New quality control marker and application of detection method in quality evaluation of curcuma aromatica

Abstract

The invention discloses application of a novel quality control marker and a detection method in quality evaluation of curcuma aromatica, and relates to the technical field of traditional Chinese medicines. The quality control marker is curcuma zedoary bicyclo-ketene. The quality detection method comprises the following steps: adding curcuma zedoary bicyclo-ketene into methanol to prepare a reference solution; the method comprises the following steps: respectively carrying out extraction treatment on radix curcumae from different producing areas by using a methanol aqueous solution to obtain different extracting solutions, and then centrifuging to take supernatant to obtain different sample solutions; and respectively carrying out ultra-high performance liquid detection on the different test solutions, and calibrating characteristic peaks by using the reference solution. Based on a spectrum-activity relationship and a serum metabolism result, the curcuma zedoary bicyclo-ketene is finally screened as the curcuma zedoary quality control marker and is positively correlated with the quality, and meanwhile, quantitative analysis on the quality control marker curcuma zedoary bicyclo-ketene shows that the curcuma zedoary bicyclo-ketene contents from different producing areas are remarkably different, so that the curcuma zedoary bicyclo-ketene contents are remarkably different. And a basis can be provided for the quality evaluation research of curcuma aromatica in different regions.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicines, and in particular to application of a new quality control marker and a detection method in quality evaluation of turmeric. Background Art

[0002] Guiyujin is one of the most common Chinese medicines. As one of the sources of turmeric, it is the root of Guangxi Curcuma (Chinese medicine name), accounting for more than 60% of China's turmeric production.

[0003] Origin is one of the key factors affecting the quality of traditional Chinese medicine. The main origin of turmeric is China's Guangxi Zhuang Autonomous Region, also known as the authentic production area. In addition, a small amount of turmeric is grown in Yunnan. However, turmeric has a variety of problems in different sources, such as geographical environment, genetic diversity and germplasm degradation, and inconsistent processing methods in the origin. This may lead to certain differences in the active ingredients of turmeric from different origins, which in turn affects its quality.

[0004] Quality control markers (QCM) of traditional Chinese medicine have always been the focus of attention in the field of traditional Chinese medicine, and are also called quality markers (Q-Marker). A reasonable QCM should be based on multiple active ingredients and combined with biological activity to reveal the quality of traditional Chinese medicine. At present, the evaluation research of QCM of turmeric mainly focuses on chemical analysis, involving HPLC fingerprint and single or multiple active ingredient contents. Due to the complexity of traditional Chinese medicine containing multiple compounds and targets, changes in the content of active ingredients do not necessarily lead to changes in drug efficacy. Therefore, it is difficult to objectively evaluate the quality of traditional Chinese medicine by single chemical component analysis.

[0005] Spectrum-effect relationship is one of the classic research methods for quality control of traditional Chinese medicine. The overall characteristics of the fingerprint spectrum can evaluate the consistency and stability of quality. At the same time, the biological assay of traditional Chinese medicine is a supplement and improvement to the quality evaluation of chemical component analysis. Modern research shows that Guiyujin has a good anti-thrombotic effect, and its anti-platelet aggregation biological activity is closely related to thrombosis, which can be used as a biological activity indicator of quality. UPLC-Q-TOF-MS E The technology has the advantages of low-energy collision dissociation of analytes and accurate determination of molecular weight. It has become a powerful tool for identifying unknown compounds. At the same time, the serum metabolism of Curcuma aromatica was studied, and the molecular docking technology was used to predict the binding interaction between blood components and targets, providing a reference for the screening of QCM. At present, there is no report on the systematic study of QCM on Curcuma aromatica from different origins. Summary of the invention

[0006] The purpose of the present invention is to provide a new quality control marker and detection method for use in the quality evaluation of Curcuma aromatica to solve the problems existing in the above-mentioned prior art. Based on the spectrum-effect relationship and serum metabolism results, the present invention finally screens Curcuma bicycloenone as a quality control marker of Curcuma aromatica, and it is positively correlated with the quality. At the same time, the quantitative analysis of Curcuma bicycloenone shows that there are significant differences in the content of Curcuma bicycloenone in different origins, which can provide a basis for the quality evaluation research of Curcuma aromatica in different regions.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The invention provides a quality control marker of Curcuma aromatica, wherein the quality control marker is Curcuma bicycloenone.

[0009] The invention also provides application of a reagent for detecting the content of zedoary bicycloenone in turmeric in preparing a quality detection kit for turmeric.

[0010] The invention also provides application of a reagent for detecting the content of curcuma bicycloenone in turmeric in preparing a kit for identifying the origin of turmeric.

[0011] The invention also provides a quality detection kit for turmeric, comprising a reagent for detecting the content of curcuma bicycloenone in turmeric.

[0012] The invention also provides a kit for identifying the origin of Curcuma aromatica, comprising a reagent for detecting the content of Curcuma bicycloenone in Curcuma aromatica.

[0013] The present invention also provides a method for constructing a fingerprint spectrum of Curcuma aromatica, comprising the following steps:

[0014] Adding zedoaryl bicycloenone into methanol to prepare a reference solution;

[0015] After extracting the turmeric from different origins with methanol and water solution, different extracts were obtained, and then the supernatants were centrifuged to obtain different test sample solutions.

[0016] The different test sample solutions are respectively subjected to ultra-high performance liquid chromatography to obtain fingerprint spectra, and the characteristic peaks are calibrated using the reference sample solution.

[0017] Furthermore, the ultra-high performance liquid chromatography detection uses an Agilent Eclipse Plus C18 chromatographic column.

[0018] Furthermore, the mobile phase A used in the ultra-high performance liquid chromatography detection is acetonitrile, the mobile phase B is methanol, and the mobile phase C is 0.5% formic acid.

[0019] The present invention also provides an application of a Curcuma aromatica fingerprint spectrum constructed according to the above-mentioned construction method in identifying the origin of Curcuma aromatica.

[0020] The present invention also provides a method for identifying the origin of Curcuma aromatica, comprising the step of comparing the characteristic peaks of Curcuma bicycloenone of the Curcuma aromatica to be tested with the Curcuma aromatica fingerprint spectrum constructed according to the above-mentioned construction method to determine the origin of the Curcuma aromatica to be tested.

[0021] The present invention discloses the following technical effects:

[0022] The present invention adopts UPLC-Q-TOF-MS E Identification of Curcuma aromatica and its serum metabolites. Eight quality control related components were screened out based on the spectrum-effect relationship combined with orthogonal partial least squares method. UPLC-Q-TOF-MS E A total of 57 components of Curcuma aromatica were identified, and 10 prototype components and 11 metabolites were identified in serum metabolism. Based on the spectrum-effect relationship and serum metabolism results, curcuma bicycloenone was finally selected as a quality control marker (QCM), and it was positively correlated with the quality. The results of the antiplatelet aggregation biopotency (AAB) experiment showed that curcuma bicycloenone had significant antiplatelet aggregation activity. The results of the molecular docking experiment showed that curcuma bicycloenone had the strongest binding effect with GP-IIb / IIIa receptors, followed by P2Y12 receptors, and P2Y1 receptors were the weakest. At the same time, the quantitative analysis of curcuma bicycloenone showed that there were significant differences in the content of curcuma bicycloenone in different origins. It can provide a basis for the quality evaluation of Curcuma aromatica in different regions. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 are chromatograms of samples and standards; wherein A is the sample chromatogram; B is the chromatogram of 10 mixed standards; 3. gingerol ketone, 4. isoprotocurenol, 5. protocurenol, 8. curcuma bicycloenone, 10. curcumenol, 15. nootkatone, 16. curcuma ketone, 17. isoprotocurenol, 18. germacron, 19. curcumol;

[0025] Figure 2 The UPLC fingerprints of 30 samples of Curcuma aromatica from different origins, where R is the reference spectrum;

[0026] Figure 3 This is the dendrogram of HCA of Curcuma aromatica from different origins;

[0027] Figure 4 It is the OPLS arrangement diagram;

[0028] Figure 5 It is the OPLS observation and prediction diagram;

[0029] Figure 6 is the scatter plot of OPLS scores;

[0030] Figure 7 is the OPLS coefficient diagram;

[0031] Figure 8 is the OPLS variable importance diagram;

[0032] Fig. 9 It is a high-resolution total ion chromatogram in positive ion mode;

[0033] Fig.10 It is a high-resolution total ion chromatogram in negative ion mode;

[0034] Fig.11 Schematic diagram of the main fragmentation pathway of curcuminol in positive ion mode;

[0035] Fig.12 Schematic diagram of the main fragmentation pathway of Curcuma bicycloenone in positive ion mode;

[0036] Fig.13 This is a schematic diagram of the serum metabolic pathway of Curcuma aromatica;

[0037] Fig.14 It is a schematic diagram of the transformation relationship of the chemical components of Curcuma aromatica;

[0038] Fig.15 The results of docking the three main receptors with the Curcuma bicycloenone molecule; A is the 3D image (left) and docking detail schematic diagram (right) of the docking of GP-IIb / IIIa with the Curcuma bicycloenone molecule; B is the 3D image (left) and docking detail schematic diagram (right) of the docking of P2Y1 with the Curcuma bicycloenone molecule; C is the 3D image (left) and docking detail schematic diagram (right) of the docking of P2Y12 with the Curcuma bicycloenone molecule. DETAILED DESCRIPTION

[0039] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0040] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0041] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0042] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0043] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0044] Example 1

[0045] 1. Materials and Methods

[0046] 1.1 Reagents and chemicals

[0047] Standard substances such as gingerolone, curcumone, isocurcumenol, curcumenol, curcumabicycloenone, nootkatone, protocurcumenone, isoprotocurcumenone, germacone, and curcumol (HPLC purity ≥ 98%) were obtained from Shanghai Yuanye Biotechnology Co., Ltd. Methanol, acetonitrile, and formic acid (HPLC grade), sodium tricitrate dihydrate (C6H5Na3O7·2H2O) (analytical grade), sodium adenosine-5-diphosphate (C6H5Na3O7·2H2O) (analytical grade), and 1,2-dihydro- ... 10 H 13 N5Na2O 10 P2, ADP), aspirin and dimethyl sulfoxide (DMSO). All aqueous solutions were prepared from The water was prepared by an integrated water purification system (18.2 MΩ, Merck, Germany). 0.9% sodium chloride injection (W / V) was purchased from Sichuan Kelong Pharmaceutical Co., Ltd.

[0048] 1.2 Instruments

[0049] KQ-500VDE dual-frequency digital ultrasonic cleaner was purchased from Kunshan Ultrasonic Instrument Co., Ltd. BSA224S precision electronic balance was purchased from Beijing Sartorius Scientific Instrument Co., Ltd. 5424R high-speed centrifuge was purchased from Eppendorf, Germany; Hei-VAP Core HL G3 rotary evaporator was purchased from Heidolph Instruments GmbH & CO.KG (Heidolph, Germany). The analysis was performed using the WatersAcquity UHPLC system of Waters, USA, which consists of a diode array detector, an online degasser, a four-pump solvent management system, and an autosampler. The chromatographic column was Agilent Eclipse PlusC18 (5μm, 4.6mm×250mm). Mass spectrometry detection was performed using a Xevo G2-XS quadrupole time-of-flight mass spectrometer (QTOF) (Waters, Manchester, UK) equipped with an electrospray ionization source (ESI). ESI-MS spectra were obtained using multiple reaction monitoring (MRM). SC-2000 platelet aggregation instrument was purchased from Beijing Houji Technology Development Co., Ltd.

[0050] 1.3 Sample collection

[0051] Thirty samples of Curcuma aromatica were collected from Yulin City, Guangxi Zhuang Autonomous Region (GX-YL), Qinzhou City, Guangxi Zhuang Autonomous Region (GX-QZ), Nanning City, Guangxi Zhuang Autonomous Region (GX-NN), Guigang City, Guangxi Zhuang Autonomous Region (GX-GG), Zhaotong City, Yunnan Province (YN-ZT), and Qujing City, Yunnan Province (YN-QJ). They were identified as authentic by Professor Luo Yi of the Guangxi Zhuang Autonomous Region Food and Drug Inspection Institute. Detailed information of all samples is shown in Table 1.

[0052] Table 1 Sample information and results of AAB and bicycloenone content in Curcuma zedoariae

[0053]

[0054]

[0055] 1.4 UPLC fingerprint and determination of bicyclic enone in Curcuma zedoaria

[0056] 1.4.1 Chromatographic conditions

[0057] Waters Acquity UPLC system, Agilent Eclipse Plus C18 chromatographic column (5μm, 4.6mm×250mm) was used. The flow rate was 0.4mL / min and the column temperature was 30℃. The injection volume was 3μL. Mobile phase A was acetonitrile, mobile phase B was methanol, and mobile phase C was 0.5% formic acid. Mobile phases A and B were mixed (A∶B=1∶1), gradient elution C: 0~5min, 15%~30% C; 5-6min, 30%-35% C; 6~12min, 35~39% C; 12~18min, 39%~58% C; 18~20min, 58%~65% C; the detection wavelength was 260nm. The above percentages are all volume fractions.

[0058] 1.4.2 Sample preparation

[0059] 1.0000 g of sample was taken and ultrasonically extracted with 10 mL of 80% methanol (efficiency 300 W, frequency 50 kHz, temperature 25° C.) for 45 min. The extract was centrifuged at 6000 rpm for 5 min and then filtered with a microfiltration membrane (0.22 μm mesh).

[0060] 1.4.3 Solution preparation

[0061] Standards were prepared in methanol and all stock solutions were stored at 4 °C.

[0062] 1.5 In vitro bioavailability assay

[0063] 1.5.1 Preparation of sample solution for AAB analysis

[0064] Take 1.0000g sample, extract by ultrasonic extraction (efficiency 300W, frequency 50kHz, temperature 25℃), extract with 10mL 80% methanol for 45min, and add weight loss after cooling. Filter the extract and then concentrate under reduced pressure. Dissolve the concentrate in a 5mL volumetric bottle with 1.5mL DMSO solution and physiological saline. Centrifuge the solution at 8000rpm for 10min, and then collect the supernatant as the preparation solution.

[0065] 1.5.2 Determination and calculation of AAB in samples

[0066] Japanese white rabbits, male, weighing about 2.5 kg, were provided by Zhuhai Besta Biotechnology Co., Ltd. (Zhuhai, China). Blood was collected from the carotid artery and placed in a centrifuge tube (3.2% trisodium citrate anticoagulant: blood = 1:9). The blood sample was centrifuged at 800 rpm for 10 min, and the upper layer was platelet-rich plasma (PRP). The remaining plasma was centrifuged at 3500 rpm for 10 min, and the upper layer was platelet-poor plasma (PPP).

[0067] 10 μL of saline and 280 μL of PPP were placed in 5 turbidity tubes. Then 10 μL of saline, 10 μL of aspirin (high concentration, 0.5 mg / mL; low concentration, 0.25 mg / mL), and 10 μL of cinnamon curcuma solution (high concentration, 20 mg / mL; low concentration, 10 mg / mL) were given as blank groups. In addition, 280 μL of PRP was placed in another 5 turbidity tubes. The saline, positive drugs, and specimens were the same as the blank group and set as the sample group.

[0068] The platelet aggregation device was incubated to 37°C, and then the instrument was zeroed using PPP. PRP was then incubated for 60 seconds, 10 μL ADP solution was added, and the maximum platelet aggregation rate was determined. Platelet inhibition rate = (maximum aggregation rate of the saline group - maximum aggregation rate of the positive drug or sample group) / maximum aggregation rate of the saline group × 100%. Each sample was measured 4 times in parallel.

[0069] AAB was calculated using BS2000 biostatistics software. Parallel line detection that required a quantitative response method was selected, and a randomized design experiment was set up with a ratio of two adjacent doses of 0.5. The estimated biopotency of the sample was 1000U / g (Chinese Pharmacopoeia 2020 edition).

[0070] 1.6 UPLC-Q-TOF-MS E Analysis of serum metabolism of Curcuma aromatica

[0071] 1.6.1 UPLC-Q-TOF-MS E condition

[0072] The chromatographic conditions and sample preparation were the same as those for fingerprint analysis. Electrospray ionization (ESI) was used in positive ion mode (ESI + ) and negative ion mode (ESI - ) was used for mass spectrometry analysis, with leucine enkeprin standard solution as the mass lock solvent. High-purity N2 was used as the auxiliary spray ionization and desolvation gas. The scanning mode detection was MSE. The mass spectrometry analysis conditions were set as follows: capillary voltage 2.5 kV, cone voltage 25 V, source temperature 120 ° C; desolvation temperature, 400 ° C; cone gas flow, 50 L / h; desolvation gas flow, 1000 L / h. Nebulizer gas flow, 600 L / h. Low energy channel collision voltage 6 V; high energy channel collision voltage 20-40 V, mass number acquisition range 100-1200.

[0073] Data collection involved multiple metabolic transformations, including desaturation (-H2), reduction (+H2), oxidation (+O), glucuronidation (+C6H8O6), acetylation (+C2H2O), sulfation (+SO3), and dealkylation. The analysis identified two types of metabolic transformations: one-phase and two-phase processes. Mass Lynx software was used to process the data.

[0074] 1.6.2 Preparation of serum samples

[0075] Take 15.0g of cinnamon turmeric sample, extract it by ultrasonic extraction (efficiency 300W, frequency 50kHz, temperature 25℃), extract it with 150mL80% methanol for 45min, and add weight loss after cooling. Filter the extract, concentrate under reduced pressure, freeze-dry, dissolve in water, and prepare a 2.0g / mL solution for oral administration of mice of the present invention.

[0076] 25 healthy male SD mice, weighing (20±2) g, were raised at room temperature of 25°C, humidity of 50%, and 12 hours per day and night, with free access to food and water for 1 week. Rats were fasted (free access to water) for 12 hours before the experiment, and randomly divided into 6 groups and weighed, with 5 mice in each group. One group was the blank group, and the rest were experimental groups. The mice were gavaged at a dose of 5 mL / kg, the blank group was gavaged with distilled water, and the experimental group was gavaged with cinnamon curcuma solution. The collected blood sample tubes were pre-coated with sodium heparin. Eye blood samples were collected from the experimental group and the blank group at 0.5, 1, 2, and 3 hours after administration, respectively. Centrifuge at 3500 rpm for 10 minutes and collect the supernatant. The supernatants of blood samples collected at different time points in the experimental group were mixed and stored in a -80°C refrigerator for later use.

[0077] 1.7 Molecular docking of Curcuma bicycloenone

[0078] Molecular docking analysis of the correlation between curcuma bicycloenone and key targets in Curcuma aromatica. First, the three-dimensional structure file of the protein target was obtained from the PDB database (https: / / www.rcsb.org / ); the SDF structure file of curcuma bicycloenone was obtained from the PubChem database, and then converted to a PDB format file through OpenBabel. AutoDockTools1.5.6 software was used to perform a series of operations (hydrogenation, water addition, setting appropriate active pockets, defining docking simulation parameters, etc.) between the protein target and curcuma bicycloenone. Finally, Discovery Studio visualization software (2019 version) was used to further process the molecular docking results.

[0079] 1.8 Statistical analysis

[0080] SPSS 26.0 (Palo Alto, CA, USA) was used for one-way ANOVA Student-Newman-Keuls (SNK). SIMCA-P 14.0 (Umetrics AB, Umea, Sweden) software was used for cluster analysis (HCA) and OPLS analysis. Modeling parameters (R2 and Q2 values) explained the accuracy of the fitted model.

[0081] 2. Results and Discussion

[0082] 2.1 Comparison of UPLC fingerprints of Curcuma aromatica from different origins

[0083] 2.1.1 Establishment and methodological investigation of UPLC fingerprint of Curcuma aromatica

[0084] A total of 21 chromatographic peaks with good separation effects were screened out as common peaks, of which 10 peaks were identified, including 3 peaks of gingerol ketone, 4 peaks of isoprotocurenol, 5 peaks of protocurenol, 8 peaks of curcuma bicycloenone, 10 peaks of curcuma alcohol, 15 peaks of nokatone, 16 peaks of curcuma ketone, 17 peaks of isoprotocurenol, 18 peaks of germacrone, and 19 peaks of curcuma alcohol. Taking germacrone as the reference peak (S), the RSD of the relative retention time and relative peak area of ​​each common peak was calculated. The RSD of precision, repeatability and stability (24h) were all <3%, indicating that the method was accurate.

[0085] 2.1.2 Similarity evaluation of fingerprint spectra of Curcuma aromatica from different origins

[0086] The common fingerprint of Curcuma aromatica was used as a reference, and the similarity was calculated using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012A version). Figure 2 . The average similarity of UPLC fingerprints was: GX-NN (0.979) > GX-YL (0.978) > GX-QZ (0.973) > GX-GG (0.959) > YN-ST (0.591) > YN-QJ (0.581), GX (0.972) > YN (0.587). The differences in the results were further analyzed by SNK analysis of One-way ANOVA (Table 2). There was a significant difference between Guangxi Zhuang Autonomous Region (GX) and Yunnan Province (YN) (P < 0.01), and there was no significant difference between different production areas in Guangxi Zhuang Autonomous Region (P > 0.01). The results are shown in Table 2.

[0087] Table 2 Results of UPLC fingerprint similarity SNK

[0088]

[0089] 2.2 Comparison of AAB of Curcuma aromatica from different origins

[0090] The average values ​​of AAB were: GX-YL (6.307U / g) > GX-QZ (6.222U / g) > GX-NN (5.943U / g) > GX-GG (4.460U / g) > YN-QJ (3.816U / g) > YN-ZT (3.662U / g), GX (5.745U / g) > YN (3.713U / g). The difference in the results was further analyzed by SNK of one-way ANOVA (Table 3). There was a significant difference between GX and YN. In GX, there was no statistically significant difference between GX-YL, GX-QZ and GX-NN. There was a significant difference between GX-GG and other production areas of GX. This shows that the quality of Guiyu Jin in GX is better than that in YN. The quality of Guiyu Jin in GX-YL, GX-QZ and GX-NN is better than that in GX-GG. The results are shown in Table 3.

[0091] Table 3 Results of SNK of AAB

[0092]

[0093] 2.3 Cluster analysis

[0094] HCA was used to compare the differences between the common peaks and AAB of Curcuma aromatica from different sources. A total of 30 samples were selected as observation samples, with the area of ​​21 common peaks as the independent variable and AAB as the dependent variable. In the dendrogram of HCA ( Figure 3 ), we can see that these samples are divided into two categories, reflecting the differences in related components and AAB between the two types of samples. GX origin is clustered into one category (category 1), and YN origin is clustered into another category (category 2), indicating that there are significant differences between GX and YN origins, but no significant differences between different origins of GX. Figure 3 .

[0095] 2.4 Screening of quality control-related components of Curcuma aromatica based on spectrum-effect relationship combined with OPLS

[0096] In order to further study the spectrum-effect relationship between UPLC fingerprint and AAB used for classification of quality-related components of Curcuma aromatica from different origins, the present invention performed OPLS. In OPLS, the first two principal components were selected. R2X(cum) was 0.487, R2Y(cum) was 0.690, and Q2(cum) was 0.587. The analysis results generated 5 parts, including permutation plot, observation and prediction plot, rating scatter plot, coefficient plot, and variable importance plot of item plot ( Figure 4-Figure 8 ). Displacement map ( Figure 4 ) shows that R2 is 0.178 and Q2 is -0.683. The values ​​on the left are lower than those on the right, indicating that the fitting model is accurate and predictive. Figure 5 ) and rating scatter plots ( Figure 6) It can be seen that the 30 samples are divided into two parts, of which GX-NN, GX-YL, GX-GG and GX-QZ are one class, and YN-QJ and YN-ZT are another class. This result is the same as HCA. From the coefficient diagram ( Figure 7 ) It can be seen that AAB is positively correlated with peaks 3, 4, 6, 8, 9, 10, 12, 13, 15, 16, 17, 19, and 21, and negatively correlated with other common peaks. From the project graph ( Figure 8 ) can be seen from the variable importance diagram that the variable importance is from high to low: Peak 11 (1.626) > Germacron (1.506) > Curcumol (1.393) > Curcumenol (1.282) > Curcuma bicycloenone (1.165) > Peak 9 (1.078) > Peak 20 (1.067) > Curcuma ketone (0.982) > Peak 13 (0.981) > Peak 21 (0.911)>isocurcumenol (0.848)>peak 14 (0.825)>peak 2 (0.822)>gingerolone (0.784)>peak 12 (0.773)>protocurcumenol (0.735)>peak 1 (0.727)>isoprotocurcumenol (0.698)>peak 7 (0.665)>peak 6 (0.664)>nokatone (0.599). The results showed that the correlation between the 21 common peaks and AAB was different. When the variable importance value was >1, peak 11, germacron, curcumol, curcumenol, curcuma bicycloenone, peak 9, and peak 20 were selected as relevant components for quality control. Curcumol, curcumenol, curcuma bicycloenone and peak 9 were positively correlated with the quality of Curcuma aromatica, while peak 11, germacrone and peak 20 were negatively correlated with the quality of Curcuma aromatica.

[0097] 2.5UPLC-Q-TOF-MS E Characterization of the components in Curcuma aromatica

[0098] For compound analysis, samples were analyzed using UPLC-Q-TOF-MS E The mass-to-charge ratio (m / z) and secondary fragment ions of the compounds were accurately obtained. Based on the mass spectrometry data and literature reports, the components could be further confirmed. High-resolution total ion chromatograms (positive and negative ion modes) were shown in Figure 2. Figure 9-10 As shown. Finally, the present invention successfully identified 57 components, as shown in Table 4, including 26 sesquiterpenes, 3 diphenylheptanes, 3 cardiac glycosides, 1 steroidal saponin, 7 phosphatidylcholines, 3 glycolipids, 2 diketones, 1 carbohydrate, 1 amino acid, 1 organic acid, and 9 fatty acids. Among them, phosphatidylcholines, cardiac glycosides, and glycolipids have not been reported in the literature before.

[0099] Table 4UPLC-Q-TOF-MS E Results of Identification of the Main Components of Curcuma aromatica

[0100]

[0101]

[0102]

[0103]

[0104] Note: #Verification of standard products; *Blood-injecting components.

[0105] Taking curcumenol as an example, in positive ion mode ( Fig.11 ), the quasi-molecular ion m / z 235.1697 can be observed, and the corresponding elemental composition is C 15 H 22 O2[M+H] + , indicating that the molecule is C 15 H 22 O2. At the same time, fragment ions such as m / z 217.1580, m / z 189.1651, m / z 175.1134, and m / z 161.0970 were observed. The ion of m / z 217.1580 is C 15 H 22 O2[M+H] + Remove one molecule of H2O to form [M+H-H2O] + The ion with m / z 189.1651 is [M+H-H2O] + , removing 1 CO molecule to form [M+H-H2O-CO] + The ion with m / z 175.1134 is [M+H-H2O] + , removing a C3H6 molecule, forming [M+H-H2O-C3H6] + The ion of m / z 161.0970 is [M+H-H2O-C3H6] + By removing one CH2 molecule, we get [M+H-H2O-C3H6-CH2] + Combining literature reports, fragment ion analysis and standard verification, it was speculated that M30 was curcuminol.

[0106] Taking Curcuma bicycloenone as an example, in positive ion mode ( Fig.12 ), the quasi-molecular ion m / z 235.1697 can be observed, and the corresponding elemental composition is C 15 H 22 O2[M+H] + , which indicates that the molecule is C 15 H 22O2. At the same time, fragment ions such as m / z 217.1580, m / z 189.165, m / z 177.1284, m / z 175.1134, m / z 161.0970, and m / z 159.1162 were observed. The fragment ion of m / z 217.1580 is C 15 H 22 O2[M+H] + Remove one molecule of H2O to form [M+H-H2O] + The fragment ion of m / z177.1284 is [M+H-H2O] + , removing a CO molecule to form [M+H-H2O-CO] + m / z175.1134[M+H] + The ion removes 1 C3H5O molecule, forming [M+H-C3H5O] + The ion of m / z 175.1134 is from [M+H-H2O-C3H5] + Remove one C3H5 molecule from [M+H-H2O-C3H5] + The fragment ion of m / z 161.0970 is [M+H-H2O-C3H5] + Removal of CH2 generates [M+H-H2O-C3H5-CH2]. The fragment ion of m / z 159.1162 is [M+H-H2O] + , remove 1 C3H5O molecule, forming [M+H-H2O-C3H5O] + Combining literature reports, fragment ion analysis and standard verification, it was speculated that M29 was a bicycloenone from Curcuma zedoaria.

[0107] Table 5UPLC-Q-TOF-MS E Analysis of serum metabolic components of Curcuma aromatica

[0108]

[0109]

[0110]

[0111] 2.6 UPLC-Q-TOF-MS E Analysis of serum metabolites of Curcuma aromatica

[0112] Ten prototype components were identified in the serum of mice given the extract of Curcuma aromatica (Table 4). These components include p-hydroxybenzoic acid, zedoalactone A / C / E, 2,3,5-trihydroxy-1-(4-hydroxyphenyl)-7-(3,5-dimethoxy-4-hydroxyphenyl) heptane, curcuma bicycloenone, linoleyl lysophosphatidylcholine, phosphatidylcholine lysophosphatidylcholine, oleoyl lysophosphatidylcholine isomer, oleoyl lysophosphatidylcholine, oleoyl lysophosphatidylcholine isomer. At the same time, 11 metabolites were detected (Table 5). These components include zedoalactone A / C / E isomers (compounds 1-5), isoprotocurenol, or protocurenol, or curcumenol, or curcuma bicycloenone, or isoprotocurenol, or curcuma bicycloenone isomer, or isoprotocurenol isomer, or curcuma alcohol isomer, or dealkylated hydrate of isoprotocurenol isomer (compounds 6-8), palmitamide (compound 9), hydroxystearamide (compound 10), acetylated hydroxystearamide (compound 11). High-resolution total ion chromatogram of serum (positive and negative ion modes). C 15 H 22 O2 (curcumenol, isocurcumenol, curcuma bicycloenone, etc.) can generate metabolites 1 to 5 (C 15 H 22 O4). Metabolites 6-8 (C 12 H 18 O4) may be caused by C 15 H 22 O2 (curcumenol, isocurcumenol, curcuma bicycloenone, etc.) are formed by dealkylation and a series of redox reactions. In addition, metabolites 6-8 (C 12 H 18 O4) may be metabolized by 1-5 (C 15 H 22 O4) is formed by dealkylation and a series of redox reactions. Fig.13 .

[0113] In addition, Guiyujin contains a large number of unstable components, and there are various transformation relationships between them, such as Fig.14 As shown. Curcumol can be synthesized from curcumone or neocurcumone. In addition, furanodiene can be converted into curcumene, and germacenone can be converted into β-elemene. In addition, furanodienone can be converted into curcumone. This suggests that the chemical composition of Curcuma aromatica is complex and metabolic transformation may occur in the body. Further research is needed in combination with pharmacokinetics.

[0114] 2.7 Verification of antiplatelet aggregation activity of Curcuma bicycloenone and molecular docking

[0115] According to the spectrum-effect relationship of relevant components and the results of serum metabolism into blood, curcuma bicycloenone was selected as QCM. In the AAB results of quality control markers, curcuma bicycloenone showed antiplatelet aggregation biological activity with an AAB of 0.387U / g. The results further verified the quality correlation between curcuma bicycloenone and cinnamon.

[0116] Modern studies have shown that GP-IIb / / IIIa, P2Y1, and P2Y12 are the main targets of ADP-induced platelet aggregation. The binding energy of the component with the target and the number of hydrogen bonds formed are key factors in evaluating the results of molecular docking. The lower the binding energy, the greater the number of hydrogen bonds, resulting in increased stability of the binding and a higher probability of interaction with the target molecule.

[0117] The results showed that the binding energy of curcuma bicycloenone to P2Y1 receptor was the strongest, followed by GP-IIb / IIIa receptor, and the weakest to P2Y12 receptor. Curcuma bicycloenone formed two hydrogen bonds with GP-IIb / IIIa receptor and P2Y12 receptor; however, it did not form a hydrogen bond with P2Y1 receptor. In the molecular docking diagram results ( Fig.15 ), Curcuma bicycloenone binds to three receptors at different sites. Among them, a stable complex is formed with the GP-IIb / IIIa receptor and the P2Y12 receptor, and at the same time, it shows good surface coordination with the P2Y1 receptor. In terms of hydrogen bonds, Curcuma bicycloenone forms stable hydrogen bonds with amino acids LEUA:421 and ARGA:422 on the GP-IIb / IIIa receptor and amino acids VALA:234 and ARGA:122 on the P2Y12 receptor. In addition to hydrogen bonds, there are also van der Waals forces, hydrophobic interactions, carbon-hydrogen bonds, etc. In summary, Curcuma bicycloenone has the highest affinity for the GP-IIb / IIIa receptor, followed by the P2Y12 receptor, while the affinity for the P2Y1 receptor is the weakest.

[0118] 2.8 Quantification and differential analysis of bicyclic enones in Curcuma zedoariae from different origins

[0119] 2.8.1 Methodological Study on Quantitative Analysis of Curcuma Bicycloketone by UPLC

[0120] The calibration curve of the analyte was determined by plotting the corresponding concentration against the peak area. The limit of detection (LOD) and limit of quantification (LOQ) of each item were determined under the conditions of signal-to-noise ratio (S / N) of 3 and 10. The precision was evaluated according to the intra-day and inter-day variations. Repeatability was evaluated for 6 samples of the same sample. The same sample solution was placed at room temperature for 0, 2, 4, 6, 8, 12, and 24 hours to evaluate its stability. Recovery experiments were carried out at three different concentration levels: low, medium, and high. The recovery rates of the sample and the standard were approximately 1:0.5 (low), 1:1 (medium), and 1:1.5 (high). The results showed that the correlation coefficient of the standard curve of Curcuma bicycloenone was >0.9999. The RSDs of intra-day precision, inter-day precision, repeatability, and stability were all <4%, and the recovery rate of the added sample was 98.90% to 100.09%. Detailed results are shown in Table 6.

[0121] Table 6 Methodological investigation of Curcuma bicycloenone

[0122]

[0123] 2.8.2 Comparison of Curcuma bicycloketone from different origins

[0124] The results of the determination of curcuma bicycloenone in Curcuma aromatica from different origins were: GX-YL 0.272~0.374mg / g, GX-QZ 0.263~0.400mg / g, GX-NN 0.211~0.336mg / g, GX-GG 10.038~10.829mg / g, YN-QJ 0.136~0.208mg / g, YN-ZT 0.145~0.230mg / g. The detailed results are shown in Table 1. The average results were: GX-GG (0.360 mg / g) > GX-QZ (0.333 mg / g) > GX-YL (0.313 mg / g) > GX-NN (0.273 mg / g) > YN-ZT (0.199 mg / g) > YN-QJ (0.172 mg / g). The differences in the results were further analyzed by SNK analysis of One-way ANOVA (Table 7). There was a significant difference between Guangxi Zhuang Autonomous Region and Yunnan Province (P<0.01), and there was no significant difference between different production areas in Guangxi Zhuang Autonomous Region (P>0.01).

[0125] Table 7 Results of SNK of bicyclic enones from Curcuma zedoariae from different origins

[0126]

[0127] There are certain differences in the content of curcuma bicycloenone in Curcuma acuminata from different origins, which may be caused by geographical factors, germplasm, harvesting, processing, cultivation and other reasons. There are certain differences in the geographical location of different origins of Curcuma acuminata, especially in Yunnan Province and Guangxi Zhuang Autonomous Region, which have certain differences in longitude, latitude and altitude, which may affect the accumulation of secondary metabolites. According to relevant literature research, the volatile oil content of Curcuma acuminata from different origins is different. Curcuma bicycloenone, as one of the volatile components of Curcuma acuminata, may be different. The genetic diversity of Curcuma acuminata germplasm from different origins is relatively rich, and there are large differences, resulting in rich phenotypic variation of Curcuma acuminata in different strains. Different strains of Curcuma acuminata not only have certain differences in medicinal properties, but also have certain differences in volatile oil content and component composition ratio. It shows that the difference in the content of curcuma bicycloenone may be caused by differences in germplasm. There are various processing methods in the origin of Curcuma acuminata, such as steaming and drying, normal pressure drying, and direct drying. Different processing methods may have different degrees of impact on curcuma bicycloenone. The harvest period of Curcuma Atractylodes is from December to March of the following year. Different harvest periods will affect the accumulation of secondary metabolites and may affect the content of curcuma bicycloenone. At present, most of Curcuma Atractylodes is cultivated products, and ethephon is one of the commonly used plant growth ripening agents in the cultivation process of Curcuma Atractylodes. Although it can increase the yield of Curcuma Atractylodes, it has a certain impact on the accumulation of secondary metabolites. It may have different degrees of impact on the accumulation of curcuma bicycloenone in plants. At present, the quality evaluation research of Curcuma Atractylodes is mainly based on indicators such as germacron, curcumol, and furandiene, and no quality research on curcuma bicycloenone has been found. In addition, the quality control indicators of Curcuma Atractylodes in the 2020 edition of the Chinese Pharmacopoeia are blank at this stage. It is recommended that the Chinese Pharmacopoeia consider using curcuma bicycloenone as an indicator for quality control of Curcuma Atractylodes.

[0128] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A quality control marker for Curcuma aromatica, characterized in that: The quality control marker is Curcuma bicycloenone.

2. Application of a reagent for detecting the content of Curcuma bicycloenone in Curcuma aromatica in the preparation of a quality detection kit for Curcuma aromatica.

3. Application of a reagent for detecting the content of Curcuma bicycloenone in Curcuma aromatica in the preparation of a kit for identifying the origin of Curcuma aromatica.

4. A quality detection kit for Curcuma aromatica, characterized in that: The invention comprises a reagent for detecting the content of curcuma bicycloenone in turmeric.

5. A kit for identifying the origin of Curcuma aromatica, characterized in that: The invention comprises a reagent for detecting the content of curcuma bicycloenone in turmeric.

6. A method for constructing a fingerprint of Curcuma aromatica, characterized in that: The following steps are involved: Adding zedoaryl bicycloenone into methanol to prepare a reference solution; After extracting the turmeric from different origins with methanol and water solution, different extracts were obtained, and then the supernatants were centrifuged to obtain different test sample solutions. The different test sample solutions are respectively subjected to ultra-high performance liquid chromatography to obtain fingerprint spectra, and the characteristic peaks are calibrated using the reference sample solution.

7. The construction method according to claim 6, characterized in that: The ultra-high performance liquid chromatography detection uses an Agilent Eclipse Plus C18 chromatographic column.

8. The construction method according to claim 6, characterized in that: The mobile phase A used in the ultra-high performance liquid chromatography detection is acetonitrile, the mobile phase B is methanol, and the mobile phase C is 0.5% formic acid.

9. An application of the Curcuma aromatica fingerprint obtained by the construction method according to any one of claims 6 to 8 in identifying the origin of Curcuma aromatica.

10. A method for identifying the origin of Curcuma aromatica, characterized in that: The method comprises the steps of comparing the characteristic peaks of curcuma bicycloenone of the turmeric herb with the turmeric herb to be tested by using the turmeric herb fingerprint obtained by the construction method according to any one of claims 6 to 8 to determine the origin of the turmeric herb to be tested.

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