CN109100431B - Basic research method of drug effect substances of Qinggong Shoutao pills - Google Patents

Abstract

The invention provides a basic research method of drug effect substances of Qinggong Shoutao pills, which belongs to the technical field of traditional Chinese medicines and is characterized in that: the method comprises the following steps: (1) LC-MS is carried out on the Qinggong birthday peach pillsⁿOverall analysis, namely performing structural identification on main chemical components in the mixture to determine the types of serial components in the mixture; (2) respectively establishing LC-MS analysis methods of series components, and combining chromatographic retention time, ultraviolet absorption spectrum characteristics, molecular weight, multi-stage cracked ion fragment information and the like to carry out rapid structure identification. On one hand, the invention can avoid the sample pretreatment method and furthest reserve the original components in the sample; on the other hand, compared with the overall analysis method, the analysis method of each series of components is respectively established, and the inhibition of the component mass spectrum signals by the component co-outflow effect, the ion inhibition effect and the like in the mass spectrum detection process is reduced to a certain extent, so that the method is beneficial to the clarification of a traditional Chinese medicine complex substance system.

Description

Basic research method of drug effect substances of Qinggong Shoutao pills

Technical Field

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a basic research method of drug effect substances of Qinggong Shoutao pills.

Background

As a treasure of Chinese nationality for thousands of years, traditional Chinese medicine plays an irreplaceable role in the health of people and the reproduction and the rest of the nationality. How to clarify the chemical components and the biological activity of the precious heritage to better serve the human beings is to give the people the most intentions to the shoulder and the most important to the people. With the development of modern technology, the research on the action mechanism and chemical substance basis of traditional Chinese medicines is also continuously and deeply carried out. However, the chemical components of Chinese herbs are very complex, contain a large amount of chemical components which need to be determined qualitatively and quantitatively, and are easy to change dynamically during the processing and compatibility, and the products may be closely related to the clinical effects. Therefore, the complexity of the chemical components of the traditional Chinese medicine becomes the bottleneck of the development and modernization of the traditional Chinese medicine.

At present, although the traditional natural product research method of extracting, separating and structurally identifying the tribasic and the natural product research method taking activity tracking as the core can systematically and deeply research chemical components or active components in natural medicines, the method has long time consumption, high cost and strong blindness, and structural information of all components in the natural medicines cannot be obtained, so that the method has a plurality of limitations. Therefore, the research of the analysis method of the complex substance system has become one of the leading subjects in the field of pharmaceutical analysis.

The liquid chromatography-mass spectrometry (LC-MS) technology matured in the late nineties of the last century organically combines excellent separation capacity with a detection method with high sensitivity and high specificity, and increasingly becomes one of the most powerful basic research means of traditional Chinese medicine substances. In modern analytical techniques, they are regarded by scientists as fast, highly sensitive, specific and informative and can be most effectively used in conjunction with techniques such as chromatography. Particularly, in recent years, with the emergence of soft ionization mass spectrometry technologies such as electrospray ionization and matrix-assisted laser desorption ionization, the application of mass spectrometry in various fields such as medicine and life science is greatly expanded. The combination of soft ionization mass spectrometry, tandem mass spectrometry and chromatography, combined with high-resolution mass spectrometry, has incomparable advantages with other analysis techniques in the aspect of structure confirmation of effective components of traditional Chinese medicines, and has become one of the best methods for researching the chemical substance basis of traditional Chinese medicines and explaining the processing principle and the scientific connotation of the compatibility principle of traditional Chinese medicines.

However, in the detection and identification process of the major and minor components of the traditional Chinese medicine, the co-outflow of the components and the ion inhibition are two problems which are difficult to overcome. That is to say, the traditional Chinese medicine complex substance system analysis based on the LC-MS technology is still limited by the key problems that the constant and trace components are difficult to detect and identify simultaneously and the like. In recent years, although the rapid development of chromatographic separation packing and the general application of multidimensional liquid phase separation technology can improve the separation selectivity between substances to a certain extent, so that the ion identification degree and attribute are enhanced, excessive chromatographic separation can cause that part of trace components, especially trace components, have too low concentration and are missed to be detected by a mass spectrum detector, and the ion inhibition effect of the co-effluent in the soft ionization technology cannot be overcome. Meanwhile, with the improvement of the detection speed and precision of the mass spectrometer, the data generated by the mass spectrometer are also increased rapidly, and how to correctly and efficiently analyze the data also becomes a great challenge in the field of basic research of traditional Chinese medicine substances. Therefore, how to construct a strategy and a method suitable for the characteristics of chemical components in traditional Chinese medicines, particularly in a traditional Chinese medicine compound, around two key problems of detection and identification of constants and trace components of the traditional Chinese medicines and rapid analysis of mass spectrum data is a current research hotspot.

The invention discloses a womb clearing and life prolonging peach pill which is a research object and consists of more than ten kidney tonifying traditional Chinese medicines selected from sharpleaf galangal fruit for tonifying kidney yang, prepared rehmannia root for tonifying kidney yin, wolfberry fruit for tonifying yin and yang, diaphragma juglandis for reinforcing kidney and arresting seminal emission and the like, has the effects of tonifying kidney and producing sperm and tonifying vitality and strengthening body, and can be used for treating dizziness and tiredness, memory deterioration, soreness and weakness of waist and knees, tinnitus and deafness, dim eyesight and lacrimation, nocturia and dribbling urination caused by kidney deficiency and aging. Because the medicinal ingredients are extremely complex and the chemical ingredients are quite abundant, the explanation of the material basis is of great significance. In the research process, firstly, a traditional Chinese medicine chemical component structure identification strategy is constructed to quickly identify the chemical components in the compound: a structure identification strategy based on traditional Chinese medicine series components aims at enriching and detecting the concentration of trace components by improving the separation effect of in-situ chromatography, and is expected to provide a methodology reference for the analysis of a traditional Chinese medicine complex substance system and the research of traditional Chinese medicine substance basis.

Disclosure of Invention

In view of the above, the invention aims to provide a basic research method of drug effect substances of Qinggong Shoutao pills.

In order to achieve the purpose, the technical scheme of the invention is realized as follows: a basic research method of drug effect substances of Qinggong Shoutao pills comprises the following steps:

(1) LC-MS is carried out on the Qinggong birthday peach pillsⁿOverall analysis, namely performing structural identification on main chemical components in the mixture to determine the types of serial components in the mixture;

(2) respectively establishing LC-MS analysis methods of series components, and combining chromatographic retention time, ultraviolet absorption spectrum characteristics, molecular weight, multi-stage cracked ion fragment information and the like to carry out rapid structure identification.

Further, the preparation of the sample solution in the LC-MS analysis method comprises the following steps:

(1) preparation of test solution of the preparation: placing 0.3g of Qinggong birthday peach pill coarse powder (intermediate without adjuvant) in 50mL conical flask, adding 25mL of methanol, weighing, ultrasonic treating at room temperature (250W, 70kHz) for 30min, standing, cooling to room temperature, weighing again, adding methanol to supplement the lost weight, filtering with 0.22 μm microporous membrane, and collecting the filtrate;

(2) preparing a test solution with each medicinal ingredient: taking the medicinal materials of Qinggong Shoutao pills, crushing, sieving with a fourth sieve, weighing 0.8g, placing in a 50mL conical flask, adding 10mL of methanol, weighing, carrying out ultrasonic treatment (250W, 70kHz) at room temperature for 30min, standing, cooling to room temperature, weighing again, adding methanol to supplement the loss weight, sieving with a 0.22 mu m microporous filter membrane, and taking the subsequent filtrate to obtain the medicine;

(3) preparation of standard solution: taking appropriate amount of above reference substances, respectively, adding methanol to obtain solution containing about 50 μ g of each reference substance per 1mL, sealing at 4 deg.C, and storing in dark place.

Further, the UPLC conditions in the LC-MS analysis method are as follows:

using Waters acquisition UPLC HSS T3, 2.1 × 100mm, 1.8 μm as chromatographic column; taking 0.1% formic acid as a mobile phase A and acetonitrile as a mobile phase B, and carrying out gradient elution, wherein the elution gradient is as follows: 0-2min, 8% B; 2-20min, 8-26% B, 20-22min, 26% B; 22-30min, 26-42% B; 30-50min, 42-60% B; 50-55min, 60-95% B; 55-58min, 95% B, 58-60min, 95-8% B; 60-65min, 8% B; room temperature; the amount of sample was 3. mu.L, and the flow rate was 0.3 mL/min.

Further, the LC-MS analysis method has the following MS conditions:

negative ion mode, capillary temperature 350 ℃, sheath gas flow rate (nitrogen) 30arb, auxiliary gas flow rate (nitrogen) 10arb, spray voltage: 3kV, capillary voltage: -35V, tube lens voltage: -110V;

the mass axis accuracy of the high-resolution mass spectrum is corrected by adopting a mixed standard solution of caffeine, sodium dodecyl sulfate, sodium taurocholate, tetrapeptide MRFA and Ultramark, and the mass precision error is within 5 ppm;

the quality detection range is as follows: m/z 100-1200; scanning a sample in a full-scanning mode, performing full scanning by using high-resolution FT, setting the resolution R as 30000, and detecting fragment ions by using an ion trap dynode;

setting dynamic exclusion, repetition times: 3, repetition time: 10s, exclude list size: 100, exclusion time: and 20 s.

Compared with the prior art, the invention has the following advantages:

the research method of the invention comprises the following steps: on one hand, the method can avoid the serious component cross caused by a sample pretreatment method, such as a sectional extraction method, and the loss of chemical components, especially trace (trace) components caused by open column chromatography, and can furthest reserve the original components in the sample; on the other hand, compared with the integral analysis method, the method for respectively establishing the analysis methods of each series of components can obtain better chromatographic separation effect, thereby obtaining more detection time in the acquisition process of trace (trace) component mass spectrum data, reducing the inhibition of component mass spectrum signals by component co-outflow effect, ion inhibition effect and the like in the mass spectrum detection process to a certain extent, and being beneficial to the clarification of a traditional Chinese medicine complex substance system.

Drawings

FIG. 1 is a plan view of basic research of Qinggong Shoutao pills.

FIG. 2 is a UPLC-LTQ-Orbitrap high resolution mass spectrum total ion flow diagram of Qinggong Shoutao pills.

FIG. 3 is a total ion flow diagram of UPLC-LTQ-Orbitrap high resolution mass spectrum of silkworm excrement medicinal material.

FIG. 4 is a UPLC-LTQ-Orbitrap high resolution mass spectrum total ion flow diagram of Angelica sinensis medicinal material.

FIG. 5 is a UPLC-LTQ-Orbitrap high resolution mass spectrum total ion flow diagram of a diaphragma juglandis medicinal material.

FIG. 6 is a UPLC-LTQ-Orbitrap high resolution mass spectrum total ion flow diagram of a medlar medicinal material.

FIG. 7 is a UPLC-LTQ-Orbitrap high resolution mass spectrum total ion flow diagram of radix Ophiopogonis.

FIG. 8 is a UPLC-LTQ-Orbitrap high resolution mass spectrometry total ion flow diagram of ginseng drug.

FIG. 9 is a UPLC-LTQ-Orbitrap high resolution mass spectrometry total ion flow diagram of radix rehmanniae Preparata crude drug.

FIG. 10 is a UPLC-LTQ-Orbitrap high resolution mass spectrum total ion flow diagram of wild jujube seed medicinal material.

FIG. 11 is a UPLC-LTQ-Orbitrap high resolution mass spectrum total ion flow diagram of radix asparagi medicinal material.

FIG. 12 shows the total ion flow diagram (negative ion mode) of Qinggong Shoutao pill and UPLC-LTQ-Orbitrap of each medicine.

FIG. 13 shows a possible cleavage pathway for dicaffeoylquinic acid components.

FIG. 14 shows the possible ESI-MS/MS cleavage pathway of ginsenoside Rc.

FIG. 15 shows the analysis of migration components in plasma of rats administered with acetonitrile precipitation (A: QINGGONGSHOUTAO pill sample; B: blank rat plasma sample; C: administered rat plasma sample).

FIG. 16 shows the analysis of migration components in plasma of rats administered with drugs after treatment by methanol precipitation (A: QINGGONGSHOUTAO pill sample; B: blank rat plasma sample; C: administered rat plasma sample).

FIG. 17 shows the migration component analysis of plasma from rats treated with solid phase extraction (A: QINGGONGSHOUTAO pill sample; B: blank rat plasma sample; C: administrated rat plasma sample).

FIG. 18 shows the analysis of migration components in urine of rats administered with acetonitrile precipitation (A: QINGGONGSHOUTAO pill sample; B: blank rat urine sample; C: administered rat urine sample).

FIG. 19 shows the analysis of migration components in urine of rats administered with methanol precipitation (A: QINGGONGSHOUTAO pill sample; B: blank rat urine sample; C: administered rat urine sample).

FIG. 20 shows the analysis of migration components in urine of rats treated by solid phase extraction (A: QINGGONGSHOUTAO pill sample; B: blank rat urine sample; C: administrated rat urine sample).

Detailed Description

1 medicinal effect substance basic research method of Qinggong Shoutao pill

1.1 materials and reagents

The Qinggong Shoutao pills and each single medicinal material are provided by entrusted manufacturers. Chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, caffeic acid, echinacoside, rutin, quercitrin, isoquercitrin, acteoside, 3, 4-dicaffeoylquinic acid, 3, 5-dicaffeoylquinic acid, notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rh1, ginsenoside Rb1, ginsenoside Rc, ginsenoside Rd, spina date seed saponin B, ginsenoside Rg3 and other reference substances are purchased from biological product verification institute of food and drug verification institute and Chengdu Puri method science and technology development Limited company, the structure of the reference substances is determined by means of 1H-NMR, 13C-NMR and the like, the purity is more than 95% detected by an HPLC normalization method, and the reference substances can be used for qualitative research.

Chromatographic grade acetonitrile and methanol were purchased from Fisher Scientific (Fair law, NJ, USA), analytical grade methanol, formic acid were purchased from Sigma Aldrich (st. louis, MO, USA), and ultrapure water was prepared by Millipore Synergy UV ultrapure water machine; 0.22 μm microporous membrane from Beijing Huazhi chromatography science and technology, Inc.

1.2 instruments

A Thermo Scientific Accela 600pump high performance liquid chromatograph equipped with an online degasser, an autosampler and a high-pressure binary gradient pump; HPLC was connected to an LTQ-Orbitrap mass spectrometer (Thermo Scientific, Bremen, Germany) via an electrospray interface; a Thermo Xcaliber 2.1 workstation; mass Frontier 7.0(Thermo Scientific). KQ-250DE model digital control ultrasonic cleaner (Kunshan ultrasonic instruments Co., Ltd.); millipore Synergy UV type ultra-pure water machine (Millipore corporation, usa); model R200D electronic analytical balance (Sartorius, germany).

1.3 preparation of sample solutions

(1) Preparation of test solution of the preparation: placing 0.3g of Qinggong birthday peach pill coarse powder (intermediate without adjuvant) in 50mL conical flask, adding 25mL of methanol, weighing, ultrasonic treating at room temperature (250W, 70kHz) for 30min, standing, cooling to room temperature, weighing again, adding methanol to supplement the lost weight, filtering with 0.22 μm microporous membrane, and collecting the filtrate;

(2) preparing a test solution with each medicinal ingredient: taking the medicinal materials of Qinggong Shoutao pills, crushing, sieving with a fourth sieve, weighing 0.8g, placing in a 50mL conical flask, adding 10mL of methanol, weighing, carrying out ultrasonic treatment (250W, 70kHz) at room temperature for 30min, standing, cooling to room temperature, weighing again, adding methanol to supplement the loss weight, sieving with a 0.22 mu m microporous filter membrane, and taking the subsequent filtrate to obtain the medicine;

(3) preparation of standard solution: taking appropriate amount of above reference substances, respectively, adding methanol to obtain solution containing about 50 μ g of each reference substance per 1mL, sealing at 4 deg.C, and storing in dark place.

1.4 LC-MS analysis method

UPLC conditions were as follows:

using Waters acquisition UPLC HSS T3, 2.1 × 100mm, 1.8 μm as chromatographic column; taking 0.1% formic acid as a mobile phase A and acetonitrile as a mobile phase B, and carrying out gradient elution, wherein the elution gradient is as follows: 0-2min, 8% B; 2-20min, 8-26% B, 20-22min, 26% B; 22-30min, 26-42% B; 30-50min, 42-60% B; 50-55min, 60-95% B; 55-58min, 95% B, 58-60min, 95-8% B; 60-65min, 8% B; room temperature; the amount of sample was 3. mu.L, and the flow rate was 0.3 mL/min.

The MS conditions were as follows:

negative ion mode, capillary temperature 350 ℃, sheath gas flow rate (nitrogen) 30arb, auxiliary gas flow rate (nitrogen) 10arb, spray voltage: 3kV, capillary voltage: -35V, tube lens voltage: -110V;

the mass axis accuracy of the high-resolution mass spectrum is corrected by adopting a mixed standard solution of caffeine, sodium dodecyl sulfate, sodium taurocholate, tetrapeptide MRFA and Ultramark, and the mass precision error is within 5 ppm;

the quality detection range is as follows: m/z 100-1200; scanning a sample in a full-scanning mode, performing full scanning by using high-resolution FT, setting the resolution R as 30000, and detecting fragment ions by using an ion trap dynode;

setting dynamic exclusion, repetition times: 3, repetition time: 10s, exclude list size: 100, exclusion time: and 20 s.

1.5 LC-MS analysis results

The obtained total ion flow graph of QING GONG SHOU TAO WAN and each medicinal taste according to LC-MS analysis method under item 1.4 is shown in FIGS. 2-12. According to literature reports, the chemical components in the Chinese medicinal preparation are preliminarily identified by combining chromatographic retention behavior, molecular weight information and mass spectrum cracking rules, the chemical component classification in the Chinese medicinal preparation is determined, and the types of serial components contained in the Chinese medicinal preparation mainly comprise: flavones, triterpenoid saponins, steroid saponins, organic acids, iridoid glycosides, phenylethanoid glycosides and the like.

Meanwhile, the chromatographic peaks in the MS chart are identified by combining literature reports and related reference products, and the results are shown in Table 1.

TABLE 1 structural identification of chemical components in Qinggong Shoutao pills based on UPLC-LTQ-Orbitrap HRMS

Δ: identifying by using a reference substance; is [ M ]⁺HCOO]^-Ion(s)

(1) Identification of organic acid Components

The Qinggong Shoutao pill contains more organic acid components, and the basic components include quinic acid (Q), caffeic acid (C), cinnamic acid (pCo), ferulic acid (F), etc. The structure of the organic acid component in the Qinggong Shoutao pill preparation is rapidly identified by comparing the chemical components in the preparation and the medicinal ingredients.

Compounds 2, 3 and 4 had very short retention times, indicating that the polarity was very high. In ESI-MS spectrum in negative ion detection mode, [ M-H ] was observed]^-Ion (m/z 515.1395), which is presumed to be C₂₂H₂₇O₁₄The mass errors are all less than 5ppm, and [ M-H-glc ] can be observed in the ESI-MS/MS spectra]^-(m/z 353)、[M-H-glc-caffeoyl]^-(m/z 191)、[M-H-glc-caffeoyl-H₂O]^-(m/z 173) plasma fragment ions. Analyzing the possible molecular formula and the fragment of the cracked ion, and identifying the fragment as caffeoylquinic acid glucoside. Combining the relative abundances of the three important daughter ions of m/z 191, m/z 173 and m/z179, the three compounds can be inferred to be 3-caffeoylquinic acid glucoside, 4-caffeoylquinic acid glucoside and 4-caffeoylquinic acid glucoside, respectively. Since the substitution positions of glucose residues are difficult to determine, they are only simply identified as caffeoylquinic acid glucoside compounds, and the substitution positions of glucose groups are not indicated.

ESI-MS spectra of compounds 5, 11 and 16 in negative ion detection mode respectively generated an excimer peak of m/z179.0339, and the molecular formula C was deduced₉H₇O₄Error is less than5ppm and produced the same daughter ions in their ESI-MS/MS spectra as the caffeic acid control, e.g., m/z 135 and m/z 161, respectively, thereby identifying them as caffeic acid isomers, and caffeic acid, respectively (accurate identification).

ESI-MS spectrum of compound 18 in negative ion detection mode gave an excimer ion peak of M/z 193.0495, and the same daughter ion as ferulic acid control, e.g. [ M-H-CO ], in its ESI-MS/MS spectrum₂]^-、[M-H-CH₃]^-And isocratic ions, thereby accurately identifying it as ferulic acid. Meanwhile, ESI-MS/MS fragment ions of the compound 9 are similar to ferulic acid, and the fragment ions are deduced to be ferulic acid glucoside by combining molecular weight information of the fragment ions.

ESI-MS spectra of compounds 6, 12, 14 in negative ion detection mode all gave the same [ M-H ]]^-Ion (m/z 353.0867) with molecular formula C₁₆H₁₇O₉The errors are all less than 5 ppm. Neochlorogenic acid (3-Caffeoylquinic acid), chlorogenic acid (5-Caffeoylquinic acid), and cryptochlorogenic acid (4-Caffeoylquinic acid) were identified by comparison with the control. The reference reports that ESI-MS is compared²The position of acylation on quinic acid can be determined by the difference of fragment ions in the spectrum. In general, [ Quinic acid-H ] is produced when the acylation is at either the 3-or 5-position]^–Ion (m/z 191), the important difference between the two being [ Caffeic acid-H ] when the acylation position is at the 3-position]^–The abundance of the ion (m/z179) is higher than that of the 5-position. And when the acylation position is 4-, [ Quinic acid-H-₂O]^-(m/z 173) is ESI-MS²The ion of the base peak of the spectrum. From this it can be concluded that compounds 6, 12, 14 are neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, respectively, which is consistent with the conclusions obtained by comparison with the control.

ESI-MS spectra of both compounds 20 and 21 gave identical [ M-H ] in negative ion detection mode]^-The mass number of the ions and the theoretical accurate molecular weight (337.0928) of the compound have errors of less than 5 ppm. At their ESI-MS²The spectra yielded the base peak ions m/z 163 and m/z 191, respectively. Of reference, 3-pCoQAMS²MS with a spectrum with a base peak of m/z 163, 4-pCoQA²MS with a base peak of m/z 173, 5-pCoQA in the spectrum²The spectral base peak was m/z 191, thereby identifying compounds 20 and 21 as 3-pCoQA (3-cinnamoylquinic acid) and 5-pCoQA (5-cinnamoylquinic acid), respectively.

ESI-MS spectra of compounds 23, 24 in negative ion detection mode both yielded the same [ M-H ]]^-Ion m/z 367.1024, deduced to be of formula C₁₇H₁₉O₉The mass errors are all lower than 5 ppm. At the same time, at their ESI-MS²The spectra yielded the base peak ions m/z193 and m/z 191, respectively. Reference, MS of 3-FQA²MS with the peak ion of m/z193, 4-FQA in spectrum²MS with the base peak ion of m/z 173, 5-FQA in the spectrum²The peak ion of the spectral base is m/z 191, thereby identifying two compounds, compound 23 and 24, as 3-feruloylquinic acid and 5-feruloylquinic acid, respectively.

Dicaffeoylquinic acid: ESI-MS spectra of Compounds 41, 44, 45 and 46 in negative ion detection mode all gave [ M-H ] of M/z 515.1184]^-Ions, their molecular formula presumed to be C₂₅H₂₃O₁₂The mass errors are all lower than 5 ppm. At the same time, at their ESI-MS²The spectrum yields m/z 353 CQA-H]^-Ions, which indicates that the dicaffeoylquinic acid compounds are all dicaffeoylquinic acid compounds. The ESI-MS/MS cleavage pathway for this class of compounds is shown in FIG. 13. They are substantially identical in terms of the species and number of fragment ions, but differ considerably in terms of the abundance of ions produced. Compounds 44, 45, 46 can be accurately identified as 3,4-DiCQA, 3,5-DiCQA and 1,5-DiCQA by comparison to the control. Among the dicaffeoylquinic acid compounds found so far, 1,3-DiCQA is the most polar and therefore will be eluted first in the reverse phase column, thus identifying compound 41 as 1, 3-DiCQA.

(2) Identification of iridoid chemical components

In ESI-MS spectrum of compound 7 in negative ion detection mode, [ M-H ] was observed]^-Ion m/z375.1295, which is presumed to be C₁₆H₂₃O₁₀The mass error is 2.40 ppm. In its multi-stage mass spectrum, [ M-H ]]^-Ion generation of [ M-H-glc ]]^-(m/z 213)、[M-H-glc-CO₂]^-(m/z 169)、[M-H-H₂O]^-(m/z 357) plasma. It was identified as 8-epistrychnic acid based on its molecular weight and cleavage fragment, in combination with the compounds reported in the literature.

(3) Identification of phenylethanoid glycosides chemical composition

Compounds 8, 25, 26, 27, 38 and 43 were all identified as phenylethanoid glycosides by analysis. The structural identification process of the compounds is illustrated by taking the compounds 25, 26 and 27 as examples. The three can generate [ M-H ] by ESI-MS spectrum in negative ion detection mode]^-The ions m/z 785.2509, m/z 785.2512, m/z785.2510, which are presumed to be C₃₅H₄₅O₂₀The mass errors are all less than 5 ppm. The three are isomers, and all can generate fragment ions of m/z 623 and m/z 461 through continuous neutral loss 162 Da. The structures of the three compounds can be identified as echinacoside and isomers thereof by combining the analysis of the cracking rule and comparison of a reference substance. Similarly, compound 8, compound 38 and compound 43 can also be identified individually.

(4) Identification of saponin components

Identification of the chemical components of the triterpenoid saponins: triterpene saponin is the main effective component in Ginseng radix. More of the components can be detected in the preparation of Qinggong Shoutao pills. Compounds 47, 49, 50, 57, 58, 61, 66 and 79 can be identified as notoginsenoside R by comparing with standard₁Ginsenoside Rg₁Ginsenoside Re and ginsenoside Rh₁Ginsenoside Rb₁Ginsenoside Rc, ginsenoside Rd and ginsenoside Rg₃. The most characteristic cleavage pathway of the compounds in the MS/MS cleavage process is [ M-H ]]^-Ions or [ M + HCOO]^-The ions sequentially lose the sugar units connected on the aglycone to generate a series of fragment ions and obtain the corresponding aglycone ions. Taking ginsenoside Rc as an example, its [ M-H ]]The excimer ion (m/z 1077.5860) first lost one of the arabinose present on the aglycone in its ESI-MS/MS spectrumResidue, resulting in a radical peak ion M/z 945 ion ([ M-H-arab)]^-) And concomitant production of M/z 783 ion ([ M-H-arab-glc)]^-) (ii) a Further removal of glucose residues by M/z 783 ion yields M/z 621([ M-H-arab-2 glc)]^-) And the ion M/z 459([ M-H-arab-3 glc)]^-) And the like. The possible cleavage pathways are shown in FIG. 14. And in combination with mass spectrum cracking data reported in the literature, the structure inference can be carried out on other compounds.

For example, the excimer ion [ M-H ] of Compound 62]^-(m/z 955.4908) and the molecular formula C₄₅H₇₃O₁₇The mass error is 1.99 ppm. At its ESI-MSⁿTwo consecutive molecules of glucose residues are lost during cleavage and further de-H₂O、CO₂Etc. to yield M/z 793[ M-H-glc]^-、m/z 613[M-H-2glc-H₂O]^-、m/z 569[M-H-2glc-CO₂-H₂O]^-And M/z 523[ M-H-2glc-CO ]₂-CO-2H₂O]^-And (3) plasma. In combination with their retention times and references, it is speculated that the structure of compound 62 may be ginsenoside Ro.

As another example, the cleavage behavior of compound 77 and compound 78 are essentially identical, indicating that they are isomers of each other. Their excimer ion m/z 783 in MS²Medium continuous loss of glucose, resulting in M/z 621[ M-H-glc]^-And M/z 459[ M-H-2glc]^-Ions. By combining the above analyses and references, it was concluded that Compound 77 is 20(S) -ginsenoside Rg₃The compound 78 is 20(R) -ginsenoside Rg₃. Therefore, other triterpenoid saponin components from the ginseng and the spina date seed medicinal materials can be sequentially identified.

Identification of chemical components of steroid saponins: the steroid saponin is a characteristic component in the ophiopogon root medicinal material. Compounds 56 and 70 were analyzed to belong to this class of compounds. Using compound 56 as an example, [ M-H ] was generated in a primary mass spectrum]^-Excimer ion (m/z 901.4807), which is assumed to be of the formula C₄₅H₇₃O₁₈The mass error is 1.77 ppm. During the fragmentation process of the multi-stage mass spectrometer, the glucose residues on the sugar chain are firstly lost to generate [ M-H-Glc]^-Ion (m/z 739), thisSubsequent loss of linkage to rhamnose to [ M-H-glc-rha]^-Ion (m/z 593). This is consistent with the order of loss of the steroid saponin compound glycosyl groups: xylose attached to glucose (fucose) at position 3 or 4 is always lost preferentially over rhamnose attached at position 2. Thereby identifying it as a Protobioside or an isomer thereof. Similarly, compound 70 can also be identified as meta-quinolone sapogenin 3-O- [ alpha-L-rhamnopyranosyl- (1 → 2)][ beta-D-xylopyranosyl- (1 → 4)]- β -D-glucopyranose.

(5) Identification of flavonoid Components

The Qinggong Shoutao pills contain a large amount of flavonoid components, and since the flavonoid components are common and have more reports in literatures, the following method is mainly adopted in the identification process of the research: (1) the literature is consulted, and flavonoid components reported in each medicine of the Qinggong Shoutao pills are systematically summarized; (2) rapidly extracting chemical components in the test solution of the Qinggong Shoutao pill preparation by adopting a high-resolution EIC data processing method; (3) finely analyzing the multistage mass spectrum fragmentation rule of each flavonoid candidate compound, and providing a basis for further determining the structure; (4) and (5) further verifying the inferred result by adopting a comparison method of reference substances. For example, using compound 33 as an example, [ M-H ] is generated in a primary mass spectrum]^-Excimer ion (m/z 609.1450), which is assumed to be of the formula C₂₇H₂₉O₁₆The mass error is-0.82 ppm. In which [ M-H ] is]^-During MS/MS cleavage of the ion, the excimer ion peak thereof also produces the aglycone ion m/z 301 through neutral loss of the rutinose residue, and further during cleavage produces m/z179[ respectively ]^{1, 2}A]^-、m/z151[^1,2A-CO]^-、m/z193[M-H-B]^-、m/z 107[^0,4A]^-Plasma: wherein M/z193[ M-H-B ]]^-Indicating that it is substituted with two hydroxyl groups on the B ring and m/z107^0,4A]^-This indicates that the aglycone has two hydroxyl groups substituted on the A ring. From this it can be concluded that compound 33 is rutin, which is consistent with the results obtained using the control comparison. And by analogy, carrying out structural identification on other flavonoid compounds.

(6) Identification of other classes of Components

In the preparation of Qinggong Shoutao pill, other ingredients such as compound 1, 10, 31, 37, 48, etc. can be found. Most of the compounds are from the Chinese wolfberry medicinal material. And (3) analyzing the accurate mass number of the primary mass spectrum, the fragment ions of the multi-stage mass spectrum and the like of each compound by combining with literature reports, and deducing the structures of the compounds.

2 identification and research of plasma and urine migration components of Qinggong Shoutao pills

2.1 materials and instruments

The coarse powder of Qinggong Shoutao pill is provided by a pharmaceutical factory; mass-spec acetonitrile, mass-spec methanol, and mass-spec formic acid were all purchased from Fisher Scientific (Fair Lawn, NJ, USA), and ultrapure water was prepared by Millipore Synergy UV ultrapure water machine.

Thermo Scientific Accela 600pump UPLC System: comprises an online degasser, an automatic sample injector and a high-pressure binary gradient pump; HPLC was connected to an LTQ-Orbitrap high resolution mass spectrometer (Thermo Scientific, Bremen, Germany) via an electrospray interface; a Thermo Xcalibur 2.1 workstation; KQ-250DE type digital control ultrasonic cleaner (ultrasonic instruments, Inc. of Kunshan, China); millipore Synergy UV type ultra-pure water machine (Millipore corporation, usa); an electronic analytical balance model R200D (one hundred thousand, Sartorius, germany); grace Pure SPE C18-Low solid phase extraction cartridge (500mg/3 mL); FRESCO 21 microcentrifuge (Thermo Fisher Scientific, USA); TARGIN VX-II model multi-tube vortex oscillator (Beijing Pedal technology, Inc.); TTL-DCI type nitrogen blowing instrument (Beijing Tongtie technology development Co., Ltd.); 50-200 μ L micropipette, 200-.

2.2 Experimental methods

2.2.1 solution preparation

Preparing a uterus cleaning and life prolonging peach pill administration sample: 36g of Qinggong birthday peach pill coarse powder is taken, 120mL of a proper amount of 0.5 percent CMC-Na solution is added to prepare a sample solution with the concentration of 300mg/mL, and the sample solution is stored at 4 ℃.

2.2.2 Experimental animals and dosing regimens

Sprague Dawley (SD) rats, weighing 220 + -10 g, purchased from Beijing Wintoli laboratory animal technology Co., Ltd, license number SCXK (Beijing) 2011-: circulating for 12h day and night, keeping the room temperature at 22-24 deg.C, and maintaining the relative humidity at 55-65%.

Collection of blood and urine samples: 8 normal male rats were acclimatized for one week in the experimental setting. Before the experiment, mice were placed in metabolic cages, fasted for 12 hours, freely drunk water, while collecting blank urine. Rats were acclimatized for 3 days and had free access to water. Fasting was for 12 hours prior to dosing, and oral gavage was administered twice daily for three days at the time of the experiment at a dose of 1.25 g/kg. After the last administration, 0.5ml of blood was collected from orbital veins of each rat at 0, 0.5,1,2,4 hours after administration (blood samples were collected as blank samples at 0 hour). The collected blood samples were placed in centrifuge tubes filled with heparin sodium and centrifuged at 3500rpm for 10 min. All supernatants were combined and frozen at-80 ℃ in a freezer.

Meanwhile, the administration urine is collected for 24h after the intragastric administration. During which food and water are supplied. All urine samples were pooled, centrifuged at 3500rpm for 10min, and the supernatant was frozen at-80 ℃ in a freezer.

2.2.3 treatment of rat biological samples

In order to identify the metabolic components in the plasma and urine of rats as much as possible, all biological samples are treated by three methods, namely a methanol precipitation method, an acetonitrile precipitation method and an SPE solid phase extraction cartridge.

(1) Acetonitrile precipitation treatment method of rat biological sample

The blood sample treatment method comprises the following steps: thawing frozen blood samples of each group at room temperature, taking 200 mu L of each of drug-containing serum and blank serum of different groups, adding 600 mu L of acetonitrile respectively, carrying out vortex oscillation for 3min, centrifuging (14300r/min) for 30min, standing, taking supernatant, drying by N2 at room temperature, re-dissolving residues by 100 mu L of initial mobile phase, carrying out vortex oscillation for 3min, centrifuging at 14000rpm for 15min, and taking supernatant for UPLC-LTQ-Orbitrap analysis.

The processing method of the urine sample comprises the following steps: thawing frozen urine samples of each group at room temperature, taking 200 mu L each of a medicine-containing urine sample and a blank urine sample, respectively adding 600 mu L acetonitrile, carrying out vortex oscillation for 3min, centrifuging (14300r/min) for 30min, standing, taking supernatant, drying at room temperature by N2, re-dissolving residues by 100 mu L initial mobile phase, carrying out vortex oscillation for 3min, centrifuging at 14000rpm for 15min, and taking supernatant for UPLC-LTQ-Orbitrap analysis.

(2) Methanol precipitation treatment method of rat biological sample

The blood sample treatment method comprises the following steps: thawing frozen blood samples at room temperature, collecting drug-containing serum and blank serum 200 μ L, respectively adding acetonitrile 600 μ L, vortex oscillating for 3min, centrifuging (14300r/min) for 30min, standing, collecting supernatant, and adding N at room temperature₂Blow-drying, re-dissolving the residue with 100 μ L of initial mobile phase, vortexing and shaking for 3min, centrifuging at 14000rpm for 15min, and collecting the supernatant for UPLC-LTQ-Orbitrap analysis.

The processing method of the urine sample comprises the following steps: thawing frozen urine samples at room temperature, collecting 200 μ L each of urine sample containing medicine and blank urine, adding 600 μ L acetonitrile, vortex oscillating for 3min, centrifuging (14300r/min) for 30min, standing, collecting supernatant, and adding N at room temperature²Blow-drying, re-dissolving the residue with 100 μ L of initial mobile phase, vortexing and shaking for 3min, centrifuging at 14000rpm for 15min, and collecting the supernatant for UPLC-LTQ-Orbitrap analysis.

(3) SPE (solid phase extraction) treatment method for rat biological sample

The blood sample treatment method comprises the following steps: the GracePure SPE solid phase extraction cartridge was taken, the cartridge was activated with 5mL of methanol first, and then 5mL of deionized water was added to balance the solid phase extraction cartridge. 1mL of the plasma sample was placed on an activated solid phase extraction cartridge, followed by washing with 2mL of water to flush the impurities, followed by 3mL of methanol and collection of the methanol eluate. Drying all the methanol eluates at room temperature with N2, re-dissolving the residue with 100 μ L of initial mobile phase, vortexing and shaking for 3min, centrifuging at 14000rpm for 15min, and collecting the supernatant for UPLC-LTQ-Orbitrap analysis.

The processing method of the urine sample comprises the following steps: dissolving frozen urine samples of each group at room temperature, placing 2mL of urine into an activated solid phase extraction column, washing impurities with 3mL of water, eluting with 3mL of methanol, and collecting methanol eluent. Drying all the methanol eluates at room temperature with N2, re-dissolving the residue with 100 μ L of initial mobile phase, vortexing and shaking for 3min, centrifuging at 14000rpm for 15min, and collecting the supernatant for UPLC-LTQ-Orbitrap analysis.

2.2.4 detection conditions

UPLC conditions: waters ACQUITY UPLC HSS T3 (2.1X 100mm, 1.8 μm) as chromatographic column; gradient elution is carried out by taking 0.1% formic acid as a mobile phase A and acetonitrile as a mobile phase B; room temperature; the amount of sample was 3. mu.L, and the flow rate was 0.3 mL/min. The elution gradient was: 0-2min, 8% B; 2-20min, 8-26% B, 20-22min, 26% B; 22-30min, 26-42% B; 30-50min, 42-60% B; 50-55min, 60-95% B; 55-58min, 95% B, 58-60min, 95-8% B; 60-65min, 8% B.

MS conditions: negative ion mode, capillary temperature 350 ℃, sheath gas flow rate (nitrogen) 30arb, auxiliary gas flow rate (nitrogen) 10arb, spray voltage: 3kV, capillary voltage: -35V, tube lens voltage: 110V. The mass axis accuracy of the high-resolution mass spectrum is corrected by adopting a mixed standard solution of caffeine, sodium dodecyl sulfate, sodium taurocholate, tetrapeptide MRFA and Ultramark, and the mass precision error is within 5 ppm. The quality detection range is as follows: m/z 100-1200; the sample was scanned by Full scan-ion list-Dynamic exclusion (FPD), Full scan (Full scan, FS) was performed with high resolution FT, resolution R was set to 30000, and fragment ions were detected with an ion trap dynode. Set Dynamic Exclusion (DE), Repeat count: 3, repetition time (repetition duration): 10s, exclude list size (Exclusion list size): 100, Exclusion time (Exclusion duration): and 20 s. The data acquisition system is Xcalibur 2.1.

2.3 results of the experiment

2.3.1 identification of migrating constituents in rat plasma after administration

To better identify the components that migrate in vivo, we have mainly used three different treatments during the processing of plasma samples: acetonitrile precipitation, methanol precipitation and solid phase extraction, thereby ensuring the reliability and comprehensiveness of the detected migration components.

The identification of plasma migration components after rat gavage is mainly carried out after high-resolution mass spectrum data are collected by using UPLC-LTQ-Orbitrap, and the figure is shown in 15-17. The related in vivo migration components are screened and identified by comparing the data of the rat plasma, the original preparation of Qinggong Shoutao pills and blank plasma.

Finally, a total of 20 transitional components were identified from the plasma of rats after administration, see table 2.

TABLE 2 determination of migration components in plasma of rats administered with Qinggong Shoutao pills

2.3.2 identification of migrating Components in rat urine after administration

In order to better identify the components migrating in vivo, three different treatment methods are mainly adopted in the treatment process of the urine sample: acetonitrile precipitation, methanol precipitation and solid phase extraction, thereby ensuring the reliability and comprehensiveness of the detected migration components.

The identification of the urine migration components after the gavage of the rats is mainly carried out after the acquisition of high-resolution mass spectrum data by using UPLC-LTQ-Orbitrap, and the figure is shown in figures 18-20. Through comparing the urine of the rat with the original preparation of Qinggong Shoutao pills and the blank urine data, the related in-vivo migration components are screened and identified.

Finally, a total of 38 transitional elements were identified from the urine of the rats after administration, see table 3.

TABLE 3 identification results of migration components in urine of rats administered with Qinggong Shoutao pills

Performing chromatographic separation, mass spectrum data acquisition and structural identification of inherent components on various components in the Qinggong birthday peach pills by using a UPLC-LTQ-Orbitrap high-resolution liquid mass spectrometer, and finally identifying 84 components such as triterpenoid saponin, flavone, steroid saponin, iridoid, phenylethanoid glycoside and the like from the Qinggong birthday peach pill preparation. The coarse powder of the Qinggong Shoutao pill is orally administrated with intragastric administration to Sprague Dawley rats in a dose of 1.25g/kg, twice a day and continuously administrated for three days, serum and accumulated urine are taken for metabolite detection, 20 migration components are identified from the plasma of the rats after continuous administration, 38 migration components are identified from the urine of the rats after continuous administration, the material basis of the Qinggong Shoutao pill is preliminarily clarified, and the basis and the premise are provided for further researching the drug effect material basis of the Qinggong Shoutao pill.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (1)

1. A basic research method of drug effect substances of Qinggong Shoutao pills is characterized in that: the method comprises the following steps:

(1) LC-MS is carried out on the Qinggong birthday peach pillsⁿOverall analysis, namely performing structural identification on main chemical components in the mixture to determine the types of serial components in the mixture;

(2) respectively establishing an LC-MS analysis method of a series of components to carry out rapid structure identification;

the preparation of the sample solution in the LC-MS analysis method comprises the following steps:

(1) preparation of test solution of the preparation: placing 0.3g of Qinggong birthday peach pill coarse powder in a 50mL conical flask, adding 25mL of methanol, weighing, performing ultrasonic treatment at room temperature for 30min, standing, cooling to room temperature, weighing again, adding methanol to supplement the weight loss, filtering with a 0.22 μm microporous membrane, and collecting the filtrate;

(2) preparing a test solution with each medicinal ingredient: taking the medicinal materials of the Qinggong birthday peach pill, crushing, sieving by a fourth sieve, weighing 0.8g, placing in a 50mL conical flask, adding 10mL of methanol, weighing, carrying out ultrasonic treatment at room temperature for 30min, standing, cooling to room temperature, weighing again, adding methanol to supplement the lost weight, filtering by a 0.22 mu m microporous filter membrane, and taking the subsequent filtrate;

(3) preparation of standard solution: respectively taking chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, caffeic acid, echinacoside, rutin, quercitrin, isoquercitrin, acteoside, 3, 4-dicaffeoylquinic acid, 3, 5-dicaffeoylquinic acid, notoginsenoside R1, ginsenoside 35Rg 1, ginsenoside Re, ginsenoside Rh1, ginsenoside Rb1, ginsenoside Rc, ginsenoside Rd, spina date seed saponin B, ginsenoside Rg3, 1, 5-dicaffeoylquinic acid and ferulic acid in proper amount, adding methanol to prepare each 1mL of solution containing about 50 μ g of each reference substance, and sealing and storing in dark at 4 deg.C to obtain the final product;

the processing method for identifying the metabolic components in the plasma and urine of the rat comprises a methanol precipitation method, an acetonitrile precipitation method and an SPE solid phase extraction column method;

the methanol precipitation method comprises the following steps:

the blood sample treatment method comprises the following steps: unfreezing each frozen blood sample at room temperature, taking 200 mu L of each drug-containing serum and blank serum of different groups, respectively adding 600 mu L of methanol, carrying out vortex oscillation for 3min, centrifuging for 30min, standing, taking supernatant, drying at room temperature by using N2, re-dissolving residues by using 100 mu L of initial mobile phase, carrying out vortex oscillation for 3min, centrifuging at 14000rpm for 15min, and taking supernatant to carry out UPLC-LTQ-Orbitrap analysis;

the processing method of the urine sample comprises the following steps: thawing frozen urine samples at room temperature, collecting 200 μ L each of urine sample containing medicine and blank urine, adding 600 μ L methanol, vortex oscillating for 3min, centrifuging for 30min, standing, collecting supernatant, and adding N at room temperature₂Drying, re-dissolving the residue with 100 μ L initial mobile phase, vortex shaking for 3min, centrifuging at 14000rpm for 15min, and collecting supernatant for UPLC-LTQ-Orbitrap analysis;

the acetonitrile precipitation method comprises the following steps:

the blood sample treatment method comprises the following steps: unfreezing each frozen blood sample at room temperature, taking 200 mu L of each drug-containing serum and blank serum of different groups, respectively adding 600 mu L of acetonitrile, carrying out vortex oscillation for 3min, centrifuging for 30min, standing, taking supernatant, drying at room temperature by using N2, re-dissolving residues by using 100 mu L of initial mobile phase, carrying out vortex oscillation for 3min, centrifuging at 14000rpm for 15min, and taking supernatant to carry out UPLC-LTQ-Orbitrap analysis;

the processing method of the urine sample comprises the following steps: unfreezing frozen urine samples of each group at room temperature, taking 200 mu L each of a medicine-containing urine sample and a blank urine sample, respectively adding 600 mu L acetonitrile, carrying out vortex oscillation for 3min, centrifuging for 30min, standing, taking supernatant, drying at room temperature by using N2, re-dissolving residues by using 100 mu L initial mobile phase, carrying out vortex oscillation for 3min, centrifuging at 14000rpm for 15min, and taking supernatant to carry out UPLC-LTQ-Orbitrap analysis;

the SPE solid phase extraction cartridge method comprises the following steps:

the blood sample treatment method comprises the following steps: taking a GracePure TMSPE solid phase extraction cartridge, firstly activating the cartridge by using 5mL of methanol, and then adding 5mL of deionized water to balance the solid phase extraction cartridge; placing 1mL of plasma sample in an activated solid phase extraction column, washing impurities with 2mL of water, eluting with 3mL of methanol, and collecting methanol eluate; drying all the methanol eluates with N2 at room temperature, re-dissolving the residue with 100 μ L of initial mobile phase, vortex shaking for 3min, centrifuging at 14000rpm for 15min, and collecting the supernatant for UPLC-LTQ-Orbitrap analysis;

the processing method of the urine sample comprises the following steps: dissolving the frozen urine samples of each group at room temperature, placing 2mL of urine into an activated solid phase extraction column, washing impurities with 3mL of water, eluting with 3mL of methanol, and collecting methanol eluent; drying all the methanol eluates with N2 at room temperature, re-dissolving the residue with 100 μ L of initial mobile phase, vortex shaking for 3min, centrifuging at 14000rpm for 15min, and collecting the supernatant for UPLC-LTQ-Orbitrap analysis;

the UPLC conditions in the LC-MS analysis method are as follows:

using WatersACQUITYLCHSST 3, 2.1 × 100mm, 1.8 μm as chromatographic column; taking 0.1% formic acid as a mobile phase A and acetonitrile as a mobile phase B, and carrying out gradient elution, wherein the elution gradient is as follows: 0-2min, 8% B; 2-20min, 8-26% B, 20-22min, 26% B; 22-30min, 26-42% B; 30-50min, 42-60% B; 50-55min, 60-95% B; 55-58min, 95% B, 58-60min, 95-8% B; 60-65min, 8% B; room temperature; the sample injection amount is 3 mu L, and the flow rate is 0.3 mL/min;

the LC-MS analysis method comprises the following MS conditions:

negative ion mode, capillary temperature 350 ℃, sheath gas flow rate 30arb, auxiliary gas flow rate 10arb, spray voltage: 3kV, capillary voltage: -35V, tube lens voltage: -110V;

the high-resolution mass spectrum mass axis accuracy is corrected by adopting a mixed standard solution of caffeine, sodium dodecyl sulfate, sodium taurocholate, tetrapeptide MRFA and Ultramark, and the mass precision error is within 5 ppm;

the quality detection range is as follows: m/z is 100-1200; scanning a sample in a full-scanning mode, performing full scanning by using high-resolution FT, setting the resolution R as 30000, and detecting fragment ions by using an ion trap dynode;

setting dynamic exclusion, repetition times: 3, repetition time: 10s, exclude list size: 100, exclusion time: and 20 s.

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