Guangdong herbal tea particle detection method

Abstract

The invention discloses a detection method of Guangdong herbal tea granules. The detection method comprises a content detection method and an identification method. The content detection method comprises the following steps: preparing a reference substance solution, preparing a test solution, respectively and precisely sucking the reference substance solution and the test solution, injecting into a liquid chromatograph, and taking octadecylsilane chemically bonded silica as a filling agent; acetonitrile is used as a mobile phase A, a phosphoric acid aqueous solution is used as a mobile phase B, and gradient elution is performed. According to the identification method, the active ingredients of the finger citron, the cherokee rose root and the roughhaired holly root are respectively identified by thin-layer chromatography.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a method for detecting traditional Chinese medicine. Background Art

[0002] Guangdong Herbal Tea Granules are a traditional Chinese medicine (TCM) primarily made from herbs such as Rhizoma Citri Reticulatae, Sesamum indicum, Citrus aurantium, Glehnia littoralis, Oroxylum bungeana, Buza leaves, Firewood, Rhizoma Citri Reticulatae, Desmodium dactylum, and Cherry Blossom Root. They are known for clearing heat and relieving summer heat, removing dampness and promoting salivation, and are used to treat colds, fever, sore throats, damp-heat stagnation, dry mouth, and yellow urine. The existing technical standard for Guangdong Herbal Tea Granules, WS3-B-3548-98-2003-2012, does not include content determination, making the identification procedure complex and the spots unclear, making it difficult to accurately assess the drug's quality.

[0003] In the prior art, Sun Caiyun, Wang Zhifang, Su Xianjun, et al. Study on the chemical components of aromatic phenolic acids in Guangdong herbal tea granules [J]. Journal of Sun Yat-sen University (Natural Science Edition), 2014, 53(04): 88-93 disclosed that 7 compounds were isolated from the methanol extract of Guangdong herbal tea granules, and their structures were determined by spectral analysis and physical constant comparison methods: chlorogenic acid (1), trans-p-coumaric acid (2), caffeic acid (3), p-hydroxybenzoic acid (4), protocatechuic acid (5), protocatechuic acid methyl ester (6), and gallic acid (7).

[0004] In 2012, Deng Jiewei of the Guangdong Institute of Testing and Analysis reported in Analytical Methods on the use of direct mass spectrometry fingerprinting to assess the quality and traceability of Guangdong herbal tea granules. A literature search revealed that 4-hydroxybenzoic acid, a phenolic compound with multiple pharmacological effects, including anti-inflammatory and antibacterial properties, underlies the "heat-purging" effect of Guangdong herbal tea and is associated with the efficacy of this formula. It is a critical quality attribute, and 4-hydroxybenzoic acid is highly water-soluble. Therefore, it is imperative to develop methods for detecting 4-hydroxybenzoic acid in Guangdong herbal tea granules and improve identification methods. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a method for detecting the content of Guangdong herbal tea granules, comprising the following steps:

[0006] Step A: Preparation of Reference Solution: Take 4-hydroxybenzoic acid reference substance and add methanol solution to prepare a solution to obtain a reference solution;

[0007] Step B Preparation of Test Solution: Take Guangdong herbal tea granules, accurately weigh them, add extraction solvent, ultrasonically treat, filter, and take the filtrate to obtain the test solution;

[0008] Step C: Accurately draw the reference solution and the test solution separately, inject them into the liquid chromatograph, determine, and calculate the content. The chromatographic conditions of the liquid chromatograph are as follows: the chromatographic column is filled with octadecylsilane bonded silica gel; acetonitrile is the mobile phase A, and phosphoric acid aqueous solution is the mobile phase B. Gradient elution is performed according to the provisions in the following table.

[0009]

[0010] The raw materials for preparing the Guangdong herbal tea granules include galangal, sesame, five-fingered orange, light bamboo leaf, wood butterfly, cloth residue leaf, fire charcoal mother, golden sand vine, sedge grass, and golden cherry root.

[0011] Guangdong herbal tea granules can be prepared by the following method: decocting 1-2 times with water of galangal, sesame, citrus aurantium, bamboo leaves, oroxylum, buzia leaves, charcoal vine, golden sand vine, stagnation herb, and golden cherry root; filtering the decoction, combining the filtrates, concentrating, and centrifuging until the solution is clear; collecting the clear solution and concentrating it into a thick paste; adding an appropriate amount of sucrose powder or soluble starch to the thick paste, forming granules, and drying the resulting granules.

[0012] Preferably, the extraction solvent in step B is water or methanol, wherein the concentration of methanol can be 10%-100%, such as 10% methanol, 20% methanol, 50% methanol, methanol.

[0013] Preferably, in step A, preparation of the reference solution is performed by adding 50% methanol to a 4-hydroxybenzoic acid reference solution containing 10 μg per ml to obtain the reference solution.

[0014] Preferably, in step B, preparation of the test solution is to take 1-4 g of Guangdong herbal tea granules, accurately weigh them, accurately add 25-100 ml of methanol, and ultrasonically treat them for 15-60 min (e.g., 15, 30, 45, 60 minutes). Take it out, let it cool to room temperature, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0015] Preferably, the column temperature in the chromatographic conditions of step C is 20-40°C, such as 30°C, 28°C, or 32°C.

[0016] Preferably, the chromatographic condition flow rate in step C is 0.5-1.5 mL / min, such as 1 mL / min, 0.9 mL / min, 1.1 mL / min.

[0017] Preferably, the chromatographic detection wavelength in step C is 240-260 nm, such as 254 nm, 250 nm, 258 nm

[0018] Preferably, the chromatographic conditions in step C are 0.05-0.3% phosphoric acid aqueous solution as mobile phase B, such as 0.1% phosphoric acid aqueous solution, 0.05% phosphoric acid aqueous solution, 0.2% phosphoric acid aqueous solution

[0019] Another object of the present invention is to provide a method for identifying Guangdong herbal tea granules, using thin layer chromatography to identify the active ingredients of five-fingered orange, golden cherry root, and cyperus rotundus, including

[0020] S1 identification of five-finger orange

[0021] Take 2-20g of Guangdong herbal tea granules, add 50ml of ethanol, heat under reflux in a water bath for 2 hours, filter, evaporate the filtrate to dryness, add 50ml of water to the residue, stir to dissolve, and shake and extract with ethyl acetate twice, each time 20ml, combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 1ml of methanol to prepare the S1 test solution;

[0022] Separately, 5 g of the control medicinal material of Citrus aurantium was taken, 50 ml of water was added, and the mixture was heated under reflux for 2 hours, cooled, and filtered. Ethanol was added to the filtrate to make the alcohol content reach 45%, and the mixture was allowed to stand for 2 hours, filtered, and the filtrate was evaporated to dryness. 1 ml of methanol was added to the residue to dissolve it, and this was used as the S1 control medicinal material solution. According to the thin layer chromatography test, 5 μl of each of the S1 test solution and the S1 control medicinal material solution were respectively spotted on the same silica gel G thin layer plate, and petroleum ether-ethyl acetate with a volume ratio of 5:5 was used as the developing solvent. The humidity was below 70%. The plate was removed, dried, and sprayed with 1% ferric chloride methanol solution until a clear color was developed. The plate was examined under sunlight. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions in the chromatogram of the control medicinal material.

[0023] S2 Golden Sakura Root Identification

[0024] Take 2-20g of Guangdong herbal tea granules, add 100ml of water to dissolve, shake and extract with ethyl acetate three times, each time 50ml, combine the ethyl acetate extracts, wash twice with water, each time 50ml, evaporate the ethyl acetate solution to dryness, and add 1ml of methanol to dissolve the residue as the S2 test solution;

[0025] Take 2.5g of golden cherry root reference medicinal material, add 50ml of water, heat and reflux for 40 minutes, let cool, centrifuge, take the supernatant, shake and extract with ethyl acetate 3 times, 50ml each time, combine the ethyl acetate extracts, wash with water 2 times, 50ml each time, evaporate the ethyl acetate solution to dryness, add 1ml of methanol to dissolve the residue to prepare S2 reference medicinal material solution, test according to thin layer chromatography, take 4μl of S2 reference medicinal material solution and 10μl of S2 test sample solution, respectively, and spot them on the same silica gel G thin layer plate to make 1cm strips. Develop twice with the upper layer solution of n-hexane-ethyl acetate-acetone-formic acid-water in the volume ratio of 3:4:3:0.3:1 as the developing solvent, or develop once with the lower layer solution of chloroform-ethyl acetate-methanol-water in the volume ratio of 20:20:40:10, which was separated below 10°C. Take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and examine under sunlight. The test sample chromatogram and the control medicinal material chromatogram under sunlight show the same blue-purple spots at the corresponding positions.

[0026] S3 identification of sorghum

[0027] Take 1-10g of Guangdong herbal tea granules, add 30ml of water to dissolve, extract twice with water-saturated n-butanol solution, 50ml each time, combine the water-saturated n-butanol extracts, add ammonia water to extract three times, 20ml each time, retain the water-saturated n-butanol solution, wash three times with n-butanol-saturated water, 20ml each time, evaporate the water-saturated n-butanol solution to dryness, add 1ml of methanol to dissolve the residue, and use it as the S3 test solution;

[0028] Take another 3g of the control medicinal material of Rhizoma Cibotii, add 50ml of water, heat and reflux for 1 hour, filter, extract twice with a saturated n-butanol solution with water, 50ml each time, combine the water-saturated n-butanol extracts, extract three times with ammonia water, 20ml each time, retain the water-saturated n-butanol solution, wash three times with n-butanol-saturated water, 20ml each time, evaporate the water-saturated n-butanol solution to dryness, add 1ml of methanol to dissolve the residue to prepare S3 control medicinal material solution, test according to the thin layer chromatography method, take 1μl of the S3 control medicinal material solution and 4μl of the S3 test sample solution, respectively, spot them on the same silica gel G thin layer plate, use ethyl acetate-methanol-glacial acetic acid-water with a volume ratio of 11:1.5:1.5:2 as the developing solvent, develop, take out, dry, spray with an appropriate amount of 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and examine under 365nm ultraviolet light. In the chromatogram of the test sample, at the corresponding position of the chromatogram of the control medicinal material, a fluorescent spot of the same color appears;

[0029] The third object of the present invention is to provide a method for detecting Guangdong herbal tea granules, characterized in that thin layer chromatography is used to identify the effective ingredients of five-fingered orange, golden cherry root, and cyperus rotundus, and liquid chromatography is used to detect the 4-hydroxybenzoic acid content of Guangdong herbal tea granules, including

[0030] Identification method

[0031] S1 identification of five-finger orange

[0032] Take 2-20g of Guangdong herbal tea granules, add 50ml of ethanol, heat under reflux in a water bath for 2 hours, filter, evaporate the filtrate to dryness, add 50ml of water to the residue, stir to dissolve, and shake and extract with ethyl acetate twice, each time 20ml, combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 1ml of methanol to prepare the S1 test solution;

[0033] Separately, 5 g of the control medicinal material of Citrus aurantium was taken, 50 ml of water was added, and the mixture was heated under reflux for 2 hours, cooled, and filtered. Ethanol was added to the filtrate to make the alcohol content reach 45%, and the mixture was allowed to stand for 2 hours, filtered, and the filtrate was evaporated to dryness. 1 ml of methanol was added to the residue to dissolve it, and this was used as the S1 control medicinal material solution. According to the thin layer chromatography test, 5 μl of each of the S1 test solution and the S1 control medicinal material solution were respectively spotted on the same silica gel G thin layer plate, and petroleum ether-ethyl acetate with a volume ratio of 5:5 was used as the developing solvent. The humidity was below 70%. The plate was removed, dried, and sprayed with 1% ferric chloride methanol solution until a clear color was developed. The plate was examined under sunlight. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions in the chromatogram of the control medicinal material.

[0034] S2 Golden Sakura Root Identification

[0035] Take 2-20g of Guangdong herbal tea granules, add 100ml of water to dissolve, shake and extract with ethyl acetate three times, each time 50ml, combine the ethyl acetate extracts, wash twice with water, each time 50ml, evaporate the ethyl acetate solution to dryness, and add 1ml of methanol to dissolve the residue as the S2 test solution;

[0036] Take 2.5g of golden cherry root reference medicinal material, add 50ml of water, heat and reflux for 40 minutes, let cool, centrifuge, take the supernatant, shake and extract with ethyl acetate 3 times, 50ml each time, combine the ethyl acetate extracts, wash with water 2 times, 50ml each time, evaporate the ethyl acetate solution to dryness, add 1ml of methanol to dissolve the residue to prepare S2 reference medicinal material solution, test according to thin layer chromatography, take 4μl of S2 reference medicinal material solution and 10μl of S2 test solution, respectively, and spot them on the same silica gel G thin layer plate to make 1cm strips. The upper layer solution of n-hexane-ethyl acetate-acetone-formic acid-water with a volume ratio of 3:4:3:0.3:1 was used as the developing solvent for secondary development; the conditions for the primary development were chloroform-ethyl acetate-methanol-water with a volume ratio of 20:20:40:10. The lower layer solution that was layered below 10°C was taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and examined under sunlight. The test sample chromatogram and the control medicinal material chromatogram under sunlight showed the same blue-purple spots at the corresponding positions.

[0037] S3 identification of sorghum

[0038] Take 2-20g of Guangdong herbal tea granules, add 30ml of water to dissolve, extract twice with water-saturated n-butanol solution, 50ml each time, combine the water-saturated n-butanol extracts, add ammonia water to extract three times, 20ml each time, retain the water-saturated n-butanol solution, wash three times with n-butanol-saturated water, 20ml each time, evaporate the water-saturated n-butanol solution to dryness, add 1ml of methanol to dissolve the residue, and use it as the S3 test solution;

[0039] Take another 3g of the control medicinal material of Rhizoma Cibotii, add 50ml of water, heat and reflux for 1 hour, filter, extract twice with a saturated n-butanol solution with water, 50ml each time, combine the water-saturated n-butanol extracts, extract three times with ammonia water, 20ml each time, retain the water-saturated n-butanol solution, wash three times with n-butanol-saturated water, 20ml each time, evaporate the water-saturated n-butanol solution to dryness, add 1ml of methanol to dissolve the residue to prepare S3 control medicinal material solution, test according to the thin layer chromatography method, take 1μl of the S3 control medicinal material solution and 4μl of the S3 test sample solution, respectively, spot them on the same silica gel G thin layer plate, use ethyl acetate-methanol-glacial acetic acid-water with a volume ratio of 11:1.5:1.5:2 as the developing solvent, develop, take out, dry, spray with an appropriate amount of 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and examine under 365nm ultraviolet light. In the chromatogram of the test sample, at the corresponding position of the chromatogram of the control medicinal material, a fluorescent spot of the same color appears;

[0040] The content detection method comprises the following steps

[0041] Step A: Preparation of Reference Solution: Take 4-hydroxybenzoic acid reference substance and add methanol solution to prepare a solution to obtain a reference solution;

[0042] Step B Preparation of Test Solution: Take Guangdong herbal tea granules, accurately weigh them, add methanol, ultrasonicate them, filter them, and take the filtrate to obtain the test solution;

[0043] Step C: Accurately draw the reference solution and the test solution separately, inject them into the liquid chromatograph, determine, and calculate the content. The chromatographic conditions of the liquid chromatograph are as follows: the chromatographic column is filled with octadecylsilane bonded silica gel; acetonitrile is the mobile phase A, and phosphoric acid aqueous solution is the mobile phase B. Gradient elution is performed according to the provisions in the following table.

[0044]

[0045] The present invention is obtained through a large number of screenings. First, the screening research of the content determination method of Guangdong herbal tea granules

[0046] 1. Instruments and test drugs

[0047] Instruments: Agilent 1260-VWD, Agilent 1260-DAD high-performance liquid chromatographs; Sartorius BCE224I-1CCN electronic balance, Sartorius BCE223I-1CCN electronic balance, Sartorius MSA6.6S-OCE-DM electronic balance.

[0048] Reagents and reagents: Gradient methanol, phosphoric acid, acetonitrile of chromatographic grade, methanol of analytical grade, distilled water, and Watsons purified water. Sample: Guangdong herbal tea granules were provided by Guangzhou Wanglaoji Pharmaceutical Co., Ltd. (see Table 1 below).

[0049] Reference substance: 4-hydroxybenzoic acid, China Food and Drug Inspection Institute, batch number: 101149-202204, content 100%.

[0050] Table 1 Information of Guangdong herbal tea granules

[0051]

[0052] 2. Chromatographic conditions and system suitability test

[0053] Chromatographic column: Filled with octadecylsilane bonded silica gel; mobile phase A: acetonitrile, mobile phase B: 0.1% aqueous phosphoric acid; gradient elution as specified in the table below; mobile phase: acetonitrile-0.1% aqueous phosphoric acid; detection wavelength: 254 nm. The theoretical plate number calculated based on the 4-hydroxybenzoic acid peak should be no less than 3000.

[0054]

[0055] 3. Preparation of Reference Solution

[0056] Take an appropriate amount of 4-hydroxybenzoic acid reference substance and add 50% methanol to prepare a solution containing 10 μg per ml.

[0057] 4. Preparation of Test Solution

[0058] Take 10 bags of Guangdong herbal tea granules, remove the packaging, and mix the contents. Accurately weigh approximately 2.5 g of the contents and place them in a 100 ml conical flask. Accurately add 50 ml of methanol and sonicate for 30 minutes. Remove the flask, allow it to cool to room temperature, and make up the lost weight with methanol. Shake well, filter, and collect the filtrate to obtain the product.

[0059] 5. Selection of chromatographic conditions

[0060] 5.1 Detection method

[0061] In the Guangdong herbal tea granules, the dosage of wuzhigan (Schisandra chinensis) accounts for 8% of the total formula. Wuzhigan has antipyretic and heat-clearing properties. Literature search revealed that 4-hydroxybenzoic acid (4-hydroxybenzoic acid) in wuzhigan is a phenolic compound with multiple pharmacological effects, including anti-inflammatory and antibacterial properties. It is the material basis for the "heat-purging" effect of Guangdong herbal tea and is relevant to the efficacy of this formula. Furthermore, 4-hydroxybenzoic acid is highly water-soluble and specific for wuzhigan, so it was selected as an indicator component for detection of wuzhigan. HPLC was used to determine the content of 4-hydroxybenzoic acid, the main component in wuzhigan.

[0062] 5.2 Exploration of analytical methods

[0063] A. Selection of chromatographic conditions

[0064] Through continuous trial and error, the mobile phase was selected as an acetonitrile-0.1% phosphoric acid solution system, and finally an Agilent Eclipse XDB-C18 4.6*250mm, 5μm chromatographic column was selected, with acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B. Gradient elution was performed as specified in the table below; the detection wavelength was 254nm, and the column temperature was 30°C.

[0065]

[0066] B. Determination of detection wavelength: Take a 4-hydroxybenzoic acid reference solution and perform ultraviolet absorption spectrum scanning between 190 and 400 nm. The results show that the maximum absorption wavelength of 4-hydroxybenzoic acid is at 254 nm. Therefore, 254 nm is selected as the wavelength for determining the content of 4-hydroxybenzoic acid in five-fingered orange.

[0067] 6. Preparation and inspection of test samples

[0068] 6.1 Examination of Extraction Solvents: The Guangdong Herbal Tea Granules extract and excipients are all water-soluble, so ultrasonic extraction was selected. Ten portions (approximately 2.5 g each) of Guangdong Herbal Tea Granules batch 2308207 were accurately weighed and placed in a conical flask. 50 ml each of 10% methanol, 20% methanol, 50% methanol, methanol, and water were accurately added and weighed. Ultrasonic extraction was performed for 30 minutes. The mixture was cooled and weighed again. The weight loss was made up with the respective solvents. The mixture was shaken well, filtered, and the filtrate was collected. Results are shown in Table 2.

[0069] Table 2 Results of investigation on solvents for extracting Guangdong herbal tea granules

[0070]

[0071] Experimental results: The content of 4-hydroxybenzoic acid was not much different in ultrasonic extraction with different solvents. Methanol was selected as the extraction solvent because it could reduce the dissolution of excipients in the test solution and protect the chromatographic column.

[0072] 6.2 Investigation of extraction time

[0073] Ten portions of Guangdong herbal tea granules from batch 2308207, approximately 2.5 g each, were accurately weighed and placed in a conical flask. 50 ml of methanol was accurately added and weighed. Ultrasonic extraction was performed for 15, 30, 45, and 60 minutes, respectively. The extract was allowed to cool and weighed again. The remaining weight was supplemented with methanol, shaken well, filtered, and the filtrate was collected. The results are shown in Table 3.

[0074] Table 3 Results of the investigation on the extraction time of Guangdong herbal tea granules

[0075]

[0076] Test results: Ultrasonic extraction can be completed in 30 minutes, so the extraction time is selected as 30 minutes.

[0077] Conclusion: The test sample was prepared by removing the packaging from 10 bags of Guangdong herbal tea granules and mixing the contents. Approximately 2.5 g of the contents was accurately weighed and placed in a 100 ml conical flask. 50 ml of methanol was accurately added and ultrasonicated for 30 min (500 W). The sample was removed and allowed to cool to room temperature. The weight loss was made up with methanol, shaken, and filtered through a 0.45 μm microporous membrane. The filtrate was obtained.

[0078] 2. Thin layer identification and screening research

[0079] 1. Identification of five-finger orange

[0080] 1.1 Instruments and Reagents

[0081] SARTORIUSBCE224I-1CCN electronic balance TP056; PTHW 2000ML heating mantle, Tianjin Kono Instrument Equipment Co., Ltd.; HHS-21-6 electric constant temperature water bath, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory; GoodSee-20E thin-layer chromatography imaging system.

[0082] Silica gel G thin layer plates were purchased from Yantai Huayang New Material Technology Co., Ltd. Petroleum ether, ethyl acetate, methanol, and ethanol were purchased from Tianjin Kangkede Technology Co., Ltd.

[0083] The control medicinal material of wuzhigan (batch number: 121496-201502) was purchased from the China Food and Drug Inspection Institute; the medicinal material of wuzhigan (batch number B230403) and Guangdong herbal tea granules (batch number 2308207) were both provided by Guangzhou Wanglaoji Pharmaceutical Co., Ltd.

[0084] 1.2 Preparation of test sample solution

[0085] Preparation of test solution: Take the contents of 2 bags of Guangdong Herbal Tea Granules (batch number 2308207), add 50 ml of ethanol, heat under reflux in a water bath for 2 hours, filter, evaporate the filtrate to dryness, add 50 ml of water to the residue, stir to dissolve, and extract with ethyl acetate by shaking twice, 20 ml each time, combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 1 ml of methanol as the test solution.

[0086] Preparation of control medicinal material solution: Take another 5g of five-fingered orange control medicinal material, add 50ml of water, heat and reflux for 2 hours, cool, filter, add ethanol to the filtrate to make the alcohol content reach 45%, let it stand for 2 hours, filter, evaporate the filtrate to dryness, add 1ml of methanol to dissolve the residue, and use it as the control medicinal material solution.

[0087] Preparation of negative test solution: Weigh the medicinal materials of Quwuzhigan according to the prescription ratio, prepare it according to the preparation process of Guangdong herbal tea granules, and prepare the negative solution according to the test sample.

[0088] 1.3 Expand the system

[0089] Developing solvent: petroleum ether (60-90°C)-ethyl acetate (5:5); sample volume: 5 μL; bandwidth: 8 mm; development distance: approximately 9 cm;

[0090] Develop: Pipette the control medicinal material solution, test sample solution, and negative solution in turn, and spot them on the same silica gel G thin layer plate according to the thin layer chromatography test. Use petroleum ether-ethyl acetate (5:5) as the developing solvent, develop, remove, dry, spray with 1% ferric chloride methanol solution until clear color is developed, and inspect under sunlight.

[0091] 1.4 Study on sample preparation methods

[0092] 1.4.1 Investigation of extraction solvent

[0093] The experiment investigated the effects of three extraction solvents (ethyl acetate, chloroform, and ethanol) on the test solution. The test solution was prepared as follows: the contents of two bags of this product (equivalent to 0.5g of five-fingered orange medicinal materials) were placed in a stoppered conical flask. 50ml of ethanol, ethyl acetate, and chloroform were added respectively. The solution was heated to reflux in a water bath for 2 hours, filtered, and the filtrate was evaporated to dryness. 50ml of water was added to the residue and stirred to dissolve. The solution was extracted twice with ethyl acetate by shaking, each time using 20ml. The ethyl acetate extracts were combined and evaporated to dryness. The residue was dissolved in 1ml of methanol to prepare the test solution.

[0094] Click on the system expansion under 1.3 to view the result. Figure 1 A.

[0095] Results: When ethanol was used as the extraction solvent, the main spot of the quinquefolia could be extracted, and other spots on the chromatogram had less interference, so it could be used as the solvent for the enrichment of the quinquefolia.

[0096] 1.4.2 Investigation of test sample preparation methods

[0097] Take 5g of this product (equivalent to 0.5g of five-fingered orange medicinal material), place it in a stoppered conical flask, add 50ml of acetone and 50ml of methanol respectively, treat with ultrasound for 30min, filter, evaporate the filtrate to dryness, and add 1ml of methanol to dissolve the residue;

[0098] Take the contents of 2 bags of this product (equivalent to 1.6g of five-fingered orange medicinal materials), crush them, place them in a stoppered conical flask, add 50ml of ethanol, ultrasonicate for 30min, filter, evaporate the filtrate to dryness, and dissolve the residue in 1ml of methanol;

[0099] Take the contents of 2 bags of this product (equivalent to 1.6g of five-fingered orange medicinal materials), crush them, place them in a stoppered conical flask, add 50ml of ethanol, heat and reflux in a water bath for 2 hours, filter, evaporate the filtrate to dryness, add 50ml of water to the residue, stir to dissolve, add to a methanol-activated solid phase extraction column, and elute with water, 40% methanol, and methanol solution in sequence. Collect the eluents, evaporate to dryness, and dissolve the residue in 1ml of methanol.

[0100] Take the contents of 2 bags of Guangdong Herbal Tea Granules (Batch No. 2308207), add 50 ml of ethanol, heat under reflux in a water bath for 2 hours, filter, evaporate the filtrate to dryness, add 50 ml of water to the residue, stir to dissolve, shake and extract with ethyl acetate twice, 20 ml each time, combine the ethyl acetate extracts, evaporate to dryness, add 1 ml of methanol to dissolve the residue, prepare the control herbal solution and the test solution according to 1.2, expand the system according to 1.3, and inspect. Figure 1 A~D.

[0101] Conclusion: The spots after ethanol reflux and ethyl acetate extraction are clearer and the operation is simple. The treatment method of the test sample remains unchanged, that is, ethanol reflux and ethyl acetate extraction.

[0102] 1.5 Investigation of the Chromatographic System

[0103] 1.5.1 Investigation of sample quantity

[0104] Investigate the effect of different sample volumes (2, 5, 10, 15 μL) on the thin layer chromatography system. Prepare the control herbal medicine and test solution according to 1.2, and expand the system according to 1.3 for inspection. Results Figure 1 E.

[0105] Conclusion: When the sample volume is 5 μL, the spots are clear under sunlight, so the sample volume is selected as 5 μL.

[0106] 1.5.2 Investigation of sampling bandwidth

[0107] Investigate the effect of bandwidth (point, 4, 6, 8, 10 mm) on the thin layer chromatography system. Prepare the control herbal solution and the test solution according to 1.2, and expand the system according to 1.3 and inspect. Figure 1 F.

[0108] Under daylight, the chromatograms of all solutions were similar. At a strip width of 8 mm, the spots were clear and well separated from each other, so a strip width of 8 mm was chosen.

[0109] 1.5.3 Investigation of the developing agent Investigate the developing system and its impact on the thin layer chromatography system.

[0110] Take the five-finger orange medicinal material solution, the test solution and the negative test solution, and develop them according to the development system and check. Figure 2 .

[0111] Conclusion: When using development system 11, the spots are clear, the Rf is moderate, and the separation is good; when using other development systems, the spot separation is poor. Development system 1 was selected as the thin layer development system in this experiment.

[0112] 1.6 Thin layer method confirmation

[0113] 1.6.1 Specificity

[0114] Prepare the control medicinal material solution and the test sample solution according to 1.2, and carry out the system development and inspection according to 1.3. Figure 3 .

[0115] The test sample shows spots of the same color at the corresponding positions of the five-fingered orange medicinal materials, while the negative sample does not have such spots. The specificity of this method is good.

[0116] 1.6.2 Solution stability study

[0117] Prepare the test solution according to 1.2, and spot the sample at 0, 1, 2, and 4 days after sample preparation. Evaluate the stability of the reference solution, test solution, and negative test solution: Carry out the system development and inspection according to 1.3. Figure 4 .

[0118] Conclusion: Based on observation under UV and visible light, the spots were clear and had moderate Rf values ​​when the samples were spotted 0, 1, and 4 days after preparation, indicating that the test solution and sample solution were stable. Spot separation was poor on the second day due to humidity.

[0119] 1.6.3 Durability test

[0120] (1) Different thin layer plates: Take the control medicinal material solution, test solution, and negative test solution and use silica gel G thin layer plates (Yantai Chemical, Merck, Germany) respectively, develop them according to the development system, and inspect them. Figure 5 .

[0121] Conclusion: When using the silica gel G thin layer plate produced by Merck, the spots of the five-fingered orange were lighter and the RF was smaller; when using the silica gel G thin layer plate produced by Yantai Chemical Factory, the main spots could be better separated, the spots were clear, and the RF was moderate, indicating that the thin layer plate had an impact on this thin layer chromatography method.

[0122] (2) Different temperatures: Take the control medicinal material solution and the test sample solution, and use the development temperature of room temperature, 4, and 35 ° C respectively, develop according to the development system, and check. Figure 6 .

[0123] Conclusion: When developed at room temperature, 4, and 35°C, the sample solution showed clear spots with moderate Rf. Therefore, temperature had little effect on this thin layer chromatography method.

[0124] (3) Different humidity: Take the control medicinal material solution and the test sample solution, saturate the thin layer plate at relative humidity of RH32%, 47%, 71%, and 88% for 10 minutes, unfold it according to the unfolding system, and inspect it. Figure 7 .

[0125] Conclusion: When developing at RH32%, 47%, and 71%, the reference solution, test solution, and negative test solution had clear spots and moderate Rf. When developing at RH88%, the test solution spots were poorly separated. Therefore, humidity has an impact on this thin layer chromatography method and the environmental humidity needs to be controlled.

[0126] 2. Identification of Prunus mume

[0127] 2.1 Instruments and reagents

[0128] HHS-21-6 electric constant temperature water bath, Shanghai Boxun Instrument Co., Ltd.; HB10 digital rotary evaporator, IKA; CAMAG double-slot developing cylinder, Karma, Switzerland; HP550-S 7-inch square plate standard heating plate; 2F-20D darkbox UV analyzer, Tianjin Kono; GoodSee-20E thin-layer chromatography imaging system, Shanghai Kezhe Biochemical Technology Co., Ltd.

[0129] Silica gel G thin layer plates were purchased from Yantai Chemical; silica gel G thin layer plates were purchased from Qingdao Ocean; high-efficiency G thin layer plates were purchased from Qingdao Ocean; and silica gel G thin layer plates were purchased from Merck, Germany. Ethyl acetate, methanol, ethanol, and glacial acetic acid were purchased from Tianjin Concord Technology Co., Ltd.; n-butanol, ammonia water, and sulfuric acid were purchased from Tianjin Fengchuan Chemical Reagent Technology Co., Ltd.; and water was purchased from Guangzhou Watsons Food and Beverage Co., Ltd.

[0130] The control medicinal materials of angelica sinensis (batch number: 121152-202205) were purchased from the China Food and Drug Administration; the medicinal materials of golden cherry root (batch number B240101), angelica sinensis (batch number B230501), sesame (batch number B230901), five-finger orange (batch number B230403), light bamboo leaf (batch number B231103), oroxylum butterfly (batch number B240101), and buza leaf (batch number B2 30403), Huotanmu medicinal materials (batch number B230501), Jinshateng medicinal materials (batch number B230501), Qianqiancao medicinal materials (batch number B230501), Guangdong herbal tea granules sucrose type (batch numbers 2308207, 2309202, 2308215), Guangdong herbal tea granules sugar-free type (batch numbers 2312007, 2312002, 2310007) were all provided by Guangzhou Wanglaoji Pharmaceutical Co., Ltd.

[0131] 2.2 Preparation of test sample solution

[0132] Preparation of the test solution: Dissolve the contents of one bag of this product in 30 ml of water. Extract twice with saturated n-butanol (50 ml each time). Combine the water-saturated n-butanol extracts and extract three times with aqueous ammonia, letting stand for 10 minutes, 20 ml each time. Retain the water-saturated n-butanol solution and wash it three times with n-butanol-saturated water (20 ml each time). Evaporate the water-saturated n-butanol solution to dryness. Dissolve the residue in 1 ml of methanol to prepare this test solution. Preparation of the control solution: Take 3 g of the control herbal material, Quegangmei, and add 50 ml of water. Heat under reflux for 1 hour, filter, and prepare the control solution by following the procedure for the test solution, starting with "Extract twice with saturated n-butanol (50 ml each time). Preparation of the negative test solution: Weigh the Quegangmei herbal compound according to the prescription ratio and prepare it according to the Guangdong herbal tea granules preparation process. Prepare the negative solution as described for the test product.

[0133] 2.3 Expand the system

[0134] Developing solvent: ethyl acetate-methanol-glacial acetic acid-water (11:1.5:1.5:2);

[0135] Spot volume: 1 μl of control herb solution, 4 μl of test sample solution; Bandwidth: 8 mm; Spreading distance: approximately 8 cm; Development: Pipette the control herb solution, test sample solution, and negative solution sequentially onto the same silica gel G thin-layer plate as described in the thin-layer chromatography method. Use ethyl acetate-methanol-glacial acetic acid-water (11:1.5:1.5:2) as the developing solvent. Develop, remove, air dry, spray with 10% sulfuric acid-ethanol solution, heat at 105°C until the spots are clearly colored (2-3 minutes), and examine under UV light (365 nm). Fluorescent spots of the same color will appear in the test sample chromatogram at the corresponding positions in the control herb chromatogram.

[0136] 2.4 Study on sample preparation methods

[0137] 2.4.1 Investigation of extraction solvent

[0138] Take the contents of 2 bags of this product, add 50 ml of water to dissolve it, extract twice with n-butanol, ethyl acetate and dichloromethane, 50 ml each time, combine the extracts with the same solvent, evaporate to dryness, and dissolve the residue with 1 ml of methanol to prepare the test solution. Take another 5g of the powder of the medicinal material of Rhizoma Cibotii, add water and reflux to extract for 2 hours, filter, and prepare the filtrate into the medicinal material solution of Rhizoma Cibotii in the same way as the preparation method of the test sample; according to the thin layer chromatography method (General Chapter 0502 of Part IV of the Chinese Pharmacopoeia), take 5μl of each of the above two solutions and spot them on the same silica gel G thin layer plate to make strips, use (1) toluene-ethyl acetate-glacial acetic acid (5:1:1) as the developing agent, develop, take out, dry, spray with 1% aluminum trichloride ethanol, and examine under ultraviolet light (365nm); (2) chloroform-ethyl acetate-methanol-water-formic acid (1:6:1.5:0.7:0.1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and examine under sunlight.

[0139] The results showed that the spots of Guangdong herbal tea and Gangmei were mainly in the n-butanol extraction part, so n-butanol was selected as the enrichment method for the test solution. Figure 8 .

[0140] 2.4.2 Investigation of purification methods:

[0141] Macroporous adsorption resin adsorption method: Take 1 bag of Guangdong herbal tea granules, add 50ml of water to dissolve, place on D101 macroporous adsorption resin (1.5*14cm), elute with 150ml of water, 200ml of 30% ethanol, 200ml of 50% ethanol and 200ml of 70% ethanol respectively, collect the eluents respectively, evaporate to dryness, add 1ml of methanol to dissolve the residue, and obtain. Prepare the medicinal solution of Gangmei and the negative control solution in the same way. According to the thin layer chromatography method (Chinese Pharmacopoeia Part IV General Rules 0502), take 5μl of each of the above two solutions, spot them on the same silica gel G thin layer plate, make them into strips, use chloroform-ethyl acetate-methanol-water-formic acid (1:6:1.5:0.7:0.1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and inspect under sunlight. The results are shown. Figure 9 .

[0142] The results showed that the sample eluted with 70% ethanol had spots of the same color at the corresponding positions of the medicinal materials, and there was no interference in the negative test. This method can be used as an alternative method for the thin layer chromatography identification of Gangmei in Guangdong herbal tea.

[0143] n-Butanol extraction method: Take the contents of 1 bag of this product, add 50 ml of water to dissolve it, extract it twice with water-saturated n-butanol solution, 30 ml each time, combine the water-saturated n-butanol extracts, extract 3 times with ammonia test solution, 20 ml each time, retain the water-saturated n-butanol solution, wash it 3 times with n-butanol-saturated water, 30 ml each time, evaporate the water-saturated n-butanol solution to dryness, add 1 ml of methanol to dissolve the residue, and use it as the test solution; prepare the negative solution of Herba Citri Rehmanniae in the same way; take 3 g of Herba Citri Rehmanniae medicinal materials, add 50 ml of water, heat and reflux for 1 hour, filter, and prepare the Herba Citri Rehmanniae medicinal material solution from "extracting it twice with water-saturated n-butanol solution, 50 ml each time" using the same method as the test solution preparation.

[0144] The results showed that the test sample had spots of the same color at the corresponding position of the medicinal material, and the negative test had no interference. Based on the previous work of the company, this method was used as the thin layer identification method for Gangmei in Guangdong herbal tea, and further optimized research was conducted. Figure 10 .

[0145] 2.4.3 Investigation of test solution preparation method

[0146] (1) Investigation on the extraction process of Medicinal Herbs of Gangmei:

[0147] Take 3g of the medicinal material of Rhizoma Cibotii, add 50ml of water, reflux extract for 1 hour, filter the extract, and shake the filtrate with saturated n-butanol solution twice, 30ml each time. Collect the n-butanol solution and extract it with ammonia water three times, 20ml each time. Wash the n-butanol solution with water saturated with n-butanol three times, 30ml each time. The ammonia solution, water washing solution, and n-butanol solution are evaporated to dryness. Dissolve the residue in 1ml of methanol to prepare the rhizoma Cibotii test solution, rhizoma Cibotii test solution, rhizoma Cibotii test solution, rhizoma Cibotii test solution, rhizoma Cibotii test solution, rhizoma Cibotii test solution, rhizoma Cibotii test solution, rhizoma Cibotii test solution, rhizoma Cibotii test solution, rhizoma Cibotii test solution, rhizoma Cibotii test solution, and rhizoma Cibotii test solution. 5μl of each of the rhizoma Cibotii test solution, rhizoma Cibotii test solution, and rhizoma Cibotii test solution were taken in turn and spotted on the same silica gel G thin layer plate according to the thin layer chromatography test. Develop with ethyl acetate-methanol-glacial acetic acid-water (11:1.5:1.5:2.5) as the developing solvent. Develop, remove, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and examine under ultraviolet light (365nm). The results showed that the same spots as those in the control solution of the medicinal material of Gangmei were found in the water solution, and the amount of water used could be reduced when eluting with water saturated with n-butanol. Figure 11 .

[0148] (2) Preparation of test solution:

[0149] Method 1: Take the contents of 1 bag of this product, add 50 ml of water to dissolve it, extract it twice with water-saturated n-butanol solution, 30 ml each time, combine the water-saturated n-butanol extracts, add ammonia water to extract 3 times, 20 ml each time, retain the water-saturated n-butanol solution, wash it 3 times with n-butanol-saturated water, 30 ml each time, evaporate the water-saturated n-butanol solution to dryness, add 1 ml of methanol to dissolve the residue, and use it as the test sample ①.

[0150] Method 2: Take the contents of 1 bag of this product, add 50 ml of water to dissolve it, extract it twice with water-saturated n-butanol solution, 30 ml each time, combine the water-saturated n-butanol extracts, extract it three times with ammonia test solution, 20 ml each time, retain the water-saturated n-butanol solution, wash it three times with n-butanol-saturated water, 30 ml each time, evaporate the water-saturated n-butanol solution to dryness, add 1 ml of methanol to dissolve the residue, and use it as test sample ②.

[0151] Method 3: Take the contents of 1 bag of this product, add 30 ml of water to dissolve it, extract it twice with water-saturated n-butanol solution, 50 ml each time, combine the water-saturated n-butanol extracts, add ammonia water to extract 3 times, let it stand for 30 minutes, 20 ml each time, retain the water-saturated n-butanol solution, wash it 3 times with n-butanol-saturated water, 20 ml each time, evaporate the water-saturated n-butanol solution to dryness, add 1 ml of methanol to dissolve the residue, and use it as test sample ③.

[0152] Prepare the control medicinal solution according to 2.2. Expand the system according to 2.3 and check the results. Figure 12 .

[0153] Conclusion: In the thin layer chromatogram, the sample obtained by the third preparation method and the control medicinal material showed three relatively obvious fluorescent spots at the same position, so method three was selected as the preparation method of the test solution.

[0154] 2.5 Investigation of the Chromatographic System

[0155] 2.5.1 Investigation of sample quantity

[0156] Investigate the effect of different sample volumes (1, 2, and 3 μl of the control herbal solution of Gynura sativa L., and 2, 4, 6, and 10 μl of the test solution) on the thin-layer chromatography system. Prepare the control herbal solution and the test solution according to 2.2, and expand the system according to 2.3 for inspection. Results Figure 13 .

[0157] Conclusion: When the sample volume of the control herb is 1μl and the sample volume of the test solution is 4ul, the spots are clear under ultraviolet light at 365nm. Therefore, the optimal sample volume of the control herb is 1μl, and the optimal sample volume of the test solution is 4μl.

[0158] 2.5.2 Investigation of sampling bandwidth

[0159] Investigate the effect of bandwidth (point, 4, 6, 8, 10 mm) on the thin layer chromatography system. Prepare the control herbal solution and the test solution according to 2.2, and expand the system according to 2.3 and inspect. Figure 14 .

[0160] Conclusion: Under UV light at 365 nm, the chromatograms of all solutions were similar. The bands narrowed as their width increased. At a band width of 8 mm, the separation between the individual spots was good, so an 8 mm band width was selected.

[0161] 2.5.3 Investigation of developing agents Investigate the developing system and its impact on the thin layer chromatography system.

[0162] Take the control medicinal material solution and the test sample solution and expand them according to the expansion system 1-5 respectively, and check. Figure 15 .

[0163] Development system 1: chloroform-ethyl acetate-methanol-water methanol-glacial acetic acid (15:40:22:10) placed below 10°C, lower layer solution.

[0164] Development system 2: ethyl acetate-methanol-glacial acetic acid (9:1:1.5)

[0165] Development system 3: ethyl acetate-methanol-glacial acetic acid-water (9:1:1.5:1);

[0166] Development system 4: ethyl acetate-methanol-glacial acetic acid-water (11:1.5:1.5:2.5);

[0167] Development system 5: ethyl acetate-methanol-glacial acetic acid-water (11:1.5:1.5:2) (1-Cyclohexylglycerin solution, 2-test solution)

[0168] Conclusion: When using development system 1, the spots were blurred and the Rf was low. When using development system 2, the spots were clear, adjacent spots were not completely separated, and the Rf was low. When using development system 3, the spots were blurred and the Rf was high. When using development system 5, three spots in the medicinal material were clearly identified as corresponding to the test sample, with clear spots, moderate Rf, and good separation. Development system 5 was selected as the thin-layer development system for this experiment.

[0169] 2.5.5 Examination of color development method

[0170] 2.5.5.1 Study on the dosage of color developer

[0171] The effects of different developer dosages on the thin-layer chromatography system were investigated. The control medicinal material solution and the test sample solution were developed in a developing system of ethyl acetate-methanol-glacial acetic acid-water (11:1:1.5:2.5). The samples were taken out, dried, and sprayed with 0.5, 1, 3, and 6 ml of 10% sulfuric acid ethanol solution. The samples were heated at 105°C or 120°C for 3 minutes and examined under a UV lamp (365 nm). Figure 16 .

[0172] Conclusion: If the amount of developer is too small, the spots will not be fully developed, and if it is too large, the color of the spots may change. When using the developer, just spray it evenly.

[0173] 2.5.5.2 Heating time investigation

[0174] The effects of different heating times on the thin-layer chromatography system were investigated. The control herbal solution and the test sample solution were developed in a developing system of ethyl acetate-methanol-glacial acetic acid-water (11:1:1.5:2.5). The samples were taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C or 120°C for 1-10 minutes, and examined under a UV lamp (365nm). Figure 17 .

[0175] Conclusion: If the heating time is too long, the color of the spots will change. If the color development time is within 5 minutes, the spots will be clear.

[0176] 2.6 Thin layer method confirmation

[0177] 2.6.1 Specificity

[0178] Prepare the test solution, control herbal solution and the negative control of Rhizoma Citri Reticulatae according to 2.2. Carry out the system development and inspection according to 2.3. Figure 18 .

[0179] Conclusion: The test sample showed three fluorescent spots of the same color at the corresponding positions of the control medicinal material of Gangmei, while the negative sample had no such spots. The specificity of this method is good.

[0180] 2.6.2 Solution stability study

[0181] Prepare the test solution and reference solution according to 2.2, spot the sample 0-3 days after sample preparation, and evaluate the stability of the reference solution and the test solution: carry out the system development and inspection according to 2.3. Figure 19 .

[0182] Conclusion: According to the observation under visible light, the spots of the samples were clear 0-3 days after preparation, and the Rf values ​​were moderate, that is, the test solution and the reference solution were stable.

[0183] 2.6.3 Durability test

[0184] (1) Different thin layer plates: Take the control medicinal material solution and the test sample solution, respectively, use silica gel G thin layer plate (Qingdao Ocean, Yantai Chemical, Merck, Germany) and high efficiency G thin layer plate (Merck, Germany), unfold according to the unfolding system, and inspect. Figure 20 .

[0185] Conclusion: Using silica gel G thin layer plates and high-efficiency G thin layer plates from different manufacturers, except for the thin layer plates from Qingdao Ocean, the main spots of Gangmei can be well separated, with clear spots and moderate RF, indicating that this method has good durability for thin layer plates.

[0186] (2) Different temperatures: Take the control medicinal material solution and the test sample solution, and use the development temperatures of 4, 25.7, and 35 °C respectively, develop according to the development system, and inspect. Figure 21 .

[0187] Conclusion: When developed at 4, 25.7, and 35°C, the spots of the test solution are clear at 4, 25.7, and 35°C, and the Rf is moderate. Therefore, temperature has little effect on this thin layer chromatography method.

[0188] (3) Different humidity: Take the control medicinal material solution and the test sample solution, saturate the thin layer plate at relative humidity of RH32%, 47%, 60%, and 88% for 10 minutes, unfold it according to the unfolding system, and inspect it. Figure 22 .

[0189] Conclusion: When developed at RH32%, 47%, and 60%, the reference solution and the test solution showed clear spots with moderate Rf values. Therefore, humidity has little effect on this thin layer chromatography method.

[0190] 3. Identification of Golden Sakura Root

[0191] 3.1 Instruments and reagents

[0192] HHS-21-6 electric constant temperature water bath, Shanghai Boxun Instrument Co., Ltd.; HB10 digital rotary evaporator, IKA; CAMAG double-slot developing cylinder, Karma, Switzerland; HP550-S 7-inch square plate standard heating plate; 2F-20D darkbox UV analyzer, Tianjin Kono; GoodSee-20E thin-layer chromatography imaging system, Shanghai Kezhe Biochemical Technology Co., Ltd.

[0193] Silica gel G thin layer plates were purchased from Yantai Chemical; silica gel G thin layer plates were purchased from Qingdao Ocean; high-efficiency G thin layer plates were purchased from Qingdao Ocean; and silica gel G thin layer plates were purchased from Merck, Germany. Chloroform, n-hexane, ethyl acetate, methanol, and ethanol were purchased from Tianjin Kangkede Technology Co., Ltd.; acetone and formic acid were purchased from Tianjin Kemiou Chemical Reagent Co., Ltd.; sulfuric acid was purchased from Tianjin Fengchuan Chemical Reagent Technology Co., Ltd.; and water was purchased from Guangzhou Watsons Food and Beverage Co., Ltd.

[0194] The control medicinal material of golden cherry root (batch number: 121115-201306) was purchased from the China Food and Drug Inspection Institute;

[0195] The medicinal materials of golden cherry root (batch number B240101), Gangmei medicinal materials (batch number B230501), Yam sesame medicinal materials (batch number B230901), Wuzhigan medicinal materials (batch number B230403), Danzhuye medicinal materials (batch number B231103), Oroxylum butterfly medicinal materials (batch number B240101), Buzhaye medicinal materials (batch number B230403), Huotanmu medicinal materials (batch number B230501), Jinshateng medicinal materials (batch number B230501), Jinqiancao herb medicinal materials (batch number B230501), Guangdong herbal tea granules sucrose type (batch numbers 2308207, 2309202, 2308215), and Guangdong herbal tea granules sugar-free type (batch numbers 2312007, 2312002, 2310007) were all provided by Guangzhou Wanglaoji Pharmaceutical Co., Ltd.

[0196] 3.2 Preparation of test sample solution

[0197] Preparation of test solution: Take the contents of 2 bags of this product, place them in a stoppered conical flask, add 100 ml of cold water to dissolve them, shake and extract with ethyl acetate 3 times, 50 ml each time, combine the ethyl acetate extracts, wash with water 2 times, 50 ml each time, evaporate the ethyl acetate solution to dryness, and add 1 ml of methanol to dissolve the residue as the test solution.

[0198] Preparation of control medicinal material solution: Take 2.5 g of golden cherry root control medicinal material, add 50 ml of water, heat and reflux for 40 minutes, cool, centrifuge, take the supernatant, and prepare the control medicinal material solution in the same way as "extract with ethyl acetate by shaking 3 times".

[0199] Preparation of negative test solution: Weigh the compound medicinal material of Qujinying root according to the prescription ratio, prepare it according to the preparation process of Guangdong herbal tea granules, and prepare the negative control solution with the same preparation method of the test solution.

[0200] 3.3 Expand the system

[0201] Developing agent: (1) chloroform-ethyl acetate-methanol-water (2:4:2:1), the lower layer solution is placed below 10℃; (2) n-hexane-ethyl acetate-acetone-formic acid-water (3:4:3:0.3:1) upper layer solution is the developing agent, secondary development

[0202] Spotting volume: 4 μl of the control solution of the golden cherry root, 8 μl of the test solution; bandwidth: 8 mm; spread: about 8 cm;

[0203] Development: Take the control medicinal material solution, test sample solution, and negative solution in turn, and spot them on the same silica gel G thin layer plate according to the thin layer chromatography test. Use (1) chloroform-ethyl acetate-methanol-water (2:4:2:1) as the lower layer solution placed below 10°C as the developing agent, or (2) n-hexane-ethyl acetate-acetone-formic acid-water (3:4:3:0.3:1) as the upper layer solution for development. Take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored (3-4 minutes), and examine under daylight. The test sample chromatogram and the control medicinal material chromatogram under daylight will show the same blue-purple spots at the corresponding positions.

[0204] 3.4 Study on sample preparation methods

[0205] 3.4.1 Cold water / hot water solubility investigation

[0206] The experiment investigated the effect of using cold water or hot water as solvent on the test solution.

[0207] The test solution was prepared as follows:

[0208] Take 2 bags of Guangdong herbal tea granules, add 100 ml of cold water or hot water respectively to dissolve, let cool, and extract with ethyl acetate twice by shaking, 50 ml each time, combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 1 ml of methanol as the test solution.

[0209] Prepare the control medicinal material solution according to 3.2.

[0210] According to the thin layer chromatography method (Chinese Pharmacopoeia Part IV General Chapter 0502), 8 μl of the golden cherry root control medicinal material solution and the two test sample solutions were taken and spotted on the same silica gel G thin layer plate. The upper layer solution of n-hexane-ethyl acetate-acetone-formic acid-water (3:4:3:0.3:1) was used as the developing solvent. The plate was developed twice, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105°C until the spots were clearly colored. In the chromatogram of the test sample, the same blue-purple main spot appeared at the corresponding position in the chromatogram of the control medicinal material. Results Figure 23 .

[0211] Results: Under the same conditions, when dissolved in cold water and hot water, there was no difference in the blue-purple spots at the same position between the sample and the control medicinal material in the thin layer chromatogram. Therefore, cold water was selected as the solvent to dissolve Guangdong herbal tea granules.

[0212] 3.4.2 Investigation of the number of extractions with ethyl acetate shaking

[0213] Take 2 bags of Guangdong herbal tea granules, add 100 ml of cold water to dissolve them, and shake and extract them 3 times with 50 ml of ethyl acetate each time. The three ethyl acetate extracts obtained were evaporated to dryness respectively, and the residue was dissolved in 1 ml of methanol to serve as the test solution.

[0214] Prepare the control medicinal material solution according to 3.2.

[0215] According to the thin layer chromatography method (Chinese Pharmacopoeia Part IV General Chapter 0502), 8 μl of the golden cherry root control medicinal material solution and the three test sample solutions were taken and spotted on the same silica gel G thin layer plate. The upper layer solution of n-hexane-ethyl acetate-acetone-formic acid-water (3:4:3:0.3:1) was used as the developing solvent. The plate was developed twice, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105°C until the spots were clearly colored. In the chromatogram of the test sample, the same blue-purple main spot appeared at the corresponding position in the chromatogram of the control medicinal material. Results Figure 24 .

[0216] Conclusion: In the thin layer chromatogram, the sample extracted with ethyl acetate for the third time still showed lighter blue-purple spots at the same position as the control medicinal material, so the number of ethyl acetate shaking extractions was selected to be 3.

[0217] 3.4.3 Investigation of ethyl acetate shaking extraction volume

[0218] Take 2 bags of Guangdong herbal tea granules, add 100 ml of cold water to dissolve, and shake and extract with ethyl acetate three times, with the volume of ethyl acetate being 50 ml / time, 80 ml / time, and 100 ml / time respectively. Combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 1 ml of methanol as the test solution.

[0219] Prepare the control medicinal material solution according to 3.2.

[0220] According to the thin layer chromatography method (Chinese Pharmacopoeia Part IV General Chapter 0502), 8 μl of the golden cherry root control medicinal material solution and the three test sample solutions were taken and spotted on the same silica gel G thin layer plate. The upper layer solution of n-hexane-ethyl acetate-acetone-formic acid-water (3:4:3:0.3:1) was used as the developing agent. The plate was developed twice, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and placed under sunlight and ultraviolet light (365nm) for inspection. Under sunlight, the test sample chromatogram and the control medicinal material chromatogram showed the same blue-purple spots at the corresponding positions. Under ultraviolet light, the test sample chromatogram and the control medicinal material chromatogram showed the same purple fluorescent spots at the corresponding positions. Results Figure 25 、 Figure 26 .

[0221] Conclusion: In the thin layer chromatogram, the samples extracted with different volumes of ethyl acetate and the control herbs showed blue-purple spots of the same color depth at the same position, so the volume of ethyl acetate shaking extraction solution was selected to be 50 ml.

[0222] 3.4.4 Investigation of the number of washing times of the ethyl acetate shaking extract

[0223] Take 2 bags of Guangdong herbal tea granules, add 100 ml of cold water to dissolve, shake and extract with ethyl acetate 3 times, 50 ml each time, combine the ethyl acetate extracts, wash with water 4 times, 50 ml each time, evaporate the ethyl acetate and the 4 water washings to dryness, and dissolve the residue in 1 ml of methanol as the test solution.

[0224] Prepare the control medicinal material solution according to 3.2.

[0225] According to the thin layer chromatography method (Chinese Pharmacopoeia Part IV General Chapter 0502), 8 μl of the golden cherry root control medicinal material solution and the four test sample solutions were taken and spotted on the same silica gel G thin layer plate. The upper layer solution of n-hexane-ethyl acetate-acetone-formic acid-water (3:4:3:0.3:1) was used as the developing agent. The plate was developed twice, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and placed under sunlight and ultraviolet light (365nm) for inspection. Under sunlight, the chromatogram of the test sample and the chromatogram of the control medicinal material showed the same blue-purple spots at the corresponding positions. Under ultraviolet light, the chromatogram of the test sample and the chromatogram of the control medicinal material showed the same purple fluorescent spots at the corresponding positions. Results Figure 27 、 Figure 28 .

[0226] Conclusion: In the thin layer chromatogram, the sample washed 4 times showed lighter blue-purple spots at the same position as the control medicinal material, and the spots after the third and fourth washings became darker in color. Therefore, the number of ethyl acetate washings was selected to be 2 times, 50 ml each time.

[0227] 3.5 Investigation of the Chromatographic System

[0228] 3.5.1 Investigation of sample quantity

[0229] Investigate the effect of different sample volumes (2, 4, 6, and 8 μl of the Golden Sakura root control herbal solution and 8, 10, 12, 14, 16, and 18 μl of the test solution) on the thin-layer chromatography system. Prepare the control herbal solution and the test solution according to 3.2, and expand the system according to 3.3 for inspection. Results Figure 29 .

[0230] Conclusion: When the spot volume of the control medicinal material is 4μl and the spot volume of the test solution is 10μl, the spots are clear under sunlight. Therefore, the optimal spot volume of the control medicinal material is 4μl and the optimal spot volume of the sample is 10μl.

[0231] 3.5.2 Investigation of sampling bandwidth

[0232] Investigate the effect of bandwidth (point, 4, 6, 8, 10 mm) on the thin layer chromatography system. Prepare the control herbal solution and the test solution according to 3.2, and expand the system according to 3.3 and inspect. Figure 30 .

[0233] Conclusion: Under daylight, the chromatograms of all solutions were similar. The bands narrowed as their width increased. At a band width of 10 mm, the separation between the individual spots was good, so a band width of 10 mm was selected.

[0234] 3.5.4 Investigation of the developing agent Investigate the developing system and its impact on the thin layer chromatography system.

[0235] Take the control medicinal material solution and the test sample solution and expand them according to the expansion system 1-10 and check. Figure 31 .

[0236] Development system 1: n-hexane-ethyl acetate (3:4);

[0237] Development system 2: n-hexane-ethyl acetate-acetone (3:4:3);

[0238] Development system 3: n-hexane-ethyl acetate-acetone (3:4:6)

[0239] Development system 4: n-hexane-ethyl acetate-acetone-formic acid (3:4:6:0.1)

[0240] Development system 5: n-hexane-ethyl acetate-acetone-formic acid (3:4:6:0.3)

[0241] Development system 6: petroleum ether-ethyl acetate-acetone (1:4:5)

[0242] Development system 7: petroleum ether-ethyl acetate-acetone-formic acid (1:4:5:0.1)

[0243] Development system 8: petroleum ether-ethyl acetate-acetone-formic acid (2:4:4:0.1)

[0244] Development system 9: n-hexane-ethyl acetate-acetone-formic acid-water (3:4:3:0.3:1)

[0245] Development system 10: chloroform-ethyl acetate-methanol-water (2:4:2:1)

[0246] Conclusion: When using development system 1, the spots were not developed. When using development system 2, the spots were clear and the Rf value was low. When using development system 3, the spots were blurred and the Rf value was larger than that of development system 2. When using development system 4, the spots were scattered and the Rf value was moderate. When using development system 5, the spots did not improve. When using development system 6, the spots were more scattered and the Rf value was larger. When using development system 7, the spots were scattered and the Rf value was high. When using development system 8, the spots were blurred and the Rf value was moderate. When using development system 9, the spots were clear and the Rf value was moderate, requiring secondary development. When using development system 10, the spots were clear, the Rf value was moderate, and the separation was good. Development system 9 or 10 was selected as the thin-layer development system for this experiment.

[0247] 3.6 Thin layer method confirmation

[0248] 3.6.1 Specificity

[0249] Prepare the test solution, control medicinal material solution and golden cherry root negative control according to 3.2. Test according to the thin layer chromatography method (Chinese Pharmacopoeia Part IV General Chapter 0502), take the golden cherry root control medicinal material solution,

[0250] 8ul of the test solution and two negative control solutions of Rhododendron chinense were spotted on the same silica gel G thin layer plate, and the upper layer solution of n-hexane-ethyl acetate-acetone-formic acid-water (3:4:3:0.3:1) was used as the developing agent. The plate was developed twice, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and examined under sunlight and ultraviolet light (365nm). Under sunlight, the chromatogram of the test sample and the chromatogram of the control medicinal material showed the same blue-purple spots at the corresponding positions, and under ultraviolet light, the chromatogram of the test sample and the chromatogram of the control medicinal material showed the same purple fluorescent spots at the corresponding positions. Results Figure 32 .

[0251] Conclusion: The test sample showed fluorescent spots of the same color at the corresponding positions of the control medicinal material, while the negative sample did not show such spots. The specificity of this method is good.

[0252] 3.6.2 Solution stability study

[0253] Prepare the test solution and reference solution according to 3.2, spot the sample at 0, 1, 2, and 3 days after sample preparation, and evaluate the stability of the reference solution and the test solution: carry out the system development and inspection according to 3.3. Figure 33 .

[0254] Conclusion: According to the observation under visible light, the spots of the samples at 0, 1, 2 and 3 days after preparation were clear and the Rf values ​​were moderate, that is, the test solution and the reference solution were stable.

[0255] 3.6.3 Durability test

[0256] (1) Different thin layer plates: Take the control medicinal material solution and the test sample solution, respectively, use silica gel G thin layer plate (Qingdao Ocean, Yantai Chemical, Merck, Germany) and high efficiency G thin layer plate (Merck, Germany), unfold according to the unfolding system, and inspect. Figure 34 .

[0257] Conclusion: Using silica gel G thin layer plates and high-efficiency G thin layer plates from different manufacturers, except for the thin layer plates from Qingdao Ocean, the main spots of the root of golden cherry can be well separated, with clear spots and moderate RF, indicating that this method has good durability for thin layer plates.

[0258] (2) Different temperatures: Take the control medicinal material solution and the test sample solution, and use the development temperatures of 4, 22.8, and 35 °C respectively, and develop them according to the development system under 3.3 and check. Figure 35 .

[0259] Conclusion: When developed at 4, 22.8, and 35°C, the spots of the test solution are clear at 4, 22.8, and 35°C, and the Rf is moderate. Therefore, temperature has little effect on this thin layer chromatography method.

[0260] (3) Different humidity: Take the control medicinal material solution and the test sample solution, saturate the thin layer plate at relative humidity of RH32%, 47%, 66%, and 88% for 10 minutes, and then develop the system according to 3.3 and inspect. Figure 36 .

[0261] Conclusion: When developed at RH32%, 47%, and 66%, the reference solution and the test solution have clear spots and moderate Rf. Therefore, humidity has little effect on this thin layer chromatography method.

[0262] Beneficial effects

[0263] 1. This invention has developed a method for detecting 4-hydroxybenzoic acid in Guangdong herbal tea granules, enabling a more comprehensive assessment of Guangdong herbal tea quality. This method utilizes high-performance liquid chromatography (HPLC) combined with specific chromatographic conditions to effectively separate 4-hydroxybenzoic acid from other components, avoiding interference and enabling accurate content determination. High sensitivity: By optimizing the mobile phase composition and gradient elution procedure, low levels of 4-hydroxybenzoic acid can be detected, making it suitable for testing Guangdong herbal tea granules of varying batches and qualities.

[0264] 2. The identification method has been improved to make the spots clearer and the identification method simpler, making it easier for inspectors to judge:

[0265] (1) Identification of five-finger orange

[0266] In terms of developing agent, the existing standard uses n-hexane-ethyl acetate-acetone-water-formic acid (3:3:2:1:0.1), while the present invention uses petroleum ether-ethyl acetate (5:5) as developing agent, which is simpler. In terms of effect, the spots of the present invention are clearer than those of the existing standard, as shown in FIG. Figure 2 A (expanded system 1) and Figure 2 K (expand system 11)

[0267] (2) Identification of Prunus mume

[0268] In terms of developing agent, the existing standard uses toluene-ethyl acetate-glacial acetic acid (5:1:1), while the present invention uses ethyl acetate-methanol-glacial acetic acid-water (11:1.5:1.5:2) without toluene, which is safer. In terms of effect, the spots of the present invention are clearer than those of the existing standard, as shown in FIG. Figure 8 Left picture and Figure 15 E

[0269] (3) Identification of Golden Sakura Root

[0270] Compared with the existing standards, the present invention has developed a developer that only requires one development; the water wash times have been optimized from 4 to 2; the spot volume has been further optimized from 8μl for both the control medicinal material solution and the test solution to 4μl for the control medicinal material solution and 10μl for the test solution, further improving the spot clarity. Figure 29 . BRIEF DESCRIPTION OF THE DRAWINGS

[0271] Figure 1 Investigation on identification of five-finger orange, among which

[0272] A. Investigation of different extraction solvents: 1-ethyl acetate, 2-chloroform, and 3-ethanol

[0273] B 1-Acetone ultrasound; 2-Methanol ultrasound; 3-Ethanol reflux Test sample preparation method 1

[0274] C 1-ethanol ultrasound; 2-ethanol reflux test sample preparation method investigation 2

[0275] D 1- Column water wash layer; 2- Column 40% methanol wash layer; 3- Column methanol wash layer; 4- Ethyl acetate extraction test sample preparation method 3E 1- Spotting volume 2μL (Wuzhigan medicinal material), 2- Spotting volume 5μL (Wuzhigan medicinal material), 3- Spotting volume 10μL (Wuzhigan medicinal material), 4- Spotting volume 15μL (Wuzhigan medicinal material), 5- Spotting volume 2μL (test sample), 6- Spotting volume 5μL (test sample), 7- Spotting volume 10μL (test sample), 8- Spotting volume 15μL (test sample) Spotting volume

[0276] F 1-point (Wuzhigan medicinal material), 2-band width 4mm (Wuzhigan medicinal material), 3-band width 6mm (Wuzhigan medicinal material), 4-band width 8mm (Wuzhigan medicinal material), 5-band width 10mm (Wuzhigan medicinal material), 6-point (test sample), 6-band width 4mm (test sample), 6-band width 6mm (test sample), 6-band width 8mm (test sample), 6-band width 10mm (test sample), strip width inspection Figure 2 A systematic investigation was carried out on the identification of five-finger oranges, including

[0277] A Development system 1: n-hexane-ethyl acetate-acetone-water-formic acid (3:3:2:1:0.1), upper solution 1-test sample, 2-Citrus sinensis medicinal material, 3-negative test sample Development system investigation 1

[0278] B Development system 2: n-hexane-ethyl acetate-formic acid (5:5:0.1) 1-negative test sample, 2-Citrus sinensis medicinal material, 3-test sample Development system investigation 2

[0279] C development system 3: petroleum ether-n-hexane-ethyl acetate-formic acid (2:3:5:0.1) 1-negative test sample, 2-Citrus sinensis medicinal material, 3-test sample development system investigation 3

[0280] D Development System 4: Petroleum ether-cyclohexane-formic acid (3:7:0.1) 1-negative test sample, 2-Citrus sinensis medicinal material, 3-test sample Development System Investigation 4

[0281] E development system 5: n-hexane-ethyl acetate-formic acid (7:3:0.1) 1-negative test sample, 2-Citrus sinensis medicinal material, 3-test sample development system investigation 5

[0282] F Development System 6: Toluene-ethyl acetate-methanol-formic acid (6:4:2:0.1) 1-negative test sample, 2-Citrus sinensis medicinal material, 3-test sample Development System Investigation 6

[0283] G Development System 7: Toluene (5ml) 1-Test Sample Development System Investigation 7

[0284] H development system 8: Ethyl acetate (5 ml) 1-Test sample development system investigation 8

[0285] I Development system 9: Toluene: ethyl acetate: formic acid (5:5:0.1) 1-Test sample development system investigation 9

[0286] J development system 10: petroleum ether: ethyl acetate: formic acid (5:5:0.02) 1-test sample development system investigation 10K development system 11: petroleum ether: ethyl acetate (5:5) 1-negative test sample, 2-Citrus sinensis medicinal material, 3-test sample development system investigation 11

[0287] L development system 12: Petroleum ether: ethyl acetate (6:4) 1-Wuzhigan medicinal material, 2-Test sample development system investigation 12M development system 13: Petroleum ether: ethyl acetate (4:6) 1-Wuzhigan medicinal material, 2-Test sample development system investigation 13N development system 14: n-hexane: ethyl acetate (5:5) 1-Wuzhigan medicinal material, 2-Test sample development system investigation 14O development system 15: n-hexane: ethyl acetate: acetone (3:3:2) 1-Wuzhigan medicinal material, 2-Test sample development system investigation 15P development system 16: Chloroform: acetone (7:3) 1-Negative, 2-Test sample development system investigation 16Q development system 17: Chloroform: ethyl acetate: methanol: water (20:40:20:10, lower layer, below 10°C) 1-Test sample development system investigation 17

[0288] R development system 18: chloroform: ethyl acetate: methanol: water (4:2:1:0.5, lower layer, below 10°C) 1-Test sample development system investigation 18

[0289] S development system 19: chloroform: ethyl acetate: methanol: water (5:1:1:1, lower layer, below 10°C) 1-Test sample development system investigation 19

[0290] T development system 20: chloroform: ethyl acetate: methanol: water (5:1:1.5:1, lower layer, below 10°C) 1-negative, 2-test sample development system inspection 20

[0291] U Development System 21: Chloroform: Acetone: Methanol: Water (5:1:1:1, lower layer, below 10°C) 1- Negative, 2- Test Sample Development System Investigation 21

[0292] V Development System 22: Chloroform: Acetone: Methanol: Water (7:1:1:1, lower layer, below 10°C) 1-Negative test sample, 2-Citrus sinensis medicinal material, 3-Test sample Development System Investigation 22

[0293] W development system 23: chloroform: ethyl acetate: methanol: water: formic acid (5:1:1.5:1:0.5, lower layer, below 10°C) 1-negative test sample, 2-Citrus sinensis medicinal material, 3-test sample development system investigation 23

[0294] X development system 24: chloroform: butanone: methanol: water (7:1:1:1, lower layer, below 10°C) 1- negative test sample, 2- five-finger orange medicinal material, 3- test sample opening system inspection 24

[0295] Y development system 25: chloroform: ethyl acetate: methanol: water: formic acid (5:1:1.5:1:0.1, lower layer, below 10°C) 1-negative test sample, 2-Citrus sinensis medicinal material, 3-test sample development system investigation 25

[0296] Z development system 26: chloroform: ethyl acetate: methanol: formic acid (5:1:1.5:0.1, lower layer, below 10°C) 1-negative test sample, 2-Citrus sinensis medicinal material, 3-test sample development system investigation 26

[0297] AA development system 27: chloroform: ethyl acetate: butanone: methanol: formic acid (5:1:1:0.5:1, lower layer, below 10°C) 1-negative test sample, 2-Citrus sinensis medicinal material, 3-test sample development system investigation 27

[0298] AB development system 28: Cyclohexane: ethyl acetate (5:5) 1-negative test sample, 2-Citrus sinensis medicinal material, 3-test sample development system investigation 28

[0299] Figure 3 Investigation on the specificity of thin-layer method for citrus aurantium

[0300] 1-negative test sample, 2-Citrus sinensis medicinal material, 3-test sample

[0301] Figure 4 Qualitative investigation of thin layer chromatography of citrus aurantium

[0302] 1-negative test sample, 2-Citrus sinensis medicinal material, 3-test sample stability 0, 1, 2, 4 days

[0303] Figure 5 Durability test of Wuzhigan thin layer method - different thin layer boards

[0304] 1-negative test sample, 2-Citrus sinensis medicinal material, 3-test sample silica gel G (Yantai Chemical), silica gel G (Merck)

[0305] Figure 6 Durability test of the thin layer method of five-finger orange - different temperatures

[0306] 1-negative test sample, 2-Citrus sinensis medicinal material, 3-test sample Citrus sinensis identification durability test (room temperature 25℃, 4℃, 35℃) Figure 7 Durability test of the thin layer method of citrus quinquefolia - humidity 1-negative test sample, 2-citrus quinquefolia medicinal material, 3-test sample citrus quinquefolia identification humidity durability (RH71%, RH32%, RH47%, RH88%)

[0307] Figure 8 Investigation on Different Extraction Solvents for Identification of Herba Citri Rehmanniae (1-herbal tea n-butanol, 2-herbal tea ethyl acetate, 3-herbal tea dichloromethane, 4-herbal tea n-butanol, 5-herbal tea ethyl acetate, 6-herbal tea dichloromethane)

[0308] Figure 9 Investigation on the Identification and Purification Method of Herba Citri Reticulatae (1-30% ethanol 2-50% ethanol 3-70% ethanol)

[0309] Figure 10 Investigation on the purification method of Identification of Herba Citri Rehmanniae (1-Negative 2-Herba Citri Rehmanniae Medicinal Materials 3-Herbal Tea Sample)

[0310] Figure 11 Identification of scutellaria baicalensis (1-scutellaria baicalensis test solution 1, 2-scutellaria baicalensis test solution 2, 3-scutellaria baicalensis-ammonia solution, 4-scutellaria baicalensis-water solution)

[0311] Figure 12 Identification of Rhizoma Cibotii 1-Control Herbs, 2-Test Sample ①, 3-Test Sample ②, 4-Test Sample ③ Preparation Method of Test Sample Solution

[0312] Figure 13 Identification of Rhizoma Citri Reticulatae Study on the spotting volume of Rhizoma Citri Reticulatae reference medicinal materials (spotting volume: 1μl, 2μl, 3μl) and 4-7 test samples (spotting volume: 2μl, 4μl, 6μl, 10μl)

[0313] Figure 14 Identification of 1- 1- 1- 1- 1- 4mm width stripe, 2- 6mm width stripe, 4- 8mm width stripe, 5- 10mm width stripe

[0314] Figure 15 A systematic study was carried out on the identification of Gangmei,

[0315] A Development system 1: chloroform-ethyl acetate-methanol-water methanol-glacial acetic acid (15:40:22:10) placed below 10°C, the lower layer solution.

[0316] B Development system 2: ethyl acetate-methanol-glacial acetic acid (9:1:1.5); Investigation of development system 2C Development system 3: ethyl acetate-methanol-glacial acetic acid-water (9:1:1.5:1); Investigation of development system 3D Development system 4: ethyl acetate-methanol-glacial acetic acid-water (11:1.5:1.5:2.5); Investigation of development system 4

[0317] E development system 5: Ethyl acetate-methanol-glacial acetic acid-water (11:1.5:1.5:2) (1-Herba sylvestris medicinal material solution, 2-test solution) development system investigation 5

[0318] Figure 16 Identification of Gangmei

[0319] 1-test solution 1, 2-control medicinal material solution colorimetric dosage (from left to right 0.5, 1, 3, 6 ml)

[0320] Figure 17 Identification of Gangmei

[0321] 1-Test solution 1, 2-Control medicinal material solution heating time investigation

[0322] Figure 18 Identification of Rhizoma Cibotii 1-Negative test solution, 2-Rhizoma Cibotii control medicinal materials, 3-Specificity investigation of test solution

[0323] Figure 19 Identification of radix serratae 1-radix serratae control medicinal material, 2-test solution From left to right: sample solution stability test 0, 1, 2, 3 days after preparation

[0324] Figure 20 Identification of radix serratae 1-radix serratae control medicinal material, 2-test solution From left to right: Qingdao Ocean Silica Gel, Yantai Chemical Silica Gel Plate, Merck Silica Gel, Merck High-Efficiency G Different Thin Layer Plates

[0325] Figure 21 Identification of 1- 1- 1- 1- 1- 1 test solution from left to right: temperature 4℃, temperature 25.7℃, temperature 35℃ different development temperature investigation

[0326] Figure 22 Identification of hyacinth 1-hyacinth control medicinal material, 2-test solution from left to right: RH32%, RH47%, RH60%, RH88% different humidity inspection

[0327] Figure 23 Identification of Golden Sakura Root 1- Golden Sakura Root reference medicinal material, 2- hot water, 3- cold water cold water / hot water solubility test

[0328] Figure 24 Identification of Golden Cherry Root 1- Golden Cherry Root reference medicinal material; 2- Ethyl acetate extraction of the first sample; 3- Ethyl acetate extraction of the second sample; 4- Ethyl acetate extraction of the third sample and the number of ethyl acetate shaking extractions

[0329] Figure 25 Identification of the root of golden cherry ethyl acetate shaking extraction volume investigation - Sunlight

[0330] Figure 26 Identification of the root of golden cherry: investigation of the volume of ethyl acetate shaking extraction - UV 365nm

[0331] Figure 27 Identification of the root of golden cherry: investigation on the number of washing times of the ethyl acetate shaking extract

[0332] Figure 28 Identification of Golden Cherry Root: Investigation of the Number of Washes of Ethyl Acetate Shaking Extract - UV 365nm 1- Golden Cherry Root reference medicinal material, 2- 1st wash sample, 3- 2nd wash sample, 4- 3rd wash sample, 5- 4th wash sample, 6- Guangdong herbal tea granule sample

[0333] Figure 29 Identification of Chrysanthemum Root 1-4 Chrysanthemum Root control herbs (spotting volume: 2μl, 4μl, 6μl, 8μl) and samples 5-11 (spotting volume: 8μl, 10μl, 12μl, 14μl, 16μl, 18μl) were examined. The developing solvent used in the upper figure was n-hexane-ethyl acetate-acetone-formic acid-water (3:4:3:0.3:1), and the developing solvent used in the lower figure was chloroform-ethyl acetate-methanol-water (2:4:2:1).

[0334] Figure 30 Identification of Golden Sakura Root 1- Golden Sakura Root reference medicinal material, 2- bandwidth 4mm, 3- bandwidth 6mm, 4- bandwidth 8mm, 5- bandwidth 10mm, the developing solvent used in the left picture is chloroform-ethyl acetate-methanol-water (2:4:2:1), the developing solvent used in the right picture is n-hexane-ethyl acetate-acetone-formic acid-water (3:4:3:0.3:1)

[0335] Figure 31 The identification system of golden cherry root is expanded, including

[0336] A Development system 1: n-hexane-ethyl acetate (3:4); Investigation of development system 1

[0337] B Development system 2: n-hexane-ethyl acetate-acetone (3:4:3); Investigation of development system 2

[0338] C development system 3: Investigation of n-hexane-ethyl acetate-acetone (3:4:6) development system 3

[0339] D Development System 4: Investigation of the Development System of n-hexane-ethyl acetate-acetone-formic acid (3:4:6:0.1)

[0340] E Development System 5: Investigation of the development system of n-hexane-ethyl acetate-acetone-formic acid (3:4:6:0.3) 5F Development System 6: Investigation of the development system of petroleum ether-ethyl acetate-acetone (1:4:5) 6

[0341] G development system 7: Investigation of petroleum ether-ethyl acetate-acetone-formic acid (1:4:5:0.1) development system 7

[0342] H development system 8: Investigation of petroleum ether-ethyl acetate-acetone-formic acid (2:4:4:0.1) development system 8

[0343] I Development System 9: Investigation of the development system of n-hexane-ethyl acetate-acetone-formic acid-water (3:4:3:0.3:1)

[0344] J Development System 10: Chloroform-ethyl acetate-methanol-water (2:4:2:1) (1-control medicinal material solution, 2-test solution) Development System Investigation 10

[0345] Figure 32 Investigation on the identification and specificity of golden cherry root-Sunlight

[0346] Figure 33 Identification of Golden Sakura Root 1- Golden Sakura Root reference medicinal material, 2- Test solution From left to right: Samples were spotted 0, 1, 2, and 3 days after preparation. The developing solvent used in the upper figure is chloroform-ethyl acetate-methanol-water (2:4:2:1), and the developing solvent used in the lower figure is n-hexane-ethyl acetate-acetone-formic acid-water (3:4:3:0.3:1).

[0347] Figure 34 Identification of Golden Cherry Root from left to right: Qingdao Ocean G plate, Merck G plate, Yantai G plate, Qingdao Ocean High G plate. 1- Golden Cherry Root reference medicinal material, 2- Different thin layer plates for test solution. The developing solvent used in the upper figure is chloroform-ethyl acetate-methanol-water (2:4:2:1), and the developing solvent used in the lower figure is n-hexane-ethyl acetate-acetone-formic acid-water (3:4:3:0.3:1).

[0348] Figure 35 The identification of golden cherry root from left to right is: temperature 4℃, temperature 22.8℃, temperature 35℃ different development temperatures. The developing solvent used in the upper figure is chloroform-ethyl acetate-methanol-water (2:4:2:1), and the developing solvent used in the lower figure is n-hexane-ethyl acetate-acetone-formic acid-water (3:4:3:0.3:1).

[0349] Figure 36 The identification of golden cherry root from left to right is: RH32%, RH47%, RH66%, RH88% different humidity inspection

[0350] Figure 37 Example 1 Chromatogram

[0351] A 4-Hydroxybenzoic acid-reference substance

[0352] B Guangdong Herbal Tea Granules (Sugar-free) 2312007

[0353] C Guangdong Herbal Tea Granules (Sugar-free) 2310007

[0354] D Guangdong Herbal Tea Granules (Sugar-free) 2312002

[0355] E Guangdong Herbal Tea Granules (with sugar) 2308215

[0356] F Guangdong Herbal Tea Granules (with sugar) 2308207

[0357] G Guangdong Herbal Tea Granules (with sugar) 2309202

[0358] Figure 38 Standard curve of 4-hydroxybenzoic acid content DETAILED DESCRIPTION

[0359] Example 1 Preparation of Guangdong Herbal Tea Granules

[0360]

prescription

[0361] Preparation: Add water to the above ten ingredients and boil twice for 1 hour each time. Filter the decoction, combine the filtrates, and concentrate to a relative density of 1.03 (45°C). Centrifuge until the solution is clear. Take the clear solution and concentrate it into a thick paste. Add an appropriate amount of sucrose powder to the thick paste, granulate it, and dry it to make 720g. Each bag contains 10g.

[0362] Example 2 Preparation of Guangdong Herbal Tea Granules

[0363]

prescription

[0364] Preparation: Add water to the above ten ingredients and boil twice, each time for 1 hour. Filter the decoction, combine the filtrates, and concentrate to a relative density of 1.03 (45°C). Centrifuge until the solution is clear. Take the clear solution and concentrate it into a thick paste. Alternatively, take the thick paste and add an appropriate amount of soluble starch, granulate it, and dry it to make 72g. Each bag contains 1g.

[0365] Example 3 Guangdong herbal tea granule content detection

[0366] Preparation of reference solution: Take an appropriate amount of 4-hydroxybenzoic acid reference substance and add 50% methanol to prepare a solution containing 10 μg per ml.

[0367] Preparation of the test solution: Take about 2.5g of Guangdong herbal tea granules, accurately weigh them, place them in a 100ml conical flask, accurately add 50ml of methanol, ultrasonically treat for 30min (power 500w), take out, let it cool to room temperature, make up the lost weight with methanol, shake well, filter through a 0.45μm microporous membrane, and take the filtrate to obtain the product.

[0368] Determination method: Accurately aspirate 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine according to the following chromatographic conditions:

[0369] Chromatographic column: octadecylsilane bonded silica gel as filler (4.6*250 mm, 5 μm); acetonitrile as mobile phase A, 0.1% phosphoric acid aqueous solution as mobile phase B, gradient elution as specified in the table below; column temperature: 30°C; flow rate: 1.0 mL / min; detection wavelength: 254 nm.

[0370]

[0371] Calculate the content, the results are shown in Table 4, and the chromatogram is shown in Figure 37

[0372] Table 4 Test results of sugar-containing and sugar-free samples of Guangdong herbal tea granules

[0373]

[0374] Example 4 Identification

[0375] ⑴ Take the contents of 2 bags of Guangdong herbal tea granules prepared according to Example 1, add 50 ml of ethanol, heat under reflux on a water bath for 2 hours, filter, evaporate the filtrate to dryness, add 50 ml of water to the residue, stir to dissolve, and extract with ethyl acetate twice by shaking, 20 ml each time, combine the ethyl acetate extracts, evaporate to dryness, and add 1 ml of methanol to dissolve the residue as the test solution. Separately take 5 g of the five-finger orange control medicinal material, add 50 ml of water, heat under reflux for 2 hours, cool, filter, add ethanol to the filtrate to make the alcohol content reach 45%, let stand for 2 hours, filter, evaporate the filtrate to dryness, and add 1 ml of methanol to dissolve the residue as the control medicinal material solution. According to the thin layer chromatography method (Chinese Pharmacopoeia, Part IV, General Chapter 0502), 5 μl of each of the two solutions was applied to the same silica gel G thin layer plate. Develop with petroleum ether-ethyl acetate (5:5) at a humidity level below 70%. Remove the plate, air dry, and spray with 1% ferric chloride in methanol until a clear color develops. Examine the plate under sunlight. The test sample chromatogram should show a spot of the same color at the corresponding position in the control chromatogram.

[0376] (2) Dissolve the contents of two bags of Guangdong herbal tea granules prepared according to Example 1 in 100 ml of water. Shake and extract with ethyl acetate three times, 50 ml each time. Combine the ethyl acetate extracts and wash with water twice, 50 ml each time. Evaporate the ethyl acetate to dryness. Dissolve the residue in 1 ml of methanol to prepare the test solution. Separately, take 2.5 g of the control medicinal material (Ceratonia odorata root), add 50 ml of water, heat and reflux for 40 minutes, cool, centrifuge, and remove the supernatant. Prepare a control medicinal material solution by the same method starting from "shake and extract with ethyl acetate three times." According to the thin layer chromatography method (Chinese Pharmacopoeia Part IV General Chapter 0502), 4 μl of the golden cherry root control medicinal material solution and 10 μl of the test sample solution were respectively spotted on the same silica gel G thin layer plate to form a 1 cm strip, and developed twice with an upper layer solution of n-hexane-ethyl acetate-acetone-formic acid-water (3:4:3:0.3:1) as a developing solvent, or developed once with a lower layer solution of chloroform-ethyl acetate-methanol-water (20:20:40:10) delaminated below 10°C as a developing solvent. The plate was taken out, dried in air, sprayed with 10% ethanolic sulfuric acid solution, heated at 105°C until the spots were clearly colored (3-4 minutes), and examined under daylight. Under daylight, the test sample chromatogram and the control medicinal material chromatogram showed identical blue-purple spots at corresponding positions.

[0377] (3) Take the contents of one bag of Guangdong herbal tea granules prepared according to Example 1, add 30 ml of water to dissolve it, extract it twice with a saturated n-butanol solution with 50 ml each time, combine the water-saturated n-butanol extracts, extract it three times with ammonia water with 20 ml each time, retain the water-saturated n-butanol solution, wash it three times with n-butanol-saturated water with 20 ml each time, evaporate the water-saturated n-butanol solution to dryness, and dissolve the residue in 1 ml of methanol to prepare the test solution. Separately, take 3 g of the control medicinal material of Rhizoma Cibotii, add 50 ml of water, heat and reflux for 1 hour, filter, and prepare the control medicinal material solution by the same method as the test solution preparation starting from "extracting it twice with a saturated n-butanol solution with 50 ml each time". According to the thin layer chromatography method (Chinese Pharmacopoeia, Part IV, General Chapter 0502), 1 μl of the control herb solution and 4 μl of the test sample solution were spotted separately on the same silica gel G thin layer plate. Developed with ethyl acetate-methanol-glacial acetic acid-water (11:1.5:1.5:2), the plate was removed, air-dried, and sprayed with an appropriate amount of 10% ethanolic sulfuric acid solution. The plate was heated at 105°C until the spots were clearly colored. The plate was then examined under ultraviolet light (365 nm). In the chromatogram of the test sample, a fluorescent spot of the same color appeared at the corresponding position in the chromatogram of the control herb.

[0378] Example 5 Content Determination Methodology Verification

[0379] Methodological investigation

[0380] Chromatographic Conditions and System Suitability Test: Column: Octadecylsilane bonded silica gel as filler; acetonitrile as mobile phase A, 0.1% aqueous phosphoric acid as mobile phase B, gradient elution as specified in the table below; acetonitrile-0.1% aqueous phosphoric acid as the mobile phase; detection wavelength: 254 nm. The theoretical plate number, calculated based on the 4-hydroxybenzoic acid peak, should be no less than 3000.

[0381]

[0382] Blank solvent inspection

[0383] Accurately pipette 10 μl of methanol, inject it into the liquid chromatograph, and record the chromatogram. The blank solvent has no interference.

[0384] Negative control test

[0385] Take 2.24g of the prescription medicinal material of Quwuzhigan, prepare the negative sample solution in the same way, record the chromatogram, and the result is negative with basically no interference.

[0386] Preparation of reference solution

[0387] Take an appropriate amount of 4-hydroxybenzoic acid reference substance and add 50% methanol to prepare a solution containing 10 μg per ml.

[0388] Preparation of test solution

[0389] Take 10 bags of Guangdong herbal tea granules, remove the packaging, and mix the contents. Accurately weigh approximately 2.5 g of the contents and place them in a 100 ml conical flask. Accurately add 50 ml of methanol and sonicate for 30 minutes. Remove the flask, allow it to cool to room temperature, and add methanol to make up the lost weight. Shake well, filter through a 0.45 μm microporous membrane, and collect the filtrate.

[0390] Investigation of linear relationships

[0391] Take 5 mg of 4-hydroxybenzoic acid reference substance, accurately weigh it, place it in a 50 ml volumetric flask, and add 50% methanol to make a solution containing 0.1 mg per 1 ml to obtain the reference substance stock solution (0.10048 mg / ml).

[0392] Accurately pipette 0.25, 0.5, 1, 2.5, 5, 10, and 25 ml of the reference stock solution into a 25 ml volumetric flask, add 50% methanol to dilute to the scale, shake well, and pipette 10 μl of each of the above solutions, inject into the liquid chromatograph, and measure the peak area. The results are shown in Table 5. Figure 38 .

[0393] Table 5 Results of the linear relationship investigation of Guangdong herbal tea granules

[0394]

[0395]

[0396] Test results: Linear equation: Y=60.757X+4.5278, r=1; the injection concentration of 4-hydroxybenzoic acid is in the range of 1.0048-100.48 μg / ml, showing a good linear relationship with the peak area.

[0397] Precision test

[0398] The same sample solution of Guangdong Herbal Tea Granules (batch 2308207) was injected into the liquid chromatograph for 6 consecutive injections, and the peak area was measured. The results are shown in Table 6.

[0399] Table 6 Precision test results of Guangdong herbal tea granules

[0400]

[0401] Test results: The RSD of the precision test was 1.51%, n=6, and the instrument precision was good.

[0402] Solution stability investigation

[0403] The same batch of test solution of Guangdong Herbal Tea Granules (2308207) was injected into the liquid chromatograph at different times, the peak area was recorded, and the RSD was calculated. The results are shown in Table 7.

[0404] Table 7 Results of the stability study of Guangdong herbal tea granules solution

[0405]

[0406] Test results: After the test solution was placed for 24 hours, the average value of the 4-hydroxybenzoic acid peak area was 623.028, and the RSD was 1.48%. The test solution was stable at room temperature for 24 hours.

[0407] Repeatability test

[0408] Take approximately 2.5 g of 6 portions of Guangdong Herbal Tea Granules (batch 2308207), accurately weigh them, and place them in a 100-ml conical flask. Accurately add 50 ml of methanol and sonicate for 30 min. Remove the flask, cool to room temperature, add methanol to make up the lost weight, shake well, filter through a 0.45 μm microporous membrane, and collect the filtrate. Take 10 μl each of the reference solution and the test solution and inject them into a liquid chromatograph. Record the chromatogram and calculate the content. The results are shown in Table 8.

[0409] Table 8 Repeatability test results of Guangdong herbal tea granules

[0410]

[0411] Test results: The average 4-hydroxybenzoic acid content was 2.04 mg / bag, and the RSD was 1.70%, which was in line with the requirements of the pharmacopoeia, indicating that the repeatability test results were good.

[0412] Sample recovery test

[0413] Take 6 portions of Guangdong herbal tea granules (batch 2308207) powder, accurately weighed, each portion is about 1.25g, and accurately added with a reference solution equivalent to 100% of the added amount. According to the method of content determination, take 10μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, record the chromatogram, and calculate the recovery rate. The results are shown in Table 9.

[0414] Table 9 Guangdong herbal tea granules sample recovery test results

[0415]

[0416] Test results: The average recovery rate of 4-hydroxybenzoic acid content determination was 94.41%, ranging from 93.94% to 94.82%, with an RSD of 0.41%, which was in line with the requirements of the pharmacopoeia and showed that the experimental method was feasible.

[0417] 7.10 Durability Test When the measurement conditions (wavelength, column temperature, flow rate, ratio of mobile phase, amount of phosphoric acid and chromatographic column) change slightly, the content change is examined.

[0418] a Wavelength change

[0419] About 2.5 g of Guangdong Herbal Tea Granules (batch 2308207) were accurately weighed and operated according to the method under the content determination item. When the wavelength changed slightly, the sample was injected and the chromatogram was recorded. The content of the sample was calculated. The results are shown in Table 10.

[0420] Table 10 Test results of durability test of Guangdong herbal tea granules

[0421]

[0422] Test results: When the wavelength changed slightly, the average value of 4-hydroxybenzoic acid content was 2.08 mg / bag, and the RSD was 3.64%, which had little effect on the content determination results.

[0423] b Column temperature change

[0424] Take about 2.5 g of the content of Guangdong Herbal Tea Granules (batch 2308207), accurately weigh it, and operate according to the method under the content determination. When the column temperature changes slightly, inject the sample separately, record the chromatogram, and calculate the content of the sample. The results are shown in Table 11.

[0425] Table 11 Results of investigation on different column temperatures of Guangdong herbal tea granules

[0426]

[0427] Test results: When the column temperature changed slightly, the average value of 4-hydroxybenzoic acid content was 2.04 mg / bag, and the RSD was 2.45%, which had little effect on the content determination results.

[0428] cFlow rate change

[0429] About 2.5 g of the contents of Guangdong Herbal Tea Granules (batch 2308207) were accurately weighed and operated according to the method under the content determination item. When the initial flow rate changed slightly, the sample was injected separately, the chromatogram was recorded, and the content of the sample was calculated. The results are shown in Table 12.

[0430] Table 12 Results of investigation on different flow rates of Guangdong herbal tea granules

[0431]

[0432] Test results: When the flow rate changes slightly, the average value of 4-hydroxybenzoic acid content is 2.02 mg / bag, and the RSD is 1.74%, which has little effect on the content determination results.

[0433] d Change in the initial mobile phase ratio

[0434] Take about 2.5 g of the content of Guangdong Herbal Tea Granules (batch 2308207), accurately weigh it, and operate according to the method under the content determination item. When the proportion of the initial mobile phase changes slightly, inject the sample separately, record the chromatogram, and calculate the content of the sample. The results are shown in Table 13.

[0435] Table 13 Investigation results of the ratio of different initial mobile phases of Guangdong herbal tea granules

[0436]

[0437] Test results: When the proportion of the mobile phase changed slightly, the average value of the 4-hydroxybenzoic acid content was 2.07 mg / bag, and the RSD was 3.29%, which had little effect on the content determination results.

[0438] eChanges in the amount of phosphoric acid used in the mobile phase

[0439] About 2.5 g of the contents of Guangdong Herbal Tea Granules (batch 2308207) were accurately weighed and operated according to the method under the content determination item. When the amount of phosphoric acid in the mobile phase changed slightly, the sample was injected separately, the chromatogram was recorded, and the content of the sample was calculated. The results are shown in Table 14.

[0440] Table 14 Investigation results of changes in phosphoric acid dosage in the mobile phase of Guangdong herbal tea granules

[0441]

[0442] Test results: When the amount of phosphoric acid in the mobile phase changes slightly, the average value of 4-hydroxybenzoic acid content is 2.02 mg / bag, RSD

[0443] The content of 1.31% has little effect on the content determination results.

[0444] f Column changes

[0445] Take about 2.5 g of the content of Guangdong Herbal Tea Granules (batch 2308207), accurately weigh it, and operate according to the method under the content determination item. When the chromatographic column changes, inject the sample separately, record the chromatogram, and calculate the content of the sample. The results are shown in Table 15.

[0446] Table 15 Investigation results of different chromatographic columns for Guangdong herbal tea

[0447]

[0448] Test results: When the chromatographic column changes, the average value of 4-hydroxybenzoic acid content is 1.97 mg / bag, and the RSD is 2.22%, which has little effect on the content determination results.

[0449] Conclusion: When the determination conditions (wavelength, column temperature, flow rate, initial mobile phase ratio, amount of mobile phase phosphoric acid and chromatographic column) change slightly, there is basically no effect on the results of 4-hydroxybenzoic acid content determination. The robustness test results of this method are good.

[0450] Intermediate precision

[0451] Six portions of Guangdong Herbal Tea Granules (batch 2308207), approximately 2.5 g each, were accurately weighed and placed in a 100-ml conical flask. 50 ml of methanol was accurately added and ultrasonicated for 30 min (500 W). The flask was removed and allowed to cool to room temperature. The weight loss was made up with methanol, shaken well, and filtered through a 0.45 μm microporous membrane. The filtrate was collected and injected into a liquid chromatograph. The chromatogram was recorded and the content was calculated. The results are shown in Table 16.

[0452] Table 16 Intermediate precision test results of five-finger orange

[0453]

[0454] Test results: The average value of 4-hydroxybenzoic acid content was 2.01 mg / bag, and the RSD was 2.09%, which was in line with the requirements of the pharmacopoeia, indicating that the intermediate precision test results were good.

[0455] The above test results show that the high performance liquid chromatography method for determining the content of 4-hydroxybenzoic acid in Guangdong herbal tea granules has a good linear relationship, the precision, repeatability, stability, sample recovery rate and intermediate precision test results meet the requirements, and the durability test results are good. Therefore, high performance liquid chromatography can be selected as the method for determining the content of 4-hydroxybenzoic acid in Guangdong herbal tea granules and included in the standard.

Claims

1. A method for detecting the content of Guangdong herbal tea granules, comprising the following steps: Step A: Preparation of Reference Solution: Take 4-hydroxybenzoic acid reference substance and add methanol solution to prepare a solution to obtain a reference solution; Step B Preparation of Test Solution: Take Guangdong herbal tea granules, accurately weigh them, add extraction solvent, ultrasonically treat, filter, and take the filtrate to obtain the test solution; Step C: Accurately aspirate the reference solution and the test solution, inject them into a liquid chromatograph, determine, and calculate the content. The chromatographic conditions of the liquid chromatograph are as follows: a chromatographic column with octadecylsilane bonded silica gel as a filler; acetonitrile as mobile phase A, and phosphoric acid aqueous solution as mobile phase B, and perform gradient elution according to the provisions in the following table. The raw materials for preparing the Guangdong herbal tea granules include rhubarb, sesame, five-fingered orange, light bamboo leaf, oroxylum, slag leaf, fire charcoal motherwort, golden sand vine, sedge grass, and golden cherry root.

2. The content detection method according to claim 1, characterized in that The extraction solvent in step B is water or methanol, wherein the concentration of methanol can be 10%-100%.

3. The content detection method according to claim 1, characterized in that Step A: Preparation of Reference Solution: Take 4-hydroxybenzoic acid reference substance and add 50% methanol to prepare a solution containing 10 μg per ml to obtain the reference solution.

4. The content detection method according to claim 1, characterized in that Step B Preparation of the test solution: Take 1-4 g of Guangdong herbal tea granules, accurately weigh, accurately add 25-100 ml of methanol, ultrasonically treat for 15-60 min, take out, let it cool to room temperature, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain the test solution.

5. The content detection method according to claim 1, characterized in that The column temperature in the chromatographic conditions of step C is 20-40°C.

6. The method for detecting the content of the compound according to claim 1, wherein The chromatographic condition flow rate in step C is 0.5-1.5 mL / min.

7. The content detection method according to claim 1, characterized in that The chromatographic conditions in step C include a detection wavelength of 240-260 nm.

8. The content detection method according to claim 1, characterized in that The chromatographic conditions of step C are as follows: 0.05-0.3% phosphoric acid aqueous solution as mobile phase B.

9. A method for identifying Guangdong herbal tea granules, using thin layer chromatography to identify the active ingredients of citrus aurantium, golden cherry root, and cyperus rotundus, characterized in that S1 identification of five-finger orange Take 2-20g of Guangdong herbal tea granules, add 50ml of ethanol, heat under reflux in a water bath for 2 hours, filter, evaporate the filtrate to dryness, add 50ml of water to the residue, stir to dissolve, and shake and extract with ethyl acetate twice, each time 20ml, combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 1ml of methanol to prepare the S1 test solution; Separately, 5 g of the control medicinal material of Citrus aurantium was taken, 50 ml of water was added, and the mixture was heated under reflux for 2 hours, cooled, and filtered. Ethanol was added to the filtrate to make the alcohol content reach 45%, and the mixture was allowed to stand for 2 hours, filtered, and the filtrate was evaporated to dryness. 1 ml of methanol was added to the residue to dissolve it, and this was used as the S1 control medicinal material solution. According to the thin layer chromatography test, 5 μl of each of the S1 test solution and the S1 control medicinal material solution were respectively spotted on the same silica gel G thin layer plate, and petroleum ether-ethyl acetate with a volume ratio of 5:5 was used as the developing solvent. The humidity was below 70%. The plate was removed, dried, and sprayed with 1% ferric chloride methanol solution until a clear color was developed. The plate was examined under sunlight. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions in the chromatogram of the control medicinal material. S2 Golden Sakura Root Identification Take 2-20g of Guangdong herbal tea granules, add 100ml of water to dissolve, shake and extract with ethyl acetate three times, each time 50ml, combine the ethyl acetate extracts, wash twice with water, each time 50ml, evaporate the ethyl acetate solution to dryness, and add 1ml of methanol to dissolve the residue as the S2 test solution; Take 2.5g of golden cherry root reference medicinal material, add 50ml of water, heat and reflux for 40 minutes, let cool, centrifuge, take the supernatant, shake and extract with ethyl acetate 3 times, 50ml each time, combine the ethyl acetate extracts, wash with water 2 times, 50ml each time, evaporate the ethyl acetate solution to dryness, add 1ml of methanol to dissolve the residue to prepare S2 reference medicinal material solution, test according to thin layer chromatography, take 4μl of S2 reference medicinal material solution and 10μl of S2 test sample solution, respectively, and spot them on the same silica gel G thin layer plate to make 1cm strips. Develop twice with the upper layer solution of n-hexane-ethyl acetate-acetone-formic acid-water in a volume ratio of 3:4:3:0.3:1 as the developing solvent, or develop once with the lower layer solution of chloroform-ethyl acetate-methanol-water in a volume ratio of 20:20:40:10, which was separated below 10°C. Take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and examine them under sunlight. The test sample chromatogram and the control medicinal material chromatogram under sunlight show the same blue-purple spots at the corresponding positions; S3 identification of sorghum Take 1-10g of Guangdong herbal tea granules, add 30ml of water to dissolve, extract twice with water-saturated n-butanol solution, 50ml each time, combine the water-saturated n-butanol extracts, add ammonia water to extract three times, 20ml each time, retain the water-saturated n-butanol solution, wash three times with n-butanol-saturated water, 20ml each time, evaporate the water-saturated n-butanol solution to dryness, add 1ml of methanol to dissolve the residue, and use it as the S3 test solution; Take another 3g of the control medicinal material of Rhizoma Cibotii, add 50ml of water, heat and reflux for 1 hour, filter, extract twice with a saturated n-butanol solution with water, 50ml each time, combine the water-saturated n-butanol extracts, extract three times with ammonia water, 20ml each time, retain the water-saturated n-butanol solution, wash three times with n-butanol-saturated water, 20ml each time, evaporate the water-saturated n-butanol solution to dryness, add 1ml of methanol to dissolve the residue to prepare S3 control medicinal material solution, test according to the thin layer chromatography method, take 1μl of the S3 control medicinal material solution and 4μl of the S3 test sample solution, respectively, spot them on the same silica gel G thin layer plate, use ethyl acetate-methanol-glacial acetic acid-water with a volume ratio of 11:1.5:1.5:2 as the developing solvent, develop, take out, dry, spray with an appropriate amount of 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and examine under 365nm ultraviolet light. In the chromatogram of the test sample, at the corresponding position of the chromatogram of the control medicinal material, a fluorescent spot of the same color appears; The raw materials for preparing the Guangdong herbal tea granules include rhubarb, sesame, five-fingered orange, light bamboo leaf, oroxylum, slag leaf, fire charcoal motherwort, golden sand vine, sedge grass, and golden cherry root.

10. A method for detecting Guangdong herbal tea granules, characterized in that: According to the identification method of claim 9, thin layer chromatography is used to identify the active ingredients of five-fingered orange, golden cherry root, and rhododendron, and according to the content detection method of right 1, liquid chromatography is used to detect the 4-hydroxybenzoic acid content of Guangdong herbal tea granules.

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