CN102772499A - Effective parts obtained by alcohol extraction of raw materials of Relingqing granules as well as preparation method and applications thereof - Google Patents
Effective parts obtained by alcohol extraction of raw materials of Relingqing granules as well as preparation method and applications thereof Download PDFInfo
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Abstract
The invention relates to effective parts obtained by alcohol extraction of raw materials of Relingqing granules as well as a preparation method and applications thereof, and particularly relates to extracts of Polygonum capitatum Buch-Ham ex D.Don. The preparation method comprises the following steps of: (1) adding 50-90% alcohol in Polygonum capitatum Buch-Ham ex D.Don overground part fresh product or dried product, carrying out reflux extraction for 1-3 times, filtering, and concentrating and drying filtrate to obtain alcoholic extract, wherein the amount of the added alcohol is 5-15 times that of the Polygonum capitatum Buch-Ham ex D.Don overground part fresh product or dried product; (2) suspending the alcoholic extract in methanol, ultrasonically treating for 0.5-5 hours, centrifuging, and loading supernate on an MCI macroporous resin column; and (3) performing gradient eluting successively by using water and 10-100% methanol, collecting all eluents, recovering solvents, drying parts obtained after elution of different solvents to obtain methanol eluates with any concentration interval or concentration point in the range of 30-50% or the mixtures of the methanol eluates, thus obtaining the effective parts namely extracts of Polygonum capitatum Buch-Ham ex D.Don. The extracts of Polygonum capitatum Buch-Ham ex D.Don disclosed by the invention have the beneficial effects as described in 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.
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.
CN1481832A (one Chinese patent application number 02129686.3; Open day on March 17th, 2004) and CN1483466A (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 ℃ 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 more efficiently product from Herba Polygoni Capitati.
Summary of the invention
The present invention seeks to expectation and obtain a kind of 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 30% methanol-eluted fractions thing (can abbreviate as: position A) in the present invention.
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 step (3) gained methanol-eluted fractions thing is that 40% methanol-eluted fractions thing (can abbreviate as: position C) in the present invention.
According to the Herba Polygoni Capitati extract of first aspect present invention, wherein step (3) gained methanol-eluted fractions thing is that 40%-50% methanol-eluted fractions thing (can abbreviate as: position D) in the present invention.
According to the Herba Polygoni Capitati extract of first aspect present invention, wherein step (3) gained methanol-eluted fractions thing is that the mixture of methanol-eluted fractions thing between the 30%-50% (can abbreviate as: position X) in the present invention.In the present invention, this position X can be the mixture of position A D dry product to the position, can also be that 30%-50% methanol-eluted fractions gained mixed liquor is through desolventizing dried dry product.
According to the Herba Polygoni Capitati extract of first aspect present invention, wherein contain for example digalloylglucose of hydrolysable tannin among the A of position.
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, wherein contain hydrolysable tannin and/or procyanidin among the C of position.
According to the Herba Polygoni Capitati extract of first aspect present invention, wherein contain for example hydrolysable tannin davidiin of hydrolysable tannin among the D of position.
According to the Herba Polygoni Capitati extract of first aspect present invention, wherein contain hydrolysable tannin and/or procyanidin among the X of position, for example digalloylglucose, trigalloylglucose, procyanidin and/or hydrolysable tannin davidiin.
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:
(a) between retention time 1-3min, show the chromatographic peak of hydrolysable tannin (for example digalloylglucose);
(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);
(c) between the retention time 3.4-4.4min between (for example about 3.9min place), the retention time 4.8-5.8min between (for example about 5.3min place), the retention time 8.2-9.2min between (for example about 8.7min place), the retention time 12.0-13.0min (for example about 12.5min place) show the chromatographic peak of hydrolysable tannin, and/or at the chromatographic peak of (for example about 9.1min place) demonstration procyanidin between (for example about 8.6min place), the retention time 8.6-9.6min between (for example about 6.7min place), the retention time 8.1-9.1min between the retention time 6.2-7.2min;
(d) chromatographic peak of (for example about 12.5min place) demonstration hydrolysable tannin (for example davidiin) between retention time 12.0-13.0min; And/or
(x) show arbitrary or whole chromatographic peak shown in above (a) to (d);
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 |
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: the chromatographic peak that (a) between retention time 1-3min, shows hydrolysable tannin (for example digalloylglucose).
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.
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: (c) between the retention time 3.4-4.4min between (for example about 3.9min place), the retention time 4.8-5.8min between (for example about 5.3min place), the retention time 8.2-9.2min between (for example about 8.7min place), the retention time 12.0-13.0min (for example about 12.5min place) show the chromatographic peak of hydrolysable tannin, and/or at the chromatographic peak of (for example about 9.1min place) demonstration procyanidin between (for example about 8.6min place), the retention time 8.6-9.6min between (for example about 6.7min place), the retention time 8.1-9.1min between the retention time 6.2-7.2min.
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: (d) chromatographic peak of (for example about 12.5min place) demonstration hydrolysable tannin (for example davidiin) between retention time 12.0-13.0min.
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:
(a) between retention time 1-3min, show the chromatographic peak of hydrolysable tannin (for example digalloylglucose);
(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);
(c) between the retention time 3.4-4.4min between (for example about 3.9min place), the retention time 4.8-5.8min between (for example about 5.3min place), the retention time 8.2-9.2min between (for example about 8.7min place), the retention time 12.0-13.0min (for example about 12.5min place) show the chromatographic peak of hydrolysable tannin, and/or at the chromatographic peak of (for example about 9.1min place) demonstration procyanidin between (for example about 8.6min place), the retention time 8.6-9.6min between (for example about 6.7min place), the retention time 8.1-9.1min between the retention time 6.2-7.2min; And/or
(d) chromatographic peak of (for example about 12.5min place) demonstration hydrolysable tannin (for example davidiin) between retention time 12.0-13.0min.
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:
(a) between retention time 1-3min, show the chromatographic peak of hydrolysable tannin (for example digalloylglucose);
(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);
(c) between the retention time 3.4-4.4min between (for example about 3.9min place), the retention time 4.8-5.8min between (for example about 5.3min place), the retention time 8.2-9.2min between (for example about 8.7min place), the retention time 12.0-13.0min (for example about 12.5min place) show the chromatographic peak of hydrolysable tannin, and/or at the chromatographic peak of (for example about 9.1min place) demonstration procyanidin between (for example about 8.6min place), the retention time 8.6-9.6min between (for example about 6.7min place), the retention time 8.1-9.1min between the retention time 6.2-7.2min;
(d) chromatographic peak of (for example about 12.5min place) demonstration hydrolysable tannin (for example davidiin) between retention time 12.0-13.0min; And/or
(x) show arbitrary or whole chromatographic peak shown in above (a) to (d);
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 |
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: the chromatographic peak that (a) between retention time 1-3min, shows hydrolysable tannin (for example digalloylglucose).
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.
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: (c) between the retention time 3.4-4.4min between (for example about 3.9min place), the retention time 4.8-5.8min between (for example about 5.3min place), the retention time 8.2-9.2min between (for example about 8.7min place), the retention time 12.0-13.0min (for example about 12.5min place) show the chromatographic peak of hydrolysable tannin, and/or at the chromatographic peak of (for example about 9.1min place) demonstration procyanidin between (for example about 8.6min place), the retention time 8.6-9.6min between (for example about 6.7min place), the retention time 8.1-9.1min between the retention time 6.2-7.2min.
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: (d) chromatographic peak of (for example about 12.5min place) demonstration hydrolysable tannin (for example davidiin) between retention time 12.0-13.0min.
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:
(a) between retention time 1-3min, show the chromatographic peak of hydrolysable tannin (for example digalloylglucose);
(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);
(c) between the retention time 3.4-4.4min between (for example about 3.9min place), the retention time 4.8-5.8min between (for example about 5.3min place), the retention time 8.2-9.2min between (for example about 8.7min place), the retention time 12.0-13.0min (for example about 12.5min place) show the chromatographic peak of hydrolysable tannin, and/or at the chromatographic peak of (for example about 9.1min place) demonstration procyanidin between (for example about 8.6min place), the retention time 8.6-9.6min between (for example about 6.7min place), the retention time 8.1-9.1min between the retention time 6.2-7.2min; And/or
(d) chromatographic peak of (for example about 12.5min place) demonstration hydrolysable tannin (for example davidiin) between retention time 12.0-13.0min.
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 30% methanol-eluted fractions thing (can abbreviate as: position A) in the present invention.
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 step (3) gained methanol-eluted fractions thing is that 40% methanol-eluted fractions thing (can abbreviate as: position C) in the present invention.
According to the Herba Polygoni Capitati extract of second aspect present invention, wherein step (3) gained methanol-eluted fractions thing is that 40%-50% methanol-eluted fractions thing (can abbreviate as: position D) in the present invention.
According to the Herba Polygoni Capitati extract of second aspect present invention, wherein step (3) gained methanol-eluted fractions thing is that the mixture of methanol-eluted fractions thing between the 30%-50% (can abbreviate as: position X) in the present invention.In the present invention, this position X can be the mixture of position A D dry product to the position, can also be that 30%-50% methanol-eluted fractions gained mixed liquor is through desolventizing dried dry product.
According to the Herba Polygoni Capitati extract of second aspect present invention, wherein contain for example digalloylglucose of hydrolysable tannin among the A of position.
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, wherein contain hydrolysable tannin and/or procyanidin among the C of position.
According to the Herba Polygoni Capitati extract of second aspect present invention, wherein contain for example hydrolysable tannin davidiin of hydrolysable tannin among the D of position.
According to the Herba Polygoni Capitati extract of second aspect present invention, wherein contain hydrolysable tannin and/or procyanidin among the X of position, for example digalloylglucose, trigalloylglucose, procyanidin and/or hydrolysable tannin davidiin.
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 30% methanol-eluted fractions thing (can abbreviate as: position A) in the present invention.
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 step (3) gained methanol-eluted fractions thing is that 40% methanol-eluted fractions thing (can abbreviate as: position C) in the present invention.
According to the method for third aspect present invention, wherein step (3) gained methanol-eluted fractions thing is that 40%-50% methanol-eluted fractions thing (can abbreviate as: position D) in the present invention.
According to the method for third aspect present invention, wherein step (3) gained methanol-eluted fractions thing is that the mixture of methanol-eluted fractions thing between the 30%-50% (can abbreviate as: position X) in the present invention.In the present invention, this position X can be the mixture of position A D dry product to the position, can also be that 30%-50% methanol-eluted fractions gained mixed liquor is through desolventizing dried dry product.
According to the method for third aspect present invention, wherein contain for example digalloylglucose of hydrolysable tannin among the A of position.
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 contain hydrolysable tannin and/or procyanidin among the C of position.
According to the method for third aspect present invention, wherein contain for example hydrolysable tannin davidiin of hydrolysable tannin among the D of position.
According to the method for third aspect present invention, wherein contain hydrolysable tannin and/or procyanidin among the X of position, for example digalloylglucose, trigalloylglucose, procyanidin and/or hydrolysable tannin davidiin.
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 said Herba Polygoni Capitati extract of second aspect in the medication of preparation clearing away heat-damp and promoting diuresis; The inducing diuresis for treating stranguria syndrome medication; Urinary system infection or calculus medication, pyelonephritis medication, detoxifcation, dissipating blood stasis medication; Antibiotic, antiinflammatory, analgesia medication, the purposes in the medicine of prostatitis medication.
Fifth 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 and optional pharmaceutically acceptable carrier.
According to the pharmaceutical composition of fifth 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.
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.
Drug action focuses on to Herba Polygoni Capitati in the present invention; Understand fully that its effect characteristics screen the drug action of the different extract parts of Herba Polygoni Capitati simultaneously, adopt the sample of the different extract parts of Herba Polygoni Capitati to carry out the screening of Herba Polygoni Capitati active component (effective site) with the drug action characteristics through antiinflammatory, analgesia, antibiotic pharmacological evaluation.
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) the bright article of Herba Polygoni Capitati aerial parts or dry product are added 65 ~ 75% alcohol reflux 2 times that 6-8 doubly measures, each about 1.5 is little), 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 30% methanol-eluted fractions thing (position A); Above step (3) gained methanol-eluted fractions thing can be 35%-40% methanol-eluted fractions thing (position B); Above step (3) gained methanol-eluted fractions thing can be 40% methanol-eluted fractions thing (position C); Above step (3) gained methanol-eluted fractions thing can be 40%-50% methanol-eluted fractions thing (position D); Above step (3) gained methanol-eluted fractions thing can be the mixture (position X) of methanol-eluted fractions thing between the 30%-50%.In the present invention, this position X can be the mixture of position A D dry product to the position, can also be that 30%-50% methanol-eluted fractions gained mixed liquor is through desolventizing dried dry product.
Contain for example digalloylglucose of hydrolysable tannin among above step (3) the gained position A; Contain for example trigalloylglucose of hydrolysable tannin among above step (3) the gained position B; Contain hydrolysable tannin and/or procyanidin among above step (3) the gained position C; Contain for example hydrolysable tannin davidiin of hydrolysable tannin among above step (3) the gained position D; Contain hydrolysable tannin and/or procyanidin among above step (3) the gained position X, for example digalloylglucose, trigalloylglucose, procyanidin and/or hydrolysable tannin davidiin.
Herba Polygoni Capitati active component provided by the present invention (effective site); Xylol causes the influence experiment of mice ear; Press 5g Herba Polygoni Capitati dry product extract obtained/kg body weight gastric infusion, continuous 5 days, test behind the last administration 60min; Significant antiinflammatory action is arranged, and the highest inflammation suppression ratio can reach 49% (effective site X); Cause pain experiment for the mice hot plate, can prolong the mice hot plate add foot reaction latent time (with matched group relatively P 0.05), the highest time-delay rate can reach 18.4% (effective site X).
The effective site that the inventive method obtains can make antiinflammatory action than the obvious enhancing of water extract.The extract of extract of the present invention and extraction process by water relatively can improve its antiphlogistic effects in highly significant ground, and its experimental result is as shown in the table:
| Group | Dosage (g/kg) | n | Inhibitory rate of intumesce (%) |
| Matched group | - | 10 | ? |
| The Herba Polygoni |
5 | 10 | 29.4 |
| Effective site A | 2.5 | 10 | 39.9 |
| Effective site B | 2.5 | 10 | 41.4 |
| Effective site C | 2.5 | 10 | 36.8 |
| Effective site D | 2.5 | 10 | 43.8 |
| Effective site X | 2.5 | 10 | 35.4 |
In the last table, " Herba Polygoni Capitati water extract " method for preparing is: the Herba Polygoni Capitati dry product is divided into secondary with 10 times of water (W/W) decocts each 1.5 hours; Filter; Merging filtrate, relative density was 1.1 when filtrating was concentrated into 20 ℃, the concentrated solution spray drying gets the above fine powder of 100 orders.This Herba Polygoni Capitati water extract also is used for the hereinafter Test Example as the contrast reagent.
In the last table, each effective site method for preparing is: (1) adds 70% alcohol reflux 2 times of 7 times of amounts with the bright article of Herba Polygoni Capitati aerial parts or dry product, and each about 1.5 hours, 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 CHP20P) 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 carry 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 30% methanol-eluted fractions thing is position A; 35%-40% methanol-eluted fractions thing is position B; 40% methanol-eluted fractions thing is position C, and 40%-50% methanol-eluted fractions thing is position D, and 30%-50% methanol-eluted fractions thing is position X.
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, Fig. 2, Fig. 3 and Fig. 4 are respectively the liquid chromatograms of position A, position B, position C and position D.Digalloylglucose representes digalloylglucose among Fig. 1, and isomer representes isomer.
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, collects the each several part eluent, reclaims solvent; Position by the different solvents eluting obtains is dry; Obtaining 30% methanol-eluted fractions thing is position A (yield 2.1%), and 35%-40% methanol-eluted fractions thing is position B (yield 3.47%), and 40% methanol-eluted fractions thing is position C (yield 1.47%); 40%-50% methanol-eluted fractions thing is position D (yield 7.16%), and 30%-50% methanol-eluted fractions thing is position X (yield 14.2%).
Hereinafter, as do not have other explanation, used position A, B, C, D, X sample are the products 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 carry 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 30% methanol-eluted fractions thing is position A; 35%-40% methanol-eluted fractions thing is position B; 40% methanol-eluted fractions thing is position C, and 40%-50% methanol-eluted fractions thing is position D, and 30%-50% methanol-eluted fractions thing is position X.The yield at each position is identical with preparation example 1 basically.
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 carry 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 30% methanol-eluted fractions thing is position A; 35%-40% methanol-eluted fractions thing is position B; 40% methanol-eluted fractions thing is position C, and 40%-50% methanol-eluted fractions thing is position D, and 30%-50% methanol-eluted fractions thing is position X.The yield at each position is identical with preparation example 1 basically.
B, pharmacodynamics test example part
Test Example 1: the experiment of mice caused by dimethylbenzene xylene ear swelling, the antiphlogistic effects of investigation Herba Polygoni Capitati extract
Animal: SPF level Kunming mouse, body weight 18～22g, male and female dual-purpose.
Reagent, dosage and medication: with the various effective sites and the control sample Herba Polygoni Capitati water extract (seeing above) of preceding text " preparation example 1: Herba Polygoni Capitati active component " gained, dosage is that to give the weight that corresponding reagent amounts to into the Herba Polygoni Capitati dry product be 5g to every kg the weight of animals.Each reagent is faced with preceding and is suspended to the concentration that is equivalent to every each gastric infusion 0.4ml of animal with normal saline, is provided with simultaneously to irritate the matched group of stomach with the volume normal saline.
Test method: mice male and female dual-purpose, random packet, 10 animals of each reagent group; Successive administration 5 days behind the last administration 60min, only is coated with auris dextra with xylene 0.1ml/ and causes inflammation, puts to death mice behind the 30min, two auricles about getting with the card punch of diameter 6mm.Use scales/electronic balance weighing immediately, with two auricle weight differences as swelling degree (mg), with the suppression ratio (%) of each reagent group of computes:
Wherein swelling degree (mg)=auris dextra sheet weight-left auricle is heavy
The result carries out statistical analysis.Experimental result is seen table 1.
Suppression ratio is carried out classification, and suppression ratio is lower than 10% and is the A level, and suppression ratio 10 ~ 20% is the AA level, and suppression ratio 20 ~ 30% is the AAA level, and suppression ratio 30 ~ 40% is the AAAA level, and suppression ratio 40 ~ 50% is the AAAAA level, and suppression ratio is the AAAAAA level greater than 50%.
Table 1: different Herba Polygoni Capitati sample xylol causes the influence of mice ear
| Reagent | The suppression ratio classification |
| Position A | AAA+* |
| Position B | AAAA** |
| Position C | AAAAA** |
| Position D | AAAA** |
| Position X | AAAAA** |
| The Herba Polygoni Capitati water extract | AAAA** |
Annotate: swelling degree (mg), compare with the solvent control animals: * P 0.05, * * P < 0.01.Each position of using preparation example 2 and preparation example 3 in the parallel test is as reagent, and the result is identical with table 1.
Test Example 2: the heat-resisting hot plate reaction experiment of mice, the analgesic effect of investigation Herba Polygoni Capitati extract
Animal, reagent, dosage and medication are identical with Test Example 1.
Test method: mice male and female dual-purpose, random packet, 10 animals of each reagent group; Successive administration 5 days behind the last administration 60min, is put into intelligent hot-plate instrument with each group mice, and the observed and recorded mice is added foot reaction latent time, and each is organized mice measurement 2 times (each about 15min at interval) and gets its meansigma methods.Experimental result is seen table 2.
Carry out classification to adding foot reaction latent time, latent time is lower than 6 seconds and is the A level, and latent time 6 ~ 7 seconds was the AA level, and latent time 7 ~ 8 seconds was the AAA level, and latent time 8 ~ 9 seconds was the AAAA level, and latent time was the AAAAA level greater than 9 seconds.
Table 2: different Herba Polygoni Capitati reagents is to the influence of the heat-resisting hot plate reaction of mice
| Reagent | Add the classification of foot reaction latent time |
| Position A | AAA |
| Position B | AAAA** |
[0145]
| Position C | AAA* |
| Position D | AAAA** |
| Position X | AAAA** |
| The Herba Polygoni Capitati water extract | AAAA** |
Annotate: add foot reaction latent time (second), compare with the solvent control animals: < 0.05**P < 0.01 for * P.Each position of using preparation example 2 and preparation example 3 in the parallel test is as reagent, and the result is identical with table 2.
Test Example 3: the antibacterial experiment of different Herba Polygoni Capitati extract
Adopt conventional antibacterial tests method; Use the MH broth bouillon that bacillus pyocyaneus, escherichia coli M421C1ST, dysentery bacterium, staphylococcus aureus, Hemolytic streptococcus are made an experiment; As reagent, test their sensitivity with each sample of preceding text " preparation example 1 " gained and Herba Polygoni Capitati water extract to these cells.
The result shows, between 1-5mg/ml, the MIC of Herba Polygoni Capitati water extract is between 10-25mg/ml to the MIC of five kinds of antibacterials for position A, position B, position C, position D, each sample of position X.
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:
(a) A sample in position shows, between retention time 1-3min, shows the chromatographic peak of hydrolysable tannin (for example digalloylglucose), referring to Fig. 1;
(b) B sample in position 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. 2;
(c) C sample in position shows; At the about 3.9min of retention time place, the about 5.3min of retention time place, the about 8.7min of retention time place, the about 12.5min of retention time place show the chromatographic peak of hydrolysable tannin; At the about 6.7min of retention time place, the about 8.6min of retention time place, the about 9.1min of retention time place show the chromatographic peak of procyanidin, referring to Fig. 3;
(d) D sample in position shows, shows the chromatographic peak of hydrolysable tannin (for example davidiin) at the about 12.5min of retention time place, referring to Fig. 4.
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:
Wherein peak 15 is Davidiin, and its chemical constitution is following:
Claims (12)
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 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.
2. according to the Herba Polygoni Capitati extract of claim 1, it is characterized in that,
Step (3) gained methanol-eluted fractions thing is 30% methanol-eluted fractions thing (position A);
Step (3) gained methanol-eluted fractions thing is 35%-40% methanol-eluted fractions thing (position B);
Step (3) gained methanol-eluted fractions thing is 40% methanol-eluted fractions thing (position C);
Step (3) gained methanol-eluted fractions thing is 40%-50% methanol-eluted fractions thing (position D); And/or
Step (3) gained methanol-eluted fractions thing is the mixture (position X) of methanol-eluted fractions thing between the 30%-50%.
3. according to the Herba Polygoni Capitati extract of claim 2, it is characterized in that,
Contain for example digalloylglucose of hydrolysable tannin among the A of position;
Contain for example trigalloylglucose of hydrolysable tannin among the B of position;
Contain hydrolysable tannin and/or procyanidin among the C of position;
Contain for example hydrolysable tannin davidiin of hydrolysable tannin among the D of position;
Contain hydrolysable tannin and/or procyanidin among the X of position, for example digalloylglucose, trigalloylglucose, procyanidin and/or hydrolysable tannin davidiin.
4. according to each Herba Polygoni Capitati extract of claim 1-3, it is measured according to Ultra Performance Liquid Chromatography-time of flight mass spectrometry coupling method, the result:
(a) chromatographic peak of demonstration hydrolysable tannin between retention time 1-3min;
(b) 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;
(c) at the chromatographic peak that shows hydrolysable tannin between the retention time 3.4-4.4min, between the retention time 4.8-5.8min, between the retention time 8.2-9.2min, between the retention time 12.0-13.0min, and/or at the chromatographic peak that shows procyanidin between the retention time 6.2-7.2min, between the retention time 8.1-9.1min, between the retention time 8.6-9.6min;
(d) between retention time 12.0-13.0min, show the chromatographic peak of hydrolysable tannin (for example davidiin); And/or
(x) show arbitrary or whole chromatographic peak shown in above (a) to (d);
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:
5. according to each Herba Polygoni Capitati extract of claim 1-4, it is characterized in that like the arbitrary embodiment of description first aspect said.
6. Herba Polygoni Capitati extract, it is measured according to Ultra Performance Liquid Chromatography-time of flight mass spectrometry coupling method, the result:
(a) chromatographic peak of demonstration hydrolysable tannin between retention time 1-3min;
(b) 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;
(c) at the chromatographic peak that shows hydrolysable tannin between the retention time 3.4-4.4min, between the retention time 4.8-5.8min, between the retention time 8.2-9.2min, between the retention time 12.0-13.0min, and/or at the chromatographic peak that shows procyanidin between the retention time 6.2-7.2min, between the retention time 8.1-9.1min, between the retention time 8.6-9.6min;
(d) chromatographic peak of demonstration hydrolysable tannin between retention time 12.0-13.0min; And/or
(x) show arbitrary or whole chromatographic peak shown in above (a) to (d);
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:
7. according to the Herba Polygoni Capitati extract of claim 6, 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.
8. according to the Herba Polygoni Capitati extract of claim 7, it is characterized in that:
Step (3) gained methanol-eluted fractions thing is 30% methanol-eluted fractions thing (position A);
Step (3) gained methanol-eluted fractions thing is 35%-40% methanol-eluted fractions thing (position B);
Step (3) gained methanol-eluted fractions thing is 40% methanol-eluted fractions thing (position C);
Step (3) gained methanol-eluted fractions thing is 40%-50% methanol-eluted fractions thing (position D);
Step (3) gained methanol-eluted fractions thing is the mixture (position X) of methanol-eluted fractions thing between the 30%-50%;
Contain for example digalloylglucose of hydrolysable tannin among the A of position;
Contain for example trigalloylglucose of hydrolysable tannin among the B of position;
Contain hydrolysable tannin and/or procyanidin among the C of position;
Wherein contain for example hydrolysable tannin davidiin of hydrolysable tannin among the D of position; And/or
Contain hydrolysable tannin and/or procyanidin among the X of position, for example digalloylglucose, trigalloylglucose, procyanidin and/or hydrolysable tannin davidiin.
9. according to each Herba Polygoni Capitati extract of claim 6-8, it is characterized in that like the arbitrary embodiment of description second aspect said.
10. prepare the method for claim 1-5 or each said Herba Polygoni Capitati extract of claim 6-9, 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.
11. each said Herba Polygoni Capitati extract of claim 1-5 or claim 6-9 is in the medication of preparation clearing away heat-damp and promoting diuresis; The inducing diuresis for treating stranguria syndrome medication; Urinary system infection or calculus medication, pyelonephritis medication, detoxifcation, dissipating blood stasis medication; Antibiotic, antiinflammatory, analgesia medication, the purposes in the medicine of prostatitis medication.
12. a pharmaceutical composition wherein comprises each said Herba Polygoni Capitati extract of claim 1-5 or claim 6-9, and optional pharmaceutically acceptable carrier.
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