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

Abstract

The invention discloses a method for detecting traditional Chinese medicine-derived components in a pinecone alcohol extract, which comprises the following steps: orally taking the pinecone alcohol extract to a healthy animal, collecting plasma and excrement samples before and after administration, carrying out UPLC-MS / MS detection analysis, comparing with the pinecone alcohol extract, and finding 8 blood-entering components in the plasma sample after administration. 18 components, which are prototype components, are found in an excrement sample after administration. The method comprehensively reflects the in-vivo metabolic process and excretion pathway of the components of the pinecones, and is of great 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 pine cone alcohol extracts. 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 symptoms such as chronic bronchitis, asthma, and cough with sputum. 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 after entering the body are still unclear, and the material basis of pharmacological efficacy needs further exploration. Therefore, it is crucial to establish a rapid and accurate detection and analysis method for the drug-derived components after pine cone administration for the research on the pharmacological material basis of pine cones, new drug development, and clinical application research. Summary of the invention

[0003] In view of the deficiencies in the prior art, the object of the present invention is to provide a method for detecting medicinal ingredients in pine cone alcohol extracts, which can quickly and accurately detect and compare the components of pine cone alcohol extracts before and after metabolism, and can more comprehensively reflect the metabolism and excretion of pine cone components in the body.

[0004] The objective of the present invention is achieved through the following technical solutions: A method for detecting medicinal components in a pine cone alcohol extract comprises the following steps: orally administering the pine cone alcohol extract to healthy animals, collecting plasma and feces samples before and after administration, performing UPLC-MS / MS detection and analysis, and comparing the samples with the pine cone alcohol extract; 8 blood-entering components were found in the plasma samples after administration, and 18 components were found in the feces samples after administration, all of which were prototype components.

[0005] 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.

[0006] Preferably, the method for preparing the pine cone alcohol extract comprises the following steps: crushing the pine cones, soaking them in 85% ethanol, and concentrating the extract under reduced pressure to obtain an extract.

[0007] The method for preparing samples for pine cone alcohol extract detection comprises the following steps: vortexing the pine cone alcohol extract and internal standard solution I at a mass volume ratio of 1 mg:10 μL to 1 mg:20 μL, centrifuging, taking the supernatant and filtering it with a microporous filter membrane for UPLC-MS / MS detection.

[0008] More preferably, the internal standard solution I is prepared by dissolving L-2-chlorophenylalanine in 70% methanol aqueous solution.

[0009] More preferably, the internal standard solution I needs to be precooled at -20°C before use.

[0010] 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.

[0011] 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).

[0012] 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.

[0013] More preferably, the internal standard solution I needs to be precooled at -20°C before use.

[0014] 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.

[0015] Flow rate: 0.35 mL / min; column temperature: 40°C; injection volume: 2 μL.

[0016] 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.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention adopts the alcohol extract of pine cone to carry out overall administration, rather than using a single compound or known component. This method is more in line with the medication mode of traditional Chinese medicine, and can more comprehensively reflect the metabolism and excretion of the components of pine cone in vivo. At the same time, the plasma and feces samples after administration are collected, and the absorption, metabolism and excretion pathways of the drug components can be more comprehensively and accurately analyzed.

[0018] 2. Using the technical solution of the present invention, 8 blood-entering components were found in the plasma samples after administration of Yunnan pine cones, and 18 components were found in the fecal samples after administration, all of which were prototype components. No metabolic components were detected in the plasma samples after administration and the fecal samples after administration.

[0019] 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.

[0020] 4. The present invention introduces plasma and feces analysis before and after administration, uses UPLC-MS / MS to track the metabolic process and excretion pathway of pine cone extract in the body, analyzes and identifies its pharmacodynamic material basis in the body, and the analysis results can be further used to comprehensively evaluate the efficacy and safety of pine cone alcohol extract, which is of great significance for guiding the clinical application and further research and development of pine cone.

[0021] 5. The present invention adopts UPLC-MS / MS technology for detection and analysis, which has the advantages and characteristics of simple operation, easy control of experimental conditions, rapid and accurate, and good reproducibility. Compared with traditional HPLC, UPLC has higher separation efficiency and shorter separation time, and can quickly separate complex components. The MS / MS detector has high sensitivity and high selectivity, and can detect target compounds in low concentrations and complex matrices. In the experiment, the detection of complex biological samples can be completed in 14 minutes. This method can fully collect the chromatographic information of the test samples, and the separation of each chromatographic peak is good, which lays the foundation for the subsequent analysis and processing of massive metabolic data.

[0022] 6. The present invention uses the pine cone alcohol extract itself as the basis for comparison, and compares through mass spectrometry fragmentation and retention time. It is independent of standard products and has stronger applicability. Even if the pine cone alcohol 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 the 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] 7. 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] 8. The detection method of the present invention is applicable to pine cones of various pine trees (such as Pinus sylvestris var. mongolica, Pinus koreaense, Pinus armandii, Pinus koraiensis, etc.). BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the TIC graph of the ethanol extract of pine cones of Yunnan pine under the positive ion mode in Example 1.

[0026] Figure 2 This is the TIC graph of blank plasma in positive ion mode in Example 1.

[0027] Figure 3 This is the TIC diagram of the administered plasma in the positive ion mode in Example 1.

[0028] Figure 4 This is the TIC graph of blank feces in positive ion mode in Example 1.

[0029] Figure 5 This is the TIC graph of the feces administered in the positive ion mode in Example 1.

[0030] Figure 6 This is the TIC graph of the ethanol extract of pine cones of Yunnan pine under the negative ion mode in Example 1.

[0031] Figure 7 This is the TIC graph of blank plasma in negative ion mode in Example 1.

[0032] Figure 8 This is the TIC diagram of the administered plasma in the negative ion mode in Example 1.

[0033] Fig. 9 This is the TIC graph of blank feces in negative ion mode in Example 1.

[0034] Fig.10 This is the TIC graph of the feces administered in the negative ion mode in Example 1.

[0035] Fig.11 It is a Venn diagram of the pine cone alcohol extract of Yunnan pine, blank plasma, administered plasma, blank feces, and administered feces in Example 1 (in the diagram: CTW is the pine cone alcohol extract of Yunnan pine, 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 pine cone alcohol extract from Yunnan pine Crush the Yunnan pine cone medicinal material, weigh about 10 kg of the crude powder, soak it in 85% ethanol solution for 3 times, each time for 24 hours, combine the extracts and concentrate them under reduced pressure to obtain an extract, thereby obtaining the Yunnan pine cone alcohol extract.

[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℃ 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), 200mg / kg Yunnan pine cone alcohol extract (pine cone alcohol extract was mixed with 0.5% CMC-Na solution) was given by gavage, 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℃ 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℃ refrigerator.

[0042] 2.3 Preparation of test samples 2.3.1 Preparation of pine cone alcohol extract test samples The alcohol extract was vortexed for 1 min, and internal standard solution I precooled at -20°C was added. For every 50 mg of alcohol 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 pine cone alcohol extract test samples of Yunnan pine, plasma test samples before and after administration, and fecal test samples before and after administration were tested by UPLC-MS / MS respectively.

[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 pine cone alcohol extract of Yunnan pine, blank plasma, drug-treated plasma, blank feces, and drug-treated feces in positive ion mode; Figure 6-10 They are the TIC graphs of pine cone alcohol extract of Yunnan pine, blank plasma, drug-treated plasma, blank feces, and drug-treated feces in negative ion mode; Fig.11 It is a Venn diagram of the alcohol extract of Pinus yunnanensis cone, blank plasma, administered plasma, blank feces, and administered feces.

[0050] Eight blood-entering components were found in the administered plasma samples, namely: 6-deoxy-6-sulfo-D-glucosyl-1,5-lactone, dioxindole-3-acetic acid, 3-hydroxyphenylacetic acid, N,N'-diaferuloylputrescine, equol (7,4'-homoisoflavanone), 7-hydroxydehydroabietic acid, 2,3-dihydrobenzofuran, and phyllanflexoid A.

[0051] Eighteen components were found in the fecal samples of the administered animals, namely: 6-hydroxyluteolin-5-glucoside, quercetin-3-O-galactoside (hyperoside), quercetin-5-O-glucoside, 5-phenyl-4-pentynoic acid methyl ester, baicalin-7-O-glucoside, irisin, coniferyl aldehyde, norspirin, baicalin (baicalin-7-O-glucoside), 4α,12-dihydroxyguaian-6,10-diene, 6-methylluteolin, 3-hydroxy-3',4',5,5',7-pentamethoxyflavone, isocurcumenol, inumakoic acid, Z-artemisinin, 7-oxodehydroabietic acid, 2,3-dihydrobenzofuran, and phyllanflexoid A.

[0052] Among them, 2,3-dihydrobenzofuran and phyllanflexoid A were detected in both plasma and feces.

[0053] The above ingredients are all prototype ingredients, and the results are shown in Table 1.

[0054] Table 1

[0055] Compound 1: t R The MS spectrum shows a quasi-molecular ion peak [M+H] at 0.6 min in positive mode. + m / z 243.02C6H 10 O8S, fragment ion [M+H] + m / z C6H8O7S 225.01. Visible fragment ion peak [M+H-C6H8O7S] + m / z 225.01 is due to the quasi-molecular ion peak [M+H] + m / z 243.02 lost 18Da (-H2O), estimated t R The compound 1 with a reaction time of 0.6 min is 6-deoxy-6-sulfo-D-glucosyl-1,5-lactone.

[0056] Compound 2: t R The MS spectrum shows a quasi-molecular ion peak [M+H] at 1.3 min in positive mode. + m / z 121.06C8H8O, fragment ion [M+H] + m / z C6H4 77.04. Visible fragment ion peak [M+H-C6H4] + m / z 77.04 is due to the quasi-molecular ion peak [M+H] + m / z 121.06 lost 44Da (-C2H4O), estimated t R The compound 2 with a reaction time of 1.3 min is 2,3-dihydrobenzofuran.

[0057] Compound 3: t R The MS spectrum shows a quasi-molecular ion peak [MH] at 2.8 min in negative mode. - m / z 206.05C 10 H9NO4, fragment ion [MH] - m / z 162.06 C9H9NO2. Visible fragment ion [MH-C9H9NO2] - m / z 162.06 is due to the quasi-molecular ion peak [MH] - m / z 206.05 obtained by loss of 44Da (-C2H4O), estimated t RCompound 3 with a reaction time of 2.8 min is dioxindole-3-acetic acid.

[0058] Compound 4: t R The MS spectrum shows a quasi-molecular ion peak [MH] at 3.3 min in negative mode. - m / z 151.04C8H8O3, fragment ion [MH] - m / z 107.05 C7H8O. Visible fragment ion [MH-C7H8O] - m / z 107.05 is caused by the quasi-molecular ion peak [MH] - m / z 151.04 obtained by loss of 44Da (-C2H4O), estimated t R Compound 4 with a reaction time of 3.3 min is 3-hydroxyphenylacetic acid.

[0059] Compound 5: t R The MS spectrum shows a quasi-molecular ion peak [MH] at 3.6 min in negative mode. - m / z 463.09C 21 H 20 O 12 , fragment ion [MH] - m / z 301.03 C 15 H 10 O7. Visible fragment ions [MHC 15 H 10 O7] - m / z 301.03 is caused by the quasi-molecular ion peak [MH] - m / z 463.09 Loss of 162Da (-C6H 10 O5) obtained, it is inferred that t R Compound 5 with a reaction time of 3.6 min is 6-hydroxyluteolin-5-glucoside.

[0060] Compound 6: t R The MS spectrum shows a quasi-molecular ion peak [MH] at 3.6 min in negative mode. - m / z 463.09C 21 H 20 O 12 , fragment ion [MH] - m / z 300.03 C 15 H9O7. Visible fragment ions [MHC 15 H9O7] - m / z 300.03 is caused by the quasi-molecular ion peak [MH] - m / z 463.09 Loss of 163Da (-C6H 11 O5) obtained, it is inferred that tR Compound 6 with a reaction time of 3.6 min is quercetin-3-O-galactoside (hyperoside).

[0061] Compound 7: t R The MS spectrum shows a quasi-molecular ion peak [M+H] at 3.8 min in positive mode. + m / z 479.08C 21 H 18 O 13 , fragment ion [M+H] + m / z 303.06 C 15 H 10 O7. Visible fragment ions [M+HC 15 H 10 O7] + m / z 303.06 is due to the quasi-molecular ion peak [M+H] + m / z 479.08 C 21 H 18 O 13 Loss of 176Da (-C6H8O6) resulted in the inferred t R Compound 7 with a reaction time of 3.8 min is quercetin-5-O-glucuronide.

[0062] Compound 8: t R The MS spectrum shows a quasi-molecular ion peak [M+H] in the positive mode. + m / z 189.09C 12 H 12 O2, fragment ion [M+H] + m / z 129.07 C 10 H8. Visible fragment ions [M+HC 10 H8] + m / z 129.07 is derived from the quasi-molecular ion peak [M+H] + m / z 189.09 C 12 H 12 O2 loses 60Da (-C2H4O2), and it is estimated that t R Compound 8 with a reaction time of 4 min is methyl 5-phenyl-4-pentynoate.

[0063] Compound 9: t R The MS spectrum shows a quasi-molecular ion peak [MH] at 4.1 min in negative mode. - m / z 447.09C 21 H 20 O 11 , fragment ion [MH] - m / z 284.03 C 15H9O6. Visible fragment ions [MHC 15 H9O6] - m / z 284.03 is caused by the quasi-molecular ion peak [MH] - m / z 447.09 Loss of 163Da (-C6H 11 O5) obtained, it is inferred that t R Compound 9 with a time of 4.1 min is baicalein-7-O-glucoside.

[0064] Compound 10: t R The MS spectrum showed a quasi-molecular ion peak [M+H] at 4.2 min in positive mode. + m / z 463.12C 22 H 22 O 11 , fragment ion [M+H] + m / z 301.09 C 16 H 12 O6. Visible fragment ions [M+HC 16 H 12 O6] + m / z 301.09 is due to the quasi-molecular ion peak [M+H] + m / z 463.12 C 22 H 22 O 11 Loss of 162Da (-C6H 10 O5) obtained, it is inferred that t R Compound 10 with a reaction time of 4.2 min is irisin.

[0065] Compound 11: t R The MS spectrum shows a quasi-molecular ion peak [M+H] at 4.6 min in positive mode. + m / z 179.07C 10 H 10 O3, fragment ion [M+H] + m / z 91.06 C7H6, fragment ion [M+H-C7H6] visible + m / z 91.06 is due to the quasi-molecular ion peak [M+H] + m / z 179.07 C 10 H 10 O3 loses 88Da (-C3H4O3), and it is estimated that t R Compound 11 with a reaction time of 4.6 min is coniferyl aldehyde.

[0066] Compound 12: t R The MS spectrum showed a quasi-molecular ion peak [M+H] at 4.9 min in the positive mode. +m / z 441.2C 24 H 28 N2O6, fragment ion [M+H] + m / z 177.05 C 10 H8O3. Visible fragment ions [M+HC 10 H8O3] + m / z 177.05 is due to the quasi-molecular ion peak [M+H] + m / z 441.2 C 24 H 28 N2O6 loses 264Da (-C 14 H 20 N2O3) and it is estimated that t R Compound 12 with a reaction time of 4.9 min is N,N'-diferuloylputrescine.

[0067] Compound 13: t R The MS spectrum shows a quasi-molecular ion peak [MH] in negative mode. - m / z 373.13C 20 H 22 O7, fragment ion [MH] - m / z 179.07 C 10 H 12 O3. Visible fragment ions [MHC 10 H 12 O3] - m / z 179.07 is due to the quasi-molecular ion peak [MH] - m / z 373.13 C 20 H 22 O7 loses 196Da (-C 10 H 12 O4) obtained, it is inferred that t R The compound 13 with a reaction time of 5 min is norchelin.

[0068] Compound 14: t R The MS spectrum shows a quasi-molecular ion peak [M+H] at 5.1 min in positive mode. + m / z 461.11C 22 H 20 O 11 , fragment ion [M+H] + m / z 285.08 C 16 H 10 O5. Visible fragment ions [M+HC 16 H 10 O5] + m / z 285.08 is due to the quasi-molecular ion peak [M+H] +m / z 461.11 obtained by loss of 176Da (-C6H8O6), estimated t R Compound 14 with a time of 5.1 min is wogonin (wogonin-7-O-glucuronide).

[0069] Compound 15: t R The MS spectrum shows a quasi-molecular ion peak [M+H] at 5.7 min in positive mode. + m / z 243.1C 15 H 14 O3, fragment ion [M+H] + m / z 133.06 C9H8O. Visible fragment ion [M+H-C9H8O] + m / z 133.06 is due to the quasi-molecular ion peak [M+H] + m / z 243.1 C 15 H 14 O3 loses 110Da (-C6H6O2), and it is estimated that t R Compound 15 with a time of 5.7 min is equol (7,4'-homoisoflavan).

[0070] Compound 16: t R The MS spectrum shows a quasi-molecular ion peak [M+H] at 5.8 min in positive mode. + m / z 237.18C 15 H 24 O2, fragment ion [M+H] + m / z 133.1 C 10 H 12 , visible fragment ions [M+HC 10 H 12 ] + m / z 133.1 is due to the quasi-molecular ion peak [M+H] + m / z 237.18 C 15 H 24 O2 loses 104Da (-C5H 11 O2) obtained, it is inferred that t R Compound 16 with a reaction time of 5.8 min is 4α,12-dihydroxyguaian-6,10-diene.

[0071] Compound 17: t R The MS spectrum shows a quasi-molecular ion peak [M+H] at 5.8 min in positive mode. + m / z 301.07C 16 H 12 O6, fragment ion [M+H]+ m / z 166.03 C8H5O4. Visible fragment ion [M+H-C8H5O4] + m / z 167.03 is due to the quasi-molecular ion peak [M+H] + m / z 301.07 C 16 H 12 O6 loses 134Da (-C8H6O2), and it is estimated that t R Compound 17 with a reaction time of 5.8 min is 6-methylluteolin.

[0072] Compound 18: t R The MS spectrum shows a quasi-molecular ion peak [M+H] at 6.2 min in positive mode. + m / z 389.12C 20 H 20 O8, fragment ion [M+H] + m / z 359.08 C 19 H 18 O7. Visible fragment ions [M+HC 19 H 18 O3] + m / z 359.08 is due to the quasi-molecular ion peak [M+H] + m / z 389.12 C 20 H 20 O8 lost 30Da (-CH2O), it is estimated that t R Compound 18 with a reaction time of 6.2 min is 3-hydroxy-3',4',5,5',7-pentamethoxyflavone.

[0073] Compound 19: t R The MS spectrum shows a quasi-molecular ion peak [M+H] at 6.9 min in positive mode. + m / z 235.17C 15 H 22 O2, fragment ion [M+H] + m / z 105.07 C7H4O. It can be seen that the fragment ion [M+H-C7H4O] is composed of the quasi-molecular ion peak [M+H] + m / z 235.17 C 15 H 22 O2 loses 130Da (-C8H 18 O), it is estimated that t R Compound 19 with a reaction time of 6.9 min is isocurcumenol.

[0074] Compound 20: t R The MS spectrum showed a quasi-molecular ion peak [M+H] at 8.1 min in positive mode. +m / z 317.21C 20 H 28 O3, fragment ion [M+H] + m / z 147.08 C 10 H 10 O. Visible fragment ions [M+HC 10 H 10 O] + m / z 147.08 is due to the quasi-molecular ion peak [M+H] + m / z 317.21 C 20 H 28 O3 loses 170Da (-C 10 H8O2) and it is inferred that t R Compound 20 with a reaction time of 8.1 min is 7-hydroxydehydroabietic acid.

[0075] Compound 21: t R The MS spectrum shows a quasi-molecular ion peak [MH] at 8.2 min in negative mode. - m / z 315.2C 20 H 28 O3, fragment ion [MH] - m / z 271.2 C 17 H 20 O3. Visible fragment ions [MHC 17 H 20 O3] - m / z 271.2 is due to the quasi-molecular ion peak [MH] - m / z 315.2 C 20 H 27 O3 loses 44Da (-C3H8), and it is estimated that t R Compound 21 with a reaction time of 8.2 min is inumakoic acid.

[0076] Compound 22: t R The MS spectrum shows a quasi-molecular ion peak [M+H] at 8.3 min in positive mode. + m / z 191.11C 12 H 14 O2, fragment ion [M+H] + m / z 117.07 C9H8. Visible fragment ion [M+H-C9H8] + m / z 117.07 is due to the quasi-molecular ion peak [M+H] + m / z 191.11 C 12 H 14 O2 loses 74Da (-C3H6O2), and it is estimated that t RCompound 22 with a reaction time of 8.3 min is Z-artemisinin.

[0077] Compound 23: t R The MS spectrum showed a quasi-molecular ion peak [M+H] at 8.6 min in positive mode. + m / z 315.2C 20 H 26 O3, fragment ion [M+H] + m / z 187.12 C 14 H 18 Visible fragment ions [M+HC 14 H 18 ] + m / z 187.12 is due to the quasi-molecular ion peak [M+H] + m / z 315.2 C 20 H 26 O3 loses 128Da (-C6H8O3), and it is estimated that t R Compound 23 with a reaction time of 8.6 min is 7-oxodehydroabietic acid.

[0078] Compound 24: t R The MS spectrum shows a quasi-molecular ion peak [M+H] at 8.7 min in positive mode. + m / z 315.19C 20 H 26 O3, fragment ion [M+H] + m / z 187.11 C 14 H 18 Visible fragment ions [M+HC 14 H 18 ] + m / z 187.12 is due to the quasi-molecular ion peak [M+H] + m / z 315.2 C 20 H 26 O3 loses 128Da (-C6H8O3), and it is estimated that t R Compound 24 with a bioactivity of 8.6 min is phyllanflexoid A.

[0079] The experimental results comprehensively reflect the direct-acting substances of pine cones in the body. By orally administering pine cone alcohol extracts, only the original components were detected in the plasma and fecal samples after administration, and no metabolites were detected, indicating that the biotransformation process of the components absorbed into the body is weak, and the basic substance that exerts the medicinal effect may be the original component, which provides a reference for the medicinal material basis of pine cones and the research and development of innovative drugs.

[0080] 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 alcohol extract, characterized in that: The following steps are involved: The pine cone alcohol extract was orally administered to healthy animals, and plasma and fecal samples were collected before and after administration. The samples were analyzed by UPLC-MS / MS and compared with the pine cone alcohol extract. Eight blood-entering components were found in the plasma samples after administration, and 18 components were found in the fecal samples after administration, all of which were prototype components.

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 preparation method of pine cone alcohol extract comprises the following steps: crushing pine cones, soaking them in 85% ethanol, and concentrating the extract under reduced pressure to obtain an extract.

4. The detection method according to claim 1, characterized in that: The sample preparation method for pine cone alcohol extract detection includes the following steps: vortexing the pine cone alcohol 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.

5. 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.

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

7. 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.

8. The detection method according to claim 7, 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.

9. 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.

10. 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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