Patent Description:
Due to strong contagiousness, rapid transmission, general population susceptibility, and lack of specific drugs, the pneumonia caused by <NUM> novel coronavirus (COVID-<NUM>) infection has led to a pandemic worldwide and has become a global super-major public health emergency. Traditional Chinese medicine has played a unique and important role in the fight against the COVID-<NUM> pandemic. The National Administration of Traditional Chinese Medicine in China has pointed out that, according to research and screening results, Jinhua Qinggan (JHQG) granules, LianhuaQingwen (LHQW) capsules, Xuebijing (XBJ) injection, Lung-Cleansing and Detoxifying Preparation, HSBD formula (Dampness Resolving and Detoxifying Preparation), and Xuanfeibaidu (XFBD) formula (Lung Diffusing and Detoxifying Preparation) have played important roles in the fight against the COVID-<NUM> pandemic.

The HSBD formula is composed of <NUM> traditional Chinese medicine components (TCMCs), including raw ephedra, almond, raw gypsum, licorice, Agastache rugosa, Magnolia officinalis, AtractylodesLancea, Amomum tsaoko, RhizomaPinelliae, Poriacocos, raw Rheum palmatum, raw Astragalus membranaceus, Semen Lepidii, and Paeoniae Radix Rubra. Clinical experiments have shown that the HSBD formula exhibits an outstanding effect on improving the symptoms of patients and increasing a conversion rate from nucleic acid positive to negative. However, most of the current studies on the HSBD formula focus on its pharmacology and efficacy, and there is no studies on its quality standards. The current production of the HSBD formula is conducted on a small scale with relatively-low requirements for QC, and there is no large-scale industrial production.

The literature "<NPL>) discloses a material basis of each component in the HSBD Composition. <NPL>) also the HSBD Composition. However, a specific QC method for the HSBD composition has not been studied by these two literatures.

<NPL>) and CHINESE PHARMACOPOEIA COMMISSION ("<NPL>) disclose methods using TLC and HPLC for qualitative and quantitative identification of crude drugs and active ingredients; however, these two literatures have not disclosed how to select detection targets from the complex Huashibaidu combination.

The technical problem to be solved by the present disclosure is to provide a QC method for an HSBD composition, which can provide a data basis for mass production and effectively ensure the stability and controllability of a product quality.

In order to solve the above technical problem, the present disclosure provides a QC method for an HSBD composition, where the HSBD composition mainly includes the following components: ephedra, fried bitter almond, raw gypsum, licorice, PogostemonisHerba, Magnolia officinalis, bran-fried AtractylodesLancea, fried Amomum tsaoko fruit, RhizomaPinelliae, Poriacocos, Rheum palmatum, Astragalus membranaceus, Semen Lepidii, Paeoniae Radix Rubra, and an adjuvant; and.

As an improvement of the above technical solution, the QC method for an HSBD composition further includes:
(<NUM>) identifying Astragalus membranaceus, Semen Lepidii, Paeoniae Radix Rubra, and Rheum palmatum by TLC.

As an improvement of the above technical solution, a determination method of the total anthraquinone content includes:.

As an improvement of the above technical solution, a determination method of the free anthraquinone content includes:.

As an improvement of the above technical solution, in the determination method of the total anthraquinone content and the determination method of the free anthraquinone content, the octadecylsilane-bonded silica gel is used as a filler, and the gradient elution is conducted with acetonitrile as a mobile phase A and a <NUM> vol% phosphoric acid aqueous solution as a mobile phase B under the following conditions: flow rate: <NUM>/min to <NUM>/min, detection wavelength: <NUM> to <NUM>, and column temperature: <NUM> to <NUM>.

As an improvement of the above technical solution, the total anthraquinone test solution is prepared as follows:
adding <NUM> to <NUM> of the HSBD composition to an Erlenmeyer flask with a stopper, adding <NUM> to <NUM> of methanol, and heating to allow extraction under reflux for <NUM> to <NUM>; taking a resulting system out, cooling, weighing, and making up for a weight loss with methanol; thoroughly shaking and filtering the system, and taking <NUM> to <NUM> of a subsequent filtrate; subjecting the subsequent filtrate to vacuum distillation to dryness to recover the solvent and obtain a residue, adding <NUM> to <NUM> of a <NUM>% hydrochloric acid solution to the residue, and subjecting a resulting mixture to an ultrasonic treatment for <NUM> to <NUM>; adding <NUM> to <NUM> of trichloromethane (TCM), and heating to reflux for <NUM> to <NUM>; cooling a resulting reflux system and transferring to a separatory funnel, washing a vessel for the reluxwith a small amount of TCM, and adding the small amount of TCM to the separatory funnel; separating to obtain a primary TCM phase and an acide phase, and subjecting the acid phase to extraction with <NUM> to <NUM> of TCM <NUM> to <NUM> times to collect TCM phases and combine with the primary TCM phase; and subjecting a combined TCM phase to vacuum distillation todryness to recover the solvent and obtain a product; dissolving the product with methanol, transferring a resulting solution to a <NUM> volumetric flask, and adding methanol to a specified scale; and thoroughly shaking, filtering, and taking a subsequent filtrate.

As an improvement of the above technical solution, the free anthraquinone test solution is prepared as follows:
adding <NUM> to <NUM> of the HSBD composition to an Erlenmeyer flask with a stopper, adding <NUM> to <NUM> of methanol, weighing, and heating to reflux for <NUM> to <NUM>; taking a resulting system out, cooling, weighing once again, and making up for a weight loss with methanol; and thoroughly shaking, filtering, and taking a subsequent filtrate.

As an improvement of the above technical solution, the anthraquinone reference solution is prepared as follows:
weighing <NUM> of an aloe emodin reference, <NUM> of a rheinic acid reference, <NUM> of an emodin reference, <NUM> of a chrysophanol reference, and <NUM> of an emodin methyl ether reference, adding each of the above to a <NUM> volumetric flask, and adding methanol to prepare an aloe emodin stock solution with <NUM>µg of the aloe emodin per mL, a rheinic acid stock solution with <NUM>µg of the rheinic acid per mL, an emodin stock solution with <NUM>µg of the emodin per mL, a chrysophanol stock solution with <NUM>µg of the chrysophanol per mL, and an emodin methyl ether stock solution with <NUM>µg of the emodin methyl ether per mL; and accurately pipetting <NUM> of the aloe emodin stock solution, <NUM> of the rheinic acid stock solution, <NUM> of the emodin stock solution, <NUM> of the chrysophanol stock solution, and <NUM> of the emodin methyl ether stock solution to a <NUM> volumetric flask, and adding methanol to prepare a mixed solution with <NUM>µg of the aloe emodin, <NUM>µg of the rheinic acid, <NUM>µg of the emodin, <NUM>µg of the chrysophanol, and <NUM>µg of the emodin methyl ether per mL.

As an improvement of the above technical solution, a determination method of the total content of ephedrine hydrochloride and pseudoephedrine hydrochloride includes:.

As an improvement of the above technical solution, in the determination method of the total content of ephedrine hydrochloride and pseudoephedrine hydrochloride, methanol and a phosphoric acid aqueous solution are adopted as mobile phases; and
the phosphoric acid aqueous solution is a mixed solution of phosphoric acid, diethylamine (DEA), triethylamine (TEA), and water, in which a volume fraction of the phosphoric acid is <NUM>% to <NUM>%, a volume fraction of the DEA is <NUM>% to <NUM>%, and a volume fraction of the TEA is <NUM>% to <NUM>%.

As an improvement of the above technical solution, a determination method of the paeoniflorin content includes:.

As an improvement of the above technical solution, a TLC identification method for the ephedra includes:.

As an improvement of the above technical solution, a TLC identification method for the licorice includes:.

As an improvement of the above technical solution, a TLC identification method for the Magnolia officinalis includes:.

As an improvement of the above technical solution, a TLC identification method for the Astragalus membranaceus includes:.

As an improvement of the above technical solution, a TLC identification method for the Semen Lepidii includes:.

As an improvement of the above technical solution, a TLC identification method for the Paeoniae Radix Rubra includes:.

As an improvement of the above technical solution, a TLC identification method for the Rheum palmatum includes:.

As an improvement of the above technical solution, the HSBD composition mainly includes the following components: <NUM> to <NUM> parts of the ephedra, <NUM> to <NUM> parts of the fried bitter almond, <NUM> to <NUM> parts of the raw gypsum, <NUM> to <NUM> parts of the licorice, <NUM> to <NUM> parts of the PogostemonisHerba, <NUM> to <NUM> parts of the Magnolia officinalis, <NUM> to <NUM> parts of the bran-fried AtractylodesLancea, <NUM> to <NUM> parts of the fried Amomum tsaoko fruit, <NUM> to <NUM> parts of the RhizomaPinelliae, <NUM> to <NUM> parts of the Poriacocos, <NUM> to <NUM> parts of the Rheum palmatum, <NUM> to <NUM> parts of the Astragalus membranaceus, <NUM> to <NUM> parts of the Semen Lepidii, <NUM> to <NUM> parts of the Paeoniae Radix Rubra, and an appropriate amount of the adjuvant; and
the HSBD composition is prepared into a traditional Chinese medicine preparation in a form selected from the group consisting of a granule, a decoction, a powder, a capsule, an oral liquid, a tablet, and a pill.

The implementation of the present disclosure has the following beneficial effects:.

To make the objectives, technical solutions, and advantages of the present disclosure clearer, the present disclosure will be further described in detail in combination with the accompanying drawings and specific embodiments.

The HSBD composition of the present disclosure mainly includes the following components: ephedra, fried bitter almond, raw gypsum, licorice, PogostemonisHerba, Magnolia officinalis, bran-fried AtractylodesLancea, fried Amomum tsaoko fruit, RhizomaPinelliae, Poriacocos, Rheum palmatum, Astragalus membranaceus, Semen Lepidii, and Paeoniae Radix Rubra.

In the HSBD composition of the present disclosure, the ephedra, PogostemonisHerba, and gypsum serve as sovereign TCMCs. The ephedra and PogostemonisHerba are hard, bitter, warm, and aromatic, and can relieve exterior syndrome, suppress asthma, and remove dampness for regulating stomach. The gypsum is hard, sweet, and cold, and can clear stagnated heat from the lung and stomach and promote the secretion of saliva or body fluid. The three sovereign TCMCs complement each other to achieve the effects of relieving exterior syndrome, dissipating coldness, resolving dampness with aromatics, clearing heat, and relieving dyspnea. The fried bitter almond, RhizomaPinelliae, Magnolia officinalis, bran-fried AtractylodesLancea, fried Amomum tsaoko fruit, and Poriacocos serve as ministerial TCMCs. The fried bitter almond, RhizomaPinelliae, and Magnolia officinalis are hard, bitter, and warm, and can promote the circulation of qi, calm the adverse-rising energy, open knots, and calm asthma. The bran-fried AtractylodesLancea and fried Amomum tsaoko fruit are pungent, bitter, and warm, and can enter the spleen and stomach meridians to eliminate dampness, strengthen spleen, and break knots caused by anger. The Poriacocos can promote diuresis and strengthen spleen. The six ministerial TCMCs together assist with the sovereign TCMCs to achieve the effects of eliminating dampness, strengthening spleen, promoting qi circulation, removing meridian obstruction, free coursing, and expelling evil spirits. The Astragalus membranaceus, Paeoniae Radix Rubra, Semen Lepidii, and Rheum palmatum serve as adjuvant TCMCs. The Astragalus membranaceus is sweet and warm, and can invigorate the lung and spleen. The Paeoniae Radix Rubra is bitter and slightly cold, and can cool blood and dissipate blood stasis, which is used to treat blood stasis caused by damage of healthy qi and stagnation of qi at a later stage of the epidemic. The Semen Lepidii is hard and cold, and can assist with the sovereign TCM component gypsum to clear lung heat and promote diuresis, thereby preventing or treating wet lung (pulmonary edema) lesions. The Rheum palmatum is bitter and cold, and can enter large intestinal meridians for bowel-relaxing. Due to the theory of "lung and large intestine being interior-exteriorly related", the Rheum palmatum can assist with the sovereign TCM component gypsum to clear lung heat, and can also assist with the Paeoniae Radix Rubra to promote blood circulation and cool blood. The four adjuvantTCMCs can achieve the effects of caring for healthy qi, purging heat, cooling blood, and promoting blood circulation to remove blood stasis. The licorice serves as a conductant TCM component, and is sweet and neutral. The licorice can reconcile the TCMCs, and the combination of Paeoniae Radix Rubra and licoriceis adopted with reference to a peony and licorice decoction to alleviate acute symptoms. Thus, the HSBD composition can play the roles of relieving exterior syndrome, resolving dampness, clearing heat, relieving dyspnea, invigorating qi, and dissipating blood stasis.

It has been clinically found that patients with severe COVID-<NUM> pneumonia have the following characteristics: (<NUM>) There is unsurfaced fever difficult to cure, or medium and low fever, or even no fever. (<NUM>) Dyspnea and fatigue are also the main manifestations. (<NUM>) Digestive system symptoms such as poor appetite, loose stool, and diarrhea are common. (<NUM>) A tongue coat is thick and greasy. The above characteristics are in line with the characteristics of dampness evil pathopoiesis: heavy turbidity, obstructing qi and damaging yang, viscosity, and downward trend. The dampness evil can cause a disease alone or a disease accompanied by cold and heat that are manifested as cold-dampness and dampness-heat, where the heat can be caused by dryness or can be caused by dampness evil and persistent depression. The dampness evil, cold-dampness, and dampness-heat each can cooperate with an epidemic toxin to cause a disease, which is common in light cold-dampness stagnating in the lung, dampness-heat accumulating in the lung, ordinary dampness-toxin stagnating in the lung, and cold-dampness obstructing the lung. If untreated timely, mistreated, or allowed to develop, the disease will hurt the ying blood, reverse the transmission to pericardium, and evolve into severe COVID-<NUM> pneumonia. Therefore, it is believed that the COVID-<NUM> pneumonia is prominently manifested as "dampness toxin epidemic", which is located in the lung and closely related to the spleen, and has the pathological characteristics of cold-heat jumble and deficiency-excess mixing and the pathological factors of toxin, dampness, heat, cold, stasis, and deficiency. For the pneumonia, the epidemic toxin is a foundation, and the core pathogenesis is a combination of epidemic toxin and dampness evil, which can be accompanied by the invasion of cold and heat into the body, the obstruction of chest and lung, the ascending and descending disorder of qi activity, the stasis of blood vessels, and the deficiency of both qi and yin. The COVID-<NUM> pneumonia has a complex pathological mechanism involving various pathological factors.

The COVID-<NUM> pneumonia is mainly caused by a lesion in the lung and involves the spleen and stomach, and its core pathogenesis is dampness toxin and stagnation of qi. A course of the COVID-<NUM> pneumonia can be divided into an early stage, a middle stage, a severe stage, and a recovery stage, and can be subjected to syndrome differentiation-based treatment accordingly, including: resolving dampness and promoting circulation of qi, dispelling filth and detoxification, clearing lung and eliminating phlegm, promoting blood circulation to remove blood stasis, bowel-relaxing, and replenishing healthy qi. Therefore, the compatibility of the HSBD composition of the present disclosure is based on the core pathogenesis, and the HSBD composition has the main therapeutic effects of relieving exterior syndrome, resolving dampness, clearing heat, relieving dyspnea, and detoxifying and also has the auxiliary effects of removing blood stasis, dredging collaterals, invigorating qi, and nourishing yin. The combination of epidemic toxin and cold-dampness causes aversion to cold with fever, which is preferably treated by relieving exterior syndrome, resolving dampness, and detoxifying; the combination of epidemic toxin and dampness-heat causes loose stool and fatigue, which is preferably treated by clearing heat, resolving dampness, invigorating qi, and nourishing yin; and when there are symptoms such as obstruction of chest and lung, dyspnea, chest tightness, and shortness of breath, it is preferably treated by relieving dyspnea, removing blood stasis, and dredging collaterals.

The HSBD composition of the present disclosure can handle the integrated core pathogenesis of TCM treatment in the "<NPL>on)", including coexisted warm-heat and dampness, impaired dispersing and descending of lung, obstruction of lung qi, turbid dampness, and stagnation of lung by heat; and the HSBD composition mainly has the effects of resolving dampness, promoting circulation of qi, freeing lung, relieving asthma, clearing heat and phlegm, invigorating qi, and promoting blood circulation. Previous clinical observations have shown that the HSBD composition of the present disclosure can improve the clinical symptoms of severe COVID-<NUM> pneumonia. For severe patients, the HSBD composition of the present disclosure can significantly alleviate the main symptoms such as cough, fatigue, thirst, and vomiting. When used in combination with Western medicine, the HSBD composition of the present disclosure can shorten a cure time, significantly improve a respiratory function of a patient, and shorten the time to get out of oxygen uptake. For ordinary patients, the HSBD composition of the present disclosure can significantly alleviate the fever symptoms and can also improve the symptoms such as appetite decrease and chest distress. The HSBD composition of the present disclosure can significantly improve the clinical symptoms such as cough, fatigue, thirst, and vomiting of severe and ordinary COVID-<NUM> pneumonia, and thus has become a drug urgently needed in the current epidemic situation for treating severe and ordinary COVID-<NUM> pneumonia.

Preferably, the HSBD composition includes the following components:
<NUM> to <NUM> parts of the ephedra, <NUM> to <NUM> parts of the fried bitter almond, <NUM> to <NUM> parts of the raw gypsum, <NUM> to <NUM> parts of the licorice, <NUM> to <NUM> parts of the PogostemonisHerba, <NUM> to <NUM> parts of the Magnolia officinalis, <NUM> to <NUM> parts of the bran-fried AtractylodesLancea, <NUM> to <NUM> parts of the fried Amomum tsaoko fruit, <NUM> to <NUM> parts of the RhizomaPinelliae, <NUM> to <NUM> parts of the Poriacocos, <NUM> to <NUM> parts of the Rheum palmatum, <NUM> to <NUM> parts of the Astragalus membranaceus, <NUM> to <NUM> parts of the Semen Lepidii, <NUM> to <NUM> parts of the Paeoniae Radix Rubra, and an appropriate amount of the adjuvant.

The HSBD composition is prepared into a TCM preparation in a form selected from the group consisting of a granule, a decoction, a powder, a capsule, an oral liquid, a tablet, and a pill.

The present disclosure provides a QC method for an HSBD composition, which can achieve a prominent detection effect on an HSBD composition of any dosage form. In the set process of the QC method of the present disclosure, the two aspects of identification and content determination of components in an HSBD composition are considered.

Specifically, for an identification method:
In a research process, the inventors use a molecular docking technique to analyze a relationship of each component in the HSBD composition with key targets for invasion, replication, assembly, and shedding and transfer of COVID-<NUM> and key targets causing lung injury and inflammatory response of a host. Analysis results show that: The ephedra can respond to targets TMPRSS2, TACE, and AAK1 (virus invasion and endocytosis regulation) for inhibiting virus invasion and shedding and key targets VEGFR2 (vascular permeability) and ALK5 (vascular permeability and pulmonary fibrosis) for tissue damage caused by the virus invasion into a host. The licorice can respond to targets FURIN (virus invasion and endocytosis regulation), TACE, and AAK1 for inhibiting virus invasion and shedding and key targets sPLA2, AMPK (oxidative stress and inflammation), CCR2 (inflammation), p38αMAPK (inflammation and apoptosis), VEGFR2, and ALK5 for tissue damage caused by the virus invasion into a host. The Magnolia officinalis can respond to a target AAK1 for inhibiting virus invasion and shedding and key targets AMPK, VEGFR2, and ALK5 for tissue damage caused by the virus invasion into a host. The Rheum palmatum can respond to a target TMPRSS2 for inhibiting virus invasion and shedding and key targets AMPK, VEGFR2, and ALK5 for tissue damage caused by the virus invasion into a host. The Astragalus membranaceus can respond to a key target VEGFR2 for tissue damage caused by the virus invasion into a host. The Paeoniae Radix Rubra can respond to targets Mpro and ACE2. Through the actions of the above components on the above targets, the HSBD composition plays an anti-COVID-<NUM> role. It can be known that the ephedra, licorice, and Magnolia officinalis can respond to a lot of targets to effectively inhibit the invasion, assembly, and shedding and transfer of the virus, and thus are core TCMCs. The Astragalus membranaceus, Semen Lepidii, Paeoniae Radix Rubra, and Rheum palmatum can only respond to few targets, but are also of great significance for anti-COVID-<NUM>. Therefore, from the perspective of efficacy, the ephedra, licorice, and Magnolia officinalis should be preferentially identified by TLC and the Astragalus membranaceus, Semen Lepidii, Paeoniae Radix Rubra, and Rheum palmatum can also be identified by TLC to establish a perfect identification method, which can provide a solid data basis for the quality monitoring of an HSBD composition.

In summary, on the basis of considering the above factors, the ephedra, licorice, Magnolia officinalis, Astragalus membranaceus, Semen Lepidii, Paeoniae Radix Rubra, and Rheum palmatum are identified by TLC in the present disclosure to establish a perfect identification method, which can provide a data basis for the quality monitoring of an HSBD composition.

For a content determination method: In a research process, the inventors use a molecular docking technique to conduct further investigation, and investigation results show that apigenin, <NUM>-methoxyherbacetin, <NUM>,<NUM>,<NUM>'-trihydroxy-<NUM>-methoxyflavone, and kaempferol in the ephedra have an impact on the above targets; rheinic acid, emodin methyl ether, <NUM>-hydroxymusizin-<NUM>-O-β-D-glucoside, procyanidin B1-<NUM>'-O-gallate, and aloe emodin in the Rheum palmatum have an impact on the above targets; and baicalein and baicalin in the Paeoniae Radix Rubra have an impact on the above targets. Therefore, the detection of components related to ephedra, Rheum palmatum, and Paeoniae Radix Rubra is a key means to achieve the QC of the HSBD composition.

In addition, the ephedra in the HSBD composition mainly plays the roles of dispersing of Lung Qi, resolving phlegm, and relieving cough; and according to the principle of the TCM formula, the ephedra is a sovereign TCM component and plays an important role in the formula. Ephedrine introduced by the ephedra is volatile and easily lost during a mass production process. Therefore, the contents of ephedrine hydrochloride and pseudoephedrine hydrochloride in the ephedra are determined in the present disclosure. Specifically, in order to make the HSBD composition effectively play its role, a total content of ephedrine hydrochloride and pseudoephedrine hydrochloride should be controlled at <NUM>/g to <NUM>/g.

The Rheum palmatum in the HSBD composition mainly plays the roles of clearing heat, purging lung, promoting blood circulation, and cooling blood, which are mainly achieved by combined anthraquinone (aloe emodin, rheinic acid, emodin, chrysophanol, and emodin methyl ether) introduced by the Rheum palmatum. However, the combined anthraquinone is easily decomposed into free anthraquinone when heated, resulting in weakened efficacy. Thus, a content of the combined anthraquinone needs to be monitored. Therefore, in the present disclosure, a content of the combined anthraquinone in a finished drug is determined and monitored to achieve the QC of the HSBD composition. Specifically, in the HSBD composition of the present disclosure, the content of the combined anthraquinone shall be no less than <NUM>%.

Further, the present disclosure also controls a content of the Paeoniae Radix Rubra by determining a content of paeoniflorin. Specifically, in the HSBD composition of the present disclosure, the content of paeoniflorin shall be <NUM>/g to <NUM>/g.

The QC method of the present disclosure is described below in three parts:.

In the present disclosure, the total anthraquinone and free anthraquinone contents in the HSBD composition are determined by HPLC, and then a combined anthraquinone content is calculated, where the combined anthraquinone content = the total anthraquinone content - the free anthraquinone content.

Specifically, in the present disclosure, the determination method of the total anthraquinone content includes:.

(<NUM>) Chromatographic conditions: chromatographic column: octadecylsilane-bonded silica gel column; mobile phase: acetonitrile-<NUM> vol% phosphoric acid aqueous solution; flow rate: <NUM>/min; detection wavelength: <NUM>; column temperature: <NUM>; and injection volume: <NUM>µL. Gradient elution was conducted in the mobile phase according to the following elution program:.

(<NUM>) Preparation of a total anthraquinone test solution: About <NUM> of the HSBD composition was accurately weighed and added to an Erlenmeyer flask with a stopper, and <NUM> of methanol was accurately added; a resulting system was weighed, heated to allow extraction under reflux for <NUM>, taken out, cooled, and weighed, then methanol was added to make up for a weight loss, a resulting mixture was thoroughly shaken and filtered, and <NUM> of a subsequent filtrate was taken and subjected to vacuum distillation to completely recover the solvent; <NUM> of a <NUM>% hydrochloric acid solution was added, a resulting mixture was subjected to an ultrasonic treatment for <NUM>, <NUM> of TCM was added, and a resulting mixture was heated to allow a reaction under reflux for <NUM>, cooled, and placed in a separatory funnel; a reaction vessel was washed with a small amount of TCM, and resulting TCM was added to the separatory funnel; a resulting TCM phase was separated, and a resulting acid phase was subjected to extraction with <NUM> of TCM <NUM> times; resulting TCM phases were combined and subjected to vacuum distillation to completely recover the solvent; a residue was dissolved with methanol and transferred to a <NUM> volumetric flask, and methanol was added to a specified scale; and a resulting mixture was thoroughly shaken and filtered, and a subsequent filtrate was taken. (<NUM>) Preparation of an anthraquinone reference solution: <NUM> of an aloe emodin reference, <NUM> of a rheinic acid reference, <NUM> of an emodin reference, <NUM> of a chrysophanol reference, and <NUM> of an emodin methyl ether reference each were weighed and added to a <NUM> volumetric flask, and methanol was added to prepare an aloe emodin stock solution with <NUM>µg of the aloe emodin per mL, a rheinic acid stock solution with <NUM>µg of the rheinic acid per mL, an emodin stock solution with <NUM>µg of the emodin per mL, a chrysophanol stock solution with <NUM>µg of the chrysophanol per mL, and an emodin methyl ether stock solution with <NUM>µg of the emodin methyl ether per mL; and <NUM> of the aloe emodin stock solution, <NUM> of the rheinic acid stock solution, <NUM> of the emodin stock solution, <NUM> of the chrysophanol stock solution, and <NUM> of the emodin methyl ether stock solution were accurately pipetted to a <NUM> volumetric flask, and methanol was added to prepare a mixed solution with <NUM>µg of the aloe emodin, <NUM>µg of the rheinic acid, <NUM>µg of the emodin, <NUM>µg of the chrysophanol, and <NUM>µg of the emodin methyl ether per mL. (<NUM>) <NUM>µL of each of the total anthraquinone test solution and the anthraquinone reference solution was accurately pipetted and injected into a liquid chromatograph to determine a total anthraquinone content in the HSBD composition.

Specifically, in the present disclosure, the determination method of the free anthraquinone content includes:.

(<NUM>) Chromatographic conditions: chromatographic column: octadecylsilane-bonded silica gel column; mobile phase: acetonitrile-<NUM> vol% phosphoric acid aqueous solution; mobile phase flow rate: <NUM>/min; detection wavelength: <NUM>; column temperature: <NUM>; and injection volume: <NUM>µL. Gradient elution was conducted in the mobile phase according to the following elution program:.

(<NUM>) Preparation of a free anthraquinone test solution: About <NUM> of the HSBD composition was accurately weighed and added to an Erlenmeyer flask with a stopper, <NUM> of methanol was accurately added, and a resulting system was weighed, heated to allow a reaction under reflux for <NUM>, taken out, cooled, and weighed once again; and methanol was added to make up for a weight loss, a resulting mixture was thoroughly shaken and filtered, and a subsequent filtrate was taken. (<NUM>) Preparation of an anthraquinone reference solution: <NUM> of an aloe emodin reference, <NUM> of a rheinic acid reference, <NUM> of an emodin reference, <NUM> of a chrysophanol reference, and <NUM> of an emodin methyl ether reference each were weighed and added to a <NUM> volumetric flask, and methanol was added to prepare an aloe emodin stock solution with <NUM>µg of the aloe emodin per mL, a rheinic acid stock solution with <NUM>µg of the rheinic acid per mL, an emodin stock solution with <NUM>µg of the emodin per mL, a chrysophanol stock solution with <NUM>µg of the chrysophanol per mL, and an emodin methyl ether stock solution with <NUM>µg of the emodin methyl ether per mL; and <NUM> of the aloe emodin stock solution, <NUM> of the rheinic acid stock solution, <NUM> of the emodin stock solution, <NUM> of the chrysophanol stock solution, and <NUM> of the emodin methyl ether stock solution were accurately pipetted to a <NUM> volumetric flask, and methanol was added to prepare a mixed solution with <NUM>µg of the aloe emodin, <NUM>µg of the rheinic acid, <NUM>µg of the emodin, <NUM>µg of the chrysophanol, and <NUM>µg of the emodin methyl ether per mL. (<NUM>) <NUM>µL of each of the free anthraquinone test solution and the anthraquinone reference solution was accurately pipetted and injected into a liquid chromatograph to determine a free anthraquinone content in the HSBD composition.

It should be noted that a same elution program is adopted in both the determination method of the total anthraquinone content and the determination method of the free anthraquinone content, and a same anthraquinone reference solution is adopted in both the determination method of the total anthraquinone content and the determination method of the free anthraquinone content.

It should also be noted that, because the HSBD composition includes a total of <NUM> active ingredients and each active ingredient introduces a large amount of chemicals to cause a great impact on the test accuracy of the total anthraquinone and free anthraquinone, the present disclosure re-designs a gradient elution program on the basis of considering the accuracy, durability, and specificity of the test method. Specifically, investigation results show that the following elution program can effectively separate the aloe emodin, rheinic acid, emodin, and chrysophanol in the HSBD composition without setting too many gradient changes: <NUM> to <NUM>: acetonitrile proportion: <NUM>%→<NUM>%, and <NUM> vol% phosphoric acid proportion: <NUM>%→<NUM>%; and during a time range of <NUM> to <NUM>, emodin methyl ether has different peak times on different columns, and thus in this time range, isocratic elution is conducted in acetonitrile-<NUM> vol% phosphoric acid (<NUM>:<NUM>) for <NUM> to ensure the durability of the test method for different columns and improve the test accuracy.

The determination methods of the total anthraquinone and free anthraquinone in the present disclosure are subjected to methodological verification below:.

A Rheum palmatum negative sample was taken to prepare a total anthraquinone negative sample solution according to the preparation method of the total anthraquinone test solution, and a Rheum palmatum negative sample was taken to prepare a free anthraquinone negative sample solution according to the preparation method the free anthraquinone test solution. <NUM>µL of each of the free anthraquinone test solution, the total anthraquinone test solution, the free anthraquinone negative sample solution, the total anthraquinone negative sample solution, and the anthraquinone reference solution was injected into a liquid chromatograph and tested according to the proposed conditions, and test results were shown in <FIG>. A peak position of each reference was shown in the following table:.

The results show that, during a separate free anthraquinone/total anthraquinone sample test process,there is no chromatographic peakat a retention time corresponding to the free anthraquinone/total anthraquinone, and there is also no impurity peak at a similar retention time. Thus, the negative sample has no interference, and the method has prominent specificity.

<NUM> of an aloe emodin reference, <NUM> of an emodin reference, <NUM> of a chrysophanol reference, and <NUM> of an emodin methyl ether reference each were accurately weighed and added to a <NUM> volumetric flask, and methanol was added to prepare an aloe emodin reference solution with <NUM>µg of the aloe emodin per mL, an emodin reference solution with <NUM>µg of the emodin per mL, a chrysophanol reference solution with <NUM>µg of the chrysophanol per mL, and an emodin methyl ether reference solution with <NUM>µg of the emodin methyl ether per mL; and <NUM> of a rheinic acid reference, <NUM> of each of the aloe emodin reference solution, the emodin reference solution, and the chrysophanol reference solution, and <NUM> of the emodin methyl ether reference solution were accurately taken and added to a <NUM> volumetric flask, and methanol was added to prepare a mixed solution with <NUM>µg of the aloe emodin, <NUM>µg of the rheinic acid, <NUM>µg of the emodin, <NUM>µg of the chrysophanol, and <NUM>µg of the emodin methyl ether per mL, which was a free anthraquinone reference stock solution. <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> of the free anthraquinone reference stock solution each were accurately pipetted and added to a <NUM> volumetric flask to prepare reference solutions respectively including <NUM>µg, <NUM>µg, <NUM>µg, <NUM>µg, and <NUM>µg of aloe emodin per mL, <NUM>µg, <NUM>µg, <NUM>µg, <NUM>µg, and <NUM>µg of rheinic acid per mL, <NUM>µg, <NUM>µg, <NUM>µg, <NUM>µg, and <NUM>µg of emodin per mL, <NUM>µg, <NUM>µg, <NUM>µg, <NUM>µg, and <NUM>µg of chrysophanol per mL, and <NUM>µg, <NUM>µg, <NUM>µg, <NUM>µg, and <NUM>µg of emodin methyl ether per mL.

<NUM>µL of each of the above <NUM> reference solutions with different concentrations was accurately pipetted and tested, and chromatographic peak areas were recorded. With a peak area as an ordinate (y) and a reference concentration as an abscissa (x), a standard curve was plotted, and results were shown in <FIG>.

It can be seen from <FIG> that a regression equation of the aloe emodin is as follows: y = <NUM>,<NUM> x + <NUM>, with a correlation coefficient R<NUM> = <NUM>, indicating that an injection quality of the aloe emodin in an injection concentration range of <NUM>µg/mL to <NUM>µg/mL has a prominent linear relationship with a peak area.

It can be seen from <FIG> that a regression equation of the rheinic acid is as follows: y = <NUM>,<NUM> x - <NUM>,<NUM>, with a correlation coefficient R<NUM> = <NUM>, indicating that an injection quality of the rheinic acid in an injection concentration range of <NUM>µg/mL to <NUM>µg/mL has a prominent linear relationship with a peak area.

It can be seen from <FIG> that a regression equation of the emodin is as follows: y = <NUM>,<NUM> x + <NUM>, with a correlation coefficient R<NUM> = <NUM>, indicating that an injection quality of the emodin in an injection concentration range of <NUM>µg/mL to <NUM>µg/mL has a prominent linear relationship with a peak area.

It can be seen from <FIG> that a regression equation of the chrysophanol is as follows: y = <NUM>,<NUM> x + <NUM>, with a correlation coefficient R<NUM> = <NUM>, indicating that an injection quality of the chrysophanol in an injection concentration range of <NUM>µg/mL to <NUM>µg/mL has a prominent linear relationship with a peak area.

It can be seen from <FIG> that a regression equation of the emodin methyl ether is as follows: y = <NUM>,<NUM> x + <NUM>, with a correlation coefficient R<NUM> = <NUM>, indicating that an injection quality of the emodin methyl ether in an injection concentration range of <NUM>µg/mL to <NUM>µg/mL has a prominent linear relationship with a peak area.

According to the preparation method of the anthraquinone reference solution, a solution with <NUM>µg of aloe emodin, <NUM>µg of rheinic acid, <NUM>µg of emodin, <NUM>µg of chrysophanol, and <NUM>µg of emodin methyl ether per mL was prepared as a total anthraquinone reference stock solution. <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> of the total anthraquinone reference stock solution each were accurately pipetted and added to a <NUM> volumetric flask to prepare reference solutions respectively including <NUM>µg, <NUM>µg, <NUM>µg, <NUM>µg, and <NUM>µg of aloe emodin per mL, <NUM>µg, <NUM>µg, <NUM>µg, <NUM>µg, and <NUM>µg of rheinic acid per mL, <NUM>µg, <NUM>µg, <NUM>µg, <NUM>µg, and <NUM>µg of emodin per mL, <NUM>µg, <NUM>µg, <NUM>µg, <NUM>µg, and <NUM>µg of chrysophanol per mL, and <NUM>µg, <NUM>µg, <NUM>µg, <NUM>µg, and <NUM>µg of emodin methyl ether per mL.

It can be seen from <FIG> that a regression equation of the aloe emodin is as follows: y = <NUM> x - <NUM>, with a correlation coefficient R<NUM> = <NUM>, indicating that an injection quality of the aloe emodin in an injection concentration range of <NUM>µg/mL to <NUM>µg/mL has a prominent linear relationship with a peak area.

It can be seen from <FIG> that a regression equation of the emodin is as follows: y = <NUM>,<NUM> x - <NUM>, with a correlation coefficient R<NUM> = <NUM>, indicating that an injection quality of the emodin in an injection concentration range of <NUM>µg/mL to <NUM>µg/mL has a prominent linear relationship with a peak area.

It can be seen from <FIG> that a regression equation of the chrysophanol is as follows: y = <NUM>,<NUM> x - <NUM>,<NUM>, with a correlation coefficient R<NUM> = <NUM>, indicating that an injection quality of the chrysophanol in an injection concentration range of <NUM>µg/mL to <NUM>µg/mL has a prominent linear relationship with a peak area.

It can be seen from <FIG> that a regression equation of the emodin methyl ether is as follows: y = <NUM>,<NUM> x + +<NUM>, with a correlation coefficient R<NUM> = <NUM>, indicating that an injection quality of the emodin methyl ether in an injection concentration range of <NUM>µg/mL to <NUM>µg/mL has a prominent linear relationship with a peak area.

<NUM>µL of the anthraquinone reference solution was accurately pipetted and tested, and peak areas of the aloe emodin, rheinic acid, emodin, chrysophanol, and emodin methyl ether were calculated. Test results were shown in Table <NUM>.

The results show that an RSD value of index peak areas resulting from <NUM> consecutive injections of a same anthraquinone reference solution is less than <NUM>%, indicating that the instrument precision is excellent.

Different experimenters were selected to conduct the content determination at different time points on different high-performance liquid chromatographs. An appropriate amount of the HSBD composition was taken and ground, and about <NUM> of the ground composition was accurately weighed to prepare a free anthraquinone test solution or a total anthraquinone test solution. <NUM> parallel samples were prepared in each group. <NUM>µL of an anthraquinone reference solution was accurately pipetted and tested, and a free anthraquinone content, a total anthraquinone content, and corresponding RSD values were calculated and compared with reproducibility investigation results. Test results were shown in Table <NUM> to Table <NUM>.

The results show that, when a same sample is tested by an operator <NUM> times at different time points on different chromatographs, an RSD value for the free anthraquinone content is <NUM>%, and when a same sample is tested by two operators <NUM> times at different time points on different chromatographs, an RSD value for the free anthraquinone content is <NUM>%; and when a same sample is tested by an operator <NUM> times at different time points on different chromatographs, an RSD value for the total anthraquinone content is <NUM>%, and when a same sample is tested by two operators <NUM> times at different time points on different chromatographs, an RSD value for the total anthraquinone content is <NUM>%. According to "<NPL>, when a content of a component to be tested in a sample is <NUM>% to <NUM>%, an intermediate precision RSD limit should be lower than <NUM>%. Therefore, the operation results of the different operators at different time points on different chromatographs show that the method has prominent intermediate precision.

<NUM>µL of each of the free anthraquinone test solution and the total anthraquinone test solution was accurately pipetted and injected at <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>, peak areas of aloe emodin, rheinic acid, emodin, chrysophanol, and emodin methyl ether in the test solutions were determined, and a relative standard deviation (RSD) value of peak areas was calculated. Determination results were shown in Table <NUM> and Table <NUM>.

The results show that, when a same free anthraquinone test solution is injected and tested at <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>, an RSD value of peak areas of each index component is lower than <NUM>%, indicating that the free anthraquinone test solution has high stability within <NUM>.

When a same total anthraquinone test solution is injected and tested at <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>, an RSD value of peak areas of the emodin methyl ether is lower than <NUM>%, but an RSD value of peak areas of each of the remaining index components is higher than <NUM>%. Thus, it is recommended that the test of the total anthraquinone test solution should be completed within <NUM>.

<NUM>µL of each of the free anthraquinone test solution and the total anthraquinone test solution was accurately pipetted and tested, and the free anthraquinone and total anthraquinone contents and corresponding RSD values were calculated. Determination results were shown in Table <NUM> and Table <NUM>.

The results show that, when a same sample is repeatedly tested <NUM> times, an RSD value for either the free anthraquinone content or the total anthraquinone content is lower than <NUM>%, indicating that the analysis method has prominent reproducibility.

<NUM> of an aloe emodin reference, <NUM> of a rheinic acid reference, <NUM> of an emodin reference, <NUM> of a chrysophanol reference, and <NUM> of an emodin methyl ether reference were accurately weighed and added to a <NUM> volumetric flask, a <NUM> volumetric flask, a <NUM> volumetric flask, a <NUM> volumetric flask, and a <NUM> volumetric flask, respectively; and methanol was added to each of the volumetric flasks to prepare an aloe emodin solution with <NUM> of aloe emodin per mL, a rheinic acid solution with <NUM> of rheinic acid per mL, an emodin solution with <NUM> of emodin per mL, a chrysophanol solution with <NUM> of chrysophanol per mL, and an emodin methyl ether solution with <NUM> of emodin methyl ether per mL, which would be used as spike-and-recovery stock solutions. <NUM> of each of the stock solutions was accurately pipetted and added to an Erlenmeyer flask, and the solvent was completely volatilized, where <NUM> replicates were set for each group; and the HSBD composition was taken to prepare a free anthraquinone test solution, a free anthraquinone content in the free anthraquinone test solution was determined, and a spiked recovery rate was calculated. Results were shown in Table <NUM> to <NUM>.

It can be seen from the table that a recovery rate of aloe emodin is <NUM>%, a recovery rate of rheinic acid is <NUM>%, a recovery rate of emodin is <NUM>%, a recovery rate of chrysophanol is <NUM>%, and a recovery rate of emodin methyl ether is <NUM>%. According to "<NPL>, when a content of a component to be tested in a sample is <NUM>% to <NUM>%, a recovery rate limit should be <NUM>% to <NUM>%. Thus, the determination method of the present disclosure has excellent accuracy.

<NUM> of an aloe emodin reference, <NUM> of a rheinic acid reference, <NUM> of an emodin reference, <NUM> of a chrysophanol reference, and <NUM> of an emodin methyl ether reference each were weighed and added to a <NUM> volumetric flask; and methanol was added to prepare an aloe emodin solution with <NUM> of aloe emodin per mL, a rheinic acid solution with <NUM> of rheinic acid per mL, an emodin solution with <NUM> of emodin per mL, a chrysophanol solution with <NUM> of chrysophanol per mL, and an emodin methyl ether solution with <NUM> of emodin methyl ether per mL, which would be used as spike-and-recovery stock solutions. <NUM> of each of the aloe emodin, emodin, and emodin methyl ether stock solutions and <NUM> of each of the rheinic acid and chrysophanol stock solutions were accurately pipetted and added to an Erlenmeyer flask, and the solvent was completely volatilized, where <NUM> replicates were set for each group; and an appropriate amount of the HSBD composition (batch No: J2004007) was taken and ground, <NUM> of the ground composition was taken and added to each of the <NUM> replicates to prepare total anthraquinone test solutions, a total anthraquinone content in each of the total anthraquinone test solutions was determined, and a spiked recovery rate was calculated. Results were shown in Table <NUM> to <NUM>.

The results show that a recovery rate of aloe emodin is <NUM>%, a recovery rate of rheinic acid is <NUM>%, a recovery rate of emodin is <NUM>%, a recovery rate of chrysophanol is <NUM>%, and a recovery rate of emodin methyl ether is <NUM>%. According to "<NPL>, when a content of a component to be tested in a sample is <NUM>% to <NUM>%, a recovery rate limit should be <NUM>% to <NUM>%. Thus, the method has excellent accuracy.

The following three chromatographic columns were adopted: Phenomenex Kinetex-EVO C18 column (<NUM> × <NUM>, <NUM>; No. JS-<NUM>), Waters HSS T3 column (<NUM> × <NUM>, <NUM>; No. JS-<NUM>), and Waters X-bridge C18 column (<NUM> × <NUM>, <NUM>; No. JS-<NUM>).

<NUM>µL of each of the free anthraquinone test solution and the total anthraquinone test solution was pipetted and tested on each of the three chromatographic columns, and the free anthraquinone and total anthraquinone contents and corresponding RSD values were calculated. Test results were shown in Table <NUM>.

The results show that the resolution effect at each of the chromatographic columns is prominent, and an RSD value for the content is lower than or equal to <NUM>%, indicating that the analysis method exhibits excellent durability during the analysis on the different chromatographic columns.

The column temperatures of <NUM>, <NUM>, and <NUM> were set to investigate the influence of the column temperature on the determination of the free/total anthraquinone contents in the HSBD composition.

<NUM>µL of each of the free anthraquinone test solution and the total anthraquinone test solution was pipetted and tested at each of the column temperatures, and the free anthraquinone and total anthraquinone contents and corresponding RSD values were calculated. Test results were shown in Table <NUM>.

The results show that an RSD value for each of the free anthraquinone content and the total anthraquinone content in the HSBD composition at a same column temperature is greater than <NUM>%, indicating that the analysis method is sensitive to the column temperature. Therefore, the column temperature is strictly controlled at <NUM>.

The flow rates of <NUM>/min, <NUM>/min, and <NUM>/min were set to investigate the influence of the flow rate on the determination of the free/total anthraquinone contents in the HSBD composition.

<NUM>µL of each of the free anthraquinone test solution and the total anthraquinone test solution was pipetted and tested at each of the flow rates, and the free anthraquinone and total anthraquinone contents and corresponding RSD values were calculated. Test results were shown in Table <NUM>.

The results show that an RSD value for each of the free anthraquinone content and the total anthraquinone content in the HSBD composition at each of the different flow rates is lower than <NUM>%; and when the flow rate is <NUM>/min, the emodin methyl ether in the total anthraquinone has a split peak, and it is recommended that the flow rate should be no less than <NUM>/min during the analysis.

<NUM> batches of the HSBD composition were taken to prepare free anthraquinone test solutions and total anthraquinone test solutions, the free anthraquinone content and total anthraquinone content in each of the <NUM> batches of the HSBD composition were determined, and a combined anthraquinone content was calculated. Experimental results were shown in Table <NUM>.

In the present disclosure, the total content of ephedrine hydrochloride and pseudoephedrine hydrochloride in the HSBD composition is determined by HPLC. Specifically, the determination method includes:.

The determination method of the ephedrine hydrochloride and pseudoephedrine hydrochloride in the present disclosure is subjected to methodological verification below:.

An ephedra negative sample was taken to prepare an ephedrine negative sample solution according to the preparation method of the ephedrine test solution; and <NUM>µL of each of the ephedrine test solution, the ephedrine negative sample solution, and the ephedrine reference solution was injected into a liquid chromatograph and tested. Test results were shown in <FIG>. A peak position of each reference was shown in the following table:.

It can be seen from <FIG> and Table <NUM> that the ephedrine negative sample solution has no obvious chromatographic peak at the corresponding retention time of ephedrine hydrochloride and pseudoephedrine hydrochloride, and thus there is no interference, indicating that the method has prominent specificity.

<NUM> of an ephedrine hydrochloride reference and <NUM> of a pseudoephedrine hydrochloride reference were taken and added to a <NUM> volumetric flask, and methanol was added to prepare a mixed reference stock solution with <NUM>µg of ephedrine hydrochloride and <NUM>µg of pseudoephedrine hydrochloride per mL; and different volumes of the reference stock solution each were accurately measured and serially diluted to obtain mixed reference solutions with ephedrine hydrochloride and pseudoephedrine hydrochloride at different concentration levels.

<NUM>µL of each of the ephedrine hydrochloride reference solutions (which included <NUM>µg, <NUM>µg, <NUM>µg, <NUM>µg, <NUM>µg, <NUM>µg, and <NUM>µg of ephedrine hydrochloride per mL, respectively, and included <NUM>µg, <NUM>µg, <NUM>µg, <NUM>µg, <NUM>µg, <NUM>µg, and <NUM>µg of pseudoephedrine hydrochloride per mL, respectively) was accurately pipetted and subjected to chromatographic assay. A peak area was determined according to the above chromatographic conditions, the ephedrine hydrochloride and pseudoephedrine hydrochloride (x) were regressed with the peak area (y), and a standard curve was plotted. Test results were shown in <FIG>.

It can be seen from <FIG> that a linear curve of ephedrine hydrochloride is as follows: y = <NUM>,<NUM> x + <NUM>, R<NUM> = <NUM>.

It can be seen from <FIG> that a linear curve of pseudoephedrine hydrochloride is as follows: y = <NUM>,<NUM> x - <NUM>,<NUM>, R<NUM> = <NUM>.

The results show that, when the ephedrine hydrochloride is in a concentration range of <NUM>µg/mL to <NUM>µg/mL, there is a prominent linear relationship between the peak area and the concentration; and when the pseudoephedrine hydrochloride is in a concentration range of <NUM>µg/mL to <NUM>µg/mL, there is a prominent linear relationship between the peak area and the concentration.

<NUM>µL of an ephedrine reference solution (ephedrine hydrochloride concentration: <NUM>µg/mL, and pseudoephedrine hydrochloride concentration: <NUM>µg/mL) was accurately pipetted and repeatedly tested <NUM> times, and peak areas of ephedrine hydrochloride and pseudoephedrine hydrochloride were calculated. Test results were shown in Table <NUM>.

The results show that an RSD value for index peak areas resulting from <NUM> consecutive injections of a same ephedrine reference solution is less than <NUM>%, indicating that the instrument precision is excellent.

<NUM>µL of a same ephedrine test solution was accurately pipetted and injected into a chromatograph at <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> to determine peak areas of ephedrine hydrochloride and pseudoephedrine hydrochloride, and an RSD value for peak areas was calculated. Test results were shown in Table <NUM>.

It can be seen from the table that RSD values (n = <NUM>) for the ephedrine hydrochloride and pseudoephedrine hydrochloride are <NUM>% and <NUM>%, respectively, which both are lower than <NUM>%, indicating that the sample has prominent stability within <NUM>.

A same batch of samples were taken to prepare ephedrine test solutions, and the ephedrine test solutions were tested. The contents of ephedrine hydrochloride and pseudoephedrine hydrochloride and the corresponding RSD values were calculated, and results were shown in Table <NUM>.

The results show that RSD values for the ephedrine hydrochloride and pseudoephedrine hydrochloride contents both are lower than <NUM>%, indicating that the method has prominent reproducibility.

The spike-and-recovery method was adopted. About <NUM> of a sample in which the ephedrine hydrochloride and pseudoephedrine hydrochloride contents were known was accurately weighed, and <NUM> parallel samples were prepared, with <NUM> in each group. The ephedrine hydrochloride and pseudoephedrine hydrochloride references were added according to sample-to-reference ratios of <NUM>:<NUM>, <NUM>:<NUM>, and <NUM>:<NUM>. <NUM> ephedrine test solutions were prepared according to the preparation method of the ephedrine test solution and tested, and a spiked recovery rate was calculated. Test results were shown in Table <NUM>.

It can be seen from the table that an average spiked recovery rate of ephedrine hydrochloride is <NUM>% and an average spiked recovery rate of pseudoephedrine hydrochloride is <NUM>%, which are in line with the relevant provisions in Chinese Pharmacopoeia. Thus, the determination method of the present disclosure has prominent accuracy.

<NUM> batches of the HSBD composition were taken to prepare test solutions. <NUM>µL of an ephedrine test solution was accurately pipetted and tested for an ephedrine hydrochloride content and a pseudoephedrine hydrochloride content, and a total content of ephedrine hydrochloride and pseudoephedrine hydrochloride was calculated. Results were shown in Table <NUM>.

In the present disclosure, the paeoniflorin content in the HSBD composition is determined by HPLC. Specifically, the determination method includes:.

The determination method of the paeoniflorin in the present disclosure is subjected to methodological verification below:.

A Paeoniae Radix Rubra negative sample was taken to prepare a paeoniflorin negative sample solution according to the preparation method of the paeoniflorin test solution; and <NUM>µL of each of the paeoniflorin test solution, the paeoniflorin reference solution, and the paeoniflorin negative sample solution was injected into a liquid chromatograph and tested. Test results were shown in <FIG>. It can be seen from the figure that the paeoniflorin negative sample solution has no obvious chromatographic peak at the same retention time as paeoniflorin, and thus it is considered that there is no interference by the negative sample.

An appropriate amount of a paeoniflorin reference was accurately weighed, and methanol was added to prepare a paeoniflorin reference solution with <NUM> of the paeoniflorin reference per mL. The paeoniflorin reference solution was diluted <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> times to prepare a series of paeoniflorin reference solutions with different concentrations. With the concentration as an abscissa and the peak area as an ordinate, a standard curve was plotted, and a regression equation was calculated. Results were shown in <FIG>.

A linear curve of paeoniflorin is as follows: y = <NUM>,<NUM>,<NUM> x - <NUM>,<NUM>, R<NUM> = <NUM>; and a linear relationship between the peak area and the concentration is prominent.

<NUM>µL of a paeoniflorin reference solution was accurately pipetted and repeatedly tested <NUM> times, and a deviation was calculated based on a paeoniflorin peak area. Test results were shown in Table <NUM>.

The results show that an RSD value is <NUM>%, indicating prominent instrument precision.

<NUM>µL of a same paeoniflorin test solution was accurately pipetted and injected into a chromatograph at <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> to determine a paeoniflorin peak area, and an RSD value was calculated. Test results were shown in Table <NUM>.

The results show that an RSD value is <NUM>%, indicating that the paeoniflorin test solution is stable within <NUM>.

A same batch of samples were taken to prepare <NUM> paeoniflorin test solutions according to the preparation method of the paeoniflorin test solution, the paeoniflorin test solutions were tested, and an RSD value for the paeoniflorin content was calculated. Test results were shown in Table <NUM>.

The results show that an RSD is <NUM>%, which is much lower than the standard requirement of <NUM>%. Thus, the paeoniflorin determination method of the present disclosure has excellent reproducibility.

The spike-and-recovery method was adopted. <NUM> of a sample was taken, and a paeoniflorin reference solution was added according to a sample-reference ratio of <NUM>:<NUM>. <NUM> parallel samples were prepared according to the preparation method of the paeoniflorin test solution, and a spiked recovery rate was calculated. Results were shown in Table <NUM>.

It can be seen from the table that an average spiked recovery rate of paeoniflorin is <NUM>%, which is in line with the relevant provisions in Chinese Pharmacopoeia. Thus, the determination method of the present disclosure has prominent accuracy.

<NUM> batches of the HSBD composition were taken to prepare paeoniflorin test solutions. <NUM>µL of a paeoniflorin test solution was accurately pipetted and tested for a paeoniflorin content. Results were shown in Table <NUM>.

An ephedra negative sample was taken to prepare an ephedra-deficient negative sample solution according to the preparation method of the ephedra test solution; <NUM>µL of the ephedra test solution, <NUM>µL of the ephedra-deficient negative sample solution, and <NUM>µL of the ephedra reference solution were taken and spotted on a same silica gel G TLC plate (Haiyang silica gel G plate), and development was conducted with a mixed solution of TCM, methanol, and a concentrated ammonia solution (<NUM>:<NUM>:<NUM>) as a developing agent at a normal temperature and humidity (T: <NUM>, RH: <NUM>%); and the silica gel G TLC plate was taken out, air-dried, sprayed with a ninhydrin solution, heated until spots were clearly stained, and observed under sunlight. Experimental results were shown in <FIG>. It can be seen from the figure that, in the TLC identification method of the present disclosure, there is no spot for an active ingredient deficient in the negative sample and there is also no impurity spot at a position nearby during TLC of the negative sample, indicating that the method has prominent specificity.

<NUM>µL of the ephedra test solution and <NUM>µL of the ephedra reference solution were taken and spotted on a same silica gel G TLC plate (Haiyang silica gel G plate), and development was conducted with a mixed solution of TCM, methanol, and a concentrated ammonia solution (<NUM>:<NUM>:<NUM>) as a developing agent at a normal temperature (T: <NUM>, RH: <NUM>%) and a low temperature (T: <NUM>, RH: <NUM>%); and the silica gel G TLC plate was taken out, air-dried, sprayed with a ninhydrin solution, heated until spots were clearly stained, and observed under sunlight. Experimental results were shown in <FIG>.

It can be seen from <FIG> that the resolution effect is excellent at both the normal temperature and the low temperature, and a main spot at a position in a chromatogram of the HSBD composition is in the same color as a main spot at a corresponding position in a chromatogram of the reference. The experimental results show that the temperature has a little impact on the TLC identification of ephedra in the HSBD composition, indicating that the TLC identification method has excellent durability for different temperatures.

<NUM>µL of the ephedra test solution and <NUM>µL of the ephedra reference solution were taken and spotted on a same silica gel G TLC plate (Haiyang silica gel G plate), and development was conducted with a mixed solution of TCM, methanol, and a concentrated ammonia solution (<NUM>:<NUM>:<NUM>) as a developing agent at a normal humidity (T: <NUM>, RH: <NUM>%), a low humidity (T: <NUM>, RH: <NUM>%), and a high humidity (T: <NUM>, RH: <NUM>%); and the silica gel G TLC plate was taken out, air-dried, sprayed with a ninhydrin solution, heated until spots were clearly stained, and observed under sunlight. Experimental results were shown in <FIG>, <FIG>.

It can be seen from <FIG>, <FIG> that the resolution effect is excellent at the normal humidity, low humidity, and high humidity, and a main spot at a position in a chromatogram of the HSBD composition is in the same color as a main spot at a corresponding position in a chromatogram of the reference. The experimental results show that the humidity has a little impact on the TLC identification of ephedra in the HSBD composition, indicating that the TLC identification method has excellent durability for different humidities.

The ephedra test solution and the ephedra reference solution each were taken and spotted at different amounts on a same silica gel G TLC plate (Haiyang silica gel G plate), and development was conducted with a mixed solution of TCM, methanol, and a concentrated ammonia solution (<NUM>:<NUM>:<NUM>) as a developing agent; and the silica gel G TLC plate was taken out, air-dried, sprayed with a ninhydrin solution, heated until spots were clearly stained, and observed under sunlight. Experimental results were shown in <FIG>.

It can be seen from <FIG> that, when the ephedra test solution is spotted at an amount of <NUM>µL and the ephedra reference solution is spotted at an amount of <NUM>µL, a main spot at a position in a test chromatogram corresponding to a reference chromatogram is clear and is not interfered. Therefore, in the ephedra identification method of the present disclosure, the ephedra test solution is spotted at an amount of <NUM>µL and the ephedra reference solution is spotted at an amount of <NUM>µL.

<NUM>µL of the ephedra test solution and <NUM>µL of the ephedra reference solution were taken and spotted on each of silica gel G TLC plates of different manufacturers (Haiyang silica gel G plate, PUKE silica gel G plate, and Merck silica gel G plate), and development was conducted with a mixed solution of TCM, methanol, and a concentrated ammonia solution (<NUM>:<NUM>:<NUM>) as a developing agent at the same temperature and humidity (T: <NUM>, RH: <NUM>%); and the silica gel G TLC plates were taken out, air-dried, sprayed with a ninhydrin solution, heated until spots were clearly stained, and observed under sunlight. Experimental results were shown in <FIG>, <FIG>, and <FIG>.

The Haiyang silica gel G plate was a TLC plate produced by Qingdao Haiyang Chemical Co. ; the PUKE silica gel G plate was a TLC plate produced by Qingdao PUKE Parting Materials Co. ; and the Merck silica gel G plate was a TLC plate produced by Merck KGaA. The TLC plates each had a specification of <NUM> × <NUM> and a thickness of <NUM> to <NUM>.

The results show that the silica gel G TLC plates of different manufacturers (Haiyang silica gel G plate, PUKE silica gel G plate, and Merck silica gel G plate) have no significant impact on the TLC identification of ephedra in the HSBD composition, indicating that the TLC identification method has prominent durability for silica gel G TLC plates of different manufacturers.

A licorice negative sample was taken to prepare a licorice-deficient negative sample solution according to the preparation method of the licorice test solution; <NUM>µL of the licorice test solution, <NUM>µL of the licorice reference solution, and <NUM>µL of the licorice-deficient negative sample solution were taken and spotted on a same silica gel G TLC plate (Haiyang silica gel G plate), and development was conducted with a mixed solution of ethyl acetate, formic acid, glacial acetic acid, and water (<NUM>:<NUM>:<NUM>:<NUM>) as a developing agent at a normal temperature and humidity (T: <NUM>, RH: <NUM>%); and the silica gel G TLC plate was taken out, air-dried, sprayed with a <NUM>% sulfuric acid-ethanol solution, heated at <NUM> until spots were clearly stained, and observed under sunlight. Experimental results were shown in <FIG>. It can be seen from the figure that, in the TLC identification method of the present disclosure, there is no spot for an active ingredient deficient in the negative sample and there is also no impurity spot at a position nearby during TLC of the negative sample, indicating that the method has prominent specificity.

<NUM>µL of the licorice test solution and <NUM>µL of the licorice reference solution were taken and spotted on a same silica gel G TLC plate (Haiyang silica gel G plate), and development was conducted with a mixed solution of ethyl acetate, formic acid, glacial acetic acid, and water (<NUM>:<NUM>:<NUM>:<NUM>) as a developing agent at a normal temperature (T: <NUM>, RH: <NUM>%) and a low temperature (T: <NUM>, RH: <NUM>%); and the silica gel G TLC plate was taken out, air-dried, sprayed with a <NUM>% sulfuric acid-ethanol solution, heated at <NUM> until spots were clearly stained, and observed under sunlight. Experimental results were shown in <FIG>.

It can be seen from <FIG> that the resolution effect is excellent at both the normal temperature and the low temperature, and a main spot at a position in a chromatogram of the HSBD composition is in the same color as a main spot at a corresponding position in a chromatogram of the reference. The experimental results show that the temperature has a little impact on the TLC identification of licorice in the HSBD composition, indicating that the TLC identification method has excellent durability for different temperatures.

<NUM>µL of the licorice test solution and <NUM>µL of the licorice reference solution were taken and spotted on a same silica gel G TLC plate (Haiyang silica gel G plate), and development was conducted with a mixed solution of ethyl acetate, formic acid, glacial acetic acid, and water (<NUM>:<NUM>:<NUM>:<NUM>) as a developing agent at a normal humidity (T: <NUM>, RH: <NUM>%), a low humidity (T: <NUM>, RH: <NUM>%), and a high humidity (T: <NUM>, RH: <NUM>%); and the silica gel G TLC plate was taken out, air-dried, sprayed with a <NUM>% sulfuric acid-ethanol solution, heated at <NUM> until spots were clearly stained, and observed under sunlight. Experimental results were shown in <FIG>, <FIG>.

It can be seen from <FIG>, <FIG> that the resolution effect is excellent at the normal humidity, low humidity, and high humidity, and a main spot at a position in a chromatogram of the HSBD composition is in the same color as a main spot at a corresponding position in a chromatogram of the reference. The experimental results show that the humidity has a little impact on the TLC identification of licorice in the HSBD composition, indicating that the TLC identification method has excellent durability for different humidities.

The licorice test solution and the licorice reference solution each were taken and spotted at different amounts on a same silica gel G TLC plate (Haiyang silica gel G plate), and development was conducted with a mixed solution of ethyl acetate, formic acid, glacial acetic acid, and water (<NUM>:<NUM>:<NUM>:<NUM>) as a developing agent at a normal temperature and humidity (T: <NUM>, RH: <NUM>%); and the silica gel G TLC plate was taken out, air-dried, sprayed with a <NUM>% sulfuric acid-ethanol solution, heated at <NUM> until spots were clearly stained, and observed under sunlight. Experimental results were shown in <FIG>.

It can be seen from <FIG> that, when the licorice test solution is spotted at an amount of <NUM>µL and the licorice reference solution is spotted at an amount of <NUM>µL, a main spot at a position in a test chromatogram corresponding to a reference chromatogram is clear and is not interfered. Therefore, in the licorice identification method of the present disclosure, the licorice test solution is spotted at an amount of <NUM>µL and the licorice reference solution is spotted at an amount of <NUM>µL.

The licorice test solution and the licorice reference solution were taken and spotted on each of silica gel G TLC plates of different manufacturers (Haiyang silica gel G plate, PUKE silica gel G plate, and Merck silica gel G plate), and development was conducted with a mixed solution of ethyl acetate, formic acid, glacial acetic acid, and water (<NUM>:<NUM>:<NUM>:<NUM>) as a developing agent at a normal temperature and humidity (T: <NUM>, RH: <NUM>%); and the silica gel G TLC plate was taken out, air-dried, sprayed with a <NUM>% sulfuric acid-ethanol solution, heated at <NUM> until spots were clearly stained, and observed under sunlight. Experimental results were shown in <FIG>, <FIG>, and <FIG>.

The results show that the silica gel G TLC plates of different manufacturers (Haiyang silica gel G plate, PUKE silica gel G plate, and Merck silica gel G plate) have no significant impact on the TLC identification of licorice in the HSBD composition, indicating that the TLC identification method has prominent durability for silica gel G TLC plates of different manufacturers.

A Magnolia officinalis negative sample was taken to prepare a Magnolia officinalis-deficient negative sample solution according to the preparation method of the Magnolia officinalis test solution; <NUM>µL of the Magnolia officinalis test solution, <NUM>µL of the magnolol reference solution, <NUM>µL of the honokiol reference solution, and <NUM>µL of the Magnolia officinalis-deficient negative sample solution were taken and spotted on a same silica gel G TLC plate (Merck silica gel G plate), and development was conducted with a mixed solution of toluene, ethyl acetate, and methanol (<NUM>:<NUM>:<NUM>) as a developing agent at a normal temperature and humidity (T: <NUM>. <NUM>, RH: <NUM>%); and the silica gel G TLC plate was taken out, air-dried, sprayed with a <NUM>% vanillin-sulfuric acid solution, heated until spots were clearly stained, and observed under sunlight. Experimental results were shown in <FIG>. It can be seen from the figure that, in the TLC identification method of the present disclosure, there is no spot for an active ingredient deficient in the negative sample and there is also no impurity spot at a position nearby during TLC of the negative sample, indicating that the method has prominent specificity.

<NUM>µL of the Magnolia officinalis test solution, <NUM>µL of the magnolol reference solution, and <NUM>µL of the honokiol reference solution were taken and spotted on a same silica gel G TLC plate (Merck silica gel G plate), and development was conducted with a mixed solution of toluene, ethyl acetate, and methanol (<NUM>:<NUM>:<NUM>) as a developing agent at a normal temperature (T: <NUM>, RH: <NUM>%) and a low temperature (T: <NUM>, RH: <NUM>%); and the silica gel G TLC plate was taken out, air-dried, sprayed with a <NUM>% vanillin-sulfuric acid solution, heated until spots were clearly stained, and observed under sunlight. Experimental results were shown in <FIG>.

It can be seen from <FIG> that the resolution effect is excellent at both the normal temperature and the low temperature, and a main spot at a position in a chromatogram of the HSBD composition is in the same color as a main spot at a corresponding position in a chromatogram of the reference. The experimental results show that the temperature has a little impact on the TLC identification of Magnolia officinalis in the HSBD composition, indicating that the TLC identification method has excellent durability for different temperatures.

<NUM>µL of the Magnolia officinalis test solution, <NUM>µL of the magnolol reference solution, and <NUM>µL of the honokiol reference solution were taken and spotted on a same silica gel G TLC plate (Merck silica gel G plate), and development was conducted with a mixed solution of toluene, ethyl acetate, and methanol (<NUM>:<NUM>:<NUM>) as a developing agent at a normal humidity (T: <NUM>, RH: <NUM>%), a low humidity (T: <NUM>. <NUM>, RH: <NUM>%), and a high humidity (T: <NUM>. <NUM>, RH: <NUM>%); and the silica gel G TLC plate was taken out, air-dried, sprayed with a <NUM>% vanillin-sulfuric acid solution, heated until spots were clearly stained, and observed under sunlight. Experimental results were shown in <FIG>, <FIG>.

It can be seen from <FIG>, <FIG> that the resolution effect is excellent at the normal humidity, low humidity, and high humidity, and a main spot at a position in a chromatogram of the HSBD composition is in the same color as a main spot at a corresponding position in a chromatogram of the reference. The experimental results show that the humidity has a little impact on the TLC identification of Magnolia officinalis in the HSBD composition, indicating that the TLC identification method has excellent durability for different humidities.

The Magnolia officinalis test solution, the magnolol reference solution, and the honokiol reference solution each were taken and spotted at different amounts on a same silica gel G TLC plate (Merck silica gel G plate), and development was conducted with a mixed solution of toluene, ethyl acetate, and methanol (<NUM>:<NUM>:<NUM>) as a developing agent at a normal temperature and humidity (T: <NUM>. <NUM>, RH: <NUM>%); and the silica gel G TLC plate was taken out, air-dried, sprayed with a <NUM>% vanillin-sulfuric acid solution, heated until spots were clearly stained, and observed under sunlight. Experimental results were shown in <FIG>.

It can be seen from <FIG> that, when the Magnolia officinalis test solution is spotted at an amount of <NUM>µL, the magnolol reference solution is spotted at an amount of <NUM>µL, and the honokiol reference solution is spotted at an amount of <NUM>µL, a main spot at a position in a test chromatogram corresponding to a reference chromatogram is clear and is not interfered. Therefore, in the Magnolia officinalis identification method of the present disclosure, the Magnolia officinalis test solution is spotted at an amount of <NUM>µL, the magnolol reference solution is spotted at an amount of <NUM>µL, and the honokiol reference solution is spotted at an amount of <NUM>µL.

<NUM>µL of the Magnolia officinalis test solution, <NUM>µL of the magnolol reference solution, and <NUM>µL of the honokiol reference solution were taken and spotted on each of silica gel G TLC plates of different manufacturers (Haiyang silica gel G plate, PUKE silica gel G plate, and Merck silica gel G plate), and development was conducted with a mixed solution of toluene, ethyl acetate, and methanol (<NUM>:<NUM>:<NUM>) as a developing agent at a normal temperature and humidity (T: <NUM>, RH: <NUM>%); and the silica gel G TLC plates were taken out, air-dried, sprayed with a <NUM>% vanillin-sulfuric acid solution, heated until spots were clearly stained, and observed under sunlight. Experimental results were shown in <FIG>, <FIG>, and <FIG>.

The results show that the silica gel G TLC plates of different manufacturers (Haiyang silica gel G plate, PUKE silica gel G plate, and Merck silica gel G plate) have no significant impact on the TLC identification of Magnolia officinalis in the HSBD composition, indicating that the TLC identification method has prominent durability for silica gel G TLC plates of different manufacturers.

An Astragalus membranaceus negative sample was taken to prepare an Astragalus membranaceus-deficient negative sample solution according to the preparation method of the Astragalus membranaceus test solution; and the Astragalus membranaceus test solution, the Astragalus membranaceus-deficient negative sample solution, and the Astragalus membranaceus reference solution were spotted on a same silica gel G TLC plate (Merck silica gel G plate) and tested according to the proposed method. Results were shown in <FIG>. It can be seen from the figure that, in the TLC identification method of the present disclosure, there is no spot for an active ingredient deficient in the negative sample and there is also no impurity spot at a position nearby during TLC of the negative sample, indicating that the method has prominent specificity.

Spotted silica gel G plates (Merck silica gel G plates) were taken and subjected to development at a normal temperature (T: <NUM>, RH: <NUM>%) and a low temperature (T: <NUM>, RH: <NUM>%), respectively, and then observed according to the proposed method. Results were shown in <FIG> and <FIG>. The results show that, at the normal temperature, a spot in either the test chromatogram or the reference chromatogram has a too-high Rf value and poor resolution (<FIG>); and at the low temperature, a spot in the test chromatogram has an appropriate Rf value and prominent resolution (<FIG>), and well corresponds to a spot at a corresponding position in the reference chromatogram. Therefore, in the method of the present disclosure, the development is conducted at <NUM> to <NUM>.

Spotted silica gel G plates (Merck silica gel G plates) were taken and subjected to development at a low temperature and low humidity (T: <NUM>, RH: <NUM>%) and a low temperature and high humidity (T: <NUM>, RH: <NUM>%), respectively, and then observed according to the proposed method. Results were shown in <FIG>. The results show that the humidity has a little impact on the TLC identification of Astragalus membranaceus in the HSBD composition, indicating that the TLC identification method has excellent durability for different humidities.

The Astragalus membranaceus test solution and the Astragalus membranaceus reference solution each were spotted at different volumes on a same silica gel G TLC plate (Merck silica gel G plate) and tested according to the proposed method. Results were shown in <FIG>, and it can be seen from the figure that, when the Astragalus membranaceus test solution is spotted at an amount of <NUM>µL to <NUM>µL and the Astragalus membranaceus reference solution is spotted at an amount of <NUM>µL to <NUM>µL, a fluorescent spot at a position in a test chromatogram corresponding to a reference chromatogram is clear. Therefore, in the method of the present disclosure, the Astragalus membranaceus test solution is spotted at an amount of <NUM>µL to <NUM>µL and the Astragalus membranaceus reference solution is spotted at an amount of <NUM>µL to <NUM>µL.

The Astragalus membranaceus test solution and the Astragalus membranaceus reference solution were spotted on each of silica gel G TLC plates of different manufacturers (Merck silica gel G plate, Haiyang silica gel G plate, and Yinlong silica gel G plate) and tested according to the proposed method. Results were shown in <FIG>, <FIG>, and <FIG>.

The Merck silica gel G plate was a TLC plate produced by Merck KGaA; the Haiyang silica gel G plate was a TLC plate produced by Qingdao Haiyang Chemical Co. ; and the Yinlong silica gel G plate was a TLC plate produced by Yantai Chemical Industry Institute. The TLC plates each had a specification of <NUM> × <NUM> and a thickness of <NUM> to <NUM>.

The results show that the <NUM> silica gel G TLC plates all can achieve a prominent resolution effect, indicating that the TLC identification method has prominent durability for silica gel G TLC plates of different manufacturers.

Specifically, the TLC identification method for Semen Lepidii is as follows:.

It should be noted that, in the existing TLC identification process for Semen Lepidii, a Semen Lepidii test solution is generally prepared by directly dissolving a relevant sample with methanol, heating to allow a reaction under reflux, and filtering. However, the large number of components in the HSBD composition in the present disclosure have a great impact on the identification method. Thus, the macroporous resin purification step is added in the present disclosure.

A Semen Lepidii negative sample was taken to prepare a Semen Lepidii-deficient negative sample solution according to the preparation method of the Semen Lepidii test solution; and <NUM>µL of the Semen Lepidii test solution, <NUM>µL of the Semen Lepidii-deficient negative sample solution, and <NUM>µL of the Semen Lepidii reference solution were spotted on a same PA film and tested according to the proposed method. Results were shown in <FIG>. It can be seen from the figure that, in the TLC identification method of the present disclosure, there is no spot for an active ingredient deficient in the negative sample and there is also no impurity spot at a position nearby during TLC of the negative sample, indicating that the method has prominent specificity.

<NUM>µL of the Semen Lepidii test solution and <NUM>µL of the Semen Lepidii reference solution were taken and spotted on a PA film, and development was conducted with a mixed solution of ethyl acetate, methanol, and water (<NUM>:<NUM>:<NUM>) as a developing agent at a normal temperature (T: <NUM>, RH: <NUM>%) and a low temperature (T: <NUM>, RH: <NUM>%); and the PA film was taken out, air-dried, sprayed with a <NUM>% aluminum chloride-ethanol solution, hot air-dried, and observed under UV light (<NUM>). Experimental results were shown in <FIG>.

It can be seen from <FIG> that the resolution effect is excellent at both the normal temperature and the low temperature, and a main spot at a position in a chromatogram of the HSBD composition is in the same color as a main spot at a corresponding position in a chromatogram of the reference. The experimental results show that the temperature has a little impact on the TLC identification of Semen Lepidii in the HSBD composition, indicating that the TLC identification method has excellent durability for different temperatures.

<NUM>µL of the Semen Lepidii test solution and <NUM>µL of the Semen Lepidii reference solution were taken and spotted on a PA film, and development was conducted with a mixed solution of ethyl acetate, methanol, and water (<NUM>:<NUM>:<NUM>) as a developing agent at a normal humidity (T: <NUM>, RH: <NUM>%), a low humidity (T: <NUM>, RH: <NUM>%), and a high humidity (T: <NUM>, RH: <NUM>%); and the PA film was taken out, air-dried, sprayed with a <NUM>% aluminum chloride-ethanol solution, hot air-dried, and observed under UV light (<NUM>). Experimental results were shown in <FIG>, <FIG>.

It can be seen from <FIG>, <FIG> that the resolution effect is excellent at the normal humidity, low humidity, and high humidity, and a main spot at a position in a chromatogram of the HSBD composition is in the same color as a main spot at a corresponding position in a chromatogram of the reference. The experimental results show that the humidity has a little impact on the TLC identification of Semen Lepidii in the HSBD composition, indicating that the TLC identification method has excellent durability for different humidities.

The Semen Lepidii test solution and the Semen Lepidii reference solution each were spotted at different volumes on a same PA film and tested according to the proposed method. Results were shown in <FIG>. It can be seen from the figure that, when the Semen Lepidii test solution and the Semen Lepidii reference solution each are spotted at an amount of <NUM>µL, a fluorescent spot at a position in a test chromatogram corresponding to a reference chromatogram is clear. Thus, the spotting amount of <NUM>µL is selected.

A Paeoniae Radix Rubra negative sample was taken to prepare a Paeoniae Radix Rubra-deficient negative sample solution according to the preparation method of the Paeoniae Radix Rubra test solution; <NUM>µL of the Paeoniae Radix Rubra test solution, <NUM>µL of the Paeoniae Radix Rubra-deficient negative sample solution, and <NUM>µL of the Paeoniae Radix Rubra reference solution were taken and spotted on a same silica gel G TLC plate (Haiyang silica gel G plate), and development was conducted with a mixed solution of TCM, ethyl acetate, methanol, and formic acid (<NUM>:<NUM>:<NUM>:<NUM>) as a developing agent at a normal temperature and humidity (T: <NUM>, RH: <NUM>%); and the silica gel G TLC plate was taken out, air-dried, sprayed with a <NUM>% vanillin-sulfuric acid solution, heated until spots were clearly stained, and observed under sunlight. Experimental results were shown in <FIG>. It can be seen from the figure that, in the TLC identification method of the present disclosure, there is no spot for an active ingredient deficient in the negative sample and there is also no impurity spot at a position nearby during TLC of the negative sample, indicating that the method has prominent specificity.

<NUM>µL of the Paeoniae Radix Rubra test solution and <NUM>µL of the Paeoniae Radix Rubra reference solution were taken and spotted on a same silica gel G TLC plate (Haiyang silica gel G plate), and development was conducted with a mixed solution of TCM, ethyl acetate, methanol, and formic acid (<NUM>:<NUM>:<NUM>:<NUM>) as a developing agent at a normal temperature (T: <NUM>, RH: <NUM>%) and a low temperature (T: <NUM>, RH: <NUM>%); and the silica gel G TLC plate was taken out, air-dried, sprayed with a <NUM>% vanillin-sulfuric acid solution, heated until spots were clearly stained, and observed under sunlight. Experimental results were shown in <FIG>.

It can be seen from <FIG> that the resolution effect is excellent at both the normal temperature and the low temperature, and a main spot at a position in a chromatogram of the HSBD composition is in the same color as a main spot at a corresponding position in a chromatogram of the reference. The experimental results show that the temperature has a little impact on the TLC identification of Paeoniae Radix Rubra in the HSBD composition, indicating that the TLC identification method has excellent durability for different temperatures.

<NUM>µL of the Paeoniae Radix Rubra test solution and <NUM>µL of the Paeoniae Radix Rubra reference solution were taken and spotted on a same silica gel G TLC plate (Haiyang silica gel G plate), and development was conducted with a mixed solution of TCM, ethyl acetate, methanol, and formic acid (<NUM>:<NUM>: <NUM>:<NUM>) as a developing agent at a normal humidity (T: <NUM>, RH: <NUM>%), a low humidity (T: <NUM>, RH: <NUM>%), and a high humidity (T: <NUM>, RH: <NUM>%); and the silica gel G TLC plate was taken out, air-dried, sprayed with a <NUM>% vanillin-sulfuric acid solution, heated until spots were clearly stained, and observed under sunlight. Experimental results were shown in <FIG>, <FIG>.

It can be seen from <FIG>, <FIG> that the resolution effect is excellent at the normal humidity, low humidity, and high humidity, and a main spot at a position in a chromatogram of the HSBD composition is in the same color as a main spot at a corresponding position in a chromatogram of the reference. The experimental results show that the humidity has a little impact on the TLC identification of Paeoniae Radix Rubra in the HSBD composition, indicating that the TLC identification method has excellent durability for different humidities.

The Paeoniae Radix Rubra test solution and the Paeoniae Radix Rubrareference solution were taken and spotted on a same silica gel G TLC plate, and development was conducted with a mixed solution of TCM, ethyl acetate, methanol, and formic acid (<NUM>:<NUM>:<NUM>:<NUM>) as a developing agent; and the silica gel G TLC plate was taken out, air-dried, sprayed with a <NUM>% vanillin-sulfuric acid solution, heated until spots were clearly stained, and observed under sunlight. Experimental results were shown in <FIG>.

It can be seen from <FIG> that, when the Paeoniae Radix Rubra test solution is spotted at an amount of <NUM>µL and the Paeoniae Radix Rubra reference solution is spotted at an amount of <NUM>µL, a main spot at a position in a test chromatogram corresponding to a reference chromatogram is clear and is not interfered. Therefore, in the Paeoniae Radix Rubra identification method of the present disclosure, the Paeoniae Radix Rubra test solution is spotted at an amount of <NUM>µL and the Paeoniae Radix Rubra reference solution is spotted at an amount of <NUM>µL.

The Paeoniae Radix Rubra test solution and the Paeoniae Radix Rubra reference solution were taken and spotted on each of silica gel G TLC plates of different manufacturers (Haiyang silica gel G plate, PUKE silica gel G plate, and Merck silica gel G plate), and development was conducted with a mixed solution of TCM, ethyl acetate, methanol, and formic acid (<NUM>:<NUM>:<NUM>:<NUM>) as a developing agent at a normal temperature and humidity (T: <NUM>, RH: <NUM>%); and the silica gel G TLC plates were taken out, air-dried, sprayed with a <NUM>% vanillin-sulfuric acid solution, heated until spots were clearly stained, and observed under sunlight. Results were shown in <FIG>, <FIG>.

The results show that the silica gel G TLC plates of different manufacturers have no significant impact on the TLC identification of Paeoniae Radix Rubra in the HSBD composition, indicating that the TLC identification method has prominent durability for silica gel G TLC plates of different manufacturers.

A Rheum palmatum negative sample was taken to prepare a Rheum palmatum-deficient negative sample solution according to the preparation method of the Rheum palmatum test solution; <NUM>µL of the Rheum palmatum test solution, <NUM>µL of the Rheum palmatum-deficient negative sample solution, and <NUM>µL of the Rheum palmatum reference solution were taken and spotted on a silica gel H TLC plate (Haiyang silica gel H plate), and development was conducted with an upper layer of a mixed solution of petroleum ether (<NUM> to <NUM>), ethyl formate, and formic acid (<NUM>:<NUM>:<NUM>) as a developing agent at a normal temperature and humidity (T: <NUM>, RH: <NUM>%); and the silica gel H TLC plate was taken out, air-dried, and observed under UV light. Results were shown in <FIG>. It can be seen from the figure that, in the TLC identification method of the present disclosure, there is no spot for an active ingredient deficient in the negative sample and there is also no impurity spot at a position nearby during TLC of the negative sample, indicating that the method has prominent specificity.

<NUM>µL of the Rheum palmatum test solution and <NUM>µL of the Rheum palmatumreference solution were taken and spotted on a same silica gel H TLC plate (Haiyang silica gel H plate), and development was conducted with an upper layer of a mixed solution of petroleum ether (<NUM> to <NUM>), ethyl formate, and formic acid (<NUM>:<NUM>:<NUM>) as a developing agent at a normal temperature (T: <NUM>, RH: <NUM>%) and a low temperature (T: <NUM>, RH: <NUM>%); and the silica gel H TLC plate was taken out, air-dried, and observed under UV light (<NUM>). Experimental results were shown in <FIG>.

It can be seen from <FIG> that the resolution effect is excellent at both the normal temperature and the low temperature, and a main spot at a position in a chromatogram of the HSBD composition is in the same color as a main spot at a corresponding position in a chromatogram of the reference. The experimental results show that the temperature has a little impact on the TLC identification of Rheum palmatum in the HSBD composition, indicating that the TLC identification method has excellent durability for different temperatures.

<NUM>µL of the Rheum palmatum test solution and <NUM>µL of the Rheum palmatum reference solution were taken and spotted on a same silica gel H TLC plate (Haiyang silica gel H plate), and development was conducted with an upper layer of a mixed solution of petroleum ether (<NUM> to <NUM>), ethyl formate, and formic acid (<NUM>:<NUM>:<NUM>) as a developing agent at a normal humidity (T: <NUM>, RH: <NUM>%), a low humidity (T: <NUM>, RH: <NUM>%), and a high humidity (T: <NUM>, RH: <NUM>%); and the silica gel H TLC plate was taken out, air-dried, and observed under UV light (<NUM>). Experimental results were shown in <FIG>, <FIG>.

It can be seen from <FIG>, <FIG> that the resolution effect is excellent at the normal humidity, low humidity, and high humidity, and a main spot at a position in a chromatogram of the HSBD composition is in the same color as a main spot at a corresponding position in a chromatogram of the reference. The experimental results show that the humidity has a little impact on the TLC identification of Rheum palmatum in the HSBD composition, indicating that the TLC identification method has excellent durability for different humidities.

<NUM>µL of the Rheum palmatum test solution and <NUM>µL of the Rheum palmatum reference solution were taken and spotted on a same silica gel H TLC plate (Haiyang silica gel H plate), and development was conducted with an upper layer of a mixed solution of petroleum ether (<NUM> to <NUM>), ethyl formate, and formic acid (<NUM>:<NUM>:<NUM>) as a developing agent at a normal temperature and humidity (T: <NUM>, RH: <NUM>%); and the silica gel H TLC plate was taken out, air-dried, and observed under UV light (<NUM>). Experimental results were shown in <FIG>.

It can be seen from <FIG> that, when the Rheum palmatum test solution is spotted at an amount of <NUM>µL and the Rheum palmatum reference solution is spotted at an amount of <NUM>µL, a main spot at a position in a test chromatogram corresponding to a reference chromatogram is clear and is not interfered. Therefore, in the Rheum palmatum identification method of the present disclosure, the Rheum palmatum test solution is spotted at an amount of <NUM>µL and the Rheum palmatum reference solution is spotted at an amount of <NUM>µL.

<NUM>µL of the Rheum palmatum test solution and <NUM>µL of the Rheum palmatum reference solution were taken and spotted on each of silica gel H TLC plates of different manufacturers (Haiyang silica gel H plate, PUKE silica gel H plate, and Yinlong silica gel H plate), and development was conducted with an upper layer of a mixed solution of petroleum ether (<NUM> to <NUM>), ethyl formate, and formic acid (<NUM>:<NUM>:<NUM>) as a developing agent at a normal temperature and humidity (T: <NUM>, RH: <NUM>%); and the silica gel H TLC plate was taken out, air-dried, and observed under UV light (<NUM>). Results were shown in <FIG>, <FIG>, and <FIG>.

The Haiyang silica gel H plate was a TLC plate produced by Qingdao Haiyang Chemical Co. ; the PUKE silica gel H plate was a TLC plate produced by Qingdao PUKE Parting Materials Co. ; and the Yinlong silica gel H plate was a TLC plate produced by Yantai Chemical Industry Institute. The TLC plates each had a specification of <NUM> × <NUM> and a thickness of <NUM> to <NUM>.

The results show that the silica gel H TLC plates of different manufacturers (Haiyang silica gel H plate, PUKE silica gel H plate, and Yinlong silica gel H plate) have no significant impact on the TLC identification of Rheum palmatum in the HSBD composition, indicating that the TLC identification method has prominent durability for silica gel H TLC plates of different manufacturers.

In summary, in the QC method for an HSBD composition of the present disclosure, based on the research of a molecular action mechanism of the HSBD composition, the analysis of specific conditions of mass production, and the large amount of experimental investigation, the ephedra, licorice, Magnolia officinalis, Astragalus membranaceus, Semen Lepidii, Paeoniae Radix Rubra, and Rheum palmatum are identified by TLC, and the contents of combined anthraquinone, ephedrine hydrochloride, pseudoephedrine hydrochloride, and paeoniflorin are determined. Through the determination of the above two aspects, a data basis is provided for the QC of mass production of the HSBD composition.

The determination method of each component in the HSBD composition of the present disclosure has excellent specificity and durability, and the accuracy and stability of the determination method can meet the requirements of large-scale production, which provides a prominent basis for ensuring the stability of the active components in the HSBD composition.

Claim 1:
A quality control (QC) method for a Huashibaidu (HSBD) composition, the HSBD composition mainly comprises the following components: ephedra, fried bitter almond, raw gypsum, licorice, PogostemonisHerba, Magnolia officinalis, bran-fried AtractylodesLancea, fried Amomum tsaoko fruit, RhizomaPinelliae, Poriacocos, Rheum palmatum, Astragalus membranaceus, Semen Lepidii, Paeoniae Radix Rubra, and an adjuvant; and
characterized in that
the QC method for an HSBD composition comprises:
(<NUM>) identifying ephedra, licorice, and Magnolia officinalis by thin-layer chromatography (TLC); and
(<NUM>) determining a total anthraquinone content, a free anthraquinone content, a total content of ephedrine hydrochloride and pseudoephedrine hydrochloride, and a paeoniflorin content in the HSBD composition by high-performance liquid chromatography (HPLC), and calculating a combined anthraquinone content, and the combined anthraquinone content = the total anthraquinone content - the free anthraquinone content;
in determination of both the total anthraquinone content and the free anthraquinone content, the chromatography column is an octadecylsilane-bonded silica gel column, and the following elution program is adopted:
mobile phase A is acetonitrile and mobile phase B is a phosphoric acid aqueous solution:
<NUM> to <NUM>: mobile phase A: from <NUM>% to <NUM>%, and mobile phase B: from <NUM>% to <NUM>%;
<NUM> to <NUM>: mobile phase A: from <NUM>% to <NUM>%, and mobile phase B: from <NUM>% to <NUM>%; and
<NUM> to <NUM>: mobile phase A: <NUM>%, and mobile phase B: <NUM>%.