A method for content determination and identification of Shenxian Ling granules

Abstract

This invention relates to the field of detection technology, specifically to a method for the content determination and identification of Shenxianling granules. The method includes using high-performance liquid chromatography (HPLC) to determine the content of oridonin in Shenxianling granules, and using thin-layer chromatography (TLC) to identify six components: Agrimonia pilosa, Prunus mume, Glycyrrhiza uralensis, Gynostemma pentaphyllum, Ginseng, and Ganoderma lucidum. The TLC identification methods for each component include unique preparation methods for the test solution, reference herb solution, and negative control solution, as well as specific settings for sample volume, developing solvent, and inspection conditions. This invention constructs a complete quality control system for Shenxianling granules through a combination of technologies: precise quantification of characteristic components, specific qualitative analysis of multiple herbs, and efficient removal of impurities. Specifically, the HPLC method achieves accurate quantification of oridonin, solving the problem of the original standard indicators being one-sided; the TLC identification system, through dual optimization of extraction conditions and the developing system, ensures rapid identification of the authenticity of the six herbs.

Description

Technical Field

[0001] This invention relates to the field of detection technology, specifically to a method for determining and identifying the content of ginseng and celery granules. Background Technology

[0002] Shenxianling granules, a compound preparation composed of eight traditional Chinese medicines including ginseng, wintergreen, and agrimony, have the effects of replenishing qi and promoting body fluid production, clearing heat and detoxifying, and are widely used in adjuvant cancer treatment. The accuracy of its quality control is directly related to its clinical efficacy and safety. However, current quality standards have significant technical bottlenecks:

[0003] The content determination method is one-sided: only ginsenoside Rg1 in ginseng is used as a quantitative indicator, but the amount of ginseng used in the prescription is only 1 / 3 of that of Rabdosia rubescens. As the most abundant active ingredient in the prescription, Rabdosia rubescens A was not measured, which makes it impossible to fully reflect the quality of the preparation.

[0004] Lack of identification methods: There are no dedicated identification methods for six Chinese medicinal herbs, namely Agrimonia pilosa, Prunus mume, Glycyrrhiza uralensis, Gynostemma pentaphyllum, and Ginseng. The existing standards rely only on thin-layer chromatography for Ginseng, which is difficult to achieve full-coverage quality control of all eight Chinese medicinal herbs and poses a risk of difficulty in distinguishing between genuine and counterfeit products and superior and inferior products.

[0005] Impurity interference: Compound preparations have complex components, and traditional extraction methods are prone to introducing impurities such as pigments and polysaccharides, which interfere with the accuracy of content determination results. Furthermore, the current impurity removal process has not been optimized for the characteristics of oridonin, affecting the detection sensitivity.

[0006] Inefficient quality control: The lack of a systematic testing system for the main active ingredients in the preparations makes it impossible to meet the requirements of the Chinese Pharmacopoeia for traditional Chinese medicines to be "safe, effective and of controllable quality", especially in terms of batch-to-batch stability evaluation and production process optimization.

[0007] To address the aforementioned issues, this invention establishes a high-performance liquid chromatography (HPLC) method for the determination of oridonin A content. Combined with a thin-layer chromatography (TLC) identification system for six traditional Chinese medicines, and through the optimization of composite impurity removal processes and specific detection conditions, a quality control technology covering the entire prescription is constructed, filling the gap in existing standards. Summary of the Invention

[0008] The purpose of this invention is to provide an HPLC method for determining the content of oridonin in Shenxianling granules, a traditional Chinese medicine preparation used for the treatment of digestive tract tumors, and a TLC method for identifying six Chinese medicinal components: Agrimonia pilosa, Prunus mume, Glycyrrhiza uralensis, Gynostemma pentaphyllum, and Ginseng. The HPLC method for determining the oridonin content in Shenxianling granules can be used for stability studies of the granules. This method is convenient to operate, highly specific, has good separation, high stability, and high efficiency, and can simultaneously determine seven components, thus achieving effective quality control of the product.

[0009] This invention provides the following technical solution:

[0010] A method for content determination and identification of Shenxianling granules includes determining the content of oridonin A in Shenxianling granules using high performance liquid chromatography (HPLC), and identifying six components—Agrimonia pilosa, Prunus mume, Glycyrrhiza uralensis, Gynostemma pentaphyllum, Panax ginseng, and Ganoderma lucidum—using thin-layer chromatography (TLC). The TLC identification method for each component includes unique preparation methods for the test solution, reference medicinal material solution, and negative control solution, as well as specific settings for the sample amount, developing solvent, and inspection conditions.

[0011] As a further technical solution, the chromatographic conditions for determining the content of oridonin by HPLC are as follows: Phyllomon C18 column (250mm×4.6μm, 5μm); mobile phase: methanol:water = 65:35; flow rate: 0.8-1.0ml / min; column temperature: 25-26℃; detection wavelength: 238nm; injection volume: 20μl.

[0012] As a further technical solution, the preparation and pretreatment method of the test solution is as follows: Grind the ginseng and ginseng granules into a fine powder, weigh 15-16g and place it in a Soxhlet extractor, add 100ml of petroleum ether, heat and reflux at 60-90℃ for 2.5h, discard the petroleum ether liquid, evaporate the petroleum ether, add 30ml of methanol and soak for 2-3h, sonicate for 20-30min, pour off the methanol extract, and repeat the same operation 5 times with 20ml of methanol. Combine the methanol extracts and evaporate to dryness. The residue is diluted to 50ml in a volumetric flask with a methanol:water = 65:35 solution, add 1.5-1.8g of compound impurity removal agent, shake for 15-20min, filter, and filter the filtrate through a 0.4μm microporous membrane to obtain the test solution.

[0013] The composite impurity removal agent is prepared by a cross-linking and calcination process of silica gel-alumina-activated carbon;

[0014] The preparation method of the composite impurity remover is as follows: silica gel (particle size 50-100μm), neutral alumina and activated carbon are mixed in a mass ratio of 3-5:2-3:1-2, 5-6% polyvinyl alcohol aqueous solution is added as a crosslinking agent, stirred at 60℃ into a paste, extruded into particles with a diameter of 1-2mm, calcined in a muffle furnace at 400-430℃ for 2-3h, cooled and passed through an 80-mesh sieve for later use;

[0015] The polyvinyl alcohol aqueous solution has a mass fraction of 20%.

[0016] As a further technical solution, the preparation method of the reference solution is as follows: accurately weigh 8 mg of oridonine standard, add 4 ml of methanol to dissolve and prepare an oridonine stock solution with a concentration of 2 mg / ml. When preparing the standard curve, dilute the oridonine stock solution with methanol to prepare reference solutions with concentrations of 100.00, 50.00, 25.00, 12.50, 6.25, 3.13, 1.56, and 0.78 μg / ml, respectively, and dispense into 1.5 ml centrifuge tubes for storage at 4°C for later use.

[0017] As a further technical solution, when using the TLC method to identify components of Agrimonia pilosa:

[0018] Preparation of test solution: Take 5g of Agrimonia pilosa, add 60ml of petroleum ether (60-90℃) and sonicate for 1h. Filter, evaporate the filtrate to dryness, dissolve the residue in 10ml of chloroform, extract with 10ml of 5% sodium hydroxide solution by shaking, discard the chloroform solution, adjust the pH of the sodium hydroxide solution to 1-2 with dilute hydrochloric acid, and extract with chloroform twice (10ml each time). Combine the chloroform solutions, wash with 10ml of water, discard the aqueous solution, and concentrate the chloroform solution to 1ml.

[0019] Control system: 5g of Agrimonia pilosa negative control preparation and 0.5g of Agrimonia pilosa reference material were prepared using the same method for negative control solution and reference material solution;

[0020] Spotting and Development: Take 10 μl each of the test solution, negative control solution and control drug solution and spot them on the same silica gel G thin layer plate. Develop the plate using the upper layer of petroleum ether (60-90℃)-ethyl acetate-acetic acid (100:9:5) as the developing solvent.

[0021] As a further technical solution, when using the TLC method to identify the components of dried plum:

[0022] Preparation of test solution: Take 15g of the powder, add 40ml of methanol and sonicate for 1h, filter, evaporate the filtrate to dryness, dissolve the residue in 40ml of water, add ether and shake to extract twice (20ml each time), combine the ether extracts and evaporate to dryness, soak the residue in petroleum ether (30-60℃) twice (20ml each time), discard the petroleum ether, dissolve the residue in 1ml of anhydrous ethanol;

[0023] Control system: 15g of the negative control preparation lacking dried plum and 1g of dried plum reference material were prepared using the same method for the negative control solution and the reference material solution;

[0024] Spotting and Development: Take 5 μl each of the test solution and negative control solution, and 10 μl of the reference drug solution, and spot them on the same silica gel G thin-layer plate. Develop with cyclohexane-chloroform-ethyl acetate-formic acid (20:5:8:0.1) as the developing solvent.

[0025] As a further technical solution, when using the TLC method to identify licorice components:

[0026] Preparation of test solution: Take 15g of the powder, add 60ml of ether and heat under reflux for 1h, filter, discard the ether solution, add 60ml of methanol to the residue and heat under reflux for 1h, filter, evaporate the filtrate to dryness, dissolve the residue in 40ml of water, extract with n-butanol 3 times (20ml each time), combine the n-butanol solutions, wash with water 3 times, discard the aqueous solution, evaporate the n-butanol solution to dryness, dissolve the residue in 1ml of methanol;

[0027] Control system: 15g of licorice-deficient negative control preparation and 1g of licorice reference material were prepared using the same method for negative control solution and reference material solution;

[0028] Spotting and development: Take 10 μl of each of the above solutions and spot them on the same silica gel G thin-layer plate. Develop with ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) as the developing solvent.

[0029] As a further technical solution, when using the TLC method to identify Gynostemma pentaphyllum components:

[0030] Preparation of test solution: Take 15g of the powder, add 60ml of methanol and sonicate for 1h, cool and filter, evaporate the filtrate to dryness, dissolve the residue in 20ml of water, extract 3 times with water-saturated n-butanol (20ml each time), combine the n-butanol solutions and evaporate to dryness, dissolve the residue in 1ml of methanol.

[0031] Control system: 15g of Gynostemma pentaphyllum negative control preparation and 1g of Gynostemma pentaphyllum reference material were prepared using the same method for negative control solution and reference material solution;

[0032] Spotting and Development: Take 5 μl of the test solution and negative control solution, and 10 μl of the reference drug solution, spot them on the same silica gel G thin layer plate, and develop with the upper layer solution of n-butanol-ethyl acetate-water (4:1:5) placed below 10℃ as the developing solvent.

[0033] As a further technical solution, when using the TLC method to identify ginseng components:

[0034] Preparation of test solution: Take 15g of the powder, add 100ml of chloroform and heat under reflux for 1h. Discard the chloroform solution, evaporate the solvent from the residue, add 1.0ml of water and stir to moisten, add 30ml of water-saturated n-butanol and sonicate for 1h. Take the supernatant, add 3 times the amount of ammonia test solution, shake well and let it separate into layers. Take the upper layer and evaporate to dryness. Dissolve the residue in 1ml of methanol.

[0035] Control system: 15g of ginseng-deficient negative control preparation and 1g of ginseng reference material were prepared using the same method for negative control solution and reference material solution;

[0036] Spotting and Development: Take 10 μl each of the test solution, negative control solution, and reference drug solution and spot them on the same silica gel G thin-layer plate. Develop the plate using the lower layer solution of chloroform-ethyl acetate-methanol-water (15:40:22:10) placed below 10°C as the developing solvent.

[0037] As a further technical solution, when identifying each component by TLC, the sample must be dried after development, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots are clearly visible, and examined under ultraviolet light (365nm); in the chromatogram of the test sample, fluorescent spots of the same color as those in the chromatogram of the reference medicinal material appear at the corresponding positions, while no spots appear at the corresponding positions in the negative control solution.

[0038] Compared with the prior art, the beneficial effects of the present invention are:

[0039] This invention achieves precise and comprehensive quality control of Shenxianling granules through multi-dimensional technological innovation, as detailed below:

[0040] I. Improved accuracy of HPLC content determination

[0041] (a) Separation mechanism of chromatographic condition optimization

[0042] Stationary phase selection: A Phenomen C18 column (250 mm × 4.6 μm, 5 μm) was used. Its octadecyl bonded phase forms a specific retention with oridonin A molecules through hydrophobic interaction. Combined with a methanol-water (65:35) mobile phase system, the resolution between the target peak and the impurity peak reached 2.8 (standard ≥1.5), effectively eliminating interference from the complex matrix.

[0043] (II) Mechanism of action of composite impurity removers

[0044] Multi-component synergistic purification: Silica gel (adsorbs pigments and sterols), neutral alumina (captures acidic impurities), and activated carbon (adsorbs polyphenols) are compounded in a specific mass ratio. Through a dual mechanism of physical adsorption and chemical action, interfering components are selectively removed. Silica gel has an adsorption capacity of up to 50 mg / g for polar impurities, while activated carbon achieves a removal rate of over 90% for phenolic acids that absorb ultraviolet light.

[0045] Structural optimization enhances efficiency: After cross-linking with polyvinyl alcohol and high-temperature calcination, the composite impurity remover forms a porous structure with uniform pore size distribution, which greatly increases the specific surface area and makes the impurity adsorption efficiency significantly higher than that of silica gel alone, while avoiding excessive adsorption of oridonin.

[0046] II. Breakthrough in the Specificity of TLC Identification System

[0047] (a) Targeted extraction for preparation of test sample solution

[0048] Polar gradient separation: A customized extraction process designed to address the differences in solubility of different components.

[0049] Agrimonia pilosa (non-polar components): After ultrasonic extraction with petroleum ether, phenolic acid components were enriched by NaOH-hydrochloric acid back-extraction, and the concentration of the target components was increased by 5 times after concentration with chloroform layer.

[0050] Licorice (polar saponins): After defatting with ether, it is extracted by reflux with methanol and then extracted with n-butanol to achieve a glycyrrhizic acid purity of over 95%, eliminating interference from fat-soluble impurities.

[0051] Acid-base environment control: For example, in the identification of dried plum, the concentration of ume acid in the test solution was increased to 2 mg / ml by stepwise defatting with ether-petroleum ether and dissolving with anhydrous ethanol, which is 2.5 times higher than the direct extraction method, and the clarity of spot color development was significantly enhanced.

[0052] (ii) Compatibility of developing solvent and inspection conditions

[0053] Selective separation mechanism:

[0054] Ginseng identification was performed using a chloroform-ethyl acetate-methanol-water (15:40:22:10) lower layer developing solvent. The polarity difference of ginsenoside Rg1 (logP=2.3) was utilized to separate it from other saponins by a distance of up to 8 cm, thus improving the spot recognition.

[0055] The identification of Gynostemma pentaphyllum was performed using the upper layer of n-butanol-ethyl acetate-water (4:1:5) as the developing solvent. By adjusting the proportion of the aqueous phase (50%), the Rf value of Gynostemma pentaphyllum saponins was controlled at 0.4-0.6, with no cross-interference with the negative control.

[0056] Enhanced fluorescence color development: After spraying with 10% sulfuric acid ethanol solution and heating at 105℃, the characteristic spots of each component show unique fluorescence under a 365nm ultraviolet lamp (e.g., glycyrrhizic acid shows yellow-green fluorescence), and the detection sensitivity reaches the μg level, which is significantly improved compared to visible light inspection.

[0057] This invention constructs a comprehensive quality control system for Shenxianling granules by combining "precise quantification of characteristic components + specific qualitative analysis of multiple medicinal ingredients + efficient removal of impurities." Specifically, HPLC achieves accurate quantification of oridonin, solving the problem of the original standard's one-sidedness; the TLC identification system, through dual optimization of extraction conditions and development system, ensures rapid identification of authenticity for the six traditional Chinese medicine ingredients; and the composite impurity removal technology improves the reliability of the detection data from the source. This approach upgrades the quality control of the preparation from "single-indicator monitoring" to "full-component coverage evaluation," not only meeting the latest requirements of the Chinese Pharmacopoeia for the quality standards of traditional Chinese medicine preparations, but also providing a referable technical paradigm for the quality improvement of similar compound preparations, possessing significant clinical application value and industrial promotion significance. Attached Figure Description

[0058] Figure 1 The graph shows the effect of calcination temperature on the polysaccharide removal rate of different composite adsorbents. Detailed Implementation

[0059] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0060] This invention provides a method for the determination and identification of the content of ginseng and celery extract granules, comprising the following steps:

[0061] High performance liquid chromatography (HPLC) was used to determine the content of oridonin in Shenxianling granules, and thin-layer chromatography (TLC) was used to identify six components: Agrimonia pilosa, Prunus mume, Glycyrrhiza uralensis, Gynostemma pentaphyllum, Ginseng, and Ganoderma lucidum. The TLC identification methods for each component included the preparation methods of the test solution, the reference medicinal material solution, and the negative control solution, as well as the specific settings of the spotting amount, developing solvent, and inspection conditions.

[0062] In this invention, the chromatographic conditions for determining the content of oridonin using the HPLC method are as follows: Phyllomon C18 column (250 mm × 4.6 μm, 5 μm); mobile phase: methanol:water = 65:35; flow rate: 0.8-1.0 ml / min; column temperature: 25-26℃; detection wavelength: 238 nm; injection volume: 20 μl.

[0063] In this invention, the preparation and pretreatment method of the test solution is as follows: Grind 15-16g of Shenxianling granules into a fine powder, place it in a Soxhlet extractor, add 100ml of petroleum ether, and extract by reflux at 60-90℃ for 2.5h. Discard the petroleum ether solution, evaporate the petroleum ether, add 30ml of methanol and soak for 2-3h, sonicate for 20-30min, pour off the methanol extract, and repeat the same operation 5 times with 20ml of methanol. Combine the methanol extracts and evaporate to dryness. The residue is diluted to a 50ml volumetric flask with a methanol:water = 65:35 solution, and 1.5-1.8g of compound impurity remover is added. Shake for 15-20 minutes, filter, and then filter the filtrate through a 0.4μm microporous membrane to obtain the test solution. The composite impurity remover is prepared by a cross-linking and calcination process of silica gel-alumina-activated carbon. The preparation method is as follows: silica gel (particle size 50-100μm), neutral alumina, and activated carbon are mixed in a mass ratio of 3-5:2-3:1-2, and 5-6% polyvinyl alcohol aqueous solution (mass fraction of 20%) is added as a cross-linking agent. The mixture is stirred into a paste at 60℃, extruded into particles with a diameter of 1-2mm, calcined in a muffle furnace at 400-430℃ for 2-3 hours, cooled, and then passed through an 80-mesh sieve for later use.

[0064] In this invention, the preparation method of the reference solution is as follows: accurately weigh 8 mg of oridonine standard, add 4 ml of methanol to dissolve it to prepare an oridonine stock solution with a concentration of 2 mg / ml. When preparing the standard curve, dilute the oridonine stock solution with methanol to prepare reference solutions with concentrations of 100.00, 50.00, 25.00, 12.50, 6.25, 3.13, 1.56, and 0.78 μg / ml, respectively, and dispense them into 1.5 ml centrifuge tubes for storage at 4°C for later use.

[0065] In this invention, when identifying the components of Agrimonia pilosa using the TLC method: the test solution is prepared by taking 5g of Agrimonia pilosa, adding 60ml of petroleum ether (60-90℃), sonicating for 1h, filtering, evaporating the filtrate to dryness, dissolving the residue in 10ml of chloroform, extracting with 10ml of 5% sodium hydroxide solution by shaking, discarding the chloroform solution, adjusting the pH of the sodium hydroxide solution to 1-2 with dilute hydrochloric acid, and then extracting twice with chloroform (10ml each time), combining the chloroform solutions. Wash with 10 ml of water, discard the aqueous solution, and concentrate the solution to 1 ml using chloroform. The control system consists of 5 g of Agrimonia pilosa negative preparation and 0.5 g of Agrimonia pilosa reference material. Prepare the negative control solution and reference material solution using the same method. For spotting and development, take 10 μl each of the test solution, negative control solution and reference material solution and spot them on the same silica gel G thin-layer plate. Develop with the upper layer of petroleum ether (60-90℃)-ethyl acetate-acetic acid (100:9:5) as the developing solvent.

[0066] In this invention, when identifying the components of dried plum by TLC: the test solution is prepared by taking 15g of the powder, adding 40ml of methanol, sonicating for 1h, filtering, evaporating the filtrate to dryness, dissolving the residue in 40ml of water, extracting twice with ether (20ml each time), combining the ether solutions and evaporating to dryness, soaking the residue twice with petroleum ether (30-60℃) (20ml each time), discarding the petroleum ether, and dissolving the residue in 1ml of anhydrous ethanol; the control system consists of 15g of a negative control preparation lacking dried plum and 1g of dried plum reference material, and preparing the negative control solution and reference material solution in the same way; the spotting and development are performed by taking 5μl each of the test solution and negative control solution and 10μl of the reference material solution and spotting them on the same silica gel G thin-layer plate, and developing with cyclohexane-chloroform-ethyl acetate-formic acid (20:5:8:0.1) as the developing solvent.

[0067] In this invention, when identifying licorice components by TLC: the test solution is prepared by taking 15g of the powder, adding 60ml of ether, heating and refluxing for 1h, filtering, discarding the ether, adding 60ml of methanol to the residue, heating and refluxing for 1h, filtering, evaporating the filtrate to dryness, dissolving the residue in 40ml of water, extracting three times with n-butanol (20ml each time), combining the n-butanol solutions, washing three times with water, discarding the aqueous solution, evaporating the n-butanol solution to dryness, and dissolving the residue in 1ml of methanol; the control system consists of 15g of licorice-deficient negative control preparation and 1g of licorice reference material, and preparing the negative control solution and reference material solution in the same way; the spotting and development are performed by taking 10μl of each of the above solutions and spotting them on the same silica gel G thin-layer plate, and developing with ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) as the developing solvent.

[0068] In this invention, when identifying Gynostemma pentaphyllum components by TLC: the test solution is prepared by taking 15g of the product powder, adding 60ml of methanol, sonicating for 1h, cooling and filtering, evaporating the filtrate to dryness, dissolving the residue in 20ml of water, extracting three times with water-saturated n-butanol (20ml each time), combining the n-butanol solutions, evaporating to dryness, and dissolving the residue in 1ml of methanol; the control system consists of 15g of Gynostemma pentaphyllum-deficient negative preparation and 1g of Gynostemma pentaphyllum reference material, and preparing the negative control solution and reference material solution in the same way; the spotting and development are performed by taking 5μl each of the test solution and negative control solution, and 10μl of the reference material solution, spotting them on the same silica gel G thin-layer plate, and developing with the upper layer solution of n-butanol-ethyl acetate-water (4:1:5) placed below 10℃ as the developing solvent.

[0069] In this invention, when identifying ginseng components by TLC: the test solution is prepared by taking 15g of the powder, adding 100ml of chloroform and heating under reflux for 1h, discarding the chloroform solution, evaporating the solvent from the residue, adding 1.0ml of water and stirring to moisten, adding 30ml of water-saturated n-butanol and sonicating for 1h, taking the supernatant, adding 3 times the amount of ammonia test solution, shaking well and allowing it to separate into layers, taking the upper layer and evaporating it to dryness, and dissolving the residue in 1ml of methanol; the control system consists of 15g of ginseng-deficient negative preparation and 1g of ginseng reference material, and preparing the negative control solution and reference material solution in the same way; the spotting and development are performed by taking 10μl each of the test solution, negative control solution, and reference material solution and spotting them on the same silica gel G thin-layer plate, and developing with the lower layer solution of chloroform-ethyl acetate-methanol-water (15:40:22:10) placed below 10℃ as the developing solvent.

[0070] In this invention, when identifying each component by TLC, the sample must be dried after development, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots are clearly visible, and examined under ultraviolet light (365nm); in the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material, while no spots appear at the corresponding positions in the negative control solution.

[0071] The materials and equipment used in this invention are all commercially available products in the field.

[0072] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments thereof.

[0073] Example 1

[0074] HPLC determination: Grind the ginseng and ginseng granules into a fine powder, weigh 15g and place it in a Soxhlet extractor. Add 100ml of petroleum ether and heat to reflux at 60℃ for 2.5h. Discard the petroleum ether solution, evaporate the extract, add 30ml of methanol and soak for 2h. Sonicate for 20min. Repeat the same operation 5 times with 20ml of methanol. Combine the methanol extracts and evaporate to dryness. Dilute the residue to a 50ml volumetric flask with a methanol:water = 65:35 solution, add 1.5g of compound impurity remover, shake for 15min, filter, and filter the filtrate through a 0.4μm microporous membrane to obtain the test solution. The concentration of oridonin A was determined to be 85μg / ml under HPLC chromatographic conditions.

[0075] The composite impurity remover is prepared by a cross-linking and calcination process of silica gel-alumina-activated carbon. The preparation method is as follows: silica gel (particle size 100μm), neutral alumina and activated carbon are mixed in a mass ratio of 3:2:1, and 5% polyvinyl alcohol aqueous solution (mass fraction of 20%) is added as a cross-linking agent. The mixture is stirred into a paste at 60°C, extruded into particles with a diameter of 1mm, calcined in a muffle furnace at 400°C for 2 hours, cooled and passed through an 80-mesh sieve for later use.

[0076] TLC identification of Agrimonia pilosa: Prepare the test solution, negative control solution and reference medicinal material solution according to the above-mentioned Agrimonia pilosa identification method. After spotting and development, the corresponding positions of the test sample chromatogram and the reference medicinal material chromatogram show the same blue-purple fluorescent spots, while the corresponding positions of the negative control solution show no spots.

[0077] Example 2

[0078] HPLC determination: Weigh 15.5g of Shenxianling granules, add 1.6g of compound impurity remover, and perform the remaining operations as in Example 1. The concentration of oridonin A was measured to be 88μg / ml.

[0079] The composite impurity remover is prepared by a cross-linking and calcination process of silica gel-alumina-activated carbon. The preparation method is as follows: silica gel (particle size 100μm), neutral alumina and activated carbon are mixed in a mass ratio of 3:2:1, and 5% polyvinyl alcohol aqueous solution (mass fraction of 20%) is added as a cross-linking agent. The mixture is stirred into a paste at 60°C, extruded into particles with a diameter of 1mm, calcined in a muffle furnace at 400°C for 2 hours, cooled and passed through an 80-mesh sieve for later use.

[0080] TLC identification of dried plum: Follow the above method for identifying dried plum. The chromatogram of the test sample and the chromatogram of the reference medicinal material show the same orange-yellow fluorescent spots at the corresponding positions, while the negative control solution shows no spots at the corresponding positions.

[0081] Example 3

[0082] HPLC determination: 16g of Shenxianling granules were weighed, 1.8g of compound impurity remover was added, and the mixture was sonicated for 30min. The remaining operations were the same as in Example 1. The concentration of oridonin was measured to be 90μg / ml.

[0083] The composite impurity remover is prepared by a cross-linking and calcination process of silica gel-alumina-activated carbon. The preparation method is as follows: silica gel (particle size 100μm), neutral alumina and activated carbon are mixed in a mass ratio of 3:2:1, and 5% polyvinyl alcohol aqueous solution (mass fraction of 20%) is added as a cross-linking agent. The mixture is stirred into a paste at 60°C, extruded into particles with a diameter of 1mm, calcined in a muffle furnace at 400°C for 2 hours, cooled and passed through an 80-mesh sieve for later use.

[0084] TLC identification of licorice: Follow the above-mentioned method for identifying licorice. The chromatogram of the test sample and the chromatogram of the reference medicinal material show the same yellow-green fluorescent spots at the corresponding positions, while the negative control solution shows no spots at the corresponding positions.

[0085] Example 4

[0086] HPLC determination: Weigh 15.2g of Shenxianling granules, add 1.7g of compound impurity remover, sonicate for 22min, and perform the remaining operations as in Example 1. The concentration of oridonin A was measured to be 87μg / ml.

[0087] The composite impurity remover is prepared by a cross-linking and calcination process of silica gel-alumina-activated carbon. The preparation method is as follows: silica gel (particle size 100μm), neutral alumina and activated carbon are mixed in a mass ratio of 3:2:1, and 5% polyvinyl alcohol aqueous solution (mass fraction of 20%) is added as a cross-linking agent. The mixture is stirred into a paste at 60°C, extruded into particles with a diameter of 1mm, calcined in a muffle furnace at 400°C for 2 hours, cooled and passed through an 80-mesh sieve for later use.

[0088] TLC identification of Gynostemma pentaphyllum: Follow the above-mentioned identification method for Gynostemma pentaphyllum. The chromatogram of the test sample and the chromatogram of the reference medicinal material show the same blue-green fluorescent spots at the corresponding positions, while the negative control solution shows no spots at the corresponding positions.

[0089] Example 5

[0090] HPLC determination: Weigh 15.8g of Shenxianling granules, add 1.7g of compound impurity remover, sonicate for 28min, and perform the remaining operations as in Example 1. The concentration of oridonin A was measured to be 89μg / ml.

[0091] The composite impurity remover is prepared by a cross-linking and calcination process of silica gel-alumina-activated carbon. The preparation method is as follows: silica gel (particle size 100μm), neutral alumina and activated carbon are mixed in a mass ratio of 3:2:1, and 5% polyvinyl alcohol aqueous solution (mass fraction of 20%) is added as a cross-linking agent. The mixture is stirred into a paste at 60°C, extruded into particles with a diameter of 1mm, calcined in a muffle furnace at 400°C for 2 hours, cooled and passed through an 80-mesh sieve for later use.

[0092] TLC identification of ginseng: Follow the above ginseng identification method. The chromatogram of the test sample and the chromatogram of the reference medicinal material show the same purple-red fluorescent spots at the corresponding positions, while the negative control solution shows no spots at the corresponding positions.

[0093] Comparative Example 1

[0094] Treatment method: No composite impurity remover was added; the remaining operations were the same as in Example 1.

[0095] Comparative Example 2

[0096] Treatment method: Ginseng was not available as a reference herb; the rest of the procedures were the same as in Example 5.

[0097] Experimental verification

[0098] HPLC Method Validation

[0099] Objective: To verify the linearity, precision, and recovery rate of oridonin A content determined by HPLC. Methods:

[0100] Linearity assessment: The reference solutions (concentrations of 0.78, 1.56, 3.13, 6.25, 12.50, 25.00, 50.00, and 100.00 μg / ml, respectively) were injected and determined under HPLC chromatographic conditions. A standard curve was plotted with peak area as the ordinate (Y) and concentration as the abscissa (X).

[0101] Precision test: Take the test solution from Example 2, inject it 6 times consecutively, and record the peak area of ​​oridonin A.

[0102] Recovery test: Take 10g of Shenxianling granules powder with known content (oridonin content of 88μg / g), make 6 portions, add 1ml of oridonin reference solution with a concentration of 100μg / ml to each portion, process according to the test solution preparation method, and determine and calculate the recovery rate.

[0103] Experimental data:

[0104] Table 1

[0105] Validate Project Test results linear relationship Standard curve equation: Y = 58234X + 1256, R² = 0.9998, linear range 0.78-100 μg / ml. Precision The peak areas were 85623, 86125, 85897, 86012, 85784, and 85998, respectively, with RSD=0.8% (n=6). Recovery rate of spiking Measured values ​​(μg / g): 175.2, 176.8, 174.9, 175.5, 176.1, 175.8, with an average recovery rate of 99.7% and an RSD of 0.6%.

[0106] TLC identification specificity verification

[0107] Objective: To verify the specificity of TLC in identifying components of Agrimonia pilosa, Prunus mume, and Glycyrrhiza uralensis, and to eliminate negative interference. Methods:

[0108] Identification and verification of Agrimonia pilosa: Take the test solution of Example 1, the negative control solution lacking Agrimonia pilosa, and the control herbal solution of Agrimonia pilosa, spot and develop according to TLC conditions, and observe the position of the spots under ultraviolet light.

[0109] Identification and verification of dried plum: Take the test solution of Example 2, the negative control solution lacking dried plum, and the control medicinal material solution of dried plum, spot and develop according to TLC conditions, and observe the color and position of the spots.

[0110] Licorice identification and verification: Take the test solution of Example 3, the negative control solution lacking licorice, and the licorice reference medicinal material solution, spot them under TLC conditions, and compare the chromatograms.

[0111] Experimental data:

[0112] Table 2

[0113] Element Test solution (example) Negative control solution results Results of the control medicinal material solution Agrimony Example 1 No fluorescent spots Blue-purple fluorescent spots Ume Example 2 No fluorescent spots Orange-yellow fluorescent spots licorice Example 3 No fluorescent spots Yellow-green fluorescent spots Gynostemma pentaphyllum Example 4 No fluorescent spots Showing blue-green fluorescent spots Ginseng Example 5 No fluorescent spots Purple-red fluorescent spots

[0114] Comparative Example 1: No composite impurity remover added

[0115] HPLC results: baseline noise was ±2 mAU, tailing factor of oridonin peak was 1.8 (standard ≤1.2), and resolution between impurity peak and target peak was 1.0 (standard ≥1.5).

[0116] TLC results: Five spots appeared in the chromatogram of Agrimonia pilosa (compared to only three spots in the control herb), while the negative control chromatogram showed two interfering spots at the corresponding positions.

[0117] Comparative Example 2: Control material lacking ginseng

[0118] TLC results: The characteristic spots of ginsenosides in the chromatogram of the test sample were missing and could not be distinguished from the negative control, resulting in identification failure.

[0119] HPLC method reliability: good linearity, precision and recovery rate both meet the requirements of the Chinese Pharmacopoeia (RSD≤2%, recovery rate 95%-105%), indicating that the method can accurately determine the content of oridonin.

[0120] TLC identification validity: The chromatograms of each component tested corresponded to the chromatograms of the reference medicinal material, and there was no negative interference, indicating that the TLC method has strong specificity and can be used for the qualitative identification of Shenxianling Granules.

[0121] Key process impact: The composite impurity removal agent is crucial for removing interference from impurities. Without a reference medicinal material, identification cannot be completed, which verifies the necessity of the formulation and process in the method of this invention.

[0122] Impurity Removal Effect Verification Test

[0123] Experimental Objective: To verify the removal ability of the composite impurity remover on interfering components such as polysaccharides and proteins, and the retention rate of oridonin. Experimental Methods:

[0124] Preparation of test solution: Take 15g of Ginseng and Astragalus membranaceus granules powder and prepare the test solution according to the method in Example 1. Divide into two groups:

[0125] Experimental group: 1.5g of compound impurity remover (Formula of Example 1) was added;

[0126] Control group: No compound impurity remover was added.

[0127] Indicator Testing:

[0128] Polysaccharide content: The absorbance was measured at a wavelength of 490 nm using the phenol-sulfuric acid method with glucose as a reference.

[0129] Protein content: The absorbance was measured at a wavelength of 595 nm using the Coomassie brilliant blue method with bovine serum albumin as a reference.

[0130] Oridonin retention rate: The concentration of oridonin before and after treatment was determined by HPLC, and the retention rate was calculated.

[0131] Experimental data:

[0132] Table 3

[0133] Group Polysaccharide removal rate (%) Protein removal rate (%) Oridonin A retention rate (%) experimental group 92.3±1.5 87.6±2.1 98.5±0.8 control group 12.7±3.2 15.4±4.3 99.2±0.5

[0134] Conclusion: The composite impurity remover showed a significantly higher removal rate of macromolecular impurities such as polysaccharides and proteins than the untreated group (p<0.01), and showed no significant adsorption loss of oridonin, proving that it can effectively retain target components while purifying samples and improving detection accuracy.

[0135] Figure 1 The curves showing the effect of different composite adsorbent calcination temperatures on their polysaccharide removal rates indicate that a high polysaccharide removal rate can be maintained within the calcination temperature range of 400-430°C.

[0136] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not describe all details exhaustively, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification.

Claims

1. A method for determining and identifying the content of ginseng and celery extract granules, characterized in that, This includes the determination of oridonin A in Shenxianling granules by high performance liquid chromatography (HPLC), and the identification of six components—Agrimonia pilosa, Prunus mume, Glycyrrhiza uralensis, Gynostemma pentaphyllum, Panax ginseng, and Ganoderma lucidum—by thin-layer chromatography (TLC). The TLC identification methods for each component include unique preparation methods for the test solution, the reference herb solution, and the negative control solution, as well as specific settings for the sample amount, developing solvent, and inspection conditions. The chromatographic conditions for determining the content of oridonin using the HPLC method were as follows: Phyllomon C18 column, 250 mm × 4.6 μm, 5 μm; mobile phase: methanol:water = 65:35; flow rate: 0.8-1.0 ml / min; column temperature: 25-26℃; detection wavelength: 238 nm; injection volume: 20 μl. The preparation and pretreatment method of the test solution is as follows: Grind the ginseng and ginseng granules into a fine powder, weigh 15-16g and place it in a Soxhlet extractor, add 100ml of petroleum ether, heat and reflux at 60-90℃ for 2.5h, discard the petroleum ether liquid, evaporate the petroleum ether, add 30ml of methanol and soak for 2-3h, sonicate for 20-30min, pour off the methanol extract, and repeat the same operation 5 times with 20ml of methanol. Combine the methanol extracts and evaporate to dryness. The residue is diluted to 50ml in a volumetric flask with a methanol:water = 65:35 solution, add 1.5-1.8g of compound impurity removal agent, shake for 15-20min, filter, and filter the filtrate through a 0.4μm microporous membrane to obtain the test solution. The composite impurity removal agent is prepared by a cross-linking and calcination process of silica gel-alumina-activated carbon; The preparation method of the composite impurity remover is as follows: 50-100μm silica gel, neutral alumina and activated carbon are mixed in a mass ratio of 3-5:2-3:1-2, 5-6% polyvinyl alcohol aqueous solution is added as a crosslinking agent, stirred at 60℃ into a paste, extruded into particles with a diameter of 1-2mm, calcined in a muffle furnace at 400-430℃ for 2-3h, cooled and passed through an 80-mesh sieve for later use; The polyvinyl alcohol aqueous solution has a mass fraction of 20%. When using the TLC method to identify components of Agrimonia pilosa: Preparation of test solution: Take 5g of Agrimonia pilosa, add 60ml of petroleum ether, sonicate at 60-90℃ for 1h, filter, evaporate the filtrate to dryness, dissolve the residue in 10ml of chloroform, extract with 10ml of 5% sodium hydroxide solution by shaking, discard the chloroform solution, adjust the pH of the sodium hydroxide solution to 1-2 with dilute hydrochloric acid, and extract with chloroform twice more by shaking, 10ml each time, combine the chloroform solutions, wash with 10ml of water, discard the aqueous solution, and concentrate the chloroform solution to 1ml; Control system: 5g of Agrimonia pilosa negative control preparation and 0.5g of Agrimonia pilosa reference material were prepared using the same method for negative control solution and reference material solution; Spotting and Development: Take 10 μl each of the test solution, negative control solution and reference drug solution and spot them on the same silica gel G thin-layer plate. Develop at 60-90℃ using the upper layer of petroleum ether-ethyl acetate-acetic acid solution (mass ratio 100:9:5) as the developing solvent.

2. The method of claim 1, wherein, The preparation method of the reference solution is as follows: Accurately weigh 8 mg of oridonine standard, add 4 ml of methanol to dissolve and prepare an oridonine stock solution with a concentration of 2 mg / ml. When preparing the standard curve, dilute the oridonine stock solution with methanol to prepare reference solutions with concentrations of 100.00, 50.00, 25.00, 12.50, 6.25, 3.13, 1.56 and 0.78 μg / ml respectively. Aliquot the solutions into 1.5 ml centrifuge tubes and store at 4℃ for later use.

3. The method of claim 1, wherein, When using the TLC method to identify components in dried plum: Preparation of test solution: Take 15g of the powder, add 40ml of methanol and sonicate for 1h, filter, evaporate the filtrate to dryness, dissolve the residue in 40ml of water, add ether and shake to extract twice, 20ml each time, combine the ether extracts and evaporate to dryness, soak the residue in petroleum ether at 30-60℃ twice, 20ml each time, desiccate the petroleum ether, dissolve the residue in 1ml of anhydrous ethanol; Control system: 15g of the negative control preparation lacking dried plum and 1g of dried plum reference material were prepared using the same method for the negative control solution and the reference material solution; Spotting and Development: Take 5 μl each of the test solution and negative control solution, and 10 μl of the reference herb solution, and spot them on the same silica gel G thin-layer plate. Develop the plate using cyclohexane-chloroform-ethyl acetate-formic acid in a mass ratio of 20:5:8:0.

1.

4. The method of claim 1, wherein, When using the TLC method to identify components in licorice: Preparation of test solution: Take 15g of the powder, add 60ml of ether and heat under reflux for 1h, filter, discard the ether solution, add 60ml of methanol to the residue and heat under reflux for 1h, filter, evaporate the filtrate to dryness, dissolve the residue in 40ml of water, extract with n-butanol 3 times, 20ml each time, combine the n-butanol solutions, wash with water 3 times, discard the aqueous solution, evaporate the n-butanol solution to dryness, dissolve the residue in 1ml of methanol; Control system: 15g of licorice-deficient negative control preparation and 1g of licorice reference material were prepared using the same method for negative control solution and reference material solution; Spotting and development: Take 10 μl of each of the above solutions and spot them on the same silica gel G thin-layer plate. Develop the plate using ethyl acetate-formic acid-glacial acetic acid-water in a mass ratio of 15:1:1:

2.

5. The method of claim 1, wherein, When using the TLC method to identify Gynostemma pentaphyllum components: Preparation of test solution: Take 15g of the powder, add 60ml of methanol and sonicate for 1h, cool and filter, evaporate the filtrate to dryness, dissolve the residue in 20ml of water, extract 3 times with 20ml of water-saturated n-butanol each time, combine the n-butanol solutions and evaporate to dryness, dissolve the residue in 1ml of methanol. Control system: 15g of Gynostemma pentaphyllum negative control preparation and 1g of Gynostemma pentaphyllum reference material were prepared using the same method for negative control solution and reference material solution; Spotting and Development: Take 5 μl each of the test solution and negative control solution, and 10 μl of the reference herb solution, spot them on the same silica gel G thin-layer plate, and develop the plate using the upper layer solution of n-butanol-ethyl acetate-water in a 4:1:5 mass ratio, which has been kept below 10°C as the developing solvent.

6. The method of claim 1, wherein, When using the TLC method to identify ginseng components: Preparation of test solution: Take 15g of the powder, add 100ml of chloroform and heat under reflux for 1h. Discard the chloroform solution, evaporate the solvent from the residue, add 1.0ml of water and stir to moisten, add 30ml of water-saturated n-butanol and sonicate for 1h. Take the supernatant, add 3 times the amount of ammonia test solution, shake well and let it separate into layers. Take the upper layer and evaporate to dryness. Dissolve the residue in 1ml of methanol. Control system: 15g of ginseng-deficient negative control preparation and 1g of ginseng reference material were prepared using the same method for negative control solution and reference material solution; Spotting and Development: Take 10 μl each of the test solution, negative control solution, and reference drug solution and spot them on the same silica gel G thin-layer plate. Develop the plate using the lower layer solution (15:40:22:10 by mass) of chloroform-ethyl acetate-methanol-water, which has been kept below 10°C as the developing solvent.

7. The method according to any one of claims 1 to 6, characterized in that, When identifying each component by the TLC method, the sample should be dried after development, sprayed with 10% sulfuric acid ethanol solution, heated at 105℃ until the spots are clearly visible, and examined under a 365nm ultraviolet lamp. In the chromatogram of the test sample, fluorescent spots of the same color as those in the chromatogram of the reference medicinal material should appear at the corresponding positions, while no spots should appear in the corresponding positions of the negative control solution.

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