CN112180000B - Method for detecting dampness-resolving and toxin-vanquishing composition - Google Patents
Method for detecting dampness-resolving and toxin-vanquishing composition Download PDFInfo
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Abstract
The invention discloses a detection method 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 quality detection method of the dampness eliminating and toxin removing composition comprises the steps of measuring the content of total anthraquinone and the content of free anthraquinone in the dampness eliminating and toxin removing composition by adopting a high performance liquid chromatography, and calculating the content of combined anthraquinone; wherein, bound anthraquinone content = total anthraquinone content-free anthraquinone content. The determination method disclosed by the invention has the advantages of stronger specificity and durability, high stability and capability of effectively ensuring the stability and controllability of the product quality.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine quality detection, in particular to a 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-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 detecting a damp-resolving and toxin-vanquishing composition, which has strong specificity and durability and can effectively ensure the stability and controllability of the product quality.
In order to solve the technical problems, the invention provides a 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 detection method of the dampness eliminating and toxin removing composition comprises the steps of measuring the content of total anthraquinone and the content of free anthraquinone in the dampness eliminating and toxin removing composition by adopting a high performance liquid chromatography, and calculating the content of combined anthraquinone;
wherein, the bound anthraquinone content is the 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 aloe-emodin, rhein, emodin, chrysophanol, and physcion with methanol to obtain mixed solution, and making into reference solution;
(2) extracting the dampness-resolving and toxin-vanquishing composition with methanol to obtain total anthraquinone sample solution;
(3) and (3) absorbing the reference substance solution and the total anthraquinone test sample solution, injecting the reference substance solution and the total anthraquinone test sample solution into a liquid chromatograph, performing gradient elution by using octadecylsilane chemically bonded silica as a filler, acetonitrile as a mobile phase A and a 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.
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 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 reference substance solution and the free anthraquinone test sample solution, injecting the reference substance solution and the free anthraquinone test sample solution into a liquid chromatograph, performing gradient elution by using octadecylsilane chemically bonded silica as a filler, acetonitrile as a mobile phase A and a phosphoric acid aqueous solution as a mobile phase B by using the liquid chromatograph, and determining the content of the free anthraquinone in the dampness eliminating and toxin removing composition.
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 traditional Chinese medicine composition.
As an improvement of the technical scheme, the reference substance solution is prepared by the following method:
weighing 1.581mg of aloe-emodin reference substance, 3.017mg of rhein reference substance, 1.604mg of emodin reference substance, 2.656mg of chrysophanol reference substance and 5.221mg of physcion reference substance, placing in a 100mL measuring flask, and adding methanol to prepare 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 1mL respectively; 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 total anthraquinone content and the method for measuring the free anthraquinone content, octadecylsilane chemically bonded silica is used as a filler, acetonitrile is used as a mobile phase A, 0.1 vol% phosphoric acid aqueous solution is used as a mobile phase B for gradient elution, the flow rate is 0.6-1 mL/min, the detection wavelength is 253-256 nm, and the column temperature is 25-35 ℃.
In the improvement of the technical scheme, 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, acetonitrile is used as a mobile phase A, 0.1 vol% phosphoric acid aqueous solution is used as a mobile phase B for gradient elution, the flow rate is 0.8mL/min, the detection wavelength is 254nm, and the column temperature is 30 ℃.
As an improvement of the technical scheme, in the method for measuring the content of the total anthraquinone, the sample volume of the reference substance solution and the total anthraquinone sample solution is 10 mu L.
As an improvement of the technical scheme, in the method for measuring the content of the free anthraquinone, the sample volume of the reference solution and the free anthraquinone sample solution is 10 mu L.
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 establishes the method for measuring the combined anthraquinone in the dampness-resolving and toxin-vanquishing composition for the first time, and the detection method has good specificity, accuracy, reliability and good stability. The content of the total anthraquinone and the content of the free anthraquinone in the dampness-eliminating and toxin-vanquishing composition can be measured by the measuring method, and the content of the combined anthraquinone can be calculated, so that the stability and the controllability of the product quality of the dampness-eliminating and toxin-vanquishing composition are ensured, and a good foundation is provided for large-scale production.
Drawings
FIG. 1 is an HPLC chart of a 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 an 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, the peak 1 is aloe-emodin peak, the peak 2 is rhein peak, the peak 3 is emodin peak, the peak 4 is chrysophanol peak, and the 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 aloe-emodin standards in the determination of total anthraquinone content;
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 standard curve diagram of physcion control in the determination of total anthraquinone content.
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 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 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 in the prescription of the dampness-resolving and toxin-vanquishing composition and key action targets of lung injury and inflammatory reaction generated by a host. The results show that: rhubarb responds to TMPRSS2 as a target for inhibiting virus invasion and shedding, AMPK (oxidative stress and inflammation), VEGFR2 (vascular permeability) and ALK5 (vascular permeability and pulmonary fibrosis) as key targets of tissue injury generated after virus invasion into a host, thereby playing a role in resisting COVID-19. Further research shows that 5 components in rhubarb, such as rhein, physcion, 6-hydroxy acid D-8-O-beta-glucoside, procyanidin B1-3' -O-gallate and aloe emodin, have effects on the target. Therefore, rhubarb belongs to a core drug in the dampness-resolving and toxin-vanquishing composition, and the detection of related components of rhubarb is one of the key means for realizing the quality control of the dampness-resolving and toxin-vanquishing composition.
Furthermore, it is difficult to test the components of 6-hydroxy acid garcinol-8-O-beta-D-glucoside, procyanidin B1-3' -O-gallate, etc. Therefore, rhein, physcion and aloe-emodin are monitored, and the content of rheum officinale in the dampness-resolving and toxin-vanquishing formula can be reflected by monitoring the 3 components.
In addition, the rhubarb in the dampness-resolving and toxin-vanquishing composition has the main functions of clearing away lung heat, promoting blood circulation 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.
In combination with the aspects, when the detection method of the dampness-resolving and toxin-vanquishing composition is formulated, the content of the combined anthraquinone in the finished product medicine is determined and monitored, so that the quality detection of the dampness-resolving and toxin-vanquishing composition is realized. 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 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, the bound anthraquinone content is the 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 a reference solution: weighing 1.581mg of aloe-emodin reference substance, 3.017mg of rhein reference substance, 1.604mg of emodin reference substance, 2.656mg of chrysophanol reference substance and 5.221mg of physcion reference substance, placing in a 100mL measuring flask, and adding methanol to prepare 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 1mL respectively; 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 reference solution, injecting into a liquid chromatograph, and measuring to obtain the total anthraquinone content in the dampness eliminating and toxin removing composition.
In the present invention, the free anthraquinone content is determined 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.
The mobile phase was subjected to gradient elution in the following order of elution:
(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.
(3) Preparation of a reference solution: weighing 1.581mg of aloe-emodin reference substance, 3.017mg of rhein reference substance, 1.604mg of emodin reference substance, 2.656mg of chrysophanol reference substance and 5.221mg of physcion reference substance, placing in a 100mL measuring flask, and adding methanol to prepare 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 1mL respectively; 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 free anthraquinone sample solution and reference solution, injecting into 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 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: within 10min to 38min, the proportion of acetonitrile is 40% → 60%, and the proportion of 0.1 vol% 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 specialization examination
Taking a rhubarb negative sample, and respectively preparing total anthraquinone/free anthraquinone negative sample solutions according to the preparation method of the total anthraquinone/free anthraquinone test sample solution. 10 microlitres 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 reference substance solution are respectively injected into a liquid chromatograph for testing, and the results are shown in figures 1 to 5. Wherein, the peak position of each reference substance is shown in the following table:
TABLE 1 Specification for determining the content of Total anthraquinone/free anthraquinone in the composition for eliminating dampness and removing toxicity
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.
2 investigation of Linear relationship
2.1 investigation of the Linear relationship for determination of free anthraquinone content
Precisely weighing 3.570mg of aloe-emodin reference substance, 2.751mg of emodin reference substance, 1.631mg of chrysophanol reference substance and 3.051mg 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 2.057mg 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, putting into 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 emodin, 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 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. 6: the regression equation for aloe-emodin is: 66,512.6130x +128.8558, and its correlation coefficient R2When the sample injection concentration is 0.9994, the linear relation between the sample injection mass and the peak area is good in the range of the sample injection concentration of 0.071 mu g/mL to 3.570 mu g/mL.
As can be seen from fig. 7, the regression equation for rhein is: 49,694.7092x-1,493.7898, and its correlation coefficient R2When the sample injection concentration is 0.9998, the linear relation between the sample injection mass and the peak area is good in the range of the sample injection concentration of 0.411 mu g/mL-20.565 mu g/mL.
As can be seen from fig. 8, the regression equation for emodin is: 50,043.3431x +269.0534, and its correlation coefficient R2When the sample injection concentration is 0.9995, the linear relation between the sample injection mass and the peak area is good in the 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 that y is 68,915.3676x +180.1324, and the correlation coefficient R thereof is2When the sample injection concentration is 0.033 mu g/mL-1.631 mu g/mL, the linear relation between the sample injection quality and the peak area is good.
As can be seen from fig. 10, the regression equation of physcion is: 51,006.3529x +707.5671, and its correlation coefficient R2When the sample injection concentration is 0.012 mug/mL-0.610 mug/mL, the linear relation between the sample injection quality and the peak area is good.
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 fig. 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: 58108.7967x-793.9060, and its correlation coefficient R2When the sample injection concentration is 0.9999, the linear relation between the sample injection mass and the peak area is good in the range of the sample injection concentration of 0.316 mu g/mL-15.807 mu g/mL.
As can be seen from fig. 12, the regression equation for rhein is: 50,539.6466x-3,498.3368, and its correlation coefficient R2When the sample injection concentration is 1.0000, the linear relation between the sample injection mass and the peak area is good when the sample injection concentration is 0.603-30.167 mu g/mL.
As can be seen from fig. 13, the regression equation for emodin is: 43,070.1473x-501.3586, and its correlation coefficient R2When the sample injection concentration is 0.9999, the linear relation between the sample injection mass and the peak area is good in the range of the sample injection concentration of 0.321 mu g/mL-16.039 mu g/mL.
As can be seen from fig. 14, the regression equation for chrysophanol is: 61,967.3466x-2,584.2033, and its correlation coefficient R2When the sample injection concentration is 0.9999, the linear relation between the sample injection mass and the peak area is good in the range of the sample injection concentration of 0.531-26.563 mu g/mL.
As can be seen from FIG. 15The regression equation of physcion is shown as follows: 30,079.1466x +505.0118, and its correlation coefficient R2When the injection concentration is between 0.104 and 5.221 mu g/mL, the linear relation between the injection mass and the peak area is good.
3 investigation of precision
3.1 precision of the instrument
Precisely absorbing 10 μ L of the reference solution, testing and analyzing, calculating peak areas of aloe-emodin, rhein, emodin, chrysophanol and physcion, and determining the results shown in Table 2.
TABLE 2 survey results of the content of free anthraquinone in dampness-eliminating and toxin-removing composition by precision survey instrument
The result shows that the same 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.
3.2 intermediate precision investigation
Selecting different experimenters to measure by different high performance liquid chromatographs at different time, taking a proper amount of the dampness-eliminating and toxin-vanquishing composition, grinding, taking about 0.5g, precisely weighing and paralleling 6 parts, respectively preparing free anthraquinone test solution and total anthraquinone test solution, precisely absorbing 10 mu L of each of the free anthraquinone test solution or the total anthraquinone test solution and the reference solution, testing and analyzing, calculating the content of free anthraquinone and total anthraquinone and RSD (content of reactive anthraquinone) and RSD (content of total anthraquinone) and comparing with the result of a repeatability investigation test, wherein the result is shown in tables 3-4.
TABLE 3 table of intermediate precision investigation results of determination of content of free anthraquinone in dampness-resolving and toxin-vanquishing composition
TABLE 4 table of intermediate precision investigation results of total anthraquinone content determination of dampness-resolving and toxin-vanquishing composition
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 total anthraquinone content RSD value is 0.75%, the RSD value of 6 data of a repeatability test is 0.92%, when the content of a component to be measured in a sample is regulated to be 0.01% -0.1% according to the 'verification guidance principle of a 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 on different dates, and the method has good intermediate precision.
4 stability survey
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.
TABLE 5 survey results of free anthraquinone content determination stability of dampness-eliminating and toxin-removing composition
TABLE 6 survey results of the total anthraquinone content determination stability of dampness-eliminating and toxin-removing composition
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 sample solution has good stability 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, the other components are all more than 3 percent, and the total anthraquinone sample solution is subjected to measurement at 13 hours.
5 repetitive investigation
Precisely sucking 10 mu L of free anthraquinone sample solution and total anthraquinone sample solution, testing, calculating the content of free anthraquinone and total anthraquinone and RSD value, and determining the results shown in tables 7 and 8.
TABLE 7 repeated investigation result table for measuring content of free anthraquinone in dampness-eliminating and toxin-removing composition
TABLE 8 repeated investigation result table for determination of total anthraquinone content of dampness-eliminating and toxin-removing composition
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.
6 accuracy survey
6.1 investigation of the accuracy of the determination of free anthraquinone content
Precisely weighing 2.455mg of aloe-emodin reference substance, 2.485mg of rhein reference substance, 1.625mg of emodin reference substance, 1.950mg of chrysophanol reference substance and 0.845mg of physcion reference substance, respectively putting the components into 250, 50, 250, 500 and 500mL measuring bottles, adding methanol to prepare solutions containing 0.00982mg of aloe-emodin, 0.04969mg of rhein, 0.00650mg of rhein, 0.00390mg of chrysophanol and 0.00169mg of physcion in each 1mL of the bottles, and taking the solutions as sample-adding and recycling mother liquor. Precisely sucking 1mL of each of the above mother liquors into a conical flask, paralleling 6 parts, 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.
TABLE 9 survey results of free anthraquinone aloe-emodin content determination sample-loading recovery rate of dampness-resolving and toxin-vanquishing composition
TABLE 10 survey results of free anthraquinone rhein content determination sample-adding recovery rate of dampness-eliminating and toxin-removing composition
TABLE 11 survey results of free anthraquinone-emodin content determination, sample loading and recovery rate of dampness-eliminating and toxin-removing composition
TABLE 12 survey results of free anthraquinone chrysophanol content determination sample-adding recovery rate of dampness-eliminating and toxin-removing composition
TABLE 13 survey results of free anthraquinone physcion content determination sample-adding recovery rate of dampness-resolving and toxin-vanquishing composition
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%, the limit of recovery rate is 80% -115% 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 recovery rate is 80% -115%, and the determination method of the invention has good accuracy
6.2 Total anthraquinone content determination accuracy survey
Precisely weighing 2.497mg of aloe-emodin reference substance, 3.960mg of rhein reference substance, 2.707mg of emodin reference substance, 2.313mg of chrysophanol reference substance and 1.995mg of physcion reference substance, respectively placing in 50mL measuring bottles, adding methanol to prepare solutions containing 0.04994mg of aloe-emodin, 0.07920mg of rhein, 0.05414mg of emodin, 0.04625mg of chrysophanol and 0.03990mg of physcion per 1mL, and taking the solutions 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, grinding, respectively taking 0.25g of the composition into the 6 conical flasks, preparing a total anthraquinone sample solution, measuring the content of total anthraquinone in the sample, and calculating the sample adding recovery rate, wherein the results are shown in tables 14-18.
TABLE 14 investigation result table of total anthraquinone aloe-emodin content determination sample-loading recovery rate of dampness-resolving and toxin-vanquishing composition
TABLE 15 survey results of total anthraquinone rhein content determination sample-adding recovery rate of dampness-resolving and toxin-removing composition
TABLE 16 investigation result table of total anthraquinone emodin content determination sample-loading recovery rate of dampness-resolving and toxin-vanquishing composition
TABLE 17 survey results of sample-adding recovery rate in determination of total anthraquinone chrysophanol content in dampness-eliminating and toxin-removing composition
TABLE 18 survey results of sample-adding recovery rate in determination of content of total anthraquinone physcion of dampness-resolving and toxin-vanquishing composition
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.
7 durability examination
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.
TABLE 19 survey results of different chromatographic columns for determining total/free anthraquinone content of dampness-eliminating and toxin-removing composition
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.
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 contents of free anthraquinone and total anthraquinone in the dampness-resolving and toxin-vanquishing composition is examined.
Taking 10 mu L of each free anthraquinone sample solution and total anthraquinone sample solution, respectively adopting the column temperature to carry out determination, calculating the content of free anthraquinone and total anthraquinone and RSD value, and the experimental result is shown in table 20.
TABLE 20 investigation result table of different column temperatures for determination of total/free anthraquinone content of dampness-resolving and toxin-vanquishing composition
The results show that the RSD values of the free anthraquinone and the total anthraquinone content values of the damp-resolving and toxin-vanquishing composition with different column temperatures are both more than 5 percent, which indicates that the analysis method is sensitive to the column temperature, so the column temperature is strictly controlled at 30 ℃.
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 contents of the free anthraquinone and the total anthraquinone in the dampness-eliminating and toxin-vanquishing composition is examined.
Taking 10 mu L of each free anthraquinone sample solution and total anthraquinone sample solution, respectively measuring by adopting the flow rates, calculating the content of free anthraquinone and total anthraquinone and RSD value, and the experimental result is shown in table 21.
TABLE 21 investigation result table of different flow rates for determination of total/free anthraquinone content of dampness-eliminating and toxin-removing composition
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, the peak of emodin methyl ether in the total anthraquinone appears, and the flow rate is controlled not to be lower than 0.8mL/min during analysis.
8 content determination of damp-resolving and toxin-vanquishing composition test samples in 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.
TABLE 22 table of the measurement results of the contents of the dampness-eliminating and toxin-removing compositions in combination with anthraquinone
In conclusion, the invention establishes the method for determining the combined anthraquinone in the dampness-resolving and toxin-vanquishing composition, and the detection method has the advantages of good specificity, accuracy, reliability and good stability. The content of the total anthraquinone and the content of the free anthraquinone in the dampness-eliminating and toxin-vanquishing composition can be measured by the measuring method, and the content of the combined anthraquinone can be calculated, so that the stability and the controllability of the product quality of the dampness-eliminating and toxin-vanquishing composition are ensured, and a good foundation is provided for large-scale production.
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 (8)
1. A 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;
the detection method of the dampness eliminating and toxin removing composition comprises the steps of measuring the content of total anthraquinone and the content of free anthraquinone in the dampness eliminating and toxin removing composition by adopting a high performance liquid chromatography, and calculating the content of combined anthraquinone;
wherein, the content of the combined anthraquinone is the content of total anthraquinone-the content of free anthraquinone;
the method for measuring the content of the total anthraquinone comprises the following steps:
(1) mixing aloe-emodin, rhein, emodin, chrysophanol, and physcion with methanol to obtain mixed solution, and making into reference solution;
(2) extracting the dampness-resolving and toxin-vanquishing composition with methanol to obtain total anthraquinone sample solution;
(3) absorbing a reference substance solution and a total anthraquinone test sample solution, injecting the reference substance solution and the total anthraquinone test sample solution into a liquid chromatograph, performing gradient elution by using octadecylsilane chemically bonded silica as a filler, acetonitrile as a mobile phase A and a 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:
taking appropriate amount of aloe-emodin, rhein, emodin, chrysophanol, and physcion, adding methanol to obtain mixed solution, and making into reference solution;
secondly, extracting the damp-resolving and toxin-vanquishing composition by using methanol to prepare a free anthraquinone test sample solution;
absorbing the reference substance solution and the free anthraquinone test sample solution, injecting the reference substance solution and the free anthraquinone test sample solution into a liquid chromatograph, performing gradient elution by using octadecylsilane chemically bonded silica as a filler, acetonitrile as a mobile phase A and a phosphoric acid aqueous solution as a mobile phase B by using the liquid chromatograph, and determining the content of the free anthraquinone in the dampness eliminating and toxin removing composition;
the determination method of the total anthraquinone content and the determination method of 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%;
38-48 min, wherein the mobile phase A is 60% and the mobile phase B is 40%;
wherein the content of the combined anthraquinone in the dampness eliminating and toxin removing composition is more than or equal to 0.016 wt%.
2. The method for detecting a damp-eliminating and toxin-vanquishing composition according to claim 1, wherein 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.
3. The method for detecting a damp-eliminating and toxin-vanquishing composition according to 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 a damp-eliminating and toxin-vanquishing composition according to claim 1, wherein the reference solution is prepared by the following steps:
weighing 1.581mg of aloe-emodin reference substance, 3.017mg of rhein reference substance, 1.604mg of emodin reference substance, 2.656mg of chrysophanol reference substance and 5.221mg of physcion reference substance, placing in a 100mL measuring flask, and adding methanol to prepare 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 1mL respectively; 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.
5. The method for detecting a composition for eliminating dampness and removing toxicity according to claim 1, wherein in the method for detecting the total anthraquinone content and the method for detecting the free anthraquinone content, octadecylsilane chemically bonded silica is used as a filler, acetonitrile is used as a mobile phase A, 0.1 vol% phosphoric acid aqueous solution is used as a mobile phase B to perform gradient elution, the flow rate is 0.6-1 mL/min, the detection wavelength is 253-256 nm, and the column temperature is 25-35 ℃.
6. The method for detecting a composition for eliminating dampness and removing toxicity according to claim 5, wherein the method for detecting the total anthraquinone content and the method for detecting the free anthraquinone content comprise the steps of performing gradient elution by using octadecylsilane chemically bonded silica as a filler, acetonitrile as a mobile phase A and 0.1 vol% phosphoric acid aqueous solution as a mobile phase B, wherein the flow rate is 0.8mL/min, the detection wavelength is 254nm, and the column temperature is 30 ℃.
7. The method for detecting a composition for eliminating dampness and removing toxicity of claim 1, wherein the total anthraquinone content is determined by a method in which the sample volume of the reference solution and the total anthraquinone sample solution is 10 μ L.
8. The method for detecting a composition for eliminating dampness and removing toxicity of claim 1, wherein the amount of the control solution and the free anthraquinone sample solution is 10 μ L in the method for measuring the content of free anthraquinone.
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