Evaluation method of quality of pinellia ternate medicine

Abstract

This invention relates to a comprehensive evaluation method for the quality of Pinellia ternata and a method for constructing a fingerprint spectrum of Pinellia ternata. The method includes: processing Pinellia ternata to obtain a test solution; preparing a reference solution containing guanosine, phenylalanine, VGTNY, tryptophan, LFSG, apigenin-6-C-arabinose-8-C-galactoside, PWVPG, NIPF, and VIYGPSVF; injecting the test solution and the reference solution separately into a liquid chromatography-mass spectrometry (LC-MS) instrument, and obtaining the fingerprint spectrum of the Pinellia ternata using LC-MS. A method for determining the content of toxic components calcium oxalate needle crystals and lectin protein is also established. The comprehensive evaluation method for the quality of Pinellia ternata provided by this invention comprehensively improves the evaluation standards for Pinellia ternata, ensuring the effectiveness and safety of Pinellia ternata in clinical use.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine quality analysis and testing technology, specifically relating to a method for evaluating the quality of Pinellia ternata. Background Technology

[0002] Pinellia ternata (Thunb.) Breit., a plant in the Araceae family, was first recorded in the *Shengji Zonglu*. It is a processed product obtained by soaking the dried tuber of Pinellia ternata in an alum solution. Pinellia ternata is known for its expectorant properties, primarily focusing on drying dampness and resolving phlegm. Clinically, it is used for coughs with damp phlegm, internal accumulation of phlegm and heat, and difficulty expectorating phlegm. Modern pharmacological studies have shown that Pinellia ternata possesses various pharmacological effects, including antitussive, expectorant, antiemetic, antitumor, and anti-inflammatory properties, but its main active components and mechanisms of action are not yet fully elucidated.

[0003] Pinellia ternata is mainly produced in Hubei, Gansu, and Guizhou provinces, and is widely distributed in East Asia. It is now widely used clinically in many countries and regions. However, the quality of processed Pinellia ternata is affected by various factors such as origin, harvesting season, processing method, and climate, resulting in significant differences in the content of its active ingredients. Currently, there is limited research on the determination of active ingredient content and quality control in processed Pinellia ternata. The 2020 edition of the Chinese Pharmacopoeia lacks standards for the determination of Pinellia ternata content. The Pharmacopoeia identifies Pinellia ternata using only microscopic identification and thin-layer chromatography to determine the presence of arginine, alanine, valine, and leucine. The 2015 edition of the Chinese Pharmacopoeia uses potentiometric titration to determine the total organic acids in the alcohol extract of Pinellia ternata, with the total acid content calculated only as succinic acid. However, Pinellia ternata has a complex composition, and the main active ingredients are not yet clearly defined. This method can only roughly reflect the total amount of organic acids in Pinellia ternata, not the specific composition of each component. Furthermore, the potentiometric titration method for determining the total organic acids in Pinellia ternata is easily affected by other acidic substances and operating conditions. Fingerprint analysis is currently an important and most intuitive method for quality control of Chinese medicinal materials. While there are reports on the determination of multiple indicator components in Pinellia ternata (Banxia) raw materials and processed slices using high-performance liquid chromatography (HPLC), the identified common peaks are mostly nucleosides and organic acids, exhibiting poor specificity and failing to adequately evaluate the active components of Pinellia ternata. Furthermore, the quality control standards for Pinellia ternata do not include tests for its toxic substances. To ensure the safety and efficacy of clinical use, we need a chemical analytical method that can comprehensively evaluate and effectively control the quality of Pinellia ternata. Summary of the Invention

[0004] This invention aims to at least partially address one of the technical problems existing in the prior art. To this end, this invention provides a method for evaluating the quality of Pinellia ternata (Qingbanxia) medicinal material. This method utilizes liquid chromatography-mass spectrometry (LC-MS) to identify common peaks in Pinellia ternata samples and establishes an LC-MS fingerprint spectrum for Pinellia ternata. It proposes using calcium oxalate needle crystals and lectin protein as control indicators for toxic components in the quality standard of Pinellia ternata, and establishes content limit testing methods for calcium oxalate needle crystals and lectin protein in Pinellia ternata to control the content of toxic components. These methods improve the evaluation standard of Pinellia ternata and provide a reference for the overall and comprehensive quality control of Pinellia ternata.

[0005] According to one aspect of the present invention, a method for evaluating the quality of Pinellia ternata medicinal material is provided, comprising: processing Pinellia ternata medicinal material to obtain a test solution; preparing a reference solution containing guanosine, phenylalanine, valine-glycine-threonine-asparagine-tyrosine (VGTNY), tryptophan, leucine-phenylalanine-serine-glycine (LFSG), apigenin-6-C-arabinose-8-C-galactoside, proline-tryptophan-valine-proline-glycine (PWVPG), asparagine-isoleucine-proline-phenylalanine (NIPF), and valine-isoleucine-tyrosine-glycine-proline-serine-valine-phenylalanine (VIYGPSVF); obtaining chromatograms of the test solution and the reference solution respectively; comparing the chromatograms of the test solution and the reference solution to determine common peaks and similarity.

[0006] Preferably, the method further includes: using calcium oxalate needle crystals and lectin protein as toxicity indicator components to comprehensively evaluate the quality of the prepared Pinellia ternata medicinal material.

[0007] Preferably, obtaining the chromatograms of the test solution and the reference solution separately includes: performing HPLC chromatographic analysis on the test solution and the reference solution respectively, wherein the HPLC chromatographic analysis conditions are: gradient elution using a mobile phase A containing 0.1% formic acid aqueous solution and a mobile phase B containing 0.1% formic acid acetonitrile solution, a flow rate of 0.3 mL / min, and a column temperature of 35 °C.

[0008] Preferably, acquiring the spectra of the test solution and the reference solution separately further includes: performing mass spectrometry analysis on the test solution and the reference solution respectively. The mass spectrometry analysis conditions are as follows: detection in positive ion mode, ion source is ESI, backflushing nitrogen temperature is 325℃, backflushing nitrogen flow rate is 11.0 L / min, nebulizer gas flow rate is 45 psig, auxiliary nitrogen temperature is 350℃, auxiliary nitrogen flow rate is 11.0 L / min, capillary voltage is 3500 V, source collision voltage is 175 V, cone voltage is 65.0 V, scanning mode is full scan, and scanning range is m / z 60~1700.

[0009] Preferably, the method of processing Pinellia ternata to obtain the test solution includes: taking Pinellia ternata powder, adding 75% ethanol for ultrasonic extraction, and obtaining the test solution.

[0010] Preferably, the preparation of the reference solution includes: dissolving guanosine, phenylalanine, valine-glycine-threonine-asparagine-tyrosine (VGTNY), tryptophan, leucine-phenylalanine-serine-glycine (LFSG), apigenin-6-C-arabinose-8-C-galactoside, proline-tryptophan-valine-proline-glycine (PWVPG), asparagine-isoleucine-proline-phenylalanine (NIPF), and valine-isoleucine-tyrosine-glycine-proline-serine-valine-phenylalanine (VIYGPSVF) in a 50% methanol aqueous solution to obtain the reference solution.

[0011] Preferably, the gradient elution is performed according to the following procedure: 0–0.5 min, mobile phase A from 92% to 92%, mobile phase B from 8% to 8%; 0.5–3.0 min, mobile phase A from 92% to 90%, mobile phase B from 8% to 10%; 3.0–4.0 min, mobile phase A from 90% to 81%, mobile phase B from 10% to 19%; 4.0–5.0 min, mobile phase A from 81% to 77%, mobile phase B from 19% to 23%; 5.0–10 min, mobile phase A from 77% to 77%, mobile phase B from 23% to 23%.

[0012] Preferably, the fingerprint spectrum of the prepared Pinellia ternata herb has 14 common fingerprint peaks in positive ion mode; among them, peak 1 is guanosine, peak 2 is phenylalanine, peak 3 is VGTNY, peak 4 is tryptophan, peak 5 is LFSG, peak 6 is apigenin-6-C-arabinose-8-C-galactoside, peak 9 is PWVPG, peak 11 is NIPF, and peak 14 is VIYGPSVF; taking peak 11 as the S peak, the relative retention time ranges of peaks 1-10 and peaks 12-14 relative to the S peak are 0.154-0.157, 0.279-0.282, 0.451-0.456, 0.504-0.510, and 0.75, respectively. The relative retention times (RSDs) of peaks 1-10 and 12-14 relative to the S peak are 2-0.758, 0.800-0.805, 0.823-0.829, 0.853-0.857, 0.876-0.880, 0.908-0.912, 1.140-1.145, 1.291-1.298, and 1.355-1.363, respectively. Taking peak 11 as the S peak, the RSDs of the relative retention times of peaks 1-10 and 12-14 relative to the S peak are 0.46%, 0.31%, 0.25%, 0.31%, 0.15%, 0.20%, 0.19%, 0.15%, 0.13%, 0.13%, 0.14%, 0.19%, and 0.20%.

[0013] Preferably, the quality of the prepared Pinellia ternata is comprehensively evaluated using calcium oxalate needle crystals as an indicator of toxicity. This includes: converting calcium oxalate in the prepared Pinellia ternata into oxalic acid and obtaining a test solution; preparing a reference solution containing oxalic acid; and determining the content of calcium oxalate needle crystals using a standard curve determined by high-performance liquid chromatography (HPLC). The HPLC conditions are as follows: column: Phenomenex Luna Omega C18 (2.1 × 100 mm, 1.6 μm); mobile phase A: 0.5% phosphoric acid aqueous solution; mobile phase B: 0.1% formic acid dissolved in acetonitrile solution; elution method: 0–5 min, 95% A, 5% B; column temperature: 28℃; flow rate: 0.2 mL / min; detection wavelength: 210 nm.

[0014] Preferably, the quality of the prepared Pinellia ternata is comprehensively evaluated using lectin protein as an indicator of toxicity, including: preparing a test solution containing lectin protein; preparing a control solution containing bovine serum albumin; and determining the content of the lectin protein by analyzing the standard curve determined by an enzyme-linked immunosorbent assay (ELISA) reader and the results of polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE conditions are as follows: protein loading amount 8 μg, separating gel concentration 15% (w / v), pH 8.8; stacking gel concentration 5% (w / v), pH 6.8; electrode buffer is tris(hydroxymethyl)aminomethane-glycine (Tirs-Gly), pH 8.3; and staining method is silver staining.

[0015] Compared with existing technologies, this invention has the following advantages: The method provided by this invention can efficiently and comprehensively evaluate the quality of Pinellia ternata. The use of liquid chromatography-mass spectrometry (LC-MS) enables better separation of active components such as nucleosides, amino acids, flavonoids, and polypeptides, which is beneficial for constructing a fingerprint spectrum of Pinellia ternata. The content of two toxic components (calcium oxalate needle crystals and lectin protein) in Pinellia ternata was determined using high-performance liquid chromatography (HPLC) and polyacrylamide gel electrophoresis, respectively, which is helpful for establishing methods for determining the content limits of major toxic components in Pinellia ternata. These methods provide a reference for the quality evaluation and further development and utilization of Pinellia ternata. Attached Figure Description

[0016] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings:

[0017] Figure 1 Fingerprint chromatograms of 15 batches of Pinellia ternata medicinal materials provided according to the present invention are shown, wherein the sample numbers are shown in Table 1, and R is the control fingerprint chromatogram;

[0018] Figure 2 The fingerprint spectrum of Pinellia ternata provided according to the present invention shows 14 common peaks.

[0019] Figure 3 The reference standard chromatogram and the fingerprint chromatogram of Pinellia ternata sample provided according to the present invention are shown.

[0020] Figure 4 The HPLC chromatograms of oxalic acid reference standard and Pinellia ternata sample provided according to the present invention are shown.

[0021] Figure 5 The SDS-PAGE images of lectin protein and Maker protein in Pinellia ternata sample provided according to the present invention are shown. Detailed Implementation

[0022] The following examples are provided to help those skilled in the art better understand the present invention. It should be noted that these examples do not constitute a limitation on the scope of protection claimed by the present invention and are merely illustrative. Unless otherwise specified, the raw materials, reagents, or devices mentioned in the following examples are commercially available or obtained through known existing methods. The origin information of the Pinellia ternata medicinal material used in this invention is shown in Table 1.

[0023]

[0024]

[0025] This invention provides a method for evaluating the quality of Pinellia ternata medicinal material, comprising the following steps:

[0026] (1) Using liquid chromatography-mass spectrometry, a fingerprint chromatogram of Pinellia ternata was constructed with guanosine, phenylalanine, valine-glycine-threonine-asparagine-tyrosine (VGTNY), tryptophan, leucine-phenylalanine-serine-glycine (LFSG), apigenin-6-C-arabinose-8-C-galactoside, proline-tryptophan-valine-proline-glycine (PWVPG), asparagine-isoleucine-proline-phenylalanine (NIPF) and valine-isoleucine-tyrosine-glycine-proline-serine-valine-phenylalanine (VIYGPSVF) as reference standards.

[0027] (2) The chromatographic peaks in the fingerprint spectrum were identified and characterized by liquid chromatography-mass spectrometry.

[0028] (3) The content of toxic components (calcium oxalate needle crystals) in Pinellia ternata was determined by high performance liquid chromatography.

[0029] (4) The content of toxic components (lectin protein) in Pinellia ternata was determined by combining BCA protein quantification method and polyacrylamide gel electrophoresis method.

[0030] In step (1), guanosine, phenylalanine, VGTNY, tryptophan, LFSG, apigenin-6-C-arabinose-8-C-galactoside, PWVPG, NIPF and VIYGPSVF reference standards were dissolved in 50% methanol aqueous solution to prepare the fingerprint chromatogram reference solution of Pinellia ternata; 100mg of Pinellia ternata powder (passed through a No. 3 sieve) was accurately weighed (error range of ±0.05mg), 1mL of 75% ethanol was added, and the mixture was soaked for 60 minutes and then weighed. The mixture was ultrasonically extracted for 60 minutes (40kHz, 30℃), cooled and weighed again. The weight loss was made up with 75% ethanol, the mixture was shaken well, and the supernatant was transferred to obtain the LC-MS fingerprint chromatogram test solution.

[0031] In step (1), a predetermined amount of the fingerprint reference solution and the LC-MS fingerprint test solution of Pinellia ternata were injected into an LC-MS liquid chromatography-mass spectrometry instrument. Octadecylsilane-bonded silica gel was used as the packing material, and a gradient elution was performed using a mobile phase A containing 0.1% formic acid aqueous solution and a mobile phase B containing 0.1% formic acid acetonitrile solution at a flow rate of 0.3 mL / min to construct the LC-MS fingerprint of Pinellia ternata. The gradient elution was carried out according to the following procedure:

[0032] From 0 to 0.5 min, mobile phase A decreased from 92% to 92%, and mobile phase B decreased from 8% to 8%.

[0033] Over 0.5–3.0 min, mobile phase A decreased from 92% to 90%, and mobile phase B decreased from 8% to 10%.

[0034] 3.0–4.0 min, mobile phase A changes from 90% to 81%, mobile phase B changes from 10% to 19%;

[0035] 4.0–5.0 min, mobile phase A changed from 81% to 77%, and mobile phase B changed from 19% to 23%;

[0036] Over 5.0–10 min, mobile phase A decreased from 77% to 77%, and mobile phase B decreased from 23% to 23%.

[0037] The chromatographic column of the liquid chromatography-mass spectrometry (LC-MS) instrument is an ACQUITY UPLC HSS T3 column with a column size of 2.1 × 100 mm and 1.8 μm.

[0038] The high-resolution mass spectrometry detection conditions were as follows: detection in positive ion mode; ESI ion source; backflushing nitrogen temperature 325℃, backflushing nitrogen flow rate 11.0 L / min, nebulizer gas flow rate 45 psig, auxiliary nitrogen temperature 350℃, auxiliary nitrogen flow rate 11.0 L / min, capillary voltage 3500 V, source collision voltage 175 V, cone voltage 65.0 V; scanning mode: full scan; scanning range: m / z 60~1700.

[0039] In step (2), the LC-MS fingerprint spectrum detected a total of 14 common fingerprint peaks in positive ion mode; among them, peak 1 was identified as guanosine, peak 2 as phenylalanine, peak 3 as VGTNY, peak 4 as tryptophan, peak 5 as LFSG, peak 6 as apigenin-6-C-arabinose-8-C-galactoside, peak 9 as PWVPG, peak 11 as NIPF, and peak 14 as VIYGPSVF. With peak 11 as the S peak, the relative retention time ranges of peaks 1-10 and peaks 12-14 relative to the S peak are: 0.154-0.157, 0.279-0.282, 0.451-0.456, 0.504-0.510, 0.752-0.758, 0.800-0.805, 0.823-0.829, 0.853-0.857, 0.876-0.880, 0.908-0.912, 1.140-1.145, 1.291-1.298, and 1.355-1.363. With peak 11 as the S peak, the RSD values ​​of the relative retention times of peaks 1-10 and peaks 12-14 relative to the S peak are 0.46%, 0.31%, 0.25%, 0.31%, 0.15%, 0.20%, 0.19%, 0.15%, 0.13%, 0.13%, 0.14%, 0.19%, and 0.20%, respectively.

[0040] In step (3), 1.26 mg of oxalic acid reference standard was dissolved in 1 mL of water to prepare a 10 mM oxalic acid reference standard solution, which was then serially diluted to concentrations of 0.625 mM, 1.25 mM, 2.5 mM, 5 mM, and 10 mM. 0.3 g of Pinellia ternata powder was added to 3 mL of water and shaken well. The solution was sonicated for 5 minutes, placed in a 60°C water bath for 20 minutes, and centrifuged. The supernatant was discarded, and 0.5 mL of 20% hydrochloric acid solution was added to the residue. The solution was sonicated for 5 minutes, placed in a 70°C water bath for 30 minutes, centrifuged, and the supernatant was collected. 2 mL of 0.2 mol / L hydrochloric acid solution was added to the precipitate, and the process was repeated twice. The supernatants were combined and brought to a final volume of 10 mL to obtain the test solution for the determination of calcium oxalate needle crystal content.

[0041] In step (3), a predetermined amount of oxalic acid reference solution and the test solution for determining the content of calcium oxalate needle crystals were injected into a high-performance liquid chromatograph (HPLC). Octadecylsilane-bonded silica gel was used as the packing material. Mobile phase A consisted of 0.5% (v / v) phosphoric acid aqueous solution and mobile phase B consisted of 0.1% (v / v) formic acid acetonitrile solution. Elution was performed under the following chromatographic conditions: 0–5 min, 95% A, 5% B; column temperature: 28℃; flow rate: 0.2 mL / min; detection wavelength: 210 nm. The HPLC column was a Phenomenex Luna Omega C18 column. The column dimensions were 2.1 × 100 mm and 1.6 μm. The standard curve for oxalic acid was y = 909215x - 40156(r 2 =0.9997 (unweighted), indicating that the oxalic acid concentration has a good linear relationship with the peak area in the range of 0.625 to 10 mM.

[0042] In step (4), 0.5 mg of bovine serum albumin (BSA) was dissolved in 1 mL of protein diluent and thoroughly mixed to prepare a 500 μg / mL protein standard solution. This solution was then serially diluted in a 96-well plate to obtain standard protein solutions with concentrations of 0, 25, 50, 100, 200, 300, 400, and 500 μg / mL. A standard curve was plotted with the standard protein concentration on the x-axis and absorbance on the y-axis. The regression equation y = 0.8828x + 0.0877 (r² = 0.9983, unweighted) was calculated, indicating a good linear relationship between protein concentration and absorbance in the range of 0–500 μg / mL.

[0043] In step (4), the protein loading amount for polyacrylamide gel electrophoresis was 8 μg, the separating gel concentration was 15% (w / v) and pH 8.8, the stacking gel concentration was 5% (w / v) and pH 6.8, the electrode buffer was tris(hydroxymethyl)aminomethane-glycine (Tirs-Gly) and pH 8.3, and the staining method was silver staining.

[0044] The following embodiments are merely examples to clearly illustrate the present invention and are not intended to limit the implementation of the invention. Those skilled in the art can make other variations or modifications based on the following description, and these variations, modifications, substitutions, and alterations arising from the principles and spirit of the present invention still fall within the protection scope of the present invention.

[0045] Example 1

[0046] Construction method of LC-MS fingerprint spectrum of Pinellia ternata

[0047] 1. Selection of liquid chromatography conditions

[0048] An ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.8 μm) was used as the elution column. Mobile phase A consisted of an aqueous solution containing 0.1% formic acid (v / v) and mobile phase B consisted of an acetonitrile solution containing 0.1% formic acid (v / v). The flow rate was 0.3 mL / min, and the column temperature was 35 °C. Gradient elution was performed according to the following procedure:

[0049] From 0 to 0.5 min, mobile phase A decreased from 92% to 92%, and mobile phase B decreased from 8% to 8%.

[0050] Over 0.5–3.0 min, mobile phase A decreased from 92% to 90%, and mobile phase B decreased from 8% to 10%.

[0051] 3.0–4.0 min, mobile phase A changes from 90% to 81%, mobile phase B changes from 10% to 19%;

[0052] 4.0–5.0 min, mobile phase A changed from 81% to 77%, and mobile phase B changed from 19% to 23%;

[0053] Over 5.0–10 min, mobile phase A decreased from 77% to 77%, and mobile phase B decreased from 23% to 23%.

[0054] As an improvement to the above technical solution, the high-resolution mass spectrometry detection conditions are as follows: detection in positive ion mode; ion source is ESI; backflushing nitrogen temperature is 325℃, backflushing nitrogen flow rate is 11.0 L / min, nebulizing gas flow rate is 45 psig, auxiliary nitrogen temperature is 350℃, auxiliary nitrogen flow rate is 11.0 L / min, capillary voltage is 3500 V, source collision voltage is 175 V, and cone voltage is 65.0 V; scanning mode: full scan; scanning range: m / z 60~1700.

[0055] 2. Preparation of reference solution

[0056] Accurately weigh 1 mg each of guanosine, phenylalanine, tryptophan, VGTNY, LFSG, apigenin-6-C-arabinose-8-C-galactoside, PWVPG, NIPF and VIYGPSVF, add them to 50% methanol aqueous solution to prepare a 1 mg / mL reference stock solution, and then take an appropriate amount of the reference stock solution to mix and prepare a mixed reference solution with a concentration of 20 μg / mL.

[0057] 3. Preparation of the test solution

[0058] Accurately weigh 100 mg of Pinellia ternata powder (passed through a No. 3 sieve), add 0.5 mL of 75% ethanol, soak for 60 minutes, weigh, and then extract ultrasonically for 30 minutes (40 kHz, 30℃). After cooling, weigh again, make up the weight loss with 75% ethanol, shake well, centrifuge at 13500 rpm for 10 minutes, and collect the supernatant. Add 0.5 mL of 75% ethanol to the precipitate again, and repeat the above operation. Combine the two supernatants to obtain the LC-MS fingerprint test solution.

[0059] 4. Fingerprint similarity evaluation

[0060] Fifteen batches of LC-MS fingerprint chromatograms in TXT format were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 version) software. The similarity of each fingerprint chromatogram was calculated, and the results are shown below. Figure 1 And Table 2.

[0061] The results showed that, using the NIPF chromatographic peak (peak 11) as the reference peak S peak, a reference chromatogram was generated by the average method, and a reference fingerprint chromatogram of Pinellia ternata was established. The superimposed fingerprint chromatogram of Pinellia ternata was obtained, and the similarity was calculated. The similarity of the fingerprint chromatograms of 15 Pinellia ternata samples was between 0.909 and 0.985, indicating that the fingerprint chromatograms of Pinellia ternata from different producing areas are relatively stable.

[0062] Table 2: Similarity of LC-MS fingerprint spectra of 15 Pinellia ternata medicinal materials

[0063]

[0064] 5. Identification of chemical composition from LC-MS fingerprints

[0065] By comparing the retention times, precise molecular weights, and fragmentation patterns of the chromatographic peaks of compounds in the reference solution and the test solution, a total of 9 compounds corresponding to chromatographic peaks were identified, namely guanosine, phenylalanine, tryptophan, VGTNY, LFSG, apigenin-6-C-arabinose-8-C-galactoside, PWVPG, NIPF, and VIYGPSVF. Detailed identification results of the 14 common peaks are available in [link to documentation]. Figure 2 , Figure 3 And Table 3.

[0066] Table 3: Identification of common peaks in the LC-MS fingerprint of Pinellia ternata

[0067]

[0068]

[0069] Example 2

[0070] Method for determining the content limit of calcium oxalate needle crystals in Pinellia ternata.

[0071] 1. Experimental Methods

[0072] 1.1 Chromatographic conditions

[0073] The chromatographic column was a Phenomenex Luna Omega C18 (2.1 × 100 mm, 1.6 μm); mobile phase A: 0.5% aqueous phosphoric acid solution; mobile phase B: 0.1% formic acid dissolved in acetonitrile solution; elution method: 0–5 min, 95% A, 5% B; column temperature: 28℃; flow rate: 0.2 mL / min; detection wavelength: 210 nm.

[0074] 1.2 Preparation of the test solution

[0075] Accurately weigh 0.3g of Pinellia ternata powder, add 3mL of water and shake well. Sonicate for 5 minutes, then place in a 60℃ water bath for 20 minutes and centrifuge. Discard the supernatant. Add 0.5mL of 20% hydrochloric acid solution to the residue, sonicate for 5 minutes, place in a 70℃ water bath for 30 minutes, centrifuge, and collect the supernatant. Add 2mL of 0.2mol / L hydrochloric acid solution to the precipitate and repeat the above treatment twice. Combine the supernatants and bring the volume to 10mL to obtain the final product.

[0076] 1.3 Preparation of reference solution

[0077] Accurately weigh 1.26 mg of oxalic acid reference standard, dissolve it in 1 mL of water to prepare a 10 mM oxalic acid reference standard solution.

[0078] 1.4 Examination of Linear Relationships

[0079] The reference solution under section 1.3 was serially diluted, and an appropriate amount of 10 mM oxalic acid reference solution was accurately pipetted to prepare oxalic acid reference solutions with concentrations of 0.625 mM, 1.25 mM, 2.5 mM, 5 mM, and 10 mM. The determination was performed under the chromatographic conditions under section 1.1. A standard curve was plotted with the reference concentration as the abscissa and the peak area as the ordinate. The regression equation was calculated as y = 909215x - 40156(r 2 =0.9997 (unweighted), indicating that the oxalic acid concentration has a good linear relationship with the peak area in the range of 0.625 to 10 mM.

[0080] 2. Sample content determination

[0081] Prepare the test solution according to the method in section 1.2, inject 5 μL of each sample, and determine the peak area of ​​oxalic acid. Calculate the oxalic acid content using the regression equation in section 1.4 and convert it to calcium oxalate content. See the detailed test results below. Figure 4 And Table 4.

[0082] Table 4: Results of Calcium Oxalate Needle Crystal Content Determination in Various Samples of Pinellia ternata

[0083]

[0084] The above results indicate that the calcium oxalate needle crystal content of commercially available Pinellia ternata ranges from 0.49% to 0.63%, with an average content of 0.58%. After alum treatment, the calcium oxalate content of Pinellia ternata of different sizes decreased from 1.33±0.11% to 0.46%–0.56%, and then remained essentially unchanged. (Liang Jun) [1] The content of calcium oxalate needle crystals in three batches of Pinellia ternata determined by HPLC ranged from 0.75% to 0.79%, with an average content of 0.77%; Dong Quanxing [2] The content of calcium oxalate needle crystals in Pinellia ternata prepared using three batches of traditional processing methods and three batches of pharmacopoeia processing methods, determined by HPLC, ranged from 0.441% to 0.720% and 0.571% to 0.592%, respectively, with average contents of 0.625% and 0.581%. Based on these results, it is recommended that the content limit for calcium oxalate needle crystals in Pinellia ternata should not exceed 0.75%.

[0085] Example 3:

[0086] Methods for determining the content limits of lectin proteins in Pinellia ternata.

[0087] 1. Experimental Methods

[0088] 1.1 Preparation of the test solution

[0089] Accurately weigh 0.5g of Pinellia ternata powder, add 1mL of 0.05M tris(hydroxymethyl)aminomethane hydrochloride (Tirs-HCl) buffer containing 0.1M sodium chloride (pH 7.8), homogenize for 2 minutes, centrifuge at 6000rpm for 30 minutes, collect the supernatant, and accurately measure the volume of the supernatant to calculate the amount of saturated ammonium sulfate needed. Add saturated ammonium sulfate to 45% saturation, mix well, centrifuge at 8000rpm for 15 minutes, discard the supernatant, and obtain the crude protein extract. Dissolve in an appropriate amount of RIPA lysis buffer, lyse on ice for 3 minutes, centrifuge at 8000rpm for 15 minutes, and collect the supernatant.

[0090] 1.2 Preparation of reference solution

[0091] Take 0.5 mg of bovine serum albumin (BSA), dissolve it in 1 mL of protein diluent, mix thoroughly, and prepare a 500 μg / mL protein standard solution.

[0092] 1.3 Examination of Linear Relationships

[0093] Add 0, 1, 2, 4, 8, 12, 16, and 20 μl of the control solution from section 1.2 to 96-well plates, and then add diluent to bring the total volume to 20 μl. The protein standard concentrations are 0, 25, 50, 100, 200, 300, 400, and 500 μg / ml, respectively. Add 20 μl of the test solution from section 1.1 to each well of the 96-well plate, add 200 μl of BCA working solution, incubate at 37°C for 30 minutes, and measure the absorbance at 562 nm using a microplate reader. Plot a standard curve with standard protein concentration on the x-axis and absorbance on the y-axis, and calculate the regression equation y = 0.8828x + 0.0877(r). 2 =0.9983 (unweighted), indicating a good linear relationship between protein concentration and absorbance in the range of 0–500 μg / ml.

[0094] 1.4 Polyacrylamide gel electrophoresis (SDS-PAGE) analysis

[0095] Take an appropriate amount of sample solution and prepare it according to the method in section 1.1. Add the sample loading buffer, heat in a water bath at 100℃ for 5 minutes, and perform SDS-PAGE analysis: the protein loading amount is 8 μg, the separating gel concentration is 15% (w / v), pH 8.8; the stacking gel concentration is 5% (w / v), pH 6.8; the electrode buffer is tris(hydroxymethyl)aminomethane-glycine (Tirs-Gly), pH 8.3; the staining method is silver staining.

[0096] 2. Sample content determination

[0097] Gel images were recorded using an Amersham Image Quant 800 imaging system (Cytiva, Tokyo, Japan). The grayscale values ​​of the target bands were measured using ImageJ software (Fiji). The lectin protein content was calculated based on the total protein content determined by the BCA method. Results are shown in [Figure number missing]. Figure 5 And Table 5.

[0098] Table 5: Results of lectin protein content determination in various samples of Pinellia ternata

[0099]

[0100] The above results indicate that the lectin protein content of commercially available Pinellia ternata ranges from 0.03% to 0.07%, with an average content of 0.058%. The lectin protein content of Pinellia ternata prepared after alum treatment significantly decreased from 0.12%–0.36% to 0.02%–0.08%, and then remained essentially unchanged. Therefore, it is recommended that the lectin protein content in Pinellia ternata should not exceed 0.08%.

[0101] In summary, this invention provides a method for constructing quality standards for Pinellia ternata, which overcomes the previous problem of poor specificity in the fingerprint chromatograms of Pinellia ternata. It optimizes the liquid chromatography method to effectively separate characteristic components in Pinellia ternata, and based on this, constructs an LC-MS fingerprint chromatogram of Pinellia ternata. The similarity of 15 batches of Pinellia ternata samples ranges from 0.909 to 0.985, indicating that the fingerprint chromatograms of Pinellia ternata from different producing areas are relatively stable. For the main toxic components in Pinellia ternata, a method for determining content limits was established, and the main toxic components (calcium oxalate needle crystals and lectin protein) in Pinellia ternata according to the pharmacopoeia were quantified. This method improves the evaluation standards for Pinellia ternata and provides a reference for the overall and comprehensive quality control of Pinellia ternata.

[0102] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0103] [1] Liang Jun, Zhang Zhenling, Wang Hengjie, et al. Study on the relationship between the numbing and spicy toxicity of different processed Pinellia ternata products and the content of calcium oxalate needle crystals [J]. Chinese Journal of Traditional Medicine, 2016, 31(10):1563-1565. DOI:10.16368 / j.issn.1674-8999.2016.10.440.

[0104] [2] Dong Quanxing. The influence of pharmacopoeia processing and traditional processing methods on the quality of Pinellia ternata [J]. Chinese Journal of Traditional and Folk Medicine, 2021, 30(18):39-41,46. DOI:10.3969 / j.issn.1007-8517.2021.18.zgmzmjyyzz202118011.

Claims

1. A method for evaluating the quality of Pinellia ternata medicinal material, characterized in that, include: Take the powdered Pinellia ternata, add 75% ethanol and extract it by ultrasonic extraction to obtain the test solution; Prepare reference solutions containing guanosine, phenylalanine, valine-glycine-threonine-asparagine-tyrosine (VGTNY), tryptophan, leucine-phenylalanine-serine-glycine (LFSG), apigenin-6-C-arabinose-8-C-galactoside, proline-tryptophan-valine-proline-glycine (PWVPG), asparagine-isoleucine-proline-phenylalanine (NIPF), and valine-isoleucine-tyrosine-glycine-proline-serine-valine-phenylalanine (VIYGPSVF). Obtain the chromatograms of the test solution and the reference solution, respectively, including: The test solution and the reference solution were respectively analyzed by HPLC. The HPLC chromatographic conditions were as follows: using an ACQUITY UPLC HSS T3 column as the elution column, and using a gradient elution with a mobile phase A containing 0.1% (v / v) formic acid aqueous solution and a mobile phase B containing 0.1% (v / v) formic acid acetonitrile solution; the gradient elution was performed according to the following procedure: From 0 to 0.5 min, mobile phase A decreased from 92% to 92%, and mobile phase B decreased from 8% to 8%. Over 0.5–3.0 min, mobile phase A decreased from 92% to 90%, and mobile phase B decreased from 8% to 10%. Over 3.0–4.0 min, mobile phase A decreased from 90% to 81%, and mobile phase B decreased from 10% to 19%. Over 4.0–5.0 min, mobile phase A decreased from 81% to 77%, and mobile phase B decreased from 19% to 23%. 5.0~10 min, mobile phase A changes from 77% to 77%, mobile phase B changes from 23% to 23%; By comparing the spectra of the test solution and the reference solution, common peaks and similarity are determined; The quality of the prepared Pinellia ternata was comprehensively evaluated using calcium oxalate needle crystals and lectin protein as indicators of toxicity.

2. The evaluation method according to claim 1, characterized in that, The gradient elution was performed at a flow rate of 0.3 mL / min and a column temperature of 35 °C.

3. The evaluation method according to claim 1, characterized in that, Obtaining the chromatograms of the test solution and the reference solution respectively, further comprising: The test solution and the reference solution were analyzed by mass spectrometry. The mass spectrometry conditions were as follows: detection in positive ion mode, ESI ion source, backflushing nitrogen temperature 325℃, backflushing nitrogen flow rate 11.0 L / min, nebulizer gas flow rate 45 psig, auxiliary nitrogen temperature 350℃, auxiliary nitrogen flow rate 11.0 L / min, capillary voltage 3500 V, source collision voltage 175 V, cone voltage 65.0 V, full scan mode, and scan range of m / z 60~1700.

4. The evaluation method according to claim 1, characterized in that, Preparation of the reference solution includes: The reference solution was prepared by dissolving guanosine, phenylalanine, valine-glycine-threonine-asparagine-tyrosine (VGTNY), tryptophan, leucine-phenylalanine-serine-glycine (LFSG), apigenin-6-C-arabinose-8-C-galactoside, proline-tryptophan-valine-proline-glycine (PWVPG), asparagine-isoleucine-proline-phenylalanine (NIPF), and valine-isoleucine-tyrosine-glycine-proline-serine-valine-phenylalanine (VIYGPSVF) in a 50% methanol aqueous solution.

5. The evaluation method according to claim 1, characterized in that, The quality of the prepared Pinellia ternata was comprehensively evaluated using calcium oxalate needle crystals as an indicator of toxicity, including: The calcium oxalate in the prepared Pinellia ternata was converted into oxalic acid to obtain the test solution; Prepare a reference solution containing oxalic acid; The content of calcium oxalate needle crystals was determined by a standard curve obtained using high performance liquid chromatography (HPLC). The HPLC conditions were as follows: chromatographic column: Phenomenex Luna Omega C18; mobile phase A: 0.5% phosphoric acid aqueous solution; mobile phase B: 0.1% formic acid dissolved in acetonitrile solution; elution method: 0-5 min, 95% A, 5% B; column temperature: 28℃; flow rate: 0.2 mL / min; detection wavelength: 210 nm.

6. The evaluation method according to claim 1, characterized in that, The quality of the prepared Pinellia ternata was comprehensively evaluated using lectin proteins as an indicator of toxicity, including: Prepare a test solution containing lectin protein; Prepare a reference solution containing bovine serum albumin; The content of the lectin protein was determined by measuring the standard curve using an ELISA reader and analyzing the results of polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE conditions were as follows: protein loading amount 8 μg, separating gel concentration 15% w / v, pH 8.8; stacking gel concentration 5% w / v, pH 6.8; electrode buffer was tris(hydroxymethyl)aminomethane-glycine (Tirs-Gly), pH 8.3; and the staining method was silver staining.

7. The evaluation method according to any one of claims 1 to 6, characterized in that, The fingerprint spectrum of the prepared Pinellia ternata medicinal material has 14 common fingerprint peaks in positive ion mode; among them, peak 1 is guanosine, peak 2 is phenylalanine, peak 3 is VGTNY, peak 4 is tryptophan, peak 5 is LFSG, peak 6 is apigenin-6-C-arabinose-8-C-galactoside, peak 9 is PWVPG, peak 11 is NIPF, and peak 14 is VIYGPSVF. With peak 11 as the S peak, the relative retention time ranges of peaks 1-10 and peaks 12-14 relative to the S peak are: 0.154-0.157, 0.279-0.282, 0.451-0.456, 0.504-0.510, 0.752-0.758, 0.800-0.805, 0.823-0.829, 0.853-0.857, 0.876-0.880, 0.908-0.912, 1.140-1.145, 1.291-1.298, and 1.355-1.

363. With peak 11 as the S peak, the RSD values ​​of the relative retention times of peaks 1-10 and peaks 12-14 relative to the S peak are 0.46%, 0.31%, 0.25%, 0.31%, 0.15%, 0.20%, 0.19%, 0.15%, 0.13%, 0.13%, 0.14%, 0.19%, and 0.20%, respectively.

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