Method for establishing characteristic chromatogram of conchae ampullaria compound traditional Chinese medicine preparation

Abstract

The application provides a characteristic spectrum establishing method of Concha Murex Trunculae compound traditional Chinese medicine preparation, and relates to the technical field of drug analysis.The Concha Murex Trunculae compound traditional Chinese medicine preparation is mixed with methanol aqueous solution (volume fraction 75%) to carry out ultrasonic extraction, and a test sample solution is obtained; the test sample solution is detected by high performance liquid chromatography to obtain a liquid chromatogram of the test sample, the liquid chromatogram of the test sample is compared with a predetermined reference spectrum, common peaks are selected from the liquid chromatogram of the test sample, and a characteristic spectrum of the Concha Murex Trunculae compound traditional Chinese medicine preparation is obtained; the reference spectrum is a liquid chromatogram of a control sample solution obtained under the same high performance liquid chromatography detection condition.The characteristic spectrum obtained by the establishing method can comprehensively reflect the components of the Concha Murex Trunculae compound traditional Chinese medicine preparation, realizes quality control of the traditional Chinese medicine preparation containing Concha Murex Trunculae, and has the characteristics of high accuracy, high stability and good repeatability, and can realize comprehensive quality monitoring of the Concha Murex Trunculae preparation.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical analysis, in particular to a method for establishing a characteristic chromatogram of a compound traditional Chinese medicine preparation containing Concha Haliotidis. Background Art

[0002] Shijueming San is included in the Catalogue of Ancient Classic Famous Prescriptions (First Batch) published by the National Medical Products Administration, and its source is "Puji Fang" (compiled by Zhu Su in the Ming Dynasty): "Shijueming San is used for treating wind-toxic qi invading the head, causing dim vision, and unfavorable head and eyes." The prescription is: one liang each of Concha Haliotidis, Notopterygii Rhizoma et Radix (removed root head), Semen Cassiae and Flos Chrysanthemi, and half liang of Glycyrrhizae Radix et Rhizoma (roasted, cut). The preparation method and administration are as follows: grind all the above herbs into powder, take two qian each time, put it in one cup of water, decoct to 60% of the original volume, take it warm after meals and before going to bed. It is a decocted powder preparation. This prescription is widely used in ophthalmology, but it is always related to the liver and gallbladder meridians, and is mostly used for eye diseases of liver heat and nebula type, which is also followed in modern clinical ophthalmology.

[0003] Subsequently, Shijueming San was improved, Schizonepetae Herba was used to replace Notopterygii Rhizoma et Radix, the prescription consists of Concha Haliotidis, Semen Cassiae, Flos Chrysanthemi, Schizonepetae Herba and Glycyrrhizae Radix et Rhizoma, and a Concha Haliotidis traditional Chinese medicine preparation is prepared. In this formula, Semen Cassiae and Concha Haliotidis are selected as the sovereign (principal) herbs. Both of them have the effect of improving eyesight and are known as "holy herbs for ophthalmology", hence the name of the prescription. Semen Cassiae, also known as Caojueming, is sweet, bitter, salty in flavor and slightly cold in nature, and attributes to the liver and large intestine meridians. It has the functions of clearing heat and improving eyesight (eliminating heat pathogens in the body and promoting vision recovery), and moistening the intestine for defecation. It is used for redness, astringency and pain of eyes, photophobia with excessive lacrimation, headache and dizziness, dim vision, and constipation. Modern studies have shown that it has effects of lowering blood pressure, lowering blood lipid, and inhibiting bacteria. Concha Haliotidis is salty in flavor and cold in nature, and attributes to the liver meridian. It has the effects of calming the liver and suppressing hyperactive yang (inhibiting excessive ascending yang in the liver), clearing liver fire and improving eyesight (clearing liver fire to promote vision recovery). It is commonly used for headache and dizziness, redness of eyes with nebula, blurred vision, and glaucoma and night blindness. Modern studies have shown that it has effects of lowering blood pressure, anti-oxidation, and neutralizing gastric acid. The combination of the two as sovereign (principal) herbs synergistically treats syndromes such as headache and dizziness, redness, swelling and pain of eyes, dryness and astringency of eyes, and blurred vision. Schizonepetae Herba is slightly warm in nature, has the effect of dispersing wind with pungent and warm properties for relieving exterior syndrome, and acts as the minister (assistant) herb to assist the sovereign (principal) herbs Concha Haliotidis and Semen Cassiae in their detoxification effect. Flos Chrysanthemi is sweet and bitter in flavor, slightly cold in nature, and attributes to the lung and liver meridians. It has the effects of dispersing wind-heat, calming the liver and improving eyesight, clearing heat and detoxifying, and is suitable for wind-heat in the liver meridian, up-flaming of liver fire, and redness, swelling and pain of eyes. The liver opens into the eyes, hyperactivity of liver fire will cause redness, swelling and pain of eyes. In this prescription, Flos Chrysanthemi is used as an assistant herb to enhance the effects of clearing heat and detoxifying, calming the liver and improving eyesight. It cooperates with Glycyrrhizae Radix et Rhizoma to guide other herbs into the liver meridian, and the compatibility of all herbs directly acts on the root cause of nourishing the liver and improving eyesight. Glycyrrhizae Radix et Rhizoma is sweet and neutral in nature, has the effects of tonifying the spleen and replenishing qi, moistening the lung to relieve cough, detoxifying, and harmonizing the properties of various herbs. When processed with honey, it is warm and tonifying; when used raw, it detoxifies. Its application in the compound preparation can moderate the drastic properties of other herbs, and enable herbs with different properties to coordinate with each other.

[0004] Due to the complex nature of Chinese medicinal materials, including their diverse sources and production areas, the quality of Chinese medicinal products varies significantly, particularly in the quantity of their active ingredients. Characteristic mapping of Chinese medicinal materials has become an effective method for controlling the quality of compound Chinese medicine formulas and their preparations in recent years. This technology provides a broader perspective for quality control research on compound Chinese medicine formulas. Characteristic mapping of Chinese medicinal materials is holistic, highlighting the complete picture of a compound Chinese medicine formula. Furthermore, characteristic maps of similar medicinal materials exhibit similarity, enabling a comprehensive evaluation of the intrinsic chemical components of Chinese medicinal materials and overall quality control, thus making the composition of Chinese medicinal preparations more controllable.

[0005] Currently, the aforementioned improved traditional Chinese medicine preparations containing abalone shell are not included in any standards, lacking quality control and thus failing to guarantee their clinical efficacy. Therefore, establishing a chromatogram that reflects the multi-component information and quality characteristics of abalone shell preparations is of great significance for ensuring the controllable quality of these preparations. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a method for establishing characteristic chromatograms of a compound traditional Chinese medicine preparation containing abalone shell. The characteristic chromatograms obtained by the method provided by this invention can comprehensively reflect the components of the compound traditional Chinese medicine preparation containing abalone shell, thereby achieving quality control of the preparation.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a method for establishing the characteristic spectrum of a compound traditional Chinese medicine preparation containing abalone shell, comprising the following steps:

[0009] The compound traditional Chinese medicine preparation of Abalone Shell was mixed with a methanol aqueous solution and subjected to ultrasonic extraction to obtain a test solution; the volume fraction of the methanol aqueous solution was 75%; the raw materials for preparing the compound traditional Chinese medicine preparation of Abalone Shell included Abalone Shell, Cassia Seed, Schizonepeta, Chrysanthemum and Licorice.

[0010] The test solution was subjected to high-performance liquid chromatography (HPLC) to obtain a HPLC chromatogram of the test sample. The HPLC chromatogram of the test sample was compared with a predetermined reference chromatogram. Common peaks were selected from the HPLC chromatogram of the test sample to obtain the characteristic chromatogram of the Shi Jue Ming compound traditional Chinese medicine preparation. The reference chromatogram was obtained by using a reference solution under the same HPLC detection conditions. The reference solution contained reference standards containing caffeic acid, chlorogenic acid, glycyrrhizin, luteolin, resveratrol, hesperidin, and cassia seed extract.

[0011] The conditions for the high-performance liquid chromatography detection include:

[0012] The chromatographic column was a Thermo Hypersil GOLD C10. 18 ;

[0013] The column temperature is 30℃;

[0014] The detection wavelength is 285nm;

[0015] The injection volume was 10 μL;

[0016] The mobile phase includes mobile phase A, mobile phase B and mobile phase C, wherein mobile phase A is acetonitrile, mobile phase B is a 0.1% (v / v) aqueous solution of phosphoric acid and mobile phase C is methanol;

[0017] The flow rate of the mobile phase is 0.3 mL / min;

[0018] The elution program is a gradient elution program;

[0019] The gradient elution procedure is as follows:

[0020] 0–9 min: The volume percentage of mobile phase A increases uniformly from 1% to 6%, the volume percentage of mobile phase B decreases uniformly from 97% to 92%, and the volume percentage of mobile phase C remains at 2%.

[0021] 9–21 min: The volume percentage of mobile phase A is maintained at 6%, the volume percentage of mobile phase B is maintained at 92%, and the volume percentage of mobile phase C is maintained at 2%.

[0022] 21–26 min: The volume percentage of mobile phase A increases uniformly from 6% to 11%, the volume percentage of mobile phase B decreases uniformly from 92% to 87%, and the volume percentage of mobile phase C remains at 2%.

[0023] 26–33 min: The volume percentage of mobile phase A is maintained at 11%, the volume percentage of mobile phase B is maintained at 87%, and the volume percentage of mobile phase C is maintained at 2%.

[0024] 33–36 min: The volume percentage of mobile phase A increases uniformly from 11% to 13%, the volume percentage of mobile phase B decreases uniformly from 87% to 85%, and the volume percentage of mobile phase C remains at 2%.

[0025] 36–42 min: The volume percentage of mobile phase A increases uniformly from 13% to 19%, the volume percentage of mobile phase B decreases uniformly from 85% to 79%, and the volume percentage of mobile phase C remains at 2%.

[0026] 42–46 min: The volume percentage of mobile phase A is maintained at 19%, the volume percentage of mobile phase B is maintained at 79%, and the volume percentage of mobile phase C is maintained at 2%.

[0027] 46–49 min: The volume percentage of mobile phase A increases uniformly from 19% to 44%, the volume percentage of mobile phase B decreases uniformly from 79% to 54%, and the volume percentage of mobile phase C remains at 2%.

[0028] 49–55 min: The volume percentage of mobile phase A is maintained at 44%, the volume percentage of mobile phase B is maintained at 54%, and the volume percentage of mobile phase C is maintained at 2%.

[0029] 55–55.5 min: The volume percentage of mobile phase A decreases uniformly from 44% to 1%, the volume percentage of mobile phase B increases uniformly from 54% to 97%, and the volume percentage of mobile phase C remains at 2%.

[0030] 55.5–60 min: The volume percentage of mobile phase A is maintained at 1%, the volume percentage of mobile phase B is maintained at 97%, and the volume percentage of mobile phase C is maintained at 2%.

[0031] Preferably, the ratio of the amount of the compound herbal preparation of abalone shell to the amount of methanol aqueous solution is 0.5g:15-20mL.

[0032] Preferably, the ultrasonic extraction frequency is 40kHz and the power is 300W.

[0033] Preferably, the ultrasonic extraction time is 90 minutes.

[0034] Preferably, the concentration of each reference standard in the reference solution is 10 μg / mL.

[0035] Preferably, the chromatographic column has a length of 100 mm and an inner diameter of 2.1 mm.

[0036] Preferably, the concentration of the test solution is 0.02 g / mL.

[0037] Preferably, the amount of abalone shell is 250 parts by weight, the amount of cassia seed is 250 parts by weight, the amount of schizonepeta is 250 parts by weight, the amount of chrysanthemum is 250 parts by weight, and the amount of licorice is 125 parts by weight.

[0038] Preferably, the preparation method of the abalone shell compound traditional Chinese medicine preparation includes the following steps:

[0039] Boil abalone shell, cassia seed, schizonepeta, chrysanthemum and licorice in water twice, filter the decoctions obtained from the two decoctions separately, and combine the filtrates.

[0040] The combined filtrate was concentrated and maltodextrin was added. After granulation and drying, the compound traditional Chinese medicine preparation of abalone shell was obtained.

[0041] Preferably, the amount of water added for the first decoction is 12 times the mass of the raw materials, and the amount of water added for the second decoction is 10 times the mass of the raw materials. The first decoction time is 2 hours, and the second decoction time is 1.5 hours.

[0042] This invention provides a method for establishing a characteristic chromatogram of a compound traditional Chinese medicine preparation containing abalone shell, comprising the following steps: mixing the compound traditional Chinese medicine preparation containing abalone shell with a methanol-water solution and performing ultrasonic extraction to obtain a test solution; the volume fraction of the methanol-water solution is 75%; the raw materials for preparing the compound traditional Chinese medicine preparation containing abalone shell, cassia seed, schizonepeta, chrysanthemum, and licorice; performing high-performance liquid chromatography (HPLC) on the test solution to obtain a HPLC chromatogram of the test sample; comparing the HPLC chromatogram of the test sample with a predetermined reference chromatogram; selecting common peaks from the HPLC chromatogram of the test sample to obtain a characteristic chromatogram of the compound traditional Chinese medicine preparation containing abalone shell; the reference chromatogram is a HPLC chromatogram of a reference solution obtained under the same HPLC detection conditions, wherein the reference solution contains reference standards containing caffeic acid, chlorogenic acid, glycyrrhizin, luteolin, resveratrol, hesperidin, and cassia seed extract. This invention, based on the prescription composition of abalone shell traditional Chinese medicine preparation, analyzes the chemical components of each component and considers their antioxidant and anti-inflammatory pharmacological effects. Corresponding reference standards can be detected in the relevant medicinal materials and finished granules. To establish more reference standards for confirmation, the test solution and reference standard solutions containing caffeic acid, chlorogenic acid, glycyrrhizin, luteolin, resveratrol, hesperidin, and cassia seed extract were subjected to high-performance liquid chromatography (HPLC). Using the HPLC chromatogram of the reference standards as a reference spectrum, common peaks were selected from the HPLC chromatogram of the test solution to construct a characteristic chromatogram of the abalone shell compound traditional Chinese medicine preparation. This invention optimizes the extraction conditions of the test solution to ensure efficient preparation; and by investigating the chromatographic conditions for HPLC detection, the optimal chromatographic parameters that best reflect the component characteristics of the abalone shell compound traditional Chinese medicine preparation were established. The characteristic chromatograms obtained by the method provided by this invention can comprehensively reflect the components of abalone shell compound traditional Chinese medicine preparations, enabling quality control of preparations containing abalone shell. This ensures the authenticity, consistency, and stability of the product, guaranteeing the clinical efficacy of the drug. The method for establishing characteristic chromatograms of abalone shell compound traditional Chinese medicine preparations provided by this invention features high accuracy, high stability, and good repeatability. In an embodiment of this invention, six different batches of abalone shell compound traditional Chinese medicine preparations were tested. The results showed that the relative retention times (RSDs) of chromatographic peaks at the same positions were all within 5%, indicating that the method has good reproducibility and high reliability. Furthermore, the obtained characteristic chromatograms have seven common peaks that achieve effective separation, effectively characterizing the quality level of the abalone shell preparation and facilitating comprehensive monitoring of its quality. Attached Figure Description

[0043] Figure 1The high performance liquid chromatogram of the control solution is shown below, where 1 is caffeic acid, 2 is chlorogenic acid, 3 is glycyrrhizin, 4 is luteolin, 5 is resveratrol, 6 is hesperidin, and 7 is cassia seed extract.

[0044] Figure 2 High-performance liquid chromatogram of the test sample extracted with water solvent by heating;

[0045] Figure 3 High-performance liquid chromatogram of the test sample extracted with 25% methanol solvent by heating;

[0046] Figure 4 High-performance liquid chromatogram of the test sample extracted with 50% methanol solvent by heating;

[0047] Figure 5 High-performance liquid chromatogram of the test sample extracted with 75% methanol solvent by heating;

[0048] Figure 6 High-performance liquid chromatogram of the test sample extracted by ultrasonication with water solvent;

[0049] Figure 7 High-performance liquid chromatogram of the test sample extracted with 25% methanol solvent by ultrasonication;

[0050] Figure 8 The high-performance liquid chromatogram of the test sample extracted with 50% methanol solvent by ultrasonication;

[0051] Figure 9 The high-performance liquid chromatogram of the test sample after ultrasonic extraction with 75% methanol solvent for 90 minutes;

[0052] Figure 10 The high-performance liquid chromatogram of the test sample after ultrasonic extraction with 75% methanol solvent for 30 minutes;

[0053] Figure 11 The high-performance liquid chromatogram of the test sample after ultrasonic extraction with 75% methanol solvent for 60 minutes;

[0054] Figure 12 The high-performance liquid chromatogram of the test sample after ultrasonic extraction with 75% methanol solvent for 120 minutes;

[0055] Figure 13 The HPLC chromatogram of the test sample is shown at a column temperature of 40℃.

[0056] Figure 14 The high-performance liquid chromatogram is for a mobile phase system of acetonitrile-water sample.

[0057] Figure 15 The high-performance liquid chromatogram of the test sample under a 68-minute mobile phase gradient elution program is shown.

[0058] Figure 16The high-performance liquid chromatogram of the test sample under a 67-minute mobile phase gradient elution program is shown.

[0059] Figure 17 The high-performance liquid chromatogram of the test sample is shown at a wavelength of 235 nm.

[0060] Figure 18 The high-performance liquid chromatogram of the test sample is shown at a wavelength of 255 nm.

[0061] Figure 19 The high-performance liquid chromatogram of the test sample is shown at a wavelength of 275 nm.

[0062] Figure 20 The high-performance liquid chromatogram of the test sample is shown at a wavelength of 305 nm.

[0063] Figure 21 The high-performance liquid chromatogram of the test sample is shown at a wavelength of 325 nm.

[0064] Figure 22 Overlapped high-performance liquid chromatography chromatograms of six consecutive injections of the test solution for precision testing;

[0065] Figure 23 High-performance liquid chromatography (HPLC) overlay chromatogram of the test solution for solution stability testing;

[0066] Figure 24 High-performance liquid chromatography (HPLC) overlay chromatogram of the test solution for repeatability testing;

[0067] Figure 25 The high performance liquid chromatography overlay chromatograms of the six batches of test solutions are shown. Peak 1 is caffeic acid, peak 2 is chlorogenic acid, peak 3 is glycyrrhizin, peak 4 is luteolin, peak 5 is resveratrol, peak 6 is hesperidin, and peak 7 is cassia seed extract.

[0068] Figure 26 High performance liquid chromatography (HPLC) overlay chromatograms of four Chinese medicinal herbs, a mixed reference solution, and a test solution;

[0069] Figure 27 The high-performance liquid chromatogram of Cassia seed as a positive control;

[0070] Figure 28 The high-performance liquid chromatogram is for the positive control of *Nepeta cataria*.

[0071] Figure 29 The high-performance liquid chromatogram is for the chrysanthemum positive control.

[0072] Figure 30 This is a high-performance liquid chromatogram of licorice as a positive control. Detailed Implementation

[0073] This invention provides a method for establishing the characteristic spectrum of a compound traditional Chinese medicine preparation containing abalone shell, comprising the following steps:

[0074] The compound traditional Chinese medicine preparation of Abalone Shell was mixed with a methanol aqueous solution and subjected to ultrasonic extraction to obtain a test solution; the volume fraction of the methanol aqueous solution was 75%; the raw materials for preparing the compound traditional Chinese medicine preparation of Abalone Shell included Abalone Shell, Cassia Seed, Schizonepeta, Chrysanthemum and Licorice.

[0075] The test solution was subjected to high-performance liquid chromatography (HPLC) to obtain a HPLC chromatogram of the test sample. The HPLC chromatogram of the test sample was compared with a predetermined reference chromatogram. Common peaks were selected from the HPLC chromatogram of the test sample solution to construct a characteristic chromatogram of the Shi Jue Ming compound traditional Chinese medicine preparation. The reference chromatogram was obtained by using a reference solution under the same HPLC detection conditions. The reference solution contained reference standards containing caffeic acid, chlorogenic acid, glycyrrhizin, luteolin, resveratrol, hesperidin, and cassia seed extract.

[0076] In this invention, unless otherwise specified, all raw materials / components used in the preparation are commercially available products well known to those skilled in the art.

[0077] This invention involves mixing a compound traditional Chinese medicine preparation containing abalone shell (in the example, abalone shell granules) with a methanol aqueous solution and then performing ultrasonic extraction to obtain a test solution.

[0078] In this invention, the raw materials for preparing the compound traditional Chinese medicine preparation containing abalone shell include abalone shell, cassia seed, schizonepeta, chrysanthemum, and licorice. Preferably, the abalone shell is 250 parts by weight, the cassia seed is 250 parts by weight, the schizonepeta is 250 parts by weight, the chrysanthemum is 250 parts by weight, and the licorice is 125 parts by weight. In this invention, the preferred method for preparing the compound traditional Chinese medicine preparation containing abalone shell includes the following steps:

[0079] Boil abalone shell, cassia seed, schizonepeta, chrysanthemum and licorice in water twice, filter the decoctions obtained from the two decoctions separately, and combine the filtrates.

[0080] The combined filtrate was concentrated and maltodextrin was added. After granulation and drying, the compound traditional Chinese medicine preparation of abalone shell was obtained.

[0081] In this invention, the amount of water added for the first decoction is preferably 12 times the mass of the raw materials, the amount of water added for the second decoction is preferably 10 times the mass of the raw materials, the first decoction time is preferably 2 hours, and the second decoction time is preferably 1.5 hours.

[0082] In this invention, the volume fraction of the methanol-water solution is preferably 75%, and the preferred ratio of the abalone shell compound traditional Chinese medicine preparation to the methanol-water solution is 0.5g:15-20mL. In the embodiments of this invention, water, a 75% (v / v) methanol-water solution, a 50% (v / v) methanol-water solution, and a 25% (v / v) methanol-water solution were used as extraction solvents. The 75% (v / v) methanol-water solution extracted the test sample, resulting in the highest number of chromatographic peaks and good separation of each peak, indicating complete extraction. In contrast, water extraction yielded no obvious chromatographic peaks, indicating incomplete extraction. Therefore, this invention selects a 75% (v / v) methanol-water solution as the extraction solvent.

[0083] In this invention, the preferred frequency of ultrasonic extraction is 40 kHz, and the preferred power is 300 W; the preferred extraction time is 90 min; the ultrasonic extraction is performed at room temperature, i.e., no additional heating is required. In the embodiments of this invention, the test sample is extracted using both ultrasonic extraction and water bath heating. In the chromatograms of the test samples extracted using water, 75% (v / v) methanol aqueous solution, 50% (v / v) methanol aqueous solution, and 25% (v / v) methanol aqueous solution as extraction solvents, the peak areas are significantly smaller than those in the chromatograms of the test samples extracted using ultrasonic extraction. Therefore, this invention sets the extraction method to ultrasonic extraction. Furthermore, in the embodiments of this invention, ultrasonic extraction is performed using 75% (v / v) methanol aqueous solution as the extraction solvent for 90 min, 30 min, 60 min, and 120 min. The peak areas in the chromatograms of the test samples extracted for 30 min and 60 min are significantly smaller. In the chromatogram of the test sample extracted for 120 min, there is interference between the chromatographic peaks. In the chromatogram of the test sample extracted for 90 min, the separation between the chromatographic peaks is better and does not interfere with detection. Therefore, this invention sets the ultrasonic extraction time to 90 min.

[0084] In this embodiment of the invention, 0.5g of the compound Chinese medicine preparation of abalone shell was accurately weighed and placed in a 25mL volumetric flask. 15mL of 75% methanol aqueous solution was added, and the mixture was sonicated for 90min. The mixture was then removed and allowed to stand at room temperature. It was then diluted to the mark with 75% methanol aqueous solution, shaken well, filtered, and the filtrate was collected to obtain the test solution.

[0085] After obtaining the test solution, the present invention performs high performance liquid chromatography on the test solution to obtain a liquid chromatogram of the test sample.

[0086] In this invention, the conditions for high-performance liquid chromatography detection include:

[0087] The chromatographic column was a Thermo Hypersil GOLD C10. 18The column length is preferably 100 mm, the inner diameter is preferably 2.1 mm, the filler is octadecylsilane bonded silica gel, and the particle size of the filler is preferably 1.9 μm;

[0088] The column temperature is 30℃;

[0089] The detection wavelength is 285nm (VWD detector);

[0090] The injection volume was 10 μL;

[0091] The concentration of the test solution (i.e., the injection concentration) is preferably 0.02 g / mL;

[0092] The mobile phase includes mobile phase A, mobile phase B and mobile phase C, wherein mobile phase A is acetonitrile, mobile phase B is a 0.1% volume fraction aqueous solution of phosphoric acid (i.e., a 0.1% phosphoric acid solution, which is prepared by diluting commercially available 85% phosphoric acid with water, wherein the volume percentage of 85% phosphoric acid is 0.1%), and mobile phase C is methanol;

[0093] The flow rate of the mobile phase is 0.3 mL / min;

[0094] The elution program is a gradient elution program;

[0095] The gradient elution procedure is as follows (listed in Table 1):

[0096] 0–9 min: The volume percentage of mobile phase A increases uniformly from 1% to 6%, the volume percentage of mobile phase B decreases uniformly from 97% to 92%, and the volume percentage of mobile phase C remains at 2%.

[0097] 9–21 min: The volume percentage of mobile phase A is maintained at 6%, the volume percentage of mobile phase B is maintained at 92%, and the volume percentage of mobile phase C is maintained at 2%.

[0098] 21–26 min: The volume percentage of mobile phase A increases uniformly from 6% to 11%, the volume percentage of mobile phase B decreases uniformly from 92% to 87%, and the volume percentage of mobile phase C remains at 2%.

[0099] 26–33 min: The volume percentage of mobile phase A is maintained at 11%, the volume percentage of mobile phase B is maintained at 87%, and the volume percentage of mobile phase C is maintained at 2%.

[0100] 33–36 min: The volume percentage of mobile phase A increases uniformly from 11% to 13%, the volume percentage of mobile phase B decreases uniformly from 87% to 85%, and the volume percentage of mobile phase C remains at 2%.

[0101] 36–42 min: The volume percentage of mobile phase A increases uniformly from 13% to 19%, the volume percentage of mobile phase B decreases uniformly from 85% to 79%, and the volume percentage of mobile phase C remains at 2%.

[0102] 42–46 min: The volume percentage of mobile phase A is maintained at 19%, the volume percentage of mobile phase B is maintained at 79%, and the volume percentage of mobile phase C is maintained at 2%.

[0103] 46–49 min: The volume percentage of mobile phase A increases uniformly from 19% to 44%, the volume percentage of mobile phase B decreases uniformly from 79% to 54%, and the volume percentage of mobile phase C remains at 2%.

[0104] 49–55 min: The volume percentage of mobile phase A is maintained at 44%, the volume percentage of mobile phase B is maintained at 54%, and the volume percentage of mobile phase C is maintained at 2%.

[0105] 55–55.5 min: The volume percentage of mobile phase A decreases uniformly from 44% to 1%, the volume percentage of mobile phase B increases uniformly from 54% to 97%, and the volume percentage of mobile phase C remains at 2%.

[0106] 55.5–60 min: The volume percentage of mobile phase A is maintained at 1%, the volume percentage of mobile phase B is maintained at 97%, and the volume percentage of mobile phase C is maintained at 2%.

[0107] HPLC characteristic chromatograms are currently the most widely used chromatographic analysis method. With advantages such as high pressure, high sensitivity, high efficiency, and automation, they are widely applied in the detection of chemical components in traditional Chinese medicine (TCM) preparations, making the composition of TCM preparations more controllable. This invention uses high-performance liquid chromatography under specific parameter conditions for detection. The resulting chromatograms can comprehensively reflect the components of TCM compound preparations, enabling quality control of TCM preparations and ensuring the clinical efficacy of drugs.

[0108] In the embodiments of this invention, the chromatograms of the test sample were examined at column temperatures of 30°C and 40°C, respectively. The results showed that at a column temperature of 40°C, the retention time of the chromatographic peaks in the chromatogram of the test sample was earlier, and the chromatographic peak separation effect was not as good as that of the chromatographic peaks in the chromatogram of the test sample at a column temperature of 30°C. Therefore, this invention selected a column temperature of 30°C.

[0109] This invention investigated the high-performance liquid chromatography (HPLC) chromatograms of test samples under different mobile phase systems. The HPLC chromatograms of test samples using an acetonitrile-water mobile phase system (acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B) were compared with those using an acetonitrile-water-methanol mobile phase system (acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, and methanol as mobile phase C). The chromatograms of the test samples using the acetonitrile-water mobile phase system did not achieve baseline separation, while the chromatograms of the test samples using the acetonitrile-water-methanol mobile phase system generally met the requirements and achieved baseline separation. Therefore, this invention selected acetonitrile-water-methanol as the mobile phase system, i.e., acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, and methanol as mobile phase C.

[0110] The embodiments of the present invention examined the chromatograms of the test sample at different detection wavelengths. The test sample solution was detected at wavelengths of 235 nm, 255 nm, 275 nm, 285 nm, 305 nm and 325 nm. The results showed that the number of chromatographic peaks and the peak area were more reasonable at 285 nm. Therefore, the present invention selected 285 nm as the detection wavelength.

[0111] After obtaining the liquid chromatogram of the test sample, the present invention compares the liquid chromatogram of the test sample with a predetermined reference chromatogram, selects common peaks from the liquid chromatogram of the test sample, and obtains the characteristic chromatogram of the Shi Jue Ming compound traditional Chinese medicine preparation; the reference chromatogram is the liquid chromatogram of the reference solution obtained under the same high performance liquid chromatography detection conditions, and the reference solution contains reference standards containing caffeic acid, chlorogenic acid, glycyrrhizin, luteolin, resveratrol, hesperidin and cassia seed extract.

[0112] In this invention, reference standards for caffeic acid, chlorogenic acid, glycyrrhizin, luteolin, resveratrol, hesperidin, and cassia seed extract are preferably mixed with a solvent to obtain a reference solution (i.e., a mixed standard solution). In this invention, the solvent is preferably an aqueous methanol solution, and the volume fraction of the aqueous methanol solution is preferably 50%; the concentration of each reference standard in the reference solution is preferably 10 μg / mL.

[0113] The present invention selects the above 7 reference standards to establish more references for confirmation; the corresponding reference standards can also be detected in the corresponding medicinal materials and finished granules, and the selected reference standards all have anti-inflammatory and antioxidant pharmacological effects.

[0114] This invention uses the same high-performance liquid chromatography (HPLC) conditions as the test solution of the compound traditional Chinese medicine preparation containing abalone shell to test the reference solution. The chromatogram of the reference solution is compared with the characteristic chromatogram of the test solution of the traditional Chinese medicine preparation to identify the chromatographic peaks in the characteristic chromatogram of the traditional Chinese medicine preparation.

[0115] In this invention, the characteristic chromatogram of the abalone shell compound traditional Chinese medicine preparation, with the chromatographic peak of luteolin as a reference, has a total of 7 common peaks, namely:

[0116] Peak 1 has a relative retention time of 0.32.

[0117] Peak 2 has a relative retention time of 0.33.

[0118] Peak 3 has a relative retention time of 0.87.

[0119] Peak 4 has a relative retention time of 1.00.

[0120] Peak 5 has a relative retention time of 1.16.

[0121] Peak 6 has a relative retention time of 1.23.

[0122] Peak 7 has a relative retention time of 1.53.

[0123] Among them, peak 1 is caffeic acid; peak 2 is chlorogenic acid; peak 3 is glycyrrhizin; peak 4 is luteolin; peak 5 is resveratrol; peak 6 is hesperidin; and peak 7 is cassia seed. Peaks 1 and 6 are associated with catnip, peaks 2, 4, and 5 with chrysanthemum, peak 3 with licorice, and peak 7 with cassia seed.

[0124] This invention provides a method for establishing characteristic chromatograms of a traditional Chinese medicine preparation made from abalone shell. The method involves ultrasonic extraction of the compound preparation from abalone shell using a 75% methanol-water solution, followed by high-performance liquid chromatography (HPLC) analysis of the sample solution. Parameters at each stage are carefully controlled. Based on the "General Rules for High-Performance Liquid Chromatography (0512)," the chromatographic data are used to generate a characteristic chromatogram of the traditional Chinese medicine preparation. The method is simple to operate, and its specificity, solution stability, precision, and repeatability meet the requirements. This invention establishes a scientific, reasonable, and feasible characteristic chromatogram, ensuring the controllable quality of the abalone shell preparation.

[0125] To further illustrate the present invention, the method for establishing the characteristic spectrum of the compound traditional Chinese medicine preparation of Abalone Shell provided by the present invention will be described in detail below with reference to examples, but these should not be construed as limiting the scope of protection of the present invention.

[0126] The instruments, equipment, and reagents used in this embodiment are as follows:

[0127] Instruments and equipment:

[0128] WatersAcquity high-performance liquid chromatograph, VWD detector, Waters audit trail software;

[0129] KQ5200DE Ultrasonic Cleaner (Kunshan Ultrasonic Instrument Co., Ltd.);

[0130] XPE105 analytical balance (METTLER);

[0131] BSA124S electronic balance (sartorius);

[0132] JC-GGC-12W Water Bath Vibrator (Qingdao Juchuang Environmental Protection Group Co., Ltd.);

[0133] Reference standards, test samples, and processed medicinal materials:

[0134] Caffeic acid (China National Institutes for Food and Drug Control, batch number: 110885-201703);

[0135] Chlorogenic acid (China National Institutes for Food and Drug Control, batch number: 110753-202119);

[0136] Glycyrrhizin (China National Institutes for Food and Drug Control, batch number: 111610-202209);

[0137] Luteolin (China National Institutes for Food and Drug Control, batch number: 111720-202312);

[0138] Resveratrol (China National Institutes for Food and Drug Control, batch number: 111535-201703);

[0139] Hesperidin (China National Institutes for Food and Drug Control, batch number: 110721-202220);

[0140] Orange-yellow cassia seed extract (China National Institutes for Food and Drug Control, batch number: 111900-202006);

[0141] The following are the preparation methods for the commissioned production of abalone shell granules for research purposes (batch numbers: 20230301, 20230302, 20230303, 20231101, 20231102, 20231103, Shenyang Feilong Pharmaceutical Co., Ltd.): Raw materials: 625g of abalone shell, 625g of cassia seed, 625g of schizonepeta, 625g of chrysanthemum, and 312.5g of licorice; Preparation method: Weigh out the abalone shell, cassia seed, schizonepeta, chrysanthemum, and licorice slices separately, add water and decoct twice. The first time, add 12 times the amount of water and decoct for 2 hours. The second time, add 10 times the amount of water and decoct for 1.5 hours. Filter, combine the filtrates, and concentrate under reduced pressure to a clear extract with a relative density of 1.05-1.12 (60℃). Add an appropriate amount of dextrin, granulate, dry, and granulate to make 1000g, which is then packaged.

[0142] The medicinal materials, including abalone shell, cassia seed, chrysanthemum, schizonepeta, and licorice, were purchased from Anguo Hongfa Chinese Medicinal Herbs Co., Ltd.

[0143] Example 1

[0144] A method for establishing the characteristic spectrum of a traditional Chinese medicine preparation containing abalone shell, comprising the following steps:

[0145] Step A, Preparation of the reference solution:

[0146] Take appropriate amounts of caffeic acid, chlorogenic acid, glycyrrhizin, luteolin, resveratrol, hesperidin, and cassia seed extract reference standards respectively, and dilute them with 50% methanol aqueous solution to prepare a mixed standard solution, i.e., reference solution, in which the concentration of each reference standard is 10 μg / mL.

[0147] Step B, Preparation of the test solution:

[0148] Accurately weigh 0.5g of Shi Jue Ming Chinese medicine granules (batch number: 20231101, Shenyang Feilong Pharmaceutical Co., Ltd.), place them in a 25mL volumetric flask, add 15mL of 75% methanol aqueous solution, sonicate (frequency 40kHz, power 300W) for 90min to dissolve, remove and let stand to room temperature, dilute with 75% methanol aqueous solution to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0149] Step C, Detection:

[0150] High-performance liquid chromatography (HPLC) was used. 10 μL of both the reference solution and the test solution were injected into the HPLC system. The chromatographic conditions were as follows: Thermo Hypersil GOLD C10000 HPLC system was used. 18 The chromatographic column was 100 mm long and 2.1 mm in inner diameter, packed with octadecylsilane-bonded silica gel with a particle size of 1.9 μm. Acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, and methanol as mobile phase C. The detection wavelength was 285 nm, and the flow rate was 0.3 mL / min. The column temperature was 30 °C, and the gradient elution program was shown in Table 1, where % represents volume percentage.

[0151] Table 1 Gradient elution program

[0152] 0 1 97 2 9 6 92 2 21 6 92 2 26 11 87 2 33 11 87 2 36 13 85 2 42 19 79 2 46 19 79 2 49 44 54 2 55 44 54 2 55.5 1 97 2 60 1 97 2

[0153] Figure 1 This is the high-performance liquid chromatogram of the reference solution. Figure 1 Peak 1 is caffeic acid, peak 2 is chlorogenic acid, peak 3 is glycyrrhizin, peak 4 is luteolin, peak 5 is resveratrol, peak 6 is hesperidin, and peak 7 is cassia seed extract.

[0154] Comparative Example 1

[0155] Water, a 50% (v / v) methanol aqueous solution, and a 25% (v / v) methanol aqueous solution were used as extraction solvents, and the mixture was sonicated for 90 minutes. The rest of the process was the same as in Example 1.

[0156] Figure 6 The high-performance liquid chromatogram of the test sample after ultrasonic extraction with water solvent for 90 minutes; Figure 7 The high-performance liquid chromatogram of the test sample after ultrasonic extraction for 90 minutes using a 25% (v / v) methanol-water solution (i.e., 25% methanol);

[0157] Figure 8 The high-performance liquid chromatogram of the test sample after ultrasonic extraction for 90 minutes using a 50% (v / v) methanol-water solution (i.e., 50% methanol). Figure 9 The high-performance liquid chromatogram of the test sample after ultrasonic extraction for 90 minutes with a 75% methanol aqueous solution (i.e., 75% methanol).

[0158] Depend on Figures 6-9 It can be seen that when water, 75% methanol, 50% methanol, and 25% methanol were used as extraction solvents, the 75% methanol extraction of the test sample showed the most chromatographic peaks and the best separation of each peak, indicating complete extraction. In contrast, no obvious chromatographic peaks were observed in the water extraction of the test sample, indicating incomplete extraction. Therefore, 75% methanol was selected as the extraction solvent.

[0159] Comparative Example 2

[0160] Water, 50% methanol aqueous solution, 25% methanol aqueous solution, and 75% methanol aqueous solution were used as extraction solvents, respectively. The mixture was heated in a water bath under reflux for 90 minutes. The rest of the process was the same as in Example 1.

[0161] Figure 2 High-performance liquid chromatogram of the test sample extracted with water solvent by heating; Figure 3 High-performance liquid chromatogram of the test sample extracted with 25% methanol solvent by heating; Figure 4 High-performance liquid chromatogram of the test sample extracted with 50% methanol solvent by heating; Figure 5 The high-performance liquid chromatograms (HPLC) of the test sample extracted with 75% methanol solvent by heating are shown. It can be seen that the peak areas in the chromatograms of the test sample extracted using water, 75% methanol, 50% methanol, and 25% methanol as extraction solvents, respectively, are significantly smaller than those in the chromatograms of the test sample extracted using ultrasonic extraction. Therefore, ultrasonic extraction was selected as the extraction method.

[0162] Comparative Example 3

[0163] The samples were ultrasonically extracted with a 75% methanol aqueous solution for 30 minutes, 60 minutes and 120 minutes respectively, with the rest being the same as in Example 1.

[0164] Figure 10 The high-performance liquid chromatogram of the test sample after ultrasonic extraction with 75% methanol solvent for 30 minutes; Figure 11The high-performance liquid chromatogram of the test sample after ultrasonic extraction with 75% methanol solvent for 60 minutes; Figure 12 The high-performance liquid chromatogram (HPLC) of the test sample extracted with 75% methanol solvent by ultrasonic extraction for 120 minutes is shown. It can be seen that the peak areas in the chromatograms of the test sample extracted for 30 min and 60 min are significantly smaller. In the chromatogram of the test sample extracted for 120 min, the peaks interfere with the detection. In the chromatogram of the test sample extracted for 90 min, the peaks show good separation and do not interfere with the detection. Therefore, the extraction time of 90 min by ultrasonic extraction was selected.

[0165] Comparative Example 4

[0166] The column temperature was 40°C, and the rest was the same as in Example 1.

[0167] Figure 13 The image shows the high-performance liquid chromatogram of the test sample at a column temperature of 40℃. Comparison reveals that at 40℃, the retention time of the chromatographic peaks in the test sample's chromatogram is earlier, and the peak separation is not as good as at a column temperature of 30℃. Therefore, a column temperature of 30℃ was selected.

[0168] Comparative Example 5

[0169] The chromatograms of the test sample under different mobile phase systems were examined. The mobile phase system in step C of Example 1 was changed to acetonitrile-water. Specifically, acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B. The gradient elution program is shown in Table 2. The rest was the same as in Example 1.

[0170] Table 2 Comparative Example 5 Gradient Elution Procedure

[0171] 0 2 98 9 7 93 21 7 93 26 12 88 33 12 88 36 14 86 42 20 80 46 20 80 49 45 55 55 45 55 55.5 2 98 60 2 98

[0172] Figure 14 The image shows the high-performance liquid chromatogram (HPLC) of the test sample under the mobile phase system of acetonitrile-water. Comparison with the HPLC chromatogram of the test sample under the mobile phase system of Example 1 (i.e., acetonitrile-water-methanol) shows that the peak resolution of the test sample under the acetonitrile-water mobile phase system does not reach baseline separation, while the peak resolution of the test sample under the acetonitrile-water-methanol mobile phase system basically meets the requirements and can achieve baseline separation. Therefore, the acetonitrile-water-methanol mobile phase system was selected, i.e., acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, and methanol as mobile phase C.

[0173] Comparative Example 6

[0174] Different mobile phase gradient elution programs were investigated. Gradient elution was performed using the 68-minute mobile phase gradient elution program shown in Table 3 and the 67-minute mobile phase gradient elution program shown in Table 4, with the rest being the same as in Example 1.

[0175] Table 3 Comparative Example: 68-minute mobile phase gradient elution program

[0176] 0 1 97 2 9 6 92 2 21 6 92 2 26 11 87 2 33 11 87 2 36 13 85 2 42 19 79 2 46 19 79 2 49 44 54 2 55 44 54 2 62 89 9 2 62.5 1 97 2 68 1 97 2

[0177] Table 4 Comparative Example: 67-minute mobile phase gradient elution program

[0178] 0 2 98 12 7 93 21 12 88 36 14 86 42 20 80 45 25 75 55 45 55 60 45 55 61 2 98 67 2 98

[0179] Figure 15 The high-performance liquid chromatogram of the test sample under a 68-minute mobile phase gradient elution program is shown. Figure 16 The high-performance liquid chromatogram of the test sample under a 67-minute mobile phase gradient elution program is shown.

[0180] The chromatogram of the test sample under the mobile phase gradient elution program with a duration of 67 minutes did not achieve baseline separation. The chromatogram of the test sample under the mobile phase gradient elution program with a duration of 68 minutes was basically consistent with the chromatogram of the test sample under the mobile phase gradient elution program with a duration of 60 minutes in Example 1. Therefore, the 60-minute mobile phase gradient elution program was selected.

[0181] Comparative Example 7

[0182] The test solution was tested at wavelengths of 235nm, 255nm, 275nm, 305nm, and 325nm, respectively, with the rest being the same as in Example 1.

[0183] Figure 17 The chromatogram is a high-performance liquid chromatography (HPLC) chromatogram at a wavelength of 235 nm. Figure 18 The image shows a high-performance liquid chromatography (HPLC) chromatogram at a wavelength of 255 nm. Figure 19 The image shows a high-performance liquid chromatography (HPLC) chromatogram at a wavelength of 275 nm. Figure 20 The chromatogram is a high-performance liquid chromatography (HPLC) chromatogram at a wavelength of 305 nm. Figure 21 The image shows the high-performance liquid chromatography (HPLC) chromatogram at a wavelength of 325 nm. Comparison with the HPLC chromatogram at 285 nm in Example 1 shows that the detection of the number of chromatographic peaks and peak area at 285 nm is more reasonable; therefore, 285 nm was selected as the detection wavelength.

[0184] Example 2

[0185] Precision test

[0186] Accurately weigh 0.5g of the traditional Chinese medicine preparation granules (Shi Jue Ming Traditional Chinese Medicine Granules (batch number: 20231101, Shenyang Feilong Pharmaceutical Co., Ltd.)), place them in a 25mL volumetric flask, add 15mL of 75% methanol, sonicate (frequency 40kHz, power 300W) for 90min, let stand to room temperature, dilute to the mark with 75% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0187] Six consecutive injections were performed, using luteolin as a reference peak. The relative retention time and relative peak area of ​​the common peaks were calculated. The results showed that the RSD values ​​of the relative retention times were all less than 2%, and the RSD of the relative peak areas, except for peak 7 (cassia seed extract), were all less than 5.0%. The peak area of ​​cassia seed extract was relatively small, resulting in an RSD of 5.90%, but its relative peak area percentage was between 90% and 110%, meeting the requirements and indicating good precision of the method. Tables 5 and 6 show the results of the relative retention time and relative peak area tests, respectively.

[0188] Table 5. Results of relative retention time in precision tests

[0189]

[0190] Table 6. Results of relative peak area in precision test

[0191]

[0192]

[0193] Figure 22 Overlapped high-performance liquid chromatography (HPLC) chromatograms of six consecutive injections of the test solution for precision testing.

[0194] Example 3

[0195] Solution stability experiment

[0196] Accurately weigh 0.5g of the traditional Chinese medicine preparation granules (Shi Jue Ming Traditional Chinese Medicine Granules (batch number: 20231101, Shenyang Feilong Pharmaceutical Co., Ltd.)), place them in a 25mL volumetric flask, add 15mL of 75% methanol, sonicate (frequency 40kHz, power 300W) for 90min, let stand to room temperature, dilute to the mark with 75% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0197] Samples were injected at 0, 2, 4, 6, 8, and 16 hours, respectively. Using luteolin as a reference peak, the relative retention time and relative peak area of ​​the common peaks were calculated. The results showed that the RSD values ​​of the relative retention time were all less than 2%, and the RSD values ​​of the relative peak area were all less than 5.0%, meeting the requirements and indicating that the test solution was basically stable within 16 hours. Tables 7 and 8 show the results of the relative retention time and relative peak area of ​​the solution stability test, respectively.

[0198] Table 7. Results of relative retention times in solution stability tests

[0199]

[0200] Table 8. Results of relative peak areas in solution stability tests

[0201]

[0202]

[0203] Figure 23 High-performance liquid chromatography (HPLC) overlay chromatogram of the test solution for solution stability testing.

[0204] Example 4

[0205] Repeatable experiments

[0206] Accurately weigh 0.5g of the traditional Chinese medicine preparation granules (Shi Jue Ming Traditional Chinese Medicine Granules (batch number: 20231101, Shenyang Feilong Pharmaceutical Co., Ltd.)), place them in a 25mL volumetric flask, add 15mL of 75% methanol, sonicate (frequency 40kHz, power 300W) for 90min, let stand to room temperature, dilute to the mark with 75% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0207] Six parallel test solutions were prepared and analyzed separately. Using luteolin as a reference peak, the relative retention time and relative peak area of ​​the common peak were calculated. The results showed that the RSD values ​​of the relative retention time were all less than 2%, and the RSD values ​​of the relative peak area were all less than 5.0%, indicating good method repeatability. Tables 9 and 10 show the results of the relative retention time and relative peak area in the repeatability test, respectively.

[0208] Table 9. Results of Relative Retention Times in Repeatability Tests

[0209]

[0210] Table 10 Results of Relative Peak Area in Repeatability Tests

[0211]

[0212]

[0213] Figure 24 High-performance liquid chromatography (HPLC) overlay chromatogram of the test solution for repeatability testing.

[0214] Example 5

[0215] Establishment of Characteristic Atlas of Abalone Shell Preparations

[0216] Accurately weigh 0.5g of each of the following traditional Chinese medicine granules (20230301, 20230302, 20230303, 20231101, 20231102, 20231103): place them in a 25mL volumetric flask, add 15mL of 75% methanol, sonicate (frequency 40kHz, power 300W) for 90min, let stand to room temperature, dilute to the mark with 75% methanol, shake well, filter, and collect the filtrate to obtain the test solution.

[0217] Take appropriate amounts of caffeic acid, chlorogenic acid, glycyrrhizin, luteolin, resveratrol, hesperidin, and cassia seed extract reference standards, respectively, and dilute them with 50% methanol solution to prepare a mixed standard solution containing 10 μg per 1 mL.

[0218] High performance liquid chromatography conditions:

[0219] Column: Thermo Hypersil GOLD C 18 The column is 100 mm long and 2.1 mm in inner diameter, with octadecylsilane-bonded silica gel as the packing material and a particle size of 1.9 μm.

[0220] Column temperature: 30℃;

[0221] Detection wavelength: 285nm;

[0222] Injection volume: 10 μL;

[0223] The concentration of the test solution injected was 0.02 g / mL.

[0224] Mobile phase: Acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and methanol was used as mobile phase C. The flow rate of the mobile phase was 0.3 mL / min.

[0225] The elution method is gradient elution, and the elution procedure for the gradient elution is shown in Table 1.

[0226] Measurement:

[0227] Accurately pipette 10 μL each of the reference solution and the test solution, inject them into the high-performance liquid chromatograph, and determine the liquid chromatograms of the test solution and the reference solution, respectively.

[0228] Liquid chromatography of the reference solution, such as Figure 1As shown, Figure 1 Peak 1 is caffeic acid, peak 2 is chlorogenic acid, peak 3 is glycyrrhizin, peak 4 is luteolin, peak 5 is resveratrol, peak 6 is hesperidin, and peak 7 is cassia seed extract.

[0229] Figure 25 The images show the high-performance liquid chromatography (HPLC) overlay chromatograms of six batches of test solutions. It can be seen that the peak positions of seven common peaks (caffeic acid, chlorogenic acid, glycyrrhizin, luteolin, resveratrol, hesperidin, and cassia seed extract) in the chromatograms of the six batches of test solutions are basically consistent (the RSD of the relative retention times of the peaks at the same positions are all within 5%). A characteristic chromatogram of the traditional Chinese medicine compound preparation can be established. Taking peak 4 (luteolin) as a reference, peak 1 in the characteristic chromatogram is caffeic acid; peak 2 is chlorogenic acid; peak 3 is glycyrrhizin; peak 5 is resveratrol; peak 6 is hesperidin; and peak 7 is cassia seed extract.

[0230] Example 6

[0231] Following the method in Example 5, the positive control chromatograms of Cassia Seed, Schizonepeta, Chrysanthemum, and Licorice in the traditional Chinese medicine compound preparation were determined respectively. By analyzing the characteristic chromatograms of the traditional Chinese medicine compound preparation and the chromatograms of the positive control chromatograms of each herb, the retention times of the chromatograms were compared, and the common peaks in the characteristic chromatograms of the traditional Chinese medicine compound preparation were finally identified. The results are shown in Table 11.

[0232] Table 11. Attribution of common peaks in the characteristic chromatograms of traditional Chinese medicine compound preparations.

[0233] 1 10.851 caffeic acid Catnip 2 11.303 chlorogenic acid chrysanthemum 3 29.515 glycyrrhizin licorice 4 34.086 Luteolin chrysanthemum 5 39.428 Resveratrol chrysanthemum 6 41.591 hesperidin Catnip 7 51.22 Orange-yellow cassia seed extract Cassia seeds

[0234] Figure 26 High performance liquid chromatography (HPLC) overlay chromatograms of the positive control and mixed control solutions of the four Chinese medicinal materials and the test solution; Figure 27 The high-performance liquid chromatogram of Cassia seed as a positive control; Figure 28 The high-performance liquid chromatogram is for the positive control of *Nepeta cataria*. Figure 29 The high-performance liquid chromatogram is for the chrysanthemum positive control. Figure 30 The high-performance liquid chromatogram of licorice as a positive control (Note: Figure 27 and Figure 28 Smaller impurity peaks are mostly very close together, and their small peaks do not interfere with the positioning of the corresponding reference peaks.

[0235] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for establishing a characteristic spectrum of a compound traditional Chinese medicine preparation containing abalone shell, characterized in that, Includes the following steps: The compound traditional Chinese medicine preparation of Abalone Shell was mixed with a methanol aqueous solution and subjected to ultrasonic extraction to obtain a test solution; the volume fraction of the methanol aqueous solution was 75%; the raw materials for preparing the compound traditional Chinese medicine preparation of Abalone Shell included Abalone Shell, Cassia Seed, Schizonepeta, Chrysanthemum and Licorice. The test solution was subjected to high-performance liquid chromatography (HPLC) to obtain a HPLC chromatogram of the test sample. The HPLC chromatogram of the test sample was compared with a predetermined reference chromatogram. Common peaks were selected from the HPLC chromatogram of the test sample to obtain the characteristic chromatogram of the Shi Jue Ming compound traditional Chinese medicine preparation. The reference chromatogram was obtained by using a reference solution under the same HPLC detection conditions. The reference solution contained reference standards containing caffeic acid, chlorogenic acid, glycyrrhizin, luteolin, resveratrol, hesperidin, and cassia seed extract. The conditions for the high-performance liquid chromatography detection include: The chromatographic column was a Thermo Hypersil GOLD C10. 18 ; The column temperature is 30℃; The detection wavelength is 285nm; The injection volume was 10 μL; The mobile phase includes mobile phase A, mobile phase B and mobile phase C, wherein mobile phase A is acetonitrile, mobile phase B is a 0.1% (v / v) aqueous solution of phosphoric acid and mobile phase C is methanol; The flow rate of the mobile phase is 0.3 mL / min; The elution program is a gradient elution program; The gradient elution procedure is as follows: 0–9 min: The volume percentage of mobile phase A increases uniformly from 1% to 6%, the volume percentage of mobile phase B decreases uniformly from 97% to 92%, and the volume percentage of mobile phase C remains at 2%. 9–21 min: The volume percentage of mobile phase A is maintained at 6%, the volume percentage of mobile phase B is maintained at 92%, and the volume percentage of mobile phase C is maintained at 2%. 21–26 min: The volume percentage of mobile phase A increases uniformly from 6% to 11%, the volume percentage of mobile phase B decreases uniformly from 92% to 87%, and the volume percentage of mobile phase C remains at 2%. 26–33 min: The volume percentage of mobile phase A is maintained at 11%, the volume percentage of mobile phase B is maintained at 87%, and the volume percentage of mobile phase C is maintained at 2%. 33–36 min: The volume percentage of mobile phase A increases uniformly from 11% to 13%, the volume percentage of mobile phase B decreases uniformly from 87% to 85%, and the volume percentage of mobile phase C remains at 2%. 36–42 min: The volume percentage of mobile phase A increases uniformly from 13% to 19%, the volume percentage of mobile phase B decreases uniformly from 85% to 79%, and the volume percentage of mobile phase C remains at 2%. 42–46 min: The volume percentage of mobile phase A is maintained at 19%, the volume percentage of mobile phase B is maintained at 79%, and the volume percentage of mobile phase C is maintained at 2%. 46–49 min: The volume percentage of mobile phase A increases uniformly from 19% to 44%, the volume percentage of mobile phase B decreases uniformly from 79% to 54%, and the volume percentage of mobile phase C remains at 2%. 49–55 min: The volume percentage of mobile phase A is maintained at 44%, the volume percentage of mobile phase B is maintained at 54%, and the volume percentage of mobile phase C is maintained at 2%. 55–55.5 min: The volume percentage of mobile phase A decreases uniformly from 44% to 1%, the volume percentage of mobile phase B increases uniformly from 54% to 97%, and the volume percentage of mobile phase C remains at 2%. 55.5–60 min: The volume percentage of mobile phase A is maintained at 1%, the volume percentage of mobile phase B is maintained at 97%, and the volume percentage of mobile phase C is maintained at 2%.

2. The method according to claim 1, characterized in that, The dosage ratio of the compound herbal preparation of abalone shell to the methanol aqueous solution is 0.5g: 15-20mL.

3. The method according to claim 1, characterized in that, The ultrasonic extraction frequency is 40kHz and the power is 300W.

4. The method according to claim 1 or 3, characterized in that, The ultrasonic extraction time was 90 minutes.

5. The method according to claim 1, characterized in that, The concentration of each reference standard in the reference solution was 10 μg / mL.

6. The method according to claim 1, characterized in that, The chromatographic column has a length of 100 mm and an inner diameter of 2.1 mm.

7. The method according to claim 1, characterized in that, The concentration of the test solution is 0.02 g / mL.

8. The method according to claim 1, characterized in that, The composition of the ingredients is as follows: 250 parts by weight of abalone shell, 250 parts by weight of cassia seed, 250 parts by weight of schizonepeta, 250 parts by weight of chrysanthemum, and 125 parts by weight of licorice.

9. The method according to claim 8, characterized in that, The preparation method of the aforementioned abalone shell compound traditional Chinese medicine preparation includes the following steps: Boil abalone shell, cassia seed, schizonepeta, chrysanthemum and licorice in water twice, filter the decoctions obtained from the two decoctions separately, and combine the filtrates. The combined filtrate was concentrated and maltodextrin was added. After granulation and drying, the compound traditional Chinese medicine preparation of abalone shell was obtained.

10. The method according to claim 9, characterized in that, The amount of water added for the first decoction is 12 times the weight of the raw materials, and the amount of water added for the second decoction is 10 times the weight of the raw materials. The first decoction time is 2 hours, and the second decoction time is 1.5 hours.

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