Analysis method for medicinal components of pinecone extract

Abstract

The invention designs a method for analyzing active components of a pinecone extract, which can systematically analyze the in-vivo metabolic process of active components of the pinecone, clarify the pharmacodynamic mechanism of the active components of the pinecone, guide the clinical application of the active components of the pinecone, improve the safety of the active components of the pinecone and promote the development of related products of the pinecone.

Description

Background Art

[0001] Pine cones have a complex chemical composition, often containing multiple active ingredients. Their efficacy often relies on the synergistic effects of these components, which may be metabolized in different regions of the digestive tract. Sequential metabolism studies can reveal the metabolic pathways and interactions of these components in the body, leading to a better understanding of the pharmacodynamic mechanisms of traditional Chinese medicine. Sequential metabolism theory offers new perspectives and methods for studying these components, particularly in understanding metabolic processes, pharmacodynamic mechanisms, and safety.

[0002] At present, modern research has not yet involved the identification of metabolites in pine cones, nor the changes in other chemical components after entering the body, and the material basis of the efficacy is still unclear. Summary of the Invention

[0003] The technical problem to be solved by the present invention is: in view of the problems mentioned in the background technology, to provide an effective analysis and detection method for the basic medicinal substances of pine cone extract.

[0004] To achieve the above object, the technical solution adopted by the present invention is:

[0005] The present invention is a method for analyzing the medicinal components of a pine cone extract, comprising the following steps:

[0006] 1. Preparation and testing of pine cone samples

[0007] Extract 10 kg of pine cone coarse powder twice with 4 L of 85% ethanol each time, soaking for 24 hours. Filter, extract the residue with hot water, and remove the hot water extract by filtration. Add alkali solution to the residue at room temperature, extract, and filter to obtain an alkaline extract. Adjust the pH of the alkaline extract to 5 with acetic acid, concentrate, filter, and remove the precipitate. Precipitate the filtrate with 1 volume of ethanol for 24 hours, filter, and obtain a precipitate. Precipitate the filtrate with 2 volume of ethanol for 24 hours, filter, and obtain a precipitate. Precipitate the filtrate with 5 volume of ethanol for 24 hours, filter, and obtain a precipitate. Combine the three precipitates to obtain the pine cone extract.

[0008] Preparation of test sample: Take an appropriate amount of pine cone extract, dissolve it in Krebs-Ringer solution, and prepare a test sample solution with a concentration of 20 mg / ml.

[0009] 50 mg of the alcohol extract was vortexed for 1 min, 600 μL of -20°C pre-cooled 70% methanol aqueous solution was added, vortexed for 15 min, centrifuged (12000 rpm, 4°C) for 3 min, and the supernatant was filtered through a microporous filter membrane (0.22 μm). The sample was subjected to UPLC-MS / MS detection.

[0010] 2. Sequential Metabolism Studies

[0011] Four rats were randomly selected and anesthetized with 10% chloral hydrate. Blood was collected from the abdominal aorta and kept warm at 37°C to replenish the lost blood volume of the experimental rats.

[0012] 2.1.1 Gut microbiota metabolome

[0013] Another rat was randomly selected and anesthetized. The abdominal cavity was opened along the midline, and a 10 cm colon was selected. Small incisions were made in the front and back. The test substance was perfused into the colon and then ligated, sealing the drug solution in the colon. The portal vein was also ligated. The carotid artery was cannulated, and blood collected from the abdominal aorta was supplied via a syringe pump. The mesenteric vein was cannulated and blood was collected. Moistened gauze was placed over the rat's abdomen and heated with a heat lamp and a heating pad on the rat's back. After blood collection, it was stored in a refrigerator at -80°C. UPLC-MS / MS analysis was performed.

[0014] 2.1.2 Intestinal bacteria-free metabolome

[0015] The operation is the same as 2.1.1, except that before the drug solution enters the colon, 37℃ saline is used to flush out the colon contents, and air is continuously injected to expel the saline.

[0016] 2.1.3 Intestinal enzyme inhibition metabolomics

[0017] The operation is the same as 2.1.2, except that aminobenzotriazole, an inhibitor of CYP450 enzyme, is added to the normal saline and the drug solution.

[0018] 2.1.4 Liver metabolome

[0019] The procedure is the same as in 2.1.1, except that no blood supply is required and ligation of the portal vein is not required.

[0020] The UPLC-MS / MS detection results of step 1, step 2.1.1, step 2.1.2, step 2.1.3, and step 2.1.4 were compared and analyzed to understand the in vivo metabolic process of the active ingredients in pine cones.

[0021] 3. Gastrointestinal Stability Study: After pre-incubation of digestive fluids (artificial gastric fluid and artificial intestinal fluid) for 5 minutes, 1 ml of the test sample solution was added to 50 ml of artificial gastric fluid and 50 ml of artificial small intestinal fluid, respectively. Samples were collected at 0 h. Incubation was carried out at 37°C with shaking. Sampling was performed at 1, 2, 3, 4, and 5 h for the artificial gastric fluid group. Sampling was performed at 1, 2, 3, 4, 5, 6, and 7 h for the artificial small intestinal fluid group. 5 ml of the sample was collected at each time. Immediately after sampling, the pH was adjusted to 6-7 with 0.1 mol / L NaOH to terminate the reaction. The volume was then adjusted to 8 ml with purified water and stored at -80°C. Plasma samples were removed from the -80°C freezer, thawed on ice, and vortexed for 10 seconds to mix. A 50 μL sample was aspirated and placed into a 1.5 mL centrifuge tube. 300 μL of 20% acetonitrile-methanol extract containing an internal standard was added and vortexed for 3 minutes. The tube was then centrifuged at 12,000 rpm at 4°C for 10 minutes. Transfer 200 μL of the supernatant to another 1.5 mL centrifuge tube and place in a -20°C refrigerator for 30 minutes. Centrifuge again at 12,000 rpm at 4°C for 3 minutes. Transfer 180 μL of the supernatant to a vial for UPLC-MS / MS analysis. Compare the correlation between the test results at different time points to determine gastrointestinal stability. The closer the correlation coefficient is to 1, the stronger the correlation between the samples and the more stable the test product is in the gastrointestinal tract.

[0022] The chromatographic conditions and mass spectrometry conditions of the present invention are as follows:

[0023] Chromatographic conditions

[0024] Chromatographic column: Agilent SB-C18 1.8 μm, 2.1 mm x 100 mm; Mobile phase: Phase A: purified water (added with 0.1% formic acid), Phase B: acetonitrile (added with 0.1% formic acid); Elution gradient: 0-9 min, 5%-95% B; 9-10 min, 95% B; 10-11 min, 95%-5% B; 11-14 min, 5% B. Flow rate: 0.35 mL / min; Column temperature: 40°C; Injection volume: 2 μL.

[0025] Mass spectrometry conditions

[0026] The electrospray ionization (ESI) source temperature 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. QQQ scanning was performed in MRM mode, with the collision gas (nitrogen) set to medium. Substances were identified based on secondary spectral information, and quantitatively analyzed using multiple reaction monitoring with a triple quadrupole mass spectrometer. Analyst 1.6.3 software was used for data processing.

[0027] Beneficial effects

[0028] 1. This study used a sequential metabolism method combined with UPLC-MS / MS to analyze its prototype components and metabolites in rats, eliminating a large number of components that cannot be absorbed into the blood, making it easier to identify the components that exert its medicinal effects.

[0029] 2. Compared with traditional blood component detection, sequential metabolism can systematically depict the dynamic changes in the body and reveal its transformation and action mechanism at different metabolic stages.

[0030] 3. Through differential multiple screening, the data analysis process can be simplified, substances with insignificant differences can be excluded, the research scope can be narrowed, substances with real biological significance can be screened out, and potential medicinal ingredients can be identified.

[0031] 4. Sequential metabolism theory provides an important theoretical framework and methodological guidance for pine cone research. Through sequential metabolism research, we can systematically analyze the in vivo metabolic processes of pine cone active ingredients, clarify their pharmacodynamic mechanisms, guide their clinical application, improve their safety, and promote the development of pine cone-related products. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Differential classification information of different experimental groups

[0033] Figure 2 Correlation analysis of pine cone extract in artificial gastric juice at different times

[0034] Figure 3 Correlation analysis of pine cone extract in artificial intestinal fluid at different times

[0035] Specific implementation cases

[0036] The following examples may enable those skilled in the art to more fully understand the present invention, but the present invention is not limited to the scope of the examples.

[0037] Example 1:

[0038] 1. Materials

[0039] 1.1 Instrument

[0040] Eppendorf 5424R centrifuge; 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).

[0041] 1.2 Drug testing

[0042] Pine cones were collected from Dali City, Dali Prefecture, Yunnan Province and identified as Pinus yunnanensis Franoh., a species of the Pinaceae family, by Professor Duan Baozhong of the Department of Pharmacognosy, College of Pharmacy, Dali University. Samples were stored in the Department of Organic Pharmaceutical Chemistry, College of Pharmacy, Dali University. Chromatographically grade methanol, acetonitrile, and formic acid were purchased from Thermo Fisher Scientific (China) Co., Ltd.; purified water was purchased from Hangzhou Wahaha Group Co., Ltd.; KREBS-RINGER buffer (sterile) (lot number R21974), artificial gastric fluid (USP) (lot number R30386), artificial small intestinal fluid (containing pancreatin and phosphate) (lot number R30384), heparin sodium (lot number S12004), and 1-aminobenzotriazole (Shanghai Yuanye Biotechnology Co., Ltd., lot number S62316) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0043] 1.3 Animals

[0044] Sixteen SPF male Sprague-Dawley rats, weighing 230–250 g, were purchased from Hunan Slake Jingda Co., Ltd. (license number: SCXK(Xiang)2019-0004). The animals were housed under the following conditions: temperature (20 ± 2)°C, humidity (60% ± 5%), 12-h light-dark cycle, and free access to food. All animal experiments were approved by the Animal Welfare and Ethics Committee of Dali University (Ethics Approval Number: 2024-PZ-066).

[0045] 2 Methods

[0046] 2.1 Preparation of pine cone extract

[0047] Extract 10 kg of pine cone coarse powder twice with 4 L of 85% ethanol each time, soaking for 24 hours. Filter, extract the residue with hot water, and remove the hot water extract by filtration. Add alkali solution to the residue at room temperature, extract, and filter to obtain an alkaline extract. Adjust the pH of the alkaline extract to 5 with acetic acid, concentrate, filter, and remove the precipitate. Precipitate the filtrate with 1 volume of ethanol for 24 hours, filter, and obtain a precipitate. Precipitate the filtrate with 2 volume of ethanol for 24 hours, filter, and obtain a precipitate. Precipitate the filtrate with 5 volume of ethanol for 24 hours, filter, and obtain a precipitate. Combine the three precipitates to obtain the pine cone extract.

[0048] Dissolve an appropriate amount of pine cone extract in KR nutrient solution to prepare a 20 mg / ml test solution. Vortex 50 mg of the ethanol extract for 1 minute, add 600 μL of -20°C pre-cooled 70% methanol in water, vortex for 15 minutes, and centrifuge (12,000 rpm, 4°C) for 3 minutes. Filter the supernatant through a 0.22 μm microporous filter membrane for UPLC-MS / MS analysis.

[0049] 2.2 Chromatographic conditions

[0050] Chromatographic column: Agilent SB-C18 1.8 μm, 2.1 mm x 100 mm; Mobile phase: Phase A: purified water (added with 0.1% formic acid), Phase B: acetonitrile (added with 0.1% formic acid); Elution gradient: 0-9 min, 5%-95% B; 9-10 min, 95% B; 10-11 min, 95%-5% B; 11-14 min, 5% B. Flow rate: 0.35 mL / min; Column temperature: 40°C; Injection volume: 2 μL.

[0051] 2.3 Mass spectrometry conditions

[0052] The electrospray ionization (ESI) source temperature 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. QQQ scanning was performed in MRM mode, with the collision gas (nitrogen) set to medium. Substances were identified based on secondary spectral information, and quantitatively analyzed using multiple reaction monitoring with a triple quadrupole mass spectrometer. Analyst 1.6.3 software was used for data processing.

[0053] 2.4 Gastrointestinal stability study

[0054] After pre-incubation of the digestive fluid (artificial gastric juice and artificial intestinal juice) for 5 minutes, add 1 ml of the test solution to 50 ml of artificial gastric juice and 50 ml of artificial small intestinal juice, and take the 0h sample. Incubate with shaking at 37°C. The sampling time points of the artificial gastric juice group are 1, 2, 3, 4, and 5h. The sampling time points of the artificial small intestinal juice group are 1, 2, 3, 4, 5, 6, and 7h. Take 5 ml of sample each time. Immediately after sampling, adjust the pH to 6-7 with 0.1 mol / LNaOH to terminate the reaction. Use pure water to make up to 8 ml and store in a -80°C refrigerator. Take out the plasma sample from the -80°C refrigerator, thaw on ice, and vortex for 10 seconds to mix. Pipette 50 μL of sample and place it in

[0055] To a 1.5 mL centrifuge tube, add 300 μL of 20% acetonitrile-methanol extract containing the internal standard. Vortex for 3 minutes and centrifuge at 12,000 rpm at 4°C for 10 minutes. Remove 200 μL of the supernatant and transfer it to another 1.5 mL centrifuge tube. Incubate in a -20°C refrigerator for 30 minutes. Centrifuge again at 12,000 rpm at 4°C for 3 minutes. Remove 180 μL of the supernatant and store in an injection vial for UPLC-MS / MS analysis using the same conditions as in 2.2 and 2.3.

[0056] 2.5 Sequential metabolic studies

[0057] Four rats were randomly selected and anesthetized with 10% chloral hydrate. Blood was collected from the abdominal aorta and kept warm at 37°C to replenish the lost blood volume of the experimental rats.

[0058] 2.5.1 Gut Microbiota Metabolome

[0059] Another rat was randomly selected for anesthesia. The abdominal cavity was cut open along the midline of the abdomen, and a 10 cm colon was selected. A small incision was made in the front and back. The test sample was perfused into the colon and then ligated to seal the drug solution in the colon. The portal vein was also ligated. The carotid artery was cannulated, and blood collected from the abdominal aorta was supplied via a syringe pump. The mesenteric vein was cannulated and blood was collected. Moistened gauze was covered on the rat's abdomen and heated with a heat lamp and a heating pad on the rat's back. After blood collection, it was stored in a refrigerator at -80°C. UPLC-MS / MS detection conditions were the same as in 2.2 and 2.3.

[0060] 2.5.2 Intestinal Bacteria Metabolome

[0061] The operation is basically the same as 2.5.1, except that before the drug solution enters the colon, 37°C saline is used to flush out the colon contents, and air is continued to be injected to expel the saline.

[0062] 2.5.3 Intestinal enzyme inhibition metabolomics

[0063] The operation is basically the same as 2.5.2, except that aminobenzotriazole, an inhibitor of CYP450 enzyme, is added to the normal saline and the drug solution.

[0064] 2.5.4 Liver metabolome

[0065] The operation is basically the same as 2.5.1, except that no blood supply is required and no portal vein ligation is required.

[0066] 3 Results

[0067] The differences between the groups were screened using the VIP screening threshold > 1.0, the difference fold screening threshold > 2.0, and the pvalue screening threshold < 0.05 as conditions. The results showed that 161 differences were screened between the intestinal flora group and the intestinal sterile group, 150 differences were screened between the intestinal sterile group and the intestinal enzyme inhibition group, and 141 differences were screened between the intestinal flora group and the liver metabolome. The differences were merged and the duplicates were removed. The main categories of the differences were terpenes, phenolic acids, alkaloids, lignin and coumarin, flavonoids, quinones, and steroids. The relative content classification information of the differences is shown in Figure 1 .

[0068] 3.1 Gastrointestinal stability

[0069] Gastrointestinal metabolism is an important factor affecting the bioavailability of oral drugs. After oral administration, drugs will be affected by various factors in the gastrointestinal tract, such as the acidic environment of gastric juice, gastrointestinal digestive enzymes, etc. UPLC-MS / MS was used to detect the components and relative contents at each time point, and the results were subjected to correlation analysis. The closer the correlation coefficient is to 1, the stronger the correlation between samples. The correlation of pine cone extract in artificial gastric juice and artificial small intestinal fluid at each time point was greater than 0.8, as shown in Figure 2. Figure 2 Generally, drugs have a retention time of less than 2 hours in the stomach and less than 4 hours in the intestine. In this study, the incubation period was 5 hours in gastric juice and 7 hours in intestinal juice, so it is believed that the pine cone extract is stable in gastrointestinal juice.

[0070] 3.2 Prototype component difference analysis

[0071] 3.2.1 Identification of phenolic acid compounds

[0072] A total of 34 phenolic acid compounds were identified in the prototype components. Compound 18 (caffeic acid) was used as an example for mass spectrometry analysis. The molecular formula of compound 18 is C 10 H 10 O4, [MH] - The quasi-molecular ion peak in this mode was m / z 179.03, and the main fragment ion was m / z 134.04. Based on the references, it was speculated to be caffeic acid. There are three possible fragmentation pathways: (1) loss of one H2O molecule followed by the continuous loss of two hydrogen radicals; (2) loss of one H2O molecule and one hydrogen radical, followed by the loss of one C2H3 molecule and one hydrogen radical; (3) loss of one H2O molecule and one hydrogen radical, followed by the loss of one CO molecule.

[0073]

[0074] 3.2.2 Identification of flavonoids

[0075] A total of 29 flavonoid compounds were identified in the prototype components, including 1 flavanol, 11 flavonoids, 4 flavonols, 4 chalcones, 2 dihydroflavonoids, 2 isoflavones, 1 aurone and 4 other flavonoids. Compound 36 (isoschaftoside) was used as an example for mass spectrometry analysis. The molecular formula of compound 36 is C 26 H 28 O 14， [MH] + The quasi-molecular ion peak in this mode is m / z 565.16, and there are two possible fragmentation pathways: (1) loss of one molecule of C6H12O6 (2) loss of one molecule of C7H14O7, and then loss of two molecules of CO.

[0076]

[0077] 3.2.3 Identification of quinone compounds

[0078] A total of 4 quinone compounds were identified in the prototype components, including 2 quinones, 1 anthraquinone, and 1 phenanthrenequinone. Compound 65 (6-hydroxyrhein) was used as an example for mass spectrometry analysis. The molecular formula of compound 65 is C 15 H8O 7， [MH] - The quasi-molecular ion peak in this mode was m / z 299.02, and the main fragment ion was m / z 255.03. According to reference

[00] , it was speculated to be 6-hydroxyrhein. There are three possible fragmentation pathways: (1) continuous loss of two CO2 molecules; (2) loss of one CO2 molecule and then loss of one CO molecule; (3) loss of one CO molecule and then loss of one CO2 molecule.

[0079]

[0080] 3.2.4 Identification of coumarin and lignin compounds

[0081] A total of 18 coumarin and lignin compounds were identified in the prototype components, including 4 coumarins and 14 lignins. Compound 84 (esculetin) was used as an example for mass spectrometry analysis. The molecular formula of compound 64 is C9H6O4, [MH] -The quasi-molecular ion peak in this mode was m / z 177.02, and the main fragment ion was m / z 133.03. According to reference

[00] , it was speculated to be aesculetin. There are two possible fragmentation pathways: (1) loss of one molecule of CO; (2) loss of one molecule of CO2.

[0082]

[0083] 3.2.5 Identification of alkaloid compounds

[0084] A total of 32 alkaloid compounds were identified in the prototype component differences, including 7 indole alkaloids, 18 alkaloids, 1 aporphine alkaloid, 3 quinoline alkaloids, 2 pyridine alkaloids, and 1 phenolamine. Compound 140 (synephrine) was used as an example for mass spectrometry analysis. The molecular formula of compound 140 is C9H 13 NO2, [MH] + The quasi-molecular ion peak in this mode was m / z 168.1, and the main fragment ion was m / z 150.09. According to reference

[00] , it was speculated to be synephrine. There are two possible fragmentation pathways: (1) loss of one H2O molecule, then one CH3 molecule, and finally one CH3N molecule; (2) loss of two H2O molecules and one C2H3N molecule, and then one CH2 molecule.

[0085]

[0086] 3.2.6 Identification of terpenoids

[0087] A total of 70 terpenoid compounds were identified in the prototype components, including 9 triterpenes, 1 triterpenoid saponin, 36 diterpenes, 4 terpenoids, 16 sesquiterpenes, and 4 sesquiterpenes. Compound 204 (maquinone aglycone) was used as an example for mass spectrometry analysis. The molecular formula of compound 204 is C 11 H 16 O5, [MH] + The quasi-molecular ion peak in this mode was m / z 229.11, and the main fragment ion was m / z 151.08. According to reference

[00] , it was speculated to be aglycone. There are two possible fragmentation pathways: (1) loss of one MeOH molecule, then one H2O molecule, and finally one CO molecule; (2) loss of one MeOH molecule, then two H2O molecules; (3) loss of one H2O molecule, then one MeOH molecule, and finally one CO molecule; (4) loss of one H2O molecule, then one MeOH molecule, and finally one H2O molecule.

[0088]

[0089] All differentially expressed compounds were compared with the components identified in pine cone extracts, and substances present in pine cone extracts were screened for potential blood transfusion. The results are shown in Table 1. Compared with the intestinal microbiome group, the levels of compounds 1, 5, and 9 were downregulated in the sterile gut group, indicating absorption by the intestinal microbiome. Compounds 8, 20, and 39 were upregulated in the sterile gut group, suggesting possible absorption by the intestinal wall. Compared with the sterile gut group, compounds 41, 46, and 49 were downregulated in the intestinal enzyme inhibition group, suggesting possible absorption by intestinal enzymes. Compounds 5, 6, and 12 were upregulated in the intestinal enzyme inhibition group, suggesting possible absorption by the intestinal wall. Compared with the intestinal microbiome group, compounds 4, 5, and 8 were downregulated in the liver metabolome group, suggesting liver metabolism. Compounds 2, 3, and 84 were upregulated in the liver metabolome group, suggesting liver absorption.

[0090] Table 1 Prototype components differences in plasma samples of rats in different groups

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102] Note: ↑: differentially expressed substance up-regulated; ↓: differentially expressed substance down-regulated; -: not detected

[0103] 3.3 Metabolite differential analysis

[0104] All differentials were compared with the components identified in pine cone extracts, and substances not detected in pine cone extracts were screened out and considered to be metabolites entering the bloodstream. The results are shown in Table 2. Compared with the intestinal bacteria group, the levels of M1, M4, and M5 were downregulated in the sterile group, indicating that these components may be metabolites of intestinal bacteria. The levels of M2, M12, and M15 were upregulated in the sterile group, indicating that these components may be metabolized in the intestinal wall and subsequently converted by the intestinal flora. Compared with the sterile group, the levels of M8, M20, and M29 were downregulated in the intestinal enzyme inhibition group, indicating that these components may be metabolites of intestinal wall enzymes. The levels of M13, M16, and M18 were upregulated in the intestinal bismuth enzyme inhibition group, indicating that these components may be substrates of CYP-450 enzyme inhibitors. Compared with the intestinal flora group, M1, M3, M4, etc. were downregulated in the liver metabolome, indicating that the above components may be converted into other substances by the liver, and M12, M24, M32, etc. were upregulated in the liver metabolome, indicating that the above may be liver metabolites.

[0105] Table 2 Metabolite differences in plasma samples of rats in different groups

[0106]

[0107]

[0108]

[0109]

[0110] Note: ↑: differentially expressed substance up-regulated; ↓: differentially expressed substance down-regulated; -: not detected

[0111] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for analyzing the medicinal components of pine cone extract, comprising the following steps: Step A: Intestinal flora metabolome detection: One rat was randomly selected and anesthetized. The abdominal cavity was cut open along the midline of the abdomen, and a 10 cm colon was selected. A small incision was made in the front and back. The pine cone extract test sample was perfused into the colon and then ligated to seal the drug solution in the colon. The portal vein was also ligated. The carotid artery was cannulated, and blood was collected from the abdominal aorta through a syringe pump. The mesenteric vein was cannulated to collect blood. Moistened gauze was covered on the abdomen of the rat and heated with a heat lamp and a heating pad on the back of the rat. After blood collection, it was stored in a refrigerator at -80°C. UPLC-MS / MS analysis was performed. The abdominal aortic blood was collected by randomly selecting rats, anesthetizing the abdominal aorta, and keeping it warm at 37°C. Step B: Intestinal sterility metabolic panel test: The operation is the same as step A, except that before the drug solution enters the colon, the colon contents are flushed out with 37°C saline, and air is continuously added to expel the saline solution. Step C: Intestinal enzyme inhibition metabolomics test: The operation is the same as step B, except that aminobenzotriazole, an inhibitor of CYP450 enzymes, is added to the normal saline and the drug solution; Step D: Liver metabolomics analysis: The procedure is the same as step A, except that the blood supply from the abdominal aorta and ligation of the portal vein are not required. Step E: UPLC-MS / MS analysis of the pine cone extract test sample; Step F: Compare and analyze the UPLC-MS / MS detection results of step A, step B, step C, step D, and step E to analyze the in vivo metabolic process of the pine cone medicinal components.

2. The method for analyzing the active ingredients of pine cone extract according to claim 1, further comprising a gastrointestinal stability test, wherein the test method is as follows: After pre-incubation of artificial gastric juice and artificial small intestinal juice for 5 minutes, 1 ml of the test solution was added to 50 ml of artificial gastric juice and 50 ml of artificial small intestinal juice, and a 0 h sample was taken; the samples were shaken and incubated at 37 ° C. The sampling time points of the artificial gastric juice group were 1, 2, 3, 4, and 5 h; the sampling time points of the artificial small intestinal juice group were 1, 2, 3, 4, 5, 6, and 7 h, and 5 ml was sampled each time. After sampling, the pH was adjusted to 6-7 with 0.1 mol / L NaOH to terminate the reaction, and the volume was made up to 8 ml with purified water and stored in a -80 ° C refrigerator; the plasma sample was taken out of the -80 ° C refrigerator, thawed on ice, and vortexed for 10 seconds to mix; 50 μL of the sample was aspirated and placed in a 1.5 mL centrifuge tube, and 300 μL was added. The 20% acetonitrile-methanol extract containing the internal standard was vortexed for 3 minutes and centrifuged at 12,000 r / min at 4°C for 10 minutes. 200 μL of the supernatant was transferred to another 1.5 mL centrifuge tube and allowed to stand in a -20°C refrigerator for 30 minutes. The extract was centrifuged again at 12,000 r / min at 4°C for 3 minutes. 180 μL of the supernatant was transferred and stored in an injection vial for UPLC-MS / MS detection. The correlation between the detection results at different time points was compared to determine the gastrointestinal stability.

3. The method for analyzing the active ingredients of a pine cone extract according to any one of claims 1 to 2, wherein the UPLC-MS / MS detection conditions are: Chromatographic conditions: Chromatographic column: Agilent SB-C18 1.8 μm, 2.1 mm x 100 mm; mobile phase: phase A: purified water (added with 0.1% formic acid), phase B: acetonitrile (added with 0.1% formic acid); elution gradient: 0–9 min, 5%–95% B; 9–10 min, 95% B; 10–11 min, 95%–5% B; 11–14 min, 5% B; flow rate: 0.35 mL / min; column temperature: 40°C; injection volume: 2 μL; Mass spectrometry conditions: The electrospray ionization (ESI) source temperature 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. The substances were qualitatively identified based on the secondary spectral information, and quantitatively analyzed by multiple reaction monitoring with a triple quadrupole mass spectrometer. The mass spectrometry data were processed using Analyst 1.6.3 software.

4. A method for analyzing the medicinal components of a pine cone extract according to any one of claims 1 to 3, wherein the preparation method of the pine cone extract sample is as follows: Preparation of pine cone extract: 10 kg of pine cone coarse powder is extracted twice with 85% ethanol, using 4 L of ethanol each time, and soaked for 24 hours; filtered, the residue is extracted with hot water, and the hot water extract is filtered to remove the hot water extract; alkali solution is added to the residue at room temperature for extraction and filtered 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% ethanol for 24 hours, filtered, and a precipitate is obtained; the filtrate is further precipitated with 2% ethanol for 24 hours, filtered, and a precipitate is obtained; the filtrate is further precipitated with 5% ethanol for 24 hours, filtered, and a precipitate is obtained; the three precipitates are combined to obtain the pine cone extract; Preparation of test sample: Take an appropriate amount of pine cone extract, dissolve it in Krebs-Ringer solution, and prepare a test sample solution with a concentration of 20 mg / ml.

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