A method for detecting traditional chinese medicine-derived components in pine cone extract

Abstract

The application discloses a detection method of traditional Chinese medicine-derived components in pine cone extract. The pine cone extract is orally administered to healthy animals, and blood plasma and fecal samples before and after administration are collected. UPLC-MS / MS is used for detection and analysis. In the blood plasma sample after administration, 17 blood components are found, including 15 prototype components and 2 metabolites. In the fecal sample after administration, 12 components are found, including 10 prototype components and 2 metabolites. The application supplements the metabolism process and excretion path of the compounds with relatively large polarity in the pine cone in vivo, 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 extract. Background Technology

[0002] Pine cones are the spherical cones of various pine trees (Pinaceae family). They are brownish-red with multiple layers of scales and have expectorant, antitussive, and antiasthmatic effects, often used to treat symptoms such as chronic bronchitis, asthma, and cough with excessive phlegm. In recent years, with in-depth research on pine cones, it has been discovered that pine cone extracts also possess various pharmacological activities, including anti-HIV activity, anti-Lassa virus activity, and anti-tumor activity. However, current research mainly 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.

[0003] Blood component analysis plays a crucial role in drug development and clinical treatment. It is mainly used to assess the absorption, distribution, metabolism, and excretion (ADME) characteristics of drugs, as well as their efficacy and safety. However, despite its importance, blood component analysis also has some drawbacks and problems: (1) Blood tests 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 is dynamic, and a blood sample from a single time period cannot accurately reflect the long-term exposure to drugs; (3) Different individuals have different abilities to absorb, metabolize, and excrete drugs, and blood tests cannot fully explain these differences; (4) Blood tests cannot accurately reflect the influence of drug interactions on drug concentration. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a method for detecting the medicinal components in pine cone extract, which can comprehensively reflect the metabolism and excretion of pine cone extract in the body.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] A method for detecting medicinal components in pine cone extract includes the following steps: pine cone extract is orally administered to healthy animals, and plasma and fecal samples are collected before and after administration. UPLC-MS / MS analysis is performed, and 17 components were found in the plasma sample after administration, including 15 original components and 2 metabolites; 12 components were found in the fecal sample after administration, including 10 original components and 2 metabolites.

[0007] The preparation method of the pine cone extract is as follows: Pine cone powder is extracted with 85% ethanol, filtered, and the residue is extracted with hot water, then filtered to remove the hot water extract. At room temperature, an alkaline extract is obtained by adding an alkaline solution to the residue. The pH of the alkaline extract is adjusted to 5 with acetic acid, concentrated, filtered, and the precipitate is removed. The filtrate is precipitated with one volume of ethanol, filtered, to obtain precipitate D; the filtrate is then precipitated with two volumes of ethanol, filtered, to obtain precipitate E; the filtrate is then precipitated with five volumes of ethanol for 24 hours, filtered, to obtain precipitate F. Precipitates D, E, and F are combined to obtain the pine cone extract.

[0008] 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.

[0009] Preferably, the sample preparation method for pine cone extract detection includes the following steps: vortexing 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 through a microporous membrane for UPLC-MS / MS detection.

[0010] More preferably, the internal standard solution I is prepared by dissolving L-2-chlorophenylalanine in a 70% methanol aqueous solution.

[0011] More preferably, internal standard solution I needs to be pre-cooled at -20°C before use.

[0012] 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.

[0013] 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).

[0014] 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.

[0015] More preferably, internal standard solution I needs to be pre-cooled at -20°C before use.

[0016] Preferred chromatographic conditions for UPLC-MS / MS detection and analysis are as follows:

[0017] The chromatographic column was an Agilent SB-C18 1.8µm, 2.1mm × 100mm;

[0018] 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%).

[0019] Elution gradient: 0~9 min, 5%~95%B; 9~10 min, 95%B; 10~11 min, 95%~5%B; 11~14 min, 5%B.

[0020] Flow rate: 0.35 mL / min; column temperature: 40 °C; injection volume: 2 μL.

[0021] 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.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. This invention targets the highly polar components in pine cones, systematically reflecting their metabolism and excretion in the body. Simultaneously collecting plasma and fecal samples after drug administration allows for a more comprehensive and accurate analysis of their absorption, metabolism, and excretion pathways.

[0024] 2. Using the technical solution described in this invention, 17 blood-entering components were found in plasma samples after drug administration, including 15 original components and 2 metabolites; 12 components were found in fecal samples after drug administration, including 10 original components and 2 metabolites.

[0025] 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.

[0026] 4. Compared with traditional high-performance liquid chromatography-mass spectrometry (HPLC-MS), this 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, better meeting the needs of modern drug research for automated, high-throughput analytical methods. The sample detection can be completed in 16 minutes. This method can acquire all chromatographic information of the sample, and the chromatographic peaks are well separated, laying the foundation for subsequent analysis and processing of massive amounts of metabolic data.

[0027] 5. This invention uses pine cone 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 extract contains unknown components, it can be compared with data in the database. This overcomes the shortcomings of existing technologies, which require consulting literature to understand the possible substances and then comparing them with standards. When encountering unknown components that have not been reported in the species, they cannot be detected, which greatly limits their application.

[0028] 6. 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] 7. The detection method described in this invention is applicable to pine cones of various pine trees and has the advantages and characteristics of being easy to operate, having easily controllable experimental conditions, being fast and accurate, and having good reproducibility. Attached Figure Description

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

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

[0032] Figure 3 This is the total ion chromatogram of plasma administered in positive ion mode in Example 1.

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

[0034] Figure 5 This is the total ion chromatogram of feces administered in positive ion mode in Example 1.

[0035] Figure 6 This is the total ion chromatogram of the Yunnan pine cone extract in negative ion mode in Example 1.

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

[0037] Figure 8 This is the total ion chromatogram of plasma administered in negative ion mode in Example 1.

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

[0039] Figure 10 This is the total ion chromatogram of feces administered in negative ion mode in Example 1.

[0040] Figure 11 This is a Venn diagram of Yunnan pine cone extract, blank plasma, administered plasma, blank stool, and administered stool in Example 1 (in the diagram: YN-DEF is Yunnan pine cone 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 extract

[0051] 10 kg of crude Yunnan pine cone powder was extracted twice with 4 L of 85% ethanol each time, soaking for 24 h. After filtration, the residue was extracted with hot water, and the hot water extract was filtered off. At room temperature, 6 L of 1 mol / L sodium hydroxide solution was added to the residue for extraction, and the extract was filtered to obtain an alkaline extract. The pH of the alkaline extract was adjusted to 5 with acetic acid, concentrated, filtered, and the precipitate was removed. The filtrate was precipitated with 1 part ethanol for 24 h, filtered, to obtain precipitate D; the filtrate was then precipitated with 2 parts ethanol for 24 h, filtered, to obtain precipitate E; the filtrate was then precipitated with 5 parts ethanol for 24 h, filtered, to obtain precipitate F. Precipitates D, E, and F were combined to obtain the Yunnan pine cone extract (YN-DEF).

[0052] 2.2 Collect plasma and fecal samples

[0053] SD rats were acclimatized for 7 days. One day before 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 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 before administration (with free access to water), and administered Yunnan pine cone extract (dissolved in distilled water) 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-administration fecal sample (i.e., administered 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-administration plasma sample (i.e., administered plasma), stored at -80℃.

[0054] 2.3 Preparation of test samples

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

[0056] Vortex the 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 extract (internal standard solution I is prepared by dissolving 1 mg 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 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 extract, blank plasma, drug-treated plasma, blank stool, and drug-treated stool under positive ion mode. Figure 6-10 These are TIC graphs of Yunnan pine cone 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 extract, blank plasma, drug-treated plasma, blank stool, and drug-treated stool.

[0070] Seventeen components were found in the blood plasma samples after administration, including 15 original components and 2 metabolites; twelve components were found in the fecal samples after administration, including 10 original components and 2 metabolites, as detailed in Table 1.

[0071] Table 1

[0072]

[0073] 4.1 Prototype components of Yunnan pine cone extract in rat plasma

[0074] Compound 1: 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 C7H6 91.06. Visible fragment ion peak [M+H-C7H6] + The m / z peak at 91.06 is due to the quasi-molecular ion peak [M+H]. + m / z 179.07 lost 88 Da (-C3H4O3), speculated t R Compound 1, with a time of 4.6 min, is Coniferaldehyde.

[0075] Compound 2: t R At 4.2 min, in positive mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. + m / z 479.12C 22 H 22 O 12 Fragment ions [M+H] + m / z C 16 H 12 O7 317.06. Visible fragment ion peak [M+HC] 16 H 12 O7] + The m / z peak at 317.6 is due to the quasi-molecular ion peak [M+H]. + m / z 479.12 lost 162 Da (-C6H) 10 O5), speculate t R Compound 2, which has a duration of 4.2 min, is Isorhamnetin-7-O-glucoside (Brassicin).

[0076] Compound 3: t RAt 1.9 min, in positive mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. + m / z 268.1C 16 H 13 NO3 fragment ions [M+H] + m / z C8H7O2 136.06. Visible fragment ion peak [M+H-C8H7O2] + The m / z peak at 136.06 is due to the quasi-molecular ion peak [M+H]. + m / z 268.1 lost 132 Da (-C8H6NO), speculated t R Compound 3, with a concentration of 1.9 min, is 7-Methoxy-3-[1-(3-pyridyl)methylidene]-4-chromanone (7-methoxy-3-(3-pyridyl)methylene-4-chromanone).

[0077] Compound 4: 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 ion peaks [M+HC] 10 H8O3] + m / z is determined by the quasi-molecular ion peak [M+H]. + m / z 441.2 lost 264 Da (-C 14 H 20 N2O3), speculate t R Compound 4, with a time of 4.9 min, is N,N'-Diferuloylputrescine.

[0078] Compound 5: t R At 2.1 min, in positive mode, the MS mass spectrometer 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] + The m / z peak at 56.05 is due to the quasi-molecular ion [M+H]. + m / z 100.08 lost 44 Da (-CH2NO), speculated t R Compound 5, with a duration of 2.1 min, is 2-Piperidone.

[0079] Compound 7: t R At 3.9 min, in negative mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. - m / z 477.06C 21 H 18 O 13 Fragment ions [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 176 Da (-C6H8O6), speculated t R Compound 7, with a duration of 3.9 min, is Tricetin-7-O-glucuronide.

[0080] Compound 8: t R At 4.5 min, in positive mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. + m / z 197.12C 11 H 16 O3, fragment ions [M+H] + m / z C9H8O 133.1. Visible fragment ion peak [M+H-C9H8O] + The m / z peak at 133.1 is due to the quasi-molecular ion peak [M+H]. + m / z 197.12 lost 64 Da (-C2H8O2), speculated t R Compound 8, which has a 4.5 min elapsed time, is Loliolide.

[0081] Compound 10: 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 20 H 26 O3, fragment ions [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 128 Da (-C9H4O), speculated t RCompound 10, with a duration of 8.7 min, is phyllanflexoid A.

[0082] Compound 11: t R At 2.3 min, in positive mode, the MS mass spectrometer 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 46 Da (-CH2O2), speculated t R Compound 11, with a time of 2.3 min, is 2-(2,3-dihydroxy-3H-indol-3-yl)acetic acid.

[0083] Compound 12: 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 13 H 14 O. Visible fragment ion peaks [M+HC] 13 H 14 O] + The m / z peak at 187.12 is due to the quasi-molecular ion peak [M+H]. + m / z 315.2 lost 128Da (-C7H) 12 O2), speculate t R Compound 12, with a duration of 8.6 min, is 7-Oxodehydroabietic acid.

[0084] Compound 13: t R At 9.9 min, in negative mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. - m / z 343.08C 18 H 16 O7, fragment ions [M+H] - m / z 328.06 C 17 H 13 O7. Visible fragment ion peaks [M+HC] 17 H 13O7] - m / z is determined by the quasi-molecular ion peak [M+H]. - m / z 343.08 lost 15 Da (-CH3), speculated t R Compound 13, with a time of 9.9 min, is Usnic acid.

[0085] Compound 14: t R At 4.2 min, in negative mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. - m / z 447.09C 21 H 20 O 11 Fragment ions [M+H] - m / z 285.04 C 14 H8O5. Visible fragment ion peaks [M+HC] 14 H8O5] - m / z is determined by the quasi-molecular ion peak [M+H]. - m / z 447.09 lost 192 Da (-C7H) 12 O6), speculate t R Compound 14, with a duration of 4.2 min, is Kaempferol-7-O-glucoside.

[0086] Compound 15: t R At 5 min, in negative mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. - m / z 181.05C9H 10 O4, fragment ions [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 74 Da (-C2H4O3), speculated t R Compound 15, which has a 5-minute time limit, is vanillic acid methyl ester.

[0087] Compound 16: t R At 4.2 min, in positive mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. + m / z 465.14C 22 H 24 O 11 Fragment ions [M+H] + m / z 303.09 C 16 H 14O6. Visible fragment ion peaks [M+HC] 16 H 14 O6] + The m / z peak at 303.09 is due to the quasi-molecular ion [M+H]. + m / z 465.14 lost 162 Da (-C6H) 10 O5), speculate t R Compound 16, with a duration of 4.2 min, is 1,2,3-Trimethoxy-7-hydroxyxanthone-glucoside.

[0088] Compound 17: t R At 6.9 min, in positive mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. + m / z 403.14C 21 H 22 O8, fragment ions [M+H] + m / z C 20 H 19 O7 373.09 visible fragment ion peak [M+HC] 20 H 19 O7] + The m / z peak at 373.09 is due to the quasi-molecular ion peak [M+H]. + m / z 403.14 lost 30 Da (-CH2O), speculate t R Compound 17, with a duration of 6.9 min, is 3',4',5,5',6,7-Hexamethoxyflavone.

[0089] 4.2 Metabolites of Pine Cone Extract from Yunnan Province in Rat Plasma

[0090] Compound 6: t R At 8.3 min, in positive mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. + m / z 473.36C 30 H 48O4, 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 duration of 8.3 min, is 3α,21β,24-trihydroxyserrat-14-en-16-one.

[0091] Compound 9: t R At 2.9 min, in positive mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. + m / z 183.07C9H 10 O4, fragment ions [M+H] + m / z C9H8O3 165.06. Visible fragment ion peak [M+H-C9H8O3] + The m / z peak at 165.05 is due to the quasi-molecular ion peak [M+H]. + m / z 183.07 lost 18 Da (-H2O), speculated t R Compound 9, with a time of 2.9 min, is Dihydrocaffeic acid.

[0092] 3.3 Prototype components of Yunnan pine cone extract in rat feces

[0093] Compound 18: t R At 5.3 min, in positive mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. + m / z 235.1C 13 H 14 O4, fragment ions [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]. + 60 Da (-C2H4O2) is lost at m / z 235.1, suggesting t R Compound 18, with a duration of 5.3 min, is Dimethyllimettin.

[0094] Compound 19: t R At 4.4 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 C 16 H 12 O6 301.07 visible fragment ion peak [M+HC] 16 H 12 O6] + The m / z peak at 301.07 is due to the quasi-molecular ion peak [M+H]. + m / z 463.12 lost 162 Da (-C6H) 10 O5), speculate t R Compound 19, with a duration of 4.4 min, is Diosmetin-7-O-galactoside.

[0095] Compound 20: t R At 5.3 min, in negative mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. - m / z 357.13C 20 H 22 O6, fragment ions [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 lost 206 Da (-C) 12 H 14 O3), speculate t R Compound 20, with a time of 5.3 min, is Clemaphenol A.

[0096] Compound 21: t R At 3.3 min, in negative mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. - m / z 401.14C 18 H 26 O 10 Fragment ions [M+H] - m / z 269.1 C 13 H 18 O6. Visible fragment ion peaks [M+HC] 13 H 18 O6] -m / z is determined by the quasi-molecular ion peak [M+H]. - m / z 401.14 lost 132 Da (-C5H8O4), speculated t R Compound 21, with a time of 3.3 min, is Benzyl-(2''-O-xylosyl) glucoside.

[0097] Compound 22: t R At 4.5 min, in positive mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. + m / z 419.1C 20 H 18 O 10 Fragment ions [M+H] + m / z 287.05 C 15 H 10 O6. Visible fragment ion peaks [M+HC] 15 H 10 O6] + The m / z peak at 287.05 is due to the quasi-molecular ion [M+H]. + m / z 429.1 lost 132 Da (-C5H8O4), speculated t R Compound 22, with a duration of 4.5 min, is Kaempferol-3-O-arabinoside.

[0098] Compound 23: t R At 2.7 min, in negative mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. - m / z 415.12C 18 H 24 O 11 Fragment ions [M+H] - m / z 193.05 C 10 H 10 O4. Visible fragment ion peaks [M+HC] 10 H 10 O4] - The m / z peak at 193.05 is due to the quasi-molecular ion peak [M+H]. - m / z 401.14 lost 222.07 Da (-C8H) 14 O7), speculate t R Compound 23, with a 2.7 min estimator, is 3-O-Feruloyl Octose.

[0099] Compound 24: t RAt 3.9 min, in positive mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. + m / z 465.1C 21 H 20 O 12 Fragment ions [M+H] + m / z 303.05 C 15 H 10 O7. Visible fragment ion peaks [M+HC] 15 H 10 O7] + The m / z peak at 303.05 is due to the quasi-molecular ion [M+H]. + m / z 465.1 lost 162 Da (-C6H) 10 O5), speculate t R Compound 24, with a duration of 3.9 min, is Quercetin-5-O-β-D-glucoside.

[0100] Compound 25: t R At 5.2 min, in negative mode, the MS mass spectrometer 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] - The m / z peak at 103.06 is due to the quasi-molecular ion [M+H]. - m / z 147.05 lost 43.99 Da (-CO2), speculated t R Compound 25, with a 5.2 min content, is Cinnamicacid (hydrogenated cinnamic acid).

[0101] Compound 28: t R At 7 min, in positive mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. + m / z 287.24C 20 H 30 O, fragment ions [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 128 Da (-C9H4O), speculated t RCompound 28, which has a 7-minute time limit, is Ent-Kaurenal.

[0102] Compound 29: t R At 4.1 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 12 O5. Visible fragment ion peaks [M+HC] 16 H 12 O] + The m / z peak at 285.08 is due to the quasi-molecular ion peak [M+H]. + m / z 461.11 is missing 176.03a (-C6H8O6), suggesting t R Compound 29, with a duration of 4.1 min, is Wogonin-7-O-Glucuronide (Wogonoside).

[0103] 3.4 Metabolites of Yunnan pine cone extract in rat feces

[0104] Compound 26: t R At 0.8 min, in positive mode, the MS mass spectrometer showed a quasi-molecular ion peak [M+H]. + m / z 162.11C7H 15 NO3, fragment ions [M+H] + m / z 85.03 C4H4O2. Visible fragment ion peak [M+H-C4H4O2] + The m / z peak at 85.03 is due to the quasi-molecular ion peak [M+H]. + m / z 162.11 lost 77 Da (-C3H) 11 NO), speculate t R Compound 26, with a concentration of 0.8 min, is L-Carnitine.

[0105] Compound 27: t R At 2.4 min, in positive mode, the MS mass spectrometer 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] +The m / z peak at 118.07 is due to the quasi-molecular ion peak [M+H]. + m / z 188.07 lost 70 Da (-C3H2O2), speculated t R Compound 27, with a time interval of 2.4 min, is 3-Indoleacrylic acid.

[0106] The experimental results systematically reflect the in vivo active substances of Yunnan pine cone extract. After oral administration of Yunnan pine cone extract to rats, a large number of the original components were detected in their plasma and fecal samples, while the metabolites were few. This suggests that the effective components of Yunnan pine cone extract are mainly the original components, providing a reference for its pharmacodynamic material basis and innovative drug research and development.

[0107] 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 ingredients in pine cone extract, comprising the following steps: orally administering pine cone extract to healthy animals, collecting blood plasma and fecal samples before and after administration, and detecting and analyzing the samples by UPLC-MS / MS, 17 blood-entering components are found in the blood plasma sample after administration, including 15 original components and 2 metabolites; 12 components are found in the fecal sample after administration, including 10 original components and 2 metabolites, characterized in that: The preparation method of the pine cone extract is as follows: pine cone coarse powder is extracted with 85% ethanol, filtered, and the residue is extracted with hot water, and the hot water extract is removed by filtration; under room temperature conditions, alkali solution is added to the residue to obtain an alkaline extract; the pH value of the alkaline extract is adjusted to 5 with acetic acid, concentrated, filtered, and the precipitate is removed; The filtrate is precipitated with 1 times the amount of ethanol, filtered, to obtain precipitate D; the filtrate is precipitated with 2 times the amount of ethanol, filtered, to obtain precipitate E; the filtrate is precipitated with 5 times the amount of ethanol for 24 hours, filtered, to obtain precipitate F; and the precipitates D, E and F are combined, which is the pine cone extract; The feces sample detection sample preparation method comprises the following steps: taking the feces sample, vacuum freeze-drying, and grinding into powder; taking the sample powder, adding internal standard solution I in a mass-volume ratio of 1 mg:20 μL~1 mg:30 μL, intermittent repeated vortex, centrifugation, taking the supernatant and filtering with a microporous filter membrane to obtain the sample; the internal standard solution I is prepared by dissolving L-2-chlorophenylalanine in 70% methanol solution; The plasma sample detection sample preparation method comprises the following steps: taking the plasma sample, thawing on ice, vortexing, vortexing the plasma and internal standard solution II in a volume ratio of 1:4~1:8, centrifugation, taking the supernatant, standing at-20℃, centrifugation again, and taking the supernatant; 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 plasma and feces samples before and after administration are taken from the same animal.

3. The method of claim 1, wherein, The pine cone extract detection sample preparation method comprises the following steps: vortexing the pine cone extract and internal standard solution I in a mass-volume ratio of 1 mg:10 μL~1 mg:20 μL, centrifugation, and filtering the supernatant with a microporous filter membrane to obtain the sample.

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

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