A detection method of traditional Chinese medicine-derived components in pine cone alcohol extract

Abstract

The application discloses a detection method of traditional Chinese medicine-derived components in pine cone alcohol extract, and comprises the following steps: orally administering the pine cone alcohol extract to healthy animals, collecting plasma and fecal samples before and after administration, detecting and analyzing the samples by UPLC-MS / MS, and comparing the samples with the pine cone alcohol extract, so that eight blood components are found in the plasma sample after administration, and 18 components are found in the fecal sample after administration, which are all prototype components. The application comprehensively reflects the metabolism process and excretion path of the components in the pine cone in the body, and has important significance for guiding the clinical application and further research and development of the pine cone.

Description

Technical Field

[0001] This invention belongs to the field of drug detection, specifically relating to a method for detecting medicinal components in pine cone alcohol extract. Background Technology

[0002] Pine cones are the brownish-red, multi-layered scaly fruits of various pine trees (Pinaceae family). They possess expectorant, antitussive, and antiasthmatic properties and are commonly used to treat symptoms such as chronic bronchitis, asthma, and excessive phlegm. In recent years, with in-depth research on pine cones, it has been discovered that pine cone extracts also exhibit various pharmacological activities, including anti-HIV, anti-Lassa virus, and anti-tumor activities. However, current research largely focuses on the chemical components of pine cones and the screening of active ingredients. The metabolic processes of pine cone components in vivo, the identification of metabolites, and the changes in other chemical components after entering the body remain unclear, and the pharmacodynamic material basis needs further exploration. Therefore, establishing rapid and accurate detection and analysis methods for the drug-derived components after pine cone administration is crucial for research on the pharmacodynamic material basis of pine cones, as well as for new drug development and clinical application research. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the present invention aims to provide a method for detecting medicinal components in pine cone alcohol extract. This method can rapidly and accurately detect and compare the components of pine cone alcohol extract before and after metabolism, and can more comprehensively reflect the metabolism and excretion of pine cone components in the body.

[0004] The objective of this invention is achieved through the following technical solution:

[0005] A method for detecting medicinal components in pine cone alcohol extract includes the following steps: administering pine cone alcohol extract orally to healthy animals, collecting plasma and fecal samples before and after administration, analyzing them by UPLC-MS / MS, and comparing them with pine cone alcohol extract. Eight blood-entering components were found in the plasma sample after administration, and 18 components were found in the fecal sample after administration, all of which are the original components.

[0006] Preferably, plasma and fecal samples before and after drug administration are taken from the same animal to avoid the influence of individual differences on the test results.

[0007] A preferred method for preparing pine cone alcohol extract includes the following steps: pulverizing pine cones, soaking them in 85% ethanol, and concentrating the extract under reduced pressure to obtain a paste.

[0008] The sample preparation method for pine cone alcohol extract includes the following steps: vortex 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, centrifuge, take the supernatant and filter it with a microporous membrane for UPLC-MS / MS detection.

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

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

[0011] A preferred method for preparing plasma samples includes 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. The supernatant is collected, allowed to stand at -20°C, centrifuged again, and used for UPLC-MS / MS detection.

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

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

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

[0015] Preferred chromatographic conditions for UPLC-MS / MS detection and analysis are as follows:

[0016] The chromatographic column was an Agilent SB-C18 1.8µm, 2.1mm × 100mm;

[0017] Mobile phases: Phase A is a mixed solution of ultrapure water and formic acid (volume ratio of formic acid to ultrapure water is 0.1%), and Phase B is a mixed solution of acetonitrile and formic acid (volume ratio of formic acid to acetonitrile is 0.1%).

[0018] Elution gradient: 0~9 min, 5%~95%B; 9~10 min, 95%B; 10~11 min, 95%~5%B; 11~14 min, 5%B.

[0019] Flow rate: 0.35 mL / min; column temperature: 40 °C; injection volume: 2 μL.

[0020] Electrospray ionization (ESI) temperature 500℃; ion spray voltage (IS) 5500V (positive ion mode) / -4500V (negative ion mode); ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) 50, 60, and 25 psi, respectively, with collision-induced ionization parameters set to high. QQQ scans used MRM mode with the collision gas (nitrogen) set to medium.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. This invention uses an alcoholic extract of pine cones for whole-body administration, rather than using a single compound or known component. This method is more in line with traditional Chinese medicine administration and can more comprehensively reflect the metabolism and excretion of pine cone components in the body. Simultaneously collecting plasma and fecal samples after administration allows for a more comprehensive and accurate analysis of the absorption, metabolism, and excretion pathways of the drug components.

[0023] 2. Using the technical solution described in this invention, eight components were found to enter the bloodstream in plasma samples after administration of Yunnan pine cone powder, and eighteen components were found in fecal samples after administration, all of which were the original components. No metabolites were detected in either the plasma or fecal samples after administration.

[0024] 3. Conventional plasma and fecal sample collection typically involves using different laboratory animals, with a separate group of animals not receiving the drug serving as a blank control group. This invention, by collecting plasma and fecal samples from the same animal and using pre-drug plasma and fecal samples as a blank control, avoids the influence of individual differences on the test results, making the analysis more accurate. It also saves on drug usage and reduces the consumption of laboratory animals.

[0025] 4. This invention introduces pre- and post-administration plasma and fecal analysis, using UPLC-MS / MS to track the metabolic process and excretion pathway of pine cone extract in vivo, and analyzes and identifies its pharmacodynamic material basis in vivo. The analytical 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 of pine cone and further research and development.

[0026] 5. This invention employs UPLC-MS / MS technology for detection and analysis, offering advantages such as ease of operation, controllable experimental conditions, rapid and accurate results, and good reproducibility. Compared to traditional HPLC, UPLC boasts higher separation efficiency and shorter separation time, enabling rapid separation of complex components. The MS / MS detector exhibits high sensitivity and selectivity, allowing the detection of target compounds at low concentrations and in complex matrices. In this experiment, the detection of complex biological samples can be completed in just 14 minutes. This method allows for the complete acquisition of chromatographic information from the sample, with good separation of each chromatographic peak, laying a foundation for subsequent analysis and processing of massive amounts of metabolic data.

[0027] 6. This invention uses the pine cone alcohol extract itself as the basis for comparison, and compares the results by mass spectrometry pyrolysis and retention time. It does not rely on standards and has greater applicability. Even if the pine cone alcohol extract contains unknown components, it can be compared with data in the database. This overcomes the shortcomings of the prior art, which requires consulting literature to understand the possible substances and then comparing them with standards. When encountering unknown components that have not been reported in this species, it is impossible to detect them, which greatly limits its application.

[0028] 7. The addition of L-2-chlorophenylalanine as an internal standard in this invention can ensure the stability of the instrument during the detection process and guarantee the accuracy of the detection results.

[0029] 8. The detection method described in this invention is applicable to the pine cones of various pine trees (such as Scots pine, Simao pine, Huashan pine, Korean pine, etc.). Attached Figure Description

[0030] Figure 1 This is the TIC diagram of the Yunnan pine cone alcohol extract in positive ion mode in Example 1.

[0031] Figure 2 This is the TIC diagram of blank plasma in positive ion mode in Example 1.

[0032] Figure 3 This is a TIC diagram of plasma administered in positive ion mode in Example 1.

[0033] Figure 4 This is the TIC diagram of blank feces in positive ion mode in Example 1.

[0034] Figure 5 This is a TIC diagram of feces administered in positive ion mode in Example 1.

[0035] Figure 6 This is the TIC diagram of the Yunnan pine cone alcohol extract under negative ion mode in Example 1.

[0036] Figure 7 This is the TIC diagram of blank plasma in negative ion mode in Example 1.

[0037] Figure 8 This is a TIC diagram of plasma administered in negative ion mode in Example 1.

[0038] Figure 9 This is the TIC diagram of blank feces in negative ion mode in Example 1.

[0039] Figure 10 This is a TIC diagram of feces administered under negative ion mode in Example 1.

[0040] Figure 11 This is a Venn diagram of Yunnan pine cone ethanol extract, blank plasma, administered plasma, blank stool, and administered stool in Example 1 (in the diagram: CTW is Yunnan pine cone ethanol extract, KBXJ is blank plasma, GYXJ is administered plasma, KBFB is blank stool, and GYFB is administered stool). Detailed Implementation

[0041] The present invention will be further described below with reference to the embodiments, but the present invention is not limited to the embodiments. Example 1

[0042] 1. Materials

[0043] 1.1 Instruments

[0044] 5424R centrifuge (Eppendorf); Biomek i5 automated workstation (Beckman Coulter); CentRiVap centrifugal concentrator (LABCONCO); KQ5200E ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); MS105DM 0.0001 g electronic balance (Mettler Toledo Instruments Co., Ltd.); MU-G02-0448 thermostatic metal mixer (Hangzhou Mio Instrument Co., Ltd.); VORTEX-5 vortex mixer (Kyllin-Bell); AB SCIEX QTRAP4500 liquid chromatography-mass spectrometry system (AB SCIEX, USA).

[0045] 1.2 Test Drugs

[0046] In this embodiment, the pine cones of *Pinus yunnanensis* were used as the experimental sample. The pine cones were collected from Dali City, Dali Prefecture, Yunnan Province, and identified by Professor Duan Baozhong of the Department of Pharmacognosy, College of Pharmacy, Dali University, as *Pinus yunnanensis* (Pinaceae family, *Pinus* genus). Pinus yunnanensisPine cones of Franoh., sampled and stored in Room 706, Department of Organic Medicinal Chemistry, School of Pharmacy, Dali University. Analytical grade sodium carboxymethyl cellulose (Tianjin Kemei Chemical Reagent Development Center), chromatographic grade methanol (Merck), chromatographic grade acetonitrile (Merck), and chromatographic grade formic acid (Aladdin). L-2-chlorophenylalanine standard (99%, Bailingwei).

[0047] 1.3 Animals

[0048] Six SPF-grade male SD rats, weighing 230–250 g, were purchased from Hunan Silek Jingda Co., Ltd., license number: SCXK (Xiang) 2019-0004. The rats were housed under the following conditions: temperature 20±2℃, humidity 60%±5%, 12-hour light and dark cycle, and free access to food.

[0049] 2 Sample Preparation

[0050] 2.1 Preparation of Yunnan pine cone alcohol extract

[0051] The Yunnan pine cone herb is pulverized, and about 10 kg of the coarse powder is weighed out and soaked in 85% ethanol solution three times, 24 hours each time. The extracts are combined and concentrated under reduced pressure to obtain an extract, which is the Yunnan pine cone ethanol extract.

[0052] 2.2 Collect plasma and fecal samples

[0053] SD rats were acclimatized for 7 days. One day before drug administration, blood was collected from the orbital cavity, centrifuged at 3500 rpm for 10 min, and the supernatant was collected as a blank control plasma sample (i.e., blank plasma), stored at -80℃. One day before drug administration, feces were collected from the rats 12 hours prior to administration as a blank control fecal sample (i.e., blank feces), stored under the same conditions. Rats were fasted for 12 hours prior to drug administration (with free access to water), and administered Yunnan pine cone ethanol extract (mixed with 0.5% CMC-Na solution) by gavage at a dose of 200 mg / kg for 7 consecutive days. Feces were collected 12 hours after administration on day 6 as the post-drug fecal sample (i.e., drug-treated feces), stored at -80℃. At 0.5 h, 1 h, 2 h, and 4 h after the last administration, blood was collected from the orbital cavity, centrifuged at 3500 rpm for 10 min, and the supernatant was collected. Plasma samples from the same rat at different time points were combined as the post-drug plasma sample (i.e., drug-treated plasma), stored at -80℃.

[0054] 2.3 Preparation of test samples

[0055] 2.3.1 Preparation of Samples for the Detection of Yunnan Pine Cone Alcoholic Extract

[0056] Vortex the ethanol extract for 1 min, add internal standard solution I pre-cooled at -20℃, add 600 μL of internal standard solution I for every 50 mg of ethanol extract (internal standard solution I is prepared by dissolving 1 mg of L-2-chlorophenylalanine standard in 1 mL of 70% methanol aqueous solution to prepare a 1000 μg / mL stock solution, and further dilute the 1000 μg / mL stock solution with 70% methanol aqueous solution to prepare a 250 μg / mL internal standard solution I), vortex for 15 min, centrifuge (12000 r / min, 4℃) for 3 min, take the supernatant and filter it through a microporous membrane (0.22 μm), place it in a sample vial for UPLC-MS / MS detection.

[0057] 2.3.2 Preparation of Plasma Samples for Detection

[0058] Remove the plasma sample from the -80℃ freezer, thaw on ice, and vortex for 10 seconds to mix. Transfer 50 μL of sample to a 1.5 mL centrifuge tube, and add 300 μL of internal standard solution II (preparation method for internal standard solution II: dissolve 1 mg L-2-chlorophenylalanine standard in 1 mL of acetonitrile-methanol solution (acetonitrile to methanol volume ratio 1:4) to prepare a 1000 μg / mL stock solution; further dilute the 1000 μg / mL stock solution with the aforementioned acetonitrile-methanol solution to a 250 μg / mL internal standard solution II). Vortex for 3 min, centrifuge at 12000 rpm, 4℃ for 10 min. Transfer 200 μL of the supernatant to another 1.5 mL centrifuge tube and incubate at -20℃ for 30 min. Centrifuge again at 12000 rpm, 4℃ for 3 min; transfer 180 μL of the supernatant to a sample vial for UPLC-MS / MS detection. Plasma was processed using this method before and after drug administration.

[0059] 2.3.3 Preparation of fecal samples for testing

[0060] Fecal samples were taken from a -80℃ freezer and freeze-dried under vacuum for 63 h in a Scientz-100F freeze dryer. They were then ground into powder using a grinder (MM 400, Retsch) at 30 Hz for 1.5 min. 50 mg of the sample powder was weighed and added to 1200 μL of pre-cooled internal standard solution I at -20℃. The mixture was vortexed every 30 min for 30 s each time, for a total of 6 vortexes. After centrifugation at 12000 rpm for 3 min, the supernatant was collected, filtered through a 0.22 μm microporous membrane, and placed in a sample vial for UPLC-MS / MS analysis. Fecal samples before and after drug administration were processed using this method.

[0061] 3. Detection methods

[0062] Samples of Yunnan pine cone alcohol extract, plasma samples before and after drug administration, and fecal samples before and after drug administration were analyzed by UPLC-MS / MS.

[0063] 3.1 UPLC testing conditions

[0064] The chromatographic column was an Agilent SB-C18 1.8µm, 2.1mm × 100mm; the mobile phase was a mixture of ultrapure water and formic acid (0.1% formic acid to ultrapure water by volume) in phase A and a mixture of acetonitrile and formic acid (0.1% formic acid to acetonitrile by volume) in phase B; the elution gradient was: 0–9 min, 5%–95% B; 9–10 min, 95% B; 10–11 min, 95%–5% B; 11–14 min, 5% B. The flow rate was 0.35 mL / min; the column temperature was 40℃; and the injection volume was 2 μL.

[0065] 3.2 Mass Spectrometry Conditions

[0066] Electrospray ionization (ESI) temperature 500℃; ion spray voltage (IS) 5500V (positive ion mode) / -4500V (negative ion mode); ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) were set to 50, 60, and 25 psi, respectively, and collision-induced ionization parameters were set to high. QQQ scans used MRM mode with the collision gas (nitrogen) set to medium.

[0067] 3.3 Qualitative analysis of substances was performed based on secondary spectral information, and quantitative analysis was performed using multiple reaction monitoring (MRM) of triple quadrupole mass spectrometry. Analyst 1.6.3 software was used to process the mass spectrometry data.

[0068] 4 Results Analysis

[0069] UPLC-MS / MS detection results are as follows Figures 1-11 As shown, where, Figure 1-5 These are TIC plots of Yunnan pine cone ethanol extract, blank plasma, drug-treated plasma, blank stool, and drug-treated stool under positive ion mode. Figure 6-10 These are TIC plots of Yunnan pine cone alcohol extract, blank plasma, drug-treated plasma, blank stool, and drug-treated stool under negative ion mode. Figure 11 It is a Venn diagram of Yunnan pine cone alcohol extract, blank plasma, drug-treated plasma, blank stool, and drug-treated stool.

[0070] Eight components were found to enter the bloodstream in the drug-treated plasma samples: 6-deoxy-6-sulfonyl-D-glucosyl-1,5-lactone, dioxindole-3-acetic acid, 3-hydroxyphenylacetic acid, N,N'-diferoyl putrescine, equol (7,4'-high isoflavone), 7-hydroxydehydrorosinic acid, 2,3-dihydrobenzofuran, and phyllanflexoid A.

[0071] Eighteen components were found in the fecal samples after administration, namely: 6-hydroxyluteolin-5-glucoside, quercetin-3-O-galactoside (hyperoside), quercetin-5-O-glucuronide, methyl 5-phenyl-4-pentyneate, baicalin-7-O-glucoside, irisin, coniferaldehyde, nortracentronin, baicalin (baicalin-7-O-glucuronide), 4α,12-dihydroxyguaian-6,10-diene, 6-methylluteolin, 3-hydroxy-3',4',5,5',7-pentamethoxyflavone, isocurcuminol, inumakoic acid, Z-artemisinin, 7-oxodehydrorosinic acid, 2,3-dihydrobenzofuran, and phyllanflexoid A.

[0072] 2,3-Dihydrobenzofuran and phyllanflexoid A were detected in both the administered plasma and feces.

[0073] All of the above components are prototype components, and the results are shown in Table 1.

[0074] Table 1

[0075]

[0076] Compound 1: t R At a time of 0.6 min, in positive mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. + m / z 243.02C6H 10 O8S, fragment ions [M+H] + m / z C6H8O7S 225.01. Visible fragment ion peak [M+H-C6H8O7S] + The m / z peak at 225.01 is due to the quasi-molecular ion peak [M+H]. + m / z 243.02 lost 18 Da (-H2O), speculated t R Compound 1, with a concentration of 0.6 min, is 6-deoxy-6-sulfono-D-glucosyl-1,5-lactone.

[0077] Compound 2: t R At 1.3 min, in positive mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. +m / z 121.06C8H8O, fragment ion [M+H] + m / z C6H4 77.04. Visible fragment ion peak [M+H-C6H4] + The m / z peak at 77.04 is due to the quasi-molecular ion peak [M+H]. + m / z 121.06 lost 44 Da (-C2H4O), speculated t R Compound 2, which has a 1.3 min elapsed time, is 2,3-dihydrobenzofuran.

[0078] Compound 3: t R At 2.8 min, in negative mode, the MS mass spectrometer showed a quasi-molecular ion peak [MH]. - m / z 206.05C 10 H9NO4, fragment ion [MH] - m / z 162.06 C9H9NO2. Visible fragment ion [MH-C9H9NO2] - The m / z peak at 162.06 is the quasi-molecular ion peak [MH]. - The value was obtained from m / z 206.05 with a loss of 44 Da (-C2H4O), and the t value is speculated to be... R Compound 3, with a duration of 2.8 min, is dioxindole-3-acetic acid.

[0079] Compound 4: t R At 3.3 min, in negative mode, the MS mass spectrometer showed a quasi-molecular ion peak [MH]. - m / z 151.04C8H8O3, fragment ion [MH] - m / z 107.05 C7H8O. Visible fragment ions [MH-C7H8O] - The m / z peak at 107.05 is due to the quasi-molecular ion [MH]. - m / z 151.04, obtained by losing 44 Da (-C2H4O), speculate t R Compound 4, with a time of 3.3 min, is 3-hydroxyphenylacetic acid.

[0080] Compound 5: t R At a time of 3.6 min, in negative mode, the MS mass spectrometer showed a quasi-molecular ion peak [MH]. - m / z 463.09C 21 H 20 O 12 Fragment ions [MH] - m / z 301.03 C 15 H 10 O7. Visible fragment ions [MHC] 15 H 10 O7]- The m / z peak at 301.03 is due to the quasi-molecular ion peak [MH]. - m / z 463.09 lost 162 Da (-C6H) 10 Obtained from O5, it is speculated that t R Compound 5, with a duration of 3.6 min, is 6-hydroxyluteolin-5-glucoside.

[0081] Compound 6: t R At a time of 3.6 min, in negative mode, the MS mass spectrometer showed a quasi-molecular ion peak [MH]. - m / z 463.09C 21 H 20 O 12 Fragment ions [MH] - m / z 300.03 C 15 H9O7. Visible fragment ions [MHC] 15 H9O7] - The m / z peak at 300.03 is due to the quasi-molecular ion peak [MH]. - m / z 463.09 lost 163Da (-C6H) 11 Obtained from O5, it is speculated that t R Compound 6, with a duration of 3.6 min, is quercetin-3-O-galactoside (hyperoside).

[0082] Compound 7: t R At 3.8 min, in positive mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. + m / z 479.08C 21 H 18 O 13 Fragment ions [M+H] + m / z 303.06 C 15 H 10 O7. Visible fragment ions [M+HC] 15 H 10 O7] + The m / z peak at 303.06 is due to the quasi-molecular ion [M+H]. + m / z 479.08 C 21 H 18 O 13 The loss of 176 Da (-C6H8O6) is used to speculate on t. R Compound 7, with a duration of 3.8 min, is quercetin-5-O-glucuronide.

[0083] Compound 8: t R At a time of 4 min, in positive mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. +m / z 189.09C 12 H 12 O2, fragment ions [M+H] + m / z 129.07 C 10 H8. Visible fragment ions [M+HC] 10 H8] + The m / z peak at 129.07 is due to the quasi-molecular ion peak [M+H]. + m / z 189.09 C 12 H 12 O2 was obtained by losing 60 Da (-C2H4O2), and it is speculated that t R Compound 8, which has a 4-min time, is methyl 5-phenyl-4-pentyne acid.

[0084] Compound 9: t R At 4.1 min, in negative mode, the MS mass spectrometer showed a quasi-molecular ion peak [MH]. - m / z 447.09C 21 H 20 O 11 Fragment ions [MH] - m / z 284.03 C 15 H9O6. Visible fragment ions [MHC] 15 H9O6] - The m / z peak at 284.03 is due to the quasi-molecular ion [MH]. - m / z 447.09 lost 163 Da (-C6H) 11 Obtained from O5, it is speculated that t R Compound 9, with a duration of 4.1 min, is baicalin-7-O-glucoside.

[0085] Compound 10: t R At 4.2 min, in positive mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. + m / z 463.12C 22 H 22 O 11 Fragment ions [M+H] + m / z 301.09 C 16 H 12 O6. Visible fragment ions [M+HC] 16 H 12 O6] + The m / z peak at 301.09 is due to the quasi-molecular ion peak [M+H]. + m / z 463.12 C 22 H 22 O 11 162 Da lost (-C6H) 10Obtained from O5, it is speculated that t R Compound 10, with a duration of 4.2 min, is irisin.

[0086] Compound 11: t R At 4.6 min, in positive mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. + m / z 179.07C 10 H 10 O3, fragment ions [M+H] + m / z 91.06 C7H6, visible fragment ion [M+H-C7H6] + The m / z peak at 91.06 is due to the quasi-molecular ion peak [M+H]. + m / z 179.07 C 10 H 10 O3 was obtained by losing 88 Da (-C3H4O3), and it is speculated that t R Compound 11, with a duration of 4.6 min, is coniferaldehyde.

[0087] Compound 12: t R At 4.9 min, in positive mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. + m / z 441.2C 24 H 28 N₂O₆, fragment ions [M+H] + m / z 177.05 C 10 H8O3. Visible fragment ions [M+HC] 10 H8O3] + The m / z peak at 177.05 is due to the quasi-molecular ion peak [M+H]. + m / z 441.2 C 24 H 28 N2O6 lost 264 Da (-C) 14 H 20 The result was obtained from N2O3, and it is speculated that t R Compound 12, with a time of 4.9 min, is N,N'-diferuloyl putrescine.

[0088] Compound 13: t R At a time of 5 min, in negative mode, the MS mass spectrometer showed a quasi-molecular ion peak [MH]. - 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]- The m / z peak at 179.07 is due to the quasi-molecular ion peak [MH]. - m / z 373.13 C 20 H 22 O7 lost 196Da (-C) 10 H 12 Obtained from O4), it is speculated that t R Compound 13, which has a 5-minute timeframe, is nortrachytic acid.

[0089] Compound 14: t R At 5.1 min, in positive mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. + m / z 461.11C 22 H 20 O 11 Fragment ions [M+H] + m / z 285.08 C 16 H 10 O5. Visible fragment ions [M+HC] 16 H 10 O5] + The m / z peak at 285.08 is due to the quasi-molecular ion peak [M+H]. + m / z 461.11 was obtained by losing 176 Da (-C6H8O6), and the t is speculated to be... R Compound 14, with a duration of 5.1 min, is baicalin (baicalin-7-O-glucuronide).

[0090] Compound 15: t R At 5.7 min, in positive mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. + m / z 243.1C 15 H 14 O3, fragment ions [M+H] + m / z 133.06 C9H8O. Visible fragment ion [M+H-C9H8O] + The m / z peak at 133.06 is due to the quasi-molecular ion [M+H]. + m / z 243.1 C 15 H 14 O3 was obtained by losing 110 Da (-C6H6O2), and it is speculated that t R Compound 15, with a duration of 5.7 min, is estrol (7,4'-homoflavane).

[0091] Compound 16: t R At 5.8 min, in positive mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. + m / z 237.18C 15 H24 O2, fragment ions [M+H] + m / z 133.1 C 10 H 12 Visible fragment ions [M+HC] 10 H 12 ] + The m / z peak at 133.1 is due to the quasi-molecular ion peak [M+H]. + m / z 237.18 C 15 H 24 O2 loss of 104 Da (-C5H) 11 Obtained from O2, it is inferred that t R Compound 16, with a concentration of 5.8 min, is 4α,12-dihydroxyguaian-6,10-diene.

[0092] Compound 17: t R At 5.8 min, in positive mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. + m / z 301.07C 16 H 12 O6, fragment ions [M+H] + m / z 166.03 C8H5O4. Visible fragment ion [M+H-C8H5O4] + The m / z peak at 167.03 is due to the quasi-molecular ion peak [M+H]. + m / z 301.07 C 16 H 12 O6 was obtained by losing 134 Da (-C8H6O2), and it is speculated that t R Compound 17, with a duration of 5.8 min, is 6-methylluteolin.

[0093] Compound 18: t R At 6.2 min, in positive mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. + m / z 389.12C 20 H 20 O8, fragment ions [M+H] + m / z 359.08 C 19 H 18 O7. Visible fragment ions [M+HC] 19 H 18 O3] + The m / z peak at 359.08 is due to the quasi-molecular ion [M+H]. + m / z 389.12 C 20 H 20O8 was obtained by losing 30 Da (-CH2O), and it is speculated that t R Compound 18, with a 6.2 min content, is 3-hydroxy-3',4',5,5',7-pentamethoxyflavone.

[0094] Compound 19: t R At 6.9 min, in positive mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. + m / z 235.17C 15 H 22 O2, fragment ions [M+H] + m / z 105.07 C7H4O. The visible fragment ion [M+H-C7H4O] is formed by the quasi-molecular ion peak [M+H]. + m / z 235.17 C 15 H 22 O2 loss of 130 Da (-C8H) 18 O), speculate t R Compound 19, with a duration of 6.9 min, is isocurcumeneol.

[0095] Compound 20: t R At 8.1 min, in positive mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. + m / z 317.21C 20 H 28 O3, fragment ions [M+H] + m / z 147.08 C 10 H 10 O. Visible fragment ions [M+HC] 10 H 10 O] + The m / z peak at 147.08 is due to the quasi-molecular ion [M+H]. + m / z 317.21 C 20 H 28 O3 loss of 170 Da (-C) 10 The result was obtained from H8O2, and it is speculated that t R Compound 20, with a duration of 8.1 min, is 7-hydroxydehydrorosinic acid.

[0096] Compound 21: t R At 8.2 min, in negative mode, the MS mass spectrometer showed a quasi-molecular ion peak [MH]. - m / z 315.2C 20 H 28 O3, fragment ions [MH] - m / z 271.2 C 17 H 20 O3. Visible fragment ions [MHC]17 H 20 O3] - The m / z peak at 271.2 is due to the quasi-molecular ion peak [MH]. - m / z 315.2 C 20 H 27 O3 was obtained by losing 44 Da (-C3H8), and it is speculated that t R Compound 21, with a time of 8.2 min, is inumakoic acid.

[0097] Compound 22: t R At 8.3 min, in positive mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. + m / z 191.11C 12 H 14 O2, fragment ions [M+H] + m / z 117.07 C9H8. Visible fragment ion [M+H-C9H8] + The m / z peak at 117.07 is due to the quasi-molecular ion peak [M+H]. + m / z 191.11 C 12 H 14 O2 was obtained by losing 74 Da (-C3H6O2), and it is speculated that t R Compound 22, with a duration of 8.3 min, is Z-artemisinin.

[0098] Compound 23: t R At 8.6 min, in positive mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. + m / z 315.2C 20 H 26 O3, fragment ions [M+H] + m / z 187.12 C 14 H 18 Visible fragment ions [M+HC] 14 H 18 ] + The m / z peak at 187.12 is due to the quasi-molecular ion peak [M+H]. + m / z 315.2 C 20 H 26 O3 was obtained by losing 128 Da (-C6H8O3), and it is speculated that t R Compound 23, with a duration of 8.6 min, is 7-oxodehydrorosinic acid.

[0099] Compound 24: t R At 8.7 min, in positive mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. + m / z 315.19C 20H 26 O3, fragment ions [M+H] + m / z 187.11 C 14 H 18 Visible fragment ions [M+HC] 14 H 18 ] + The m / z peak at 187.12 is due to the quasi-molecular ion peak [M+H]. + m / z 315.2 C 20 H 26 O3 was obtained by losing 128 Da (-C6H8O3), and it is speculated that t R Compound 24, with a duration of 8.6 min, is phyllanflexoid A.

[0100] The experimental results comprehensively reflect the direct-acting substances of pine cones in vivo. After oral administration of pine cone ethanol extract, only the original components were detected in plasma and fecal samples after administration, and no metabolites were detected. This suggests that the biotransformation process of the absorbed components in the body is weak, and the basic substances that exert the pharmacological effect may be the original components. This provides a reference for the pharmacodynamic material basis of pine cones and the research and development of innovative drugs.

[0101] The above are merely specific application examples of the present invention. The present invention also has other implementation methods. All technical solutions formed by equivalent substitution or equivalent transformation fall within the protection scope claimed by the present invention.

Claims

1. A method for detecting traditional Chinese medicine-derived components in pine cone alcohol extract, characterized in that, Comprising the following steps: The pine cone alcohol extract is orally administered to healthy animals, and blood plasma and fecal samples before and after administration are collected, UPLC-MS / MS detection analysis is performed, comparison is made with the pine cone alcohol extract, 8 blood-entering components are found in the blood plasma sample after administration, and 18 components are found in the fecal sample after administration, all of which are prototype components; The preparation method of the pine cone alcohol extract comprises the following steps: the pine cone is crushed, 85% ethanol is used for immersion, and the extract is reduced pressure concentrated to extract powder, thereby obtaining the pine cone alcohol extract; The preparation method of the fecal sample detection sample comprises the following steps: the fecal sample is taken, vacuum freeze-dried, and ground into powder; the sample powder is taken, the internal standard solution I is added in a mass-volume ratio of 1 mg:20 μL~1 mg:30 μL, intermittent repeated vortex is performed, centrifugation is performed, the supernatant is taken, and filtration is performed with a microporous filter membrane, thereby obtaining the sample; the internal standard solution I is prepared by dissolving L-2-chlorophenylalanine in 70% methanol solution; The preparation method of the blood plasma sample detection sample comprises the following steps: the blood plasma sample is taken, thawed on ice, vortexed and uniformly mixed, the blood plasma and the internal standard solution II are vortexed in a volume ratio of 1:4~1:8, centrifugation is performed, the supernatant is taken, and the supernatant is placed at-20℃, again centrifugation is performed, and the supernatant is taken, thereby obtaining the sample; the internal standard solution II is prepared by dissolving L-2-chlorophenylalanine in a 1:4 acetonitrile-methanol solution; The chromatographic detection conditions are as follows: the chromatographic column is Agilent SB-C18 1.8 µm, 2.1 mm×100 mm; The mobile phase is as follows: the A phase is a 0.1% ultrapure water-formic acid mixed solution with a volume ratio of formic acid to ultrapure water, and the B phase is a 0.1% acetonitrile-formic acid mixed solution with a volume ratio of formic acid to acetonitrile; the elution gradient is as follows: 0~9 min, 5%~95% B; 9~10 min, 95% B; 10~11 min, 95%~5% B; 11~14 min, 5% B; the flow rate is 0.35 mL / min; the column temperature is 40℃; and the injection amount is 2 μL.

2. The detection method according to claim 1, characterized in that, The blood plasma and fecal samples before and after administration are taken from the same animal.

3. The method of claim 1, wherein, The preparation method of the pine cone alcohol extract detection sample comprises the following steps: the pine cone alcohol extract and the internal standard solution I are vortexed in a mass-volume ratio of 1 mg:10 μL~1 mg:20 μL, centrifugation is performed, the supernatant is taken, and filtration is performed with a microporous filter membrane, thereby obtaining the sample.

4. The method of claim 1, wherein, The mass spectrometric detection conditions are as follows: the electrospray ion source temperature is 500℃; the ion spray voltage is 5500 V / -4500 V; the ion source gas I, gas II and gas curtain are 50, 60 and 25 psi respectively, the collision-induced ionization parameter is high, and the QQQ scan uses the MRM mode, and the collision gas is moderate.

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