Method for detecting traditional Chinese medicine-derived components in pinecone extract

Abstract

The invention discloses a method for detecting traditional Chinese medicine-derived components in a pinecone extract, which comprises the following steps: orally taking the pinecone extract to a healthy animal, collecting plasma and excrement samples before and after administration, detecting and analyzing by adopting UPLC-MS / MS (Ultra Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry), and finding 17 blood-entering components in the plasma sample after administration, including 15 prototype components and 2 metabolites; 12 components are found in an excrement sample after administration, 10 prototype components and 2 metabolites are included, the in-vivo metabolic process and excretion pathway of compounds with large polarity in the pinecones are supplemented, and the application has important significance for guiding clinical application and further research and development of the pinecones.

Description

Technical Field

[0001] The invention belongs to the field of drug detection, and particularly relates to a method for detecting drug-derived components in a pine cone extract. Background Art

[0002] Pine cones are cones of various pine trees in the Pinaceae family. They are brown in color and have multiple layers of scales. They have the effects of expectoration, cough relief, and asthma relief. They are often used to treat chronic bronchitis, asthma, cough with sputum, and other symptoms. In recent years, with the in-depth study of pine cones, it has been found that pine cone extracts also have multiple pharmacological activities such as anti-HIV activity, anti-Lassa virus activity, and anti-tumor activity. However, current research is mostly focused on the study of the chemical components contained in pine cones and the screening of active ingredients. The metabolic process of pine cone components in the body, the identification of metabolites, and other changes in chemical components that occur after entering the body are still unclear.

[0003] Blood component testing plays a vital role in drug development and clinical treatment. It is mainly used to evaluate the absorption, distribution, metabolism and excretion (ADME) characteristics of drugs, as well as the efficacy and safety of drugs. However, although blood component testing is very important, it also has some shortcomings and problems: (1) Blood testing can only provide information on the concentration of drugs in the blood, but cannot fully reflect the distribution of drugs in various tissues throughout the body; (2) The concentration of drugs in the body changes dynamically, and a single blood sample cannot accurately reflect the long-term exposure of drugs; (3) Different individuals have different abilities to absorb, metabolize and excrete drugs, and blood testing cannot fully explain these differences; (4) Blood testing cannot accurately reflect the effects of drug interactions on drug concentrations. Summary of the invention

[0004] In view of the deficiencies in the prior art, the object of the present invention is to provide a method for detecting the medicinal components in pine cone extracts, which can comprehensively reflect the metabolism and excretion of the pine cone extracts in the body.

[0005] The objective of the present invention is achieved through the following technical solutions: A method for detecting medicinal components in a pine cone extract comprises the following steps: orally administering the pine cone extract to healthy animals, collecting plasma and feces samples before and after administration, and performing UPLC-MS / MS detection and analysis, wherein 17 blood-entering components are found in the plasma samples after administration, including 15 prototype components and 2 metabolites; and 12 components are found in the feces samples after administration, including 10 prototype components and 2 metabolites.

[0006] The preparation method of the pine cone extract is as follows: extract the pine cone coarse powder with 85% ethanol, filter, extract the residue with hot water, and filter to remove the hot water extract. Under room temperature, add alkali solution to the residue to extract to obtain an alkaline extract. Adjust the pH value of the alkaline extract to 5 with acetic acid, concentrate, filter, and remove the precipitate. The filtrate is precipitated with 1 times the amount of ethanol, filtered, and precipitate D is obtained; the filtrate is precipitated with 2 times the amount of ethanol, filtered, and precipitate E is obtained; the filtrate is precipitated with 5 times the amount of ethanol for 24 hours, filtered, and precipitate F is obtained. Precipitation D, E, and F are combined to obtain the pine cone extract.

[0007] Preferably, the plasma and feces samples before and after administration are taken from the same animal to avoid the influence of individual differences on the test results.

[0008] Preferably, the method for preparing samples for pine cone extract detection comprises the following steps: vortexing the pine cone extract and internal standard solution I at a mass volume ratio of 1 mg:10 μL to 1 mg:20 μL, centrifuging, and filtering the supernatant with a microporous filter membrane for UPLC-MS / MS detection.

[0009] More preferably, the internal standard solution I is prepared by dissolving L-2-chlorophenylalanine in 70% methanol aqueous solution.

[0010] More preferably, the internal standard solution I needs to be precooled at -20°C before use.

[0011] Preferably, the method for preparing a plasma sample for detection comprises the following steps: taking a plasma sample, thawing it on ice, vortexing it, vortexing the plasma and internal standard solution II at a volume ratio of 1:4 to 1:8, and centrifuging it. Taking the supernatant and letting it stand at -20°C, centrifuging it again, and taking the supernatant for UPLC-MS / MS detection.

[0012] More preferably, the internal standard solution II is prepared by dissolving L-2-chlorophenylalanine in acetonitrile-methanol solution (the volume ratio of acetonitrile to methanol is 1:4).

[0013] Preferably, the method for preparing a stool sample for detection comprises the following steps: taking a stool sample, vacuum freeze-drying, and grinding it into a powder; taking the sample powder, adding an internal standard solution I at a mass volume ratio of 1 mg: 20 μL to 1 mg: 30 μL, intermittently and repeatedly vortexing (preferably, vortexing once every 30 minutes, each time lasting 30 seconds, and vortexing 6 times in total), centrifuging, and filtering the supernatant with a microporous filter membrane for UPLC-MS / MS analysis.

[0014] More preferably, the internal standard solution I needs to be precooled at -20°C before use.

[0015] Preferably, the chromatographic conditions for UPLC-MS / MS detection and analysis are as follows: The chromatographic column was Agilent SB-C18 1.8 µm, 2.1 mm × 100 mm; Mobile phase: Phase A is a mixed solution of ultrapure water and formic acid (the volume ratio of formic acid to ultrapure water is 0.1%), and phase B is a mixed solution of acetonitrile and formic acid (the volume ratio of formic acid to acetonitrile is 0.1%). Elution gradient: 0~9min, 5%~95%B; 9~10min, 95%B; 10~11min, 95%~5%B; 11~14min, 5%B.

[0016] Flow rate: 0.35 mL / min; column temperature: 40°C; injection volume: 2 μL.

[0017] The temperature of the electrospray ionization source (ESI) was 500°C; the ion spray voltage (IS) was 5500V (positive ion mode) / -4500V (negative ion mode); the ion source gas I (GSI), gas II (GSII) and curtain gas (CUR) were 50, 60 and 25 psi respectively, and the collision induced ionization parameter was set to high. The QQQ scan used the MRM mode, and the collision gas (nitrogen) was set to medium.

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention detects a part of the components with greater polarity in pine cones, which can systematically reflect the metabolism and excretion of these components in the body. At the same time, plasma and fecal samples after administration are collected, which can more comprehensively and accurately analyze their absorption, metabolism and excretion pathways.

[0019] 2. By using the technical solution described in the present invention, 17 blood-entering components were found in the plasma samples after administration, including 15 prototype components and 2 metabolites; 12 components were found in the fecal samples after administration, including 10 prototype components and 2 metabolites.

[0020] 3. Conventional plasma and feces samples are usually collected from different experimental animals, and a group of animals that are not given medication are set up as a blank control group. The present invention collects plasma and feces samples from the same animal, and uses the plasma and feces samples before medication as blank controls, thereby avoiding the influence of individual differences on the test results, making the test analysis results more accurate, saving the amount of drugs, and reducing the consumption of experimental animals.

[0021] 4. Compared with traditional high performance liquid chromatography and mass spectrometry (HPLC-MS), the present invention uses UPLC-MS / MS technology for detection and analysis, which significantly improves the repeatability and reliability of quantitative analysis and the accuracy of qualitative analysis, and better adapts to the needs of modern drug research for automated and high-throughput analysis methods. In the experiment, the sample detection can be completed in 16 minutes. This method can fully collect the chromatographic information of the detected sample, and the chromatographic peaks are well separated, laying the foundation for the subsequent analysis and processing of massive metabolic data.

[0022] 5. The present invention uses the pine cone extract itself as the basis for comparison, and compares it through mass spectrometry fragmentation and retention time. It is independent of standard products and has stronger applicability. Even if the pine cone extract contains unknown components, it can be compared with the data in the database. It overcomes the defect that the prior art must first consult literature to understand the possible substances contained, and then further compare with standard products. When encountering unknown components that have not been reported in the plant of this genus, it is impossible to detect them, which greatly limits the application.

[0023] 6. The present invention adds L-2-chlorophenylalanine as an internal standard, which can ensure the stability of the instrument during the detection process and ensure the accuracy of the detection results.

[0024] 7. The detection method of the present invention is applicable to pine cones of various pine trees, and has the advantages and characteristics of simple operation, easy control of experimental conditions, rapidity, accuracy and good reproducibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the total ion current chromatogram of the Yunnan pine cone extract under positive ion mode in Example 1.

[0026] Figure 2 This is the total ion current chromatogram of blank plasma in positive ion mode in Example 1.

[0027] Figure 3 This is the total ion current chromatogram of the administered plasma in positive ion mode in Example 1.

[0028] Figure 4 This is the total ion current chromatogram of blank feces in positive ion mode in Example 1.

[0029] Figure 5 This is the total ion current chromatogram of the feces administered in Example 1 under the positive ion mode.

[0030] Figure 6 This is the total ion current chromatogram of the Yunnan pine cone extract under negative ion mode in Example 1.

[0031] Figure 7 This is the total ion current chromatogram of blank plasma in negative ion mode in Example 1.

[0032] Figure 8 This is the total ion current chromatogram of the administered plasma in negative ion mode in Example 1.

[0033] Fig. 9 This is the total ion current chromatogram of blank feces in negative ion mode in Example 1.

[0034] Fig.10 This is the total ion current chromatogram of the feces administered in the negative ion mode in Example 1.

[0035] Fig.11 It is a Venn diagram of the Yunnan pine cone extract, blank plasma, administered plasma, blank feces, and administered feces in Example 1 (in the diagram: YN-DEF is the Yunnan pine cone extract, KBXJ is blank plasma, GYXJ is administered plasma, KBFB is blank feces, and GYFB is administered feces). DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited to the embodiments. Example 1

[0037] 1 Materials 1.1 Instrument 5424R centrifuge (Eppendorf); Biomek i5 automated workstation (Beckman Coulter); CentriVap centrifugal concentrator (LABCONCO); KQ5200E ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); MS105DM 1 / 100,000 electronic balance (Mettler Toledo Instrument Co., Ltd.); MU-G02-0448 constant temperature metal mixer (Hangzhou Miou Instrument Co., Ltd.); VORTEX-5 vortex mixer (Kyllin-Bell); AB SCIEX QTRAP4500 liquid chromatography-mass spectrometer (AB SCIEX, USA).

[0038] 1.2 Drug testing In this example, pine cones of Yunnan pine were selected as test samples. Pine cones of Yunnan pine were collected from Dali City, Dali Prefecture, Yunnan Province, and were identified as Yunnan pine (Pinus yunnanensis, a plant of the genus Pinus in the Pinaceae family) by Professor Duan Baozhong of the Pharmacognosy Teaching and Research Section of the School of Pharmacy of Dali University. Pinus Yunnanensis Franoh.) pine cones, sample specimens are stored in Room 706, Organic Pharmaceutical Chemistry Teaching and Research Section, School of Pharmacy, Dali University. Analytical grade sodium carboxymethyl cellulose (Tianjin Kemeiou Chemical Reagent Development Center), chromatographic grade methanol (Merck), chromatographic grade acetonitrile (Merck), chromatographic grade formic acid (Aladdin). L-2-chlorophenylalanine standard (99%, J&K).

[0039] 1.3 Animals Six SPF male SD rats, weighing 230-250 g, were purchased from Hunan Slake Jingda Company, license number: SCXK (Xiang) 2019-0004. The breeding conditions were: temperature 20±2℃, humidity 60%±5%, 12h light and dark cycle, and free diet.

[0040] 2. Sample preparation 2.1 Preparation of Yunnan pine cone extract 10kg of Yunnan pine cone powder was extracted twice with 85% ethanol, 4L of ethanol each time, and soaked for 24h. Filter, extract the residue with hot water, and filter to remove the hot water extract. At room temperature, add 6L of 1mol / L sodium hydroxide solution to the residue for extraction and filter to obtain an alkaline extract. The pH value of the alkaline extract was adjusted to 5 with acetic acid, concentrated, filtered, and the precipitate was removed. The filtrate was precipitated with 1 times the amount of ethanol for 24h, filtered, and precipitate D was obtained; the filtrate was precipitated with 2 times the amount of ethanol for 24h, filtered, and precipitate E was obtained; the filtrate was precipitated with 5 times the amount of ethanol for 24h, filtered, and precipitate F was obtained. Precipitation D, E, and F were combined to obtain Yunnan pine cone extract (YN-DEF).

[0041] 2.2 Collection of plasma and fecal samples SD rats were adaptively fed for 7 days. Blood was collected from the eye sockets 1 day before administration, centrifuged at 3500r / min for 10min, and the supernatant was collected as a blank control plasma sample (i.e., blank plasma) and stored in a -80°C refrigerator. 12h feces of rats were collected 1 day before administration as blank control fecal samples (i.e., blank feces), and the storage conditions were the same as above. Fasting for 12h before administration (free drinking water), Yunnan pine cone extract (pine cone extract dissolved in distilled water) was given by gavage at 200mg / kg, and the drug was administered continuously for 7 days. Feces 12h after administration on the 6th day were collected as fecal samples after administration (i.e., administration feces), and stored in a -80°C refrigerator. Blood was collected from the eye sockets 0.5h, 1h, 2h, and 4h after the last administration, centrifuged at 3500r / min for 10min, and the supernatant was collected. The plasma samples of the same rat at different time points were combined as plasma samples after administration (i.e., administration plasma), and stored in a -80°C refrigerator.

[0042] 2.3 Preparation of test samples 2.3.1 Preparation of Yunnan pine cone extract test samples The extract was vortexed for 1 min, and internal standard solution I precooled at -20°C was added. For every 50 mg of the extract, 600 μL of internal standard solution I was added (preparation method of internal standard solution I: 1 mg of L-2-chlorophenylalanine standard was dissolved in 1 mL of 70% methanol aqueous solution to prepare a 1000 μg / mL mother solution, and the 1000 μg / mL mother solution was further diluted with 70% methanol aqueous solution to prepare 250 μg / mL internal standard solution I), vortexed for 15 min, centrifuged (12000 r / min, 4°C) for 3 min, and the supernatant was filtered with a microporous filter membrane (0.22 μm) and placed in an injection bottle for UPLC-MS / MS detection.

[0043] 2.3.2 Preparation of plasma samples for testing Take out the plasma sample from the -80℃ refrigerator, thaw on ice, and vortex for 10s to mix. Take 50μL of sample, put it into a 1.5mL centrifuge tube, and add 300μL of internal standard solution II (preparation method of internal standard solution II: 1mg L-2-chlorophenylalanine standard is dissolved in 1mL acetonitrile-methanol solution (the volume ratio of acetonitrile to methanol is 1:4) to prepare 1000μg / mL mother solution, and 1000μg / mL mother solution is further diluted with the aforementioned acetonitrile-methanol solution to 250μg / mL internal standard solution II). Vortex for 3min, 12000r / min, 4℃, centrifuge for 10min. Take 200μL of supernatant, put it into another 1.5mL centrifuge tube, and let it stand in a -20℃ refrigerator for 30min. Centrifuge again at 12000r / min, 4℃ for 3min; take 180μL of supernatant and put it into a sample injection bottle for UPLC-MS / MS detection. The plasma before and after administration was processed by this method.

[0044] 2.3.3 Preparation of stool samples for testing Fecal samples were taken out from a -80℃ freezer and placed in a freeze dryer (Scientz-100F) for vacuum freeze drying for 63h; ground into powder using a grinder (MM 400, Retsch) (30Hz, 1.5min); 50mg of sample powder was weighed and 1200μL of internal standard solution I precooled at -20℃ was added; vortexed once every 30min, each time for 30s, for a total of 6 times; centrifuged at 12000r / min for 3min, the supernatant was aspirated, filtered with a microporous filter membrane (0.22μm), and placed in a sample injection bottle for UPLC-MS / MS analysis. The feces before and after administration were processed using this method.

[0045] 3. Detection Methods The Yunnan pine cone extract test samples, plasma sample test samples before and after administration, and fecal sample test samples before and after administration were tested by UPLC-MS / MS.

[0046] 3.1 UPLC detection conditions The chromatographic column was Agilent SB-C18 1.8µm, 2.1mm×100mm; the mobile phase was phase A, which was a mixed solution of ultrapure water and formic acid (the volume ratio of formic acid to ultrapure water was 0.1%), and phase B, which was a mixed solution of acetonitrile and formic acid (the volume ratio of formic acid to acetonitrile was 0.1%); the elution gradient was: 0-9min, 5%-95%B; 9-10min, 95%B; 10-11min, 95%-5%B; 11-14min, 5%B. The flow rate was 0.35mL / min; the column temperature was 40℃; and the injection volume was 2μL.

[0047] 3.2 Mass spectrometry conditions The temperature of the electrospray ionization source (ESI) was 500 °C; the ion spray voltage (IS) was 5500 V (positive ion mode) / -4500 V (negative ion mode); the ion source gas I (GSI), gas II (GSII) and curtain gas (CUR) were set to 50, 60 and 25 psi respectively, and the collision induced ionization parameter was set to high. The QQQ scan used the MRM mode, and the collision gas (nitrogen) was set to medium.

[0048] 3.3 The substances were qualitatively identified based on the secondary spectrum information, and quantitatively analyzed using multiple reaction monitoring of triple quadrupole mass spectrometry. The mass spectrometry data were processed using Analyst 1.6.3 software.

[0049] 4 Results Analysis UPLC-MS / MS test results are as follows Figure 1-Figure 11 As shown, Figure 1-5 They are the TIC graphs of Yunnan pine cone extract, blank plasma, drug-treated plasma, blank feces, and drug-treated feces in positive ion mode; Figure 6-10 They are the TIC graphs of Yunnan pine cone extract, blank plasma, drug-treated plasma, blank feces, and drug-treated feces in negative ion mode; Fig.11 It is a Venn diagram of Pinus yunnanensis pine cone extract, blank plasma, administered plasma, blank feces, and administered feces.

[0050] In the administered plasma samples, 17 blood-entering components were found, including 15 prototype components and 2 metabolites; in the administered fecal samples, 12 components were found, including 10 prototype components and 2 metabolites, as shown below (Table 1).

[0051] Table 1

[0052] 4.1 Prototype components of Yunnan pine cone extract in rat plasma Compound 1: t RAt 4.6 min, in the positive mode, the MS mass spectrum showed a quasi-molecular ion peak [M+H] + m / z 179.07C 10 H 10 O3, fragment ion [M+H] + m / z C7H6 91.06. Visible fragment ion peak [M+H-C7H6] + m / z 91.06 is due to the quasi-molecular ion peak [M+H] + m / z 179.07 lost 88Da (-C3H4O3), estimated t R The compound 1 with a reaction time of 4.6 min is Coniferaldehyde.

[0053] Compound 2: t R At 4.2 min, in the positive mode, the MS mass spectrum showed a quasi-molecular ion peak [M+H] + m / z 479.12C 22 H 22 O 12 , fragment ion [M+H] + m / z C 16 H 12 O7 317.06. Visible fragment ion peaks [M+HC 16 H 12 O7] + m / z 317.6 is due to the quasi-molecular ion peak [M+H] + m / z 479.12 Loss of 162Da (-C6H 10 O5), it is estimated that t R Compound 2 with a reaction time of 4.2 min is Isorhamnetin-7-O-glucoside (Brassicin).

[0054] Compound 3: t R At 1.9 min, in the positive mode, the MS mass spectrum showed a quasi-molecular ion peak [M+H] + m / z 268.1C 16 H 13 NO3 fragment ion [M+H] + m / z C8H7O2 136.06. Visible fragment ion peak [M+H-C8H7O2] + m / z 136.06 is due to the quasi-molecular ion peak [M+H] + m / z 268.1 lost 132Da (-C8H6NO), estimated t RCompound 3 with a reaction time of 1.9 min is 7-Methoxy-3-[1-(3-pyridyl)methylidene]-4-chromanone.

[0055] Compound 4: t R At 4.9 min, in the positive mode, the MS mass spectrum showed a quasi-molecular ion peak [M+H] + m / z 441.2C 24 H 28 N2O6, fragment ion [M+H] + m / z 177.05 C 10 H8O3. Visible fragment ion peak [M+HC 10 H8O3] + m / z is determined by the quasi-molecular ion peak [M+H + m / z 441.2 Loss of 264Da (-C 14 H 20 N2O3), it is estimated that t R Compound 4 with a reaction time of 4.9 min is N,N'-Diferuloylputrescine.

[0056] Compound 5: t R At 2.1 min, in the positive mode, the MS mass spectrum showed a quasi-molecular ion peak [M+H] + m / z 100.08C5H9NO, fragment ion [M+H] + m / z C4H7 56.05. Visible fragment ion peak [M+H-C4H7] + m / z 56.05 is due to the quasi-molecular ion peak [M+H] + m / z 100.08 lost 44Da (-CH2NO), estimated t R Compound 5 with a reaction time of 2.1 min is 2-Piperidone.

[0057] Compound 7: t R At 3.9 min, in negative mode, the MS spectrum showed a quasi-molecular ion peak [M+H] - m / z 477.06C 21 H 18 O 13 , fragment ion [M+H] - m / z 301.03 C 15 H 10 O7. Visible fragment ion peaks [M+HC 15 H 10 O7]- m / z is determined by the quasi-molecular ion peak [M+H - m / z 477.06 lost 176Da (-C6H8O6), estimated t R Compound 7 with a reaction time of 3.9 min is Tricetin-7-O-glucuronide.

[0058] Compound 8: t R At 4.5 min, in the positive mode, the MS mass spectrum showed a quasi-molecular ion peak [M+H] + m / z 197.12C 11 H 16 O3, fragment ion [M+H] + m / z C9H8O 133.1. Visible fragment ion peak [M+H-C9H8O] + m / z 133.1 is due to the quasi-molecular ion peak [M+H] + m / z 197.12 lost 64Da (-C2H8O2), estimated t R The compound 8 with a reaction time of 4.5 min is Loliolide.

[0059] Compound 10: t R At 8.7 min, in the positive mode, the MS mass spectrum showed a quasi-molecular ion peak [M+H] + m / z 315.19C 20 H 26 O3, fragment ion [M+H] + m / z 187.11 C 11 H 22 O2. Visible fragment ion peaks [M+HC 11 H 22 O2] + m / z is determined by the quasi-molecular ion peak [M+H + m / z 315.19 lost 128Da (-C9H4O), estimated t R Compound 10 with a bioactivity of 8.7 min is phyllanflexoid A.

[0060] Compound 11: t R At 2.3 min, in the positive mode, the MS mass spectrum showed a quasi-molecular ion peak [M+H] + m / z 208.06C 10 H9NO4, fragment ion [M+H] + m / z 162.06 C9H7NO2. Visible fragment ion peak [M+H-C9H7O2] +m / z is determined by the quasi-molecular ion peak [M+H + m / z 208.06 lost 46Da (-CH2O2), estimated t R Compound 11 with a reaction time of 2.3 min is 2-(2,3-dihydroxy-3H-indol-3-yl) acetic acid.

[0061] Compound 12: t R At 8.6 min, in the positive mode, the MS mass spectrum showed a quasi-molecular ion peak [M+H] + m / z 315.2C 20 H 26 O3, fragment ion [M+H] + m / z 187.12 C 13 H 14 O. Visible fragment ion peak [M+HC 13 H 14 O] + m / z 187.12 is due to the quasi-molecular ion peak [M+H] + m / z 315.2 Loss of 128Da (-C7H 12 O2), it is estimated that t R Compound 12 with a reaction time of 8.6 min is 7-Oxodehydroabietic acid.

[0062] Compound 13: t R At 9.9 min, in negative mode, the MS spectrum showed a quasi-molecular ion peak [M+H] - m / z 343.08C 18 H 16 O7, fragment ion [M+H] - m / z 328.06 C 17 H 13 O7. Visible fragment ion peaks [M+HC 17 H 13 O7] - m / z is determined by the quasi-molecular ion peak [M+H - m / z 343.08 lost 15Da (-CH3), estimated t R Compound 13 with a reaction time of 9.9 min is Usnic acid.

[0063] Compound 14: t R At 4.2 min, in negative mode, the MS mass spectrum showed a quasi-molecular ion peak [M+H] - m / z 447.09C21 H 20 O 11 , fragment ion [M+H] - m / z 285.04 C 14 H8O5. Visible fragment ion peak [M+HC 14 H8O5] - m / z is determined by the quasi-molecular ion peak [M+H - m / z 447.09 Loss of 192Da (-C7H 12 O6), it is estimated that t R Compound 14 with a reaction time of 4.2 min is Kaempferol-7-O-glucoside.

[0064] Compound 15: t R At 5 min, in negative mode, the MS spectrum showed a quasi-molecular ion peak [M+H] - m / z 181.05C9H 10 O4, fragment ion [M+H] - m / z 107.01 C7H8O. Visible fragment ion peak [M+H-C7H8O7] - m / z is determined by the quasi-molecular ion peak [M+H - m / z 181.05 lost 74Da (-C2H4O3), estimated t R Compound 15 with a reaction time of 5 min is Vanillic acidmethyl ester.

[0065] Compound 16: t R At 4.2 min, in the positive mode, the MS mass spectrum showed a quasi-molecular ion peak [M+H] + m / z 465.14C 22 H 24 O 11 , fragment ion [M+H] + m / z 303.09 C 16 H 14 O6. Visible fragment ion peak [M+HC 16 H 14 O6] + m / z 303.09 is due to the quasi-molecular ion peak [M+H] + m / z 465.14 Loss of 162Da (-C6H 10 O5), it is estimated that t RCompound 16 with a reaction time of 4.2 min is 1,2,3-Trimethoxy-7-hydroxyxanthone-glucoside.

[0066] Compound 17: t R At 6.9 min, in the positive mode, the MS mass spectrum showed a quasi-molecular ion peak [M+H] + m / z 403.14C 21 H 22 O8, fragment ion [M+H] + m / z C 20 H 19 O7 373.09 visible fragment ion peak [M+HC 20 H 19 O7] + m / z 373.09 is due to the quasi-molecular ion peak [M+H] + m / z 403.14 lost 30Da (-CH2O), estimated t R Compound 17 with a reaction time of 6.9 min is 3',4',5,5',6,7-Hexamethoxyflavone.

[0067] 4.2 Metabolites of Yunnan pine cone extract in rat plasma Compound 6: t R At 8.3 min, in the positive mode, the MS mass spectrum showed a quasi-molecular ion peak [M+H] + m / z 473.36C 30 H 48 O4, refer to the literature (Zhang Z, ElSohly HN, Jacob MR, Pasco DS, Walker LA, Clark AM. Natural products inhibiting Candida albicans secreted aspartic proteases from Lycopodium cernuum. J Nat Prod. 2002 Jul;65(7):979-85.), speculated t R Compound 6 with a bioactivity of 8.3 min is 3α,21β,24-trihydroxyserrat-14-en-16-one.

[0068] Compound 9: t R At 2.9 min, in the positive mode, the MS mass spectrum showed a quasi-molecular ion peak [M+H]+ m / z 183.07C9H 10 O4, fragment ion [M+H] + m / z C9H8O3 165.06. Visible fragment ion peak [M+H-C9H8O3] + m / z 165.05 is due to the quasi-molecular ion peak [M+H] + m / z 183.07 lost 18Da (-H2O), estimated t R Compound 9 with a reaction time of 2.9 min is dihydrocaffeic acid.

[0069] 3.3 Prototype components of Yunnan pine cone extract in rat feces Compound 18: t R At 5.3 min, in the positive mode, the MS mass spectrum showed a quasi-molecular ion peak [M+H] + m / z 235.1C 13 H 14 O4, fragment ion [M+H] + m / z 175.07 C 11 H 10 O2, visible fragment ion peak [M+HC 11 H 10 O2] + m / z is determined by the quasi-molecular ion peak [M+H] + m / z 235.1 lost 60Da (-C2H4O2), estimated t R Compound 18 with a reaction time of 5.3 min is Dimethyllimettin.

[0070] Compound 19: t R At 4.4 min, in the positive mode, the MS mass spectrum showed a quasi-molecular ion peak [M+H] + m / z 463.12C 22 H 22 O 11 , fragment ion [M+H] + m / z C 16 H 12 O6 301.07 visible fragment ion peak [M+HC 16 H 12 O6] + m / z 301.07 is due to the quasi-molecular ion peak [M+H] + m / z 463.12 Loss of 162Da (-C6H 10 O5), it is estimated that t RCompound 19 with a reaction time of 4.4 min is diosmetin-7-O-galactoside.

[0071] Compound 20: t R At 5.3 min, in negative mode, the MS mass spectrum showed a quasi-molecular ion peak [M+H] - m / z 357.13C 20 H 22 O6, fragment ion [M+H] - m / z 151.04 C8H8O3. Visible fragment ion peak [M+H-C8H8O3] - m / z is determined by the quasi-molecular ion peak [M+H - m / z 357.13 Loss of 206Da (-C 12 H 14 O3), it is estimated that t R Compound 20 with a reaction time of 5.3 min is Clemaphenol A.

[0072] Compound 21: t R At 3.3 min, in negative mode, the MS spectrum showed a quasi-molecular ion peak [M+H] - m / z 401.14C 18 H 26 O 10 , fragment ion [M+H] - m / z 269.1 C 13 H 18 O6. Visible fragment ion peak [M+HC 13 H 18 O6] - m / z is determined by the quasi-molecular ion peak [M+H - m / z 401.14 lost 132Da (-C5H8O4), inferred t R Compound 21 with a melting point of 3.3 min is Benzyl-(2''-O-xylosyl) glucoside.

[0073] Compound 22: t R At 4.5 min, in the positive mode, the MS mass spectrum showed a quasi-molecular ion peak [M+H] + m / z 419.1C 20 H 18 O 10 , fragment ion [M+H] + m / z 287.05 C 15 H 10O6. Visible fragment ion peak [M+HC 15 H 10 O6] + m / z 287.05 is due to the quasi-molecular ion peak [M+H] + m / z 429.1 lost 132Da (-C5H8O4), estimated t R Compound 22, whose reaction time was 4.5 min, was Kaempferol-3-O-arabinoside.

[0074] Compound 23: t R At 2.7 min, in negative mode, the MS mass spectrum showed a quasi-molecular ion peak [M+H] - m / z 415.12C 18 H 24 O 11 , fragment ion [M+H] - m / z 193.05 C 10 H 10 O4. Visible fragment ion peaks [M+HC 10 H 10 O4] - m / z 193.05 is due to the quasi-molecular ion peak [M+H] - m / z 401.14 Loss of 222.07Da (-C8H 14 O7), it is speculated that t R Compound 23 with a reaction time of 2.7 min is 3-O-Feruloyl Octose.

[0075] Compound 24: t R At 3.9 min, in the positive mode, the MS mass spectrum showed a quasi-molecular ion peak [M+H] + m / z 465.1C 21 H 20 O 12 , fragment ion [M+H] + m / z 303.05 C 15 H 10 O7. Visible fragment ion peaks [M+HC 15 H 10 O7] + m / z 303.05 is due to the quasi-molecular ion peak [M+H] + m / z 465.1 Loss of 162Da (-C6H 10 O5), it is estimated that t RCompound 24 with a reaction time of 3.9 min is Quercetin-5-O-β-D-glucoside.

[0076] Compound 25: t R At 5.2 min, in negative mode, the MS mass spectrum showed a quasi-molecular ion peak [M+H] - m / z 147.05C9H8O2, fragment ion [M+H] - m / z 103.06 C8H8. Visible fragment ion peak [M+H-C8H8] - m / z 103.06 is due to the quasi-molecular ion peak [M+H] - m / z 147.05 lost 43.99Da (-CO2), estimated t R Compound 25 with a reaction time of 5.2 min is Cinnamicacid (hydrocinnamic acid).

[0077] Compound 28: t R At 7 minutes, in the positive mode, the MS mass spectrum showed a quasi-molecular ion peak [M+H] + m / z 287.24C 20 H 30 O, fragment ion [M+H] + m / z 159.12 C 11 H 26 . Visible fragment ion peaks [M+HC 11 H 26 ] + m / z 159.12 is due to the quasi-molecular ion peak [M+H] + m / z 287.24 lost 128Da (-C9H4O), estimated t R Compound 28 with a reaction time of 7 min is Ent-Kaurenal.

[0078] Compound 29: t R At 4.1 min, in the positive mode, the MS mass spectrum showed a quasi-molecular ion peak [M+H] + m / z 461.11C 22 H 20 O 11 , fragment ion [M+H] + m / z 285.08 C 16 H 12 O5. Visible fragment ion peak [M+HC 16 H 12 O] + m / z 285.08 is due to the quasi-molecular ion peak [M+H] +m / z 461.11 lost 176.03a (-C6H8O6), inferred t R Compound 29 with a time of 4.1 min is Wogonin-7-O-Glucuronide (Wogonoside).

[0079] 3.4 Metabolites of Yunnan pine cone extract in rat feces Compound 26: t R At 0.8 min, in the positive mode, the MS mass spectrum showed a quasi-molecular ion peak [M+H] + m / z 162.11C7H 15 NO3, fragment ion [M+H] + m / z 85.03 C4H4O2. Visible fragment ion peak [M+H-C4H4O2] + m / z 85.03 is due to the quasi-molecular ion peak [M+H] + m / z 162.11 Loss of 77Da (-C3H 11 NO), it is speculated that R The compound 26 with a reaction time of 0.8 min is L-Carnitine.

[0080] Compound 27: t R At 2.4 min, in the positive mode, the MS mass spectrum showed a quasi-molecular ion peak [M+H] + m / z 188.07C 11 H9NO2, fragment ion [M+H] + m / z 118.07 C8H7N. Visible fragment ion peak [M+H-C8H7N] + m / z 118.07 is due to the quasi-molecular ion peak [M+H] + m / z 188.07 lost 70Da (-C3H2O2), estimated t R Compound 27 with a reaction time of 2.4 min is 3-Indoleacrylic acid.

[0081] The experimental results systematically reflect the in vivo active substances of Yunnan pine cone extract. By orally administering Yunnan pine cone extract to rats, a large amount of prototype components were detected in their plasma and fecal samples, with fewer metabolites, indicating that the effective components of Yunnan pine cone extract are mainly prototype components, which provides a reference for its pharmacological material basis and innovative drug research and development.

[0082] The above are only specific application examples of the present invention. The present invention has other implementation modes. Any technical solutions formed by equivalent replacement or equivalent transformation fall within the protection scope required by the present invention.

Claims

1. A method for detecting medicinal components in pine cone extract, comprising the following steps: orally administering pine cone extract to healthy animals, collecting plasma and feces samples before and after administration, and performing UPLC-MS / MS detection and analysis. 17 blood-entering components were found in the plasma samples after administration, including 15 prototype components and 2 metabolites; 12 components were found in the feces samples after administration, including 10 prototype components and 2 metabolites, characterized in that: The preparation method of the pine cone extract is as follows: extracting pine cone coarse powder with 85% ethanol, filtering, extracting the residue with hot water, filtering and removing the hot water extract; adding alkali solution to the residue at room temperature to obtain an alkaline extract; adjusting the pH value of the alkaline extract to 5 with acetic acid, concentrating, filtering, and removing precipitation; The filtrate was precipitated with 1 times the amount of ethanol, filtered to obtain precipitate D; the filtrate was precipitated with 2 times the amount of ethanol again, filtered to obtain precipitate E; the filtrate was precipitated with 5 times the amount of ethanol again for 24 hours, filtered to obtain precipitate F, and precipitates D, E, and F were combined to obtain the pine cone extract.

2. The detection method according to claim 1, characterized in that: Plasma and feces samples before and after administration were obtained from the same animals.

3. The detection method according to claim 1, characterized in that: The sample preparation method for pine cone extract detection includes the following steps: vortexing the pine cone extract and internal standard solution I in a mass volume ratio of 1 mg:10 μL to 1 mg:20 μL, centrifuging, and filtering the supernatant with a microporous filter membrane.

4. The detection method according to claim 1, characterized in that: The fecal sample test sample preparation method includes the following steps: taking a fecal sample, vacuum freeze-drying, and grinding it into powder; taking the sample powder, adding internal standard solution I at a mass volume ratio of 1 mg:20 μL~1 mg:30 μL, intermittently and repeatedly vortexing, centrifuging, and taking the supernatant and filtering it with a microporous filter membrane.

5. The detection method according to claim 3 or 4, characterized in that: The internal standard solution I is prepared by dissolving L-2-chlorophenylalanine in 70% methanol aqueous solution.

6. The detection method according to claim 1, characterized in that: The sample preparation method for plasma sample detection includes the following steps: taking a plasma sample, thawing it on ice, vortexing it to mix it, vortexing the plasma and internal standard solution II at a volume ratio of 1:4 to 1:8, centrifuging it, taking the supernatant and letting it stand at -20°C, centrifuging it again, and taking the supernatant.

7. The detection method according to claim 6, characterized in that: The internal standard solution II is prepared by dissolving L-2-chlorophenylalanine in an acetonitrile-methanol solution with a volume ratio of 1:

4.

8. The detection method according to claim 1, characterized in that: The chromatographic detection conditions were as follows: the chromatographic column was Agilent SB-C18 1.8 μm, 2.1 mm × 100 mm; Mobile phase: Phase A is a mixed solution of ultrapure water and formic acid with a volume ratio of formic acid to ultrapure water of 0.1%, and phase B is a mixed solution of acetonitrile and formic acid with a volume ratio of formic acid to acetonitrile of 0.1%; elution gradient: 0~9min, 5%~95%B; 9~10min, 95%B; 10~11min, 95%~5%B; 11~14min, 5%B; flow rate 0.35mL / min; column temperature 40℃; injection volume 2μL.

9. The detection method according to claim 1, characterized in that: The mass spectrometry detection conditions were as follows: electrospray ion source temperature of 500°C; ion spray voltage of 5500V / -4500V; ion source gas I, gas II and curtain gas of 50, 60 and 25psi respectively, collision induced ionization parameter of high; QQQ scanning using MRM mode, collision gas of medium.

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