CN109557203B - Method for detecting prototype components and metabolic components in rhizoma bletillae extract - Google Patents

Method for detecting prototype components and metabolic components in rhizoma bletillae extract Download PDF

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CN109557203B
CN109557203B CN201811437131.0A CN201811437131A CN109557203B CN 109557203 B CN109557203 B CN 109557203B CN 201811437131 A CN201811437131 A CN 201811437131A CN 109557203 B CN109557203 B CN 109557203B
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黄勇
李勇军
郑林
潘洁
巩仔鹏
李月婷
孙慧园
杨畅
陆苑
刘亭
马雪
刘春花
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Guizhou Medical University
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Abstract

The invention discloses a detection method of prototype components and metabolic components in a bletilla striata extract body, which comprises the following steps: after the rhizoma bletillae extract is orally taken by a rat, biological samples of serum, urine, excrement and bile are collected, and the prototype component and the metabolic component in the rat body after the rhizoma bletillae extract is orally taken are determined by adopting ultra-high performance liquid chromatography-quadrupole time of flight tandem mass spectrometry (UHPLC-Q-TOF-MS) technology detection and metabolic software analysis, so that the main existing form of the chemical component in the rhizoma bletillae extract in the body is determined, and a reference is provided for the research and quality control of the rhizoma bletillae innovative medicine.

Description

Method for detecting prototype components and metabolic components in rhizoma bletillae extract
Technical Field
The invention relates to the field of drug analysis, in particular to a detection method of prototype components and metabolic components in a bletilla striata extract.
Background
The oral administration is the main means of traditional Chinese medicine treatment, and modern researches prove that the oral administration can enter a blood circulation system through epithelial cells of gastrointestinal tracts and is distributed to various tissues and organs or target points, and can generate curative effect when reaching a certain blood concentration. In actual research work, many traditional Chinese medicines and compound medicines thereof are not detected as prototype medicine components in blood, but the medicine effect is still very obvious, and the possibility that metabolic components generated after the medicines are subjected to in vivo biotransformation play a therapeutic role is presumed. Therefore, the research on the in vivo metabolism of the traditional Chinese medicine has an important role in determining the existence form of the components absorbed into the body of the traditional Chinese medicine and the metabolic pathway and mechanism thereof, and further truly clarifying the substance basis of the effect of the traditional Chinese medicine.
The Bletilla striata is a dry tuber of Bletilla striata (Thunb.) Reichb.F of an orchid family plant, has the effects of astringing to stop bleeding, clearing heat and promoting diuresis, and reducing swelling and promoting granulation, is loaded in Chinese pharmacopoeia of the calendar edition, is mainly produced in Guizhou province, Sichuan province, Hunan province, Hubei province and other provinces, is widely applied to treating traumatic bleeding, phthisis hematemesis, digestive tract hemorrhage, pyocutaneous disease and pyogenic infections, chapped skin and the like, and has obvious curative effect. The rhizoma bletillae mainly contains a plurality of compounds such as bibenzyls, dihydrophenanthrenes, diphenanthrenes, glycosides and rhizoma bletillae polysaccharides, and has the effects of stopping bleeding, promoting wound healing, resisting ulcer, preventing intestinal adhesion, resisting bacteria, resisting tumors and the like. However, except that the rhizoma bletillae polysaccharide is proved to have the hemostatic effect, other action substances and action mechanisms of the rhizoma bletillae for hemostasis are not clear, and particularly, the research on the substances directly acting in vivo of the rhizoma bletillae is blank, so that the deep development of the rhizoma bletillae product is severely restricted. Therefore, the components absorbed into the body by the bletilla striata extract and the existing form thereof are researched, the structure type of the metabolite is analyzed, the chemical components directly acting in the body in the bletilla striata medicinal material are determined, and the foundation can be laid for the research and quality control of innovative medicaments of the bletilla striata medicinal material.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for detecting prototype components and metabolic components in a bletilla striata extract body,
the technical scheme of the invention is as follows:
a method for detecting prototype components and metabolic components in a rhizoma bletillae extract body comprises the steps of preparing and collecting one or a combination of serum, urine, excrement and bile biological samples after a rat orally takes the rhizoma bletillae extract, and determining the prototype components and the metabolic components in the rat body after the rhizoma bletillae extract is orally taken by adopting ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF-MS) technology detection and metabolic software analysis.
The preparation method of the rat serum sample after oral administration of the bletilla striata extract comprises the following steps: collecting blood from abdominal aorta after last administration, placing whole blood in 37 deg.C constant temperature water bath until yellow liquid is separated out at upper layer, centrifuging with a desk centrifuge, and collecting upper layer serum; mixing the sera of the same group to eliminate individual difference, and storing at-20 deg.C for use; taking rat serum, placing the rat serum in a centrifuge tube, supplementing methanol, carrying out vortex mixing oscillation, then carrying out ultrasonic treatment and centrifugal treatment in sequence, taking supernatant, drying the supernatant at 37 ℃ in nitrogen, adding methanol into a dried sample, adding methanol secondary precipitated protein according to the treatment method, adding 50% methanol aqueous solution to dissolve residues, carrying out vortex mixing oscillation, then carrying out ultrasonic treatment and centrifugal treatment in sequence, and taking supernatant UHPLC-Q-TOF MS for sample injection analysis.
The preparation method of the rat urine sample after oral administration of the bletilla striata extract comprises the following steps: collecting urine in 12, 24, 48 and 72h time periods respectively, recording the urine volume in each time period, and storing at-20 ℃ for later use; mixing urine at each time point in equal amount before sample treatment; the method comprises the following steps of putting rat urine into a centrifuge tube, supplementing methanol, carrying out vortex mixing and oscillation, carrying out ultrasonic treatment and centrifugal treatment in sequence, taking supernate, carrying out nitrogen blow-drying at 37 ℃, adding methanol into a blow-dried sample, adding methanol secondary precipitated protein according to the treatment method, adding 50% methanol aqueous solution to dissolve residues, carrying out vortex mixing and oscillation, carrying out ultrasonic treatment and centrifugal treatment in sequence, and taking supernate UHPLC-Q-TOF MS for sample injection analysis.
The preparation method of the rat fecal sample after oral administration of the bletilla striata extract comprises the following steps: collecting feces in 12, 24, 48 and 72h time periods respectively, recording the weight of the feces after drying in each time period, and storing at-20 ℃ for later use; before sample treatment, the feces at each time point are mixed in equal amount; taking dried rat feces, preparing 25% homogenate by using normal saline, sequentially carrying out ultrasonic treatment and centrifugal treatment, separating supernatant liquid, taking homogenate, supplementing methanol, carrying out vortex mixing, sequentially carrying out ultrasonic treatment and centrifugal treatment, drying supernatant liquid at 37 ℃ by using nitrogen, adding methanol into a dried sample, carrying out secondary protein precipitation according to the treatment method, dissolving residues by using a 50% methanol aqueous solution, carrying out ultrasonic treatment and centrifugal treatment sequentially after vortex mixing and oscillation, and taking supernatant liquid UHPLC-Q-TOF MS for sample injection analysis.
The preparation method of the rat bile sample after oral administration of the bletilla striata extract comprises the following steps: collecting bile after 0-4 h, 4-12 h, 12-24 h and 24-48 h in sections; storing the sample in a refrigerator at the temperature of-20 ℃ for later use; before sample treatment, bile at each time point is mixed in equal amount; adding 1% formic acid water into rat bile, extracting with ethyl acetate for 3 times, mixing extractive solutions, blow-drying at 37 deg.C with nitrogen, adding 50% methanol water solution to dissolve residue, vortex mixing, vibrating, sequentially ultrasonic processing and centrifuging, and sampling supernatant UHPLC-Q-TOF MS for analysis.
Further, the chromatographic conditions of UHPLC-Q-TOF-MS detection and analysis are as follows: the column was a Waters BEH C18(2.1 mm. times.50 mm,1.7 μm) column; mobile phase: 0.01% aqueous formic acid (a) -0.01% formic acid acetonitrile (B), gradient elution: 0-2 min, 5% B, Curve: initial; 2-5 min, 5% -15% of B, Curve: 6; 5-8 min, 15% -15% of B, Curve: 1; 8-10 min, 15% -45% of B, Curve: 6; 10-14 min, 45% -95% of B, Curve: 6; 14-15 min, 95% -5% of B; flow rate: 0.5 mL/min; column temperature: 35 ℃; the sample injection volume is 1 mu L; the mass spectrum conditions are as follows: an electrospray ion source, wherein the scanning mode is negative ion scanning (ESI-, m/z 50-1200); capillary voltage: 1.5 KV; taper hole voltage: 30V; ion source temperature: 100 ℃; the temperature of desolventizing gas is 300 ℃; the flow rate of the taper hole is 50L/h; collision energy is 20-30V; the flow rate of the desolventizing agent is 10L/min; and (3) data acquisition mode: MSE Continum; correcting sodium formate; and (3) correction mode: a sensitivity; the mass spectrum data acquisition and processing software comprises: masslynx V4.1 workstation; the scanning mode is MS central mode.
According to the method, 10 components in the bletilla striata extract are detected from a rat serum, urine, excrement and bile biological sample after oral administration of the bletilla striata extract, wherein the 10 components comprise 5 prototype components: gastodin, α -isobutylmalic acid, gymnoside I, dactylorhin A and militarine, and 5 metabolic components including hydrolysis, desugarization, glucuronidation, sulfation metabolites of the prototypic component.
The invention has the following beneficial effects: the technical scheme of the invention uses the time-of-flight mass spectrometer with the advantages of high repeatability, high flux, high ion transmission efficiency and the like to detect prototype components and metabolic components in the body of the rhizoma bletillae extract rat, so as to determine the components absorbed into the body and the existence form thereof, analyze the structure type of the metabolic products and clarify substances directly acting in the body. The rhizoma bletillae directly absorbs the components and metabolites in blood, is an effective component group of the rhizoma bletillae extract which exerts the drug effect in vivo, and the chemical components provide references for further developing the quality control, the pharmacokinetic research and the development of innovative drugs of the rhizoma bletillae.
Drawings
FIG. 1 shows UPLC chromatograms of control (A) and rhizoma Bletillae extract (B);
FIG. 2 shows the chemical structures of seven main components in the bletilla striata extract;
FIG. 3 is ESI-total ion flow chromatogram of blank serum sample (A) and rhizoma Bletillae extract administration serum sample (B);
FIG. 4 is an ESI-total ion flow chromatogram of a blank urine sample (C) and a rhizoma bletillae extract administration urine sample (D);
FIG. 5 is an ESI-total ion flow chromatogram of a blank stool sample (E) and a rhizoma bletillae extract administration serum sample (F);
FIG. 6 shows ESI-total ion flow chromatograms of blank bile sample (G) and rhizoma Bletillae extract administration bile sample (H).
FIG. 7 major metabolites and possible metabolic pathways of bletilla striata extract (1: desugarization; 2: hydrolysis; 3: double hydrolysis; 4: desugared sulfation; S: serum; U: urine; F: feces; B: bile).
Detailed Description
The following further describes embodiments of the present invention with reference to the drawings. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other. In the following, reference to a numerical value or a numerical ratio, unless otherwise specified, refers to a mass value or a mass ratio.
1. Material
1.1 reagent
The milerine reference (batch number: PS180413-03, content ≥ 98%) was purchased from Kjeldahl Biotech Co., Ltd; the gastodin reference (batch No. 110807-; the alpha-isobutylmalic and gymnoside I reference substances are all self-made in laboratories (the purity is more than or equal to 95 percent), and the structures and the purities of the alpha-isobutylmalic and gymnoside I reference substances are confirmed by IR, 1H NMR, MS and HPLC. Rhizoma bletillae extract (self-made), acetonitrile as chromatographic purity (Merck, Germany), formic acid as chromatographic purity, water as purified water, and other reagents as analytical purity.
1.2 instruments
UPLC-Q-TOF (Xevo G2-XS, Volter, USA, electrospray quadrupole tandem time-of-flight mass spectrometer, MassLynx V4.1 mass spectrometry workstation, UNIFI database), KQ-300DE numerical control ultrasonic cleaner (Volter technologies, Inc. of Sichuan), NA-5L Nitrogen air integrated machine (Kyowa technologies, Inc. of Beijing), EL204 electronic balance (Mettler-Torledo instruments, Inc.), GILSON pipettor (Kabo of Denmark), metabolism cage (imported by Techniplast, Italy).
1.3 Experimental animals
Healthy SD rats, both male and female, having a body weight of (220 ± 20) g, were provided by Chongqing Tengxin biotechnology limited [ certification number: SCXK (Cho) 2015 + 0001 ]. After the rats are introduced into a laboratory, the rats are subpackaged by 8 according to each rearing cage, female rats and male rats are separately reared, the illumination in an animal room is sufficient, an air conditioning and ventilation equipment system is good, the temperature is controlled to be 18-25 ℃, and the relative humidity is 50-60%. The laboratory regularly disinfects and sterilizes the animal room, and the rat is raised in the animal room for 1 week and used for animal experiments after adapting to the environment of the animal room.
2. Method of producing a composite material
2.1 preparation of bletilla striata extract
Weighing rhizoma bletilla, extracting with 4 times of 95% ethanol solution under reflux for 3 times, 2 hr/time, filtering, mixing filtrates, concentrating to obtain extract, dissolving the extract with water, subjecting to D101 macroporous adsorbent resin column chromatography, eluting with water, eluting with 80% ethanol solution, collecting eluate, concentrating under reduced pressure, and microwave vacuum drying the residue to obtain effective components of rhizoma bletilla with extraction rate of 6.82%.
2.2 chromatographic conditions
The guard column was Waters VanGuard BEH C18(2.1 mm. times.5 mm,1.71.7 μm); the column was a Waters BEH C18(2.1 mm. times.50 mm,1.7 μm) column; mobile phase: 0.01% aqueous formic acid (a) -0.01% formic acid acetonitrile (B), gradient elution: 0-2 min, 5% B, Curve: initial; 2-5 min, 5% -15% of B, Curve: 6; 5-8 min, 15% -15% of B, Curve: 1; 8-10 min, 15% -45% of B, Curve: 6; 10-14 min, 45% -95% of B, Curve: 6; 14-15 min, 95% -5% of B, Curve: 1; flow rate: 0.5 mL/min; column temperature: 35 ℃; the injection volume was 1. mu.L.
2.3 Mass Spectrometry conditions
An electrospray ion source; the scanning mode is negative ion scanning (ESI-, m/z 50-1200); the capillary voltage is 1.5 kV; the ion source temperature is 100 ℃; the voltage of the taper hole is 30V; the temperature of desolventizing gas is 300 ℃; the flow rate of the taper hole is 50L/h; collision energy is 20-30V; the flow rate of the desolventizing agent is 10L/min; and (3) data acquisition mode: MSE Continum; correcting sodium formate; and (3) correction mode: a sensitivity; the mass spectrum data acquisition and processing software comprises: masslynx V4.1 workstation. The scanning mode is MS central mode.
2.4 Collection of biological samples
2.4.1 Collection of serum
12 healthy SD rats with similar growth conditions, half male and half female, and body weight (220 +/-20) g, are selected and normally bred for one week to adapt to the laboratory environment. Randomly divided into 2 groups (administration group and blank group), 6 groups/group, and fed in metabolism cage, and fasting for 12h before administration, and free drinking water. The effective parts of rhizoma bletillae and 1% CMC-Na solution are respectively administered. The preparation is administered by gavage for 3 days at a dose of 44 g.Kg < -1 > (crude drug) each time, 2 times per day, and feeding 1% CMC-Na solution with the same volume as the blank group. Collecting blood from abdominal aorta 30min after last administration, placing whole blood in 37 deg.C constant temperature water bath until yellow liquid is separated out at upper layer, centrifuging with desktop centrifuge at 5000r min-1(2683g) for 10min, and collecting upper layer serum. Mixing the sera of the same group to eliminate individual difference, and storing at-20 deg.C for use.
2.4.2 Collection of urine and feces
Selecting 6 healthy SD rats with similar growth conditions, half male and half female, and weight (220 +/-20) g, normally feeding for one week to adapt to laboratory environment, feeding in a metabolism cage, fasting for 12h before administration, and freely drinking water. Urine and feces are collected for 24 hours respectively, and then rhizoma bletillae effective parts and 1% CMC-Na solution are given respectively. The preparation is administered by gavage for 3 days at a dose of 44 g.Kg < -1 > (crude drug) each time, 2 times per day, and feeding 1% CMC-Na solution with the same volume as the blank group. Urine and feces were collected for 12, 24, 48, 72h periods, respectively, and the volume of urine and the weight of dried feces were recorded for each period. Storing at-20 deg.C for use. Before sample treatment, urine and feces were mixed in equal amounts at each time point.
2.4.3 Collection of bile
Taking 12 healthy SD rats, randomly dividing into 2 groups (drug administration group and blank group), fasting for 12h before operation, performing bile duct intubation under Uratan anesthesia, selecting a silicone rubber tube (1.5mm) with small inner diameter, finding out a bile duct, cutting a small opening, inserting the bile duct intubation, fixing the bile duct intubation, and suturing. The rat limbs were fixed to a metabolic cage while maintaining smooth bile flow. According to the weight ratio of 44 g.Kg-1The rats are infused with the dose of rhizoma bletillae effective parts, and the blank group is infused with 1% CMC-Na solution with the same volume as the rats. And collecting bile after 0-4 h, 4-12 h, 12-24 h and 24-48 h in a segmented manner. The samples were stored in a-20 ℃ freezer for future use. Before sample treatment, bile was mixed in equal amounts at each time point.
2.5 treatment of biological samples
2.5.1 method for processing serum and urine samples
Taking 1mL of rat serum or urine, placing the rat serum or urine in a 5mL inlet plastic centrifuge tube, supplementing 4mL of methanol, performing vortex mixing and oscillation for 2min, performing ultrasonic treatment for 5min at 15000rpm, centrifuging for 10min, taking supernatant, drying the supernatant with nitrogen at 37 ℃, adding 1mL of methanol into a dried sample, adding 1mL of methanol for secondary protein precipitation according to the treatment method, adding 200 mu L of 50% methanol aqueous solution to dissolve residues, performing vortex mixing and oscillation for 2min, performing ultrasonic treatment for 5min at 15000rpm, centrifuging for 10min, and taking supernatant UHPLC-Q-TOF for sample injection analysis.
2.5.2 bile sample processing method
Adding 1mL of 1% formic acid water into 1mL of rat bile, adding 2mL of ethyl acetate, extracting for 3 times, combining extracts, drying the extracts at 37 ℃ with nitrogen, adding 200 mu L of 50% methanol water solution to dissolve residues, carrying out vortex mixing and oscillation for 2min, carrying out ultrasonic treatment for 5min, carrying out centrifugation for 10min at 15000rpm, and taking supernatant fluid to carry out sample injection analysis by UHPLC-Q-TOF MS.
2.5.3 fecal sample treatment method
Taking 1g of dried rat feces, preparing 25% homogenate with physiological saline, performing ultrasound for 5min, then performing 5000rpm (centrifuging for 10min, separating supernatant, taking 1mL of homogenate, supplementing 4mL of methanol, performing vortex mixing for 1min, performing ultrasound for 5min, performing 15000rpm (centrifuging for 10min, drying supernatant with nitrogen at 37 ℃), adding 1mL of methanol into a dried sample, performing secondary protein precipitation according to the treatment method, dissolving residues with 200 muL of 50% methanol aqueous solution, performing vortex mixing and oscillation for 2min, performing ultrasound for 5min, performing 15000rpm, centrifuging for 10min, and taking supernatant, performing sample injection analysis by using UHPLC-Q-TOF MS.
3. Metabolism of bletilla striata extract in rat
3.1 analysis of serum, urine, feces and bile samples
Taking a bletilla striata extract as an experimental object, collecting serum, urine, excrement and bile of a rat after drug administration, and detecting each biological sample by using a high-resolution quadrupole-time-of-flight tandem mass spectrometer to obtain blank serum and drug-containing serum maps; blank urine and medicine-containing urine maps; blank stool and medicated stool atlas; blank bile and medicated bile. The components give better response signals in ESI-mode.
3.2 identification and analysis of metabolites in serum, urine, feces and bile
The MassLynx V4.1 mass spectrum workstation and the UNIFI database are combined to rapidly analyze the metabolites of the rhizoma bletillae after the rhizoma bletillae is perfused into the rat, the biotransformation rule of the rhizoma bletillae effective part in the rat is researched, the possible metabolic pathways of the active or potential active ingredients in the medicine are presumed, and clues are provided for clarifying the material basis and the action mechanism of the pharmacological action of the rhizoma bletillae.
3.2.1 prototype compositional characterization
C1: at 0.55min there is M/z 331.1018[ M + HCOO]-Peak compounds, showing major fragment ion peaks at m/z 285.0990, 123.0443, with a chemical formula predicted by Single Mass Analysis as C14H19O9T is determined from the same as the gastodin controlRC1 at 0.55min is gastodin.
C2: at 4.23min there is M/z 189.0756[ M-H ]]-Peak Compounds, showing the major fragment ion peak of m/z 129.0556, the chemical formula predicted by Single Mass Analysis is classified as C8H13O5T is determined as the same as the control of α -isobutylmalic acidRC2 at 4.23min is alpha-isobutylmalic acid.
C4: at 8.72min there was M/z 457.1710[ M-H ]]-Peak Compounds, showing the major fragment ion peak of m/z 285.1025, the chemical formula predicted by Single Mass Analysis is classified as C21H29O11From which T was determined, as in the gymsoside I controlRC4 at 8.72min was gymsoside I.
C5: at 9.32min there was M/z 887.3223[ M-H ]]-Peak compounds, showing major fragment ion peaks of 725.2758, 457.1754, 189.0765, with a chemical formula predicted by Single Mass Analysis as C40H55O22T was determined as in the dactylorhin A controlRC5 at 9.32min is dactylorhin A.
C6: as can be seen from the difference map obtained by Masslynx V4.1 software, M/z 725.2648[ M-H ] exists at 10.06min]Peak Compound, showing the major fragment ion peak of m/z 457.1797, the chemical formula predicted by Single Mass Analysis being C34H45O17Identical to the mileanine control, thus determining that C6 with a TR of 10.06min is mileanine.
3.2.2 identification of ester bond hydrolysates
M2: at 3.31min there was M/z 351.1286[ M-H ]]-Peak compounds, showing major fragment ion peaks at m/z 189.0765, 129.0556, predicted by Single Mass AnalysisHas the chemical formula of C14H23O10M2 is presumed to be a metabolite of hydrolysis of ester bonds at C-1 and C-4 positions of dactylorhin A or a metabolite of hydrolysis of ester bonds at C-4 position of M4.
M4: a compound with M/z 619.2284[ M-H ] -peak at 6.32min showed major fragment ion peaks of M/z 439.1647, 189.0793, and a loss of one molecule of glucose in formula M4 predicted by Single Mass Analysis showed a fragment ion peak of M/z 439.1647. As is known from literature, the compounds esterified at the C-4 position generate ions which lose one molecule of glucose, while the compounds esterified at the C-1 position generate substantially no ions [1], so M4 is presumed to be a metabolite of the hydrolysis of the ester bond at the C-1 position of dactylorhin A.
C4: c3 was identified as gymnoside I. Analyzing the chemical structure of the milearine, so that the C-1 position and the C-4 position of the milearine are ester bonds; analysis of the chemical structure of gymoside I revealed that gymoside I C-1 was an ester bond. C4 is presumed to be a metabolite of the hydrolysis of the ester bond at the C-4 position of mileanine.
C2: c2 was identified as α -isobutylmethalic acid. C2 is presumed to be a metabolite of hydrolysis of the ester bond at C-1 position of gymnoside I or a metabolite of hydrolysis of the ester bond at C-1 and C-4 positions of mileanine.
C1: c1 was identified as gastodin. C1 is presumed to be a metabolite of hydrolysis of the ester bond at C-1 position of gymnoside I or a metabolite of hydrolysis of the ester bond of militarine or hydrolysis of the ester bond at C-4 position of M4 or hydrolysis of the ester bond of dactylorhin A.
3.2.3 identification of desugarized product
C6: c6 was identified as mileanine. The molecular weight of milearine is 162 lower than that of dactylorhin A, and analysis of the chemical structure of dactylorhin A reveals that the C-2 position of dactylorhin A is substituted by glucosyl, so C6 is assumed to be a metabolite of dactylorhin A with a C-2 position of glycosidic bond broken and a loss of anhydroglucose.
C2: c2 was identified as α -isobutylmethalic acid. The molecular weight is 162% lower than that of M2, and C2 is assumed to be a metabolite that the glycosidic bond at the-2 position of M2C is broken to lose one anhydroglucose.
3.2.4 desulphation product identification
C6: at 0.58min there is M/z 203.0023[ M-H ]]-Compounds of peaks showingmajor fragment ion peak of m/z 123.0443, chemical formula predicted by Single Mass Analysis as C7H7O5S, M1 is presumed to be a metabolite of sulfation after desugaring of the gastrodin. Since the specific information is not comprehensive enough, the site where metabolism and transformation occur cannot be determined.
3.2.5 sulfation product identification
M5: at 7.93min there was M/z 321.0447[ M-H ]]-Peak compounds, more than 80 Mass% diphenoanthrene 5, showed a major fragment ion peak of m/z 241.0866 with a chemical formula C predicted by Single Mass Analysis15H13O6S, M5 is presumed to be a metabolite of dihydrophenanthrene 5 sulfation. Since the specific information is not comprehensive enough, the site where metabolism and transformation occur cannot be determined.
3.2.6 glucuronic acid product identification
M3: at 6.22min there was M/z 417.1174[ M-H ]]-Peak Compounds, 176 greater in Mass than dihydrophenanthrene 5, show a major fragment ion peak at m/z 241.0834, with a chemical formula C predicted by Single Mass Analysis21H21O9M3 is presumed to be a dihydrophenanthrene 5 glucuronidated metabolite. Since the specific information is not comprehensive enough, the site where metabolism and transformation occur cannot be determined.
3.3 summary of information on major metabolites in serum, urine, feces and bile
After the treatment by using Masslynx V4.1 software, the information of the main metabolites of the bletilla striata extract in rat serum, urine, feces and bile is shown in Table 1. Including 5 prototype components: gastodin (C1), α -isobutylmalic acid (C2), gymsoside I (C4), dactylorhin A (C5), and militarine (C6), and 5 metabolic components including hydrolysis, desugarization, glucuronidation, sulfation metabolites of the prototype components.
TABLE 1 Main metabolites in serum, urine, feces and bile of rats orally administered with bletilla striata extract (S: serum U: urine F: feces B: bile;. vs. standard)
Figure BDA0001884044480000111
3.3 metabolic pathway analysis
In this study, 10 components including 5 prototype components were detected from biological samples of serum, urine, feces, bile from rhizoma bletillae extract administered orally to rats: gastodin (C1), α -isobutylmalic acid (C2), gymnoside I (C4), dactylorhinA (C5), and militarine (C6); and 5 metabolic components, which show that the effective parts of the bletilla striata can be subjected to biotransformation in vivo, and the metabolic products mainly comprise hydrolysis, desugarization, glucuronidation and sulfation. In the research, 5 prototype components are identified in the metabolite, and the chemical structures of the components are analyzed to know that gymnoside I (C4), dactylorhin A (C5) and amitarine (C6) are benzyl succinate glycoside compounds and are monoester or diester derivatives formed by 2-isobutyl malic acid and 4-glucosyl benzyl alcohol; alpha-isobutylmalic acid (C2) is 2-isobutylmalic acid; gastodin (C1). The benzyl succinate glycoside compounds are mainly subjected to the breakage of glycosidic bonds and glucosyloxybenzyl. Therefore, metabolic transformation relation may exist among the benzyl succinate glycosides. The content of the Militarine in the effective parts of the bletilla striata is up to 26 percent, and the Militarine basically disappears from chromatograms containing drug serum, urine, feces and bile. The rhizoma bletillae extract is suggested to be absorbed into the body to generate a violent biotransformation process, so that the metabolite of the rhizoma bletillae extract can be the active ingredient of the rhizoma bletillae extract besides the original ingredient.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.

Claims (1)

1. A detection method for prototype components and metabolic components in a bletilla striata extract body is characterized in that: after the common bletilla pseudobulb extract is orally taken by a rat, one or a combination of serum, urine, excrement and bile biological samples is prepared and collected, and the prototype component and the metabolic component in the rat body after the common bletilla pseudobulb extract is orally taken are determined by adopting ultra-high performance liquid chromatography-quadrupole time of flight tandem mass spectrometry (UHPLC-Q-TOF-MS) technology detection and metabolic software analysis;
the preparation method of the rat serum sample after oral administration of the bletilla striata extract comprises the following steps: collecting blood from abdominal aorta after last administration, placing whole blood in 37 deg.C constant temperature water bath until yellow liquid is separated out at upper layer, centrifuging with a desk centrifuge, and collecting upper layer serum; mixing the sera of the same group to eliminate individual difference, and storing at-20 deg.C for use; taking rat serum, placing the rat serum in a centrifuge tube, supplementing methanol, carrying out vortex mixing oscillation, then carrying out ultrasonic treatment and centrifugal treatment in sequence, taking supernatant, drying the supernatant at 37 ℃ in nitrogen, adding methanol into a dried sample, adding methanol secondary precipitated protein according to the treatment method, adding 50% methanol aqueous solution to dissolve residues, carrying out vortex mixing oscillation, then carrying out ultrasonic treatment and centrifugal treatment in sequence, and taking supernatant UHPLC-Q-TOF MS for sample injection analysis;
the preparation method of the rat urine sample after oral administration of the bletilla striata extract comprises the following steps: collecting urine in 12, 24, 48 and 72h time periods respectively, recording the urine volume in each time period, and storing at-20 ℃ for later use; mixing urine at each time point in equal amount before sample treatment; taking rat urine, placing the rat urine in a centrifuge tube, supplementing methanol, carrying out vortex mixing and oscillation, then carrying out ultrasonic treatment and centrifugal treatment in sequence, taking supernatant, drying the supernatant at 37 ℃ in nitrogen, adding methanol into a dried sample, adding methanol secondary precipitated protein according to the treatment method, adding 50% methanol aqueous solution to dissolve residues, carrying out vortex mixing and oscillation, then carrying out ultrasonic treatment and centrifugal treatment in sequence, and taking supernatant UHPLC-Q-TOF MS for sample injection analysis;
the preparation method of the rat fecal sample after oral administration of the bletilla striata extract comprises the following steps: collecting feces in 12, 24, 48 and 72h time periods respectively, recording the weight of the feces after drying in each time period, and storing at-20 ℃ for later use; before sample treatment, the feces at each time point are mixed in equal amount; taking dried rat feces, preparing 25% homogenate by using normal saline, sequentially carrying out ultrasonic treatment and centrifugal treatment, separating supernatant liquid, taking homogenate, supplementing methanol, carrying out vortex mixing, sequentially carrying out ultrasonic treatment and centrifugal treatment, drying supernatant liquid by blowing nitrogen at 37 ℃, adding methanol into a dried sample, carrying out secondary protein precipitation according to the treatment method, dissolving residues by using 50% methanol aqueous solution, carrying out vortex mixing oscillation, sequentially carrying out ultrasonic treatment and centrifugal treatment, and taking supernatant liquid UHPLC-Q-TOF MS for sample injection analysis;
the preparation method of the rat bile sample after oral administration of the bletilla striata extract comprises the following steps: collecting bile after 0-4 h, 4-12 h, 12-24 h and 24-48 h in sections; storing the sample in a refrigerator at the temperature of-20 ℃ for later use; before sample treatment, bile at each time point is mixed in equal amount; adding 1% formic acid water into rat bile, adding ethyl acetate, extracting for 3 times, mixing the extracts, drying at 37 deg.C with nitrogen, adding 50% methanol water solution to dissolve residues, vortex mixing, vibrating, sequentially performing ultrasonic treatment and centrifugal treatment, and sampling and analyzing supernatant UHPLC-Q-TOF MS;
the chromatographic conditions of UHPLC-Q-TOF-MS detection and analysis are as follows: the chromatographic column is a Waters BEH C18, 2.1mm × 50mm,1.7 μm column; mobile phase: 0.01% aqueous formic acid a-0.01% formic acid acetonitrile B, gradient elution: 0 → 2min, 5% B; 2 → 5min, 5% → 15% B; 5 → 8min, 15% → 15% B; 8 → 10min, 15% → 45% B; 10 → 14min, 45% → 95% B; 14 → 15min, 95% → 5% B; flow rate: 0.5 mL/min; column temperature: 35 ℃; the sample injection volume is 1 mu L; the mass spectrum conditions are as follows: an electrospray ion source adopts a scanning mode of negative ion scanning, ESI-, and m/z is 50-1200; capillary voltage: 1.5 KV; taper hole voltage: 30V; ion source temperature: 100 ℃; the temperature of desolventizing gas is 300 ℃; the flow rate of the taper hole is 50L/h; collision energy is 20-30V; the flow rate of the desolventizing agent is 10L/min; and (3) data acquisition mode: MSE Continum; correcting sodium formate; and (3) correction mode: a sensitivity; the mass spectrum data acquisition and processing software comprises: masslynx V4.1 workstation; the scanning mode is an MS central mode;
10 components in the bletilla striata extract are detected from rat serum, urine, excrement and bile biological samples after oral administration of the bletilla striata extract, and the 10 components comprise 5 prototype components: gastodin, α -isobutylmalic acid, gymnoside I, dactylorhin A and militarine, and 5 metabolic components including hydrolysis, desugarization, glucuronidation, sulfation metabolites of the prototypic component.
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