Quality detection method of five-color nine-ingredient granules

Abstract

The invention discloses a quality detection method of five-color nine-ingredient granules, and belongs to the field of traditional Chinese medicine preparation analysis. According to the quality detection method, curcuma zedoary, polygonum cuspidatum and liquorice in the five-color nine-ingredient granules are identified by thin-layer chromatography, the content of polydatin in the five-color nine-ingredient granules is determined by high-performance liquid chromatography, and thin-layer chromatography detection conditions, test solution extraction conditions, high-performance liquid chromatography mobile phase conditions and the like are investigated and optimized. The method is high in specificity and good in accuracy, precision and stability, so that the quality control standard of the five-color nine-ingredient granule preparation is established, the quality of the five-color nine-ingredient granules is effectively controlled, and the clinical curative effect and safety of the medicine are guaranteed.

Description

Technical Field

[0001] The present invention relates to a method for quality inspection of a five-color and nine-flavor granule, belonging to the technical field of analysis of traditional Chinese medicine preparations. Background Art

[0002] Flat warts are viral skin diseases caused by human papillomavirus (HPV) infection. Adolescents are susceptible, and they tend to occur in exposed areas such as the face, back of the hand, and forearm. They appear as flat, raised papules of brown or normal skin color, round or oval in shape, ranging from the size of a grain of rice to a soybean. Long-existing flat warts can fuse into patches, and in severe cases, they can spread throughout the body. This disease is often chronic, prone to recurrence, and has a certain degree of infectivity. It can auto-inoculate or be mutually infected among people through direct or indirect contact.

[0003] Clinically, the treatment of flat warts mainly relies on physical therapy and drug therapy. The mechanism of action of drug therapy is clear, but there are problems such as long treatment time, incomplete treatment, and easy recurrence of skin lesions. Physical therapy, surgical resection and other means can directly destroy skin warts, with significant short-term curative effects, but they cannot inhibit and eliminate the virus, cannot reduce the recurrence rate, and have disadvantages such as large wound surfaces, easy infection, difficult healing, painful treatment, easy formation of scars and pigmentation.

[0004] In the previously authorized patent CN116211985B of the applicant, a traditional Chinese medicine composition, a traditional Chinese medicine preparation and their applications for treating flat warts are disclosed. The traditional Chinese medicine composition is composed of the following raw materials: polygonum cuspidatum, oldenlandia diffusa, arnebia euchroma, isatis root, oyster, curcuma aromatica, safflower, zedoary turmeric and licorice. The prepared pharmaceutical composition has the effects of clearing heat and detoxifying, promoting qi circulation and removing blood stasis, and has a good therapeutic effect on flat warts caused by qi stagnation and blood stasis and scattered flat papules on the skin. However, the preparation method of the traditional Chinese medicine composition is the decoction method, with complex components, difficult quality control, short storage time, inconvenient to carry, and unstable clinical curative effects. Therefore, it is necessary to prepare it into a granule preparation - a five-color and nine-flavor granule.

[0005] However, there is currently no literature report on a comprehensive analysis of the chemical components of the traditional Chinese medicine compound preparation containing polygonum cuspidatum and zedoary turmeric required to be protected by the present invention. In the prior art, only the quality analysis of single components in this traditional Chinese medicine preparation is carried out, and there is no relatively comprehensive and systematic quality control method to reflect the quality status of the main reference sample components in the traditional Chinese medicine compound preparation of the present invention, and it is impossible to effectively control its production process and product quality, and it is unable to better ensure its clinical curative effects. Summary of the Invention

[0006] To solve the problems of the prior art, according to the characteristics and preparation process of traditional Chinese medicinal materials in the prescription, the present invention repeatedly studies and screens the identification test and content determination of the granule, selects Polygonum cuspidatum, the monarch drug, and Curcuma zedoaria and Glycyrrhiza uralensis Fisch. from several other medicinal materials as reference medicinal materials for quality control, thereby providing a detection method for the five-color and nine-flavor granule preparation; the method is a specific and feasible method for component identification and content determination. The established identification method has strong specificity, and the content determination method has good specificity, accuracy, precision, linearity and range, and stability, providing detection indicators, detection means and technical methods for production units and testing institutions, etc., so as to better guide production, make the production process control more strict and reasonable, and effectively control the quality of the five-color and nine-flavor granules, thereby ensuring the clinical efficacy and safety of the drug.

[0007] The present invention provides a quality detection method for a five-color and nine-flavor granule. The five-color and nine-flavor granule is prepared from raw medicinal materials and auxiliary materials with a prescription ratio of 10-30 parts of Polygonum cuspidatum, 20-40 parts of Hedyotis diffusa Willd., 5-20 parts of Arnebia euchroma (Royle) Johnst., 10-30 parts of Isatis tinctoria L., 20-40 parts of Oyster, 5-20 parts of Curcuma aromatica Salisb., 8-18 parts of Carthamus tinctorius L., 5-30 parts of Curcuma zedoaria (Christm.) Rosc., and 1-5 parts of Glycyrrhiza uralensis Fisch. The method includes identifying Curcuma zedoaria, Polygonum cuspidatum and Glycyrrhiza uralensis Fisch. in the five-color and nine-flavor granule by thin-layer chromatography, and determining the content of polydatin in the five-color and nine-flavor granule by high-performance liquid chromatography; wherein:

[0008] The steps for identifying Curcuma zedoaria by the thin-layer chromatography method include:

[0009] 1.1) Add water to the five-color and nine-flavor granule according to the ratio of 1.5 g: 25 ml, perform ultrasonic treatment, extract the supernatant with ethyl acetate, evaporate to dryness, dissolve the residue in methanol to obtain a test solution;

[0010] 1.2) Add methanol to the reference medicinal material of Curcuma zedoaria (Curcuma wenyujin Y. H. Chen et C. Ling) according to the ratio of 0.5 g: 30 ml, perform ultrasonic treatment, centrifuge, evaporate the supernatant to dryness, dissolve the residue in water, extract with dichloromethane, discard the dichloromethane solution, extract the aqueous phase with ethyl acetate, evaporate to dryness, and dissolve the residue in methanol to obtain a reference medicinal material solution;

[0011] 1.3) Add water to the negative control sample prepared from other medicinal materials except Curcuma zedoaria, namely Polygonum cuspidatum, Hedyotis diffusa Willd., Arnebia euchroma (Royle) Johnst., Isatis tinctoria L., Oyster, Curcuma aromatica Salisb., Carthamus tinctorius L., and Glycyrrhiza uralensis Fisch. according to the same ratio as the prescription ratio and auxiliary materials according to the ratio of 1.5 g: 25 ml, perform ultrasonic treatment, centrifuge, extract the supernatant with ethyl acetate, evaporate to dryness, and dissolve the residue in methanol to obtain a negative control solution;

[0012] 1.4) Pipette 5 - 10 μl of each of the above - mentioned test solution and negative control solution, and 3 - 5 μl of the reference crude drug solution respectively onto the same silica gel G thin - layer plate. Add the developing solvent, develop, take out, air - dry, spray with the color - developing agent, and heat - blow until the spots are clear. Examine under daylight. In the chromatogram of the test sample, spots of the same color should appear at the corresponding positions as in the chromatogram of the reference crude drug; in the chromatogram of the negative control, spots of the same color should not appear at the corresponding positions as in the chromatogram of the reference crude drug.

[0013] The steps for identifying Polygonum cuspidatum by thin - layer chromatography are as follows:

[0014] 2.1) Take the Wuse Jiuwei Granules, add water according to the ratio of 1.5 g:25 ml, perform ultrasonic treatment, extract the supernatant with ethyl acetate, evaporate to dryness, dissolve the residue in methanol to obtain the test solution.

[0015] 2.2) Weigh accurately emodin reference substance, dissolve it in methanol to prepare a solution containing 0.1 mg of emodin per 1 ml as the reference solution.

[0016] 2.3) Take the negative control sample prepared from other medicinal materials (except Polygonum cuspidatum) such as Hedyotis diffusa, Arnebia euchroma, Isatis tinctoria, Oyster, Curcuma aromatica Salisb., Carthamus tinctorius, Rhizoma zedoariae, and Glycyrrhiza uralensis Fisch. according to the same ratio as the prescription ratio and excipients. Add water according to the ratio of 1.5 g:25 ml, perform ultrasonic treatment, centrifuge, extract the supernatant with ethyl acetate, evaporate to dryness, dissolve the residue in methanol to obtain the negative control solution.

[0017] 2.4) Pipette 5 - 10 μl of each of the test solution in step 2.1) and the negative control solution in step 2.3), and 10 μl of the reference solution in step 2.2) respectively onto the same silica gel G thin - layer plate. Add the developing solvent, develop, take out, air - dry, and examine under an ultraviolet lamp at 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color should appear at the corresponding positions as in the chromatogram of the reference substance; in the chromatogram of the negative control, fluorescent spots of the same color should not appear at the corresponding positions as in the chromatogram of the reference substance.

[0018] The steps for identifying Glycyrrhiza uralensis Fisch. by thin - layer chromatography are as follows:

[0019] 3.1) Take the Wuse Jiuwei Granules, add water according to the ratio of 1.5 g:25 ml, perform ultrasonic treatment, extract the supernatant with dichloromethane, discard the dichloromethane layer, extract the aqueous phase with water - saturated n - butanol, wash with 0.1 - 0.2 mol / L sodium hydroxide solution, discard the washing solution, then wash with 0.2 - 1 mol / L hydrochloric acid solution, discard the washing solution, evaporate to dryness, dissolve the residue in methanol to obtain the test solution.

[0020] 3.2) Weigh accurately liquiritin reference substance, dissolve it in methanol to prepare a solution containing 0.1 mg of liquiritin per 1 ml as the reference solution.

[0021] 3.3) The negative control sample prepared from polygonum cuspidatum, oldenlandia diffusa, arnebia euchroma (Royle) Johnst, isatis root, oyster, curcuma aromatica Salisb., safflower, and zedoary turmeric, excluding licorice, according to the same ratio as the prescription ratio and excipients, is added to water at a ratio of 1.5 g:25 ml, ultrasonically treated, centrifuged, the supernatant is extracted with dichloromethane, the dichloromethane solution is discarded, the aqueous phase is extracted with water-saturated n-butanol and then washed with 0.1 - 0.2 mol / L sodium hydroxide solution, the washing solution is discarded, then washed with 0.2 - 1 mol / L hydrochloric acid solution, the washing solution is discarded, evaporated to dryness, and the residue is dissolved in methanol to obtain the negative control solution;

[0022] 3.4) Pipette 5 - 10 μl each of the test solution in step 3.1), the negative control solution in step 3.3), and 10 μl of the reference solution in step 3.2), respectively, spot them on the same silica gel G thin-layer plate, add the developing agent, develop, take out, dry in air, spray with the color-developing agent, heat at 105 °C until the spots are clear; view under an ultraviolet lamp at 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference substance; in the chromatogram of the negative control, fluorescent spots of the same color do not appear at the corresponding positions as in the chromatogram of the reference substance;

[0023] The determination of the content of polydatin in the Wuse Jiuwei Granules by high performance liquid chromatography includes the following steps:

[0024] 4.1) Chromatographic conditions and system suitability test

[0025] Using octadecylsilane chemically bonded silica gel as the filler; using acetonitrile as mobile phase A and water as mobile phase B; the detection wavelength is 306 nm, and the number of theoretical plates calculated based on the polydatin peak should be not less than 5000;

[0026] 4.2) Preparation of the test solution

[0027] Accurately weigh the Wuse Jiuwei Granules, place them in a stoppered conical flask, accurately add 70 v / v% methanol to make a solution containing 50 mg per 1 mL, weigh, ultrasonically treat, cool, weigh again, make up the lost weight with 70 v / v% methanol, shake well, filter, and take the subsequent filtrate to obtain;

[0028] 4.3) Preparation of the reference solution

[0029] Accurately weigh the polydatin reference substance, place it in a volumetric flask, dissolve it with 70 v / v% methanol and dilute to the mark, shake well, and make a reference solution containing 60 μg of polydatin per 1 mL to obtain;

[0030] 4.4) Determination

[0031] Precisely pipette 10 μL each of the reference substance solution and the test solution, inject into a high performance liquid chromatograph, and determine, thus obtaining the results.

[0032] Further, the prescription ratio is as follows: 15 parts of Polygonum cuspidatum, 30 parts of Hedyotis diffusa, 30 parts of Oyster, 15 parts of Isatis indigotica, 10 parts of Lithospermum erythrorhizon, 12 parts of Carthamus tinctorius, 10 parts of Curcuma zedoaria, 10 parts of Curcuma aromatica, and 3 parts of Licorice.

[0033] Further, the developing agent in step 1.4) is composed of petroleum ether - ethyl acetate - formic anhydride at 60 - 90 °C with a volume ratio of 20:20:1, and the color developing agent is a 10 v / v% sulfuric acid - ethanol solution containing 2 v / v% vanillin.

[0034] Further, the developing agent in step 2.4) is composed of petroleum ether - ethyl acetate at 60 - 90 °C with a volume ratio of 2:1.

[0035] Further, the developing agent in step 3.4) is composed of ethyl acetate - formic anhydride - water with a volume ratio of 16:1:1, and the color developing agent is a 10 v / v% sulfuric acid - ethanol solution.

[0036] Further, the mobile phase in step 4.1) adopts gradient elution as shown in the following table:

[0037]

[0038]

[0039] Further, the column temperature in step 4.1) is set at 30 °C, and the flow rate of the mobile phase is set at 1.0 mL / min.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The present invention selects Polygonum cuspidatum as the monarch drug and Curcuma zedoaria and Licorice among several other drugs as the reference medicinal materials for quality control, provides a specific and feasible method for component identification and content determination of the Five - Color and Nine - Flavor Granules. The present invention has investigated and optimized the thin - layer chromatography detection conditions, the extraction conditions of the test solution, the mobile phase of the high - performance liquid chromatography method, etc., making the adopted method have strong specificity, good accuracy, precision, and stability, thereby establishing a quality control standard for the Five - Color and Nine - Flavor Granule preparation to effectively control the quality of the Five - Color and Nine - Flavor Granules, and thus ensuring the clinical efficacy and safety of the drug. Description of the Drawings

[0042] Figure 1 It is the thin - layer identification diagram of Curcuma zedoaria, wherein: 1 - reference medicinal material of Curcuma zedoaria, 2, 3, 4 - test samples, 5 - negative control;

[0043] Figure 2Thin layer identification diagram of Polygonum cuspidatum, where: 1, 2, 3 - test samples, 4 - emodin reference substance, 5 - negative control;

[0044] Figure 3 Thin layer identification diagram of Glycyrrhiza uralensis Fisch., where: 1, 2, 3 - test samples, 4 - liquiritin reference substance, 5 - negative control;

[0045] Figure 4 Chromatogram of polydatin reference substance;

[0046] Figure 5 Chromatogram of polydatin in the test sample of the granule prepared in Example 1;

[0047] Figure 6 Chromatogram of the negative sample without Polygonum cuspidatum. Detailed implementation mode

[0048] To make the present invention more obvious and understandable, preferred embodiments are hereby described in detail in conjunction with the accompanying drawings as follows.

[0049] Example 1. A five - color and nine - flavor granule for treating flat warts

[0050] The five - color and nine - flavor granule comprises the following raw materials in parts by mass:

[0051] Polygonum cuspidatum 10 - 30 parts, Hedyotis diffusa 20 - 40 parts, Arnebia euchroma 5 - 20 parts, Isatis indigotica 10 - 30 parts, Oyster 20 - 40 parts, Curcuma aromatica 5 - 20 parts, Safflower 8 - 18 parts, Rhizoma zedoariae 5 - 30 parts, Glycyrrhiza uralensis Fisch. 1 - 5 parts.

[0052] Preparation method:

[0053] Step 1: For the nine kinds of Chinese herbal medicine pieces in the prescription, decoct the oyster piece first, and then add the remaining pieces;

[0054] Step 2: Decoct with water for 1 hour, then filter the decoction to obtain filtrate A;

[0055] Step 3: Continue to decoct with water for 1 hour and then filter to obtain filtrate B;

[0056] Step 4: Combine filtrate A and filtrate B, concentrate, and let stand for 12 hours;

[0057] Step 5: Take the supernatant and concentrate it to an appropriate amount, pass it through a sieve to obtain a clear paste, mix the clear paste evenly with dextrin, make the obtained soft material into wet granules, dry, sieve and size the granules, and finally mix them, and package them in bags of 12 g each to obtain the product.

[0058] Example 2. Thin layer identification study of Rhizoma zedoariae

[0059] Take 1.5 g of the sample prepared in Example 1, add 25 ml of water, and treat with ultrasound for 30 minutes. The supernatant is extracted with ethyl acetate and evaporated to dryness. The residue is dissolved with 1 ml of methanol to prepare the test solution.

[0060] Take 0.5 g of Curcuma zedoaria (Wenyujin) as a control medicinal material, add 30 ml of methanol, ultrasonically treat for 30 minutes, centrifuge, evaporate the supernatant, dissolve the residue in 25 ml of water, extract with dichloromethane, discard the dichloromethane liquid, extract the aqueous phase with ethyl acetate, evaporate to dryness, and dissolve the residue in 1 ml of methanol as a control medicinal material solution.

[0061] Pipette 5-10 μl of the test sample solution and 3-5 μl of the control drug solution, spot them separately on the same silica gel G thin layer plate, use petroleum ether (60-90℃)-ethyl acetate-anhydrous formic acid (20:20:1) as the developing agent, develop, take out, dry, spray with 10 v / v% sulfuric acid-ethanol solution containing 2 v / v% vanillin, blow with hot air until the spots are clear, and examine under sunlight.

[0062] For the specificity test, 1.5 g of negative control sample prepared by the prescription ratio of other medicinal ingredients except Curcuma zedoariae was taken, 25 ml of water was added, ultrasonic treatment was performed for 30 minutes, centrifuged, the supernatant was extracted with ethyl acetate, evaporated to dryness, and 1 ml of methanol was added to the residue to dissolve it as the negative control solution. After development, in the chromatogram of the test sample, a spot of the same color appeared at the corresponding position of the chromatogram of the control medicinal material; in the chromatogram of the negative control, a spot of the same color did not appear at the corresponding position of the chromatogram of the control medicinal material.

[0063] This indicates that the negative control lacking Curcuma zedoaria has no interference with the experiment. Figure 1 As shown, 1-Zhuzhu control medicinal material, 2, 3, 4-test samples, 5-negative control.

[0064] Embodiment 3, thin layer identification research of Curcuma zedoaria

[0065] Take 1.5 g of the sample prepared in Example 1, add 25 ml of water, and ultrasonically treat for 30 minutes. Centrifuge to take the supernatant, extract with ethyl acetate, evaporate to dryness, and dissolve with 1 ml of methanol as the test solution.

[0066] Take 0.5 g of Curcuma zedoaria (Wenyujin) as a control medicinal material, add 30 ml of methanol, ultrasonically treat for 30 minutes, centrifuge, evaporate the supernatant, dissolve the residue in 25 ml of water, extract with dichloromethane, discard the dichloromethane liquid, extract the aqueous phase with ethyl acetate, evaporate to dryness, and dissolve the residue in 1 ml of methanol as a control medicinal material solution.

[0067] Absorb 10 μl of the above-mentioned test solution and 5 μl of the control crude drug solution, and spot them respectively on the same silica gel G thin-layer plate. Use petroleum ether (60 - 90 °C) - ethyl acetate (1:1) or dichloromethane - ethyl acetate - anhydrous formic acid (20:10:1) as the developing agent, develop, spray with 10 v / v% sulfuric acid - ethanol solution or 10 v / v% sulfuric acid - ethanol solution containing 2 v / v% vanillin, heat at 105 °C until the spots are clear, and examine under daylight.

[0068] The results show that only changing the developing agent or changing both the developing agent and the color-developing agent simultaneously results in poor developing effect or unclear color development.

[0069] Example 4. TLC Identification Study of Polygonum cuspidatum

[0070] Take 1.5 g of the sample prepared in Example 1, add 25 ml of water, ultrasonically treat for 30 minutes, extract the supernatant with ethyl acetate, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution.

[0071] Precisely weigh 1 mg of emodin reference substance, dissolve it in 10 ml of methanol to prepare a solution containing 0.1 mg of emodin per 1 ml as the reference substance solution.

[0072] Absorb 5 - 10 μl of the above-mentioned test solution and 10 μl of the reference substance solution, and spot them respectively on the same silica gel G thin-layer plate. Use petroleum ether (60 - 90 °C) - ethyl acetate (2:1) as the developing agent, develop, take out, air dry, and examine under an ultraviolet lamp (365 nm).

[0073] Specificity test: Take 1.5 g of the negative control sample prepared from other medicinal flavors according to the prescription ratio except Polygonum cuspidatum, add 25 ml of water, ultrasonically treat for 30 minutes, centrifuge, extract the supernatant with ethyl acetate, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the negative control solution. After development, in the test sample chromatogram, at the corresponding position to the reference substance chromatogram, there are fluorescent spots showing the same color; in the negative control chromatogram, at the corresponding position to the control crude drug chromatogram, there are no fluorescent spots showing the same color.

[0074] It shows that the negative control without Polygonum cuspidatum has no interference with the experiment, and the test results are as Figure 2 shown, where 1, 2, 3 - test sample, 4 - emodin reference substance, 5 - negative control.

[0075] Example 5. TLC Identification Study of Polygonum cuspidatum

[0076] Take 1.5 g of the sample prepared in Example 1, add 25 ml of water, ultrasonically treat for 30 minutes, extract the supernatant with ethyl acetate, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution.

[0077] Accurately weigh 1 mg of emodin reference substance, dissolve it in 10 ml of methanol to prepare a solution containing 0.1 mg of emodin per 1 ml as the reference substance solution.

[0078] Absorb 10 μl of the above-mentioned test solution and 10 μl of the reference substance solution, respectively spot them on the same silica gel G thin-layer plate, use petroleum ether (30 - 60 °C)-ethyl formate-formic acid upper layer (15:5:1) or petroleum ether (60 - 90 °C)-ethyl acetate (4:1) as the developing agent, develop, take out, air dry, and examine under an ultraviolet lamp (365 nm).

[0079] As a result, the developing effect is poor or the color development is not clear.

[0080] Example 6. TLC Identification Study of Licorice

[0081] Take 1.5 g of the sample prepared in Example 1, add 25 ml of water, ultrasonically treat for 30 minutes, centrifuge, extract the supernatant with dichloromethane, discard the dichloromethane solution, extract the aqueous phase with water-saturated n-butanol and then wash with 0.1 mo1 / L sodium hydroxide solution, discard the washing solution, wash again with 0.6 mol / L hydrochloric acid solution, discard the washing solution, evaporate to dryness, dissolve the residue in 1 ml of methanol as the test solution.

[0082] Accurately weigh 1 mg of liquiritin reference substance, dissolve it in methanol to prepare a solution containing 0.l mg of liquiritin per 1 ml as the reference substance solution.

[0083] Absorb 5 - 10 μl of the above-mentioned test solution and 10 μl of the reference substance solution, respectively spot them on the same silica gel G thin-layer plate, use ethyl acetate-anhydrous formic acid-water (16:1:1) as the developing agent, develop, take out, air dry, spray with 10 v / v% sulfuric acid-ethanol solution, heat at 105 °C until the spots are clear, and examine under an ultraviolet lamp (365 nm).

[0084] Specificity test: Take 1.5 g of the negative control sample prepared from other medicinal flavors except licorice according to the prescription ratio, add 25 ml of water, ultrasonically treat for 30 minutes, centrifuge, extract the supernatant with dichloromethane, discard the dichloromethane solution, extract the aqueous phase with water-saturated n-butanol and then wash with 0.1 mol / L sodium hydroxide solution, discard the washing solution, wash again with 0.6 mol / L hydrochloric acid solution, discard the washing solution, evaporate to dryness, dissolve the residue in 1 ml of methanol as the negative control solution. After development, in the test sample chromatogram, at the corresponding position to the reference substance chromatogram, there are fluorescent spots showing the same color; in the negative control chromatogram, at the corresponding position to the reference medicinal material chromatogram, there are no fluorescent spots showing the same color.

[0085] It shows that the negative control without licorice has no interference with the experiment, and the test results are as Figure 3 shown, where 1, 2, 3 - test sample, 4 - liquiritin reference substance, 5 - negative control.

[0086] Example 7, TLC Identification Study of Licorice

[0087] Take 3 g of the sample prepared in Example 1, add 25 ml of water, ultrasonicate for 10 minutes, centrifuge, extract the supernatant once with 20 ml of dichloromethane, discard the dichloromethane liquid, saturate the aqueous phase with water-saturated n-butanol twice, 20 ml each time, combine the n-butanol, wash the n-butanol liquid with 10 ml of 0.1% sodium hydroxide solution, add 10 ml of hydrochloric acid solution (0.5 ml of concentrated hydrochloric acid is diluted to 10 ml with water), shake well, extract twice with ethyl acetate, 20 ml each time, combine the ethyl acetate liquid, evaporate to dryness, add 1 ml of methanol to dissolve the residue, and use it as the test solution.

[0088] Accurately weigh 1 mg of liquiritin reference substance, add methanol to dissolve it, and prepare a solution containing 0.1 mg of liquiritin per 1 ml as the reference substance solution.

[0089] Pipette 10 μl of the test solution and 10 μl of the reference solution, spot them separately on the same silica gel G thin layer plate, use ethyl acetate-anhydrous formic acid-water (16:1:1) as the developing agent, develop, take out, dry, spray with 10 v / v% sulfuric acid-ethanol solution, heat at 105°C until the spots are clear, and examine under ultraviolet light (365 nm).

[0090] As a result, the background color was too dark, resulting in unclear spots of the indicator components.

[0091] Example 8, Content Determination of Five-Color Nine-Flavor Granules

[0092] 8.1 Chromatographic conditions and system suitability test

[0093] Chromatographic column: octadecylsilane bonded silica gel as filler; acetonitrile as mobile phase A, water as mobile phase B, gradient elution according to the ratio in Table 1; detection wavelength is 306nm; injection volume: 10μl; flow rate: 1.0mL / min; column temperature: 30℃; the theoretical plate number is calculated based on the polydatin peak and should not be less than 5000.

[0094] Table 1 Gradient elution table

[0095] Time (min) Mobile phase A (v / v%) Mobile phase B (v / v%) 0～10 20 80 10～10.1 20→80 80→20 10.1～12 80 20 12～13 80→20 20→80 13～20 20 80

[0096] 8.2 Test solution

[0097] Take 1 g of the sample powder prepared in Example 1, accurately weigh it, place it in a 150 ml conical flask with a stopper, accurately add 20 ml of 70 v / v% methanol, weigh it, ultrasonically treat it for 30 minutes, cool it, weigh it again, make up the lost weight with 70 v / v% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0098] 8.3 Reference solution

[0099] Take the polydatin reference substance, accurately weigh it, place it in a 100 ml volumetric flask, dissolve it with 70 v / v% methanol and dilute it to the mark, shake well, and prepare a reference substance solution containing 60 μg of polydatin per 1 mL, thus obtaining.

[0100] 8.4 Determination

[0101] Precisely pipette 10 μL each of the reference substance solution and the test solution, inject them into a high performance liquid chromatograph, determine, record the chromatogram, and calculate the content based on the peak area of polydatin by the external standard method, thus obtaining.

[0102] The test results are as Figure 4 and Figure 5 shown.

[0103] (1) Specificity test

[0104] Refer to the proportion of medicinal herbs in the prescription of Example 1, self - prepare a group of herbs without polygonum cuspidatum, and prepare a negative control sample without polygonum cuspidatum according to the preparation method of Example 1. Take 1 g of the powder and prepare a negative control solution according to the preparation method of the test solution under item 8.2.

[0105] According to the chromatographic conditions under item 8.1, precisely pipette 10 μl of the negative control solution and inject it into the liquid chromatograph. The results show that there is no interference in the negative control solution at the position of the chromatographic peak corresponding to polydatin.

[0106] The test results are as Figure 6 shown, indicating that the content determination of polydatin in the five - color and nine - flavor granule provided by the present invention has high specificity.

[0107] (2) Investigation of linear relationship

[0108] Accurately weigh 6.0 mg of the polydatin reference substance, place it in a 50 ml volumetric flask, dissolve it with 70 v / v% methanol and dilute it to the mark. Precisely pipette different volumes of the reference substance solution respectively, and prepare a series of reference substance solutions with concentrations of 6, 12, 24, 48, 96 μg / mL with 70 v / v% methanol. Precisely pipette 10 μL and inject it into a high performance liquid chromatograph for determination. The results are shown in Table 2. Using the reference substance concentration (X) as the abscissa and the average integral value of the peak area (Y) as the ordinate for linear regression, the regression equation is Y = 42931.01614X - 27685.87500, and the linear range of polydatin is 6.315 - 101.04 μg / mL (R 2 = 0.99997).

[0109] Table 2 Investigation of linear relationship

[0110]

[0111] (3) Precision test

[0112] Accurately weigh the polydatin reference substance, prepare the reference substance solution according to the method under item 8.3, continuously inject samples for determination 5 times according to the chromatographic conditions under item 8.1, record the relative retention time and relative peak area of polydatin, and calculate the RSD value. The results are shown in Table 3.

[0113] Table 3 Results of precision test

[0114]

[0115]

[0116] As can be seen from Table 3, the content determination method of polydatin in the five-color nine-flavor granule provided by the present invention has high precision.

[0117] (4) Stability test

[0118] Take the five-color nine-flavor granule sample prepared in Example 1, prepare the test solution according to item 8.2, place it at room temperature for 24 h, and after 0 h, 2 h, 4 h, 6 h, 10 h, 12 h, 24 h and placing it in the refrigerator for 24 h, respectively, inject it into the high performance liquid chromatograph according to the chromatographic conditions under item 8.1, record the peak area of polydatin and calculate the RSD value. The results show that polydatin in the test solution is stable within 24 h, as shown in Table 4.

[0119] Table 4 Results of stability test

[0120] Time (h) Retention time Peak area 0 8.547 2735551 2 8.548 2731940 4 8.543 2716606 6 8.569 2724113 10 8.564 2727184 12 8.614 2730798 24 8.538 2750102 24(4℃) 8.520 2721305 Average value 8.560 2730899 RSD (%) 0.328 0.376

[0121] (5) Repeatability test

[0122] Take the five-color nine-flavor granule sample prepared in Example 1, prepare the test solution according to the method under item 8.2, prepare 6 portions in parallel, determine the peak area of polydatin, and calculate the content and RSD value. The results are shown in Table 5, indicating that the method has good repeatability.

[0123] Table 5 Results of repeatability test

[0124]

[0125] (6) Recovery test by addition

[0126] Using the standard addition method, 0.5 g of the Wuse Jiuwei Granules sample prepared in Example 1 was accurately weighed and placed in a 20-ml volumetric flask. 5 ml of the reference solution was accurately measured and added, and about 15 ml of 70 v / v% methanol solution was added. It was sonicated (power 380 W, frequency 37 kHz) for 30 minutes, cooled to room temperature, diluted to the mark with 70 v / v% methanol solution, shaken well, filtered, and the subsequent filtrate was taken to obtain the spiked test solution at 100% concentration level. Six portions were prepared in the same manner. According to the chromatographic conditions under item 8.1, 10 μl of each of the above spiked test solutions was accurately drawn and injected into the high-performance liquid chromatograph. The chromatogram was recorded, and the recovery rate was calculated according to the following formula. The results are shown in Table 6:

[0127]

[0128] Table 6 Results of Spike Recovery

[0129]

[0130] From the above results, it can be seen that the average recovery rate of polydatin in the 6 spiked test samples was 98.2%, and the RSD was 0.107%, meeting the requirements.

[0131] Example 9. Content Determination of Wuse Jiuwei Granules

[0132] Chromatographic Conditions and System Suitability Test: Agilent Zorbax Eclipse Plus C18 (column length 25 cm, inner diameter 4.6 mm, particle size 5 μm) was used as the chromatographic column; acetonitrile-water (20:80) was used as the mobile phase, the detection wavelength was 306 nm; the column temperature was 30 °C; the flow rate was 1.0 ml / min.

[0133] Test Solution: 1 g of the sample powder prepared in Example 1 was accurately weighed and placed in a stoppered conical flask. 20 ml of water, 30 v / v% methanol aqueous solution, 50 v / v% methanol aqueous solution, 70 v / v% methanol aqueous solution, and methanol were accurately added respectively, weighed, sonicated (37 kHz, 380 w) for 30 minutes, cooled to room temperature, weighed again, and the lost weight was made up with the corresponding solvent, shaken well, filtered, and the subsequent filtrate was taken to obtain the solution.

[0134] Reference Solution: Polydatin reference substance was accurately weighed and placed in a 100-ml volumetric flask, dissolved and diluted to the mark with 70 v / v% methanol, shaken well, to obtain a reference solution containing 60 μg of polydatin per 1 mL.

[0135] 10 μL of the reference solution and the test solution were accurately drawn respectively and injected into the high-performance liquid chromatograph for determination. The content of polydatin was calculated by the external standard method. The results are shown in Table 7.

[0136] Table 7 Results of Investigation of Extraction Solvents

[0137]

[0138] The results showed that the extraction effects of 50 v / v% methanol and 70 v / v% methanol on polydatin were basically the same, and the extraction effect of 70 v / v% methanol was slightly higher. Therefore, 70 v / v% methanol solution was selected as the extraction solvent.

[0139] Example 10: Content determination of Wuse Jiuwei Granules

[0140] Chromatographic conditions and system suitability test: An Agilent Zorbax Eclipse Plus C18 column (column length 25 cm, inner diameter 4.6 mm, particle size 5 μm) was used as the chromatographic column; acetonitrile - water (20:80) was used as the mobile phase, the detection wavelength was 306 nm; the column temperature was 30 °C; the flow rate was 1.0 ml / min.

[0141] Test solution: Take 1 g of the sample powder prepared in Example 1, accurately weigh it, place it in a stoppered conical flask, accurately add 20 ml of 70 v / v% methanol, weigh it, extract it by ultrasonic treatment (37 KHz, 380 w) for 30 minutes and by heating under reflux for 30 minutes respectively, let it cool, weigh it again, make up the lost weight with 70 v / v% methanol, shake well, filter, and take the subsequent filtrate, that is obtained.

[0142] Reference solution: Take the reference substance of polydatin, accurately weigh it, place it in a 100 ml volumetric flask, dissolve it with 70 v / v% methanol and dilute to the mark, shake well, and prepare a reference solution containing 60 μg of polydatin per 1 mL, that is obtained.

[0143] Accurately pipette 10 μL of the reference solution and the test solution respectively, inject them into the high - performance liquid chromatograph for determination, and calculate the content of polydatin by the external standard method respectively. The results are shown in Table 8.

[0144] Table 8 Results of the investigation on extraction methods

[0145] Extraction solvent Ultrasonic treatment Heating under reflux Content (mg / g) 1.100 1.133

[0146] The results showed that the extraction effects of heating under reflux and ultrasonic treatment were basically equivalent. Considering the simplicity of the operation, ultrasonic treatment was selected as the extraction method.

[0147] Example 11: Content determination of Wuse Jiuwei Granules

[0148] Chromatographic conditions and system suitability test: An Agilent Zorbax Eclipse Plus C18 column (column length 25 cm, inner diameter 4.6 mm, particle size 5 μm) was used as the chromatographic column; acetonitrile - water (20:80) was used as the mobile phase, the detection wavelength was 306 nm; the column temperature was 30 °C; the flow rate was 1.0 ml / min.

[0149] Test solution: Accurately weigh 1 g of the sample powder prepared in Example 1, place it in a stoppered conical flask, accurately add 20 ml of 70 v / v% methanol, weigh it, ultrasonically treat (37 KHz, 380 w) for 15 minutes, 30 minutes, and 45 minutes, allow to cool, weigh again, make up the lost weight with 70 v / v% methanol, shake well, filter, and take the subsequent filtrate, which is the test solution.

[0150] Reference solution: Take polygonum cuspidatum glycoside reference substance, accurately weigh it, place it in a 100 ml volumetric flask, dissolve it with 70 v / v% methanol and dilute to the mark, shake well, and prepare a reference solution containing 60 μg of polygonum cuspidatum glycoside per 1 mL, which is the reference solution.

[0151] Accurately pipette 10 μL each of the reference solution and the test solution, inject them into a high performance liquid chromatograph for determination, and calculate the content of polygonum cuspidatum glycoside by the external standard method respectively. The results are shown in Table 9.

[0152] Table 9 Results of the investigation on extraction time

[0153] Extraction time 15 minutes 30 minutes 45 minutes Content (mg / g) 1.051 1.100 0.930

[0154] The results show that the extraction efficiency is the highest at 30 minutes, so 30 minutes is selected as the extraction time.

[0155] Example 12, Content determination of Wuse Jiuwei Granules

[0156] Chromatographic conditions and system suitability test: Use Agilent Zorbax Eclipse Plus C18 (column length 25 cm, inner diameter 4.6 mm, particle size 5 μm) as the chromatographic column; use acetonitrile - water (20:80) as the mobile phase, the detection wavelength is 306 nm; the column temperature is 30 °C; the flow rate is 1.0 ml / min.

[0157] Test solution: Accurately weigh 1 g of the sample powder prepared in Example 1, place it in a stoppered conical flask, accurately add 10 ml, 20 ml, and 50 ml of 70 v / v% methanol respectively, weigh it, ultrasonically treat (37 KHz, 380 w) for 30 minutes, allow to cool, weigh again, make up the lost weight with 70 v / v% methanol, shake well, filter, and take the subsequent filtrate, which is the test solution.

[0158] Reference solution: Take polygonum cuspidatum glycoside reference substance, accurately weigh it, place it in a 100 ml volumetric flask, dissolve it with 70 v / v% methanol and dilute to the mark, shake well, and prepare a reference solution containing 60 μg of polygonum cuspidatum glycoside per 1 mL, which is the reference solution.

[0159] Accurately pipette 10 μL each of the reference solution and the test solution, inject them into a high performance liquid chromatograph for determination, and calculate the content of polygonum cuspidatum glycoside by the external standard method respectively. The results are shown in Table 10.

[0160] Investigation Results of the Dosage of Extraction Solvent in Table 10

[0161] Solvent dosage 10ml 20ml 50ml Content (mg / g) 1.092 1.100 1.079

[0162] The results show that the extraction efficiency is the highest with 20 ml. Therefore, 20 ml is selected as the dosage of extraction solvent.

[0163] As described above, it is only a preferred embodiment of the present invention and does not impose any formal or substantial limitations on the present invention. It should be noted that for those of ordinary skill in the art of this technology, several improvements and supplements can still be made without departing from the present invention, and these improvements and supplements should also be regarded as within the protection scope of the present invention.

Claims

1. A quality detection method for five-color and nine-flavor granules, characterized in that: The five-color and nine-flavor granules are prepared from raw materials and auxiliary materials in the following prescription ratios: 10-30 parts of Polygonum cuspidatum, 20-40 parts of Hedyotis diffusa, 5-20 parts of Lithospermum chinense, 10-30 parts of Radix Isatidis, 20-40 parts of Concha Ostreae, 5-20 parts of Curcuma acuminata, 8-18 parts of Carthamus tinctorius, 5-30 parts of Curcuma zedoariae, and 1-5 parts of Licorice. The method comprises the steps of identifying Curcuma zedoariae, Polygonum cuspidatum, and Licorice in the five-color and nine-flavor granules by thin layer chromatography, and determining the content of Polydatin in the five-color and nine-flavor granules by high performance liquid chromatography; wherein: The step of identifying Curcuma zedoaria by thin layer chromatography comprises: 1.1) Take Wu Se Jiu Wei Granules and add water in a ratio of 1.5 g: 25 ml, ultrasonically treat, extract the supernatant with ethyl acetate, evaporate to dryness, and dissolve the residue with methanol as the test solution; 1.2) Take Curcuma zedoaria (Wen Yujin) as a control medicinal material, add methanol at a ratio of 0.5 g: 30 ml, ultrasonically treat, centrifuge, evaporate the supernatant to dryness, dissolve the residue in water, extract with dichloromethane, discard the dichloromethane liquid, extract the aqueous phase with ethyl acetate, evaporate to dryness, and dissolve the residue in methanol to serve as the control medicinal material solution; 1.3) Take the other medicinal ingredients except Curcuma zedoaria, Polygonum cuspidatum, Hedyotis diffusa, Lithospermum officinale, Radix Isatidis, Oyster, Curcuma australis, Carthamus tinctorius, Licorice in the same proportion as the prescription and the auxiliary materials to prepare a negative control sample in a ratio of 1.5g:25ml, add water, ultrasonic treatment, centrifuge, extract the supernatant with ethyl acetate, evaporate to dryness, and add methanol to dissolve the residue as the negative control solution; 1.4) Take 5-10 μl of the test solution and negative control solution, and 3-5 μl of the control medicinal material solution, and spot them on the same silica gel G thin layer plate, add a developing agent, develop, take out, dry, spray with a color developer, and blow with hot air until the spots are clear; check under sunlight, and in the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatogram of the control medicinal material; in the chromatogram of the negative control, spots of the same color do not appear at the corresponding positions of the chromatogram of the control medicinal material; The step of identifying Polygonum cuspidatum by thin layer chromatography comprises: 2.1) Take Wu Se Jiu Wei Granules in a ratio of 1.5 g: 25 ml and add water, ultrasonically treat, extract the supernatant with ethyl acetate, evaporate to dryness, and dissolve the residue with methanol as the test solution; 2.2) Accurately weigh the emodin reference substance and add methanol to dissolve it to prepare a 1 ml solution containing 0.1 mg emodin as the reference substance solution; 2.3) Take other medicinal herbs except Polygonum cuspidatum, including Hedyotis diffusa, Lithospermum officinale, Radix Isatidis, Concha oyster, Curcuma australis, Carthamus tinctorius, Rhizoma Curcumae, and Radix Glycyrrhizae in the same proportion as the prescription and the excipients to prepare a negative control sample in a ratio of 1.5 g: 25 ml, add water, ultrasonic treatment, centrifuge, extract the supernatant with ethyl acetate, evaporate to dryness, and add methanol to dissolve the residue as the negative control solution; 2.4) 5-10 μl of the test solution in step 2.1) and the negative control solution in step 2.3) and 10 μl of the reference solution in step 2.2) are respectively spotted on the same silica gel G thin layer plate, and a developing agent is added, the plate is developed, taken out, dried, and examined under a UV lamp at 365 nm. In the chromatogram of the test sample, a fluorescent spot of the same color is shown at the corresponding position of the chromatogram of the reference; in the chromatogram of the negative control, a fluorescent spot of the same color is not shown at the corresponding position of the chromatogram of the reference; The step of identifying liquorice by thin layer chromatography comprises: 3.1) Take Wu Se Jiu Wei Granules in a ratio of 1.5 g: 25 ml and add water, ultrasonically treat, extract the supernatant with dichloromethane, discard the dichloromethane solution, extract the aqueous phase with water-saturated n-butanol, wash with 0.1-0.2 mol / L sodium hydroxide solution, discard the washing solution, then wash with 0.2-1 mol / L hydrochloric acid solution, discard the washing solution, evaporate to dryness, add methanol to dissolve the residue, and use it as the test solution; 3.2) Accurately weigh the liquiritin reference substance, add methanol to dissolve it, and prepare a 1 ml solution containing 0.1 mg liquiritin as the reference substance solution; 3.3) Take the negative control sample prepared by taking the other medicinal ingredients except licorice, including Polygonum cuspidatum, Hedyotis diffusa, Lithospermum chinense, Radix Isatidis, Oyster, Curcuma acuminata, Carthamus tinctorius, and Rhizoma Curcumae in the same proportion as the prescription and the auxiliary materials in the ratio of 1.5g:25ml, add water, ultrasonic treatment, centrifuge, extract the supernatant with dichloromethane, discard the dichloromethane solution, extract the aqueous phase with water-saturated n-butanol, wash with 0.1-0.2mol / L sodium hydroxide solution, discard the washing solution, and then wash with 0.2-1mol / L hydrochloric acid solution, discard the washing solution, evaporate to dryness, and add methanol to the residue to dissolve it as the negative control solution; 3.4) Take 5-10 μl of the test solution in step 3.1) and the negative control solution in step 3.3) and 10 μl of the reference solution in step 3.2) and spot them on the same silica gel G thin layer plate, add a developing agent, develop, take out, dry, spray with a color developer, and heat at 105°C until the spots are clear; examine under a UV lamp at 365nm. In the chromatogram of the test sample, a fluorescent spot of the same color appears at the corresponding position of the chromatogram of the reference; in the chromatogram of the negative control, a fluorescent spot of the same color does not appear at the corresponding position of the chromatogram of the reference; The high performance liquid chromatography method for determining the content of polydatin in the five-color and nine-flavor granules comprises the following steps: 4.1) Chromatographic conditions and system suitability test Octadecylsilane bonded silica gel is used as filler; acetonitrile is used as mobile phase A, and water is used as mobile phase B; the detection wavelength is 306nm, and the theoretical plate number is calculated based on the polydatin peak, which should be no less than 5000; 4.2) Preparation of test solution Take the Wu Se Jiu Wei granules and weigh them accurately, place them in a stoppered conical bottle, accurately add 70 v / v% methanol to make a solution containing 50 mg per 1 mL, weigh them, treat with ultrasound, let them cool, weigh them again, make up the lost weight with 70 v / v% methanol, shake them well, filter them, and take the filtrate to obtain; 4.3) Preparation of reference solution Take the polydatin reference substance, weigh it accurately, put it in a volumetric flask, add 70v / v% methanol to dissolve and dilute to the scale, shake well, and make a reference substance solution containing 60μg polydatin per 1mL. 4.4) Determination Accurately pipette 10 μL of reference solution and test solution respectively, inject into high performance liquid chromatography, and measure to obtain the result.

2. The quality inspection method according to claim 1, characterized in that: The developing agent in step 1.4) is composed of 60-90°C petroleum ether-ethyl acetate-anhydrous formic acid in a volume ratio of 20:20:1, and the color developer is a 10v / v% sulfuric acid-ethanol solution containing 2v / v% vanillin.

3. The quality inspection method according to claim 1, characterized in that: The developing agent in the step 2.4) is composed of 60-90° C. petroleum ether-ethyl acetate in a volume ratio of 2:

1.

4. The quality inspection method according to claim 1, characterized in that: The developing agent in step 3.4) is composed of ethyl acetate-anhydrous formic acid-water in a volume ratio of 16:1:1, and the color developer is a 10 v / v% sulfuric acid-ethanol solution.

5. The quality inspection method according to claim 1, characterized in that: The mobile phase in step 4.1) adopts the gradient elution shown in the following table:

6. The quality inspection method according to claim 1, characterized in that: The column temperature in step 4.1) was set to 30°C, and the mobile phase flow rate was set to 1.0 mL / min.

Images