 9 is a glycyrrhizic acid peak, peak 10 is a honokiol peak, and peak 11 is a magnolol peak. Selecting the peak corresponding to the chrysophanol-8-O-glucoside reference peak as an S peak, and calculating the relative retention time of each characteristic peak and the S peak, wherein the relative retention time is within +/-10% of a specified value; the specified values are: 0.43 (peak 2), 0.60 (peak 3), 0.82 (peak 5), 0.96 (peak 6), 1.25 (peak 8).

Determination of chromatographic conditions

3.1 investigation of different elution gradients

Gradient 1: a Waters BEH C18 column (2.1X 150mm, 1.7 μm) was used as a chromatographic column; acetonitrile is used as a mobile phase A, 0.05% formic acid is used as a mobile phase B, and gradient elution is carried out according to the table 2; detection wavelength: 235nm for 0-18 min, 250nm for 18-80 min, 30 ℃ for column temperature, 0.3mL per minute for flow rate, and 1 μ L for sample injection, the results are shown in FIG. 1.

Gradient 2: a Waters BEH C18 column (2.1X 150mm, 1.7 μm) was used as a chromatographic column; acetonitrile was used as mobile phase a, 0.05% formic acid was used as mobile phase B, and gradient elution was performed according to table 3; detection wavelength: 235nm at 0-18 min, 250nm at 18-80 min, 30 ℃ at column temperature, 0.3mL per minute at flow rate, and 1 μ L at sample rate, and the results are shown in FIG. 1.

Gradient 3: a Waters BEH C18 column (2.1X 150mm, 1.7 μm) was used as a chromatographic column; acetonitrile is taken as a mobile phase A, 0.05 percent formic acid is taken as a mobile phase B, and gradient elution is respectively carried out according to the table 4; detection wavelength: 235nm at 0-18 min, 250nm at 18-65 min, 30 ℃ at column temperature, 0.3mL per minute at flow rate, and 1 μ L at sample rate, as shown in FIG. 1.

Comparing 3 different elution gradients, the chromatographic peaks of the gradient 2 and the gradient 3 are more, and the characteristic peak information of more medicinal materials can be contained, such as: when gradient 2 and gradient 3 are used, the paeoniflorin component in radix Paeoniae Rubra is eluted at acetonitrile ratio of about 15%, but not eluted at gradient 1. In comparison of gradient 2 and gradient 3, the resolution of each chromatographic peak in gradient 3 is better than that in gradient 2, and in conclusion, gradient 3 was selected as the optimum chromatographic elution condition.

3.2 investigation of different columns

The ultra-high chromatographic columns of different manufacturers are investigated, and the method comprises the following steps: agilent SB C18 (2.1X 150mm, 1.7 μm), Phenomenex Luna Omega C18 (2.1X 150mm, 1.6 μm), Waters BEH C18 (2.1X 150mm, 1.7 μm) three different manufacturers of chromatographic columns, determined to be the most suitable chromatographic column. Gradient elution was performed as specified in table 4 using acetonitrile as mobile phase a and 0.05% formic acid as mobile phase B at a flow rate of 0.3mL per minute; the sample injection amount is 1 mu L; detection wavelength: 235nm in 0-18 min and 250nm in 18-60 min; the results are shown in FIG. 2.

The results show that the chromatographic columns of three different manufacturers have different separation effects on each chromatographic peak, and the Waters BEH chromatographic column has better separation effect and better peak type on each chromatographic peak. Thus, Waters BEH C18 (2.1X 150mm, 1.7 μm) was chosen as analytical column.

3.3 determination of the optimum absorption wavelength

Investigating different absorption wavelengths; the absorption wavelengths were 235nm, 240nm, 250nm and 300nm, respectively. A Waters BEH C18(2.1 mm. times.150 mm, 1.7 μm) column was used; gradient elution was performed as specified in table 4 using acetonitrile as mobile phase a and 0.05% formic acid as mobile phase B; the flow rate is 0.3mL per minute; the sample injection amount is 1 mu L; the results are shown in FIG. 3.

By comparing the 4 detection wavelength chromatograms, it can be found that when 235nm and 240nm are selected as the detection wavelengths, the base line is not stable after 35 minutes; when the wavelengths of 250nm and 300nm are selected, the response value of the whole chromatographic peak becomes low.

Furthermore, by consulting the literature of the medicinal materials such as red peony root, rhubarb, magnolia bark, liquorice and the like, the best absorption wavelength of paeoniflorin is about 230nm, the maximum absorption of magnolol and honokiol which are main components in the magnolia bark are about 250nm and 290nm respectively is found, and the detection wavelength of glycyrrhizic acid in the liquorice is about 250nm generally.

In order to reflect the characteristic peaks of the medicinal materials as much as possible, ensure the response values of the various spectral peaks and ensure the base line to be stable, the detection wavelength is 235nm when the test time is selected to be 0-18 minutes; when the test time is 18-65 minutes, the detection wavelength is 250nm.

3.4 investigation of the mobile phase

(1) Investigating the type of the buffer solution (mobile phase B) in the mobile phase, and respectively selecting a 0.1% phosphoric acid solution, a 0.1% acetic acid solution and a 0.05% formic acid solution; a Waters BEH C18(2.1 mm. times.150 mm, 1.7 μm) column was used; acetonitrile was used as mobile phase a, and gradient elution was performed as specified in table 4; the flow rate is 0.3mL per minute; the detection wavelength is 235nm at 0-18 minutes and 250nm at 18-65 minutes; the amount of sample was 1. mu.L, and the results are shown in FIG. 4.

(2) Investigating the concentration of a buffer solution (mobile phase B) in the mobile phase, and respectively selecting a 0.05% formic acid solution, a 0.1% formic acid solution and a 0.2% formic acid solution; a Waters BEH C18(2.1 mm. times.150 mm, 1.7 μm) column was used; acetonitrile was used as mobile phase a, and gradient elution was performed as specified in table 4; the flow rate is 0.3mL per minute; the detection wavelength is 235nm at 0-18 minutes and 250nm at 18-65 minutes; the amount of sample was 1. mu.L, and the results are shown in FIG. 5.

Comparing three chromatograms of 0.1% phosphoric acid solution, 0.1% acetic acid solution and 0.05% formic acid solution, finding that the peak information of the 0.05% formic acid solution is complete and the separation degree is optimal; by comparing formic acid solutions with different concentrations, the influence of three formic acid solutions with different concentrations on overall peak information is small, but the baseline fluctuation of a 0.05% formic acid solution is small when the wavelength is switched. In summary, 0.05% formic acid solution was used as buffer (mobile phase B).

Examination of preparation method of test solution

4.1 examination of extraction solvent

The influence of different solvents on the characteristic spectrum of the dampness-resolving and toxin-vanquishing composition is considered, 100% methanol, 70% methanol, 50% methanol, 20% methanol, 95% ethanol, 70% ethanol, 50% ethanol and 20% ethanol (the concentrations are volume concentrations) are respectively used as extraction solvents, the influence of different extraction solvents on the characteristic spectrum of the dampness-resolving and toxin-vanquishing composition is compared by observing the peak types and the separation degrees of 11 characteristic peaks in the dampness-resolving and toxin-vanquishing composition and calculating the total peak area/sample weighing amount of the 11 characteristic peaks, and the optimal extraction solvent is selected.

Specifically, a proper amount of the dampness-resolving and toxin-vanquishing composition is taken, ground, precisely weighed to be about 1.0g, eight groups are arranged in parallel, two parts of each group are placed in a conical flask with a plug, 100% methanol, 70% methanol, 50% methanol, 20% methanol, 95% ethanol, 70% ethanol, 50% ethanol and 25mL of 20% ethanol are precisely added respectively, the weight is weighed, the reflux treatment is carried out for 30 minutes, the mixture is taken out, the mixture is cooled, the weight is weighed again, the loss weight is compensated by using a corresponding solvent, the mixture is shaken up, the filtration is carried out, a subsequent filtrate is taken, the sample injection analysis is carried out according to the chromatographic condition of 2.1 items, and the result is shown in a figure 6 and a.

The results show that: the extraction is incomplete when 95% ethanol, 20% methanol and 20% ethanol are used as extraction solvents, the extraction effect of other solvents on each characteristic peak is similar, and the peak type and the separation effect of each characteristic peak have no obvious difference, wherein when 70% methanol and 70% ethanol are used as extraction solvents, the total peak area/sample weighing amount of 11 characteristic peaks is the largest, and when 70% methanol is used as an extraction solvent, the theoretical plate number of each characteristic peak is more excellent, so 70% methanol is selected as the extraction solvent.

4.2 examination of extraction methods

The influence of different extraction modes on the characteristic spectrum of the dampness-resolving and toxin-vanquishing composition is investigated, two extraction modes of ultrasonic and reflux are respectively investigated, the peak types and the separation degrees of 11 characteristic peaks are observed, the total peak area/sample weighing of the 11 characteristic peaks is calculated, and the influence of different extraction modes on the characteristic spectrum of the dampness-resolving and toxin-vanquishing composition is compared.

Specifically, a proper amount of the dampness-resolving and toxin-vanquishing composition is taken, ground, precisely weighed, taken about 1.0g, parallelly arranged in 2 groups, placed in a conical flask with a plug, precisely added with 25mL of 70% methanol, weighed, respectively treated by ultrasonic treatment (power 250W, frequency 40 kHz) for 60 minutes, heated and refluxed for 60 minutes, taken out, cooled, weighed again, complemented by 70% methanol to reduce the weight, shaken well, filtered, and a subsequent filtrate is taken; the sample injection analysis was carried out under the chromatographic conditions of item 2.1, and the results are shown in FIG. 7 and Table 6.

The results show that: different extraction modes are adopted, the peak type and the separation effect of each characteristic peak are not obviously different, but the value of the total peak area/sample weighing amount of the reflux treatment is higher than that of the ultrasonic treatment, and the reflux treatment is selected to ensure that the extraction is completely considered.

4.3 extraction time study

And (3) investigating the influence of the extraction time on the characteristic spectrum of the dampness-resolving and toxin-vanquishing composition, and comparing the influence of different extraction times on the characteristic spectrum of the dampness-resolving and toxin-vanquishing composition through the total peak area/sample weighing of 11 characteristic peaks.

Specifically, a proper amount of dampness-resolving and toxin-vanquishing particles are taken, ground, precisely weighed, taken about 1.0g, parallelly arranged in 3 groups, placed in a conical flask with a plug, precisely added with 25mL of 70% methanol, weighed, respectively heated and refluxed for 30 minutes, 60 minutes and 90 minutes, taken out, cooled, weighed again, complemented by 70% methanol to reduce the weight, shaken evenly, filtered, and a subsequent filtrate is taken. The sample injection analysis was carried out under the chromatographic conditions of item 2.1, and the results are shown in FIG. 8 and Table 7.

The results show that: different extraction times are adopted, the total peak area/sample weighing amount of 11 characteristic peaks has no obvious difference, certain deviation exists between two samples in 30-minute extraction, and the reflux extraction time is selected to be 60 minutes in order to ensure the durability and complete extraction of the method in consideration of the influence of experimental environment.

4.4 determination of preparation method of test solution

According to the experimental result, the pretreatment method of the damp-resolving and toxin-vanquishing composition characteristic spectrum sample is determined as follows:

taking a proper amount of the dampness-resolving and toxin-vanquishing composition, grinding, taking about 1.0g, precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of 70% methanol, weighing, heating and refluxing for 60 minutes, taking out, cooling, weighing again, supplementing the loss weight with 70% methanol, shaking up, filtering, and taking the subsequent filtrate.

Methodology validation

5.1 specialization examination

The damp-resolving and toxin-vanquishing composition lacking each medicine is prepared according to the preparation method of the test solution in item 2.3 to obtain a negative sample solution lacking each medicine.

Respectively taking semen Lepidii, Glycyrrhrizae radix, radix Paeoniae Rubra, cortex Magnolia officinalis, Poria, radix et rhizoma Rhei, herba Ephedrae, herba Agastaches and radix astragali as reference medicinal materials, respectively taking Gypsum Fibrosum, parched semen Alpiniae Oxyphyllae, bran-parched rhizoma Atractylodis, rhizoma Pinelliae Preparata, and parched semen Armeniacae amarum as reference medicinal materials, and preparing reference solution of each medicinal material according to the preparation method of 2.2 reference solutions of reference medicinal materials.

Taking appropriate amount of penoniflorin reference substance, glycyrrhizin reference substance, chrysophanol-8-O-glucoside reference substance, glycyrrhizic acid reference substance, honokiol reference substance, and magnolol reference substance, and preparing reference substance solution of each reference substance according to 2.2 reference substance solutions.

Injecting 1 μ L of the test solution, the negative sample solution without each medicinal material, the reference substance solution of each medicinal material, and the reference substance solution of each reference substance into a liquid chromatograph, and analyzing by sample injection under 2.1 chromatographic conditions, with the results shown in FIGS. 9-22.

As can be seen from FIGS. 9 to 21: peak 4 (glycyrrhizin), Peak 8, Peak 9 (glycyrrhizic acid) are characteristic components of Glycyrrhrizae radix; peak 1 (paeoniflorin) and Peak 2 are characteristic components of radix Paeoniae Rubra; peak 3, Peak 10 (honokiol), Peak 11 (magnolol) are characteristic components of Magnolia officinalis; peak 5, Peak 6, Peak 7 (chrysophanol-8-O-glucoside) are characteristic components of radix et rhizoma Rhei; the peak 9 (glycyrrhizic acid) is detected in the rhizoma pinellinae praeparata, and the component is determined to be the component of the liquorice due to the use of the liquorice in the rhizoma pinellinae praeparata processing process and by combining the liquorice map; no component is eluted from gypsum, tuckahoe, fried bitter almond and bran-fried rhizoma atractylodis under the chromatographic condition; main components in the medicinal materials of the pepperweed seed, the ephedra, the parched tsaoko nut, the patchouli and the astragalus are eluted in the first 20 minutes, but the chromatographic peak separation effect in the medicinal materials in the first 20 minutes is poor, and the medicinal materials are easily influenced by the background, so that no characteristic peak belongs to the medicinal materials under the determined chromatographic condition.

As can be seen from FIG. 22, the chromatogram of the test sample had the same chromatographic peak at the retention time corresponding to that of the chromatogram of the control sample, and the test sample had no interference, which indicates that the method is well-specified.

5.2 precision investigation

Taking a proper amount of the dampness-resolving and toxin-vanquishing composition, grinding, taking about 1.0g, precisely weighing, preparing according to a preparation method of a test solution determined under 4.4 items, carrying out sample injection analysis under 2.1 items of chromatographic conditions, carrying out continuous sample injection for 6 times, taking a chromatographic peak of chrysophanol-8-O-glucoside (peak 7) as a reference peak S, calculating the relative retention time and the relative peak area of each characteristic peak and the S peak, and calculating the RSD value. The results are shown in tables 8 and 9.

The result shows that the relative retention time RSD of each characteristic peak and the S peak is within the range of 0.02% -0.16%, the relative peak area RSD is within the range of 0.45% -1.64%, and the relative peak area RSD is less than 3.0% by taking the chromatographic peak of chrysophanol-8-O-glucoside (peak 7) as a reference peak S, and the instrument precision is good.

5.3 repeatability test

Taking a proper amount of the dampness-resolving and toxin-vanquishing composition, grinding, taking about 1.0g of the dampness-resolving and toxin-vanquishing composition, taking 6 parts of the dampness-resolving and toxin-vanquishing composition in parallel, precisely weighing, preparing according to a preparation method of a test solution determined under 4.4 items, carrying out sample injection analysis under 2.1 items of chromatographic conditions, taking chrysophanol-8-O-glucoside (peak 7) as a reference peak S, calculating the relative retention time and the relative peak area of each characteristic peak and the S peak, and calculating the RSD value. The results are shown in tables 10 and 11.

The result shows that the relative retention time RSD of each characteristic peak and the S peak is in the range of 0.02% -0.38%, the relative peak area RSD is in the range of 0.33% -1.70%, and the relative peak area RSD is less than 3.0% by taking the chromatographic peak of chrysophanol-8-O-glucoside (peak 7) as a reference peak S, and the method is good in repeatability.

5.4 stability Studies

Taking a proper amount of the dampness-resolving and toxin-vanquishing composition, grinding, taking about 1.0g, precisely weighing, preparing according to a preparation method of a test solution determined under item 4.4, respectively carrying out sample injection analysis for 0, 8, 12, 17, 22 and 24 hours under chromatographic conditions under item 2.1, taking chrysophanol-8-O-glucoside (peak 7) as a reference peak S, calculating the relative retention time and the relative peak area of each characteristic peak and the S peak, and calculating the RSD value. The results are shown in tables 12 and 13.

The result shows that the same test solution is analyzed in 0, 8, 12, 17, 22 and 24 hours respectively, the relative retention time RSD of each characteristic peak and the S peak is in the range of 0.04-0.60% by taking chrysophanol-8-O-glucoside (peak 7) as a reference peak S, and the relative peak area RSD is in the range of 0.70-2.75% and is less than 3.0%, which indicates that the test solution is relatively stable in 24 hours.

5.5 intermediate precision investigation

Different analysts operate on different instruments at different time, a proper amount of the dampness-resolving and toxin-vanquishing composition is taken, ground, about 1.0g of the dampness-resolving and toxin-vanquishing composition is taken, 6 parts of the dampness-resolving and toxin-vanquishing composition are accurately weighed and prepared according to a preparation method of a test solution determined under 4.4 items, sample injection analysis is carried out according to 2.1 items, a chromatographic peak of chrysophanol-8-O-glucoside (peak 7) is taken as a reference peak S, the relative retention time and the relative peak area of each characteristic peak and the S peak are recorded, and an RSD value is calculated, and the result is shown in a figure 23, a table 14 and a table 15.

The result shows that when the chrysophanol-8-O-glucoside (peak 7) chromatographic peak is taken as a reference peak S, the relative retention time RSD of each characteristic peak and the S peak is in the range of 0.14-6.39%, the relative peak area RSD is in the range of 0.89-3.36%, the relative peak area RSD value is less than 5.0%, the relative retention time of the peak 1 (paeoniflorin) chromatographic peak is 6.39%, the retention time reference of a reference substance with paeoniflorin is recommended, and the relative retention time of other various chromatographic peaks is less than 3.0%, so that the middle precision of each characteristic peak is good.

5.6 durability examination

(1) Investigation of different column temperatures

The influence of different column temperatures on the characteristic spectrum of the dampness-resolving and toxin-vanquishing composition is examined, and the column temperatures of 25 ℃, 30 ℃ and 35 ℃ are respectively examined.

Specifically, a proper amount of the dampness-resolving and toxin-vanquishing composition is taken, ground, about 1.0g of the dampness-resolving and toxin-vanquishing composition is taken, precisely weighed and prepared according to a preparation method of a test solution determined under item 4.4, the chromatographic conditions are the same as those under item 2.1 except that the column temperatures are respectively 28 ℃, 30 ℃ and 32 ℃, the chromatographic peak of chrysophanol-8-O-glucoside (peak 7) is taken as a reference peak S, the relative retention time and the relative peak area of each characteristic peak and the S peak are calculated, and the RSD value is calculated. The results are shown in FIG. 24, Table 16 and Table 17.

The results show that the relative retention time RSD of each characteristic peak and the S peak is in the range of 0.08-0.74% and the relative peak area RSD is in the range of 0.63-3.02% under different column temperatures, and the results show that when the column temperature is +/-2 ℃, the influence of the change of the column temperature on the relative retention time and the relative peak area of each characteristic peak is small, and the column temperature has good durability.

(2) Investigation of different flow rates

The influence of different flow rates on the characteristic spectrum of the dampness-resolving and toxin-vanquishing composition is examined, and the flow rates are respectively 0.28mL per minute, 0.30mL per minute and 0.32mL per minute.

Specifically, a proper amount of the dampness-resolving and toxin-vanquishing composition is taken, ground, about 1.0g of the dampness-resolving and toxin-vanquishing composition is taken, precisely weighed and prepared according to a preparation method of a test solution determined under item 4.4, except that the flow rates are respectively 0.28mL/min, 0.30mL/min and 0.32mL/min, other chromatographic conditions are the same as those under item 2.1, a chromatographic peak of chrysophanol-8-O-glucoside (peak 7) is taken as a reference peak S, the relative retention time and the relative peak area of each characteristic peak and the S peak are calculated, and the RSD value is calculated. The results are shown in FIG. 25, Table 18 and Table 19.

The results show that under different flow rates, the relative retention time RSD of each characteristic peak and the S peak is in the range of 0.13% -3.40%, and the relative peak area RSD is in the range of 0.22% -2.73%, and the results show that when the flow rate is +/-0.02 mL/min, the change of the flow rate has small influence on the relative retention time and the relative peak area of each characteristic peak, and the flow rate has good durability.

(3) Investigation of different chromatographic columns

The influence of different chromatographic columns on the characteristic spectrum of the dampness-resolving and toxin-vanquishing composition is examined, and the influence is respectively examined by different Waters BEH C18 columns (the numbers are BH-195, BH-155 and BH-216 respectively) of the same brand.

Specifically, a proper amount of the dampness-resolving and toxin-vanquishing composition is taken, ground, about 1.0g of the dampness-resolving and toxin-vanquishing composition is taken, precisely weighed and prepared according to a preparation method of a test solution determined under item 4.4, other chromatographic conditions are the same as those under item 2.1 except different chromatographic columns, a chromatographic peak of chrysophanol-8-O-glucoside (peak 7) is taken as a reference peak S, the relative retention time and the relative peak area of each characteristic peak and the S peak are calculated, and the RSD value is calculated. The results are shown in FIG. 26, Table 20 and Table 21.

The result shows that when the chromatographic peak of chrysophanol-8-O-glucoside (peak 7) is taken as a reference peak S, the relative retention time RSD of each characteristic peak and the S peak is in the range of 0.07-0.89%, the relative peak area RSD is in the range of 0.98-9.54%, and the relative peak areas of the peak 3 and the peak 4 (liquiritigenin) fluctuate greatly, which indicates that chromatographic columns of different batches have small influence on the relative retention time of each characteristic peak and large influence on the relative peak area.

5.7 Small knot

The chrysophanol-8-O-glucoside (peak 7) is taken as a reference peak S, the relative retention time and the relative peak area of each characteristic peak and the S peak are less influenced by the column temperature and the flow rate, chromatographic columns of the same brand and different batches have less influence on the relative retention time of each chromatographic peak, and have larger influence on the relative peak area. Therefore, only the relative retention time of each characteristic peak is specified, and the relative peak area is not required.

Sample assay

6.1 determination of different batches of dampness-resolving and toxin-vanquishing compositions

Respectively taking 15 batches of small samples (serial numbers S1-S15) of the wet detoxification composition and 6 batches of production samples (serial numbers P1-P6); preparing a test solution according to the test solution preparation method determined under item 4.4, precisely absorbing the test solution, carrying out sample injection determination under the chromatographic condition of item 2.1, obtaining 15 batches of the wet-removing and toxin-vanquishing composition sample characteristic map superposition pictures as shown in 27, obtaining 6 batches of production sample characteristic superposition pictures as shown in 28, taking chrysophanol-8-O-glucoside (peak 7) as a reference peak S, and calculating the relative retention time and the relative peak area of each characteristic peak and the S peak as shown in tables 22-25.

The results show that: the relative retention time RSD value of a small sample of the 15 batches of the wet toxin-vanquishing composition is in the range of 0.06% -0.22%, and the relative peak area RSD value is in the range of 46.36% -111.79%. The relative retention time RSD value of the characteristic spectrum of the 6 batches of damp-eliminating and toxin-vanquishing composition production samples is in the range of 0.02% -0.25%, and the relative peak area RSD value is in the range of 5.03% -92.99%. The relative retention time of each characteristic peak of the production sample of the dampness-eliminating and toxin-vanquishing composition and the small sample of the dampness-eliminating and toxin-vanquishing composition are in the same range, which indicates the consistency of the production of the dampness-eliminating and toxin-vanquishing composition and the quality of the small sample.

6.2 creation of feature maps

Respectively matching the characteristic spectrums of 15 batches of small samples of the dampness-resolving and toxin-vanquishing composition and 6 batches of production samples of the dampness-resolving and toxin-vanquishing composition by using a traditional Chinese medicine chromatography fingerprint similarity evaluation system, generating a reference spectrum by taking chrysophanol-8-O-glucoside (peak 7) as a reference peak S according to an average method, establishing a reference characteristic spectrum (figure 29) of the small samples of the dampness-resolving and toxin-vanquishing composition, and establishing a reference characteristic spectrum (figure 30) of the production samples of the dampness-resolving and toxin-vanquishing composition.

The research results show that the characteristic spectrum standard of the dampness-resolving and toxin-vanquishing composition is determined by taking the chromatographic peak of chrysophanol-8-O-glucoside (peak 7) as a reference peak S and referring to the characteristic spectrum results: 11 characteristic peaks are presented in the chromatogram of the test sample, wherein 6 peaks respectively correspond to the retention time of the peaks of the corresponding reference sample, the peak corresponding to the peak of the chrysophanol-8-O-glucoside reference sample is the S peak, the relative retention time of each characteristic peak and the S peak is calculated, the relative retention time is within +/-10% of a specified value, and the specified value is as follows: 0.43 (peak 2), 0.60 (peak 3), 0.82 (peak 5), 0.96 (peak 6), 1.25 (peak 8).

In conclusion, the invention establishes the characteristic spectrum of the dampness-resolving and toxin-vanquishing composition for the first time, the characteristic spectrum can fully display the chemical component characteristics of the dampness-resolving and toxin-vanquishing composition, the characteristic peak information content is rich, and the quality information of the dampness-resolving and toxin-vanquishing composition can be comprehensively reflected, so that the aim of comprehensively and effectively controlling the product quality of the dampness-resolving and toxin-vanquishing composition can be achieved. Meanwhile, the characteristic map construction method provided by the invention has the advantages of good reproducibility, accuracy, reliability and good stability.

While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.

Claims (12)

1. A construction method of a characteristic spectrum of a dampness-resolving toxin-vanquishing composition is characterized in that the dampness-resolving toxin-vanquishing composition mainly comprises the following components: ephedra, fried bitter almond, gypsum, liquorice, patchouli, mangnolia officinalis, bran-fried rhizoma atractylodis, fried grass nut, rhizoma pinellinae praeparata, poria cocos, rheum officinale, astragalus membranaceus, semen lepidii and red paeony root;

the construction method of the dampness-eliminating and toxin-vanquishing composition characteristic spectrum comprises the following steps:

(1) respectively taking appropriate amount of penoniflorin reference substance, glycyrrhizin reference substance, chrysophanol-8-O-glucoside reference substance, glycyrrhizic acid reference substance, honokiol reference substance, and magnolol reference substance, adding solvent, and dissolving or extracting to obtain reference substance solution;

(2) extracting the dampness-resolving and toxin-vanquishing composition with an extraction solvent to obtain a test solution;

(3) injecting a preset amount of reference substance solution and test solution into a liquid chromatograph, and performing gradient elution by using octadecylsilane chemically bonded silica as a filler, acetonitrile as a mobile phase A and formic acid solution as a mobile phase B in the liquid chromatograph to establish a characteristic spectrum of the dampness-resolving and toxin-vanquishing composition.

2. The method for constructing the characteristic spectrum of the dampness-eliminating and toxin-vanquishing composition according to claim 1, wherein in the step (2), the extraction solvent is 50-100% methanol or 50-70% ethanol;

the extraction method is ultrasonic extraction or reflux extraction.

3. The method for constructing the characteristic map of the dampness-eliminating and toxin-vanquishing composition according to claim 1 or 2, wherein the step (2) comprises:

taking the dampness-resolving and toxin-vanquishing composition, grinding, taking 0.5-1.5 g of the composition, precisely weighing, placing the composition into a conical flask with a plug, adding 20-30 mL of 50-70% methanol, weighing, heating and refluxing for 30-90 minutes, taking out, cooling, weighing again, complementing the weight loss by using 50-70% methanol, shaking uniformly, filtering, taking the subsequent filtrate, and preparing the test solution.

4. The method for constructing the characteristic map of the dampness-eliminating and toxin-vanquishing composition according to claim 1 or 2, wherein the step (2) comprises:

taking the dampness-resolving and toxin-vanquishing composition, grinding, taking 0.5-1.5 g of the composition, precisely weighing, placing the composition into a conical flask with a plug, adding 20-30 mL of 50-70% methanol, weighing, carrying out ultrasonic treatment with the power of 200-300 kW and the frequency of 35-45 kHz for 30-90 minutes, taking out, cooling, weighing again, complementing the loss weight with 50-70% methanol, shaking uniformly, filtering, and taking a subsequent filtrate to prepare a sample solution.

5. The method for constructing the characteristic spectrum of the dampness-eliminating and toxin-vanquishing composition according to claim 1, wherein in the step (3), the concentration of the formic acid solution is 0.05-0.2% by volume.

6. The method for constructing the characteristic spectrum of the dampness-eliminating and toxin-vanquishing composition according to claim 1 or 5, wherein the formic acid solution is 0.05% by volume.

7. The method for constructing the characteristic map of the dampness-eliminating and toxin-vanquishing composition according to claim 1, wherein the gradient elution is performed according to the following procedure:

0-1 min, wherein the content of mobile phase A is from 3% → 6%, and the content of mobile phase B is from 97% → 94%;

1-3 min, the mobile phase A is 6% → 11%, and the mobile phase B is 94% → 89%;

3-5 min, wherein the mobile phase A is 11% → 13%, and the mobile phase B is 89% → 87%;

5-15 min, wherein the mobile phase A is from 13% → 16%, and the mobile phase B is from 87% → 84%;

15-22 min, wherein the mobile phase A is 16% → 19%, and the mobile phase B is 84% → 81%;

22-30 min, wherein the mobile phase A is 19% → 23%, and the mobile phase B is 81% → 77%;

30-35 min, wherein the mobile phase A is 23% → 27%, and the mobile phase B is 77% → 73%;

35-40 min, wherein the mobile phase A is from 27% → 34%, and the mobile phase B is from 73% → 66%;

40-45 min, wherein the mobile phase A is 34% → 36%, and the mobile phase B is 66% → 64%;

45-50 min, wherein the mobile phase A is 36% → 50%, and the mobile phase B is 64% → 50%;

50-65 min, the mobile phase A is 50% → 60%, and the mobile phase B is 50% → 40%.

8. The method for constructing a characteristic spectrum of a dampness-resolving and toxin-vanquishing composition according to claim 1, wherein in the step (3), 1 to 3 μ L of each of the reference solution and the test solution is respectively absorbed, octadecylsilane chemically bonded silica is used as a filler, the column length is 150mm, the inner diameter is 2.1mm, and the particle size is 1.7 μm; acetonitrile is used as a mobile phase A, and 0.05% -0.2% formic acid solution is used as a mobile phase B; the flow rate is 0.28-0.32 mL/min; the column temperature is 28-32 ℃, and the detection wavelength is 235-250 nm.

9. The method according to claim 8, wherein in the step (3), 1 μ L of each of the control solution and the test solution is separately sucked, octadecylsilane chemically bonded silica is used as a filler, the column length is 150mm, the inner diameter is 2.1mm, and the particle size is 1.7 μm; acetonitrile is taken as a mobile phase A, and a 0.05% formic acid solution is taken as a mobile phase B; the flow rate is 0.3 mL/min; the column temperature is 30 ℃, and the detection wavelength is 235-250 nm.

10. The method for constructing the characteristic spectrum of the dampness-resolving and toxin-vanquishing composition according to claim 1, wherein in the step (3), when the detection time is 0-18 minutes, the detection wavelength is 235 nm; when the detection time is 18-65 minutes, the detection wavelength is 250nm.

11. The method for constructing the characteristic spectrum of the dampness-resolving and toxin-vanquishing composition according to claim 1, wherein the characteristic spectrum of the dampness-resolving and toxin-vanquishing composition comprises 11 characteristic peaks; wherein, the peak corresponding to the chrysophanol-8-O-glucoside reference peak is an S peak, and the relative retention time of each characteristic peak and the S peak is within +/-10% of a specified value; the prescribed values include: peak 2 was 0.43, Peak 3 was 0.60, Peak 5 was 0.82, Peak 6 was 0.96, Peak 8 was 1.25.

12. The method for constructing the characteristic spectrum of the dampness-eliminating and toxin-vanquishing composition according to claim 1, wherein the dampness-eliminating and toxin-vanquishing composition mainly comprises the following components: 3-60 parts of ephedra, 4.5-90 parts of fried bitter almond, 7.5-150 parts of gypsum, 1.5-30 parts of liquorice, 5-100 parts of pogostemon cablin, 5-100 parts of mangnolia officinalis, 7.5-150 parts of bran-fried rhizoma atractylodis, 5-100 parts of fried grass nut, 4.5-90 parts of rhizoma pinellinae praeparata, 7.5-150 parts of poria cocos, 2.5-50 parts of rheum officinale, 5-100 parts of astragalus membranaceus, 5-100 parts of semen lepidii, 5-100 parts of red paeony root and a proper amount of auxiliary materials;

the dampness eliminating and toxin removing composition is prepared into a traditional Chinese medicine preparation which is granules, decoction, powder, capsules, oral liquid, tablets or pills.

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