A method for constructing a characteristic map of Tripterygium wilfordii for distinguishing its processed products and counterfeits and its application

Abstract

The present invention discloses a method for constructing a characteristic spectrum of Trillium gracile for distinguishing Trillium gracile and its processed products and counterfeits, and its application. By optimizing column temperature and flow rate, optimizing chromatographic columns and other conditions, the present invention allows the Trillium gracile characteristic spectrum to contain more information, with a more stable baseline and better chromatographic peak separation. Through multi-batch testing, common peaks are selected and identified using reference substances. On this basis, Trillium gracile and Trillium gracile can be quickly distinguished through different chromatographic peaks. Simultaneously, through mathematical and statistical analysis, characteristic peaks that can distinguish Trillium gracile from vinegar Trillium gracile slices are optimized, and relative peak areas are optimized, providing a rapid and information-rich characteristic spectrum construction method for quality control analysis of distinguishing Trillium gracile from vinegar Trillium gracile slices.

Description

Technical Field

[0001] The present invention relates to a method for constructing and applying a characteristic map of Trigonella ternata, and in particular to a method for constructing and applying a characteristic map of Trigonella ternata for distinguishing Trigonella ternata and its processed products and counterfeits, belonging to the technical field of traditional Chinese medicine component analysis. Background Art

[0002] Sparganium is the dried tuber of Sparganium stoloniferum Buch.-Ham., a plant of the family Sparganium. It is mainly produced in Zhejiang, Jiangxi, and Hunan provinces. Sparganium has the effects of promoting blood circulation and promoting qi, eliminating stagnation and relieving pain. Modern research shows that Sparganium contains chemical components such as phenylpropanoids and organic acids (Li Yao, Zhao Jinshuang, Zhao Hui, et al. Research Progress on the Traditional Chinese Medicine Sparganium [J]. Journal of Liaoning University of Traditional Chinese Medicine, 2018, 20(9):92-94.), and has pharmacological effects such as anti-platelet aggregation and anti-thrombosis, anti-inflammatory and analgesic (Feng Yaru, Zhang Wenting, Li Erwen, et al. Research Progress on the Chemical Composition and Pharmacological Effects of Sparganium [J]. Chinese Herbal Medicine, 2017, 48(22):4804-4818.). It is often used clinically to treat various congestion diseases caused by blood stasis.

[0003] As a commonly used Chinese medicinal material, Sanleng and its processed products are also commonly used in many Chinese patent medicines. However, the current 2020 edition of the "Chinese Pharmacopoeia" only includes properties, identification, inspection and extraction items under "Sanleng" and its processed product "Vinegar Sanleng", which is not enough to fully describe the quality of Chinese medicinal materials, and it is difficult to effectively control the quality of Sanleng and its processed products.

[0004] The existing technology for the quality control of Trigonella ternata is mostly studied by fingerprint analysis. Liu Jia et al. established an HPLC fingerprint of Trigonella ternata decoction, and the similarity between 20 batches of samples and the control spectrum was between 0.537 and 0.955. The HPLC fingerprint of Trigonella ternata established by this method took up to 60 minutes, and the chromatographic peaks identified were low-specificity components such as vanillic acid, 4-coumaric acid, and ferulic acid, and the chromatographic peak separation was poor (Liu Jia, Liu Junbiao, Liu Yongfu, et al. Establishment of HPLC fingerprint of Trigonella ternata decoction and determination of component content [J]. Chinese Materia Medica, 2022, 45(7): 1674-1678.).

[0005] Chang Yiling et al. established the HPLC fingerprint of Trillium, which took 70 minutes and had only 10 common peaks. The chromatographic peak separation was also poor (Chang Yiling, Wu Qinan, Liang Qiaoli, et al. Research on the HPLC fingerprint of Trillium [J]. Journal of Nanjing University of Chinese Medicine, 2010, 2: 135-136.).

[0006] The above studies on the fingerprint or characteristic spectrum of Trillium are mostly based on the analysis of organic acid components and components with similar polarity. Their material specificity is not strong and cannot fully reflect the quality of Trillium. Or the spectrum has problems such as poor separation of chromatographic peaks, small number of common peaks or unstable baseline. There is no research on the identification of genuine products based on this.

[0007] The 2020 edition of the Chinese Pharmacopoeia lists the processed product of Trillium wilfordii as "Vinegar Trillium wilfordii," which is prepared by sautéing clean Trillium wilfordii slices according to the vinegar-fried method (General Chapter 0213 of the 2020 edition of the Chinese Pharmacopoeia) until the color darkens. Distinguishing Trillium wilfordii from Vinegar Trillium wilfordii is difficult based solely on odor, properties, or thin layer analysis.

[0008] Mao Shujie et al. used colorimetry to determine the flavonoid content in different processed products of Trillium gracile and found that the flavonoid content in the vinegar-fried product was higher than that in the raw product. However, the determination was relatively rough and the information obtained was limited. It could not fully reflect the specific flavonoid components in Trillium gracile and vinegar Trillium gracile. The specificity was not strong and the research role in distinguishing the two was limited (Mao Shujie, Shen Hong, Jie Jingping, et al. Determination of flavonoid content in different processed products of Trillium gracile [J]. China Journal of Traditional Chinese Medicine, 1999, 24(01): 29-30.).

[0009] Liu Junbiao established a fingerprint map and identified 17 common peaks in the samples of Trillium and vinegar Trillium, but they only contained organic acid components such as gallic acid, protocatechuic acid, and ferulic acid, lacked certain specificity, and the chromatographic peak separation was poor, and the analysis time was long (Liu Junbiao. Study on the anti-thrombotic effect and active ingredients of Trillium based on the control of processing and compatibility [D]. Jiangxi University of Traditional Chinese Medicine, 2022).

[0010] Both Sparganium and Sparganium stoloniferum have a long history of use. The names of the two medicinal materials are similar, but their origins are different. In all previous editions of the Chinese Pharmacopoeia, the origin of Sparganium is recorded as Sparganium stoloniferum Buch.-Ham. of the Sparganaceae family, while Sparganium stoloniferum is recorded as Bolboschoenus yagara (Ohwi) YC Yang & M. Zhan of the Cyperaceae family. However, in their use, the names of the medicinal materials and plants are often reversed. There are few studies on the identification of genuine and counterfeit Sparganium. Luo Ying et al. identified Sparganium and its counterfeits by molecular identification (Luo Ying, Zeng Xiaoxuan, Chen Keli, et al. Molecular identification of Sparganium and its counterfeits based on ITS2 sequences [J]. Chinese Pharmacist, 2016, 19(10): 1813-1815.).

[0011] Molecular identification techniques can be used to distinguish Trillium and its counterfeits. However, molecular identification techniques are not universally applicable, and the organic reagents used are highly toxic, making them less practical for distinguishing genuine products from counterfeits. To ensure the safety of Trillium and its processed products, or preparations using it as a raw material, it is crucial to establish a comprehensive, accurate, and rapid quality control method.

[0012] The characteristic spectrum of traditional Chinese medicine is a comprehensive and quantifiable multi-index quality control model that can comprehensively reflect the types and quantities of chemical components of traditional Chinese medicine. By evaluating the stability and consistency of the quality of traditional Chinese medicine and its preparations, its quality is controlled, evaluated and described as a whole, so that the intrinsic quality of traditional Chinese medicine can be systematically, holistically and exclusively reflected and controlled. Therefore, the present invention provides a method for constructing a characteristic spectrum of Tripterygium wilfordii and its processed products using UPLC as an analytical means, and mathematical and statistical analysis is performed on this basis to achieve the purpose of simultaneously identifying and distinguishing Tripterygium wilfordii and its processed products and counterfeits, thereby providing a new analytical means for controlling the intrinsic quality of Tripterygium wilfordii. Compared with the prior art, the present invention shows a characteristic peak component group other than the organic acid component group, and on this basis, Tripterygium wilfordii and its processed products are distinguished, and Tripterygium wilfordii and its counterfeits are distinguished, with the advantages of a large number of characteristic peaks, strong specificity, higher chromatographic peak separation, high stability, good repeatability, simpler operation, and environmental protection. Summary of the Invention

[0013] Purpose of the invention: The purpose of the present invention is to provide a method for constructing a triangular characteristic spectrum with a large number of characteristic peaks, strong specificity, higher chromatographic peak separation, high stability and good repeatability; another purpose of the present invention is to provide an application of a triangular characteristic spectrum.

[0014] Technical solution: The method for constructing a triangular characteristic map of the present invention comprises the following steps:

[0015] (1) Prepare the test solution;

[0016] (2) preparing a reference solution, wherein the reference includes a reference medicinal material and a reference substance, wherein the reference substance is ferulic acid and 4-coumaric acid;

[0017] (3) The reference solution and the test solution were respectively drawn into an ultra-high performance liquid chromatograph, and the ultra-high performance liquid chromatogram was obtained; the detection elution conditions of the ultra-high performance liquid chromatogram were as follows: acetonitrile as mobile phase A, 0.05% phosphoric acid solution or water as mobile phase B; elution was performed according to the following gradient: 0 min → 10 min → 38 min → 44 min, mobile phase A: 15% → 20% → 40% → 40%;

[0018] (4) Confirm the common peaks by comparing with the reference and construct the characteristic spectrum.

[0019] "Reference substances" and "reference medicinal materials" refer to standard substances used for identification, inspection, content determination and calibration of the performance of testing instruments.

[0020] This method detects multiple batches of test sample solutions, selects common peaks, verifies the presence of each common peak in the control medicinal material by referring to the reference medicinal material spectrum, and confirms the composition of the effective ingredient in combination with the characteristic peaks of the reference material.

[0021] Wherein, in step (1), the test sample is a medicinal material, decoction piece, extract, standard decoction, formula granule of Trigonella ternata, or a reference sample, preparation intermediate or finished product of a classic prescription containing Trigonella ternata. "Medicinal material" or "raw drug" refers to a raw material of Chinese medicine that has not been processed or made into a finished product. "Decoction piece" refers to a Chinese medicine that has been processed according to needs and is used for formulation, or a Chinese medicine that can be directly used in clinical Chinese medicine.

[0022] Furthermore, in step (1), the test sample is extracted with water and then extracted with ethyl acetate to obtain a test sample solution; and / or, in step (2), the control medicinal material is extracted with water and then extracted with ethyl acetate to obtain a control medicinal material solution.

[0023] Specifically, the preparation method of the test solution is as follows:

[0024] Take the test sample, add water, heat to reflux, cool, shake, filter, add ethyl acetate and shake to extract, combine the ethyl acetate extracts, evaporate to dryness, add 50% methanol to dissolve the residue and make up to volume, shake, filter, take the filtrate as the test solution, shake, filter, and take the filtrate to obtain.

[0025] Specifically, the preparation method of the control medicinal material solution is as follows:

[0026] Take the reference medicinal material of Trillium, add water, heat to reflux, cool, shake, filter, add ethyl acetate and shake to extract, combine the ethyl acetate solution, evaporate to dryness, add 50% methanol to dissolve the residue to volume, shake, filter, and take the filtrate as the reference solution of the reference medicinal material.

[0027] Furthermore, in step (2), the reference substance is dissolved in methanol to obtain a reference substance solution.

[0028] Take the reference substance and add methanol to prepare a solution containing 10 μg of 4-coumaric acid and 10 μg of ferulic acid per 1 ml, which is used as the reference substance solution.

[0029] Furthermore, in step (3), the chromatographic column is an octadecylsilane bonded silica gel column; the column temperature is 33-47° C.; the flow rate is 0.28-0.32 ml; the detector is an ultraviolet detector; and the detection wavelength is 200 nm-400 nm.

[0030] Specifically, the chromatographic conditions of the ultra-high performance liquid chromatography are as follows:

[0031] Syncronis C18 (Thermo, 100 × 2.1 mm, 1.7 μm); gradient elution with acetonitrile as mobile phase A and 0.05% phosphoric acid solution as mobile phase B; flow rate 0.3 ml / min; column temperature 35°C; detection wavelength 330 nm. The theoretical plate number calculated based on the 4-coumaric acid peak should be no less than 5000.

[0032]

[0033] Furthermore, in step (4), the triangular characteristic spectrum has 12 characteristic peaks, among which peak 2 and peak 3 are characteristic peaks of 4-coumaric acid and ferulic acid, respectively; taking peak 7 as the reference peak S, the relative retention times of the remaining characteristic peaks are: peak 1 is 0.22±10%, peak 4 is 0.67±10%, peak 5 is 0.90±10%, peak 6 is 0.92±10%, peak 8 is 1.31±10%, peak 9 is 1.46±10%, peak 10 is 1.64±10%, peak 11 is 1.69±10%, and peak 12 is 1.74±10%.

[0034] On the other hand, the present invention provides an application of a characteristic map of Trillium constructed by the above method in distinguishing Trillium and its processed products.

[0035] Furthermore, in the ultra-high performance liquid chromatography (ULCP) spectrum of the test solution, if the relative peak area ratio of peak 9 to peak 10 is less than 0.34, the test sample is vinegar-scented Trillium slices; otherwise, it is not vinegar-scented Trillium slices.

[0036] Specifically, the method for distinguishing Tripterygium wilfordii and its processed products, that is, the method for quality inspection of Tripterygium wilfordii and its processed products are as follows:

[0037] (1) Preparation of test solution: Extract Trigonella ternata or its processed product to obtain the test solution;

[0038] (2) Preparation of reference solution: taking a ferulic acid reference substance and a 4-coumaric acid reference substance, adding a solvent and dissolving them to obtain the reference solution;

[0039] (3) Establishment of the chromatographic method: The reference solution and the test solution are respectively aspirated, injected into an ultra-high performance liquid chromatograph, and measured to obtain the characteristic spectrum. The elution conditions for the ultra-high performance liquid chromatography detection are: acetonitrile as mobile phase A, 0.05% phosphoric acid solution or water as mobile phase B; elution is performed according to the following gradient: 0 min → 10 min → 38 min → 44 min, mobile phase A: 15% → 20% → 40% → 40%;

[0040] (4) Establishment and comparison of characteristic maps: Determine the decoction pieces of Trigonella ternata and the corresponding vinegar Trigonella ternata decoction pieces, generate control characteristic maps respectively, and compare the raw and processed products by combining mathematical and statistical analysis methods.

[0041] The above-mentioned method for distinguishing Trillium and its processed products is used to evaluate the quality of Trillium and its processed products, to identify Trillium, vinegar Trillium or medicines containing Trillium or vinegar Trillium as raw materials.

[0042] On the other hand, the present invention provides an application of a triangular feature map constructed by the above method in distinguishing triangular and its counterfeit products.

[0043] Furthermore, if the ultra-high performance liquid chromatography (ULCP) spectrum of the test solution shows 12 peaks corresponding to the characteristic spectrum of Trillium, then the test sample is Trillium or its processed product; otherwise, it is not Trillium or its processed product, that is, it is a counterfeit Trillium.

[0044] Specifically, the detection method for distinguishing three-edged swords and their counterfeits is as follows:

[0045] (1) Preparation of test solution: extract the test solution from Tripterygium wilfordii and the counterfeit product;

[0046] (2) Preparation of reference solution: taking a ferulic acid reference substance and a 4-coumaric acid reference substance, adding a solvent and dissolving them to obtain the reference solution;

[0047] (3) Establishment of the chromatographic method: The reference solution and the test solution are respectively aspirated, injected into an ultra-high performance liquid chromatograph, and measured to obtain the characteristic spectrum. The elution conditions for the ultra-high performance liquid chromatography detection are: acetonitrile as mobile phase A, 0.05% phosphoric acid solution or water as mobile phase B; elution is performed according to the following gradient: 0 min → 10 min → 38 min → 44 min, mobile phase A: 15% → 20% → 40% → 40%;

[0048] (4) Establishment and comparison of characteristic spectra: Determine the characteristic spectra of Trillium spicata and its counterfeit Trillium spicata, and compare the counterfeit products.

[0049] The method for distinguishing Trillium and its counterfeit product, Trillium chinense, is as follows: Trillium chinense and Trillium chinense share nine common characteristic peaks, Peaks 1, 4, and 8 are missing from the spectrum of Trillium chinense, and compared with Trillium chinense, Peaks A, B, C, and D are present. Using Peak 7 as the reference peak S, their relative retention times are 0.43±10%, 0.78±10%, 0.95±10%, and 1.09±10%, respectively. The characteristic spectrum established using the method of this application can distinguish Trillium chinense and Trillium chinense decoction pieces.

[0050] The present invention optimizes column temperature and flow rate, chromatographic column conditions, and other factors to ensure that the characteristic spectrum of Trillium contains more information, a more stable baseline, and better chromatographic peak separation. Through multi-batch testing, common peaks are selected and identified using reference substances. On this basis, Trillium and Trillium can be quickly distinguished through different chromatographic peaks. Simultaneously, through mathematical and statistical analysis, characteristic peaks that can distinguish Trillium from vinegar Trillium slices are optimized, and relative peak areas are optimized, providing a rapid and information-rich characteristic spectrum construction method for quality control analysis of Trillium and vinegar Trillium slices.

[0051] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0052] (1) The present invention adopts ultra-high performance liquid chromatography and reasonably controls the chromatographic conditions, uses acetonitrile as mobile phase A and 0.05% phosphoric acid solution as mobile phase B for gradient elution, and determines a total of 12 characteristic peaks, which constitute the overall picture of the characteristic spectrum of Trillium, and more comprehensively reflects the characteristics of Trillium medicinal materials, avoiding the defects of single or a few peak determinations, and can control the entire intrinsic quality of Trillium medicinal materials and decoction pieces, and can provide a new analytical means for the intrinsic quality control of Trillium;

[0053] (2) The present invention establishes a high performance liquid chromatography method, using the relative peak area difference of the characteristic peaks of Trigonum multiflorum slices and vinegar Trigonum multiflorum slices as the discrimination point, providing a new idea for rapid identification of Trigonum multiflorum and its processed products;

[0054] (3) The present invention establishes a high-performance liquid chromatography method, using the difference in the number of characteristic peaks of Trillium and Trillium as the discrimination point, to establish a high-performance liquid chromatography method to quickly identify Trillium and its counterfeit products, thereby preventing the mixing of Trillium and its counterfeit products in clinical practice and ensuring the safety of medication;

[0055] (4) The method of the present invention is simple, reproducible, accurate, reliable, and easy to operate. Compared with HPLC, it saves time, consumes less solvent, and has little pollution to the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1A and Figure 1B The liquid chromatograms of the reference medicinal material, reference substance and the decoction pieces of Trigonella ternata are shown in Figure 2. Figure 1A This is the liquid chromatogram of the solution of Trigonum multiflorum slices and reference substance. Figure 1B This is a comparison chart of Trillium slices and Trillium control medicinal materials;

[0057] Figure 2 Chromatograms for investigating the durability of different chromatographic columns for Tripterygium wilfordii decoction pieces;

[0058] Figure 3 Chromatograms for investigating the durability of Tripterygium wilfordii slices at different column temperatures;

[0059] Figure 4 Chromatograms for investigating the durability of Tripterygium wilfordii slices at different flow rates;

[0060] Figure 5 This is an overlay of characteristic spectra of multiple batches of Tripterygium wilfordii medicinal materials;

[0061] Figure 6 This is the reference characteristic spectrum of Trigonella ternata medicinal material;

[0062] Figure 7 This is an overlay of characteristic spectra of multiple batches of Trillium decoction pieces;

[0063] Figure 8 This is the reference characteristic spectrum of Trillium decoction pieces;

[0064] Figure 9 This is an overlay of characteristic spectra of multiple batches of vinegar Trillium slices;

[0065] Figure 10 This is the reference characteristic spectrum of vinegar Trigonella Root slices;

[0066] Figure 11 This is a comparison of the characteristic spectra of Trillium and Trillium with vinegar;

[0067] Figure 12 This is the OPLS-DA graph of the slices of Trillium and Trillium with vinegar, where SL represents Trillium and CSL represents Trillium with vinegar;

[0068] Figure 13 is the VIP score graph in OPLS-DA, where F1 to F12 represent peaks 1 to 12;

[0069] Figure 14 This is a comparison chart of the characteristic spectra of Trillium and its counterfeit product. DETAILED DESCRIPTION

[0070] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0071] Example 1 Construction of UPLC Characteristic Spectrum Detection Method for Trillium and Its Processed Products and Counterfeit Products 1. Instruments, Reagents and Samples

[0072] Agilent 1290 UPLC ultra-high performance liquid chromatograph (Agilent); OpenLAB CDS workstation (Agilent); METTLER TOLEDO XP6 micrometer balance (Mettler-Toledo (Shanghai) Instrument Co., Ltd.); ME204E electronic analytical balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.); KQ-250B ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); GKC114 temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.); Milli-Q water system (Millipore); TGL-16C centrifuge (Shanghai Anting Scientific Instrument Factory); CenLee 20K centrifuge (Anhui Zhuoyi Scientific Instrument Co., Ltd.). Methanol (chromatographic grade, Thermo Fisher); acetonitrile (chromatographic grade, Thermo Fisher); ultrapure water; phosphoric acid (chromatographic grade, Aladdin); all other reagents were of analytical grade.

[0073] 4-Coumaric acid (No. 112037-202102) and ferulic acid (No. 110773-201614) were purchased from the China Food and Drug Administration.

[0074] The medicinal materials of Trillium were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd., with batch numbers YC 1~17; the decoction pieces of Trillium were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd., with batch numbers YP 1~17; and the decoction pieces of vinegared Trillium were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd., with batch numbers CYP 1~17.

[0075] 2. Preparation of reference solution

[0076] Take 2.0g of Trigonella ternata reference medicinal material, place it in a stoppered conical flask, add 50ml of water, heat and reflux for 90 minutes, let cool, shake well, filter, take 40ml of the filtrate, add ethyl acetate and shake to extract twice, 40ml each time, combine the ethyl acetate, evaporate to dryness, dissolve the residue in 50% methanol, transfer to a 5ml volumetric flask, add 50% methanol to the scale, shake well, filter, and take the filtrate as the reference solution of the reference medicinal material. Separately, take appropriate amounts of 4-coumaric acid reference substance and ferulic acid reference substance, accurately weigh them, and add methanol to make solutions containing 10μg of 4-coumaric acid and 10μg of ferulic acid per 1ml respectively, as the reference solution of the reference substance.

[0077] 3. Preparation of test solution

[0078] Take 2.0g of Trigonella ternata slices (passed through No. 3 sieve), place in a stoppered conical flask, add 50ml of water, heat and reflux for 90 minutes, cool, shake, filter, take 40ml of the filtrate, add ethyl acetate and shake to extract twice, 40ml each time, combine the ethyl acetate, evaporate to dryness, dissolve the residue in 50% methanol, transfer to a 5ml volumetric flask, add 50% methanol to the scale, shake, filter, take the filtrate as the test solution, shake, filter, and take the filtrate.

[0079] 4. Determination of chromatographic conditions

[0080] Syncronis C18 (Thermo, 100 × 2.1 mm, 1.7 μm); acetonitrile as mobile phase A, 0.05% phosphoric acid solution as mobile phase B, gradient elution according to Table 1; flow rate 0.3 ml / min; column temperature 35°C; detection wavelength 330 nm. The theoretical plate number calculated based on the 4-coumaric acid peak should be no less than 5000.

[0081] Table 1 Gradient elution table

[0082]

[0083] 5. Test results

[0084] Accurately pipette 5 μl of each reference solution and test solution, inject into liquid chromatograph, record the chromatogram, and see the results. Figure 1A and Figure 1B .Depend on Figure 1A and Figure 1B It can be seen that the characteristic spectrum obtained by this method has good peak shape and separation, large amount of information and stable baseline; there are chromatographic peaks in the Trillium slices that correspond to the retention times of ferulic acid and 4-coumaric acid reference substances.

[0085] Example 2 Methodological Verification of UPLC Characteristic Spectra of Tripterygium wilfordii and Its Processed Products and Counterfeit Products 1. Precision Investigation

[0086] The same test solution in Example 1 was injected 6 times continuously, and Peak 7 was used as the reference peak. The RSD values ​​of the relative retention time and relative peak area of ​​each common characteristic peak were calculated (Table 2, Table 3), all of which were less than 2%, indicating that the characteristic spectrum method had good precision.

[0087] Table 2 Precision test results (relative retention time)

[0088]

[0089] Table 3 Precision test results (relative peak area)

[0090]

[0091] 2. Repeatability inspection

[0092] Take the same batch of sample powder in Example 1, accurately weigh 6 portions, and prepare 6 test sample solutions in parallel according to the test sample preparation method in Example 1. The samples were injected separately, and Peak 7 was used as the reference peak. The RSD values ​​of the relative retention time and relative peak area of ​​each common peak were calculated (Table 4, Table 5), all of which were less than 2%, indicating that the construction method has good reproducibility.

[0093] Table 4 Repeatability test results (relative retention time)

[0094]

[0095] Table 5 Repeatability test results (relative peak area)

[0096]

[0097] 3. Stability inspection

[0098] The same sample solution was injected and analyzed at 0, 2, 4, 6, 8, 10, 12, and 24 h, respectively. Peak 7 was used as the reference peak, and the RSD values ​​of the relative retention time and relative peak area of ​​each common peak were calculated (Table 6 and Table 7), all of which were less than 2%, indicating that the sample solution had good stability within 24 hours.

[0099] Table 6 Stability test results (relative retention time)

[0100]

[0101] Table 7 Stability test results (relative peak area)

[0102]

[0103]

[0104] 4. Durability inspection

[0105] 1) Investigation of different chromatographic columns

[0106] Three different brands of chromatographic columns were compared: ACQUITY The effects of three chromatographic columns, HSS T3 (Waters, 100×2.1mm, 1.8μm), Syncronis C18 (Thermo, 100×2.1mm, 1.7μm), and CORTECS UPLC T3 (Waters, 100×2.1mm, 1.6μm), on the durability of the characteristic spectrum are shown in the table. Figure 2The results showed that the trifoliate sample could obtain a better separation effect on the Syncronis C18 (Thermo, 100×2.1mm, 1.7μm) chromatographic column. Therefore, it is recommended to use the Syncronis C18 (Thermo, 100×2.1mm, 1.7μm) ultra-high performance liquid chromatography column in this method.

[0107] 2) Investigation at different temperatures

[0108] A Syncronis C18 (Thermo, 100 mm × 2.1 mm, 1.7 μm) column was used to investigate the sample separation effects at column temperatures of 33°C, 35°C, and 37°C. The results are shown in Table 1. Figure 3 The results showed that the analytical method had good separation effects for each characteristic peak when the column temperature was 33°C to 37°C, and the relative retention times were all within the specified range, indicating good durability and that the system suitability requirements could be met with small column temperature fluctuations.

[0109] 3) Investigation of different flow rates

[0110] A Syncronis C18 (Thermo, 100 mm × 2.1 mm, 1.7 μm) column was used to investigate the sample separation effects at flow rates of 0.28 ml / min, 0.30 ml / min, and 0.32 ml / min. The results are shown in the table. Figure 4 The results showed that the separation effect of each characteristic peak was good when the flow rate was in the range of 0.28ml / min to 0.32ml / min, and the relative retention times were all within the specified range, indicating that the method was robust and could meet the system suitability requirements when the flow rate varied.

[0111] Example 3 Investigation and establishment of characteristic spectra of multiple batches of Tripterygium wilfordii

[0112] 1. Preparation of reference solution

[0113] The relevant method is the same as that in Example 1.

[0114] 2. Preparation of test solution

[0115] The relevant method is the same as that in Example 1.

[0116] 3. Construction of feature maps

[0117] (1) Chromatographic conditions

[0118] The relevant method is the same as that in Example 1.

[0119] (2) Testing and results

[0120] 5 μl of reference solution and test solution were accurately aspirated respectively, injected into liquid chromatograph, and chromatograms were recorded. The chromatograms were then imported into the “Chinese Herbal Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)” recommended by the Chinese Pharmacopoeia Committee for result analysis. The time window width was selected as 0.1 min, and the reference spectrum was generated with the median. After multi-point correction, the Mark peaks were matched and the characteristic spectra of 17 batches of Trillium herb samples were superimposed. Figure 5 The reference characteristic spectrum generated by fitting is shown in Figure 6 Taking peak 7 as the reference peak, the relative retention time and relative peak area of ​​the common peaks in the characteristic spectrum were calculated and shown in Tables 8 and 9.

[0121] Table 8 Relative retention time of 17 batches of Tripterygium wilfordii

[0122]

[0123] Table 9 Relative peak areas of 17 batches of Trigonella ternata

[0124]

[0125]

[0126] Based on the test results of multiple batches of Trigonella ternata medicinal materials, it is stipulated that 12 characteristic peaks should be present in the characteristic spectrum of Trigonella ternata medicinal materials, and the retention times of the 12 characteristic peaks in the chromatogram of the reference medicinal material should correspond to each other. Two of the peaks should correspond to the retention times of the corresponding reference peaks. Taking Peak 7 as the S peak, the relative retention times of the remaining characteristic peaks and the S peak are calculated. The relative retention times should be within the range of ±10% of the specified values. The specified values ​​are: 0.22 (Peak 1), 0.67 (Peak 4), 0.90 (Peak 5), 0.92 (Peak 6), 1.31 (Peak 8), 1.46 (Peak 9), 1.64 (Peak 10), 1.69 (Peak 11), and 1.74 (Peak 12).

[0127] Example 4 Investigation of characteristic spectra of multiple batches of Tripterygium wilfordii slices

[0128] 1. Preparation of reference solution

[0129] The relevant method is the same as that in Example 1.

[0130] 2. Preparation of test solution

[0131] The relevant method is the same as that in Example 1.

[0132] 3. Construction of feature maps

[0133] (1) Chromatographic conditions

[0134] The relevant method is the same as that in Example 1.

[0135] (2) Testing and results

[0136] 5 μl of reference solution and test solution were accurately aspirated respectively, injected into liquid chromatograph, chromatograms were recorded, and the chromatograms were imported into the “Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)” recommended by the State Pharmacopoeia Committee for result analysis. The time window width was selected as 0.1 min, and the reference spectrum was generated with the median. After multi-point correction, the Mark peak was matched, and the characteristic spectrum of 17 batches of Trillium decoction pieces samples was superimposed. Figure 7 The reference characteristic spectrum generated by fitting is shown in Figure 8 Taking peak 7 as the reference peak, the relative retention time and relative peak area of ​​the common peaks in the characteristic spectrum were calculated and shown in Tables 10 and 11.

[0137] Table 10 Relative retention time of 17 batches of Tripterygium wilfordii slices

[0138]

[0139] Table 11 Relative peak areas of 17 batches of Tripterygium wilfordii slices

[0140]

[0141]

[0142] According to the measurement results of multiple batches of Trillium slices, it is stipulated that 12 characteristic peaks should be present in the characteristic spectrum of Trillium slices, and the retention times of the 12 characteristic peaks in the chromatogram of the reference medicinal material should correspond to each other. Two of the peaks should correspond to the retention times of the corresponding reference peaks. Taking Peak 7 as the S peak, the relative retention times of the remaining characteristic peaks and the S peak are calculated, and the relative retention times should be within the range of ±10% of the specified values. The specified values ​​are: 0.22 (peak 1), 0.67 (peak 4), 0.90 (peak 5), 0.92 (peak 6), 1.31 (peak 8), 1.46 (peak 9), 1.64 (peak 10), 1.69 (peak 11), and 1.74 (peak 12).

[0143] Example 5 Investigation of characteristic spectra of multiple batches of vinegar-sweetened Tripterygium wilfordii slices

[0144] 1. Preparation of reference solution

[0145] The relevant method is the same as that in Example 1.

[0146] 2. Preparation of test solution

[0147] The relevant method is the same as that in Example 1.

[0148] 3. Construction of feature maps

[0149] (1) Chromatographic conditions

[0150] The relevant method is the same as that in Example 1.

[0151] (2) Testing and results

[0152] 5 μl of reference solution and test solution were accurately aspirated respectively, injected into liquid chromatograph, and chromatograms were recorded. The chromatograms were then imported into the “Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)” recommended by the Chinese Pharmacopoeia Committee for result analysis. The time window width was selected as 0.1 min, and the reference spectrum was generated with the median. After multi-point correction, the Mark peaks were matched and the characteristic spectra of 17 batches of vinegar Trillium slices were superimposed. Figure 9 The reference characteristic spectrum generated by fitting is shown in Figure 10 Taking peak 7 as the reference peak, the relative retention time and relative peak area of ​​the common peaks in the characteristic spectrum were calculated and shown in Tables 12 and 13.

[0153] Table 12 Relative retention time of 17 batches of vinegar Tripterygium wilfordii slices

[0154]

[0155]

[0156] Table 13 Relative peak areas of 17 batches of vinegar-cured Tripterygium wilfordii slices

[0157]

[0158] According to the measurement results of multiple batches of vinegar Trillium slices, it is stipulated that 12 characteristic peaks should be present in the characteristic spectrum of vinegar Trillium slices, and the retention times of the 12 characteristic peaks in the chromatogram of the reference medicinal material should correspond to each other. Two of the peaks should correspond to the retention times of the corresponding reference peaks. Taking peak 7 as the S peak, the relative retention times of the remaining characteristic peaks and the S peak are calculated, and the relative retention times should be within the range of ±10% of the specified values. The specified values ​​are: 0.22 (peak 1), 0.67 (peak 4), 0.90 (peak 5), 0.92 (peak 6), 1.31 (peak 8), 1.46 (peak 9), 1.64 (peak 10), 1.69 (peak 11), and 1.74 (peak 12).

[0159] Example 6 Comparison of characteristic spectra of Tripterygium wilfordii and Tripterygium wilfordii slices with vinegar

[0160] 1. Preparation of reference solution

[0161] The relevant method is the same as that in Example 1.

[0162] 2. Preparation of test solution

[0163] Take about 2 g of Trillium slices and vinegar Trillium slices respectively, and the rest of the relevant methods are the same as in Example 1.

[0164] 3. Construction of feature maps

[0165] (1) Chromatographic conditions

[0166] The relevant method is the same as that in Example 1.

[0167] (2) Testing and results

[0168] 5 μl of each test solution was accurately drawn and injected into the liquid chromatograph. The characteristic spectra of the Tripterygium wilfordii slices and the corresponding vinegar Tripterygium wilfordii slices were respectively imported into the Chinese medicine fingerprint similarity software to generate the control characteristic spectra. The two spectra were compared. Figure 11 OPLS-DA analysis was performed on the relative peak areas of the 12 characteristic peaks of Trillium slices and the corresponding vinegar Trillium slices. It was found that peak 10 made a greater contribution to distinguishing Trillium slices from vinegar Trillium slices. Figure 12 、 Figure 13 At the same time, it was found that the peak area ratio ranges of Peak 9 / Peak 10 of the two did not overlap (the range of Peak 9 / Peak 10 of Sparganium slices was 0.36-0.52, and the range of Peak 9 / Peak 10 of Vinegar Sparganium slices was 0.25-0.31), which can be used to distinguish Sparganium and Vinegar Sparganium slices. The results are shown in Tables 14 and 15. Based on the 2-fold SD value of the peak area ratio of Peak 9 / Peak 10 of multiple batches of Sparganium slices, it is stipulated that the ratio of Peak 9 to Peak 10 of Sparganium slices shall not be less than 0.34, which is used to distinguish Sparganium and Vinegar Sparganium slices.

[0169] Table 14 Peak area ratios of characteristic peaks and peak 10 in the characteristic spectrum of Tripterygium wilfordii decoction pieces

[0170]

[0171]

[0172] Table 15 Peak area ratios of characteristic peaks and peak 10 in the characteristic spectrum of vinegar Tripterygium wilfordii slices

[0173]

[0174] Example 7 Comparison of the characteristic spectra of Trillium and Trillium chinense

[0175] 1. Preparation of reference solution

[0176] The relevant method is the same as that in Example 1.

[0177] 2. Preparation of test solution

[0178] The relevant method is the same as that in Example 1.

[0179] 3. Construction of feature maps

[0180] (1) Chromatographic conditions

[0181] The relevant method is the same as that in Example 1.

[0182] (2) Testing and results

[0183] Accurately pipette 5 μl of reference solution and test solution respectively, inject into liquid chromatograph, record chromatogram, and compare the characteristic spectrum results. Figure 14 . From the results, it can be seen that Trillium and Jing Trillium can be distinguished based on the number of chromatographic peaks. Trillium and Jing Trillium have 9 common characteristic peaks. Peaks 1, 4, and 8 are missing in the spectrum of Jing Trillium. Compared with Trillium, there are 4 more peaks: Peak A, Peak B, Peak C, and Peak D. Taking Peak 7 as the reference peak S, their relative retention times are 0.43±10%, 0.78±10%, 0.95±10%, and 1.09±10%, respectively. The characteristic spectrum established by this method can distinguish Trillium and Jing Trillium decoction pieces.

[0184] The above-described embodiments merely illustrate several implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the claims.

Claims

1. A method for constructing a triangular feature map, characterized in that: The following steps are involved: (1) preparing a test solution; the test sample is a medicinal material, decoction piece, standard decoction, or formula granule of Trigonella ternata; the test sample is extracted with water and then extracted with ethyl acetate to obtain a test solution; (2) preparing a reference solution, wherein the reference includes a reference medicinal material and a reference substance, wherein the reference substance is ferulic acid and 4-coumaric acid; (3) The reference solution and the test solution were respectively drawn, injected into an ultra-high performance liquid chromatograph, and measured to obtain a corresponding ultra-high performance liquid chromatogram; the detection conditions of the ultra-high performance liquid chromatograph were as follows: acetonitrile as mobile phase A, 0.05% phosphoric acid solution as mobile phase B; elution was performed according to the following gradient: 0 min → 10 min → 38 min → 44 min, mobile phase A: 15% → 20% → 40% → 40%; the chromatographic column was Syncronis C18, 100×2.1 mm, 1.7 μm; the detection wavelength was 330 nm, and the detector was a UV detector; (4) The common peaks were confirmed by comparing with the reference materials and the characteristic spectrum was constructed. The characteristic spectrum of Sanling had 12 characteristic peaks, among which peak 2 and peak 3 were the characteristic peaks of 4-coumaric acid and ferulic acid, respectively.

2. The method for constructing the characteristic spectrum of Tripterygium wilfordii according to claim 1, wherein in step (2), the control medicinal material is extracted with water and then extracted with ethyl acetate to obtain a control medicinal material solution.

3. The method for constructing a triangular characteristic spectrum according to claim 1, wherein: In step (2), the reference substance is dissolved in methanol to obtain a reference substance solution.

4. The method for constructing a triangular characteristic map according to claim 1, wherein: In step (3), the column temperature is 33-47°C; the flow rate is 0.28-0.32 ml.

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