CN107870216B - Spectral efficiency analysis method for bacteriostasis of polygonum capitatum parts with different polarities - Google Patents
Spectral efficiency analysis method for bacteriostasis of polygonum capitatum parts with different polarities Download PDFInfo
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Abstract
The invention relates to a spectrum effect analysis method of the bacteriostatic action of polygonum capitatum parts with different polarities, which concretely comprises the following steps of material providing, medicine extraction and separation, UP L C analysis and mass spectrum analysis, determination of the minimum bacteriostatic concentration, and partial least squares regression analysis between each peak of the polygonum capitatum parts with different polarities and 1/MIC, wherein the invention can comprehensively analyze and determine the chemical components of the polygonum capitatum parts with different polarities by a UP L C-TOF-MS mass spectrum combination technology, combines with a pharmacodynamic test, analyzes, screens and determines bacteriostatic substances in the polygonum capitatum by a P L SR method, and clarifies the drug effect substance basis of the polygonum capitatum on the treatment of urinary tract infection of escherichia coli and pseudomonas aeruginosa from the molecular chemical level.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a method for analyzing a medicine of polygonum capitatum, and more particularly relates to a method for analyzing the spectrum effect of different polarity parts of the polygonum capitatum. The method of the invention can lay a solid foundation for the deep development of the polygonum capitatum.
Background
Polygonum capitatum (Polygonum capitatum Buch-Ham ex D. Don) is a perennial herb of Polygonum of Polygonaceae. Has the efficacies of clearing heat and promoting diuresis, and inducing diuresis for treating stranguria, and has obvious effect on treating urinary system infection clinically. The Chinese patent medicine preparation 'Relinqing granules' prepared by taking the granules as raw materials enters the national basic medicine catalogue in 2002 and enters the national basic medical insurance catalogue in 2004.
The polygonum capitatum is a common medicine in minority regions, and is mainly used for symptoms such as pyelonephritis, urinary tract infection, diuresis and stranguria treatment, related pharmacological studies are rare, only a few reports such as anyou and the like exist at present, anyou adopts a rat bacterial pyelonephritis model to carry out experiments, and as a result, WBC and B L D in rat urine of a polygonum capitatum water extract group are obviously reduced compared with a control group, and the polygonum capitatum water extract has a certain anti-inflammatory effect on pyelonephritis, the anyou and the like observe the death condition of a mouse within 5 days by injecting escherichia coli liquid into an abdominal cavity of the mouse, the death rate of the control group is 100%, the death rate of the polygonum capitatum group is respectively 20% and 50%, the polygonum capitis capable of resisting the infection caused by escherichia coli, the anyou and the like are administrated to a rabbit by stomach irrigation with polygonum capitatum water extract, and as a result, the polygonum capitatum water extract group has no significant difference in body temperature but can reduce the fever of the neisseria caused by injecting paratyphoid vaccine, the polygonum capitis subjected to a rabbit urinary tract infection, the rabbit urinary tract infection caused by a bacteriostatic effect of the polygonum capitis detected by a rabbit urinary tract, the polygonum capitis detected by a bacteriostatic effect of a diluted by a dendranthera in vitro by a dendranthera, the rat urine test method of the polygonum capitis 6310, the polygonum capitis 3610, the polygonum capitis shown by using a diluted sarcina, the sar.
Chinese patent application publication No. CN1054899A (Chinese patent application No. 90107810.7, published as 1991, 10 and 2) discloses a process for producing urinary tract medicine granule and syrup. The production process is that the four seasons grass is used as raw material and decocted by water for 30-60 minutes, the extracting solution is filtered and concentrated, the supernatant fluid is decompressed and concentrated into paste, then the paste is extracted by 60-70% ethanol, the ethanol extracting solution is dried in vacuum to obtain the four seasons red extractum, and the four seasons red extractum is further prepared into medicinal granules or syrup.
A Chinese patent medicine named as Relinqing granules is collected in the Ministry of health of the people's republic of China (the seventeenth volume) of the drug Standard-Chinese medicine preparation compiled by the pharmacopoeia Committee of the Ministry of public health of the people's republic of China in 1998, and is prepared by decocting polygonum capitatum in water twice, filtering and concentrating.
CN 1481832a (chinese patent application No. 02129686.3, published 2004 year 3 month 17) and CN1483466A (chinese patent application No. 03146381.9, published day 2004 year 3 month 24) disclose polygonum capitatum extracts, which are prepared essentially by the following steps: a. decocting fresh or dried herba Polygoni Capitati with water twice or reflux-extracting with alcohol-water mixture for two to three times, each for 1-2 hr, mixing decoctions, filtering, concentrating to relative density of 1.2 at 20 deg.C, and spray drying or drying under reduced pressure to obtain final product; or b, extracting the polygonum capitatum herb and the water extraction dregs thereof by supercritical carbon dioxide extraction. The extract is believed to be useful for antibacterial, anti-inflammatory, analgesic, diuretic, urinary system calculus, pyelonephritis, and prostatitis.
Although the polygonum capitatum preparation is widely applied clinically, pharmacodynamic studies of various effective parts of the polygonum capitatum preparation are still necessary to be deeply developed, for example, spectral efficiency analysis of bacteriostasis of different polar parts of the polygonum capitatum is still a necessary means for guiding clinical application of the polygonum capitatum preparation, a fingerprint of the separated and purified effective parts in a polygonum capitatum extract is established, contribution values of chemical components represented by all peaks of different parts to the bacteriostasis are clarified, and the pharmacodynamic material basis of the polygonum capitatum bacteriostasis is disclosed, which is still expected by technical personnel in the field.
Disclosure of Invention
The invention aims to research pharmacodynamics of various effective parts of polygonum capitatum, such as spectrum effectiveness analysis of bacteriostasis of different polar parts of polygonum capitatum, guide clinical application of polygonum capitatum preparations, establish a fingerprint of the separated and purified effective parts in polygonum capitatum extracts, clarify contribution values of chemical components represented by all peaks of different parts to bacteriostasis and disclose pharmacodynamic material bases of polygonum capitatum bacteriostasis. Through the method, the invention has surprisingly found that the main bacteriostatic active sites of polygonum capitatum on escherichia coli and pseudomonas aeruginosa are A, B, C, D four strong-polarity sites, and the main bacteriostatic components of polygonum capitatum are 6-gallic acid acyl glucose, 3, 6-digallic acid acyl glucose, 1,3, 6-trigallic acid acyl glucose and davidiin. The present invention has been completed based on this finding.
Therefore, in one aspect of the invention, a method for analyzing the spectrum effect of the bacteriostatic action of the polygonum capitatum at different polar parts is provided, which comprises the following steps:
(1) material supply:
providing an ultra-high performance liquid chromatography system and a liquid chromatography-mass spectrometer, providing a polygonum capitatum medicinal material, and providing a strain for bacteriostatic detection;
(2) extracting and separating the medicines:
(21) adding 60-80% ethanol 6-8 times the amount of the polygonum capitatum medicinal material into the polygonum capitatum medicinal material, extracting for 2 times, each time for 1-2 hours, filtering, combining filtrates, recovering ethanol, concentrating into thick paste, drying under reduced pressure, and crushing into fine powder to obtain polygonum capitatum ethanol extract;
(22) taking another polygonum capitatum alcohol extract sample, adding a proper amount of methanol, carrying out ultrasonic extraction for 3 times, combining filtrates, centrifuging, taking supernate, carrying out reduced pressure distillation to remove the solvent, dissolving with 20-40% methanol, loading into an MCI type macroporous resin column, and carrying out chromatographic separation;
(23) performing gradient elution by using 30%, 35% -40%, 40% -50%, 60% and 60% -80% of methanol in volume fraction respectively, collecting eluates of each part, and volatilizing to obtain seven parts with different polarities A-G;
(24) accurately weighing seven parts A-G, respectively dissolving the seven parts A-G in DMSO, and preparing a solution with the mass concentration of 25mg/m L for later use;
(3) UP L C analysis and mass spectrometry analysis:
(31) chromatographic and mass spectrometric conditions:
chromatographic conditions are as follows: c18A chromatographic column with the column temperature of 40 ℃, the flow rate of 0.35L/min, the sample injection amount of 2 mu L and the mobile phase using 0.1 percent formic acid water solution (A) to 0.1 percent formic acid acetonitrile solution (B) for gradient elution;
mass spectrum conditions: electrospray ion source (ESI), dry gas temperature 180 deg.C, capillary voltage 4500 eV, negative ion detection mode, spray pressure 0.25MPa, dry gas (N)2) The flow velocity is 8L/min, the scanning range m/z is 100-2000, and the collision energy is 10 eV;
(32) establishing fingerprint spectrums of different polar parts:
establishing UHP L C-TOF-MS chromatograms of different polar parts according to the conditions of (31) chromatographic and mass spectrum conditions, and determining compounds corresponding to peaks of different polar parts through mass spectrum so as to determine main chemical compositions in each part;
(4) determination of minimum inhibitory concentration:
(41) preparing bacterial suspension and preparing an agar plate culture medium for experiments according to a minimum inhibitory concentration determination method;
(42) according to a minimum inhibitory concentration determination method, determining the minimum inhibitory concentrations of the parts with different polarities obtained in the step (24);
(5) partial least squares regression analysis between each peak and 1/MIC of different polarity parts of polygonum capitatum:
for each site, analysis was performed by averaging using the partial least squares regression analysis (P L SR) of SPSS16.0 software, and the relationship between each substance peak and the pharmacodynamic index was analyzed using the peak area of each substance peak as an independent variable and 1/MIC as a dependent variable, to determine the substance peak positively correlated with the test strain and the site where these substances exist.
In one embodiment of the method of the present invention, the ultra-high performance liquid chromatography system is a 1290Infinity model ultra-high performance liquid chromatography system, in one embodiment, the liquid chromatography-mass spectrometer is an Agilent TOF L C/MS spectrometer, in one embodiment, the Polygonum capitatum drug is Polygonum capitatum collected in Guizhou.
In one embodiment of the method, in the step (21), the polygonum capitatum medicinal material is added with 7 times of 70% ethanol for extraction for 2 times, each time lasts for 1.5h, the filtration is carried out, the filtrates are combined, the ethanol is recovered and concentrated into thick paste with the relative density of 1.20-1.25 (60 ℃), and the thick paste is dried under reduced pressure and crushed into fine powder, so that 70% ethanol extract of polygonum capitatum is obtained. In one embodiment of the method of the present invention, in the step (21), 0.05% thioglycerol and 0.002% glacial acetic acid are added into the ethanol solution used for the polygonum capitatum medicinal material.
In one embodiment of the method of the present invention, in step (22), 45g of polygonum capitatum 70% ethanol extract sample is taken, an appropriate amount of methanol is added, ultrasonic extraction is performed for 3 times, filtrates are combined, centrifugation is performed for 5min (3000rpm), the supernatant is taken, vacuum distillation is performed to remove the solvent, and 30% methanol is used for dissolving and loading the supernatant into an MCI type macroporous resin column (1BV ═ 1L) for chromatographic separation.
In one embodiment of the method of the present invention, wherein in step (23), methanol with volume fractions of 30%, 35% to 40%, 40% to 50%, 60% to 80% is used for gradient elution, and the eluates of each part are collected and evaporated to dryness to obtain seven parts A to G.
In one embodiment of the method of the present invention, in step (24), 25.00mg of each of the seven sites A to G is accurately weighed and dissolved in 1m L DMSO to prepare a solution with a mass concentration of 25mg/m L.
In one embodiment of the process of the present invention, wherein said chromatography column is an ACQUITY BEH C18A column of 2.1mm × 100mm, 1.7 μm.
In one embodiment of the method of the invention, wherein the elution procedure of the gradient elution is: 0-0.5 min, 95% A, 0.5-20 min, 95% A → 81.5% A; 20-30 min, 81.5% A → 95% A; 30-32 min, 95% A.
In one embodiment of the method, the bacterial suspension is prepared by the steps of firstly inoculating experimental strains on a flat plate, culturing for 24 hours in a constant temperature and humidity incubator at 37 ℃, selecting characteristic strains, inoculating the characteristic strains in a liquid culture medium, shaking for 24 hours at 37 ℃, and diluting the characteristic strains to 0.5 McGreek concentration by a McGreek turbidimetric method to obtain 10^6-10^8CFU/m L bacterial suspension for later use.
In one embodiment of the method, the agar plate culture medium for experiments is prepared by adding 20g of purified agar powder into a 1L nutrient broth culture medium, adding 1L double distilled water, adjusting the pH value to 7.4, sterilizing at 121 ℃ for 30min, taking out, cooling to about 45 ℃, pouring the culture medium into a sterile culture dish on an ultraclean workbench, and condensing to obtain the agar plate culture medium for experiments.
In one embodiment of the method, the process of determining the minimum inhibitory concentration of different polar parts comprises the steps of firstly adding 50 mu L liquid culture medium and 50 mu L bacterial suspension into each hole of a sterile 96-hole plate on an ultraclean workbench, accurately measuring 100 mu L of a drug to be detected by using a liquid transfer gun, adding the drug to be detected into a first hole of the 96-hole plate, uniformly mixing, sucking 100 mu L, transferring the drug to a second hole, uniformly mixing, sucking 100 mu L, discarding the drug until the last hole is uniformly mixed, keeping 100 mu L in each hole, taking gentamicin sulfate as a positive control and DMSO as a negative control, culturing at 37 ℃ for 24h, respectively transferring the drug to an agar plate culture medium, and taking the concentration of the drug in the micro hole without bacterial growth after 24h as the minimum inhibitory concentration.
In the above-described method steps of the present invention, although the specific steps described therein may be distinguished in some detail or in language specific to the steps described in the preparation examples of the detailed description section below, those skilled in the art can nevertheless fully summarize the above-described method steps in light of the detailed disclosure of the invention as it extends throughout the present application.
Any embodiment of any aspect of the invention may be combined with other embodiments, as long as they do not contradict. Furthermore, in any embodiment of any aspect of the invention, any feature may be applicable to that feature in other embodiments, so long as they do not contradict. The invention is further described below.
All documents cited herein are incorporated by reference in their entirety and to the extent such documents do not conform to the meaning of the present invention, the present invention shall control. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even though such terms and phrases are intended to be described or explained in greater detail herein, reference is made to the term and phrase as being inconsistent with the known meaning and meaning as is accorded to such meaning throughout this disclosure.
The invention aims to establish fingerprint spectrums of a plurality of effective parts separated and purified in a dry paste extracted from polygonum capitatum, clarify contribution values of chemical components represented by all peaks at different parts to bacteriostatic effects and reveal the drug effect substance basis of polygonum capitatum bacteriostatic action, UP L C-TOF-MS is adopted to establish fingerprint spectrums of polygonum capitatum at different polar parts, chemical component analysis is carried out on each peak, the minimum bacteriostatic concentration (MIC) of each polygonum capitatum at different polar parts to escherichia coli and pseudomonas aeruginosa is measured by a 96-well plate method, partial least squares regression analysis (P L SR) is utilized to establish the spectrum effect relation between the polygonum capitatum fingerprint spectrums and the bacteriostatic action, the result shows that the invention establishes fingerprint spectrums of the polygonum capitatum at different polar parts and determines that 9 and 13 peaks which are positively correlated to the bacteriostatic effects of escherichia coli and pseudomonas aeruginosa by the spectrum effect analysis, the result of the invention establishes the compounds represented by 1-5 and 15 and 19 as the main components of polygonum capitatum, the study result of the inhibitory activity of the polygonum capitatum to the principle P capitatum, the polysaccharide gallic acid, the polysaccharide.
Polygonum capitatum Canitatum is perennial herb of Polygonum of Polygonaceae, and is mainly distributed in Jiangxi, Hubei, Guangxi, Sichuan, Guizhou, Yunnan and Tibet regions (Wanghiping, Caoang, Yangxiwei, chemical component research of aerial parts of Polygonum capitatum [ J ] Chinese herbal medicine, 2013, 44 (1): 24-30.). The whole herb is used as a medicine, has the effects of clearing heat and promoting diuresis, inducing diuresis for treating strangurtia, activating blood and relieving pain, is a common medicinal material in Guizhou Miao region, takes local standard of 2003 edition Guizhou province, and has remarkable curative effect on urinary system infection caused by bacteria such as Escherichia coli and the like by a single preparation 'Relinqing granule' which takes Polygonum capitatum as a raw material and is prepared by Guizhou Weimen pharmaceutical industry, Inc., the authors find that Polygonum capitatum has better inhibiting effect on Escherichia coli and Pseudomonas aeruginosa in the early stage research, and the current literature reports mainly carry out research and analysis on chemical components and content of Polygonum capitatum (Panxitin, Zhali, Xiuyu, etc.. the main components and a cluster analysis method are used for comprehensive quality evaluation of Polygonum capitatum in different production places [ J, China Experimental journal, 2012, 18 (10): 153-157), and in addition, the research on the components and analysis results are still found by a standard comparison method, zhangjin, Liaoliancai, etc. Polygonum capitatum bacteriostasis substance basic research based on spectrum effect relationship [ J ] Chinese medicinal materials, 2016, 39 (9): 2037-2040).
The experiment can comprehensively analyze and determine chemical components of different polarity parts of the polygonum capitatum by using a UP L C-TOF-MS mass spectrometry combined technology, combines a pharmacodynamic test, and finally analyzes, screens and determines antibacterial substances in the polygonum capitatum by using a P L SR method, and clarifies pharmacodynamic substance basis of the polygonum capitatum on the treatment of urinary tract infection of escherichia coli and pseudomonas aeruginosa from a molecular chemistry level.
Drawings
FIG. 1 shows that the different polarity parts of Polygonum capitatum are UHP L C.
Detailed Description
The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible.
Example 1: spectrum efficiency of different polarity parts of polygonum capitatum for bacteriostasisAnalytical method
1. Material
1290 ultra-efficient high liquid chromatography system model Infinity and model TOF L C/MS combination (Agilent, USA), HWS-150 isothermal incubator (Beijing Kogawa Yongxing instruments, Inc.).
Polygonum capitatum is collected from Guizhou Shuicheng, and is identified as dry whole herb of Polygonum capitatum of Polygonum of Polygonaceae by Guiyang Hokko medicinal plant cultivation and identification professor Wei sublimation in church and research laboratory.
Escherichia coli CMCC44102 (batch No. 44102-3a24-2) and Pseudomonas aeruginosa CMCC10104 (batch No. 10104-2a21-1) were purchased from the Chinese food and drug testing institute.
MCI type macroporous resin column (mitsubishi chemical corporation, japan), nutrient broth culture medium (shanghai bo co micro technology ltd., lot No. 3103627), nutrient agar (shanghai bo micro technology ltd., lot No. 160825), dimethyl sulfoxide (DMSO, beijing solibao technology ltd., lot No. 520C0314), methanol, acetonitrile, and formic acid as chromatographies, and other reagents as analytically pure.
2. Method and results
2.1 extraction and separation of the drug
Extracting polygonum capitatum with 7 times of 70% ethanol (0.05% thioglycerol and 0.002% glacial acetic acid) for 2 times (1.5 h each time), filtering, mixing filtrates, recovering ethanol, concentrating to obtain soft extract with relative density of 1.20-1.25 (60 deg.C), drying under reduced pressure, and pulverizing into fine powder to obtain 70% ethanol extract of polygonum capitatum;
taking another 45g of polygonum capitatum 70% alcohol extract sample, adding a proper amount of methanol, carrying out ultrasonic extraction for 3 times, combining filtrates, centrifuging for 5min (3000rpm), taking supernate, carrying out reduced pressure distillation to remove the solvent, dissolving with 30% methanol, and carrying out chromatography separation on the supernate by using an MCI type macroporous resin column (1BV is 1L);
performing gradient elution by using 30%, 35% -40%, 40% -50%, 60% and 60% -80% of methanol in volume fraction respectively, collecting eluates of each part, and volatilizing to obtain 7 parts A-G;
accurately weighing 25.00mg of each of A to G, dissolving the A to G in 1m L DMSO, and preparing a solution with the mass concentration of 25mg/m L for later use.
2.2 UP L C analysis and Mass Spectrometry analysis of different parts of Polygonum capitatum
2.2.1 chromatographic and Mass Spectroscopy conditions
ACQUITY BEH C18A chromatographic column (2.1mm × 100mm, 1.7 μm), a column temperature of 40 ℃, a flow rate of 0.35L/min, a sample injection amount of 2 μ L, and gradient elution with a mobile phase of 0.1% formic acid aqueous solution (A) and 0.1% formic acid acetonitrile solution (B) (0-0.5 min, 95% A, 0.5-20 min, 95% A → 81.5% A; 20-30 min, 81.5% A → 95% A; 30-32 min, 95% A).
The mass spectrum conditions are electrospray ionization (ESI), dry gas temperature 180 deg.C, capillary voltage 4500 eV, negative ion detection mode, spray pressure 0.25MPa, and dry gas (N)2) The flow velocity is 8L/min, the scanning range m/z is 100-2000, and the collision energy is 10 eV.
2.2.2 establishment of fingerprint spectra of different polarity parts
UHP L C-TOF-MS chromatograms of different polarity sites were established under the conditions of item 2.2.1, the results are shown in FIG. 1 and Table 1, and the compounds corresponding to the peaks of different polarity sites were determined by mass spectrometry, the results are shown in Table 2.
Table 1: compounds contained in different polar parts and peak areas thereof
Table 2: mass spectrometry data for all compounds in different polar regions
As is apparent from table 1 and fig. 1, the seven polar regions separated from methanol are more thoroughly separated, and the main components of the different polar regions are effectively enriched, especially the davidiin obtained by first separating the D region has a purity of more than 90%.
The molecular formula of each peak and the secondary mass spectrum data thereof are obtained by mass spectrum analysis in the table 2, so that the chemical composition in the polygonum capitatum can be determined, and the main components of each part can be obtained by analysis, wherein A is mainly digallacyl hydrolysable tannin; b is mainly galloyl hydrolysable tannin; c is mainly galloyl hydrolysable tannin and procyanidin; more than 90% of D is the hydrolysable tannin davidiin; e mainly contains flavonoid glycoside and lignans; f mainly contains flavonoid glycoside components, especially flavonoid C-glycoside components; g mainly contains flavone monoglycoside and aglycone.
As described above, in site E, the main substance ingredient was quercetin-3-O-B-D-glucopyranoside No. 20, which had a retention time of about 10.48 min. The inventor finds that in the experiment, when trace thioglycerol and glacial acetic acid are added into an extracting solution, namely an ethanol solution, in the process of extracting an alcohol extract of a polygonum capitatum medicinal material, the obtained part E is obviously enriched with the substance No. 20. However, in the supplementary test, when (a) thioglycerol is not added, (b) glacial acetic acid is not added, or (c) neither thioglycerol nor glacial acetic acid is added to the extract solution, the test is performed according to the method of example 1 for seven sites obtained in each of the three cases (a) - (c), and it is shown that A, B, C, D, F, G obtained in the three cases are basically consistent with the results in the above-mentioned fig. 1, table 1 and table 2 of example 1 in the graphs and data provided in the modes of fig. 1, table 1 and table 2, for example, the peak areas of peak 8 and peak 19 in the data represented in table 1 are respectively in the range of 59730-60825 and in the range of 1382340-1385630 for the D sites obtained in the three cases; however, the amount of substance 20 in the part E obtained in the three cases without the simultaneous addition of thioglycerol and glacial acetic acid is significantly reduced, for example, the peak area of the peak 20 in the data represented in table 1 of the part E obtained in the three cases is in the range of 22730 to 26825, about 11% of the peak area of the peak 20 is obtained when the two substances are added simultaneously, and the retention time of the peak 20 in the part E in fig. 1 is still 10.48 min. The result shows that the part E can effectively enrich the substance No. 20 only when thioglycerol and glacial acetic acid are simultaneously added into the extraction solvent of the polygonum capitatum medicinal material.
2.3 MIC determination
2.3.1 preparation of bacterial suspension and preparation of plates
The experimental strain is firstly inoculated on a flat plate, cultured in a constant temperature and humidity incubator at 37 ℃ for 24h, selected characteristic strain is inoculated in a liquid culture medium, shaken at 37 ℃ for 24h, diluted to 0.5 McLeeb concentration by adopting a McLeeb method, namely 10^6-10^8CFU/m L bacterial suspension for later use, 20g of purified agar powder is added into a 1L nutrient broth culture medium, 1L double distilled water is added, the PH value is adjusted to 7.4, the mixture is sterilized at 121 ℃ for 30min and then taken out, when the culture medium is cooled to about 45 ℃, the culture medium is poured into a sterile culture dish on an ultraclean workbench to be condensed, and the experimental agar plate culture medium is obtained.
2.3.2 MIC assay
Adding 50 mu L liquid culture medium and 50 mu L bacterial suspension into each hole of a sterile 96-hole plate on an ultraclean workbench, accurately measuring 100 mu L of a drug to be detected by using a liquid transfer gun, adding the drug to be detected into the first hole of the 96-hole plate, uniformly mixing the drug to be detected into the second hole by absorbing 100 mu L, uniformly mixing the drug to the last hole by analogy, absorbing 100 mu L, keeping 100 mu L of each hole to obtain the concentration of 12.5-0.012 mg/m L, taking gentamicin sulfate as a positive control and DMSO as a negative control, culturing the drug at 37 ℃ for 24h, respectively transferring the drug to an agar plate culture medium, and taking the micro-hole without bacterial growth after 24h as the Minimum Inhibitory Concentration (MIC) (the test method can refer to Hulu, Zhang Jinliang, Liang, etc., rush, etc., and researching the antibacterial substance basis of the spectrum effect relationship of polygonum capitatum, wherein the results of the A, the Pseudomonas aeruginosa, the A, the E, and the E are respectively.
TABLE 3 MIC (g/L) for E.coli and P.aeruginosa in different polarity sites
| Separation site | Escherichia coli | Pseudomonas aeruginosa |
| A | 0.78 | 3.12 |
| B | 1.56 | 1.56 |
| C | 3.12 | 1.56 |
| D | 3.12 | 1.56 |
| E | 6.25 | 6.25 |
| F | 3.12 | 3.12 |
| G | 6.25 | 3.12 |
2.4P L SR analysis of peaks and 1/MIC of different fractions of Polygonum capitatum
The P L SR of the software SPSS16.0 is adopted to carry out analysis through equalization, the peak area of each peak is taken as independent variable, 1/MIC is taken as dependent variable analysis to obtain the relation result between each peak and the drug effect index, see table 4. 9 peaks which are positively correlated with the antibacterial effect of Escherichia coli can be obtained from table 4, the A part mainly exists, the peak which contributes to the larger is the peak with the number of 1-5, the peak which positively correlates with the antibacterial effect of Pseudomonas aeruginosa is 13, the B, C, D parts mainly exist in a concentrated mode, the peaks which contribute to the larger are the peaks with the numbers of 19 and 15, the mass spectrometry result is combined to obtain the maximum contribution value of 6-galloylglucose and 3, 6-digalliylglucose to Escherichia coli, and the contribution value of 1,3, 6-tripicolylglucose and davidiin to Pseudomonas aeruginosa.
Table 4: partial correlation coefficient of each peak with Escherichia coli and Pseudomonas aeruginosa
Example 2: spectral efficiency analysis method for bacteriostasis of polygonum capitatum parts with different polarities
Substantially as described with reference to example 1, except that: in the step (21), adding 8 times of 60% ethanol into the polygonum capitatum medicinal material, extracting for 2 times, each time for 1 hour, filtering, combining the filtrates, recovering ethanol, concentrating into thick paste, drying under reduced pressure, and pulverizing into fine powder to obtain polygonum capitatum ethanol extract; in step (22): taking a polygonum capitatum alcohol extract sample, adding a proper amount of methanol, carrying out ultrasonic extraction for 3 times, combining filtrates, centrifuging, taking supernate, carrying out reduced pressure distillation to remove the solvent, dissolving with 40% methanol, loading into an MCI type macroporous resin column, and carrying out chromatographic separation.
Example 3: spectral efficiency analysis method for bacteriostasis of polygonum capitatum parts with different polarities
Substantially as described with reference to example 1, except that: in the step (21), adding 80% ethanol 6 times the amount of the polygonum capitatum medicinal material into the polygonum capitatum medicinal material, extracting for 2 times, each time for 2 hours, filtering, combining the filtrates, recovering the ethanol, concentrating into thick paste, drying under reduced pressure, and crushing into fine powder to obtain polygonum capitatum ethanol extract; in step (22): taking a polygonum capitatum alcohol extract sample, adding a proper amount of methanol, carrying out ultrasonic extraction for 3 times, combining filtrates, centrifuging, taking supernate, carrying out reduced pressure distillation to remove the solvent, dissolving with 20% methanol, loading into an MCI type macroporous resin column, and carrying out chromatographic separation.
Example 4: spectral efficiency analysis method for bacteriostasis of polygonum capitatum parts with different polarities
Substantially as described with reference to example 1, except that: in the step (21), adding 75% ethanol in an amount which is 7 times that of the polygonum capitatum medicinal material into the polygonum capitatum medicinal material, extracting for 2 times, each time for 1 hour, filtering, combining the filtrates, recovering the ethanol, concentrating into thick paste, drying under reduced pressure, and crushing into fine powder to obtain polygonum capitatum ethanol extract; in step (22): taking a polygonum capitatum alcohol extract sample, adding a proper amount of methanol, carrying out ultrasonic extraction for 3 times, combining filtrates, centrifuging, taking supernate, carrying out reduced pressure distillation to remove the solvent, dissolving with 30% methanol, loading into an MCI type macroporous resin column, and carrying out chromatographic separation.
The three examples of the above examples 2 to 4 each give three lots of A, B, C, D, E, F, G with different polarity, and the three lots of seven sites are substantially identical to the results of fig. 1, table 2, table 3 and table 4 of the example 1 in the maps and data provided in the manners of fig. 1, table 2, table 3 and table 4, respectively, and for example, the peak areas of the peak No. 8 and the peak No. 19 in the data characterized in table 1 of the obtained D site are in the ranges of 59160 to 60786 and 1381825 to 1385743, respectively.
The invention obtains fingerprint spectra of polygonum capitatum different polarity parts and the bacteriostasis effect on escherichia coli and pseudomonas aeruginosa through UHP L C-TOF-MS and a liquid dilution method, and discloses spectral efficiency relation analysis of each chemical component of polygonum capitatum different polarity parts on escherichia coli and pseudomonas aeruginosa through a P L SR method, thereby obtaining the correlation between each peak of different polarity parts and the bacteriostasis, the spectral efficiency analysis result shows that the peak with larger contribution of the polygonum capitatum bacteriostasis activity exists in A, B, C, D and other polar parts, and simultaneously determines that the main action substances of polygonum capitatum on escherichia coli and pseudomonas aeruginosa have strong polarity correlation of 1-5 and 15, 19 in the bacteriostasis effect, the main action substances of the polygonum capitatum are compounds represented by 1-5 and 15, 19 (6-glucose gallate, 3, 6-glucose digallic acid, 1,3, 6-glucose trigallic acid and davidiin and isomers) the invention obtains the maximum effective peak area of polygonum capitatum and provides the maximum effective index for inhibiting infection of pseudomonas aeruginosa through the clinical application of the related polysaccharide in L through a simple combined technology, and the related experiments of the related effective peak area of polygonum capitatum.
Claims (12)
1. A method for analyzing the spectrum effectiveness of the bacteriostatic action of different polarity parts of polygonum capitatum comprises the following steps:
(1) material supply: providing an ultra-high performance liquid chromatography system and a liquid chromatography-mass spectrometer, providing a polygonum capitatum medicinal material, and providing a strain for bacteriostatic detection;
(2) extracting and separating the medicines:
(21) adding 60-80% ethanol 6-8 times the amount of the polygonum capitatum medicinal material into the polygonum capitatum medicinal material, extracting for 2 times, each time for 1-2 hours, filtering, combining filtrates, recovering ethanol, concentrating into thick paste, drying under reduced pressure, and crushing into fine powder to obtain polygonum capitatum ethanol extract; 0.05% of thioglycerol and 0.002% of glacial acetic acid are added into the 60-80% ethanol;
(22) taking another polygonum capitatum alcohol extract sample, adding a proper amount of methanol, carrying out ultrasonic extraction for 3 times, combining filtrates, centrifuging, taking supernate, carrying out reduced pressure distillation to remove the solvent, dissolving with 20-40% methanol, loading into an MCI type macroporous resin column, and carrying out chromatographic separation;
(23) performing gradient elution by using 30%, 35% -40%, 40% -50%, 60% and 60% -80% of methanol in volume fraction respectively, collecting eluates of each part, and volatilizing to obtain seven parts A-G with different polarities;
(24) accurately weighing seven parts A-G, respectively dissolving the seven parts A-G in DMSO, and preparing a solution with the mass concentration of 25mg/m L for later use;
(3) UP L C analysis and mass spectrometry analysis:
(31) chromatographic and mass spectrometric conditions:
chromatographic conditions are as follows: c18A chromatographic column with the column temperature of 40 ℃, the flow rate of 0.35L/min and the sample injection amount of 2 mu L, wherein the mobile phase is subjected to gradient elution by using a mobile phase A and a mobile phase B, the mobile phase A is 0.1% formic acid aqueous solution, and the mobile phase B is 0.1% formic acid acetonitrile solution;
the elution procedure for the gradient elution was: 0-0.5 min, 95% mobile phase A; 0.5-20 min, 95% of mobile phase A → 81.5% of mobile phase A; 20-30 min, 81.5% of mobile phase A → 95% of mobile phase A; 30-32 min, 95% of mobile phase A;
the mass spectrum conditions comprise electrospray ion source, drying gas temperature of 180 ℃, capillary voltage of 4500 eV, negative ion detection mode, spraying pressure of 0.25MPa, drying gas nitrogen flow rate of 8L/min, and scanning rangem/z100 to 2000, collision energy of 10 eV;
(32) establishing fingerprint spectra of different polar parts, namely establishing UHP L C-TOF-MS chromatograms of the different polar parts according to the conditions of (31) chromatogram and mass spectrum, and determining compounds corresponding to peaks of the different polar parts through mass spectrum so as to determine main chemical compositions in the parts;
(4) determination of minimum inhibitory concentration:
(41) preparing bacterial suspension and preparing an agar plate culture medium for experiments according to a minimum inhibitory concentration determination method;
(42) according to a minimum inhibitory concentration determination method, determining the minimum inhibitory concentrations of the parts with different polarities obtained in the step (24);
(5) partial least squares regression analysis between each peak and 1/MIC of different polarity parts of polygonum capitatum: for each part, the analysis is carried out by averaging the part by using the partial least squares regression analysis method of SPSS16.0 software, the relation between each substance peak and the pharmacodynamic index is obtained by analyzing the peak area of each substance peak as an independent variable and 1/MIC as a dependent variable, and the substance peak which is positively correlated with the test strain and the part where the substances exist are determined.
2. The method of claim 1, wherein the ultra high performance liquid chromatography system is a 1290Infinity model ultra high performance liquid chromatography system.
3. The method according to claim 1, wherein the LC-MS is an Agilent TOF L C/MS.
4. The method according to claim 1, wherein the Polygonum capitatum is Polygonum capitatum collected from Guizhou.
5. The method according to claim 1, wherein the bacteria for bacteriostasis detection comprises Escherichia coli and Pseudomonas aeruginosa.
6. The method according to claim 1, wherein in the step (21), the polygonum capitatum medicinal material is added with 7 times of 70% ethanol for extraction for 2 times, each time lasts for 1.5h, the filtration is carried out, the filtrates are combined, the ethanol is recovered and concentrated to thick paste with the relative density of 1.20-1.25 at 60 ℃, the thick paste is dried under reduced pressure and crushed into fine powder, and 70% ethanol extract of polygonum capitatum is obtained; the ethanol used for extraction was added with 0.05% thioglycerol and 0.002% glacial acetic acid.
7. The method according to claim 1, wherein in step (22), 45g of 70% alcoholic extract sample of Polygonum capitatum is taken, added with an appropriate amount of methanol, ultrasonically extracted for 3 times, the filtrates are combined, centrifuged at 3000rpm for 5min, the supernatant is taken, distilled under reduced pressure to remove the solvent, dissolved with 30% methanol and loaded into MCI type macroporous resin column for chromatography and separation.
8. The method according to claim 1, wherein in the step (24), 25.00mg of each of the seven parts A-G is accurately weighed and dissolved in 1m L DMSO to prepare a solution.
9. The method according to claim 1, wherein the chromatographic column is an ACQUITY BEH C18A column of 2.1mm × 100mm, 1.7 μm.
10. The method of claim 1, said bacterial suspension being formulatedThe operation process is as follows: the strain for experiment is firstly inoculated on a flat plate and cultured in a constant temperature and humidity incubator at 37 ℃ for 24 hours; selecting characteristic strains, inoculating the strains into a liquid culture medium, and shaking at 37 ℃ for 24 h; diluting to 0.5 McLeod by McLeod's turbidimetry to obtain 106-108CFU/m L bacterial suspension for use.
11. The method of claim 1, wherein the agar plate medium for experiments is prepared by adding 20g of purified agar powder to 1L nutrient broth medium, adding 1L double distilled water and adjusting pH =7.4, sterilizing at 121 deg.C for 30min, taking out, cooling to about 45 deg.C, pouring the medium into a sterile petri dish on a clean bench, and condensing to obtain the agar plate medium for experiments.
12. The method of claim 1, wherein the determination of the minimum inhibitory concentration of the different polar regions comprises adding 50 μ L liquid culture medium and 50 μ L bacterial suspension into each well of a sterile 96-well plate on a clean bench, accurately weighing 100 μ L of the drug to be tested by a pipette, adding the drug to be tested into the first well of the 96-well plate, mixing the drug to be tested into the second well by sucking 100 μ L, mixing the drug to be tested into the last well by sucking 100 μ L, keeping each well at 100 μ L, using gentamicin sulfate as a positive control and DMSO as a negative control, culturing the drug at 37 ℃ for 24h, transferring the drug to an agar plate culture medium, and determining the concentration of the drug in the micro-well without bacterial growth after 24h as the minimum inhibitory concentration.
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Denomination of invention: Spectral analysis method of bacteriostasis of different polar parts of Polygonum capitatum Effective date of registration: 20230223 Granted publication date: 20200728 Pledgee: Agricultural Bank of China Limited by Share Ltd. Guiyang Xinhua Branch Pledgor: GUIZHOU WARMEN PHARMACEUTICAL Co.,Ltd. Registration number: Y2023990000128 |
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