CN107870216A - The spectrum effect of polygonum capitatum opposed polarity position bacteriostasis learns analysis method - Google Patents
The spectrum effect of polygonum capitatum opposed polarity position bacteriostasis learns analysis method Download PDFInfo
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Abstract
The present invention relates to the spectrum of polygonum capitatum opposed polarity position bacteriostasis effect to learn analysis method.Specifically, comprise the following steps：Material is provided, and medicine is extracted with separating, UPLC analyses and mass spectral analysis, the measure of minimum inhibitory concentration, the partial least-squares regressive analysis between each peak in polygonum capitatum opposed polarity position and 1/MIC.The present invention can be analyzed comprehensively by UPLC TOF MS mass spectrometric hyphenated techniques to be determined the chemical composition at polygonum capitatum opposed polarity position and combines the test of pesticide effectiveness, using the Analysis and Screening of PLSR methods, antibacterial substance in polygonum capitatum is determined, polygonum capitatum is illustrated in treatment EHEC, pseudomonas aeruginosa urinary tract infections effective substance from molecular chemistry level.
Description
Technical field
The invention belongs to pharmaceutical technology field, and in particular to the Pharmaceutical Analysis method of polygonum capitatum, be more particularly to polygonum capitatum
The spectrum effect of opposed polarity position bacteriostasis learns analysis method.The inventive method can lay solid for the deep development of polygonum capitatum
Basis.
Background technology
Polygonum capitatum (Polygonum capitatum Buch-Ham ex D.Don) is planted for polygonaceae Polygonum perennial herb
Thing.The effect of with clearing heat and promoting diuresis, inducing diuresis for treating strangurtia, clinically treating urinary system infection contamination has significant effect.Using its as
Chinese patent medicine preparation made of raw material " Relinqing Granula " enters National essential drugs list for 2002, and it is basic to enter country within 2004
Medical insurance catalogue.
Polygonum capitatum is the common medicine of minority area, is mainly used in the diseases such as pyelonephritis, urinary tract infection, inducing diuresis for treating strangurtia.
Related pharmaceutical research is rare, only appoints several reports such as light friend at present.Ren Guangyou uses rat bacterial pyelonephritis mould
Type is tested, as a result the WBC in polygonum capitatum water extract group rat urine and BLD and the obvious reduction of control group, display
Polygonum capitatum water extract has certain antiinflammatory action to pyelonephritis.Ren Guangyou etc. is seen with mouse peritoneal injection Escherichia coli bacteria liquid
Dead mouse situation in 5 days is examined, as a result the control group death rate is 100%, and the polygonum capitatum group death rate is respectively 20% and 50%, is shown
Show polygonum capitatum water extract to resist caused by Escherichia coli to infect.Ren Guangyou etc. gives house with polygonum capitatum water extract gastric infusion
Rabbit, as a result, polygonum capitatum water extract group is compared with control group, and there was no significant difference for body temperature, but can reduce the secondary wound of intravenous injection typhoid fever
The heating body temperature of rabbit caused by cold vaccine.Ren Guangyou etc., with polygonum capitatum water extract difference gastric infusion rabbit, rat, with sky
White group and furosemide control group compare urine volume.As a result show polygonum capitatum water extract to rabbit and rat without obvious diuresis.Xu Ying
Spring et al. have detected In Vitro Bacteriostatic of the polygonum capitatum to 10 plants of neisseria gonorrhoeaes (gonococcus) using agar dilution, as a result
Polygonum capitatum has bacteriostatic activity to gonococcus.Its minimal inhibitory concentration scope to 10 plants of gonococcus is 8~32g/L, and average value is
11.2g/L。
Chinese patent application prospectus CN1054899A (Chinese Patent Application No. 90107810.7, publication date 1991
On October 2) in disclose a kind of miganling instant herbal medicine, syrup production technology.The production technology is using four seasons grass as starting material with water
Decoct 30-60 minutes, extract solution filtering and concentrating, its supernatant is concentrated under reduced pressure into cream, then extracted with 60-70% ethanol, by second
Alcohol extract is dried in vacuo, and is obtained the four seasons red medicinal extract, is further configured to electuary or syrup, it is believed that should be with removing toxic substances, the scattered stasis of blood, profit
The effect of urinating, be treating stranguria.
The Ministry of Health of the People's Republic of China compiled in 1998 of pharmacopoeia commission of Ministry of Health of the People's Republic of China《Medicine mark
Standard-Traditional Chinese medicine historical preparation》The Chinese patent medicine preparation of entitled " Relinqing Granula " is recorded in (the 17th), it is by by head
After flower knotweed adds water to cook twice made of filtering and concentrating.
CN 1481832A (Chinese Patent Application No. 02129686.3, publication date on March 17th, 2004) and CN
1483466A (Chinese Patent Application No. 03146381.9, publication date on March 24th, 2004) discloses Herba Polygoni Capitati extract, its base
It is to be prepared by following steps in sheet：A. decocted with moisture by the fresh goods or dry product of polygonum capitatum herb or mixed with alcohol water twice
Thing point two to refluxing extraction three times, each 1-2 hours, merging decoction liquor, filtering and concentrating to relative density at 20 DEG C is 1.2,
It is spray-dried or is dried under reduced pressure to obtain；Or b. is obtained by polygonum capitatum herb and its water extraction dregs of a decoction through carbon dioxide supercritical extraction
Arrive.It is believed that this extract can be used for antibacterial, anti-inflammatory, analgesia, diuresis, treatment calculi in urinary system, treatment pyelonephritis and preceding
Row adenositis.
Although polygonum capitatum preparation has clinically been widely used, but the pharmacodynamic study of its various active component is still
Be have carry out in a deep going way it is necessary, such as polygonum capitatum opposed polarity position bacteriostasis spectrum effect credit analysis, be still to instruct head
The necessary means of flower knotweed preparation clinical practice, establish the finger-print of the active component isolated and purified in Herba Polygoni Capitati extract, explain
The chemical composition that bright all peaks of different parts represent discloses the antibacterial effective substance base of polygonum capitatum to the contribution margin of fungistatic effect
Plinth, it is still that those skilled in the art urgently expect.
The content of the invention
It is an object of the present invention to the pharmacodynamic study of the various active components to polygonum capitatum, such as it is different to polygonum capitatum
The spectrum effect credit analysis of polar fraction bacteriostasis, to instruct polygonum capitatum preparation clinical practice, establishes in Herba Polygoni Capitati extract and divides
From the finger-print of the active component of purifying, contribution of the chemical composition of all peaks representatives of different parts to fungistatic effect is illustrated
Value, disclose the antibacterial effective substance of polygonum capitatum.By the inventive method, have now surprisingly been found that, have learned that polygonum capitatum
Main antibacterial active site to EHEC, pseudomonas aeruginosa is tetra- highly polar positions of A, B, C, D, and it mainly presses down
Bacterium composition be 6- gallic acid acyls glucose, the gallic acid acyl glucose of 3,6- bis-, the gallic acid acyl glucose of 1,3,6- tri- and
davidiin.The present invention is accomplished based on this discovery.
Therefore, in one aspect of the invention, there is provided a kind of spectrum effect of polygonum capitatum opposed polarity position bacteriostasis is learned
The method of analysis, it comprises the following steps：
(1) material provides：
There is provided ultra performance liquid chromatography system and liquid chromatograph-mass spectrometer, there is provided medicinal material of polygonum capilalum, there is provided antibacterial inspection
Survey bacterial strain；
(2) medicine extraction is with separating：
(21) take medicinal material of polygonum capilalum to add 6~8 times of 60~80% ethanol of amount to extract 2 times, 1~2 hour every time, filter, merge
Filtrate, reclaim ethanol and be condensed into thick paste, be dried under reduced pressure, be ground into fine powder, produce polygonum capitatum alcohol-extracted extract；
(22) polygonum capitatum alcohol-extracted extract sample separately is taken, adds methanol appropriate, ultrasonic extraction 3 times, filtrate merges, and centrifugation, takes
Clear liquid, which is evaporated under reduced pressure, removes solvent, after being dissolved with 20~40% methanol, is loaded into MCI type macroporous resin columns, carries out chromatography；
(23) respectively with volume fraction be 30%, 35%~40%, 40%, 40%~50%, 50%, 60%, 60%~
80% methanol carries out gradient elution, collects each several part eluent, and volatilizes, and obtains the position of A~G totally seven opposed polarities；
(24) seven positions of A~G are accurately weighed, are dissolved in respectively in DMSO, are made into the solution that mass concentration is 25mg/mL,
It is standby；
(3) UPLC analyses and mass spectral analysis：
(31) chromatogram and Mass Spectrometry Conditions：
Chromatographic condition：C18Chromatographic column, 40 DEG C, flow velocity 0.35L/min of column temperature, the μ L of sample size 2, mobile phase use 0.1% first
The formic acid acetonitrile solution (B) of aqueous acid (A) -0.1% carries out gradient elution；
Mass Spectrometry Conditions：Electric spray ion source (ESI), 180 DEG C of dry gas temperature, the 500eV of capillary voltage 4, anion
Detection pattern, atomisation pressure 0.25MPa, dry gas (N2) flow velocity 8L/min, scanning range m/z 100~2 000, collision energy
10eV；
(32) opposed polarity position finger-print is established：
The UHPLC-TOF-MS chromatograms at opposed polarity position are established by condition under " (31) chromatogram and Mass Spectrometry Conditions " item, and
Compound corresponding to each peak in opposed polarity position is determined by mass spectrum, so that it is determined that the primary chemical composition in each position；
(4) measure of minimum inhibitory concentration：
(41) lowest bacteria fogging-resistant concentration determining method is pressed, carries out the preparation of bacteria suspension and the system of experiment Agar Plating
It is standby；
(42) lowest bacteria fogging-resistant concentration determining method is pressed, opposed polarity position is minimum antibacterial dense obtained by determination step (24)
Degree；
(5) partial least-squares regressive analysis between each peak in polygonum capitatum opposed polarity position and 1/MIC：
For each position, pass through its average using the partial least-squares regressive analysis method (PLSR) of SPSS16.0 softwares
Change analyzed, using the peak area at its each material peak as independent variable, 1/MIC be used as dependent variable, analyze obtain each material peak and
Relation between pharmacodynamics index, it is determined that the position present in test strain into positively related material peak and these materials.
In an embodiment of the inventive method, the ultra performance liquid chromatography system is that 1290Infinity types surpass
Imitate high liquid chromatography system.In one embodiment, the liquid chromatograph-mass spectrometer is the TOF of Agilent companies
LC/MS combined instruments.In one embodiment, the medicinal material of polygonum capilalum is the polygonum capitatum for picking up from Guizhou.In an embodiment
In, the antibacterial detection bacterial strain includes but is not limited to EHEC, pseudomonas aeruginosa.
In an embodiment of the inventive method, wherein in step (21), medicinal material of polygonum capilalum is taken to add 7 times of 70% second of amount
Alcohol extracting 2 times, each 1.5h, filtration, merging filtrate, and it is 1.20~1.25 (60 DEG C) to reclaim ethanol and be concentrated into relative density
Thick paste, be dried under reduced pressure, be ground into fine powder, produce the alcohol-extracted extract of polygonum capitatum 70%.In an embodiment of the inventive method
In, wherein in step (21), take and 0.05% thioglycerol and 0.002% ice second are with the addition of in ethanol solution used in medicinal material of polygonum capilalum
Acid.
In an embodiment of the inventive method, wherein in step (22), the alcohol-extracted extract sample of polygonum capitatum 70% is taken
45g, add methanol appropriate, ultrasonic extraction 3 times, filtrate merges, centrifugation 5min (3000rpm), takes supernatant to be evaporated under reduced pressure removing molten
Agent, dissolved with 30% methanol and be loaded into MCI types macroporous resin column (1BV=1L) chromatography.
It is respectively 30%, 35% with volume fraction wherein in step (23) in an embodiment of the inventive method
~40%, 40%, 40%~50%, 50%, 60%, 60%~80% methanol carries out gradient elution, collects each several part elution
Liquid, and volatilize, obtain A~G totally seven positions.
In an embodiment of the inventive method, wherein in step (24), it is each accurately to weigh seven positions of A~G
25.00mg is dissolved in 1mL DMSO, is made into the solution that mass concentration is 25mg/mL.
In an embodiment of the inventive method, wherein the chromatographic column is ACQUITY BEH C18Chromatographic column, its
Specification is 2.1mm × 100mm, 1.7 μm.
In an embodiment of the inventive method, wherein the elution program of the gradient elution is：0~0.5min,
95%A, 0.5~20min, 95%A → 81.5%A；20~30min, 81.5%A → 95%A；30~32min, 95%A.
In an embodiment of the inventive method, wherein the operating process of the preparation of the bacteria suspension is：Experiment is used
Strain is inoculated on flat board first, and 24h is cultivated in 37 DEG C of constant temperature and humidity incubators；Select feature strain and be connected to fluid nutrient medium
In, under the conditions of 37 DEG C, shake 24h；0.5 Maxwell concentration is diluted to using Maxwell turbidimetry, produces 10^6-10^8CFU/
ML bacteria suspensions, it is standby.
In an embodiment of the inventive method, wherein the preparation process of the experiment Agar Plating
For：20g purified agar powder is added into 1L nutrient broth mediums, 1L distilled waters is added and adjusts PH=7.4；In 121 DEG C of bars
Under part, taken out after the 30min that sterilizes；When culture medium is cooled to 45 DEG C or so, culture medium poured on superclean bench sterile
Condensed in culture dish, produce experiment Agar Plating.
In an embodiment of the inventive method, the process of the minimum inhibitory concentration at measure opposed polarity position is：
On superclean bench, 50 μ L fluid nutrient mediums and 50 μ L bacteria suspension are first added into the every hole of 96 sterile orifice plates, will be to be measured
The first hole that medicine is added to the μ L of liquid-transfering gun accurate measuring 100 in 96 orifice plates mixes, and 100 μ L of absorption move to the second hole and mixed
It is even, mix 100 μ L of absorption to last hole by that analogy and discard, every hole is kept 100 μ L；Using gentamicin sulphate to be positive right
According to DMSO is negative control；37 DEG C are cultivated 24h, respectively transferred species to Agar Plating, the micropore without bacterial growth after 24h
The concentration of middle medicine is then minimum inhibitory concentration.
In the step of above method of the present invention, although the specific steps of its description are in some details or language describes
The step of described in the upper preparation example with following detailed description part, is otherwise varied, however, those skilled in the art's root
Approach described above step can be summarized completely according to the detailed disclosure of full text of the present invention.
Any embodiment of the either side of the present invention, can be combined with other embodiments, as long as they are not
Contradiction occurs.In addition, in any embodiment of either side of the present invention, any technical characteristic goes for other realities
The technical characteristic in scheme is applied, as long as they are not in contradiction.The invention will be further described below.
All documents recited in the present invention, their full content are incorporated herein by reference, and if these are literary
When offering expressed implication and the inconsistent present invention, it is defined by the statement of the present invention.In addition, the various terms that use of the present invention and
Phrase has well known to a person skilled in the art general sense, nonetheless, the present invention remain desirable at this to these terms and
Phrase is described in more detail and explains, the term and phrase referred to is if any inconsistent with common art-recognized meanings, with institute's table of the present invention
The implication stated is defined.
It is an object of the invention to establish in polygonum capitatum extraction dry cream the fingerprint image of several active components isolated and purified
Spectrum, the chemical composition of all peaks representatives of different parts is illustrated to the contribution margin of fungistatic effect, discloses the antibacterial drug effect thing of polygonum capitatum
Matter basis.The present invention establishes the finger-print at polygonum capitatum opposed polarity position using UPLC-TOF-MS, and carries out chemistry to each peak
Constituent analysis, each polygonum capitatum opposed polarity position is measured to EHEC, pseudomonas aeruginosa most by 96 well plate methods
Low Mlc (MIC), polygonum capitatum finger-print and bacteriostasis are established using partial least-squares regressive analysis method (PLSR)
Spectrum effect relationship.As a result show, the present invention establishes polygonum capitatum opposed polarity position finger-print, and learns relationship analysis by composing to imitate
Determine polygonum capitatum to EHEC, pseudomonas aeruginosa fungistatic effect into positively related peak be 9 and 13 respectively, its
Main antipathogenic composition is the compound representated by 1~5 and 15,19.The study result show that polygonum capitatum is uncommon to large intestine angstrom
Bacterium, the main antibacterial active site of pseudomonas aeruginosa are tetra- highly polar positions of A, B, C, D, and its main antipathogenic composition is 6-
Gallic acid acyl glucose, the gallic acid acyl glucose of 3,6- bis-, the gallic acid acyl glucose of 1,3,6- tri- and davidiin.This
Invention result of study is laid a good foundation for the antimicrobial mechanism of further investigation polygonum capitatum.
Polygonum capitatum Polygonum capitatum plant for polygonaceae Polygonace-ae Polygonum Polygonum perennial herbs
Thing, it is distributed mainly on Jiangxi, Hubei, Guangxi, Sichuan, Guizhou, Yunnan and Tibet region (the big heads of Wang Hongping, Cao Fang, Yang Xiu
Chemical constitution study [J] Chinese herbal medicines of flower knotweed aerial part, 2013,44 (1)：24-30.).With all herbal medicine, there is heat-clearing profit
The effect of wet, inducing diuresis for treating strangurtia, promoting blood circulation and stopping pain, it is the medicinal herbs most in use in Miao in Guizhou area, and has taken in 2003 editions Guizhou Province place
Standard, large intestine is being treated using the single preparations of ephedrine " Relinqing Granula " that polygonum capitatum is raw material by GuiZhou WeiMen Pharmacy Co., Ltd
The bacterial urinary system infection contaminations such as angstrom uncommon bacterium are evident in efficacy, and author has found that polygonum capitatum is uncommon to large intestine angstrom in early-stage Study
Bacterium, pseudomonas aeruginosa have good inhibiting effect, and at present in document report primarily directed to the chemical composition of polygonum capitatum
And its content is researched and analysed and (Pan Wenting, Zhang Liyan, Xie Yu, waits principal components and clustering methodology to different sources polygonum capitatum
Quality evaluation [J] Chinese experimental pharmacology of traditional Chinese medical formulae magazines, 2012,18 (10)：153-157), additionally, it is observed that having with standard
Product Comparison Method probes into that its antipathogenic composition its analysis result is still indefinite (Hu Lu, Zhang Jin, Lin Liangcai, to wait based on spectrum effect relationship
Polygonum capitatum bacteriostasis material base studies [J] Chinese medicines, 2016,39 (9)：2037-2040).
Present invention experiment can be analyzed comprehensively by UPLC-TOF-MS mass spectrometric hyphenated techniques determines polygonum capitatum opposed polarity
The chemical composition at position simultaneously combines the test of pesticide effectiveness, finally using the Analysis and Screening of PLSR methods, determine antibacterial substance in polygonum capitatum, from
Molecular chemistry level illustrates polygonum capitatum in treatment EHEC, pseudomonas aeruginosa urinary tract infections effective substance.This
The PLSR methods that invention uses are the advantages of one kind integrate multiple linear regression, principal component analysis and canonical correlation analysis, can
(phase beauty, Wang Xiaoxia, Sun Qihui, asarums not homopolarity is waited preferably to disclose the correlation between each chromatographic peak and pharmacodynamics index
Property position HPLC finger-prints and its antalgic and inflammation relieving activity spectrum effect relationship research [J] Liaoning Journal of Traditional Chinese Medicine, 2016,43 (12)：
2603-2607；Lv Shao Wa, Dong Shuyu, Guo Yuyan, wait application progress [J] of data analysis techniques in Chinese medicine spectrum effect relationship
Chinese experimental pharmacology of traditional Chinese medical formulae magazine, 2015,21 (15)：226-230).Author is thought for complicated component and active ingredient not simultaneously
For the Chinese medicine of determination, Chinese medicine spectrum effect relationship is analyzed to determine its active ingredient using suitable evaluation method, while also may be used
Research foundation is provided for the application of polygonum capitatum substitute antibiotics.
Brief description of the drawings
Fig. 1：Polygonum capitatum opposed polarity position UHPLC.
Embodiment
The present invention can be further described by the following examples, however, the scope of the present invention and unlimited
In following embodiments.One of skill in the art, can be with it is understood that on the premise of without departing substantially from the spirit and scope of the present invention
Various change and modification are carried out to the present invention.The present invention carries out general to the material and test method that are arrived used in experiment
And/or specific description.Although for realize many materials used in the object of the invention and operating method be it is known in the art that
But the present invention is still described in detail as far as possible herein.
Embodiment 1：The spectrum effect of polygonum capitatum opposed polarity position bacteriostasis learns analysis method
1st, material
(U.S. Agilent is public for the super effect high liquid chromatography system of 1290Infinity types and 6230 type TOF LC/MS combined instruments
Department), HWS-150 types constant incubator (Ke Wei Yongxings, Beijing Instrument Ltd.).
Polygonum capitatum, Shuicheng, Guizhou Province is picked up from, through Guiyang College of Traditional Chinese Medicine's medicinal plant cultivation and identification teaching and research room Wei Shenghua professors
It is accredited as polygonaceae arsesmart polygonum capitatum Polygonum capitatum drying herb.
EHEC CMCC44102 (lot number 44102-3a24-2) and pseudomonas aeruginosa CMCC10104 (lot numbers
10104-2a21-1) it is purchased from National Institute for Food and Drugs Control.
MCI types macroporous resin column (Mitsubishi chemical company), nutrient broth medium (win micro- limited public affairs of science and technology in Shanghai
Department, lot number 3103627), nutrient agar (Shanghai Bo Wei Science and Technology Ltd.s, lot number 160825), dimethyl sulfoxide (DMSO) (DMSO, north
Jing Suolaibao Science and Technology Ltd.s, lot number 520C0314), methanol, acetonitrile, formic acid are chromatographically pure, and other reagents are that analysis is pure.
2nd, method and result
2.1st, medicine extraction is with separating
Medicinal material of polygonum capilalum is taken to add 7 times of 70% ethanol of amount (interior plus 0.05% thioglycerol and 0.002% glacial acetic acid) extractions 2
It is secondary, each 1.5h, filtration, merging filtrate, and reclaim ethanol and be concentrated into the thick paste that relative density is 1.20~1.25 (60 DEG C),
It is dried under reduced pressure, is ground into fine powder, produces the alcohol-extracted extract of polygonum capitatum 70%；
The alcohol-extracted extract sample 45g of polygonum capitatum 70% separately is taken, adds methanol appropriate, ultrasonic extraction 3 times, filtrate merges, centrifugation
5min (3000rpm), take supernatant to be evaporated under reduced pressure and remove solvent, dissolved with 30% methanol and be loaded into MCI type macroporous resin columns (1BV
=1L) chromatography；
It is respectively 30%, 35%~40%, 40%, 40%~50%, 50%, 60%, 60%~80% with volume fraction
Methanol carry out gradient elution, collect each several part eluent, and volatilize, obtain A~G totally 7 positions；
Accurately weigh each 25.00mg of A~G to be dissolved in 1mL DMSO, be made into the solution that mass concentration is 25mg/mL, it is standby
With.
2.2nd, the UPLC analyses and mass spectral analysis of polygonum capitatum different parts
2.2.1, chromatogram and Mass Spectrometry Conditions
ACQUITY BEH C18Chromatographic column (2.1mm × 100mm, 1.7 μm), 40 DEG C, flow velocity 0.35L/min of column temperature, sample introduction
Measure 2 μ L, the aqueous formic acid of mobile phase 0.1% (A) -0.1% formic acid acetonitrile solution (B) gradient elution (0~0.5min, 95%A,
0.5~20min, 95%A → 81.5%A；20~30min, 81.5%A → 95%A；30~32min, 95%A).
Mass Spectrometry Conditions are electric spray ion source (ESI), 180 DEG C of dry gas temperature, the 500eV of capillary voltage 4, bear from
Sub- detection pattern, atomisation pressure 0.25MPa, dry gas (N2) flow velocity 8L/min, scanning range m/z 100~2 000, impact energy
Measure 10eV.
2.2.2, opposed polarity position finger-print is established
The UHPLC-TOF-MS chromatograms at opposed polarity position are established by condition under 2.2.1 items；As a result Fig. 1 and table 1 are seen；And
By mass spectrum determine each peak in opposed polarity position corresponding to compound, the results are shown in Table 2.
Table 1：Compound and its peak area contained by opposed polarity position
Table 2：The analytical data of mass spectrum of all compounds in opposed polarity position
By table 1 with can significantly see in Fig. 1, seven polar fractions being obtained by separating methanol separate more thorough
Bottom, and the principal component at opposed polarity position also obtained effective enrichment, particularly D positions davidiin isolated first its
Purity reaches more than 90%.
Each peak molecular formula and its second order mses data are obtained by the mass spectral analysis in table 2, thus may determine that in polygonum capitatum
Chemical composition, while can also analyze to obtain the main component at each position, A is mainly two nutgall acyl condensed tannins；B master
To be three nutgall acyl condensed tannins；C is mainly three nutgall acyl condensed tannins and OPC constituents；D 90% with
On be condensed tannin davidiin；E mainly contains flavonoid glycoside and lignan component；F mainly contains flavonoid glycoside composition, special
It is not flavones C- methods of glycosides；G mainly contains flavones monoglycosides and aglycon constituents.
As described above, in the E of position, main matter composition is the Quercetin -3-O-B-D- glucopyranosides of No. 20, its
Retention time is about 10.48min.The present inventor in experiments it is found that, this experiment to medicinal material of polygonum capilalum carry out alcohol-extracted extract carry
When micro thioglycerol and glacial acetic acid are added into extract solution i.e. ethanol solution during taking, No. 20 materials are presented in gained position E
Significant enrichment.But in the experiment of supplement and in the extract solution (a) do not add thioglycerol, (b) do not add glacial acetic acid,
Or when not added both (c) thioglycerol glacial acetic acid, seven positions that three kinds of situations of (a)~(c) respectively obtain, according to this reality
The method for applying example 1 is tested, and shows that A, B, C, D, F, G obtained by three kinds of situations are provided in Fig. 1, table 1, the mode of table 2
It is basically identical with the result in 1 above-mentioned Fig. 1 of this implementation column, table 1, table 2 in collection of illustrative plates and data, such as D positions obtained by three kinds of situations
In the data that table 1 characterizes the peak area at No. 8 peaks and No. 19 peaks respectively in the range of 59730~60825 and 1382340~
In the range of 1385630；But do not add No. 20 amount of substance in position E obtained by three kinds of situations of thioglycerol and glacial acetic acid simultaneously and show
Write and reduce, for example, E positions No. 20 peaks in the data that table 1 characterizes obtained by three kinds of situations peak area in 22730~26825 scopes
It is interior, only simultaneously when adding above two material No. 20 peak peak areas 11% or so, and now No. 20 peaks in Fig. 1 E
Retention time still at 10.48min.This result shows, sulphur is added simultaneously only in medicinal material of polygonum capilalum Extraction solvent
During for glycerine and glacial acetic acid, position E could effectively be enriched with No. 20 materials.
2.3rd, MIC is determined
2.3.1, bacteria suspension is prepared prepares with flat board
Experiment is inoculated on flat board first with strain, and 24h is cultivated in 37 DEG C of constant temperature and humidity incubators.Select feature strain
It is connected in fluid nutrient medium, under the conditions of 37 DEG C, shakes 24h.0.5 Maxwell concentration is diluted to using Maxwell turbidimetry, i.e.,
10^6-10^8CFU/mL bacteria suspensions are standby；20g purified agar powder is added into 1L nutrient broth mediums, adds 1L distilled waters
And adjust PH=7.4.Under the conditions of 121 DEG C, taken out after the 30min that sterilizes.When culture medium is cooled to 45 DEG C or so, in ultra-clean work
Make platform and pour into culture medium in sterile petri dish to condense, produce experiment Agar Plating.
2.3.2 MIC is determined
50 μ L fluid nutrient mediums and 50 μ L bacteria suspension are added in superclean bench is first into 96 sterile orifice plates per hole,
The first hole that medicine to be measured is added in 96 orifice plates with the μ L of liquid-transfering gun accurate measuring 100 is mixed, and draws 100 μ L and moves to second
Hole mixes, and mixing 100 μ L of absorption to last hole by that analogy discards, and every hole is kept 100 μ L.Produce concentration for 12.5~
0.012mg/mL；Using gentamicin sulphate as positive control, DMSO is negative control；37 DEG C of culture 24h, difference transferred species to agar
Plating medium, then for minimal inhibitory concentration of drug (MIC), (test method refers to the micropore without bacterial growth after 24h：Recklessly
Dew, Zhang Jin, Lin Liangcai, wait based on spectrum effect relationship polygonum capitatum bacteriostasis material base research [J] Chinese medicines, 2016,39
(9)：2037-2040), 3 are shown in Table.As a result it is respectively A to EHEC, pseudomonas aeruginosa fungistatic effect to show each position>B
>C=D=F>E=G；B=C=D>A=F=G>E.
Table 3：Opposed polarity position is to EHEC, the MIC (g/L) of pseudomonas aeruginosa
| Separated part | EHEC | 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 |
2.4：Each peak of polygonum capitatum different component and 1/MIC PLSR are analyzed
Analyzed using the PLSR of SPSS16.0 softwares by its equalization, using its each peak peak area as independent variable,
1/MIC analyzes to obtain the relational result between each peak and pharmacodynamics index as dependent variable, is shown in Table 4.Can be obtained from table 4 with greatly
The uncommon bacterium fungistatic effect of intestines angstrom has 9 into positively related peak, is primarily present A positions, wherein it is 1-5 peaks to contribute larger peak；It is right
Pseudomonas aeruginosa fungistatic effect has 13 into positively related peak, and main concentrate has B, C, D position, contributes larger peak to be
19th, No. 15 peaks.6- gallic acid acyl glucose and the gallic acid acyl glucose of 3,6- bis- can be obtained with reference to mass spectrometry results
It is maximum to the contribution margin of EHEC；The gallic acid acyl glucose of 1,3,6- tri- and davidiin are to pseudomonas aeruginosa
Contribution margin is larger.
Table 4：Each peak and EHEC, the partial correlation coefficient of pseudomonas aeruginosa
Embodiment 2：The spectrum effect of polygonum capitatum opposed polarity position bacteriostasis learns analysis method
Carried out substantially with reference to embodiment 1, unlike：In step (21), medicinal material of polygonum capilalum is taken to add 8 times of 60% second of amount
Alcohol extracting 2 times, 1 hour every time, filtration, merging filtrate, reclaim ethanol and be condensed into thick paste, be dried under reduced pressure, be ground into fine powder, i.e.,
Obtain polygonum capitatum alcohol-extracted extract；In step (22)：Polygonum capitatum alcohol-extracted extract sample is taken, adds methanol appropriate, ultrasonic extraction 3 times, filter
Liquid merges, centrifugation, takes supernatant to be evaporated under reduced pressure and removes solvent, after being dissolved with 40% methanol, is loaded into MCI type macroporous resin columns, enters
Row chromatography.
Embodiment 3：The spectrum effect of polygonum capitatum opposed polarity position bacteriostasis learns analysis method
Carried out substantially with reference to embodiment 1, unlike：In step (21), medicinal material of polygonum capilalum is taken to add 6 times of 80% second of amount
Alcohol extracting 2 times, 2 hours every time, filtration, merging filtrate, reclaim ethanol and be condensed into thick paste, be dried under reduced pressure, be ground into fine powder, i.e.,
Obtain polygonum capitatum alcohol-extracted extract；In step (22)：Polygonum capitatum alcohol-extracted extract sample is taken, adds methanol appropriate, ultrasonic extraction 3 times, filter
Liquid merges, centrifugation, takes supernatant to be evaporated under reduced pressure and removes solvent, after being dissolved with 20% methanol, is loaded into MCI type macroporous resin columns, enters
Row chromatography.
Embodiment 4：The spectrum effect of polygonum capitatum opposed polarity position bacteriostasis learns analysis method
Carried out substantially with reference to embodiment 1, unlike：In step (21), medicinal material of polygonum capilalum is taken to add 7 times of 75% second of amount
Alcohol extracting 2 times, 1 hour every time, filtration, merging filtrate, reclaim ethanol and be condensed into thick paste, be dried under reduced pressure, be ground into fine powder, i.e.,
Obtain polygonum capitatum alcohol-extracted extract；In step (22)：Polygonum capitatum alcohol-extracted extract sample is taken, adds methanol appropriate, ultrasonic extraction 3 times, filter
Liquid merges, centrifugation, takes supernatant to be evaporated under reduced pressure and removes solvent, after being dissolved with 30% methanol, is loaded into MCI type macroporous resin columns, enters
Row chromatography.
Above example 2 to 4 three examples of embodiment each get three batches of A, B, C, D, E, F, G opposed polarity parts, three batches seven
Individual position Fig. 1, table 1, table in the collection of illustrative plates and data that Fig. 1, table 1, table 2, table 3, the mode of table 4 are provided with implementation column 1 respectively
2nd, the result in table 3, table 4 is basically identical, such as gained D positions peak area at No. 8 peaks and No. 19 peaks in the data that table 1 characterizes
Respectively in the range of 59160~60786 and in the range of 1381825~1385743.
Polygonum capitatum opposed polarity position fingerprint image is obtained by UHPLC-TOF-MS and liquid dilution method in present invention research
Spectrum and the fungistatic effect to EHEC, pseudomonas aeruginosa, and polygonum capitatum opposed polarity position is disclosed using PLSR methods
Each chemical composition is analyzed the spectrum effect relationship of EHEC, pseudomonas aeruginosa, so as to obtain each peak in opposed polarity position
With antibacterial correlation, show that the bacteriostatic activity of polygonum capitatum contributes larger peak to be present in A, B, C, D etc. by spectrum effect analysis result
The larger position of polarity, while also determine polygonum capitatum and its in EHEC, pseudomonas aeruginosa bacteriostasis is mainly made
It is polarity strong 1-5 and 15 with material, compound (6- gallic acid acyls glucose, the gallic acid acyls of 3,6- bis- representated by 19
Glucose, tetra- kinds of compounds of the gallic acid acyl glucose of 1,3,6- tri- and davidiin and its isomer).The present invention passes through
Mass spectrometric hyphenated technique specify that the relation combination test of pesticide effectiveness of chemical composition and its amount in polygonum capitatum and obtain peak by PLSR methods
The simple correlation coefficient matrix of area and 1/MIC, the bigger contribution to pharmacodynamics index of its correlation is bigger, wherein separating first
The davidiin compounds arrived are contributed maximum in the inhibitory action to pseudomonas aeruginosa；It is clear and definite to the end by the present invention
Flower knotweed and its preparation clinically treat urinary tract infections caused by EHEC, pseudomonas aeruginosa etc. it is main effectively into
Point, foundation and the guidance of correlation are also provided for its quality standard.
Claims (10)
1. the method for the spectrum effect credit analysis of polygonum capitatum opposed polarity position bacteriostasis, it comprises the following steps：
(1) material provides：There is provided ultra performance liquid chromatography system and liquid chromatograph-mass spectrometer, there is provided medicinal material of polygonum capilalum, carry
For antibacterial detection bacterial strain；
(2) medicine extraction is with separating：
(21) take medicinal material of polygonum capilalum to add 6~8 times of 60~80% ethanol of amount to extract 2 times, 1~2 hour every time, filter, merging filtrate,
Recovery ethanol is simultaneously condensed into thick paste, is dried under reduced pressure, is ground into fine powder, produces polygonum capitatum alcohol-extracted extract；
(22) polygonum capitatum alcohol-extracted extract sample separately is taken, adds methanol appropriate, ultrasonic extraction 3 times, filtrate merges, and centrifugation, takes supernatant
It is evaporated under reduced pressure and removes solvent, after being dissolved with 20~40% methanol, is loaded into MCI type macroporous resin columns, carries out chromatography；
(23) it is 30%, 35%~40%, 40%, 40%~50%, 50%, 60%, 60%~80% to use volume fraction respectively
Methanol carry out gradient elution, collect each several part eluent, and volatilize, obtain the position of A~G totally seven opposed polarities；
(24) seven positions of A~G are accurately weighed, are dissolved in respectively in DMSO, are made into the solution that mass concentration is 25mg/mL, it is standby；
(3) UPLC analyses and mass spectral analysis：
(31) chromatogram and Mass Spectrometry Conditions：
Chromatographic condition：C18Chromatographic column, 40 DEG C, flow velocity 0.35L/min of column temperature, the μ L of sample size 2, mobile phase use 0.1% formic acid water
The formic acid acetonitrile solution (B) of solution (A) -0.1% carries out gradient elution；
Mass Spectrometry Conditions：Electric spray ion source (ESI), 180 DEG C of dry gas temperature, the 500eV of capillary voltage 4, anionic textiles
Pattern, atomisation pressure 0.25MPa, dry gas (N2) flow velocity 8L/min, scanning range m/z 100~2 000, collision energy
10eV；
(32) opposed polarity position finger-print is established：Opposed polarity portion is established by condition under " (31) chromatogram and Mass Spectrometry Conditions " item
The UHPLC-TOF-MS chromatograms of position, and compound corresponding to each peak in opposed polarity position is determined by mass spectrum, so that it is determined that respectively
Primary chemical composition in individual position；
(4) measure of minimum inhibitory concentration：
(41) lowest bacteria fogging-resistant concentration determining method is pressed, carries out the preparation of bacteria suspension and the preparation of experiment Agar Plating；
(42) lowest bacteria fogging-resistant concentration determining method is pressed, the minimum inhibitory concentration at opposed polarity position obtained by determination step (24)；
(5) partial least-squares regressive analysis between each peak in polygonum capitatum opposed polarity position and 1/MIC：For each position,
Analyzed using the partial least-squares regressive analysis method (PLSR) of SPSS16.0 softwares by its equalization, with its each material peak
Peak area as independent variable, 1/MIC obtains the relation between each material peak and pharmacodynamics index as dependent variable, analysis, it is determined that
With the position present in test strain into positively related material peak and these materials.
2. method according to claim 1, the ultra performance liquid chromatography system is the super high liquid phase color of effect of 1290 Infinity types
Spectra system.
3. method according to claim 1, the liquid chromatograph-mass spectrometer is the TOF LC/MS combinations of Agilent companies
Instrument.
4. method according to claim 1, the medicinal material of polygonum capilalum is the polygonum capitatum for picking up from Guizhou.
5. method according to claim 1, the antibacterial detection bacterial strain includes but is not limited to EHEC, P. aeruginosa
Bacterium.
6. in method according to claim 1, wherein step (21), take medicinal material of polygonum capilalum to add 7 times of 70% ethanol of amount to extract 2 times,
Each 1.5h, filtration, merging filtrate, and reclaim ethanol and be concentrated into the thick paste that relative density is 1.20~1.25 (60 DEG C), subtract
Press dry it is dry, be ground into fine powder, produce the alcohol-extracted extract of polygonum capitatum 70%；Such as wherein taken in step (21) used in medicinal material of polygonum capilalum
0.05% thioglycerol and 0.002% glacial acetic acid are with the addition of in ethanol solution.
7. in method according to claim 1, wherein step (22), the alcohol-extracted extract sample 45g of polygonum capitatum 70% is taken, adds methanol to fit
Amount, ultrasonic extraction 3 times, filtrate merge, centrifugation 5min (3000rpm), take supernatant to be evaporated under reduced pressure and remove solvent, with 30% methanol
Dissolving is loaded into MCI types macroporous resin column (1BV=1L) chromatography.
8. in method according to claim 1, wherein step (23), be respectively 30% with volume fraction, 35%~40%,
40%th, 40%~50%, 50%, 60%, 60%~80% methanol carries out gradient elution, collects each several part eluent, and wave
It is dry, obtain A~G totally seven positions.
9. method according to claim 1, accurate to claim wherein in step (24) in an embodiment of the inventive method
Take seven each 25.00mg in position of A~G to be dissolved in 1mL DMSO, be made into the solution that mass concentration is 25mg/mL.
10. method according to claim 1, wherein：
The chromatographic column is ACQUITY BEH C18Chromatographic column, its specification are 2.1mm × 100mm, 1.7 μm；
The elution program of the gradient elution is：0~0.5min, 95%A, 0.5~20min, 95%A → 81.5%A；20~
30min, 81.5%A → 95%A；30~32min, 95%A；
The operating process of the preparation of the bacteria suspension is：Experiment is inoculated on flat board first with strain, is trained in 37 DEG C of constant temperature and humidities
Support in case and cultivate 24h；Select feature strain to be connected in fluid nutrient medium, under the conditions of 37 DEG C, shake 24h；Using Maxwell than turbid
Method is diluted to 0.5 Maxwell concentration, produces 10^6-10^8CFU/mL bacteria suspensions, standby；
The experiment is with the preparation process of Agar Plating：20g purified agars are added into 1L nutrient broth mediums
Powder, add 1L distilled waters and adjust PH=7.4；Under the conditions of 121 DEG C, taken out after the 30min that sterilizes；Treat that culture medium is cooled to 45 DEG C
During left and right, culture medium is poured into sterile petri dish on superclean bench and condensed, produce experiment Agar Plating；
And/or
The process of the minimum inhibitory concentration at measure opposed polarity position is：It is first every to 96 sterile orifice plates on superclean bench
50 μ L fluid nutrient mediums and 50 μ L bacteria suspension are added in hole, medicine to be measured is added to 96 with the μ L of liquid-transfering gun accurate measuring 100
The first hole in orifice plate mixes, and draws 100 μ L and move to the mixing of the second hole, is mixed to last hole draw 100 μ L by that analogy
Discard, every hole is kept 100 μ L；Using gentamicin sulphate as positive control, DMSO is negative control；37 DEG C of culture 24h, respectively
Transferred species is to Agar Plating, and the concentration of medicine is then minimum inhibitory concentration in the micropore without bacterial growth after 24h.
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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 |