Method for identifying Hubei radix ophiopogonis

Abstract

The invention relates to the technical field of detection and analysis of chemical components of traditional Chinese medicines, and particularly discloses a method for identifying Hubei radix ophiopogonis. According to the method, eight characteristic peaks are determined by adopting an ultrasonic-assisted ethanol solution extraction technology, an UPLC (Ultra Performance Liquid Chromatography) and an evaporative light detector, and are used as characteristic chromatograms of the Hubei radix ophiopogonis medicinal material and the preparation thereof, so that the Hubei radix ophiopogonis can be quickly and efficiently identified and distinguished from the Zhejiang radix ophiopogonis, the Sichuan radix ophiopogonis and the liriope muscari radix ophiopogonis, and the method is an identification method with very high practicability, and is suitable for popularization and application. Meanwhile, the contents of three main components of fructose, cane sugar and glucose in the Hubei radix ophiopogonis medicinal material and the preparation thereof can be measured, so that the quality control of the Hubei radix ophiopogonis and the preparation thereof reaches a higher level. The method has the characteristics of better sensitivity, convenience in operation, low cost, high universality and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of detection and analysis of chemical components of traditional Chinese medicines, and particularly relates to a method for identifying Ophiopogon japonicus. Background Art

[0002] Hubei Ophiopogon comes from the dried root of Liriope spicata (Thunb.) Lour.var.prolifera YTMa, a plant of the Liliaceae family. It has been used as medicine since the Song Dynasty. In the early years, Ophiopogon medicinal materials were mixed. Since the 1995 edition of the Chinese Pharmacopoeia, Ophiopogon medicinal materials have been divided into two types: Ophiopogon and Wild Ophiopogon. From the perspective of function and indications, the two are basically the same, both of which have the effects of nourishing yin, promoting body fluid, moistening the lungs and clearing the heart. It is used for dry cough due to lung dryness, tuberculosis due to yin deficiency, sore throat due to throat numbness, thirst due to loss of body fluid, internal heat and thirst, restlessness and insomnia, and constipation due to dry intestines. Ophiopogon japonicus is divided into Hubei Ophiopogon and Liriope muscari (Decne.) Baily. Ophiopogon japonicus (Lf) Ker-Gawl can be divided into Sichuan Ophiopogon and Zhejiang Ophiopogon according to the place of origin. Sichuan Ophiopogon is mainly produced in Santai County, Sichuan, Zhejiang Ophiopogon is mainly produced in Yuyao and Hangzhou, Zhejiang, Hubei Ophiopogon is mainly produced in the Hanjiang River Basin such as Xiangyang, Hubei, and Short-stalked Ophiopogon is mainly produced in Quanzhou, Hui'an, Fujian. Sichuan Ophiopogon has the highest output, followed by Hubei Ophiopogon.

[0003] The current edition of the Chinese Pharmacopoeia only stipulates the source, properties, identification, inspection, and extracts of Hubei Ophiopogon japonicus. It lacks a highly sensitive, characteristic, and quantifiable quality control method, resulting in uneven quality of Hubei Ophiopogon japonicus and its preparations in the market. The national standards for formula granules such as Sichuan Ophiopogon japonicus and Zhejiang Ophiopogon japonicus issued by the State Pharmacopoeia Commission use the technology of ultra-high liquid phase plus electrospray detector, which has a high detection cost and low universality, limiting the application of the technology. At the same time, since Hubei Ophiopogon japonicus has similar properties and effects to the other three types of Ophiopogon japonicus, several types of Ophiopogon japonicus are often mixed, and it is difficult for non-experienced professionals to distinguish them. Therefore, it is very necessary to find a method that can not only conduct an overall qualitative and quantitative evaluation of the quality of Hubei Ophiopogon japonicus, but also simply and quickly identify and distinguish Hubei Ophiopogon japonicus from the other three types of Ophiopogon japonicus. Summary of the invention

[0004] The purpose of the present invention is to provide a method for identifying Hubei Ophiopogon japonicus in view of the above-mentioned deficiencies in the prior art, which can identify and distinguish Hubei Ophiopogon japonicus from Zhejiang Ophiopogon japonicus, Sichuan Ophiopogon japonicus, and Breviscapus ophiopogon japonicus, and can also accurately detect the content of various sugar components in Hubei Ophiopogon japonicus, which can more accurately reflect the quality of Hubei Ophiopogon japonicus and its preparations, is conducive to quality control, and has lower cost.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] The first object of the present invention is to provide a method for identifying Ophiopogon japonicus, the method comprising the following steps:

[0007] S1. Prepare solution:

[0008] Test solution: Take an appropriate amount of Ophiopogon japonicus or its preparation sample to be tested, grind it into powder, add an appropriate amount of ethanol for ultrasonic extraction, shake well, filter, and take the filtrate as the test solution;

[0009] Reference substance solution: Take appropriate amounts of three reference substances, including fructose, sucrose, and D-anhydrous glucose, and dissolve them in ethanol to prepare a mixed reference substance solution;

[0010] Reference solution for reference medicinal materials: Take an appropriate amount of standard Hubei Ophiopogon japonicus medicinal materials, add an appropriate amount of ethanol and perform ultrasonic extraction.

[0011] Shake well, filter, and take the filtrate as the reference solution of the control medicinal material;

[0012] S2. Implement testing:

[0013] Inject the test solution, mixed reference solution, and reference medicinal material reference solution prepared in step S1 into an ultra-high pressure high performance liquid chromatograph, respectively, and record the chromatogram;

[0014] The chromatographic parameters set on the high performance liquid chromatograph are: using one of a BEH AMIDE column or a Poroshell 120h HILIC-Z column as a chromatographic column; using acetonitrile as mobile phase A, using water as mobile phase B for gradient elution, setting the flow rate to 0.1-0.3 mL / min, using an evaporative light scattering detector for detection, and setting the column temperature to 25-30° C.;

[0015] The gradient elution procedure is as follows: 0-5 min, the volume ratio of mobile phase A: mobile phase B is 85:15; 5-12 min, the volume ratio of mobile phase A: mobile phase B is changed from 85:15 to 80:20; 12-17 min, the volume ratio of mobile phase A: mobile phase B is changed from 80:20 to 78:22; 17-30 min, the volume ratio of mobile phase A: mobile phase B is changed from 78:22 to 75:25; 30-35 min, the volume ratio of mobile phase A: mobile phase B is changed from 75:25 to 40:60;

[0016] S3. Analysis of the graph:

[0017] The spectrum of the test solution is analyzed with the characteristic spectrum of the control medicinal material reference solution as the standard, wherein the characteristic spectrum of the control medicinal material reference solution includes 8 common peaks, with peak 5 as the reference peak, and the relative retention times of the remaining characteristic peaks should be within ±10% of the specified value, and the specified values ​​are: peak 1 with a relative retention time of 0.36, peak 2 with a relative retention time of 0.45, peak 3 with a relative retention time of 0.51, peak 4 with a relative retention time of 0.55, peak 6 with a relative retention time of 1.55, peak 7 with a relative retention time of 1.67, and peak 8 with a relative retention time of 2.21. Peak 2 is a fructose peak, peaks 3 and 4 are isomers of each other and are glucose peaks, peak 4 is a sucrose peak, peak 7 is a sucrose trisaccharide peak, and peak 8 is a nastose peak;

[0018] S4. Identification of Radix Ophiopogonis

[0019] Calculate the logarithmic ratio of the peak areas of Peak 7 and Peak 6: log peak area of ​​Peak 7 / log peak area of ​​Peak 6. If it is greater than 0.91, it is Hubei Ophiopogon japonicus medicinal material. If it is less than 0.91, it is not Hubei Ophiopogon japonicus medicinal material.

[0020] Furthermore, in step S1, the ultrasonic power is 250-300 W, and the concentration of the test solution is 5-30 mg / mL.

[0021] Further, in step S1, the preparation method of the test solution specifically includes taking an appropriate amount of the Ophiopogon japonicus medicinal material or its preparation to be tested, grinding it, accurately weighing it, placing it in a stoppered conical flask, accurately adding 10 ml of 70% ethanol, weighing it, ultrasonically extracting it for 10 minutes, with an ultrasonic power of 300 W and a frequency of 53 kHz, cooling it, weighing it again, making up the lost weight with 70% ethanol, shaking it well, filtering it, and taking the filtrate to obtain it.

[0022] Furthermore, in step S1, the preparation method of the control medicinal material reference solution specifically includes taking 0.2 g of Hubei Ophiopogon japonicus control medicinal material, placing it in a stoppered conical flask, adding 10 ml of 70% ethanol, ultrasonically extracting for 10 minutes, cooling and shaking, filtering, and taking the filtrate as the control medicinal material reference solution.

[0023] Furthermore, in step S2, the flow rate is set to 0.2 mL / min.

[0024] Furthermore, in step S2, the column temperature is set to 25°C.

[0025] Furthermore, in step S2, the chromatographic column is a BEH Amide column with specifications of 100×2.1 mm and 1.7 μm.

[0026] Furthermore, it is used to determine the contents of fructose, sucrose and glucose, three main components in the test samples of Hubei Ophiopogon japonicus and its preparations, specifically including accurately aspirating 1μl and 3μl of the reference solution, 2μl of the test solution and the control medicinal material reference solution, respectively, injecting into the ultra-high performance liquid chromatography, determining, and calculating with the external standard two-point method logarithmic equation to obtain the result.

[0027] Furthermore, the concentrations of the reference solution are: fructose is 1.2 mg / mL, sucrose is 0.2 mg / mL, and D-anhydrous glucose is 0.3 mg / mL; the concentration of the reference medicinal material reference solution is 20 mg / mL.

[0028] Furthermore, the Ophiopogon japonicus medicinal material or preparation sample to be tested is at least one of Ophiopogon japonicus from Hubei, Ophiopogon japonicus from Zhejiang, Ophiopogon japonicus from Sichuan and Ophiopogon japonicus from Breviscapus.

[0029] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects:

[0030] (1) The method for identifying Hubei Ophiopogon japonicus provided by the present invention can quickly and efficiently identify and distinguish Hubei Ophiopogon japonicus from Zhejiang Ophiopogon japonicus, Sichuan Ophiopogon japonicus, and Breviscapus ophiopogon japonicus. It is a very practical identification method, which enables the quality control of Hubei Ophiopogon japonicus and its preparations to reach a higher level.

[0031] (2) The method provided by the present invention has good separation of characteristic peaks, high sensitivity and strong reproducibility. It can also be used for quality control of Hubei Ophiopogon japonicus medicinal materials, Hubei Ophiopogon japonicus decoction pieces, Hubei Ophiopogon japonicus standard decoction and Hubei Ophiopogon japonicus formula granules, and has a very wide range of applications.

[0032] (3) The method provided by the present invention is low-cost and is beneficial to the production operations of pharmaceutical manufacturers. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is the color spectrum of experimental group 1 in the embodiment of the present invention;

[0034] Figure 2 is the color spectrum of control group 1 in the embodiment of the present invention;

[0035] Figure 3 is the color spectrum of control group 2 in the embodiment of the present invention;

[0036] Figure 4 is the color spectrum of control group 3 in the embodiment of the present invention;

[0037] Figure 5 It is the specific property investigation spectrum in Example 1;

[0038] Figure 6is a chromatogram of the ACQUITY BEH AMIDE column in Example 1 of the present invention;

[0039] Figure 7 is a chromatogram of the Poroshell 120h HILIC-Z column in Example 1 of the present invention;

[0040] Figure 8 It is the characteristic spectrum of 24 batches of Hubei Ophiopogon japonicus standard decoction dry paste powder in Example 1 of the present invention;

[0041] Fig. 9 This is the characteristic spectrum generated in Example 1 of the present invention, in which Peak 2: fructose; Peak 3 and Peak 4: glucose; Peak 5 (S): sucrose; Peak 7: kestose; Peak 8: nastose. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical scheme and advantages of the present invention clearer, the specific embodiments of the present invention are further described in detail below in conjunction with specific examples and drawings. If no specific technology or conditions are specified in the examples, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0043] In the present invention, "Hubei Ophiopogon japonicus" refers to Hubei Ophiopogon japonicus medicinal materials and preparation products (decoction pieces, decoctions, granules, etc.) processed from Hubei Ophiopogon japonicus medicinal materials.

[0044] Reagent and instrument of the present invention:

[0045] Instruments: High performance liquid chromatograph (Agilent 1290); WSK-A fully automatic air generator (Tianjin Jinfen Analytical Instrument Manufacturing Co., Ltd.), evaporative light detector (Alltech ELSD 6100), 1 / 10,000 electronic balance (Sartorius); 1 / 100,000 electronic balance (Sartorius); DZKW-S-8 water bath (Beijing Yongguangming Medical Instrument Co., Ltd.); SK8200XS ultrasonic cleaner (Shanghai Kedao Ultrasonic Instrument Co., Ltd.).

[0046] Chromatographic columns: ACQUITY BEH AMIDE (2.1×100 mm, 1.7 μm); Poroshell 120hHILIC-Z (2.1×100 mm, 2.7 μm);

[0047] Reagents: Radix Ophiopogonis (Hubei Radix Ophiopogonis) control drug (batch number: 121136-201803); fructose (batch number: 100231-202309), D-anhydrous glucose (batch number: 110833-202109), sucrose (batch number 111507-202406), nesitose (batch number: 111891-201704), all purchased from China Food and Drug Inspection Institute, saccharide (batch number: DST231213-087, purchased from Desit Biotechnology). Acetonitrile was chromatographically pure from Fisher Chemical, water was ultrapure water obtained by filtration through ELGA ultrapure water system, and other reagents were chromatographically pure.

[0048] Trial drugs: 48 batches of Hubei Ophiopogon japonicus and other Ophiopogon japonicus medicinal materials, standard decoction dry paste powder of formula granules made from 48 batches of medicinal materials, 4 batches of finished formula granules of Ophiopogon japonicus (Hubei Ophiopogon japonicus) (C210621022, C263824011, C263824021, C263824031. Maltodextrin (batch number: F21042004, COFCO Biochemical Energy (Gongzhuling) Co., Ltd.).

[0049] The preparation method of the standard decoction dry paste powder is as follows: weigh 100 g of Ophiopogon japonicus medicinal slices, place them in a decoction pot, add 1000 mL of water, soak for 30 minutes, boil over high heat, and then simmer for 60 minutes over low heat. After the extract is filtered through a 200-mesh filter, 800 mL of water is added to the residue, boil over high heat, and then simmer for 40 minutes over low heat. The two filtrates are combined and concentrated by rotary evaporator to a solid-liquid ratio of about 1:1. The concentration temperature is set to 65°C, the concentrate is placed in a tray, dried in a low-temperature vacuum, and crushed to obtain the extract.

[0050] The preparation method of the formula granules of Radix Ophiopogonis (Hubei Radix Ophiopogonis) is as follows: take an appropriate amount of Radix Ophiopogonis (Hubei Radix Ophiopogonis), add water and boil twice, add 10 times the amount of water for the first time, soak for 30 minutes, and boil for 90 minutes; add 8 times the amount of water for the second time, boil for 60 minutes, filter the decoction, concentrate the filtrate into a clear paste, add an appropriate amount of auxiliary materials, dry (or dry and crush), then add an appropriate amount of auxiliary materials, mix well, and granulate to obtain the paste.

[0051] The batch numbers and origins of 48 batches of Hubei Ophiopogon japonicus and other Ophiopogon japonicus medicinal materials used in the present invention are as follows:

[0052] Table 1. Batch numbers and origins of 48 batches of Hubei Ophiopogon japonicus and other Ophiopogon japonicus medicinal materials

[0053]

[0054]

[0055] Example 1

[0056] The method for identifying Ophiopogon japonicus provided in this embodiment is specifically as follows:

[0057] 1. Determination method

[0058] 1.1. Preparation of reference solution: Take appropriate amount of fructose, sucrose and D-anhydrous glucose reference substances, weigh accurately, add 70% ethanol to prepare mixed solutions containing 1.2 mg / mL fructose, 0.2 mg / mL sucrose and 0.3 mg / mL D-anhydrous glucose per 1 ml as reference solution.

[0059] 1.2. Preparation of reference medicinal material reference solution: Take 0.2 g of Radix Ophiopogonis (Hubei Radix Ophiopogonis) reference medicinal material, place it in a stoppered conical flask, add 10 ml of 70% ethanol, ultrasonically treat (power 300 W, frequency 53 kHz) for 10 minutes, cool and shake well, filter, and take the filtrate as the reference medicinal material solution.

[0060] 1.3. Preparation of test solution: Take an appropriate amount of the product, grind it into powder, weigh it accurately, put it in a stoppered conical flask, accurately add 10 ml of 70% ethanol, weigh it, treat it with ultrasound (power 300 W, frequency 53 kHz) for 10 minutes, let it cool, weigh it again, make up the lost weight with 70% ethanol, shake it well, filter it, and take the filtrate.

[0061] 1.4. Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.7 μm); acetonitrile was used as mobile phase A, water was used as mobile phase B, and gradient elution was performed according to the provisions in Table 2 below; the flow rate was 0.3 mL per minute, the column temperature was 25°C, and the evaporative light scattering detector was used for detection. The theoretical plate number calculated based on the sucrose peak should not be less than 5000.

[0062] Table 2 Gradient elution program

[0063]

[0064] 1.5. Determination method: Accurately pipette 1μl and 3μl of reference solution, 2μl of test solution and reference medicinal material solution respectively, inject into liquid chromatograph, determine, and calculate with external standard two-point method logarithmic equation to get the result.

[0065] During the method development phase, the inventors conducted the following studies:

[0066] (1) Investigation of elution gradient program.

[0067] The same batch of Hubei Ophiopogon japonicus standard decoction dry paste powder was taken, and the test sample solution was prepared according to item 1.3 of Example 1, and the experimental group 1 was measured according to the chromatographic conditions of items 1.4 and 1.5 of Example 1. The control group was measured according to the following chromatographic conditions.

[0068] The differences between control group 1, control group 2, control group 3 and experimental group 1 are that the elution gradient programs, column temperatures and flow rates are different. The elution gradient programs, column temperatures and flow rates of each group are shown in the table below.

[0069] Table 3. Elution gradient program for control group 1

[0070]

[0071] Table 4. Elution gradient program for control group 2

[0072]

[0073]

[0074] Table 5 Elution gradient program of control group 3

[0075]

[0076] The chromatograms of experimental group 1, control group 1, control group 2 and control group 3 are shown in Figure 1-4 ; From the chromatograms of experimental group 1, control group 1, control group 2 and control group 3, it can be seen that the peak theoretical plate number, tailing factor, peak shape and separation degree of the chromatogram of the stir-fried barley sprout standard decoction dry paste powder obtained in experimental group 1 are higher than those of the three control groups, and the stability is better. Finally, the experimental group conditions were selected as the elution gradient program of this method.

[0077] (2) Investigation of the preparation method of the test solution

[0078] (2.1) Investigation of extraction solvent

[0079] Take the same batch of Hubei Ophiopogon japonicus standard decoction dry paste powder, grind it finely, take 5 portions, each about 0.1g, accurately weigh, put it in a stoppered conical bottle, accurately add water, 30% ethanol, 50% ethanol, 70% ethanol, 80% ethanol, 90% ethanol 10ml, plug it tightly, weigh it, ultrasonic treatment (power 250W, frequency 35kHz) for 30 minutes, take it out, let it cool, weigh it again, and then use water, 10% ethanol, 50% ethanol, 70% ethanol, 80% ethanol, 90% ethanol to make up the lost weight, shake it well, filter it, take the filtrate, and get the test solution, inject it into the ultra-high performance liquid chromatography for determination, and calculate the content of fructose, sucrose, and glucose respectively. The results in Table 6 show that when the extraction solvent is 70% methanol, there are fewer impurities, the separation of each peak is better, and there is less interference; while the content of sugar components above 80% ethanol is relatively low, and 70% ethanol is finally determined as the preparation solvent of this product.

[0080] Table 6. Investigation of extraction solvents

[0081]

[0082]

[0083] (2.2) Investigation of extraction methods

[0084] Take the same batch of Hubei Ophiopogon japonicus standard decoction dry paste powder, grind it finely, take 5 portions, each about 0.1g, accurately weigh, accurately add 70% ethanol 10mL, weigh the weight, ultrasonic extraction for 60 minutes, ultrasonic 30 minutes, ultrasonic 10 minutes, heat reflux 1h, cool, use 70% ethanol to make up the lost weight, shake well, filter, take the filtrate, and get the test solution. Inject into ultra-high liquid chromatography for determination, and calculate the content of fructose, sucrose, and glucose respectively. The results in Table 7 show that when ultrasonic for 10 minutes, the content of fructose, sucrose, and glucose in the test solution is basically the same as the other three methods. Considering the ease of operation and time saving, the extraction method is determined to be ultrasonic for 10 minutes.

[0085] Table 7. Extraction method investigation

[0086]

[0087] (2.3) Sample weighing inspection

[0088] Take the same batch of Hubei Ophiopogon japonicus standard decoction dry paste powder, grind it, weigh about 0.05g, 0.1g, 0.2g, 0.3g respectively, accurately weigh, accurately add 10mL of 70% ethanol respectively, weigh the weight, ultrasonically extract for 10 minutes respectively, cool, make up the lost weight with 70% ethanol, shake well, filter, take the filtrate, and get the test solution. Inject into ultra-high liquid chromatography for determination, and calculate the content of fructose, sucrose, and glucose respectively. The results in Table 8 show that the sample weight is 0.1g, and the content of fructose, sucrose, and glucose in the test solution is higher than that of the sample weight of 0.2g and 0.3g, which is basically consistent with the sample weight of 0.05g. Considering that a higher precision balance is required to weigh 0.05g, the experimental error may also be large, so the sample weight of 0.1g is selected.

[0089] Table 8 Sample weighing inspection

[0090]

[0091] (3) Methodological investigation

[0092] (3.1) Specificity investigation

[0093] Inject 70% ethanol solution of dextrin (negative control), together with reference substance solution, reference medicinal material reference substance solution and test solution into the liquid chromatograph and record the chromatogram. Figure 5 . Figure 5The results showed that the negative control solution did not have any peaks, while the peaks of the test solution, reference solution, and control medicinal material solution all had good peak shapes, indicating that this method has good specificity.

[0094] (3.2) Linear investigation

[0095] Accurately weigh 90.053 mg, 25.148 mg and 14.964 mg of fructose, D-glucose and sucrose as three reference substances, respectively, dissolve in 70% ethanol and make up to 20 mL, shake well to obtain mixed reference substance mother solutions ① of fructose, glucose and sucrose, with concentrations of 4.4981 mg / ml, 1.2561 mg / ml and 0.7482 mg / ml, respectively. Accurately pipette 5 mL of the above-mentioned mother solution of reference substance into a 10 mL volumetric flask, add 70% ethanol to make up to the mark, shake well, and use it as DZ-5 and the mother solution for the next batch of solutions. Repeat this step twice to obtain reference substance solutions DZ-1 and DZ-3. Accurately weigh 67.540 mg, 18.861 mg, and 11.223 mg of three kinds of reference substances, fructose, D-glucose, and sucrose, respectively, add 70% ethanol to dissolve and make up to 20 mL, shake well, and obtain a mixed reference substance mother solution ② of fructose, glucose, and sucrose with concentrations of 3.3762 mg / ml, 0.9421 mg / ml, and 0.5611 mg / ml (DZ-6), respectively. Accurately pipette 5mL of the mother solution of the above-mentioned reference substance solution into a 10mL volumetric flask, add 70% ethanol to the mark, shake well, and use it as DZ-4. Repeat this step to obtain DZ-2. Take the above-mentioned 6 different concentrations of the reference substance solution and perform chromatographic analysis under the chromatographic conditions of the content determination to obtain the peak area of ​​the chromatographic peak of each concentration. Use the LOG concentration as the abscissa and the corresponding LOG chromatographic peak area as the ordinate to establish a standard curve, and obtain the linear relationship and range (injection volume 2μl):

[0096] Fructose: Y = 2.2415X + 2.4634, r = 0.9991;

[0097] Glucose: Y = 1.9086X + 2.7998, r = 0.9991;

[0098] Sucrose: Y = 1.6096X + 3.6406, r = 0.9993;

[0099] Table 9. Data related to the establishment of standard curve

[0100]

[0101] The results showed that the fructose reference solution had a good linear relationship in the concentration range of 1.1245 mg / ml-6.7472 mg / ml, the glucose reference solution had a good linear relationship in the concentration range of 0.3140 mg / ml-1.8842 mg / ml, and the sucrose had a good linear relationship in the concentration range of 0.1871 mg / ml-1.1223 mg / ml, with r>0.999, which met the linearity requirements of method validation.

[0102] (3.3) Repeatability study

[0103] Weigh about 0.1g of the same batch of Hubei Ophiopogon japonicus standard decoction dry paste powder accurately, prepare 6 test sample solutions according to the preparation method under 1.3 above, analyze according to the chromatographic conditions under 1.4 and 1.5, and determine the peak area. By calculation, the average fructose content in Hubei Ophiopogon japonicus standard decoction dry paste powder is 116.28mg / g, and the RSD value is 0.81%; the average glucose content is 29.48mg / g, and the RSD value is 1.23%; the average sucrose content is 19.62mg / g, and the RSD value is 1.27%. The results show that the detection method has good repeatability.

[0104] Table 10. Relevant data of repeatability test

[0105]

[0106]

[0107] (3.4) Intermediate precision study

[0108] Different analysts used another Waters ultra-high performance liquid chromatograph at different times to conduct repeatable experiments. According to calculations, the average fructose content in the Hubei Ophiopogon japonicus standard decoction dry paste powder was 115.37 mg / g, with an RSD value of 0.61%; the average glucose content was 29.31 mg / g, with an RSD value of 0.75%; the average sucrose content was 19.31 mg / g, with an RSD value of 1.17%. The RSDs of the three components between different instruments were 0.55%, 0.39%, and 1.12%, respectively, which met the requirements of the analysis.

[0109] Table 11. Intermediate precision related data

[0110] Sample weight (g) Fructose content mg / g Glucose content mg / g Sucrose content mg / g 0.1049 114.98 29.19 19.08 0.1011 115.49 29.02 19.39 0.1036 114.32 29.16 19.13 0.1046 116.18 29.58 19.48 0.1052 116.08 29.47 19.15 0.1019 115.16 29.45 19.64 Mean 115.37 29.31 19.31 RSD% 0.61 0.75 1.17 RSD% of different instruments 0.55 0.39 1.12

[0111] (3.5) Accuracy test

[0112] Take the same batch of Hubei Ophiopogon japonicus standard decoction dry paste powder (the average content of fructose, glucose and sucrose are 116.28mg / g, 29.48mg / g and 19.62mg / g, respectively), accurately weigh about 0.05g in 6 stoppered conical bottles. Accurately pipette ①12ml of the linear reference substance mother solution into a 100mL volumetric flask, add 70% ethanol to dissolve and dilute to volume, draw 10mL of this solution into a 100mL volumetric flask, add 70% ethanol to dissolve and dilute to volume, shake well, and use it as the reference substance solution for spiking (the concentrations of fructose, glucose and sucrose are 5.3978 mg / ml, 1.5074mg / ml, 0.89784mg / ml). Accurately pipette 10ml of the reference solution into the 6 volumetric flasks containing the samples, and prepare the test solution for recovery determination according to the preparation method under 1.3, and analyze according to the chromatographic conditions under 1.4 and 1.5. Calculate the recovery of fructose, glucose and sucrose. The results showed that the average recovery was 98.98% and the RSD value was 1.27%, which met the requirements. This detection method is accurate and reliable in detecting the content of fructose, glucose and sucrose in the dry paste powder of Hubei Ophiopogon japonicus standard decoction.

[0113] Table 12. Fructose accuracy test results

[0114]

[0115] Table 13. Glucose accuracy test results

[0116]

[0117] Table 14. Sucrose accuracy test results

[0118]

[0119] (3.6) Durability test

[0120] The above Hubei Ophiopogon japonicus standard decoction dry paste powder test solution was taken and measured using two different chromatographic columns (①BEH AMIDE (2.1×100mm, 1.7μm); ②Poroshell120hHILIC-Z (2.1×100mm, 2.7μm) according to the determined chromatographic conditions to determine the durability of the method for different chromatographic columns. The results are shown in Figure 6 , Figure 7 As can be seen from the chart, the peak shapes of the spectra obtained by the two chromatographic columns are quite different, so it is recommended to choose the ACQUITY BEH AMIDE (2.1×100mm, 1.7μm) chromatographic column.

[0121] (4) Characteristic spectrum and characteristic peak determination

[0122] The above method was used to complete the content determination of 24 batches of Hubei Ophiopogon japonicus standard decoction dry paste powder. At the same time, the chromatographic peaks of the content determination of 24 batches of Hubei Ophiopogon japonicus standard decoction were superimposed using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software System (2012 Edition)" issued by the National Pharmacopoeia Committee to form a common peak pattern diagram (see Figure 8 ), the results showed that 8 common peaks were present in all 24 batches of Hubei Ophiopogon japonicus standard decoction dry paste powder test samples, and the reference characteristic spectrum was established by median calculation, among which peak 5 was the sucrose reference substance chromatographic peak, with a large peak area and good separation, so the peak was marked as S peak, peak 2 was the fructose reference substance chromatographic peak, and peaks 3 and 4 were D-anhydrous glucose reference substance chromatographic peaks (isomers). The reference medicinal materials also had corresponding 8 peaks, which were marked, and the relative retention time and RSD value were calculated. The results are shown in Table 14 (peaks 2, 3, 4, and 5 have been compared with the reference substance and are no longer counted).

[0123] Table 15. Relative retention time of 24 batches of Hubei Ophiopogon japonicus standard decoction dry paste powder

[0124] sample Peak 1 Peak 6 Peak 7 Peak 8 HBMDYC01 0.37 1.54 1.66 2.20 HBMDYC02 0.36 1.56 1.69 2.24 HBMDYC03 0.36 1.56 1.69 2.24 HBMDYC04 0.36 1.56 1.69 2.24 HBMDYC05 0.36 1.56 1.69 2.24 HBMDYC07 0.36 1.56 1.69 2.24 HBMDYC08 0.36 1.55 1.67 2.20 HBMDYC10 0.36 1.57 1.69 2.23 HBMDYC11 0.35 1.55 1.69 2.23 HBMDYC12 0.36 1.56 1.69 2.24 HBMDYC18 0.36 1.56 1.69 2.24 HBMDYC19 0.36 1.56 1.69 2.24 HBMDYC20 0.36 1.56 1.69 2.24 HBMDYC21 0.36 1.55 1.67 2.20 HBMDYC22 0.36 1.57 1.69 2.23 HBMDYC30 0.35 1.55 1.69 2.23 HBMDYC38 0.35 1.55 1.69 2.23 HBMDYC39 0.36 1.56 1.69 2.23 HBMDYC40 0.36 1.56 1.69 2.24 HBMDYC41 0.36 1.56 1.69 2.24 HBMDYC42 0.36 1.56 1.69 2.24 HBMDYC43 0.36 1.56 1.69 2.24 HBMDYC44 0.36 1.56 1.69 2.24 HBMDYC45 0.36 1.56 1.69 2.24 Mean 0.36 1.55 1.69 2.24 RSD% 0.59 0.52 0.64 0.57

[0125] As shown in Table 15, there is no significant difference in the relative retention time of the 8 characteristic peaks. The average value of the relative retention time is taken as the measured value. Excluding the 4 characteristic peaks that have been marked with reference substances, the relative retention times of the remaining 4 characteristic peaks are: 0.36 (peak 1), 1.56 (peak 6), 1.68 (peak 7), 2.23 (peak 8). The relative retention times are all within the range of ±10% of the specified value. The reference characteristic spectra of 24 batches of Hubei Ophiopogon japonicus standard decoction dry paste powder generated by the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software System (2012 Edition)" are as follows Fig. 9 As shown in the figure, the eight characteristic peaks have clear shapes and good separation. This method can be used to accurately control the quality of Hubei Ophiopogon japonicus medicinal materials and their preparations.

[0126] (5) Identification of Hubei Ophiopogon japonicus and other Ophiopogon japonicus medicinal materials

[0127] 48 batches of Hubei Ophiopogon japonicus and other Ophiopogon japonicus medicinal materials were collected and their characteristic spectra were determined by the above method. The results in Table 15 show that the logarithmic ratio of peak area between peak 6 and peak 7 of Ophiopogon japonicus standard decoction dry paste powder from different sources is as follows: Hubei Ophiopogon japonicus: 0.91-1.05; Zhejiang Ophiopogon japonicus: 0.80-0.90; Short-stalked Ophiopogon japonicus: 0.84-0.89; Sichuan Ophiopogon japonicus: 0.75-0.83. Therefore, by comparing the logarithmic ratio of peak area between peak 6 and peak 7 (greater than 0.91), Hubei Ophiopogon japonicus can be distinguished from other Ophiopogon japonicus medicinal materials.

[0128] Table 16 Peak area logarithm ratio of peak 7 to peak 6 in Hubei Ophiopogon japonicus and other Ophiopogon japonicus standard decoctions

[0129]

[0130] In summary, the present invention discloses a quality control method for Hubei Ophiopogon japonicus medicinal materials and preparations thereof based on a one-test-multiple-evaluation method, which can not only simultaneously determine the contents of the three main components (fructose, sucrose, and glucose) in Hubei Ophiopogon japonicus medicinal materials and preparations thereof, but can also be used as a characteristic spectrum of Hubei Ophiopogon japonicus medicinal materials and preparations thereof, and can be applied to the identification of Hubei Ophiopogon japonicus medicinal materials and other Ophiopogon japonicus medicinal materials and preparations thereof. The method adopts ultrasonic-assisted ethanol solution extraction technology and UPLC and evaporative light detector, has better sensitivity, strong universality and repeatability, convenient operation, low cost, and good application prospects.

[0131] In the absence of conflict, the above embodiments and features in the embodiments may be combined with each other.

[0132] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for identifying Ophiopogon japonicus, characterized in that: The method comprises the following steps: S1. Prepare solution: Test solution: Take an appropriate amount of Ophiopogon japonicus or its preparation sample to be tested, grind it into powder, add an appropriate amount of ethanol for ultrasonic extraction, shake well, filter, and take the filtrate as the test solution; Reference substance solution: Take appropriate amounts of three reference substances, including fructose, sucrose, and D-anhydrous glucose, and dissolve them in ethanol to prepare a mixed reference substance solution; Reference solution for reference medicinal materials: Take an appropriate amount of standard Hubei Ophiopogon japonicus medicinal materials, add an appropriate amount of ethanol and perform ultrasonic extraction. Shake well, filter, and take the filtrate as the reference solution of the control medicinal material; S2. Implement testing: Inject the test solution, mixed reference solution, and reference medicinal material reference solution prepared in step S1 into an ultra-high pressure high performance liquid chromatograph, respectively, and record the chromatogram; The chromatographic parameters set on the high performance liquid chromatograph are: using one of a BEH AMIDE column or a Poroshell 120h HILIC-Z column as a chromatographic column; using acetonitrile as mobile phase A, using water as mobile phase B for gradient elution, setting the flow rate to 0.1-0.3 mL / min, using an evaporative light scattering detector for detection, and setting the column temperature to 25-30° C.; The gradient elution procedure is as follows: 0-5 min, the volume ratio of mobile phase A: mobile phase B is 85:15; 5-12 min, the volume ratio of mobile phase A: mobile phase B is changed from 85:15 to 80:20; 12-17 min, the volume ratio of mobile phase A: mobile phase B is changed from 80:20 to 78:22; 17-30 min, the volume ratio of mobile phase A: mobile phase B is changed from 78:22 to 75:25; 30-35 min, the volume ratio of mobile phase A: mobile phase B is changed from 75:25 to 40:60; S3. Analysis of the graph: The spectrum of the test solution is analyzed with the characteristic spectrum of the control medicinal material reference solution as the standard, wherein the characteristic spectrum of the control medicinal material reference solution includes 8 common peaks, with peak 5 as the reference peak, and the relative retention times of the remaining characteristic peaks should be within ±10% of the specified value, and the specified values ​​are: peak 1 with a relative retention time of 0.36, peak 2 with a relative retention time of 0.45, peak 3 with a relative retention time of 0.51, peak 4 with a relative retention time of 0.55, peak 6 with a relative retention time of 1.55, peak 7 with a relative retention time of 1.67, and peak 8 with a relative retention time of 2.

21. Peak 2 is a fructose peak, peaks 3 and 4 are isomers of each other and are glucose peaks, peak 4 is a sucrose peak, peak 7 is a sucrose trisaccharide peak, and peak 8 is a nastose peak; S4. Identification of Radix Ophiopogonis Calculate the logarithmic ratio of the peak areas of Peak 7 and Peak 6: log peak area of ​​Peak 7 / log peak area of ​​Peak 6. If it is greater than 0.91, it is Hubei Ophiopogon japonicus medicinal material. If it is less than 0.91, it is not Hubei Ophiopogon japonicus medicinal material.

2. The method according to claim 1, characterized in that In step S1, the ultrasonic power is 250-300 W, and the concentration of the test solution is 5-30 mg / mL.

3. The method according to claim 2, characterized in that In step S1, the preparation method of the test solution specifically includes taking an appropriate amount of the Ophiopogon japonicus medicinal material or its preparation to be tested, grinding it, accurately weighing it, placing it in a stoppered conical flask, accurately adding 10 ml of 70% ethanol, weighing it, ultrasonically extracting it for 10 minutes, the ultrasonic power is 300 W, the frequency is 53 kHz, cooling it, weighing it again, making up the lost weight with 70% ethanol, shaking it well, filtering it, and taking the filtrate to obtain it.

4. The method according to claim 3, characterized in that In step S1, the preparation method of the control medicinal material reference solution specifically includes taking 0.2 g of Hubei Ophiopogon japonicus control medicinal material, placing it in a stoppered conical flask, adding 10 ml of 70% ethanol, ultrasonically extracting for 10 minutes, cooling and shaking, filtering, and taking the filtrate as the control medicinal material reference solution.

5. The method according to claim 2, characterized in that In step S2, the flow rate was set to 0.2 mL / min.

6. The method according to claim 2, characterized in that In step S2, the column temperature was set to 25°C.

7. The method according to claim 2, characterized in that In step S2, the chromatographic column is a BEH Amide column with specifications of 100×2.1 mm and 1.7 μm.

8. The method according to claim 4, characterized in that It is used to determine the contents of fructose, sucrose and glucose, three main components in the samples of Hubei Ophiopogon japonicus and its preparations. Specifically, 1μl and 3μl of the reference solution, 2μl of the test solution and the reference medicinal material solution are accurately aspirated respectively, injected into the ultra-high performance liquid chromatography, determined, and calculated by the external standard two-point method logarithmic equation to obtain the result.

9. The method according to claim 8, characterized in that The concentrations of the reference solution are: fructose is 1.2 mg / mL, sucrose is 0.2 mg / mL, and D-anhydrous glucose is 0.3 mg / mL; the concentration of the reference medicinal material reference solution is 20 mg / mL.

10. The method according to any one of claims 1 to 9, characterized in that The Ophiopogon japonicus medicinal material or preparation sample to be tested is at least one of Ophiopogon japonicus from Hubei, Ophiopogon japonicus from Zhejiang, Ophiopogon japonicus from Sichuan and Ophiopogon japonicus from Breviscapus.

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