CN102805767A - Heat stranguria removal granule raw material polygonum capitatum extract with anti-gonococcus effect - Google Patents
Heat stranguria removal granule raw material polygonum capitatum extract with anti-gonococcus effect Download PDFInfo
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- CN102805767A CN102805767A CN2012103016604A CN201210301660A CN102805767A CN 102805767 A CN102805767 A CN 102805767A CN 2012103016604 A CN2012103016604 A CN 2012103016604A CN 201210301660 A CN201210301660 A CN 201210301660A CN 102805767 A CN102805767 A CN 102805767A
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- methanol
- herba polygoni
- polygoni capitati
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 87
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Abstract
The invention discloses a heat stranguria removal granule raw material polygonum capitatum extract with an anti-gonococcus effect, in particular to a polygonum capitatum extract. A preparation method of the polygonum capitatum extract comprises the following steps of: (1) adding 50-90 percent ethanol in an amount which is 5-15 times of the amount of fresh or dry product of the underground part of polygonum capitatum into the fresh or dry product of the underground part of polygonum capitatum to perform reflux extraction for one to three times, filtering, concentrating filtrate and drying to obtain alcoholic extract; (2) suspending the alcoholic extract in methanol, ultrasonically treating for 0.5-5 hours, centrifuging, and loading supernatant to an MCI macro-porous resin column; and (3) performing gradient elution by using water and 10-100 percent methanol in sequence, collecting each part of eluent, recovering solvent and drying a part obtained by elution of different solvents to obtain a methanol eluent or mixture thereof. The polygonum capitatum extract has the beneficial effects mentioned as the specification.
Description
Technical field
The invention belongs to the field of Chinese medicines, be used to prepare the clear particulate raw material of Chinese medicine pyretic stranguria the extract that is the medical material Herba Polygoni Capitati.Particularly, the present invention relates to by the clear raw material of pyretic stranguria is the effective site that alcohol extract obtained of medical material Herba Polygoni Capitati, and the method for preparing of this effective site and purposes.This effective site has the effect of good resistance gonococcus.
Background technology
Herba Polygoni Capitati (Polygonum capitatum Buch-Ham ex D.Don) is a Polygonaceae Polygonum herbaceos perennial.Effect with clearing away heat-damp and promoting diuresis, inducing diuresis for treating stranguria syndrome, treating urinary system infection clinically has significant effect.Got into national basic medical insurance catalogue in 2004 with its Chinese patent medicine preparation " the clear granule of pyretic stranguria " that is raw material is processed, entering Guizhou Province essential drug list in 2012.
Herba Polygoni Capitati is the medicine commonly used of minority area, is mainly used in diseases such as pyelonephritis, urinary tract infection, inducing diuresis for treating stranguria syndrome.Relevant pharmaceutical research is rare, and several pieces of reports such as Ren Guangyou are only arranged at present.Ren Guangyou adopts rat bacterial pyelonephritis model to experimentize, and WBC in the Herba Polygoni Capitati water extract group rat urine and BLD and the apparent in view minimizing of matched group as a result shows that the Herba Polygoni Capitati water extract has certain antiinflammatory action to pyelonephritis.Ren Guangyou etc. observe dead mouse situation in 5 days with mouse peritoneal injection Escherichia coli bacteria liquid, and the matched group mortality rate is 100% as a result, and Herba Polygoni Capitati group mortality rate is respectively 20% and 50%, show that the Herba Polygoni Capitati water extract can resist the infection that escherichia coli cause.Ren Guangyou etc. give rabbit with Herba Polygoni Capitati water extract gastric infusion, the result, Herba Polygoni Capitati water extract group and matched group relatively, body temperature there was no significant difference, but can reduce the heating body temperature of the rabbit that the intravenous injection TAB causes.Ren Guangyou etc. are with Herba Polygoni Capitati water extract respectively gastric infusion rabbit, rat, with blank control group and furosemide matched group urine amount relatively.The result shows that the Herba Polygoni Capitati water extract does not have obvious diuresis to rabbit and rat.People such as Xu Yingchun adopt agar dilution to detect the external bacteriostatic activity of Herba Polygoni Capitati to 10 strain Neisseria gonorrhoeae (gonococcus), and Herba Polygoni Capitati has bacteriostatic activity to gonococcus as a result.It is 8 ~ 32g/L to the gonococcal minimal inhibitory concentration scope of 10 strains, and meansigma methods is 11.2g/L.
One Chinese patent application prospectus CN1054899A discloses a kind of miganling instant herbal medicine, syrup production technology in (one Chinese patent application numbers 90107810.7, open day on October 2nd, 1991).This production technology is to be that the raw material water decocted 30-60 minute with four seasons grass; Extracting liquid filtering concentrates, and its supernatant concentrating under reduced pressure is become cream, reuse 60-70% ethanol extraction; With the ethanol extract vacuum drying; Herba Polygoni Capitati extractum, further be mixed with electuary or syrup, it is believed that it has detoxifcation, dissipating blood stasis, diuresis, treating stranguria effect.
Recorded the Chinese patent medicine preparation of " the clear granule of pyretic stranguria " by name in the Ministry of Health of the People's Republic of China " drug standard-Chinese traditional patent formulation preparation " (the 17) that Ministry of Health of the People's Republic of China's committee of pharmacopeia was compiled in 1998, it is processed through twice after-filtration of Herba Polygoni Capitati decocte with water concentrated.
CN 1481832A (one Chinese patent application number 02129686.3; Open day on March 17th, 2004) and CN 1483466A (one Chinese patent application number 03146381.9; Open day on March 24th, 2004) Herba Polygoni Capitati extract is disclosed; It is prepared by following steps basically: a. is decocted at twice by the aquatic foods article of Herba Polygoni Capitati herb or dry product water or divides reflux, extract, two to three times with alcohol-water mixture, each 1-2 hour, merges decoction liquor; Relative density during filtering and concentrating to 20 ° C is 1.2, and spray drying or drying under reduced pressure obtain; Perhaps, b. carries medicinal residues by Herba Polygoni Capitati herb and water thereof and obtains through carbon dioxide supercritical fluid extraction.It is believed that this extract can be used for antibiotic, antiinflammatory, analgesia, diuresis, treatment urinary system calculus, treatment pyelonephritis and prostatitis.
Although many reports about the preparation Herba Polygoni Capitati extract are arranged at present, yet those skilled in the art expect still to obtain clinical effective product from Herba Polygoni Capitati.
Summary of the invention
The present invention seeks to expectation and obtain a kind of clinical effective product from Herba Polygoni Capitati.The inventor finds, uses the alcohol extraction Herba Polygoni Capitati, the eluate that the extract that obtains is obtained through specific macroporous resin eluting, and it has the effect of desirable aspect biological activity.The present invention is based on this discovery and be accomplished.
For this reason, first aspect present invention provides a kind of Herba Polygoni Capitati extract, and it is prepared by the method that may further comprise the steps basically:
(1) the bright article of Herba Polygoni Capitati aerial parts or dry product are added 5-15 and measure doubly that (for example 5-12 doubly measures; For example 6-10 doubly measures) 50 ~ 90% (for example 60 ~ 80%, for example 65 ~ 75%, for example about 70%) alcohol reflux 1-3 time (for example 2 times); Each 1-3 hour (for example about 1.5 hours); Filter, make filtrating concentrating, dry, get pure extractum;
(2) pure extractum is suspended in the methanol, and supersound process 0.5 ~ 5 hour (for example 0.5 ~ 3 hour, for example 1 ~ 2 hour; For example about 1.5 hours), centrifugal, supernatant (for example is loaded into the MCI macroporous resin column; Every 100g extractum uses the amount of resin to be the 0.5-4 liter; 1-3 liter for example, 1.5-2.5 liter for example, for example 2 liters) on;
(3) water, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 100% methanol carry out gradient elution successively, and (for example every kind of solvent load is 0.5 ~ 5 times of column volume, for example 0.5 ~ 2.5 times of column volume; For example 0.5 ~ 2 times of column volume, for example 1 times of column volume), collect the each several part eluent; Reclaim solvent, dry by the position that the different solvents eluting obtains, obtain between the 30%-50% arbitrarily concentration at interval or the methanol-eluted fractions thing of concentration point; Perhaps their mixture promptly gets.
According to the Herba Polygoni Capitati extract of first aspect present invention, wherein step (3) gained methanol-eluted fractions thing is that 35%-40% methanol-eluted fractions thing (can abbreviate as: position B) in the present invention.
According to the Herba Polygoni Capitati extract of first aspect present invention, wherein contain for example trigalloylglucose of hydrolysable tannin among the B of position.
According to the Herba Polygoni Capitati extract of first aspect present invention, it is measured according to Ultra Performance Liquid Chromatography-time of flight mass spectrometry coupling method (can abbreviate UPLC-TOF-MS as in the present invention), the result:
Between the retention time 3.4-4.4min between (for example about 3.9min place) and the retention time 4.8-5.8min (for example about 5.3min place) show the chromatographic peak of hydrolysable tannin (for example trigalloylglucose);
Wherein, the assay method of UPLC-TOF-MS is following:
(i) test liquid preparation: take by weighing an amount of Herba Polygoni Capitati extract powder, add the suspension that 70% methanol is processed the about 5mg/ml of concentration, ultrasonic making as far as possible dissolved, and 10 times of suspension dilutions are filtered, and sample introduction 2 μ l make Mass Spectrometer Method;
(ii) chromatograph and mass spectral analysis condition:
Chromatographic column: Acquity BEH C18 post (2.1 * 100mm, 1.7 μ m), column temperature: 40 ℃; Flow velocity: 0.35ml/min; Sample size: 2 μ l; Mass spectrum condition: ion source: ESI source; Dry gas temperature: 180 ℃; Capillary voltage: 4500eV; Detecting pattern: negative ion mode; Atomisation pressure: 2.5bar; Dry gas (N2) flow velocity: 8L/min; Sweep limits: 100-2000amu; Collision energy: 10ev; With 0.1% aqueous formic acid is mobile phase A, and 0.1% formic acid acetonitrile solution is a Mobile phase B, and the according to the form below regulated procedure is carried out gradient elution:
| Time (min) | A(%) | B(%) |
| ?0 | 95 | 5 |
| ?0.5 | 95 | 5 |
| ?20 | 81.5 | 18.5 |
| ?28 | 0 | 100 |
| ?30 | 0 | 100 |
| ?30.1 | 95 | 5 |
| ?32 | 95 | 5 |
Herba Polygoni Capitati extract according to first aspect present invention; It is measured according to said Ultra Performance Liquid Chromatography-time of flight mass spectrometry coupling method, the result: (b) at the chromatographic peak of (for example about 5.3min place) demonstration hydrolysable tannin (for example trigalloylglucose) between (for example about 3.9min place) and the retention time 4.8-5.8min between the retention time 3.4-4.4min.
According to the Herba Polygoni Capitati extract of first aspect present invention, it is measured according to said Ultra Performance Liquid Chromatography-time of flight mass spectrometry coupling method, the result:
Between the retention time 3.4-4.4min between (for example about 3.9min place) and the retention time 4.8-5.8min (for example about 5.3min place) show the chromatographic peak of hydrolysable tannin (for example trigalloylglucose).
According to the Herba Polygoni Capitati extract of first aspect present invention, it has the said characteristic of the arbitrary embodiment of second aspect present invention.
Second aspect present invention provides a kind of Herba Polygoni Capitati extract, and it is measured according to Ultra Performance Liquid Chromatography-time of flight mass spectrometry coupling method (can abbreviate UPLC-TOF-MS as in the present invention), the result:
(b) between the retention time 3.4-4.4min between (for example about 3.9min place) and the retention time 4.8-5.8min (for example about 5.3min place) show the chromatographic peak of hydrolysable tannin (for example trigalloylglucose);
Wherein, the assay method of UPLC-TOF-MS is following:
(i) test liquid preparation: take by weighing an amount of Herba Polygoni Capitati extract powder, add the suspension that 70% methanol is processed the about 5mg/ml of concentration, ultrasonic making as far as possible dissolved, and 10 times of suspension dilutions are filtered, and sample introduction 2 μ l make Mass Spectrometer Method;
(ii) chromatograph and mass spectral analysis condition:
Chromatographic column: Acquity BEH C18 post (2.1 * 100mm, 1.7 μ m), column temperature: 40 ℃; Flow velocity: 0.35ml/min; Sample size: 2 μ l; Mass spectrum condition: ion source: ESI source; Dry gas temperature: 180 ℃; Capillary voltage: 4500eV; Detecting pattern: negative ion mode; Atomisation pressure: 2.5bar; Dry gas (N2) flow velocity: 8L/min; Sweep limits: 100-2000amu; Collision energy: 10ev; With 0.1% aqueous formic acid is mobile phase A, and 0.1% formic acid acetonitrile solution is a Mobile phase B, and the according to the form below regulated procedure is carried out gradient elution:
| Time (min) | A(%) | B(%) |
| ?0 | 95 | 5 |
| ?0.5 | 95 | 5 |
| ?20 | 81.5 | 18.5 |
| ?28 | 0 | 100 |
| ?30 | 0 | 100 |
| ?30.1 | 95 | 5 |
| ?32 | 95 | 5 |
Herba Polygoni Capitati extract according to second aspect present invention; It is measured according to said Ultra Performance Liquid Chromatography-time of flight mass spectrometry coupling method, the result: (b) at the chromatographic peak of (for example about 5.3min place) demonstration hydrolysable tannin (for example trigalloylglucose) between (for example about 3.9min place) and the retention time 4.8-5.8min between the retention time 3.4-4.4min.
According to the Herba Polygoni Capitati extract of second aspect present invention, it is measured according to said Ultra Performance Liquid Chromatography-time of flight mass spectrometry coupling method, the result:
Between the retention time 3.4-4.4min between (for example about 3.9min place) and the retention time 4.8-5.8min (for example about 5.3min place) show the chromatographic peak of hydrolysable tannin (for example trigalloylglucose).
According to the Herba Polygoni Capitati extract of second aspect present invention, it is prepared by the method that may further comprise the steps basically:
(1) the bright article of Herba Polygoni Capitati aerial parts or dry product are added 5-15 and measure doubly that (for example 5-12 doubly measures; For example 6-10 doubly measures) 50 ~ 90% (for example 60 ~ 80%, for example 65 ~ 75%, for example about 70%) alcohol reflux 1-3 time (for example 2 times); Each 1-3 hour (for example about 1.5 hours); Filter, make filtrating concentrating, dry, get pure extractum;
(2) pure extractum is suspended in the methanol, and supersound process 0.5 ~ 5 hour (for example 0.5 ~ 3 hour, for example 1 ~ 2 hour; For example about 1.5 hours), centrifugal, supernatant (for example is loaded into the MCI macroporous resin column; Every 100g extractum uses the amount of resin to be the 0.5-4 liter; 1-3 liter for example, 1.5-2.5 liter for example, for example 2 liters) on;
(3) water, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 100% methanol carry out gradient elution successively, and (for example every kind of solvent load is 0.5 ~ 5 times of column volume, for example 0.5 ~ 2.5 times of column volume; For example 0.5 ~ 2 times of column volume, for example 1 times of column volume), collect the each several part eluent; Reclaim solvent, dry by the position that the different solvents eluting obtains, obtain between the 30%-50% arbitrarily concentration at interval or the methanol-eluted fractions thing of concentration point; Perhaps their mixture promptly gets.
According to the Herba Polygoni Capitati extract of second aspect present invention, wherein step (3) gained methanol-eluted fractions thing is that 35%-40% methanol-eluted fractions thing (can abbreviate as: position B) in the present invention.
According to the Herba Polygoni Capitati extract of second aspect present invention, wherein contain for example trigalloylglucose of hydrolysable tannin among the B of position.
According to the Herba Polygoni Capitati extract of second aspect present invention, it has the said characteristic of the arbitrary embodiment of first aspect present invention.
Third aspect present invention provides the method for preparing first aspect present invention or the said Herba Polygoni Capitati extract of the arbitrary embodiment of second aspect, and it consists essentially of following steps:
(1) the bright article of Herba Polygoni Capitati aerial parts or dry product are added 5-15 and measure doubly that (for example 5-12 doubly measures; For example 6-10 doubly measures) 50 ~ 90% (for example 60 ~ 80%, for example 65 ~ 75%, for example about 70%) alcohol reflux 1-3 time (for example 2 times); Each 1-3 hour (for example about 1.5 hours); Filter, make filtrating concentrating, dry, get pure extractum;
(2) pure extractum is suspended in the methanol, and supersound process 0.5 ~ 5 hour (for example 0.5 ~ 3 hour, for example 1 ~ 2 hour; For example about 1.5 hours), centrifugal, supernatant (for example is loaded into the MCI macroporous resin column; Every 100g extractum uses the amount of resin to be the 0.5-4 liter; 1-3 liter for example, 1.5-2.5 liter for example, for example 2 liters) on;
(3) water, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 100% methanol carry out gradient elution successively, and (for example every kind of solvent load is 0.5 ~ 5 times of column volume, for example 0.5 ~ 2.5 times of column volume; For example 0.5 ~ 2 times of column volume, for example 1 times of column volume), collect the each several part eluent; Reclaim solvent, dry by the position that the different solvents eluting obtains, obtain between the 30%-50% arbitrarily concentration at interval or the methanol-eluted fractions thing of concentration point; Perhaps their mixture promptly gets.
According to the method for third aspect present invention, wherein step (3) gained methanol-eluted fractions thing is that 35%-40% methanol-eluted fractions thing (can abbreviate as: position B) in the present invention.
According to the method for third aspect present invention, wherein contain for example trigalloylglucose of hydrolysable tannin among the B of position.
According to the method for third aspect present invention, wherein the gained Herba Polygoni Capitati extract has first aspect present invention or the said characteristic of the arbitrary embodiment of second aspect.
Fourth aspect present invention provides first aspect present invention or the purposes of the said Herba Polygoni Capitati extract of second aspect in the anti-gonococcal medicine of preparation.
Fifth aspect present invention provides the purposes of trigalloylglucose in the anti-gonococcal medicine of preparation.
Sixth aspect present invention provides a kind of pharmaceutical composition, wherein comprises described Herba Polygoni Capitati extract of the arbitrary embodiment of first aspect present invention or second aspect or trigalloylglucose and optional pharmaceutically acceptable carrier.
According to the pharmaceutical composition of sixth aspect present invention, it is the dosage form of oral or drug administration by injection.In one embodiment, said pharmaceutical composition is the form of tablet, capsule, granule, pill, oral solutions, injection (liquid drugs injection and/or powder pin) etc.
According to the pharmaceutical composition of sixth aspect present invention, it is to be used for anti-gonococcal medicine.
Arbitrary technical characterictic that arbitrary embodiment had of the arbitrary aspect of the present invention or this arbitrary aspect is suitable for arbitrary embodiment of other arbitrary embodiment or other arbitrary aspect equally; As long as they can be not conflicting; Certainly at where applicable each other, necessary words can be done suitably to modify to individual features.Do further to describe with characteristics to various aspects of the present invention below.
All documents that the present invention quoted from, their full content is incorporated this paper by reference into, and if the expressed implication of these documents and the present invention when inconsistent, be as the criterion with statement of the present invention.In addition; Various terms and phrase that the present invention uses have the general sense of well known to a person skilled in the art; Nonetheless; The present invention still hopes at this more detailed explanation and explanation to be done in these terms and phrase, and term of mentioning and phrase are as the criterion with the implication that the present invention was explained if any inconsistent with known implication.
In the present invention, the inventive method obtains " Herba Polygoni Capitati extract ", and this term also can be described as " Herba Polygoni Capitati active component " of the present invention or " Herba Polygoni Capitati effective site " of the present invention.
In the present invention; The MCI macroporous resin can be the MCIGEL series of being produced by MIT; MCI GEL series reverse phase separation filler designs on Diaion of Mitsubishi Chemical and Sepabeads macroporous adsorbent resin basis; Because based on the HPLC HPLC isolation technics in modern times, smaller particles has higher chromatographic separation performance, be widely used for separating of natural product and tunning.The MCI macroporous resin has two kinds of fillers: polystyrene and acrylic type, and the resin particle of polystyrene type is 4 μ m-100 μ m through scope, the grain of methacrylate type resin is 4 μ m-31 μ m through scope.MCI series not only has the resin of macroporous type in addition, also has anionic and model resin such as cationic.MCI GEL anti-phase fine separation filler is a kind of resinae filler, can be used for separating saponin component, generally with methanol-water or ethanol water elution.Moreover MCI generally is used for the chlorophyll in the little composition of place to go polarity, and is fine to petroleum ether part and the chlorophyllous effect in chloroform part place to go.In the present invention; The preferred MCI macroporous resin that uses is a polystyrene type, and it includes but not limited to MCI GEL CHP 10M, MCI GEL CHP 5C, MCI GEL CHP55A, MCI GEL CHP 55Y, MCI GEL CHP 20Y, MCI GEL CHP 20P, MCI GEL CHP 20SS.In addition, in the present invention, as not specifying in addition that preferably using model is the macroporous resin of MCI GEL CHP 20P (75 ~ 150 μ m), it is the most widely used model.It is a kind of porous polystyrene high polymer; The anti-phase adsorption that it showed is extensive; Except the polar sugar of height, aminoacid are not had the adsorption basically, most of secondary metabolites there is absorption in various degree, this makes it can remove effectively on the one hand water solublity is disturbed sugar greatly; Aminoacid can separate dissimilar chemical compounds again on the other hand.
The object of the invention is to overcome the deficiency of prior art, and a kind of Herba Polygoni Capitati effective content of anti inflammation (effective site) and application of high-efficiency low-toxicity is provided.
In one embodiment, invention realizes above-mentioned purpose through following technical scheme:
The invention provides a kind of Herba Polygoni Capitati active component (effective site), is to be obtained by following steps:
(1) Herba Polygoni Capitati aerial parts aquatic foods article or dry product are added 65 ~ 75% alcohol reflux 2 times (each about 1.5 is little) that 6-8 doubly measures, filter, making filtrating concentrating, dry, get pure extractum;
(2) pure extractum is suspended in the methanol, about 1.5 hours of supersound process), centrifugal, supernatant is loaded on MCI macroporous resin (the MCI GEL CHP 20P) post (every 100g extractum uses the amount of resin to be about 2 liters);
(3) water; 10%, 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 100% methanol carries out gradient elution (for example every kind of solvent load is about 1 times of column volume) successively, collects the each several part eluent, reclaims solvent; Position by the different solvents eluting obtains is dry; Obtain between the 30%-50% arbitrarily concentration at interval or the methanol-eluted fractions thing of concentration point, perhaps their mixture promptly gets.
Above step (3) gained methanol-eluted fractions thing can be 35%-40% methanol-eluted fractions thing (position B).
Contain for example trigalloylglucose of hydrolysable tannin among above step (3) the gained position B.
According to any aspect of the present invention, wherein in the Herba Polygoni Capitati extract of position B trigalloylglucose content more than 30%, for example 30-70%, for example 50-70%.
In the present invention, during the dosage representing to test with g/kg, as do not have other explanation, be meant that every kg the weight of animals gives the corresponding reagent weight that (extract for example of the present invention) amounted to into the Herba Polygoni Capitati dry product.
Herba Polygoni Capitati active component of the present invention can become various common dosage forms and slow releasing agent, controlled release agent, targeting preparation etc. with acceptable adjuvant combined preparation in the medicine manufacturing.
Description of drawings
Fig. 1 is the liquid chromatogram of position B.
Fig. 2-6 has shown the fungistatic effect of extract of the present invention respectively.
The specific embodiment
In order more to be expressly understood technology contents of the present invention, the special following examples of lifting specify, but embodiment of the present invention is not limited thereto.
A, extract prepare routine part
Preparation example 1: Herba Polygoni Capitati active component
(1) with Herba Polygoni Capitati aerial parts dry product 10Kg, add 70% alcohol reflux 2 times of 7 times of amounts, each about 1.5 hours, filter, making filtrating concentrating, dry, pure extractum (885g); (2) pure extractum (45g) is suspended in the methanol, about 1.5 hours of supersound process, centrifugal, supernatant is loaded on MCI macroporous resin (the MCI GEL CHP 20P) post (every 100g extractum uses the amount of resin to be about 2 liters); (3) water; 10%, 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 100% methanol carries out gradient elution (every kind of solvent load is about 1 times of column volume) successively; Collect the each several part eluent, reclaim solvent, dry by the position that the different solvents eluting obtains; Obtaining 35%-40% methanol-eluted fractions thing is position B (yield 6.38, trigalloylglucose content are 62.6%%).
Hereinafter, as do not have other explanation, used position B sample is the product with this preparation example 1.
Preparation example 2: Herba Polygoni Capitati active component
(1) with Herba Polygoni Capitati aerial parts dry product 10Kg, add 60% alcohol reflux 3 times of 6 times of amounts, each about 1 hour, filter, making filtrating concentrating, dry, pure extractum (935g); (2) pure extractum (45g) is suspended in the methanol, about 2 hours of supersound process, centrifugal, supernatant is loaded on MCI macroporous resin (the MCI GEL CHP 20Y) post (every 100g extractum uses the amount of resin to be about 3 liters); (3) water; 10%, 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 100% methanol carries out gradient elution (every kind of solvent load is about 2 times of column volumes) successively; Collect the each several part eluent, reclaim solvent, dry by the position that the different solvents eluting obtains; Obtaining 35%-40% methanol-eluted fractions thing is position B (yield 5.28%, trigalloylglucose content are 73.7%).
In addition, again through chromatogram purification, obtain the trigalloylglucose of 96.6% purity by above extract.
Preparation example 3: Herba Polygoni Capitati active component
(1) with Herba Polygoni Capitati aerial parts dry product 10Kg, add 80% alcohol reflux 1 time 3 hours of 10 times of amounts, filter, making filtrating concentrating, dry, pure extractum (818g); (2) pure extractum (45g) is suspended in the methanol, about 1 hour of supersound process, centrifugal, supernatant is loaded on MCI macroporous resin (the MCI GEL CHP 55A) post (every 100g extractum uses the amount of resin to be about 1 liter); (3) water; 10%, 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 100% methanol carries out gradient elution (every kind of solvent load is about 0.5 times of column volume) successively; Collect the each several part eluent, reclaim solvent, dry by the position that the different solvents eluting obtains; Obtaining 35%-40% methanol-eluted fractions thing is position B (yield 8.93%, trigalloylglucose content are 36.7%).
B, pharmacodynamics test: anti-gonococcus test is investigated
1, material and method
Bacterial strain: gonococcus (Neisseria gonorrhoeae) 1 strain
Ingredient: Herba Polygoni Capitati component 1 to 9; Be designated as F1 to F9 respectively; They are respectively that preceding text prepare routine 1 step (3) water, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 100% methanol carries out the collected each several part eluent of gradient elution successively, and the position that after reclaiming solvent processing, obtains, the different solvents eluting obtains is dry.Wherein:
10-20% methanol-eluted fractions thing can be described as component 1 or F1 in the present invention; 30% methanol-eluted fractions thing can abbreviate position A as in the present invention, or is called component 2 or F2; 35%-40% methanol-eluted fractions thing can abbreviate position B as in the present invention, or is called component 3 or F3, and it is the Herba Polygoni Capitati extract of first aspect present invention; 40% methanol-eluted fractions thing can abbreviate position C as in the present invention, or is called component 4 or F4; 40%-50% methanol-eluted fractions thing can abbreviate position D as in the present invention, or is called component 5 or F5; 50% methanol-eluted fractions thing can abbreviate position E as in the present invention, or is called component 6 or F6; 50%-60% methanol-eluted fractions thing can abbreviate position F as in the present invention, or is called component 7 or F7; 60%-80% methanol-eluted fractions thing can abbreviate position G as in the present invention, or is called component 8 or F8; 80-100% methanol-eluted fractions thing can be described as component 9 or F9 in the present invention.
Wherein F1-F5 is with DW (deionized water) dissolving, and F6-F9 is with 50% ethanol (EtOH) dissolving, and concentration is 40mg/ml.Select a kind of antibiotic else: tetracycline (Tet, concentration is 10mg/ml)
Method of testing: gonococcus is made into the bacteria suspension of 0.5 Maxwell turbidity with normal saline, and the coating chocolate is dull and stereotyped, pastes the round scraps of paper of the about 6mm of diameter, adds each sample of F1 to F9, DW, 50% ethanol, Tet on each scraps of paper respectively.Totally three flat boards, applied sample amount is respectively 10 μ l, 3 μ l, 1 μ l.
2, result
Like Fig. 2, Fig. 3, shown in Figure 4, three figure have shown the result of the test of applied sample amount 10 μ l, 3 μ l, 1 μ l respectively.Among each figure, as shown in the figurely begin, counterclockwise be followed successively by the reagent of F1-F9, DW, 50% ethanol, Tet from the F1 position.The result of three figure of figure shows that the antibacterial circle diameter of F3 reagent is obviously greater than other extract sample or blank solvent (water or alcoholic solution).
On this basis, the inventor has repeated single test, and applied sample amount is respectively 10 μ l and 3 μ l results see Fig. 5.Among the figure, left side applied sample amount 10 μ l, applied sample amount 3 μ l in right side begin from F1 position shown in the figure, counterclockwise are followed successively by F1-F9, DW, 50% ethanol, Tet.The result shows that still the antibacterial circle diameter of F3 obviously greater than other extract sample or blank solvent (water or alcoholic solution), has good fungistatic effect.
Through measuring, antibacterial circle diameter is the result see the following form:
It is identical with scraps of paper diameter that antibacterial circle diameter 6mm is, no bacteriostasis.Antibacterial circle diameter is big more, and bacteriostasis is strong more.When the application of sample amount was 10 μ l, F3 (400 μ g) had good bacteriostasis, and F2, F4-6 have faint bacteriostasis, and other extract does not have bacteriostasis.When application of sample amount 3 μ l, F3 (120 μ g) has certain bacteriostasis, and other extract does not have bacteriostasis.
Represent that with the difference of inhibition zone radius and scraps of paper radius the antibacterial result of each component is referring to Fig. 6.First half left side has shown the difference result of calculation of inhibition zone radius and scraps of paper radius among the figure; First half right side has shown computational methods; Lower Half has shown the antibacterial result of each material with the rectangular histogram mode; Show that F3 has the fungistatic effect that can compare with tetracycline, for example under 3ul dosage in addition its fungistatic effect better than tetracycline; And a little less than the fungistatic effect of other each material.
The inventor is in other test; Use obtains the trigalloylglucose of preparation example acquisition as reagent; Make an experiment by top said anti-gonococcus test investigation; The result with the F3 same dose under, its antibacterial circle diameter and F3 are basic identical, show that trigalloylglucose also has significant anti-gonococcus effect.
C, chemical analysis example part
Use the routine 1 gained alcohol extract of UPLC-TOF-MS method test preparation and each position.
The assay method of UPLC-TOF-MS is following:
(i) test liquid preparation: take by weighing an amount of Herba Polygoni Capitati extract powder, add the suspension that 70% methanol is processed the about 5mg/ml of concentration, ultrasonic making as far as possible dissolved, and 10 times of suspension dilutions are filtered, and sample introduction 2 μ l make Mass Spectrometer Method;
(ii) chromatograph and mass spectral analysis condition:
Chromatographic column: Acquity BEH C18 post (2.1 * 100mm, 1.7 μ m), column temperature: 40 ℃; Flow velocity: 0.35ml/min; Sample size: 2 μ l; Mass spectrum condition: ion source: ESI source; Dry gas temperature: 180 ℃; Capillary voltage: 4500eV; Detecting pattern: negative ion mode; Atomisation pressure: 2.5bar; Dry gas (N2) flow velocity: 8L/min; Sweep limits: 100-2000amu; Collision energy: 10ev; With 0.1% aqueous formic acid is mobile phase A, and 0.1% formic acid acetonitrile solution is a Mobile phase B, and the according to the form below regulated procedure is carried out gradient elution:
| Time (min) | A(%) | B(%) |
| ?0 | 95 | 5 |
| ?0.5 | 95 | 5 |
| ?20 | 81.5 | 18.5 |
| ?28 | 0 | 100 |
| ?30 | 0 | 100 |
| ?30.1 | 95 | 5 |
| ?32 | 95 | 5 |
The result:
Position B sample shows, at the chromatographic peak of retention time about 3.9min place and the about 5.3min of retention time place demonstration hydrolysable tannin (for example trigalloylglucose), referring to Fig. 1;
Each effective site of Herba Polygoni Capitati provided by the invention, the position material and the characterization data thereof that are wherein comprised see the following form:
Claims (10)
1. Herba Polygoni Capitati extract, it is prepared by the method that may further comprise the steps basically:
(1) the bright article of Herba Polygoni Capitati aerial parts or dry product are added 50 ~ 90% alcohol reflux 1-3 time that 5-15 doubly measures, each 1-3 hour, filter, making filtrating concentrated, dry, get pure extractum;
(2) pure extractum is suspended in the methanol, supersound process 0.5 ~ 5 hour, centrifugal, supernatant is loaded on the MCI macroporous resin column;
(3) water; 10%, 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 100% methanol carries out gradient elution successively, collects the each several part eluent, reclaims solvent; Position by the different solvents eluting obtains is dry; Obtain between the 30%-50% arbitrarily concentration at interval or the methanol-eluted fractions thing of concentration point, perhaps their mixture promptly gets.
2. according to the Herba Polygoni Capitati extract of claim 1, wherein:
Step (3) gained methanol-eluted fractions thing is 35%-40% methanol-eluted fractions thing (can be described as position B); And/or
Contain for example trigalloylglucose of hydrolysable tannin among the B of position.
3. according to each Herba Polygoni Capitati extract of claim 1 to 2, it is measured according to Ultra Performance Liquid Chromatography-time of flight mass spectrometry coupling method, the result:
At the chromatographic peak that shows hydrolysable tannin (for example trigalloylglucose) between the retention time 3.4-4.4min and between the retention time 4.8-5.8min;
Wherein, the assay method of UPLC-TOF-MS is following:
(i) test liquid preparation: take by weighing an amount of Herba Polygoni Capitati extract powder, add the suspension that 70% methanol is processed the about 5mg/ml of concentration, ultrasonic making as far as possible dissolved, and 10 times of suspension dilutions are filtered, and sample introduction 2 μ l make Mass Spectrometer Method;
(ii) chromatograph and mass spectral analysis condition:
Chromatographic column: Acquity BEH C18 post (2.1 * 100mm, 1.7 μ m), column temperature: 40 ℃; Flow velocity: 0.35ml/min; Sample size: 2 μ l; Mass spectrum condition: ion source: ESI source; Dry gas temperature: 180 ℃; Capillary voltage: 4500eV; Detecting pattern: negative ion mode; Atomisation pressure: 2.5bar; Dry gas (N2) flow velocity: 8L/min; Sweep limits: 100-2000amu; Collision energy: 10ev; With 0.1% aqueous formic acid is mobile phase A, and 0.1% formic acid acetonitrile solution is a Mobile phase B, and the according to the form below regulated procedure is carried out gradient elution:
4. according to the Herba Polygoni Capitati extract of claim 3; It is measured according to said Ultra Performance Liquid Chromatography-time of flight mass spectrometry coupling method, result: (b) at the chromatographic peak that shows hydrolysable tannin (for example trigalloylglucose) between the retention time 3.4-4.4min and between the retention time 4.8-5.8min.
5. Herba Polygoni Capitati extract, it is measured according to Ultra Performance Liquid Chromatography-time of flight mass spectrometry coupling method, the result: at the chromatographic peak that shows hydrolysable tannin between the retention time 3.4-4.4min and between the retention time 4.8-5.8min;
Wherein, the assay method of UPLC-TOF-MS is following:
(i) test liquid preparation: take by weighing an amount of Herba Polygoni Capitati extract powder, add the suspension that 70% methanol is processed the about 5mg/ml of concentration, ultrasonic making as far as possible dissolved, and 10 times of suspension dilutions are filtered, and sample introduction 2 μ l make Mass Spectrometer Method;
(ii) chromatograph and mass spectral analysis condition:
Chromatographic column: Acquity BEH C18 post (2.1 * 100mm, 1.7 μ m), column temperature: 40 ℃; Flow velocity: 0.35ml/min; Sample size: 2 μ l; Mass spectrum condition: ion source: ESI source; Dry gas temperature: 180 ℃; Capillary voltage: 4500eV; Detecting pattern: negative ion mode; Atomisation pressure: 2.5bar; Dry gas (N2) flow velocity: 8L/min; Sweep limits: 100-2000amu; Collision energy: 10ev; With 0.1% aqueous formic acid is mobile phase A, and 0.1% formic acid acetonitrile solution is a Mobile phase B, and the according to the form below regulated procedure is carried out gradient elution:
6. according to the Herba Polygoni Capitati extract of claim 5; It is measured according to said Ultra Performance Liquid Chromatography-time of flight mass spectrometry coupling method, the result: at the chromatographic peak of retention time about 3.9min place and the about 5.3min of retention time place demonstration hydrolysable tannin (for example trigalloylglucose).
7. according to each Herba Polygoni Capitati extract of claim 5 to 6, it prepares by may further comprise the steps (1) method to (3) basically:
(1) the bright article of Herba Polygoni Capitati aerial parts or dry product are added 50 ~ 90% alcohol reflux 1-3 time that 5-15 doubly measures, each 1-3 hour, filter, making filtrating concentrated, dry, get pure extractum;
(2) pure extractum is suspended in the methanol, supersound process 0.5 ~ 5 hour, centrifugal, supernatant is loaded on the MCI macroporous resin column;
(3) water; 10%, 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 100% methanol carries out gradient elution successively, collects the each several part eluent, reclaims solvent; Position by the different solvents eluting obtains is dry; Obtain between the 30%-50% arbitrarily concentration at interval or the methanol-eluted fractions thing of concentration point, perhaps their mixture promptly gets.
Further, wherein step (3) gained methanol-eluted fractions thing is 35%-40% methanol-eluted fractions thing (can be described as position B); Further, wherein contain for example trigalloylglucose of hydrolysable tannin among the B of position.
8. each the method for Herba Polygoni Capitati extract of preparation claim 1 to 7, it consists essentially of following steps:
(1) the bright article of Herba Polygoni Capitati aerial parts or dry product are added 50 ~ 90% alcohol reflux 1-3 time that 5-15 doubly measures, each 1-3 hour, filter, making filtrating concentrated, dry, get pure extractum;
(2) pure extractum is suspended in the methanol, supersound process 0.5 ~ 5 hour, centrifugal, supernatant is loaded on the MCI macroporous resin column;
(3) water, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 100% methanol carries out gradient elution successively, collects the each several part eluent; Reclaim solvent; Position by the different solvents eluting obtains is dry, obtains between the 30%-50% arbitrarily concentration at interval or the methanol-eluted fractions thing of concentration point, perhaps their mixture; Promptly get
Further, wherein step (3) gained methanol-eluted fractions thing is 35%-40% methanol-eluted fractions thing (can be described as position B); Further, wherein contain for example trigalloylglucose of hydrolysable tannin among the B of position.
9. each Herba Polygoni Capitati extract or the trigalloylglucose purposes in the anti-gonococcal medicine of preparation of claim 1 to 9.
10. pharmaceutical composition wherein comprises each Herba Polygoni Capitati extract or trigalloylglucose and optional pharmaceutically acceptable carrier of claim 1 to 7; Further, it is with being to be used for anti-gonococcal medicine; Further, it is the dosage form of oral or drug administration by injection; For example it is the form of tablet, capsule, granule, pill, oral solutions, injection (liquid drugs injection and/or powder pin) etc.
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| CN103983732A (en) * | 2014-05-29 | 2014-08-13 | 贵州威门药业股份有限公司 | Determination method for two components in plasma of Relinqing granules |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103432213A (en) * | 2013-09-09 | 2013-12-11 | 贵州威门药业股份有限公司 | Harvesting and processing method of polygonum capitatum serving as medicinal raw material of relinqing granules |
| CN103432213B (en) * | 2013-09-09 | 2014-11-05 | 贵州威门药业股份有限公司 | Harvesting and processing method of polygonum capitatum serving as medicinal raw material of relinqing granules |
| CN103983732A (en) * | 2014-05-29 | 2014-08-13 | 贵州威门药业股份有限公司 | Determination method for two components in plasma of Relinqing granules |
| CN108181416A (en) * | 2017-12-15 | 2018-06-19 | 中国农业科学院茶叶研究所 | A kind of method for measuring 1,2,6 three nutgall acyl-β of tealeaves-D-Glucose and application thereof |
| CN107870216A (en) * | 2017-12-27 | 2018-04-03 | 贵州威门药业股份有限公司 | The spectrum effect of polygonum capitatum opposed polarity position bacteriostasis learns analysis method |
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| CN102805767B (en) | 2014-04-16 |
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