HPLC (High Performance Liquid Chromatography) fingerprint spectrum determination and quality judgment method of Wenning codonopsis pilosula

Abstract

The invention discloses an HPLC (High Performance Liquid Chromatography) fingerprint spectrum determination and quality judgment method of Wenning codonopsis pilosula, and the fingerprint spectrum determination method of the Wenning codonopsis pilosula comprises the following steps: S1, dissolving 5-hydroxymethylfurfural, lobetyolin, codonopsis pilosula glycoside I and syringin into methanol to obtain a reference substance solution; s2, dissolving a to-be-detected product in methanol to obtain a to-be-detected product solution; and S3, analyzing the reference substance solution and the to-be-detected substance solution by adopting high performance liquid chromatography to obtain a high performance liquid chromatogram. Data support is provided for resource development and utilization of codonopsis pilosula, expansion of medicinal parts and clinical application.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine detection, and in particular to a method for determining the HPLC fingerprint of Weining Codonopsis pilosula and judging its quality. Background Art

[0002] Weining Codonopsis pilosula (Franch.) Nannf. is a local medicinal plant of the genus Codonopsis in the Campanulaceae family. It is a Geographical Indication product of Weining County, Guizhou Province, named after its place of origin. It has long been recorded in the "Guiyang Prefecture Chronicle" as "the best Codonopsis in Weining, Guizhou Province." Weining Codonopsis is primarily produced in Weining County, Guizhou Province, and the surrounding high-altitude mountainous areas. Benefiting from the unique local natural conditions, its rhizomes are large, firm, and of superior quality. Weining Codonopsis is not only a key Qi-tonifying herb in Traditional Chinese Medicine, but has also been included in local standards (such as the "Geographical Indication Product Weining Codonopsis"), highlighting its status as an authentic medicinal material. While not as potent as ginseng, its mild nature and flavor make it widely applicable, making it particularly well-suited for daily medicinal preparations such as soups, porridge, and pastes.

[0003] In recent years, with the expansion of Chinese medicinal herb cultivation, the medicinal value of Codonopsis pilosula, a plant with both medicinal and edible properties, has been continuously explored. Clinical application research has gradually increased, demonstrating significant economic development value in both the medical and food industries. Furthermore, the standardized large-scale cultivation further ensures the sustainable development of traditional Chinese medicinal materials. Fingerprinting is an effective method for authenticity verification, species differentiation, and quality control, providing a scientific basis for the identification and quality evaluation of Weining Codonopsis pilosula. However, current research on the quality of Weining Codonopsis pilosula is limited. Summary of the Invention

[0004] The present invention aims to solve at least one of the above-mentioned technical problems in the prior art. To this end, the present invention aims to provide a HPLC fingerprint determination and quality judgment method for Weining Codonopsis pilosula.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] The first aspect of the present invention provides a method for determining the fingerprint of Codonopsis pilosula, comprising the following steps:

[0007] S1: Dissolve 5-hydroxymethylfurfural, codonopsis pilosula, codonopsis pilosula I and syringin in methanol to obtain a reference solution;

[0008] S2: dissolving the test substance in methanol to obtain a test substance solution;

[0009] S3: Analyze the reference solution and the test solution by high performance liquid chromatography to obtain a high performance liquid chromatogram;

[0010] The conditions of the high performance liquid chromatography method are as follows: formic acid solution as mobile phase A and acetonitrile as mobile phase B;

[0011] The mobile phase was passed through the chromatographic column using gradient elution, followed by UV detection;

[0012] Calculated based on the volume ratio of mobile phase B in the mobile phase, the gradient elution procedure is:

[0013] 0-7 min, isocratic elution, the volume proportion of the mobile phase B in the mobile phase is 3-7%;

[0014] From 7 to 80 minutes, the volume proportion of the mobile phase B in the mobile phase increases from 3 to 7% to 24 to 28%;

[0015] From 80 to 108 minutes, the volume proportion of the mobile phase B in the mobile phase increases from 24% to 28% to 86% to 90%;

[0016] From 108 to 113 min, the volume proportion of the mobile phase B in the mobile phase increased from 86% to 90% to 100% B;

[0017] 113-120 min, isocratic elution, the volume proportion of the mobile phase B in the mobile phase is 100%.

[0018] In the present invention, a fingerprint of Codonopsis pilosula in Weining is established and four peaks of 5-hydroxymethylfurfural, codonopsis argynin, codonopsis glycoside I and syringin are identified, and the above-mentioned specific HPLC analysis method is adopted, which can greatly shorten the detection time and ensure the integrity of the main information of the spectrum; multiple common peaks of Codonopsis pilosula in Weining are selected, which can better reflect the overall chemical composition of Codonopsis pilosula in Weining and effectively discover the differences between different Codonopsis pilosula, such as different original plants; with the help of standard substances, the main peaks in the fingerprint are successfully further attributed, and the two peaks with the largest proportion in the fingerprint are attributed to two indicator components in Codonopsis pilosula in Weining: codonopsis glycoside I and codonopsis glycoside I, especially the attribution of codonopsis glycoside I, which provides a new idea for the quality control of Codonopsis pilosula in Weining (and even Codonopsis pilosula).

[0019] In some embodiments of the present invention, in the reference solution, the concentrations of 5-hydroxymethylfurfural, codonopsis pilosula, codonopsis pilosula I and syringin are 0.1-0.5 mg / mL, respectively.

[0020] In some embodiments of the present invention, the mass concentration of the test substance is 0.08-0.15 g / mL.

[0021] In some embodiments of the present invention, the test product is the underground rhizome of Codonopsis pilosula (Franch.) Nannf.; the Codonopsis pilosula (Franch.) Nannf.

[0022] In some embodiments of the present invention, in S2, the sample to be tested is dissolved in methanol and ultrasonically extracted to obtain a sample solution; the power of the ultrasound is 230-250 W; the frequency of the ultrasound is 30-50 kHz; and the time of the ultrasound is 20-40 min.

[0023] In some embodiments of the present invention, the volume concentration of the formic acid solution is 0.05-0.2%, such as 0.08-0.15%, or 0.1%.

[0024] In some embodiments of the present invention, the detection wavelength of the ultraviolet detection is 230-270 nm, such as 240-260 nm, 250-258 nm, or 254 nm.

[0025] In some embodiments of the present invention, the gradient elution procedure is calculated based on the volume ratio of mobile phase B in the mobile phase:

[0026] 0-7 min, isocratic elution, the volume proportion of the mobile phase B in the mobile phase is 3-7%;

[0027] From 7 to 50 minutes, the volume proportion of the mobile phase B in the mobile phase increases from 3 to 7% to 12% to 16%;

[0028] From 50 to 80 minutes, the volume proportion of the mobile phase B in the mobile phase increases from 12% to 16% to 24% to 28%;

[0029] From 80 to 108 minutes, the volume proportion of the mobile phase B in the mobile phase increases from 24% to 28% to 86% to 90%;

[0030] From 108 to 113 min, the volume proportion of the mobile phase B in the mobile phase increased from 86% to 90% to 100% B;

[0031] 113-120 min, isocratic elution, the volume proportion of the mobile phase B in the mobile phase is 100%.

[0032] In some embodiments of the present invention, the gradient elution procedure is calculated based on the volume ratio of mobile phase B in the mobile phase:

[0033] 0-7 min, isocratic elution, the volume proportion of the mobile phase B in the mobile phase is 3-7%;

[0034] From 7 to 40 minutes, the volume proportion of the mobile phase B in the mobile phase increases from 3 to 7% to 11% to 13%;

[0035] 40-60 min, the volume proportion of the mobile phase B in the mobile phase increases from 11%-13% to 16-18%;

[0036] From 60 to 80 minutes, the volume proportion of the mobile phase B in the mobile phase increases from 16 to 18% to 24 to 28%;

[0037] From 80 to 108 minutes, the volume proportion of the mobile phase B in the mobile phase increases from 24% to 28% to 86% to 90%;

[0038] From 108 to 113 min, the volume proportion of the mobile phase B in the mobile phase increased from 86% to 90% to 100% B;

[0039] 113-120 min, isocratic elution, the volume proportion of the mobile phase B in the mobile phase is 100%.

[0040] In some embodiments of the present invention, the gradient elution procedure is calculated based on the volume ratio of mobile phase B in the mobile phase:

[0041] 0-7 min, isocratic elution, the volume proportion of the mobile phase B in the mobile phase is 3-7%;

[0042] From 7 to 40 minutes, the volume proportion of the mobile phase B in the mobile phase increases from 3 to 7% to 11% to 13%;

[0043] 40-50 min, the volume proportion of the mobile phase B in the mobile phase increases from 11%-13% to 13.5-15%;

[0044] 50-60 min, the volume proportion of the mobile phase B in the mobile phase increases from 13.5-15% to 16-18%;

[0045] From 60 to 80 minutes, the volume proportion of the mobile phase B in the mobile phase increases from 16 to 18% to 24 to 28%;

[0046] From 80 to 108 minutes, the volume proportion of the mobile phase B in the mobile phase increases from 24% to 28% to 86% to 90%;

[0047] From 108 to 113 min, the volume proportion of the mobile phase B in the mobile phase increased from 86% to 90% to 100% B;

[0048] 113-120 min, isocratic elution, the volume proportion of the mobile phase B in the mobile phase is 100%.

[0049] In some embodiments of the present invention, the gradient elution program is calculated based on the volume ratio of mobile phase B in the mobile phase: 0-7 min, 5%; 7-40 min, 5%-12.4%; 40-50 min, 12.4%-14%; 50-60 min, 14%-17.2%; 60-80 min, 17.2%-26%; 80-108 min, 26%-88%; 108-113 min, 88%-100%; 113-120 min, 100%.

[0050] In some embodiments of the present invention, the chromatographic column is a chromatographic column using octadecylsilane bonded silica gel as a filler.

[0051] In some embodiments of the present invention, the particle size of the filler in the chromatographic column is 3.5 to 7.0 μm; the size is 4.6×250 mm; and the model is Phenomenex lux cellulose-2.

[0052] In some embodiments of the present invention, the column temperature of the chromatographic column is 20-40°C, such as 25-35°C, or 30°C.

[0053] In some embodiments of the present invention, the injection volume during the HPLC detection process is 5 to 20 μL, such as 8 to 15 μL, or 10 μL.

[0054] In some embodiments of the present invention, the flow rate of the mobile phase during the high performance liquid chromatography detection process is 0.5 to 2 mL / min, such as 0.8 to 1.7 mL / min, 0.8 to 1.2 mL / min, or 1.0 mL / min.

[0055] In some embodiments of the present invention, there should be 23 peaks in the fingerprint, the chromatographic peak corresponding to 5-hydroxymethylfurfural is peak 3, the chromatographic peak corresponding to syringin is peak 8, the chromatographic peak corresponding to codonopsis pilosula I is peak 13, and the chromatographic peak corresponding to codonopsis pilosula is peak 18.

[0056] In some embodiments of the present invention, the fingerprint spectrum is characterized by taking peak 13 as the reference peak and specifying a value of 1.000, and calculating the relative retention times of the remaining characteristic peaks relative to the reference peak. The relative retention times should be within the range of ±5% of the specified value. The specified values are: peak 1: 0.105, peak 2: 0.114, peak 3: 0.134, peak 4: 0.191, peak 5: 0.244, peak 6: 0.330, peak 7: 0.452, peak 8: 0. 493, peak 9: 0.556, peak 10: 0.597, peak 11: 0.692, peak 12: 0.722, peak 13: 1.000, peak 14: 1.095, peak 15: 1.222, peak 16: 1.363, peak 17: 1.375, peak 18: 1.400, peak 19: 1.555, peak 20: 1.625, peak 21: 1.798, peak 22: 2.086, peak 23: 2.269.

[0057] A second aspect of the present invention provides a method for determining the quality of Codonopsis pilosula, comprising the following steps:

[0058] S1: Detecting Codonopsis pilosula using the fingerprint determination method of Weining Codonopsis pilosula to obtain a fingerprint;

[0059] S2: Principal component analysis was performed based on the common peak areas, and the quality of Codonopsis pilosula was determined based on the comprehensive score.

[0060] In some embodiments of the present invention, in S2, the higher the comprehensive score, the better the quality of Codonopsis pilosula.

[0061] In some embodiments of the present invention, in S2, the larger the peak area of codonopsis pilosula I or codonopsis pilosula, the higher the comprehensive score.

[0062] The beneficial effects of the present invention are:

[0063] The present invention uses the dried rhizome of Weining Codonopsis as the research object and establishes an HPLC fingerprint. The test method is accurate, precise, reproducible and stable, with rich chromatographic peak information and good separation. The established fingerprint can effectively characterize the quality of Weining Codonopsis. The present invention also identifies four peaks through comparison with reference substances, namely 5-hydroxymethylfurfural (P.4), syringin (P.8), codonoside I (P.13), and codonopsis argynin (P.18). The similarity evaluation results show that the similarities of Weining Codonopsis from different origins are all above 0.94, which can more comprehensively reflect the component information contained in Weining Codonopsis and better characterize the quality of Weining Codonopsis. The results show that its overall quality is relatively stable; the similarity evaluation results can preliminarily reflect the differences in chemical composition. Taking the Weining Codonopsis control fingerprint as a reference, the similarities of the remaining Codonopsis are all between (0.78 and 0.978), reflecting that the chemical composition differences between the varieties are large.

[0064] Cluster analysis and principal component analysis of Weining Codonopsis revealed that samples of the same origin were mostly clustered together, and samples from the same origin were also mostly clustered together, indicating that this method can distinguish them. The principal component scores indicate that Weining Codonopsis, as a type of Codonopsis, is of higher quality than the average of mainstream Codonopsis, and that the Weining Codonopsis produced in Wenge Township, Dafang County, is significantly higher than the Codonopsis produced in Weining County.

[0065] To further analyze the causes and markers of sample differences, an OPLS-DA model with supervised mode discrimination was established and validated using a permutation test. The results showed that the model was reliable, free of overfitting, and suitable for discriminating between-group differences. A total of five significantly influential differential components were screened, including codonopsis pilosula I and codonopsis pilosula.

[0066] The present invention draws the HPLC fingerprint of its rhizome part, and combines the chemical pattern recognition method to find the difference of chemical composition of Weining Codonopsis compared with other varieties and the compound that causes quality difference, which provides a certain basis for the identification of Weining Codonopsis. The Chinese Pharmacopoeia (2020 edition) contains the plants of Campanulaceae Codonopsis C.pilosula, Codonopsis pilosula var.modesta and Codonopsis tangshen C., and Weining Codonopsis is the mainstream variety of Codonopsis, and the relative content of chemical components is relatively high, and its research in pharmacodynamics and other aspects needs to be continued. The present invention provides data support for the resource development and utilization of Codonopsis, the expansion of medicinal parts and clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 This is the HPLC reference fingerprint of the Weining Codonopsis pilosula of the present invention.

[0068] Figure 2 This is an overlay of the HPLC fingerprints of Weining Codonopsis pilosula of the present invention.

[0069] Figure 3 This is an overlay of the HPLC fingerprints of Weining Codonopsis pilosula of the present invention.

[0070] Figure 4 The HPLC fingerprint of the mixed reference substance of the present invention is shown in FIG.

[0071] Figure 5 This is the dendrogram of the cluster analysis of the present invention.

[0072] Figure 6 This is the OPLS-DA score diagram of the present invention.

[0073] Figure 7 This is the OPLS-DA permutation test diagram of the present invention.

[0074] Figure 8 It is the common peak VIP value of the present invention. DETAILED DESCRIPTION

[0075] The present invention is further described in detail below using specific examples. Unless otherwise specified, the raw materials, reagents, and apparatus used in the examples and comparative examples are available from conventional commercial sources or by conventional methods. Unless otherwise specified, all experimental or testing methods are conventional in the art.

[0076] In the following examples, the instruments used were: high performance liquid chromatograph (Shimadzu LC-20AD); electronic analytical balance (XSE105METTLE TOLEDO); rotary evaporator (N-1300EYELA); and electronic constant temperature water bath (SB-1300EYELA).

[0077] All reagents used, including the reference substances codonopsis pilosulfoside, codonopsis pilosulfoside I, and syringin, were isolated and identified and stored at the School of Pharmacy, Sun Yat-sen University. The reference substance 5-hydroxyfurfural was purchased from Guangzhou Shuopu Biotechnology Co., Ltd. with a mass fraction greater than 98%. Acetonitrile was chromatographically pure; water was ultrapure; all other reagents were analytically pure.

[0078] Six batches of Codonopsis pilosula from Weining, as well as one batch each from Jilin, Lu, Tai, Banqiao, Wen, and Minxian were identified as the dried rhizomes of Codonopsis pilosula (Franch.) Nannf., Codonopsis pilosula (Franch.) Nannf., Codonopsis pilosula (Franch.) Nannf., Codonopsis pilosula (Franch.) Nannf. var. modesta (Latinf.) LTShen, and Codonopsis pilosula (Franch.) Nannf. Banqiao was identified as the dried rhizomes of Codonopsis tangshen Oliv. Sample information is provided in Table 1.

[0079] Table 1

[0080]

[0081] Example 1

[0082] This example performs fingerprint detection of Weining Codonopsis pilosula, and the specific process is as follows:

[0083] (1) Preparation of reference solution: Weigh 1.53 mg of 5-hydroxymethylfurfural, 3.15 mg of codonopsis pilosula, 4.31 mg of codonopsis pilosula I, and 2.88 mg of syringoside into a 10 mL volumetric flask, and then add methanol to the mark to obtain a mixed solution with concentrations of 0.153 mg / mL, 0.315 mg / mL, 0.431 mg / mL, and 0.288 mg / mL, respectively. Shake well, filter through a 0.45 μm microporous membrane, and store the filtrate at 4°C until testing.

[0084] (2) Preparation of test solution: Weigh the rhizome of Codonopsis pilosula in Weining, dry it in an oven at 50℃ for 6h, process it with a Codonopsis pilosula grinder, sieve it (40-60 mesh), take 10g and weigh it accurately, place it in a stoppered conical flask, accurately add 100mL of methanol, soak it for 10-15min, ultrasonically treat it (power 240W, frequency 40kHz) for 30min, reduce the pressure on the filtrate to recover the solvent until it is almost dry, then add a small amount of methanol to dissolve it and transfer it to a volumetric flask, add methanol to the scale (10mL), shake it well, filter it with a 0.45μm microporous filter membrane, take the filtrate, and store it at 4℃ for testing.

[0085] (3) HPLC detection: Accurately pipette 10 μL of reference solution and test solution respectively, inject into liquid chromatograph, and record the chromatogram. The chromatographic conditions are as follows: chromatographic column: Phenomenex lux cellulose-2 column (250×4.6 mm, 5 μm); the mobile phase is 0.1% formic acid aqueous solution (A)-acetonitrile (B), gradient elution (0-7 min, 5% B; 7-40 min, 5%-12.4% B; 40-50 min, 12.4%-14% B; 50-60 min, 14%-17.2% B; 60-80 min, 17.2%-26% B; 80-108 min, 26%-88% B; 108-113 min, 88%-100% B; 113-120 min, 100% B); volume flow rate 1.0 mL / min; detection wavelength 254 nm; column temperature 30°C, injection volume 10 μL.

[0086] Precision test

[0087] Accurately aspirate the same sample solution and inject it 6 times continuously. Taking Codonopsis pilosula I as the reference peak, the RSD value of the relative retention time of each common peak is <1.00%, and the RSD value of the relative peak area is <3.00%, indicating that the instrument has good precision.

[0088] Repeatability test

[0089] Accurately weigh 6 portions of the same sample, prepare the test solution according to the above method, inject the sample, and use Codonopsis pilosula I as the reference peak. The RSD value of the relative retention time of each common peak is <1.00%, and the RSD value of the relative peak area is <3.00%, indicating that the method has good repeatability.

[0090] Stability test

[0091] Accurately draw the same portion of the test solution and measure it at 0, 4, 8, 12, 16 and 24 h, respectively. Taking Codonopsis pilosula I as the reference peak, the RSD value of the relative retention time of each common peak is calculated to be less than 1.00%, and the RSD value of the relative peak area is less than 3.00%, indicating that the test solution is stable within 24 h.

[0092] Fingerprint establishment

[0093] Twelve batches of Codonopsis pilosula samples (DS1 to DS12) were taken, and the test solution was prepared according to the above method. The sample was injected and analyzed according to the chromatographic conditions of the above method, and the chromatogram was recorded. The original data was uniformly integrated by Chromeleon 7 software, exported in the form of cdf file, and imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 version). The DS1 sample was selected as a reference to generate the Weining Codonopsis pilosula control fingerprint, and the WNR sample was selected as a reference to generate the Codonopsis pilosula control fingerprint. The median method was used, and the time width was set to 0.5 min. After multi-point correction and Mark peak matching, the HPLC overlay spectrum and the control fingerprint spectrum were generated respectively. A total of 23 common peaks were calibrated, see Figures 1 to 3 .

[0094] Identification of common peaks

[0095] By comparing the retention time and UV absorption spectrum of the chromatographic peaks with those of the reference substance, a total of four common peaks were identified: 5-hydroxymethylfurfural (P.4), syringin (P.8), codonoside I (P.13), and codonopsis pilosula (P.18), in order of retention time. Peak 13, with its large peak area, good peak shape, and good resolution, was selected as the reference peak (S), with a specified value of 1.000. The relative retention times of the remaining characteristic peaks and the reference peak were calculated, and their relative retention times should be within the range of ±5% of the specified value; the specified values are: Peak 1: 0.105, Peak 2: 0.114, Peak 3: 0. 134, peak 4: 0.191, peak 5: 0.244, peak 6: 0.330, peak 7: 0.452, peak 8: 0.493, peak 9: 0.556, peak 10: 0.597, peak 11: 0.692, peak 12: 0.722, peak 13: 1.000, peak 14: 1.095, peak 15: 1.222, peak 16: 1.363, peak 17: 1.375, peak 18: 1.400, peak 19: 1.555, peak 20: 1.625, peak 21: 1.798, peak 22: 2.086, peak 23: 2.269. The HPLC chart of the mixed reference substance is shown in Figure 4 .

[0096] Example 2

[0097] This embodiment performs fingerprint detection of Codonopsis pilosula, and the specific process is as follows:

[0098] (1) Preparation of reference solution: 5-HMF, syringin, codonopsis pilosula I, and codonopsis pilosula glycoside reference standards were accurately weighed and added with methanol to prepare a mixed reference solution with a mass concentration of each component of approximately 0.2 mg / mL.

[0099] (2) Preparation of test solution: Weigh other rhizomes of Codonopsis pilosula, place them in an oven at 50℃ and dry them for 6h, process them with a Codonopsis pilosula grinder, sieve them (40-60 mesh), take 10g and weigh it accurately, place it in a stoppered conical flask, accurately add 100mL of methanol, soak it for 10-15min, ultrasonically treat it (power 240W, frequency 40kHz) for 30min, decompress the filtrate to recover the solvent until it is almost dry, then add a small amount of methanol to dissolve it and transfer it to a volumetric flask, add methanol to the scale (10ml), shake it well, filter it with a 0.45μm microporous filter membrane, take the filtrate, and store it at 4℃ for testing.

[0100] (3) High performance liquid chromatography detection: Accurately pipette 10 μL of each test solution, inject it into the liquid chromatograph, and record the chromatogram. The chromatographic conditions are as follows: chromatographic column: Phenomenex lux cellulose-2 column (250×4.6 mm, 5 μm); the mobile phase is 0.1% formic acid aqueous solution (A)-acetonitrile (B), gradient elution (0-7 min, 5% B; 7-40 min, 5%-12.4% B; 40-50 min, 12.4%-14% B; 50-60 min, 14%-17.2% B; 60-80 min, 17.2%-26% B; 80-108 min, 26%-88% B; 108-113 min, 88%-100% B; 113-120 min, 100% B); volume flow rate 1.0 mL / min; detection wavelength 254 nm; column temperature 30°C, injection volume 10 μL.

[0101] (4) Analyze the chromatogram: There are 21 common characteristic peaks in the fingerprint of Codonopsis pilosula. The chromatographic peak corresponding to 5-hydroxymethylfurfural is Peak 3, the chromatographic peak corresponding to syringin is Peak 8, the chromatographic peak corresponding to Codonopsis pilosula I is Peak 13, and the chromatographic peak corresponding to Codonopsis pilosulae acetylidine is Peak 18. Peak 13 is used as the reference peak and the specified value is 1.000. The relative retention time of the remaining characteristic peaks and the reference peak is calculated. The relative retention time should be within the range of ±5% of the specified value; the specified values are: Peak 1: 0.103, Peak 2: 0.114, Peak 3: 0. 133, peak 4: 0.191, peak 5: 0.245, peak 6: 0.323, peak 7: 0.450, peak 8: 0.492, peak 9: 0.555, peak 10: 0.597, peak 11: 0.691, peak 12: 0.721, peak 13: 1.000, peak 14: 1.081, peak 15: 1.221, peak 16: 1.364, peak 18: 1.400, peak 19: 1.556, peak 20: 1.629, peak 22: 2.089, peak 23: 2.272.

[0102] Example 3

[0103] Similarity analysis of Weining Codonopsis pilosula

[0104] Using sample DS1 as a reference, a control fingerprint of Weining Codonopsis was generated, and the similarities of the fingerprints of six batches of Weining Codonopsis were calculated (see Table 2). The HPLC fingerprint results showed that the six batches of Weining Codonopsis samples had high similarities, ranging from 0.943 to 0.995. Among them, two batches of Codonopsis from Wenge Township, Dafang County, Guizhou Province (DS1 and DS2) and two batches of Codonopsis from Wenge Township, Dafang County, Guizhou Province (DS5 and DS6) showed extremely high similarities, both greater than 0.975. However, two batches of Codonopsis from Haila Town, Weining County, Guizhou Province (DS3 and DS4) showed slight differences from the other batches of Weining Codonopsis. In particular, sample DS4 showed a similarity between DS1, DS2, DS5, and DS6, ranging from 0.94 to 0.96.

[0105] Table 2

[0106]

[0107] Similarity comparison of Codonopsis pilosula fingerprints

[0108] Samples of Codonopsis pilosula from six common origins of the Chinese medicinal herb Codonopsis pilosula were prepared according to the aforementioned method. Samples were injected under the same chromatographic conditions as described above, and similarity analysis was performed using the Weining Codonopsis pilosula control fingerprint as a reference. (See Table 3.) The results showed that the similarity between samples of Codonopsis pilosula from different origins ranged from 0.78 to 0.978. Dongdang (DS7) from Changbai Mountain, Jilin Province, and Taidang (DS9) from Wutai Mountain, Shanxi Province, showed some differences from the other Codonopsis pilosula samples. Dongdang (DS7) in particular had a similarity of approximately 0.9 with the other Codonopsis pilosula samples. HPLC analysis of DS7 and DS9 revealed that the peak areas for P.13 (codonoside I) and P.18 (codonoside acetyl) were significantly lower than the average values. This result demonstrates that similarity analysis can distinguish Codonopsis pilosula of different qualities to a certain extent.

[0109] Table 3

[0110]

[0111] Cluster analysis

[0112] The peak areas of the 21 common chromatographic peaks of the fingerprints of 6 batches of Weining Codonopsis and 6 batches of Codonopsis from other production areas were combined into a 21×12 matrix and imported into SPSS26.0 software for cluster analysis. The common peak area was used as the variable, the Ward clustering method was used, the squared Euclidean distance was used as the metric, and the Z score was used for data normalization to obtain the cluster analysis dendrogram. Figure 5 .according to Figure 5As can be seen, first, Banqiao Codonopsis (DS10), a Sichuan Codonopsis pilosula, exhibits significant differences from all other Codonopsis varieties, clearly due to differences in their origin. Dongdang (DS7) also differs significantly from the other Codonopsis varieties. Combined with the HPLC fingerprint, the peak areas for P.13 (codonoside I) and P.18 (codonoside acetyl) in Dongdang (DS7) are significantly lower than the average, likely due to differences between wild and cultivated species, consistent with the similarity evaluation results. Wendang (DS11) also differs significantly from the other Codonopsis varieties, primarily due to differences in their origin. Finally, they are divided into two major groups. Ludang (DS8), Taidang (DS9), and Baitiaodang (DS12), all cultivated Codonopsis varieties, show minimal differences, while their differences from Weining Codonopsis are more likely due to differences in their chemical composition, which is more likely due to differences in origin. Overall, the cluster analysis results enable further differentiation of Codonopsis varieties from different origins and regions, building on the similarity evaluation.

[0113] Principal Component Analysis (PCA)

[0114] Principal component analysis (PCA) was performed using SPSS 26.0 software, using the peak areas of the common peaks of Codonopsis pilosula as variables. Eigenvalues and variance contributions are shown in Table 4. Using an eigenvalue > 1 as the extraction criterion, three principal components were identified, with a cumulative variance contribution of 87.39%. The selected principal components each reflect the vast majority of chemical information in the fingerprint and are representative.

[0115] Table 4

[0116]

[0117] The principal component factor loading matrix is shown in Tables 5 and 6. P.7, P.9, P.11, P.12, P.14, P.15, P.16, and P.18 belong to component 1; P.1, P.8, and P.10 belong to component 2; P.13, P.19, P.20, and P.23 belong to component 3; and P.2 and P.5 belong to component 4.

[0118] Table 5

[0119] Ingredients P.1 P.2 P.5 P.7 P.8 P.9 P.10 P.11 P.12 Ingredient 1 0.569 -0.466 0.203 0.898 0.27 0.955 0.148 0.919 0.668 Ingredient 2 0.687 0.46 0.068 0.26 0.743 0.221 0.903 0.296 0.651 Ingredient 3 -0.344 0.264 -0.062 0.002 -0.358 -0.06 -0.104 0.064 0.242 Ingredient 4 0.059 0.622 0.952 0.22 -0.009 0.104 0.207 -0.08 -0.141

[0120] Table 6

[0121]

[0122]

[0123] Variance contribution rates were used to comprehensively evaluate the quality of Codonopsis pilosula. The principal component scores are shown in Table 7. As shown in Table 7, Sichuan Codonopsis pilosula and Codonopsis pilosula (DS10 and DS11) ranked at the top, while DS7 ranked last with a score of 0.13. Combined with the HPLC chromatogram showing that the peak areas of P.13 (codonoside I) and P.18 (codonoside acetyl) were below average, it can be inferred that the remaining common peak components were also not high. The remaining Codonopsis pilosula were interspersed with Weining Codonopsis pilosula. Notably, the two Weining Codonopsis pilosula varieties, DS1 and DS2, from Wenge Township, Dafang County, scored higher than the four Weining Codonopsis pilosula varieties from Weining County, ranking third and fourth.

[0124] Table 7

[0125] Codonopsis pilosula Principal component 1 score Principal component 2 score Principal component 3 score Principal component 4 score Comprehensive score DS10 7.71 4.07 0.39 1.50 5.13 DS11 4.73 2.37 0.48 0.08 3.07 DS2 4.05 1.41 2.77 0.27 2.82 DS1 3.95 1.07 2.27 -0.36 2.55 DS6 2.14 1.12 2.94 1.22 1.90 DS3 2.14 1.22 1.86 1.35 1.79 DS12 2.49 1.11 0.30 0.93 1.67 DS8 2.60 0.95 0.56 0.25 1.66 DS9 1.80 2.15 0.30 1.00 1.60 DS4 1.29 0.97 1.53 1.32 1.24 DS5 0.79 0.45 2.02 0.84 0.88 DS7 -0.19 0.37 0.27 0.91 0.13

[0126] As the only indicative component of Codonopsis pilosula, dangshenin (dangshenin) is specified in the pharmacopoeia, its content in Codonopsis pilosula is undoubtedly a significant indicator of the quality of the product. The peak area of P.18 (dangshenin) in the fingerprints of the 12 batches of Codonopsis pilosula can, to a certain extent, reflect its content. Table 8 shows the 12 batches of Codonopsis pilosula ranked from highest to lowest by P.18 peak area.

[0127] Table 8

[0128] Codonopsis pilosula P.18 peak area Codonopsis pilosula P.18 peak area DS10 8492448 DS12 4318074 DS1 5659742 DS8 3955783 DS6 5007357 DS4 3733071 DS11 4944847 DS5 2981299 DS3 4856579 DS9 2852674 DS2 4389899 DS7 1722352

[0129] This indicates that the average content of dangshen glycosides in Weining Codonopsis is high, placing it at a premium level among mainstream Codonopsis. Specifically, the Weining Codonopsis produced in Wenge Township, Dafang County, is significantly higher than that in Weining County. Combined with principal component analysis, the two results were found to be highly consistent, further strengthening the credibility of using principal component analysis to compare Codonopsis quality.

[0130] Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA)

[0131] To further analyze the causes and markers of sample differences, an OPLS-DA model with supervised mode discrimination was established. The common peak areas of the two samples were imported into SIMCA14.1 software for analysis. The OPLS-DA score diagram is shown in Figure 6 The results showed that the explanatory rate parameters of the Codonopsis pilosula model, R²X = 0.755 and R²Y = 0.766, both greater than 0.5, indicating that the constructed model has a strong explanatory rate, stability, and high predictive power. The model indicates that DS1-DS6 are grouped together, DS8, DS9, and DS12 are grouped together, and DS7, DS11, and DS10 are significantly different from the other varieties.

[0132] The permutation test (n=200) was used to verify the current model. The permutation test diagram is shown in Figure 7The results showed that the test parameters R2 = (0.0, 0.346), Q2 = (0.0, -0.653), R2 < 0.5, Q2 < 0, indicating that the two models are relatively reliable, without overfitting, and can be used to discriminate differences between groups.

[0133] The variable importance projection value (VIP) was used to screen the compounds that caused the differences. The larger the VIP, the greater the weight of the impact on the differences between groups. The VIP value diagram is shown in Figure 8 Using a VIP > 1.05 threshold, five common peaks were identified: P.19, P.13, P.5, P.12, and P.18. Their representative compounds include codonopsis pilosula I and codonopsis pilosula glycosides. This suggests that these compounds are markers of quality differences in Codonopsis pilosula.

[0134] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A fingerprint determination method for Codonopsis pilosula, characterized in that: The following steps are involved: S1: Dissolve 5-hydroxymethylfurfural, codonopsis pilosula, codonopsis pilosula I and syringin in methanol to obtain a reference solution; S2: dissolving the test sample in methanol to obtain a test sample solution; S3: Analyze the reference solution and the test solution by high performance liquid chromatography to obtain a high performance liquid chromatogram; The conditions of the high performance liquid chromatography method are as follows: formic acid solution as mobile phase A and acetonitrile as mobile phase B; The mobile phase was passed through the chromatographic column using gradient elution, followed by UV detection; Calculated based on the volume ratio of mobile phase B in the mobile phase, the gradient elution procedure is: 0-7 min, isocratic elution, the volume proportion of the mobile phase B in the mobile phase is 3-7%; From 7 to 80 minutes, the volume proportion of the mobile phase B in the mobile phase increases from 3 to 7% to 24 to 28%; From 80 to 108 minutes, the volume proportion of the mobile phase B in the mobile phase increases from 24% to 28% to 86% to 90%; From 108 to 113 min, the volume proportion of the mobile phase B in the mobile phase increased from 86% to 90% to 100% B; 113-120 min, isocratic elution, the volume proportion of the mobile phase B in the mobile phase is 100%.

2. The fingerprint determination method of Codonopsis pilosula according to claim 1, wherein: In the reference solution, the concentrations of 5-hydroxymethylfurfural, codonopsis pilosula, codonopsis pilosula I and syringin are 0.1-0.5 mg / mL, respectively.

3. The fingerprint determination method of Codonopsis pilosula according to claim 1, wherein: The mass concentration of the test product is 0.08-0.15 g / mL.

4. The fingerprint determination method of Codonopsis pilosula according to claim 1, wherein: The test product is the underground rhizome of Codonopsis pilosula (Franch.) Nannf.; the Codonopsis pilosula (Franch.) Nannf.

5. The fingerprint determination method of Codonopsis pilosula according to claim 1, wherein: The detection wavelength of the ultraviolet detection is 230-270 nm.

6. The fingerprint determination method of Codonopsis pilosula according to claim 1, wherein: The column temperature of the chromatographic column is 20-40°C.

7. The fingerprint determination method of Codonopsis pilosula according to claim 1, wherein: The flow rate of the mobile phase during the high performance liquid chromatography detection process is 0.5 to 2 mL / min.

8. The fingerprint determination method of Codonopsis pilosula according to claim 1, wherein: There should be 23 peaks in the fingerprint spectrum, the chromatographic peak corresponding to 5-hydroxymethylfurfural is peak 3, the chromatographic peak corresponding to syringin is peak 8, the chromatographic peak corresponding to codonopsis pilosula I is peak 13, and the chromatographic peak corresponding to codonopsis pilosula is peak 18.

9. The fingerprint determination method of Codonopsis pilosula according to claim 1, wherein: In the fingerprint, peak 13 is used as the reference peak, with a prescribed value of 1.000, and the relative retention times of the remaining characteristic peaks and the reference peak are calculated, and the relative retention times should be within the range of ±5% of the prescribed value; the prescribed values are: peak 1: 0.105, peak 2: 0.114, peak 3: 0.134, peak 4: 0.191, peak 5: 0.244, peak 6: 0.330, peak 7: 0.452, peak 8: 0.493, peak 9: 0.556, peak 10: 0.597, peak 11: 0.692, peak 12: 0.722, peak 13: 1.000, peak 14: 1.095, peak 15: 1.222, peak 16: 1.363, peak 17: 1.375, peak 18: 1.400, peak 19: 1.555, peak 20: 1.625, peak 21: 1.798, peak 22: 2.086, peak 23: 2.

269.

10. A method for judging the quality of Codonopsis pilosula, characterized in that: The following steps are involved: S1: Detecting Codonopsis pilosula using the fingerprint determination method of Weining Codonopsis pilosula to obtain a fingerprint; S2: Principal component analysis was performed based on the common peak areas, and the quality of Codonopsis pilosula was determined based on the comprehensive score.

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