CN111735889B

CN111735889B - Quality detection method of dampness-resolving and toxin-vanquishing composition

Info

Publication number: CN111735889B
Application number: CN202010834335.9A
Authority: CN
Inventors: 程学仁; 魏梅; 李定发; 李振雨; 霍文杰; 陈向东; 何广铭; 何民友; 潘礼业; 索彩仙; 杨晓东; 吴文平; 黄跃前; 何嘉莹; 童培珍
Original assignee: Guangdong Yifang Pharmaceutical Co Ltd
Current assignee: Guangdong Yifang Pharmaceutical Co Ltd
Priority date: 2020-08-19
Filing date: 2020-08-19
Publication date: 2021-02-09
Anticipated expiration: 2040-08-19
Also published as: CN111735889A

Abstract

The invention discloses a quality detection method of a dampness-resolving and toxin-vanquishing composition, which 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 quality detection method of the dampness-eliminating and toxin-vanquishing composition comprises the following steps: determining total anthraquinone content, free anthraquinone content, ephedrine hydrochloride content, pseudoephedrine hydrochloride content and paeoniflorin content in the dampness eliminating and toxin removing composition by high performance liquid chromatography, and calculating combined anthraquinone content; wherein, bound anthraquinone content = total anthraquinone content-free anthraquinone content. The determination method disclosed by the invention is high in specificity and stability and high in durability, and can effectively ensure the stability and controllability of the product quality of the dampness-resolving and toxin-vanquishing composition in a large-scale production process.

Description

Quality detection method of dampness-resolving and toxin-vanquishing composition

Technical Field

The invention relates to the technical field of traditional Chinese medicine quality detection, in particular to a quality detection method 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-eliminating and toxin-vanquishing composition medicine flavor for resisting novel coronavirus pneumonia (COVID-19)' (Chinese modern traditional medicine, No. 3 of 2020). However, no specific quality testing method for drugs has been studied.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a quality detection method of the dampness-resolving and toxin-vanquishing composition, which has good specificity and stability and strong durability and can effectively ensure the stability and controllability of the product quality of the dampness-resolving and toxin-vanquishing composition in the large-scale production process.

In order to solve the technical problems, the invention provides a quality detection method of a dampness-resolving and toxin-vanquishing composition, which 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 quality detection method of the dampness-eliminating and toxin-vanquishing composition comprises the following steps: determining the total anthraquinone content, free anthraquinone content, ephedrine hydrochloride and pseudoephedrine hydrochloride content and paeoniflorin content in the dampness eliminating and toxin removing composition by high performance liquid chromatography, and calculating the content of bound anthraquinone;

wherein, bound anthraquinone content = total anthraquinone content-free anthraquinone content.

As an improvement of the technical scheme, the method for measuring the total anthraquinone content comprises the following steps:

(1) mixing appropriate amount of aloe-emodin, rhein, emodin, chrysophanol, and physcion with methanol to obtain mixed solution, and making into anthraquinone reference solution;

(2) extracting the dampness-resolving and toxin-vanquishing composition with methanol to obtain total anthraquinone sample solution;

(3) and (2) absorbing the anthraquinone reference substance solution and the total anthraquinone sample solution, injecting the anthraquinone reference substance solution and the total anthraquinone sample solution into a liquid chromatograph, performing gradient elution on a chromatographic column of the liquid chromatograph by using octadecylsilane chemically bonded silica as a filler, and determining the total anthraquinone content in the dampness eliminating and toxin removing composition by using acetonitrile as a mobile phase A and a phosphoric acid aqueous solution as a mobile phase B in the liquid chromatograph.

As an improvement of the technical scheme, the method for measuring the content of the free anthraquinone comprises the following steps:

taking appropriate amount of aloe-emodin, rhein, emodin, chrysophanol, and physcion, adding methanol to obtain mixed solution, and making into anthraquinone reference solution;

secondly, extracting the damp-resolving and toxin-vanquishing composition by using methanol to prepare a free anthraquinone test sample solution;

and (III) absorbing the anthraquinone reference substance solution and the free anthraquinone test sample solution, injecting the anthraquinone reference substance solution and the free anthraquinone test sample solution into a liquid chromatograph, performing gradient elution on a chromatographic column of the liquid chromatograph by using octadecylsilane chemically bonded silica as a filler, and determining the content of the free anthraquinone in the dampness resolving and toxin removing composition by using acetonitrile as a mobile phase A and a phosphoric acid aqueous solution as a mobile phase B of the liquid chromatograph.

As an improvement of the technical scheme, the method for measuring the total anthraquinone content and the method for measuring the free anthraquinone content are carried out by adopting the following elution procedures: 0-10 min, the mobile phase A is 35% → 40%, and the mobile phase B is 65% → 60%;

10-38 min, wherein the mobile phase A is from 40% → 60%, and the mobile phase B is from 60% → 40%;

and (3) 38-48 min, wherein the mobile phase A is 60% and the mobile phase B is 40%.

As an improvement of the technical scheme, the total anthraquinone sample solution is prepared by the following method:

taking 0.2-0.5 g of dampness-eliminating and toxin-removing composition, placing the composition in a conical flask with a plug, adding 20-30 mL of methanol, heating and refluxing for extraction for 20-30 minutes, taking out, cooling, weighing again, complementing the weight loss with methanol, shaking up, filtering, taking 10-15 mL of subsequent filtrate, recovering the solvent under reduced pressure until the filtrate is dry, adding 10-15 mL of 8% hydrochloric acid solution, performing ultrasonic treatment for 2-5 minutes, adding 10-20 mL of trichloromethane, heating and refluxing for 1-3 hours, cooling, placing the filtrate in a separating funnel, washing a container with a small amount of trichloromethane, merging the chloroform layer, extracting the acid solution with trichloromethane for 2-5 times, 10-15 mL each time, merging the trichloromethane solution, recovering the solvent under reduced pressure until the solvent is dry, adding methanol to dissolve the residue, transferring the residue to a 10mL measuring flask, adding methanol to filter, shaking up, and taking the subsequent filtrate to obtain the composition.

As an improvement of the technical scheme, the free anthraquinone test solution is prepared by the following method:

taking 0.2-0.5 g of the dampness-eliminating and toxin-vanquishing composition, placing the composition into a conical flask with a plug, adding 25-30 mL of methanol, weighing, heating and refluxing for 20-60 minutes, taking out, cooling, weighing again, supplementing the lost weight with methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the composition;

as an improvement of the technical scheme, the anthraquinone reference substance solution is prepared by the following method:

weighing 1.608mg of aloe-emodin reference substance, 3.038mg of rhein reference substance, 1.625mg of emodin reference substance, 2.667mg of chrysophanol reference substance and 5.274mg of physcion reference substance, placing in a 100mL measuring flask, adding methanol to obtain mother liquor containing 15.807 μ g of aloe-emodin, 30.167 μ g of rhein, 16.039 μ g of emodin, 26.563 μ g of chrysophanol and 52.213 μ g of physcion per 1 mL; precisely sucking 1mL of the aloe-emodin, rhein, emodin and chrysophanol mother liquor and 0.1mL of the physcion mother liquor respectively, placing into a 10mL measuring flask, and adding methanol to obtain a mixed solution containing 1.581 μ g of aloe-emodin, 3.017 μ g of rhein, 1.604 μ g of emodin, 2.656 μ g of chrysophanol and 0.522 μ g of physcion per 1 mL.

In the improvement of the technical scheme, in the method for measuring the content of the total anthraquinone and the method for measuring the content of the free anthraquinone, octadecylsilane chemically bonded silica is used as a filler in a chromatographic column of the liquid chromatograph, the column temperature is 25-35 ℃, the liquid chromatograph is subjected to gradient elution by using acetonitrile as a mobile phase A and 0.1vol% phosphoric acid aqueous solution as a mobile phase B, the flow rate is 0.6-1 mL/min, and the detection wavelength is 253-256 nm.

As an improvement of the technical scheme, the method for measuring the total content of the ephedrine hydrochloride and the pseudoephedrine hydrochloride comprises the following steps:

(1) weighing appropriate amount of ephedrine hydrochloride reference substance and pseudoephedrine hydrochloride reference substance, and adding methanol to obtain mixed solution containing 10 μ g of each 1mL to obtain ephedrine reference substance solution;

(2) extracting the dampness-resolving and toxin-vanquishing composition with hydrochloric acid solution to obtain ephedrine test solution;

(3) absorbing the ephedrine reference solution and the ephedrine test solution, injecting into a liquid chromatograph, wherein a chromatographic column of the liquid chromatograph adopts polar ether connected phenyl bonded silica gel as a filler, and the column temperature is 25-35 ℃; the liquid chromatograph takes a mixed solution of methanol and phosphoric acid aqueous solution as a mobile phase, the flow rate is 0.6-1 mL/min, and the detection wavelength is 205-215 nm; determining the total content of ephedrine hydrochloride and pseudoephedrine hydrochloride in the composition for eliminating dampness and removing toxic substance.

As an improvement of the technical proposal, in the method for measuring the total content of the ephedrine hydrochloride and the pseudoephedrine hydrochloride, methanol and phosphoric acid aqueous solution are taken as mobile phases;

the phosphoric acid aqueous solution is a mixed solution of phosphoric acid, diethylamine, triethylamine and water; wherein the volume fraction of the phosphoric acid is 0.090-0.094%, the volume fraction of the diethylamine is 0.01-0.04%, and the volume fraction of the triethylamine is 0.02-0.06%.

As an improvement of the technical scheme, the ephedrine test solution is prepared by the following method:

taking 0.2-0.5 g of dampness-eliminating and toxin-vanquishing composition, grinding, weighing, placing in a conical flask with a plug, adding 50mL of 0.1mol/L hydrochloric acid solution, sealing the plug, weighing, ultrasonically treating for 30 minutes, cooling, weighing again, supplementing the lost weight with 0.1mol/L hydrochloric acid solution, shaking uniformly, centrifuging for 5 minutes, taking the supernatant, filtering, weighing 25mL of subsequent filtrate, placing on a solid phase extraction column taking a mixed type cation exchange reversed phase adsorbent as a filler, sequentially eluting with 6mL of each of 0.lmol/L hydrochloric acid solution and methanol, discarding the eluent, eluting with 10mL of newly prepared mixed solution of acetonitrile and concentrated ammonia test solution with a volume ratio of 95:5, collecting the eluent, placing in a 10mL measuring flask, adding the mixed solution to scale, and shaking uniformly to obtain the composition.

As an improvement of the technical scheme, the method for measuring the content of paeoniflorin comprises the following steps:

(1) weighing appropriate amount of penoniflorin reference substance, adding methanol to obtain solution containing 0.13mg per 1mL to obtain penoniflorin reference substance solution;

(2) extracting dampness eliminating and toxin removing composition with methanol to obtain paeoniflorin test solution;

(3) sucking penoniflorin reference solution and penoniflorin test solution, and injecting into liquid chromatograph; the chromatographic column of the liquid chromatograph takes octadecyl bonded silica gel as a filler, and the column temperature is 30-35 ℃; the liquid chromatograph takes methanol-0.05 mol/L potassium dihydrogen phosphate aqueous solution as a mobile phase, the flow rate is 0.8-1.2 mL/min, and the detection wavelength is 225-230 nm; determining the content of paeoniflorin in the dampness-eliminating and toxin-vanquishing composition.

As an improvement of the technical scheme, the paeoniflorin test sample solution is prepared by the following method:

weighing 0.8-1.5 g of the dampness-eliminating and toxin-vanquishing composition, placing the weighed composition in a conical flask with a plug, adding 25mL of methanol, weighing, heating and refluxing for 30 minutes, cooling, weighing again, supplementing the lost weight with methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the composition.

As an improvement of the technical scheme, the dampness eliminating and toxin removing 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.

The implementation of the invention has the following beneficial effects:

the invention respectively detects the contents of total anthraquinone and free anthraquinone, the total contents of ephedrine hydrochloride and pseudoephedrine hydrochloride and the content of paeoniflorin in the dampness eliminating and toxin removing composition by high performance liquid chromatography, and calculates the content of combined anthraquinone. The detection methods in the invention have excellent specificity and durability, the accuracy and the stability of the detection methods can meet the requirements of large-scale production, and the stability and the controllability of the product quality of the dampness-eliminating and toxin-removing composition can be effectively ensured.

Drawings

FIG. 1 is an HPLC plot of an anthraquinone control solution; wherein, peak 1 is aloe-emodin peak, peak 2 is rhein peak, peak 3 is emodin peak, peak 4 is chrysophanol peak, and peak 5 is physcion peak;

FIG. 2 is a HPLC chart of a free anthraquinone negative sample solution;

FIG. 3 is an HPLC chart of total anthraquinone negative sample solution;

FIG. 4 is an HPLC chart of a free anthraquinone test solution; wherein, peak 1 is aloe-emodin peak, peak 2 is rhein peak, peak 3 is emodin peak, peak 4 is chrysophanol peak, and peak 5 is physcion peak;

FIG. 5 is an HPLC chart of a total anthraquinone test sample solution; wherein, peak 1 is aloe-emodin peak, peak 2 is rhein peak, peak 3 is emodin peak, peak 4 is chrysophanol peak, and peak 5 is physcion peak;

FIG. 6 is a graph of the aloe-emodin control calibration curve in the determination of free anthraquinone content;

FIG. 7 is a graph of a rhein control standard in the determination of free anthraquinone content;

FIG. 8 is a graph of a standard emodin control for free anthraquinone content determination;

FIG. 9 is a graph of a standard chrysophanol control in free anthraquinone content determination;

FIG. 10 is a graph of the calibration curve of the physcion control in the determination of the content of free anthraquinone;

FIG. 11 is a graph of the aloe-emodin standard in a total anthraquinone control;

FIG. 12 is a graph of the standard Rhein control in the determination of total anthraquinone content;

FIG. 13 is a graph of the standard emodin control for total anthraquinone content determination;

FIG. 14 is a graph of a standard chrysophanol control in total anthraquinone content determination;

FIG. 15 is a graph of the calibration curve of physcion control in the determination of total anthraquinone content;

FIG. 16 is an HPLC chart of ephedrine control with different mobile phases;

FIG. 17 is an HPLC chart of a solution of a Ephedra negative sample;

FIG. 18 is an HPLC chart of ephedrine control solution; wherein, peak 1 is ephedrine hydrochloride peak, and peak 2 is pseudoephedrine hydrochloride peak;

FIG. 19 is an HPLC chart of the ephedrine test solution; wherein, peak 1 is ephedrine hydrochloride peak, and peak 2 is pseudoephedrine hydrochloride peak;

FIG. 20 is a graph showing the standard ephedrine hydrochloride profile of ephedrine reference;

FIG. 21 is a graph of the standard ephedrine hydrochloride profile of ephedrine control;

FIG. 22 is an HPLC chart of a peony negative sample solution;

FIG. 23 is an HPLC chart of a paeoniflorin control solution; wherein, the peak 1 is a paeoniflorin peak;

FIG. 24 is an HPLC chart of a paeoniflorin test sample solution; wherein, the peak 1 is a paeoniflorin peak;

FIG. 25 is a standard graph of a paeoniflorin control.

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.

In the invention, the dampness-resolving 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 invention provides a detection method of a dampness-resolving and toxin-vanquishing composition, which can achieve a good detection effect on the dampness-resolving and toxin-vanquishing composition of any dosage form. In the research process of the prescription of the dampness-resolving and toxin-vanquishing composition, the inventor adopts a molecular docking technology to analyze key targets of invasion, replication, assembly, shedding and transfer of various traditional Chinese medicines and COVID-19 and key action targets of lung injury and inflammatory reaction generated by a host. The results show that: ephedra is responsive to TMPRSS2, TACE, AAK1 (viral entry, endocytosis regulation), which are targets for inhibiting viral entry and shedding, VEGFR2 (vascular permeability) and ALK5 (vascular permeability, pulmonary fibrosis), which are critical targets for tissue damage following viral entry into the host. Rhubarb is responsive to TMPRSS2, a target for inhibiting virus entry and shedding, AMPK (oxidative stress, inflammation), VEGFR2 and ALK5, a key target for tissue damage after virus entry into the host. Red peony root responds to Mpro and ACE2 and thus acts against COVID-19. Further research shows that 4 components of apigenin, 3-methoxyhollyhock aglycone, 3,5, 4' -trihydroxy-8-methoxyflavone, kaempferol and the like in the ephedra play a role in the target; rheinic acid, physcion, 6-hydroxy acid garcinoside-8-O-beta-D-glucoside, procyanidin B1-3' -O-gallate and aloe emodin in rhubarb have effect on the target 5 components; 2 components of baicalein, baicalin and the like in the red paeony root have effects on the targets. Therefore, from the perspective of efficacy, the ephedra herb, the rhubarb and the red paeony root belong to core drugs in the dampness-resolving and toxin-vanquishing composition, and the detection of relevant components of the ephedra herb, the rhubarb and the red paeony root is a key means for realizing the quality control of the dampness-resolving and toxin-vanquishing composition.

In addition, the ephedra in the composition for eliminating dampness and detoxifying plays an important role in the formula, and belongs to monarch drugs in the principle of traditional Chinese medicine formula, and the ephedra mainly plays roles in ventilating lung qi, eliminating phlegm and relieving cough. Meanwhile, ephedrine introduced by the ephedra is volatile and is easy to lose in the process of large-scale production. Therefore, the invention determines the content of ephedrine hydrochloride and ephedrine hydrochloride in the ephedra. Specifically, in order to effectively exert the effect of the composition for eliminating dampness and detoxifying, the content of ephedrine hydrochloride and pseudoephedrine hydrochloride should be controlled to be 0.7-2.7 mg/g.

For rhubarb in the composition for eliminating dampness and removing toxicity, the main functions are clearing lung heat, activating blood and cooling blood; it is mainly realized by combining anthraquinone (aloe-emodin, rhein, emodin, chrysophanol and physcion) introduced into radix et rhizoma Rhei. However, bound anthraquinone readily decomposes to free anthraquinone upon heating, resulting in a decrease in efficacy. For this purpose, the content of the bound anthraquinone needs to be monitored. Therefore, the invention selects to measure and monitor the content of the combined anthraquinone in the finished product medicine so as to realize the quality control of the dampness-resolving and toxin-vanquishing composition. Specifically, in the dampness-eliminating and toxin-vanquishing composition of the present invention, the content of the conjugated anthraquinone is controlled to be not less than 0.016 wt%.

The invention also controls the content of the red paeony root by measuring the content of the paeoniflorin. Specifically, in the dampness eliminating and toxin removing composition, the content of paeoniflorin is 3-14 mg/g.

The measurement method of the present invention is explained in three sections as follows:

method for measuring content of total anthraquinone and free anthraquinone

The content of the total anthraquinone and the content of the free anthraquinone in the dampness eliminating and toxin removing composition are measured by the high performance liquid chromatography, and then the content of the combined anthraquinone is calculated. Wherein, bound anthraquinone content = total anthraquinone content-free anthraquinone content.

Specifically, in the present invention, the method for measuring the total anthraquinone content comprises:

(1) chromatographic conditions are as follows: taking an octadecylsilane chemically bonded silica chromatographic column as a chromatographic column, taking acetonitrile-0.1 vol% phosphoric acid aqueous solution as a mobile phase, wherein the flow rate is 0.8mL/min, the detection wavelength is 254nm, and the column temperature is 30 ℃; the amount of sample was 10. mu.L.

The mobile phase was subjected to gradient elution in the following order of elution:

(2) preparing a total anthraquinone test solution: taking about 0.5g of dampness-eliminating and toxin-removing composition, precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of methanol, weighing, heating and refluxing for extraction for 30 minutes, taking out, cooling, weighing again, complementing the weight loss by methanol, shaking up, filtering, taking 10mL of subsequent filtrate, recovering the solvent under reduced pressure until the solvent is dry, adding 10mL of 8% hydrochloric acid solution, carrying out ultrasonic treatment for 2 minutes, adding 10mL of trichloromethane, heating and refluxing for 1 hour, cooling, placing in a separating funnel, washing the container by a small amount of trichloromethane, merging into the separating funnel, separating and taking a trichloromethane layer, extracting the acid liquor by trichloromethane for 3 times, 10mL each time, merging trichloromethane liquid, recovering the solvent under reduced pressure until the solvent is dry, adding methanol to dissolve the residue, transferring to a 10mL measuring flask, adding methanol to the scale, shaking up, filtering, and taking the subsequent filtrate.

(3) Preparation of anthraquinone reference solution: weighing 1.608mg of aloe-emodin reference substance, 3.038mg of rhein reference substance, 1.625mg of emodin reference substance, 2.667mg of chrysophanol reference substance and 5.274mg of physcion reference substance, placing in a 100mL measuring flask, adding methanol to obtain mother liquor containing 15.807 μ g of aloe-emodin, 30.167 μ g of rhein, 16.039 μ g of emodin, 26.563 μ g of chrysophanol and 52.213 μ g of physcion per 1 mL; precisely sucking 1mL of the aloe-emodin, rhein, emodin and chrysophanol mother liquor and 0.1mL of the physcion mother liquor respectively, placing into a 10mL measuring flask, and adding methanol to obtain a mixed solution containing 1.581 μ g of aloe-emodin, 3.017 μ g of rhein, 1.604 μ g of emodin, 2.656 μ g of chrysophanol and 0.522 μ g of physcion per 1 mL.

(4) Precisely absorbing 10 μ L of each of the total anthraquinone sample solution and the anthraquinone reference solution, injecting into a liquid chromatograph, and measuring to obtain the content of the total anthraquinone in the dampness eliminating and toxin removing composition.

Specifically, in the present invention, the method for measuring the content of free anthraquinone is as follows:

(1) chromatographic conditions are as follows: an octadecylsilane chemically bonded silica chromatographic column is used as a chromatographic column, acetonitrile-0.1 vol% phosphoric acid aqueous solution is used as a mobile phase, the flow rate of the mobile phase is 0.8mL/min, the detection wavelength is 254nm, the column temperature is 30 ℃, and the sample injection amount is 10 mu L.

(2) preparation of free anthraquinone test solution: precisely weighing about 0.5g of dampness-eliminating and toxin-removing composition, placing the composition into a conical flask with a plug, precisely adding 25mL of methanol, weighing, heating and refluxing for 30 minutes, taking out, cooling, weighing again, supplementing the lost weight with methanol, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the composition.

(4) Precisely absorbing 10 μ L of each of the free anthraquinone sample solution and anthraquinone reference solution, injecting into a liquid chromatograph, and measuring to obtain the content of free anthraquinone in the dampness eliminating and toxin removing composition.

The method for measuring the total anthraquinone content and the method for measuring the free anthraquinone content are both performed by using the same elution procedure, and the method for measuring the total anthraquinone content and the method for measuring the free anthraquinone content use the same anthraquinone reference solution.

It should be noted that, because the dampness-resolving and toxin-vanquishing composition contains 14 kinds of bulk drugs in total, the finished product has complex components, and has a great influence on the test accuracy of total anthraquinone and free anthraquinone. For this reason, the present invention redesigns the gradient elution procedure taking into account the accuracy, durability and specificity of the test method. Specifically, the research finds that: in 10min to 38min, the proportion of acetonitrile is 40% → 60%, and the proportion of 0.1vol% phosphoric acid is 60% → 40%, so that aloe-emodin, rhein, emodin and chrysophanol in the dampness-eliminating and toxin-removing composition can be effectively separated, and no large gradient change is required. In addition, within 38 min-48 min, the peak-appearing time of the physcion on different chromatographic columns is different, so that within the time range, the isocratic elution of acetonitrile-0.1 vol% phosphoric acid (60: 40) is set for 10min, the durability of the test method on different chromatographic columns is ensured, and the test accuracy is also improved.

The methodology of the determination of total anthraquinone and free anthraquinone in the present invention was verified as follows:

1.1 specialization examination

Taking a rhubarb negative sample to prepare a total anthraquinone negative sample solution according to the preparation method of the total anthraquinone sample solution, and taking the rhubarb negative sample to respectively prepare free anthraquinone negative sample solutions according to the preparation method of the free anthraquinone sample solution. Respectively injecting 10 mu L of each of the free anthraquinone sample solution, the total anthraquinone sample solution, the free anthraquinone negative sample solution, the total anthraquinone negative sample solution and the anthraquinone reference substance solution into a liquid chromatograph, and testing according to the formulated conditions, wherein the results are shown in figures 1-5. Wherein, the peak position of each reference substance is shown in the following table:

the result shows that the chromatogram of the test sample has the same chromatographic peak at the corresponding retention time with the chromatogram of the reference sample, the free anthraquinone negative sample has no interference, the total anthraquinone negative sample has no interference, and the method has good specificity.

1.2 Linear relationship investigation

1.2.1 investigation of the Linear relationship for determination of free anthraquinone content

Precisely weighing 3.632mg of aloe-emodin reference substance, 2.787mg of emodin reference substance, 1.638mg of chrysophanol reference substance and 3.082mg of physcion reference substance, placing in 50mL measuring bottles, and adding methanol to obtain solutions containing 71.405 μ g of aloe-emodin, 55.015 μ g of emodin, 32.629 μ g of chrysophanol and 61.024 μ g of physcion per 1 mL; precisely measuring 2.071mg of rhein reference substance, precisely sucking 5mL of each of the aloe-emodin, emodin and chrysophanol reference substance solutions and 1mL of physcion reference substance, placing in a 100mL measuring flask, and adding methanol to obtain a mixed solution containing 3.570 μ g of aloe-emodin, 20.565 μ g of rhein, 2.751 μ g of rhein, 1.631 μ g of chrysophanol and 0.610 μ g of physcion per 1mL of the mixed solution as a mother solution of free anthraquinone reference substance. Precisely sucking 5mL, 3mL, 1mL, 0.5mL and 0.2mL of free anthraquinone control mother liquor, placing each of the mother liquor in 10mL measuring bottles, respectively, to prepare a control solution containing 0.071 μ g, 0.179 μ g, 0.357 μ g, 1.071 μ g and 1.785 μ g of aloe-emodin in each 1mL, 0.411 μ g, 1.028 μ g, 2.057 μ g, 6.170 μ g and 10.283 μ g of rhein in each 1mL, 0.055 μ g, 0.138 μ g, 0.275 μ g, 0.825 μ g and 1.375 μ g of emodin in each 1mL, 0.033 μ g, 0.082 μ g, 0.163 μ g, 0.489 μ g and 0.816 μ g of phenol in each 1mL, and 0.030 μ g, 0.183 μ g, 0.305 μ g and 0.061 μ g of physcion in each 1 mL.

Precisely sucking 10 μ L of the 6 reference solutions with different concentrations, testing, analyzing, and recording the chromatographic peak area. The results are shown in fig. 6 to 10, in which the peak area is plotted as ordinate (y) and the reference concentration is plotted as abscissa (x), and a standard curve is drawn.

As can be seen from fig. 6: the regression equation for aloe-emodin is: y =66,512.6130x +128.8558, correlation coefficient R thereof²=0.9994, which shows that the linear relation between the injection quality and the peak area of the aloe-emodin is good in the injection concentration range of 0.071 mug/mL-3.570 mug/mL.

As can be seen from fig. 7, the regression equation for rhein is: y =49,694.7092x-1,493.7898, correlation coefficient R thereof²=0.9998, which shows that the injection quality of rhein has good linear relation with the peak area within the injection concentration range of 0.411 mug/mL-20.565 mug/mL.

As can be seen from fig. 8, the regression equation for emodin is: y =50,043.3431x +269.0534, correlation coefficient R thereof²=0.9995, which shows that the emodin has good linear relation between the injection quality and the peak area within the injection concentration range of 0.055 mu g/mL-2.751 mu g/mL.

As can be seen from fig. 9, the regression equation of chrysophanol is y =68,915.3676x +180.1324, and the correlation coefficient R thereof²=0.9995, which shows that the injection quality and peak area of chrysophanol are linearly related in the injection concentration range of 0.033-1.631 mug/mLThe product was satisfactory.

As can be seen from fig. 10, the regression equation of physcion is: y =51,006.3529x +707.5671, correlation coefficient R thereof²And =0.995, which shows that the linear relation between the injection quality and the peak area of the physcion is good in the injection concentration range of 0.012 mug/mL-0.610 mug/mL.

1.2.2 Total anthraquinone content determination Linear relationship investigation

According to the preparation method of anthraquinone reference solution, every 1mL of solution containing 15.807 μ g of aloe-emodin, 30.167 μ g of rhein, 16.039 μ g of emodin, 26.563 μ g of chrysophanol and 5.221 μ g of physcion is prepared as total anthraquinone reference mother liquor. Precisely sucking 5mL, 3mL, 1mL, 0.5mL and 0.2mL of total anthraquinone reference mother liquor, placing each of the mother liquor in 10mL measuring bottles, respectively, preparing aloe-emodin containing 0.316 μ g, 0.790 μ g, 1.581 μ g, 4.742 μ g and 7.903 μ g in each 1mL, emodic acid containing 0.603 μ g, 1.508 μ g, 3.017 μ g, 9.050 μ g and 15.084 μ g in each 1mL, chrysophanic acid containing 0.321 μ g, 0.802 μ g, 1.604 μ g, 4.812 μ g and 8.109 μ g in each 1mL, chrysophanol containing 0.531 μ g, 1.328 μ g, 2.656 μ g, 7.969 μ g and 13.282 μ g in each 1mL, and physcion containing 0.104 μ g, 0.261 μ g, 0.522 μ g, 1.566 μ g and 2.611 μ g in each 1 mL.

Precisely sucking 10 μ L of the 6 reference solutions with different concentrations, testing, analyzing, and recording the chromatographic peak area. The results are shown in FIGS. 11 to 15, in which the peak area is plotted as the ordinate (y) and the control concentration is plotted as the abscissa (x), and a standard curve is plotted.

As can be seen from fig. 11, the regression equation for aloe-emodin is: y =58108.7967x-793.9060, correlation coefficient R thereof²=0.9999, which shows that the linear relation between the sample injection mass and the peak area of the aloe-emodin is good in the range of the sample injection concentration of 0.316-15.807 mug/mL.

As can be seen from fig. 12, the regression equation for rhein is: y =50,539.6466x-3,498.3368, correlation coefficient R thereof²And =1.0000, which shows that the linear relation between the injection quality and the peak area of the rhein is good in the injection concentration range of 0.603-30.167 mug/mL.

As can be seen from fig. 13, the regression equation for emodin is: y =43,070.1473x-501.3586, coefficient of correlation R thereof²=0.9999, which shows that the emodin has good linear relation between the injection quality and the peak area within the injection concentration range of 0.321 mu g/mL-16.039 mu g/mL.

As can be seen from fig. 14, the regression equation for chrysophanol is: y =61,967.3466x-2,584.2033, correlation coefficient R thereof²=0.9999, which shows that the injection quality of chrysophanol has a good linear relation with the peak area within the injection concentration range of 0.531-26.563 mug/mL.

As can be seen from fig. 15, the regression equation of physcion is: y =30,079.1466x +505.0118, correlation coefficient R thereof²=1.000, which shows that the linear relation between the injection quality and the peak area of the physcion is good in the injection concentration range of 0.104-5.221 mug/mL.

1.3 precision investigation

1.3.1 precision of the Instrument

Precisely absorbing 10 μ L of anthraquinone reference substance solution, testing and analyzing, calculating peak areas of aloe-emodin, rhein, emodin, chrysophanol and physcion, and determining results shown in Table 2.

The result shows that the same anthraquinone reference substance solution is continuously injected into 5 needles, the peak area RSD value of each index is less than 3.0 percent, and the precision of the instrument is good.

1.3.2 intermediate precision investigation

Selecting different experimenters to measure at different times and different high performance liquid chromatographs, taking a proper amount of the dampness-eliminating and toxin-vanquishing composition, grinding the composition into fine powder, taking about 0.5g of the composition, precisely weighing the fine powder, paralleling 6 parts, respectively preparing a free anthraquinone sample solution and a total anthraquinone sample solution, precisely absorbing 10 mu L of an anthraquinone reference solution, testing and analyzing, calculating the content of the free anthraquinone and the total anthraquinone and the RSD value, and comparing with the result of a repeatability investigation test, wherein the result is shown in tables 3-4.

The results show that the same batch of samples are operated on different instruments by different personnel at different times, and the measurement is repeated for 6 times, the RSD value of the content of free anthraquinone is 0.49 percent, and the RSD value of 6 data in the repeated test is 0.45 percent; the RSD value of the total anthraquinone content is 0.75%, the RSD value of 6 data of the repeated tests is 0.92%, when the content of the component to be measured in the sample is 0.01% -0.1% according to the verification guidance principle of the medicine quality standard analysis method of the year edition of Chinese pharmacopoeia 2015, the limit of the intermediate precision RSD is less than 6%, therefore, different analysts operate under different chromatographs at different dates, and the method has good intermediate precision.

1.4 stability Studies

Precisely sucking 10 μ L of free anthraquinone sample solution and total anthraquinone sample solution, respectively injecting at 0, 2.5, 6, 9.5, 13, 17, 20.5, and 25.5 hr, measuring peak areas of aloe-emodin, rhein, emodin, chrysophanol, and physcion in the sample solution, calculating peak areas RSD, and finding out results shown in tables 5 and 6.

The results show that the peak area RSD value of each index component is less than 3.0% when the same free anthraquinone sample solution is subjected to sample injection measurement for 0.0, 2.5, 6.0, 9.5, 13.0, 17.0, 20.5 and 25.5 hours, which indicates that the stability of the free anthraquinone sample solution is good within 24 hours.

The same total anthraquinone sample solution is subjected to sample injection measurement at 0.0 hour, 2.5 hours, 6.0 hours, 9.5 hours, 13.0 hours, 17.0 hours, 20.5 hours and 25.5 hours respectively, the RSD value of each index component except the physcion peak area is less than 3.0 percent, and the other components are all more than 3 percent, and the total anthraquinone sample solution is recommended to finish the measurement at 13 hours.

1.5 repeatability test

Precisely absorbing 10 mu L of free anthraquinone test sample solution and total anthraquinone test sample solution, testing, calculating the content of free anthraquinone and total anthraquinone and RSD value, and determining the result shown in table 7 and table 8.

The result shows that the same batch of samples are repeatedly measured for 6 times, and the RSD value of each index content of free anthraquinone and total anthraquinone is less than 3 percent, which indicates that the repeatability of the analysis method is good.

1.6 accuracy survey

1.6.1 investigation of the accuracy of the determination of free anthraquinone content

Precisely weighing 2.497mg of aloe-emodin reference substance, 2.502mg of rhein reference substance, 1.646mg of emodin reference substance, 1.958mg of chrysophanol reference substance and 0.854mg of physcion reference substance, respectively putting into 250, 50, 250, 500 and 500mL measuring bottles, adding methanol to prepare solutions containing 0.00982 mug of aloe-emodin, 0.04969 mug of rhein, 0.00650 mug of emodin, 0.00390 mug of chrysophanol and 0.00169 mug of physcion per 1mL, and taking the solutions as sample-adding and recycling mother liquor. Precisely sucking 1mL of the mother liquor into a conical flask, parallelly placing 6 parts of the mother liquor, and volatilizing the solvent; and preparing a free anthraquinone test sample solution from the dampness-resolving and toxin-vanquishing composition, measuring the content of free anthraquinone in the test sample, and calculating the sample loading recovery rate, wherein the results are shown in tables 9-13.

As can be seen from the table, the recovery rate of aloe-emodin is 112.08%, the recovery rate of rhein is 86.17%, the recovery rate of emodin is 107.31%, the recovery rate of chrysophanol is 97.79%, the recovery rate of physcion is 105.93%, when the content of the component to be measured in the sample is 0.001% -0.01% according to the verification and guidance principle of the drug quality standard analysis method of the national pharmacopoeia 2015 edition, the limit of the recovery rate is 80% -115%, and the accuracy of the measuring method is good.

1.6.2 Total anthraquinone content determination accuracy survey

2.540mg of aloe-emodin reference substance, 3.988mg of rhein reference substance, 2.743mg of emodin reference substance, 2.322mg of chrysophanol reference substance and 2.015mg of physcion reference substance are precisely weighed and respectively placed in 50mL measuring bottles, methanol is added to respectively prepare solutions containing 0.04994 mu g of aloe-emodin, 0.07920 mu g of rhein, 0.05414 mu g of emodin, 0.04625 mu g of chrysophanol and 0.03990 mu g of physcion in each 1mL of the bottles, and the solutions are used as sample adding and recycling mother liquor. Precisely sucking 1mL of the aloe-emodin, emodin and physcion mother liquor and 2mL of rhein and chrysophanol into a conical flask, parallelly adding 6 parts of rhein and chrysophanol, and volatilizing the solvent; and then taking a proper amount of the dampness-eliminating and toxin-vanquishing composition (batch number: J2004007), grinding, respectively taking 0.25g of the composition into the 6 conical flasks, preparing a total anthraquinone test sample solution, measuring the content of total anthraquinone in the test sample, and calculating the sample adding recovery rate, wherein the results are shown in tables 14-18.

The result shows that the recovery rate of aloe-emodin is 95.05%, the recovery rate of rhein is 88.68%, the recovery rate of emodin is 101.25%, the recovery rate of chrysophanol is 98.83%, the recovery rate of physcion is 101.53%, when the content of the component to be detected in the sample is 0.001% -0.01% according to the verification and guidance principle of the drug quality standard analysis method of the 2015 edition of Chinese pharmacopoeia, the limit of the recovery rate is 80% -115%, and the method has good accuracy.

1.7 durability examination

1.7.1 investigation of different chromatography columns

Three kinds of chromatographic columns were selected, namely a Philomen Kinetex-EVO C18 chromatographic column (4.6 mm. times.150 mm, 5 μm; No. JS-091), a Waters HSS T3 chromatographic column (4.6 mm. times.150 mm, 5 μm; No. JS-136), and a Waters X-bridge C18 chromatographic column (4.6 mm. times.150 mm, 5 μm; No. JS-154).

Taking 10 mu L of each of the free anthraquinone test sample solution and the total anthraquinone test sample solution, respectively adopting the three chromatographic columns for determination, calculating the content of the free anthraquinone and the total anthraquinone and the RSD value, and the experimental results are shown in Table 19.

The result shows that the chromatographic columns have good separation effect, and the content RSD value is less than or equal to 5 percent, which indicates that the analysis method has good analysis durability under different chromatographic columns.

1.7.2 investigation of different column temperatures

Column temperatures of 28 ℃, 30 ℃ and 32 ℃ are respectively set, and the influence of the column temperatures on the determination of the free anthraquinone content and the total anthraquinone content of the dampness-resolving and toxin-vanquishing composition is examined.

Taking 10 mu L of each of the free anthraquinone sample solution and the total anthraquinone sample solution, respectively adopting the column temperature to carry out measurement, calculating the content of the free anthraquinone and the total anthraquinone and the RSD value, and the experimental result is shown in a table 20.

The results show that the content values of free anthraquinone and total anthraquinone in the damp-resolving and toxin-vanquishing composition at the same column temperature are both greater than 5 percent, which indicates that the analysis method is sensitive to the column temperature, and therefore, the column temperature is strictly controlled at 30 ℃.

1.7.3 investigation of different flow rates

The flow rates are respectively set to be 0.7mL/min, 0.8mL/min and 0.9mL/min, and the influence of the flow rates on the determination of the free anthraquinone content and the total anthraquinone content of the dampness-eliminating and toxin-vanquishing composition is examined.

Taking 10 mu L of each of the free anthraquinone test sample solution and the total anthraquinone test sample solution, respectively adopting the flow rates to carry out determination, calculating the content of the free anthraquinone and the total anthraquinone and the RSD value, and the experimental results are shown in a table 21.

The results show that the content values of free anthraquinone and total anthraquinone in the damp-eliminating and toxin-removing composition with three different flow rates have RSD values less than 5 percent. However, when the flow rate is 0.7mL/min, there is a peak of emodin methyl ether in the total anthraquinones, and it is recommended to control the flow rate not less than 0.8mL/min in the analysis.

1.8 content determination of damp-resolving and toxin-vanquishing composition samples of different batches

Taking 10 batches of the damp-eliminating and toxin-vanquishing composition, respectively preparing a free anthraquinone sample solution and a total anthraquinone sample solution, measuring the content of free anthraquinone and the content of total anthraquinone in the 10 batches of the damp-vanquishing composition, and calculating the content of bound anthraquinone; the results are shown in Table 22.

Method for measuring total content of ephedrine hydrochloride and pseudoephedrine hydrochloride

The content of ephedrine hydrochloride and the total content of pseudoephedrine hydrochloride in the dampness-resolving and toxin-vanquishing composition are determined by high performance liquid chromatography. Specifically, the measurement method comprises:

(1) chromatographic conditions are as follows: polar ether is connected with phenyl bonded silica gel as a filling agent, a mixed solution of methanol and 0.092 vol% phosphoric acid (containing 0.04 vol% triethylamine and 0.02 vol% diethylamine) (1.5: 98.5) is used as a mobile phase, the flow rate is 0.8mL/min, the detection wavelength is 210nm, the column temperature is 35 ℃, and the number of theoretical plates is not less than 3000 according to ephedrine hydrochloride.

In general, when the contents of ephedrine hydrochloride and pseudoephedrine hydrochloride in ephedra are measured, a mixed solution of methanol and 0.092 vol% phosphoric acid (containing 0.04% triethylamine and 0.02% di-n-butylamine) (1.5: 98.5) is used as a mobile phase, and di-n-butylamine is replaced by diethylamine in the present invention. Experiments show that the flow in the invention has stronger retention capacity relative to the target substance, can effectively prolong the retention time and optimize the separation effect of the ephedrine hydrochloride peak and the pseudoephedrine hydrochloride peak (figure 16).

(2) Preparing ephedrine solution for test: taking about 0.5g of damp-eliminating and toxin-vanquishing composition, grinding, weighing, placing into a conical flask with a plug, adding 50mL of 0.1mol/L hydrochloric acid solution, sealing the plug, weighing, carrying out ultrasonic treatment (power 250W and frequency 40 kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 0.1mol/L hydrochloric acid solution, shaking uniformly, centrifuging for 5 minutes (4000 rpm), taking supernatant, filtering, weighing 25mL of subsequent filtrate, placing on a solid phase extraction column (taking a mixed type cation exchange reverse phase adsorbent as a filler, 150mg and 6mL, using methanol and water for prewashing respectively 6 mL), eluting with 0.lmol/L hydrochloric acid solution and methanol respectively, discarding eluent, eluting with 10mL of newly prepared acetonitrile-concentrated ammonia test solution (95: 5), collecting eluent, placing into a 10mL measuring flask, adding the mixed solution to dilute to scale, shaking uniformly, obtaining the product;

(3) preparing ephedrine reference solution: weighing appropriate amount of ephedrine hydrochloride reference substance and pseudoephedrine hydrochloride reference substance, and adding methanol to obtain mixed solution containing 10 μ g of each 1 mL;

(4) and (3) sucking 10 mu L each of the ephedrine test solution and the ephedrine reference solution prepared in the steps (2) and (3), injecting into a liquid chromatograph, and measuring to obtain the total content of ephedrine hydrochloride and pseudoephedrine hydrochloride in the dampness-resolving and toxin-vanquishing composition.

The methodology of the determination method of ephedrine hydrochloride and pseudoephedrine hydrochloride in the invention is verified as follows:

2.1 specialization examination

Preparing ephedrine negative sample solutions from the ephedrine negative samples according to the preparation method of the ephedrine test solution; the ephedrine test solution, ephedrine negative sample solution and ephedrine reference solution each 10 μ L are injected into liquid chromatograph for testing, and the results are shown in FIGS. 17-19. Wherein, the peak position of each reference substance is shown in the following table:

as can be seen from FIGS. 17-19 and Table 23, the ephedrine negative sample solution has no obvious chromatographic peak in the corresponding retention time of ephedrine hydrochloride and pseudoephedrine hydrochloride, so that the method has no interference and good specificity.

2.2 Linear relationship investigation

Placing ephedrine hydrochloride reference substance 2.115mg and pseudoephedrine hydrochloride reference substance 2.196mg in 10mL volumetric flask, adding methanol to obtain mixed reference substance stock solution containing ephedrine hydrochloride 211.500 μ g and pseudoephedrine hydrochloride 219.161 μ g per 1 mL; precisely measuring the reference stock solutions with different volumes, and gradually diluting to obtain mixed reference stock solutions of ephedrine hydrochloride and pseudoephedrine hydrochloride with different concentration levels.

Separately, 10. mu.L of ephedrine hydrochloride control solutions (1 mL containing ephedrine hydrochloride of 1.058. mu.g, 2.115. mu.g, 4.230. mu.g, 10.575. mu.g, 21.150. mu.g, 52.875. mu.g and 105.750. mu.g, pseudoephedrine hydrochloride of 1.096. mu.g, 2.192. mu.g, 4.383. mu.g, 10.958. mu.g, 21.916. mu.g, 54.790. mu.g and 109.580. mu.g) of different concentrations were precisely sucked, and peak areas were measured under the above-mentioned chromatographic conditions, and ephedrine hydrochloride and pseudoephedrine hydrochloride (x) were regressed with peak areas (y) to draw standard curves, and the results are shown in FIGS. 20 and 21.

As can be seen from FIG. 20, the linear curve of ephedrine hydrochloride is: y =26,038.4703x +198.2720, R²=0.9999；

As can be seen from fig. 21, the linear curve of pseudoephedrine hydrochloride is: y =26,339.9956x-1,062.3701, R²=1.0000；

The result shows that the linear relation between the peak area and the concentration of the ephedrine hydrochloride is good within the concentration range of 1.058-105.750 mu g/mL; the pseudoephedrine hydrochloride has good linear relation between peak area and concentration within the concentration range of 1.096-109.580 mu g/mL.

2.3 precision investigation

Precisely sucking 10 μ L of ephedrine reference solution (ephedrine hydrochloride concentration: 10.575 μ g/mL; pseudoephedrine hydrochloride concentration: 10.958 μ g/mL) and repeating sample injection for 6 times, and calculating according to the peak areas of ephedrine hydrochloride and pseudoephedrine hydrochloride, and determining the results shown in Table 24.

The result shows that the same ephedrine reference solution is continuously injected for 6 times, the RSD value of each index peak area is less than 2.0 percent, and the instrument precision is good.

2.4 stability Studies

Precisely sucking 10 μ L of the same ephedrine test solution, injecting for 1 time in 0, 2, 4, 8, 12, 14h, respectively, measuring peak areas of ephedrine hydrochloride and pseudoephedrine hydrochloride, and calculating peak area RSD value, the results are shown in Table 25.

As can be seen from the table, the relative standard deviation RSD (n = 6) of ephedrine hydrochloride and pseudoephedrine hydrochloride is 1.08% and 1.07%, respectively, and both are less than 3.0%, indicating that the stability of the sample is good within 14 hours.

2.5 repeatability test

And (4) preparing ephedrine test solution from the same batch of samples, and determining. The contents of ephedrine hydrochloride and pseudoephedrine hydrochloride and RSD values were calculated, and the results are shown in Table 26.

The results show that the relative standard deviation RSD of the contents of ephedrine hydrochloride and pseudoephedrine hydrochloride is less than 2.0 percent, which indicates that the method has good repeatability.

2.6 accuracy survey

By adopting a sample adding recovery method, about 0.25g of a sample with known content is taken, precisely weighed, 9 parts of the sample are weighed in parallel, and 3 parts of the sample are taken as a group, ephedrine hydrochloride and pseudoephedrine hydrochloride reference substances are added according to the sample content of 0.5:1.0, 1.0:1.0 and 1.5:1.0 respectively, 9 parts of ephedrine test solution are prepared according to the ephedrine test sample preparation method, and the sample adding recovery rate is respectively measured and calculated, and the result is shown in a table 27.

As can be seen from the table, the average sample adding recovery rate of ephedrine hydrochloride is 94.96%, and the average sample adding recovery rate of pseudoephedrine hydrochloride is 96.59%, which accords with the relevant regulations of Chinese pharmacopoeia, and the determination method of the invention has good accuracy.

2.7 determination of different batches of test samples of dampness-resolving and toxin-vanquishing composition

Taking 3 batches of the dampness-resolving and toxin-vanquishing composition, respectively preparing ephedrine test solution, precisely sucking 10 μ L of ephedrine test solution, measuring ephedrine hydrochloride content and pseudoephedrine hydrochloride content, and calculating total content, the results are shown in Table 28.

Method for measuring paeoniflorin content

The content of paeoniflorin in the dampness eliminating and toxin removing composition is determined by high performance liquid chromatography. Specifically, the measurement method comprises:

(1) chromatographic conditions are as follows: octadecyl bonding silica gel is used as a filling agent, a methanol-0.05 mol/L potassium dihydrogen phosphate solution is used as a (35: 65) mobile phase, the flow rate is 1.0 mL/min, the detection wavelength is 230nm, and the column temperature is 30 ℃;

(2) preparation of a paeoniflorin test solution: weighing about 1.0g of dampness-eliminating and toxin-removing composition, placing into a conical flask with a plug, adding 25mL of methanol, weighing, heating and refluxing for 30 minutes, cooling, weighing again, supplementing the weight loss with methanol, shaking up, filtering, and taking the subsequent filtrate;

(3) preparation of paeoniflorin reference solution: weighing appropriate amount of penoniflorin as control, and adding methanol to obtain solution containing 0.13mg per 1 mL;

(4) sucking 10 μ L each of the paeoniflorin test sample solution and the paeoniflorin reference solution prepared in the steps (2) and (3), injecting into a liquid chromatograph, and determining the content of the paeoniflorin in the dampness eliminating and toxin removing composition.

The method for determining paeoniflorin in the invention is verified by the following methodology:

3.1 specialization examination

Preparing a paeoniflorin negative sample solution from a red paeony root negative sample according to a paeoniflorin test sample solution preparation method; 10 μ L each of the paeoniflorin test solution, the paeoniflorin control solution, and the paeoniflorin negative test solution is injected into a liquid chromatograph for testing, and the results are shown in FIGS. 22-24. As can be seen from the figure, the paeoniflorin-negative sample solution has no obvious chromatographic peak at the same retention time as paeoniflorin, so that the negative sample solution is considered to be non-interference.

3.2 Linear relationship investigation

Taking a proper amount of paeoniflorin reference substance, precisely weighing, and adding methanol to obtain a paeoniflorin reference substance solution containing 0.53mg per 1 mL. Diluting the solution by 2, 4, 8, 16, 32 and 64 times to obtain serial paeoniflorin control solutions with different concentrations. The concentration is used as the abscissa and the peak area is used as the ordinate, a standard curve is drawn, a regression equation is calculated, and the result is shown in figure 25.

The linear curve of paeoniflorin is: y =12,804,471.2418x-1,628.2577, R²= 0.9996; the peak area has a good linear relation with the concentration.

3.3 precision investigation

Precisely sucking 10 μ L of paeoniflorin control solution, repeatedly sampling and measuring for 6 times, calculating deviation according to paeoniflorin peak area, and obtaining measurement results shown in Table 29.

The results show a relative standard deviation of 1.08%, indicating good instrument precision.

3.4 stability Studies

Precisely sucking 10 μ L of the same paeoniflorin sample solution, injecting for 1 time in 0, 4, 8, 12, 18, 24h, respectively, measuring the peak area of paeoniflorin, and calculating relative standard deviation, wherein the result is shown in Table 30.

The result shows that the relative standard deviation is 0.84%, and the paeoniflorin test solution is stable within 24 h.

3.5 repeatability test

Taking the same batch of samples, preparing 6 parts of paeoniflorin test sample solution according to the preparation method of the paeoniflorin test sample solution, and measuring to obtain the relative standard deviation of the content of the paeoniflorin, wherein the result is shown in a table 31.

The result shows that the relative standard deviation is 1.1 percent and is far less than 5 percent of the standard requirement, and the paeoniflorin determination method has good repeatability.

3.6 accuracy survey

Adopting a sample adding and recovering method, taking 0.5g of sample, adding a paeoniflorin reference substance solution according to the proportion of 1:1, preparing a sample according to the preparation method of the paeoniflorin test substance solution, measuring 6 parts in parallel, and calculating the sample adding and recovering rate. The results are shown in Table 32.

As can be seen from the table, the average sample adding recovery rate of paeoniflorin is 98.86%, which accords with the relevant regulations of Chinese pharmacopoeia, and the determination method of the invention has good accuracy.

3.7 determination of different batches of test samples of dampness-resolving and toxin-vanquishing composition

3 batches of the dampness-resolving and toxin-vanquishing compositions were taken to prepare paeoniflorin test solution, and 10 μ L of the paeoniflorin test solution was precisely absorbed to determine the content of paeoniflorin, and the results are shown in table 33.

In conclusion, the invention respectively detects the contents of total anthraquinone and free anthraquinone, the total content of ephedrine hydrochloride and pseudoephedrine hydrochloride and the content of paeoniflorin in the dampness eliminating and toxin removing composition by high performance liquid chromatography, and calculates the content of combined anthraquinone. The detection methods in the invention have excellent specificity and durability, the accuracy and the stability of the detection methods can meet the requirements of large-scale production, and the stability and the controllability of the product quality of the dampness-eliminating and toxin-removing composition can be effectively ensured.

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

1. A quality detection method of a dampness-resolving and toxin-vanquishing composition is characterized in that the dampness-resolving 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-vanquishing composition is prepared into granules;

wherein, bound anthraquinone content = total anthraquinone content-free anthraquinone content;

the content of the combined anthraquinone in the dampness eliminating and toxin removing composition is more than or equal to 0.016wt%, the total content of ephedrine hydrochloride and pseudoephedrine hydrochloride is 0.7-2.7 mg/g, and the content of paeoniflorin is 3-14 mg/g;

the method for measuring the content of the total anthraquinone comprises the following steps:

(3) absorbing anthraquinone reference substance solution and total anthraquinone sample solution, injecting the solution into a liquid chromatograph, performing gradient elution on a chromatographic column of the liquid chromatograph by using octadecylsilane chemically bonded silica as a filler, and using acetonitrile as a mobile phase A and phosphoric acid aqueous solution as a mobile phase B by using the liquid chromatograph, and determining the content of the total anthraquinone in the dampness eliminating and toxin removing composition;

the method for measuring the content of the free anthraquinone comprises the following steps:

(III) absorbing the anthraquinone reference substance solution and the free anthraquinone test sample solution, injecting the anthraquinone reference substance solution and the free anthraquinone test sample solution into a liquid chromatograph, performing gradient elution on a chromatographic column of the liquid chromatograph by using octadecylsilane chemically bonded silica as a filler, and determining the content of the free anthraquinone in the dampness resolving and toxin removing composition by using acetonitrile as a mobile phase A and a phosphoric acid aqueous solution as a mobile phase B of the liquid chromatograph;

wherein, when the content of the total anthraquinone and the content of the free anthraquinone are measured, the following elution procedures are adopted:

0-10 min, the mobile phase A is 35% → 40%, and the mobile phase B is 65% → 60%;

2. The method for detecting the quality of the dampness-eliminating and toxin-removing composition as claimed in claim 1, wherein the total anthraquinone sample solution is prepared by the following method:

3. The method for detecting the quality of the dampness-eliminating and toxin-removing composition as claimed in claim 1, wherein the free anthraquinone sample solution is prepared by the following method:

taking 0.2-0.5 g of the dampness-eliminating and toxin-vanquishing composition, placing the composition into a conical flask with a plug, adding 25-30 mL of methanol, weighing, heating and refluxing for 20-60 minutes, taking out, cooling, weighing again, supplementing the lost weight with methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the traditional Chinese medicine composition.

4. The method for detecting the quality of the damp-eliminating and toxin-vanquishing composition according to claim 1, wherein the anthraquinone reference solution is prepared by the following steps:

5. The method for detecting the quality of the dampness-eliminating and toxin-vanquishing composition according to claim 1, wherein in the method for measuring the total anthraquinone content and the method for measuring the free anthraquinone content, octadecylsilane chemically bonded silica is used as a filler in a chromatographic column of the liquid chromatograph, and the column temperature is 25-35 ℃; the liquid chromatograph is used for gradient elution by taking acetonitrile as a mobile phase A and 0.1vol% phosphoric acid aqueous solution as a mobile phase B, the flow rate is 0.6-1 mL/min, and the detection wavelength is 253-256 nm.

6. The method for detecting the quality of the composition for eliminating dampness and removing toxicity of claim 1, wherein the method for measuring the total content of ephedrine hydrochloride and pseudoephedrine hydrochloride comprises the following steps:

7. The method for detecting the quality of the composition for eliminating dampness and removing toxicity of claim 6, wherein the total content of ephedrine hydrochloride and pseudoephedrine hydrochloride is determined by using methanol and phosphoric acid aqueous solution as mobile phase;

8. The method for detecting the quality of the dampness-resolving and toxin-vanquishing composition as claimed in claim 6, wherein the ephedrine test solution is prepared according to the following method:

9. The method for detecting the quality of the dampness-eliminating and toxin-vanquishing composition according to claim 1, wherein the method for measuring the paeoniflorin content comprises:

10. The method for detecting the quality of the dampness-resolving and toxin-vanquishing composition as claimed in claim 9, wherein the paeoniflorin test solution is prepared by the following method: