A method for identifying Ophiopogon japonicus from Hubei

Abstract

This invention relates to the field of chemical component detection and analysis technology of traditional Chinese medicine, specifically disclosing a method for identifying Hubei Ophiopogon japonicus. This method employs ultrasound-assisted ethanol solution extraction technology and UPLC with an evaporative light detector to determine eight characteristic peaks, serving as characteristic spectra of Hubei Ophiopogon japonicus and its preparations. This allows for rapid and efficient identification and differentiation between Hubei Ophiopogon japonicus and Zhejiang Ophiopogon japonicus, Sichuan Ophiopogon japonicus, and *Ophiopogon japonicus var. spp.*, making it a highly practical identification method. Simultaneously, it can also determine the content of three main components—fructose, sucrose, and glucose—in Hubei Ophiopogon japonicus and its preparations, thus achieving a higher level of quality control for Hubei Ophiopogon japonicus and its preparations. This method features improved sensitivity, convenient operation, low cost, and strong universality.

Description

Technical Field

[0001] This invention belongs to the field of detection and analysis technology of chemical components of traditional Chinese medicine, and specifically relates to a method for identifying Ophiopogon japonicus from Hubei. Background Technology

[0002] Hubei Ophiopogon japonicus is a medicinal herb derived from the dried tuberous root of *Liriope spicata* (Thunb.) Lour. var. *prolifera* YTMa, a plant belonging to the Liliaceae family. Its medicinal use has been recorded since the Song Dynasty. In earlier times, various Ophiopogon japonicus herbs were used interchangeably. However, since the 1995 edition of the *Chinese Pharmacopoeia*, Ophiopogon japonicus herbs have been divided into two types: *Mai Dong* and *Shan Mai Dong*. In terms of function and indications, the two are essentially the same, both possessing the effects of nourishing yin and promoting body fluid production, moistening the lungs and clearing the heart. It is used for dry cough due to lung dryness, consumptive cough due to yin deficiency, sore throat, thirst due to fluid depletion, internal heat and thirst, irritability and insomnia, and constipation due to intestinal dryness. Ophiopogon japonicus is divided into Hubei Ophiopogon japonicus and short-leaved Ophiopogon japonicus (Liriope muscari (Decne.) Baily), while Ophiopogon japonicus (Lf.) Ker-Gawl can be divided into Sichuan Ophiopogon japonicus and Zhejiang Ophiopogon japonicus according to their place of origin. Sichuan Ophiopogon japonicus is mainly produced in Santai County, Sichuan Province, while Zhejiang Ophiopogon japonicus is mainly produced in Yuyao and Hangzhou, Zhejiang Province. Hubei Ophiopogon japonicus is mainly produced in the Han River basin, including Xiangyang, Hubei Province, while short-leaved Ophiopogon japonicus is mainly produced in Quanzhou and Huian, Fujian Province. Currently, Sichuan Ophiopogon japonicus has the highest production volume, followed by Hubei Ophiopogon japonicus.

[0003] The current edition of the Chinese Pharmacopoeia only specifies the source, properties, identification, inspection, and extractives of Hubei Ophiopogon japonicus, lacking highly sensitive, characteristic, and quantifiable quality control methods. This leads to inconsistent quality of Hubei Ophiopogon japonicus and its preparations in the market. Furthermore, the national standards for granules of Sichuan Ophiopogon japonicus and Zhejiang Ophiopogon japonicus issued by the National Pharmacopoeia Commission employ ultra-high performance liquid chromatography with electrospray ionization (UHPLC) detection, which is costly and lacks universality, limiting its application. Meanwhile, because Hubei Ophiopogon japonicus shares similar properties and efficacy with the other three types of Ophiopogon japonicus, they are frequently used interchangeably, making it difficult for non-experienced professionals to distinguish them. Therefore, it is essential to find a method that can provide a comprehensive qualitative and quantitative evaluation of the quality of Hubei Ophiopogon japonicus while also providing a simple and rapid method for identifying and differentiating it from the other three types. Summary of the Invention

[0004] The purpose of this invention is to address the aforementioned shortcomings of the prior art by providing a method for identifying Hubei Ophiopogon japonicus. This method can distinguish Hubei Ophiopogon japonicus from Zhejiang Ophiopogon japonicus, Sichuan Ophiopogon japonicus, and Duan Ting Shan Ophiopogon japonicus. It can also accurately detect the content of various sugar components in Hubei Ophiopogon japonicus, thus more accurately reflecting the quality of Hubei Ophiopogon japonicus and its preparations. This is beneficial for quality control and reduces costs.

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

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

[0007] S1. Preparation of solution:

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

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

[0010] Reference solution: Take an appropriate amount of standard Hubei Ophiopogon japonicus, add an appropriate amount of ethanol, and extract by ultrasonication.

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

[0012] S2. Conduct testing:

[0013] The test solution, mixed reference solution, and reference medicinal material solution prepared in step S1 were injected into an ultra-high pressure high performance liquid chromatograph, and the chromatograms were recorded.

[0014] The chromatographic parameters set on the high performance liquid chromatograph are as follows: using either a BEH AMIDE column or a Poroshell 120h HILIC-Z column as the chromatographic column; using acetonitrile as mobile phase A and 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; and setting the column temperature to 25–30 °C.

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

[0016] S3, Analysis of the spectrum:

[0017] The spectrum of the test sample solution was analyzed using the characteristic spectrum of the reference solution of the control medicinal material as the standard. The characteristic spectrum of the reference solution of the control medicinal material includes 8 common peaks, with peak 5 as the reference peak. The relative retention times of the remaining characteristic peaks should be within ±10% of the specified values. 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 the fructose peak, peaks 3 and 4 are isomers of each other and are glucose peaks, peak 4 is the sucrose peak, peak 7 is the fructosaccharide peak, and peak 8 is the sucrose peak.

[0018] S4. Identification of Ophiopogon japonicus medicinal material to be tested

[0019] Calculate the logarithmic ratio of the peak areas of peak 7 and peak 6: log7 peak area / log6 peak area. 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 250-300W, and the concentration of the test sample solution is 5-30mg / mL.

[0021] Further, in step S1, the preparation method of the test solution specifically includes taking an appropriate amount of the Ophiopogon japonicus herb or its preparation to be tested, grinding it finely, 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, replenishing the lost weight with 70% ethanol, shaking it well, filtering it, and taking the filtrate to obtain the test solution.

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

[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 dimensions of 100×2.1mm and 1.7μm.

[0026] Furthermore, this method is used to determine the content of three main components—fructose, sucrose, and glucose—in samples of Hubei Ophiopogon japonicus and its preparations. Specifically, 1 μl and 3 μl of the reference solution, 2 μl each of the test solution and the reference medicinal material solution are precisely pipetted into an ultra-high performance liquid chromatograph, and the results are calculated using the logarithmic equation of the external standard two-point method.

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

[0028] Furthermore, the tested Ophiopogon japonicus medicinal material or its preparation sample is at least one of Hubei Ophiopogon japonicus, Zhejiang Ophiopogon japonicus, Sichuan Ophiopogon japonicus, and Short-stemmed Ophiopogon japonicus.

[0029] Compared with the prior art, the beneficial effects of the technical solution provided by the present invention are as follows:

[0030] (1) The present invention provides a method for identifying Hubei Ophiopogon japonicus, which can quickly and efficiently identify and distinguish Hubei Ophiopogon japonicus from Zhejiang Ophiopogon japonicus, Sichuan Ophiopogon japonicus, and short-stemmed Ophiopogon japonicus. It is a highly practical identification method that enables Hubei Ophiopogon japonicus and its preparations to reach a higher level of quality control.

[0031] (2) The present invention provides a method for identifying Hubei Ophiopogon japonicus, which 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 slices, Hubei Ophiopogon japonicus standard decoction and Hubei Ophiopogon japonicus formula granules, etc., and has a very wide range of applications.

[0032] (3) The method provided by the present invention has low cost and is beneficial to the production operations of pharmaceutical manufacturing enterprises. Attached Figure Description

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

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

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

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

[0037] Figure 5 This is the specificity assessment graph in Example 1;

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

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

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

[0041] Figure 9 The image shows the characteristic spectrum generated in Example 1 of this invention. Peak 2: fructose; peaks 3 and 4: glucose; peak 5 (S): sucrose; peak 7: sucrose trisaccharide; peak 8: nitrite. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the specific embodiments and accompanying drawings are described in further detail below. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0043] In this invention, "Hubei Ophiopogon japonicus" refers to Ophiopogon japonicus medicinal material from Hubei Province and preparations (processed slices, decoctions, granules, etc.) made from Ophiopogon japonicus medicinal material from Hubei Province.

[0044] The reagents and instruments of this 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); 0.01% electronic balance (Sartorius); 0.01% 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 (2.1×100mm, 1.7μm); Poroshell 120hHILIC-Z (2.1×100mm, 2.7μm);

[0047] Reagents: *Ophiopogon japonicus* (Hubei Ophiopogon japonicus) reference material (batch number: 121136-201803); fructose (batch number: 100231-202309), D-anhydrous glucose (batch number: 110833-202109), sucrose (batch number: 111507-202406), and nicotinic acid (batch number: 111891-201704), all purchased from the China National Institutes for Food and Drug Control; fructosyl sucrose (batch number: DST231213-087, purchased from Dest Biotech). Acetonitrile was Fisher Chemical chromatographic grade, and water was ultrapure water obtained by filtration through an ELGA ultrapure water system. All other reagents were chromatographic grade.

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

[0049] The preparation method of standard decoction dry powder is as follows: Weigh 100g of Ophiopogon japonicus medicinal slices, place them in a decoction pot, add 1000mL of water, soak for 30min, bring to a boil over high heat, then simmer over low heat for 60min. After filtering the extract through a 200-mesh filter, add 800mL of water to the dregs, bring to a boil over high heat, then simmer over low heat for 40min. Combine the two filtrates, concentrate them in a rotary evaporator until the material-to-liquid ratio is about 1:1, set the concentration temperature to 65℃, place the concentrated liquid in a tray, dry it under low temperature vacuum, and pulverize it to obtain the final product.

[0050] The preparation method of Ophiopogon japonicus (Hubei Ophiopogon japonicus) formula granules is as follows: Take an appropriate amount of Ophiopogon japonicus (Hubei Ophiopogon japonicus), add water and decoct twice. For the first decoction, add 10 times the amount of water, soak for 30 minutes, and decoct for 90 minutes. For the second decoction, add 8 times the amount of water and decoct for 60 minutes. Filter the decoction, concentrate the filtrate into a clear paste, add an appropriate amount of excipients, dry (or dry and pulverize), add an appropriate amount of excipients again, mix well, and granulate to obtain the final product.

[0051] The batch numbers and places of origin of the 48 batches of Hubei Ophiopogon japonicus and other Ophiopogon japonicus-derived medicinal materials used in this invention are as follows:

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

[0053]

[0054]

[0055] Example 1

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

[0057] 1. Measurement Method

[0058] 1.1 Preparation of reference solutions: Take appropriate amounts of fructose, sucrose, and D-anhydrous glucose reference standards, accurately weigh them, and 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, respectively, as reference solutions.

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

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

[0061] 1.4 Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.7 μm); acetonitrile was used as mobile phase A, and water as mobile phase B, with gradient elution as specified in Table 2; the flow rate was 0.3 mL / min, the column temperature was 25℃, and detection was performed using an evaporative light scattering detector. 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 1 μl and 3 μl of the reference solution, 2 μl each of the test solution and the reference medicinal material solution, inject them into the liquid chromatograph, determine the result, and calculate the result using the logarithmic equation of the external standard two-point method.

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

[0066] (1) Investigation of the elution gradient procedure.

[0067] The same batch of Hubei Ophiopogon japonicus standard decoction dry extract powder was used to prepare the test sample solution according to item 1.3 of Example 1. Experimental group 1 was determined according to the chromatographic conditions of items 1.4 and 1.5 of Example 1. The control group was determined 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 program, column temperature and flow rate are different. The elution gradient program, column temperature and flow rate 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 for control group 3

[0075]

[0076] The chromatograms of experimental group 1, control group 1, control group 2 and control group 3 are shown in the figure. 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 theoretical plate number, tailing factor, peak shape and resolution of the dry extract powder of the stir-fried germinated rice standard decoction 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 powder, grind it into a fine powder, take 5 portions, each about 0.1g, accurately weigh them, place them in stoppered conical flasks, and accurately add 10ml of water, 30% ethanol, 50% ethanol, 70% ethanol, 80% ethanol, and 90% ethanol respectively. Seal tightly, weigh, and sonicate (power 250W, frequency 35kHz) for 30 minutes. Remove, cool, and weigh again. Then add water, 10% ethanol, 50% ethanol, 70% ethanol, 80% ethanol, and 90% ethanol respectively to make up the lost weight, shake well, filter, and take the filtrate to obtain the test solution. Inject into an ultra-high performance liquid chromatograph for determination, and calculate the contents of fructose, sucrose, and glucose respectively. Table 6 shows that when the extraction solvent is 70% methanol, there are fewer impurities, better peak separation, and less interference; while the content of sugar components is relatively low when the extraction solvent is above 80% ethanol. Finally, 70% ethanol was determined to be the preparation solvent for this product.

[0080] Table 6. Investigation of extraction solvents

[0081]

[0082]

[0083] (2.2) Examination of extraction methods

[0084] Take the same batch of Hubei Ophiopogon japonicus standard decoction dry extract powder, grind it into a fine powder, take 5 portions, each about 0.1g, accurately weigh them, accurately add 10mL of 70% ethanol, weigh them, and extract them by ultrasonication for 60 minutes, 30 minutes, and 10 minutes respectively. Heat under reflux for 1 hour, cool, replenish the lost weight with 70% ethanol, shake well, filter, and collect the filtrate to obtain the test solution. Inject into an ultra-high performance liquid chromatograph for determination, and calculate the contents of fructose, sucrose, and glucose respectively. Table 7 shows that when ultrasonicated for 10 minutes, the contents of fructose, sucrose, and glucose in the test solution are basically consistent with the other three methods. Considering the simplicity of operation and time saving, the extraction method of ultrasonication for 10 minutes was determined.

[0085] Table 7. Examination of Extraction Methods

[0086]

[0087] (2.3) Sample size assessment

[0088] Take the same batch of Hubei Ophiopogon japonicus standard decoction dry extract powder, grind it into a fine powder, and weigh approximately 0.05g, 0.1g, 0.2g, and 0.3g respectively. Accurately weigh each sample, add 10mL of 70% ethanol to each, weigh again, and ultrasonically extract for 10 minutes. After cooling, replenish the lost weight with 70% ethanol, shake well, filter, and collect the filtrate to obtain the test solution. Inject into an ultra-high performance liquid chromatograph (UHPLC) for determination, and calculate the contents of fructose, sucrose, and glucose. Table 8 shows that the contents of fructose, sucrose, and glucose in the test solution with a sample weight of 0.1g are higher than those with sample weights of 0.2g and 0.3g, and are basically consistent with those with a sample weight of 0.05g. Considering that a more precise balance is required for a sample weight of 0.05g, and the experimental error may be larger, a sample weight of 0.1g was chosen.

[0089] Table 8 Sample Size Survey

[0090]

[0091] (3) Methodological investigation

[0092] (3.1) Specificity Examination

[0093] A 70% ethanol solution of dextrin (negative control), along with the reference solution, the reference herb solution, and the test solution, were injected separately into the liquid chromatograph, and the chromatograms were recorded. (See figure) Figure 5 . Figure 5The results showed that the negative control solution had no peaks, while the peaks in the test solution, reference solution, and reference herb solution were all well-shaped, indicating that the method has good specificity.

[0094] (3.2) Linear Examination

[0095] Accurately weigh 90.053 mg, 25.148 mg, and 14.964 mg of fructose, D-glucose, and sucrose, respectively. Dissolve them in 70% ethanol and bring the volume to 20 mL. Shake well to obtain a mixed reference stock solution of fructose, glucose, and sucrose ① with concentrations of 4.4981 mg / mL, 1.2561 mg / mL, and 0.7482 mg / mL, respectively. Accurately pipette 5 mL of the above reference solution stock solution into a 10 mL volumetric flask, add 70% ethanol to the mark, shake well, and this is DZ-5, which will also be the stock solution for the next batch of solutions. Repeat this step twice to obtain reference solutions DZ-1 and DZ-3. Separately, accurately weigh 67.540 mg, 18.861 mg, and 11.223 mg of fructose, D-glucose, and sucrose, respectively, dissolve them in 70% ethanol, and dilute to 20 mL. Shake well to obtain a mixed reference stock solution ② of fructose, glucose, and sucrose, with concentrations of 3.3762 mg / mL, 0.9421 mg / mL, and 0.5611 mg / mL, respectively (DZ-6). Accurately pipette 5 mL of the above reference solution stock solution into a 10 mL volumetric flask, add 70% ethanol to the mark, and shake well. This is DZ-4. Repeat this step to obtain DZ-2. Take the above 6 reference solutions of different concentrations and perform chromatographic analysis under the content determination chromatographic conditions to obtain the peak area of ​​each concentration. Establish a standard curve with LOG concentration as the abscissa and the corresponding LOG peak area as the ordinate. The linear relationship and range are as follows (injection volume 2 μl):

[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. Relevant data for establishing the standard curve

[0100]

[0101] The results showed that fructose reference solution exhibited good linearity in the concentration range of 1.1245 mg / ml to 6.7472 mg / ml, glucose reference solution in the concentration range of 0.3140 mg / ml to 1.8842 mg / ml, and sucrose in the concentration range of 0.1871 mg / ml to 1.1223 mg / ml, with r > 0.999, meeting the linearity requirements for method validation.

[0102] (3.3) Repeatability test

[0103] Approximately 0.1 g of the same batch of Hubei Ophiopogon japonicus standard decoction powder was accurately weighed and prepared into six test solutions according to the preparation method described in section 1.3 above. The peak areas were determined by chromatographic analysis under the conditions described in sections 1.4 and 1.5. Calculations showed that the average fructose content in the Hubei Ophiopogon japonicus standard decoction powder was 116.28 mg / g, with an RSD of 0.81%; the average glucose content was 29.48 mg / g, with an RSD of 1.23%; and the average sucrose content was 19.62 mg / g, with an RSD of 1.27%. The results indicate that the detection method has good repeatability.

[0104] Table 10. Relevant data from repeatability tests

[0105]

[0106]

[0107] (3.4) Intermediate precision test

[0108] Different analysts conducted repeatability experiments at different times using a different Waters ultra-high performance liquid chromatograph. Calculations showed that the average fructose content in the dried extract powder of the Hubei Ophiopogon japonicus standard decoction was 115.37 mg / g with an RSD of 0.61%; the average glucose content was 29.31 mg / g with an RSD of 0.75%; and the average sucrose content was 19.31 mg / g with an RSD of 1.17%. The RSDs for the three components across different instruments were 0.55%, 0.39%, and 1.12%, respectively, meeting the analytical requirements.

[0109] Table 11. Relevant data on intermediate precision

[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 assessment

[0112] Take approximately 0.05 g of the dried extract powder of the same batch of Hubei Ophiopogon japonicus standard decoction (with average contents of fructose, glucose, and sucrose of 116.28 mg / g, 29.48 mg / g, and 19.62 mg / g, respectively) and accurately weigh it into six stoppered conical flasks. Accurately transfer 12 ml of the linear reference standard stock solution ① into a 100 mL volumetric flask, dissolve it in 70% ethanol, and dilute to volume. Pipette 10 mL of this solution into a 100 mL volumetric flask, dissolve it in 70% ethanol, and dilute to volume. Shake well to obtain the reference solution for spiking (with fructose, glucose, and sucrose concentrations of 5.3978 mg / g, respectively). (mg / ml, 1.5074mg / ml, 0.89784mg / ml). Accurately transfer 10ml of the reference solution to each of the six volumetric flasks containing the sample, and prepare the test solution for recovery determination according to the preparation method under section 1.3. Analyze under the chromatographic conditions under sections 1.4 and 1.5. Calculate the recoveries of fructose, glucose, and sucrose. The results show that the average recovery rate is 98.98%, and the RSD is 1.27%, which meets the requirements. This detection method is accurate and reliable for detecting the content of fructose, glucose, and sucrose in the dry extract powder of Hubei Ophiopogon japonicus standard decoction.

[0113] Table 12. Results of Fructose Accuracy Test

[0114]

[0115] Table 13. Results of the glucose accuracy test

[0116]

[0117] Table 14. Results of Sucrose Accuracy Test

[0118]

[0119] (3.6) Durability test

[0120] The above-mentioned Hubei Ophiopogon japonicus standard decoction dry extract powder test solution was taken and subjected to chromatographic analysis under the determined chromatographic conditions using two different chromatographic columns (①BEH AMIDE (2.1×100mm, 1.7μm); ②Poroshell 120hHILIC-Z (2.1×100mm, 2.7μm)). This was to determine the robustness of the method to different chromatographic columns. The results are shown in […]. Figure 6 , Figure 7 As can be seen from the chart, the peak shapes of the chromatograms obtained by the two columns differ significantly. Therefore, it is recommended to choose the ACQUITY BEH AMIDE (2.1×100mm, 1.7μm) column.

[0121] (4) Determination of characteristic spectrum and characteristic peaks

[0122] The above method was used to determine the content of 24 batches of Hubei Ophiopogon japonicus standard decoction dry extract powder. Simultaneously, the chromatographic peaks of the 24 batches of Hubei Ophiopogon japonicus standard decoction were superimposed using the "Software System for Evaluation of Chromatographic Fingerprint Similarity of Traditional Chinese Medicine (2012 Edition)" issued by the National Pharmacopoeia Commission to form a common peak pattern diagram (see...). Figure 8 The results showed that all 24 batches of Hubei Ophiopogon japonicus standard decoction powder samples exhibited 8 common peaks. A reference characteristic chromatogram was established based on the median. Peak 5 was the chromatographic peak of sucrose reference standard, with a large peak area and good resolution; therefore, this peak was labeled as peak S. Peak 2 was the chromatographic peak of fructose reference standard, and peaks 3 and 4 were the chromatographic peaks of D-anhydrous glucose reference standard (isomers). The reference medicinal materials also had corresponding 8 peaks. These 8 peaks were labeled, and the relative retention times and RSD values ​​were calculated. The results are shown in Table 14 (peaks 2, 3, 4, and 5 were compared with reference standards and are not included in the statistics).

[0123] Table 15. Relative retention times of 24 batches of Hubei Ophiopogon japonicus standard decoction 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] Table 15 shows that there was no significant difference in the relative retention times of the eight characteristic peaks. Using the average relative retention time as the measured value, and excluding the four characteristic peaks already labeled with reference standards, the relative retention times of the remaining four characteristic peaks were: 0.36 (peak 1), 1.56 (peak 6), 1.68 (peak 7), and 2.23 (peak 8). All relative retention times were within ±10% of the specified values. The characteristic chromatograms of 24 batches of Hubei Ophiopogon japonicus standard decoction dry extract powder generated by the "Software System for Similarity Evaluation of Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)" are shown below. Figure 9 As shown, the eight characteristic peaks are clearly shaped and well separated. This method can effectively achieve precise quality control of Hubei Ophiopogon japonicus medicinal materials and their preparations.

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

[0127] Forty-eight batches of standard decoction powders made from Hubei Ophiopogon japonicus and other Ophiopogon japonicus-like medicinal materials were collected, and their characteristic spectra were determined using the method described above. Table 15 shows that the logarithmic ratio of the peak areas of peaks 6 and 7 for the standard decoction powders from different sources ranged as follows: Hubei Ophiopogon japonicus: 0.91-1.05; Zhejiang Ophiopogon japonicus: 0.80-0.90; Short-stemmed Ophiopogon japonicus: 0.84-0.89; Sichuan Ophiopogon japonicus: 0.75-0.83. Therefore, by comparing the logarithmic ratio of the peak areas of peaks 6 and 7 (greater than 0.91), Hubei Ophiopogon japonicus can be distinguished from other Ophiopogon japonicus-like medicinal materials.

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

[0129]

[0130] In summary, this invention discloses a quality control method for Hubei Ophiopogon japonicus medicinal materials and their preparations based on a single-measurement, multi-evaluation approach. This method can simultaneously determine the content of three main components (fructose, sucrose, and glucose) in Hubei Ophiopogon japonicus medicinal materials and their preparations, and can also serve as a characteristic chromatogram for Hubei Ophiopogon japonicus medicinal materials and their preparations, applicable to the identification of Hubei Ophiopogon japonicus from other Ophiopogon japonicus medicinal materials and their preparations. This method employs ultrasonic-assisted ethanol solution extraction technology and UPLC with an evaporative light detector, resulting in better sensitivity. The method has strong universality and repeatability, is convenient to operate, and has low cost, showing promising application prospects.

[0131] Where there is no conflict, the above embodiments and features described herein can 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 principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for identifying Hubei Ophiopogon japonicus, characterized in that, The method includes the following steps: S1. Preparation of solution: Test solution: Take an appropriate amount of the Ophiopogon japonicus or its preparation sample to be tested, grind it into a fine powder, add an appropriate amount of ethanol for ultrasonic extraction, shake well, filter, and take the filtrate as the test solution. Reference solution: Take appropriate amounts of three reference standards, namely fructose, sucrose, and D-anhydrous glucose, dissolve them in ethanol to prepare a mixed reference solution; Reference solution: Take an appropriate amount of standard Hubei Ophiopogon japonicus, add an appropriate amount of ethanol, and extract by ultrasonication. Shake well, filter, and take the filtrate as a reference solution for the control medicinal material; S2. Conduct testing: The test solution, mixed reference solution, and reference medicinal material solution prepared in step S1 were injected into an ultra-high pressure high performance liquid chromatograph, and the chromatograms were recorded. The chromatographic parameters set on the high performance liquid chromatograph are as follows: using either a BEH AMIDE column or a Poroshell 120h HILIC-Z column as the chromatographic column; using acetonitrile as mobile phase A and 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; and setting the column temperature to 25–30 °C. The gradient elution program is as follows: 0-5 min, mobile phase A:mobile phase B volume ratio is 85:15; 5-12 min, mobile phase A:mobile phase B volume ratio is changed from 85:15 to 80:20; 12-17 min, mobile phase A:mobile phase B volume ratio is changed from 80:20 to 78:22; 17-30 min, mobile phase A:mobile phase B volume ratio is changed from 78:22 to 75:25; 30-35 min, mobile phase A:mobile phase B volume ratio is changed from 75:25 to 40:

60. S3, Analysis of the spectrum: The spectrum of the test sample solution was analyzed using the characteristic spectrum of the reference solution of the control medicinal material as the standard. The characteristic spectrum of the reference solution of the control medicinal material includes 8 common peaks, with peak 5 as the reference peak. The relative retention times of the remaining characteristic peaks should be within ±10% of the specified values. 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 the fructose peak, peaks 3 and 4 are isomers of each other and are glucose peaks, peak 4 is the sucrose peak, peak 7 is the fructosaccharide peak, and peak 8 is the sucrose peak. S4. Identification of Ophiopogon japonicus medicinal material to be tested Calculate the logarithmic ratio of the peak areas of peak 7 and peak 6: log7 peak area / log6 peak area. 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 as described in claim 1, characterized in that, In step S1, the ultrasonic power is 250-300W, and the concentration of the test sample solution is 5-30mg / mL.

3. The method as described in 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 or its preparation to be tested, grinding it finely, 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, replenishing the lost weight with 70% ethanol, shaking it well, filtering it, and taking the filtrate to obtain the test solution.

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

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

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

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

8. The method as described in claim 4, characterized in that, This method is used to determine the content of three main components—fructose, sucrose, and glucose—in samples of Hubei Ophiopogon japonicus and its preparations. Specifically, 1 μl and 3 μl of the reference solution, 2 μl each of the test solution and the reference medicinal material solution are precisely pipetted into an ultra-high performance liquid chromatograph, and the results are calculated using the logarithmic equation of the external standard two-point method.

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

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

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