DRUG FOR TREATING UROLITHIASIS-RELATED DISEASE, AND PREPARATION METHOD THEREFOR
20240115645 ยท 2024-04-11
Assignee
Inventors
- Hong ZHANG (Sanhe City, langfang city, Hebei, CN)
- Kuiwu WANG (Hangzhou, Zhejiang, CN)
- Qian GUI (Beijing, CN)
Cpc classification
A61K31/7048
HUMAN NECESSITIES
A61K2236/15
HUMAN NECESSITIES
A61K2236/331
HUMAN NECESSITIES
A61K2236/51
HUMAN NECESSITIES
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
International classification
A61K31/7048
HUMAN NECESSITIES
Abstract
Provided in the present invention is a pharmaceutically active ingredient extract extracted from Polygala japonica Houtt., which extract comprises a flavonol compound of the following formula (I) structure as a first active ingredient, and optionally comprises a xanthone compound selected from the following formula (II) as a second active ingredient and a glycolipid compound selected from the following formula (III-1) as a third active ingredient. An animal experiment confirms that the drug of the present invention has significantly better effects than potassium sodium hydrogen citrate in typical test indicators such as calcium oxalate crystal aggregation, renal interstitial inflammatory cell infiltration and renal tubule dilation lesions, showing that the drug has high potential and market prospects in the treatment of urolithiasis and urinary tract infections or kidney damage caused by urolithiasis, and as an adjuvant drug after surgical treatment of urolithiasis.
##STR00001##
Claims
1. A pharmaceutically active ingredient extract extracted from the plant Polygala japonica Houtt., wherein the extract comprises a flavonol compound of the following formula (I) as a first active ingredient ##STR00033## wherein, R.sub.1 is selected from the group consisting of OH, OGlc, OGal, OApi, ORha, OGlcGlc, OGlcGal, OGlcApi, OGlcRha, OGalGlc, OGalGal, OGalApi, OGalRha, OGlcGlcApi, OGalGlcApi, OGlcGalApi, OGalGalApi, OGalRhaGal, OGalRhaGlc, OGlcRhaGlc, and OGlcRhaGal; R.sub.2 is a substituent selected from the group consisting of OH, OMe, OGlc, OGal, OApi, and ORha; R.sub.3 is a substituent selected from the group consisting of H, OH, OMe, OGlc, OGal, OApi, and ORha; R.sub.4 is a substituent selected from the group consisting of OH, and OMe, the pharmaceutically active ingredient extract optionally comprises a xanthone compound selected from the following formula (II) as a second active ingredient, and a glycolipid compound selected from the following formula (III) as a third active ingredient ##STR00034## wherein, R.sub.5 is a substituent selected from the group consisting of OGal, OApi, ORha, OGlcGlc, OGlcGal, OGlcApi, OGlcRha, OGalGlc, OGalGal, OGalApi, OGalRha, OApiGlc, OApiGal, OApiApi, and OApiRha; R.sub.6 is a substituent selected from the group consisting of OH, and OMe; ##STR00035## wherein, R.sub.7 and R.sub.8 are each independently selected from the group consisting of H, and CH3; and wherein, in the definition of R.sub.1-R.sub.6 in the above formula (I) and formula (II), Glc represents glucosyl, Gal represents galactosyl, Api represents celosyl, and Rha represents rhamnose.
2. The pharmaceutically active ingredient extract extracted from the plant Polygala japonica Hotta. of claim 1, wherein the flavonol compound of formula (I) as the first active ingredient is preferably selected from one or more of the compounds of the following general formula ##STR00036## wherein, R.sub.1 is selected from the group consisting of OH, OGlc, OGal, OGlcGlc, OGlcGal, OGlcRha, OGalGlc, OGalGal, OGalRha, OGlcGlcApi, OGalGlcApi, OGlcGalApi, and OGalGalApi, ##STR00037## wherein, R.sub.1 is selected from the group consisting of OH, OGlc, OGal, OGlcGlc, OGlcGal, OGlcApi, OGlcRha, OGalGlc, OGalGal, OGalApi, OGalRha, OGlcGlcApi, OGalGlcApi, OGlcGalApi, OGalGalApi, OGalRhaGal, OGalRhaGlc, OGlcRhaGlc, and OGlcRhaGal, ##STR00038## wherein, R.sub.1 is selected from the group consisting of OH, OGlc, OGal, OGlcApi, OGalApi. ##STR00039## wherein, R is selected from the group consisting of OH, OGlc, OGal, OGlcRha, and OGalRha, ##STR00040## wherein, R is selected from the group consisting of OH, OGlc, OGal, OGlcRha, and OGalRha, ##STR00041## wherein, R is selected from the group consisting of OH, OGal, and OGalApi; the xanthone compound of formula (II) as the second active ingredient is preferably selected from one or more of the following formulas (II-1), (III-2) and (II-3) ##STR00042## (II-1, polygalaxanthone III) ##STR00043## (II-2, polygalaxanthone XI) ##STR00044## (II-3, polygalaxanthone VIII) the glycolipid compound as the third active ingredient is preferably a compound of the following formula (III-1) or (III-2): ##STR00045## (III-1, 3,6-disinapoyl sucrose) ##STR00046## (III-2, Tenuifoliside C).
3. The pharmaceutically active ingredient extract extracted from the plant Polygala japonica Houtt. of claim 2, wherein, the first active ingredient is selected from the flavonol compounds of the above general formulas F-7K, F-7Q, F-74Q, and F-74K.
4. The pharmaceutically active ingredient extract extracted from the plant Polygala japonica Houtt. of claim 3, wherein the first active ingredient is preferably at least one selected from the following compounds: ##STR00047##
5. The pharmaceutically active ingredient extract extracted from the plant Polygala japonica Houtt. of claim 1, wherein: the total content of the flavonol compound of the formula (I) as the first active ingredient, and optionally the xanthone of the formula (II) as the second active ingredient and the glycolipid of formula (III) accounts for 30-100% of the total extract of Polygala japonica Houtt., wherein, the component content (%) is the HPLC% content calculated by using the HPLC integral area normalization method according to the common method in the art.
6. The pharmaceutically active ingredient extract extracted from the plant Polygala japonica Houtt. of claim 5, wherein, the total content of the flavonol compound of formula (I) as the first active ingredient accounts for 20-100% of the total extract of Polygala japonica Houtt.
7. The pharmaceutically active ingredient extract extracted from the plant Polygala japonica Houtt. of claim 6, wherein, the total content of the flavonol compound of formula (I) as the first active ingredient accounts for 75-100% of the total extract of Polygala japonica Houtt.
8. A method for preparing the pharmaceutically active ingredient extract extracted from the plant Polygala japonica Houtt. of claim 1, comprising the following steps (1) pretreatment of Polygala japonica Houtt. the raw material of Polygala japonica Houtt. is obtained by taking the whole herb of Polygala japonica Houtt. or the aboveground part of Polygala japonica Houtt., or commercially available pharmaceutical materials of Polygala japonica Houtt., washing and crushing; (2) rough extraction of effective parts of Polygala japonica Houtt. the total alcohol extract of Polygala japonica Houtt. is obtained by taking some of the raw material of Polygala japonica Houtt. obtained in step (1), heating and refluxing with ethanol with a concentration of 20-95% (v/v) that is about 6 to 12 times the weight of the raw material of Polygala japonica Houtt. for 1 to 3 hours each time, and repeatedly refluxing to extract 1 to 3 times, combining the obtained alcohol extract, and concentrating; or, the total water extract of Polygala japonica Houtt. is obtained by taking some of the raw material of Polygala japonica Houtt. obtained in step (1), heating them to boil with deionized water about 6 to 15 times the weight of Polygala japonica Houtt. and keeping boiling for 1 to 3 hours, repeatedly extracting for 1 to 3 times, combining the obtained water extract, and concentrating; (3) refining of effective parts of Polygala japonica Houtt. the required pharmaceutically active ingredient extract of Polygala japonica Houtt. is obtained by filtering or centrifuging the total water extract or total alcohol extract of Polygala japonica Houtt. in step (2), after concentrating the filtrate or supernatant, separating with a macroporous adsorption resin chromatographic column or polyamide resin chromatographic column, and sequentially gradient eluting with different ratios of water/ethanol until the eluent is colorless, collecting the 0-95% ethanol gradient eluent, and drying under reduced pressure.
9. The method for preparing the pharmaceutically active ingredient extract extracted from the plant Polygala japonica Houtt. of claim 8, wherein in the step (3), the macroporous resin is selected from type D101, type HPD100, type HPD200 or type AB-8 macroporous resin, and the polyamide resin is selected from 100-200 mesh SCR polyamide resin.
10. The method for preparing the pharmaceutically active ingredient extract extracted from the plant Polygala japonica Houtt. of claim 8, wherein in the step (1), the Polygala japonica Houtt. is preferably the stem and leaf part of Polygala japonica Houtt.
11. The method for preparing the pharmaceutically active ingredient extract extracted from the plant Polygala japonica Houtt. of claim 8, wherein in the step (3), the macroporous resin is selected from type D101 or type AB-8 macroporous resin, and the polyamide resin is selected from 100-200 mesh SCR polyamide resin.
12. The method for preparing the pharmaceutically active ingredient extract extracted from the plant Polygala japonica Houtt. of claim 8, wherein in the step (3), the gradient eluting is performed by sequentially eluting with water, 25% ethanol, 50% ethanol, 75% ethanol, and 95% ethanol until the eluent is colorless.
13. A method for treating or preventing urolithiasis and urinary tract infections or kidney damage caused by urolithiasis in a subject, wherein the method comprises administering to the subject an effective amount of the pharmaceutically active ingredient extract extracted from the plant Polygala japonica Houtt. of claim 1 or as an adjuvant drug after surgical treatment of urolithiasis.
14. A pharmaceutical composition comprising at least one compound selected from the following formulas as an active ingredient: ##STR00048##
15. The pharmaceutical composition of claim 14, further comprising a pharmaceutically acceptable carrier, excipient or auxiliary material.
16. A method for treating or preventing urolithiasis and urinary tract infections or kidney damage caused by urolithiasis in a subject, wherein the method comprises administering to the subject an effective amount of the pharmaceutical composition of claim 14 or as an adjuvant drug after surgical treatment of urolithiasis.
17. A method for treating or preventing urolithiasis and urinary tract infections or kidney damage caused by urolithiasis in a subject, wherein the method comprises administering to the subject an effective amount of any one of the following compounds or as an adjuvant drug after surgical treatment of urolithiasis, ##STR00049##
Description
DESCRIPTION OF THE FIGURES
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DETAILED DESCRIPTION OF THE INVENTION
[0078] Examples of the present invention are described in detail below, and the illustration of Examples is shown in the accompanying drawings. The Examples described below by referring to the drawings are exemplary only for explaining the present invention and should not be construed as limiting the present invention.
I. PREPARATION EXAMPLES
Preparation Example 1: Components Analysis of Alcohol Extract From The Whole Herb of Polygala Japonica Houtt.
[0079] The whole herb of Polygala japonica Houtt. was weighed, in which times the amount of 75% ethanol was added, the obtained solution was heated to reflux, extracted 3 times with each time for 3 hours, filtered while hot, and then the alcohol extracts were combined;
[0080] The alcohol extract was concentrated to an extract concentrate with a relative density of 1.1-1.3 g/ml.
[0081] The fingerprint analysis was performed for the obtained alcohol extract concentrate of Polygala japonica Houtt. by using HPLC.
[0082] HPLC test conditions: Mobile phase: acetonitrile (A), 0.1% formic acid aqueous solution (B), binary gradient separation;
[0083] Flow rate: 0.8 mL min.sup.?1;
[0084] Detection wavelength: 330 nm;
[0085] Column temperature: 20? C.;
[0086] Injection volume: 20 ?L.
[0087] Chromatogram was recorded for 90 min.
Preparation Example 2: Components Analysis of Water Extract from the whole herb of Polygala japonica Houtt.
[0088] The whole herb of Polygala japonica Houtt. was weighed, in which 10 times the amount of deionized water was added, the obtained solution was heated to reflux, extracted twice with each time for 3 hours, filtered while hot, and then the water extracts were combined; the extract is a dark brown liquid.
[0089] The water extract was concentrated to an extract concentrate with a relative density of 1.1-1.3 g/ml.
[0090] The fingerprint analysis was performed for the obtained water extract concentrate of Polygala japonica Houtt. by using HPLC (see
[0091] Comparing the components obtained by the water extraction method and the alcohol extraction method, it can be found that: due to the relatively large polarity of water, the content of impurities with lower polarity such as chlorophyll in the extract obtained by the water extraction method will be less, whereas the high-polar tannin components will be more. The extract is dark brownish yellow. However, the content of low-polar components (such as chlorophyll, etc.) in the alcohol extract is more, while the content of high-polar components (such as tannin, etc.) is significantly reduced. The alcohol extract is greenish, but turns brownish yellow after cooling.
[0092] Whole herb extraction consumes a lot of pharmaceutical plants. Considering that most of the commercially available Polygala japonica Houtt. are the aboveground parts, it is also beneficial to protect the pharmaceutical plant resources by only selecting the aboveground parts and keeping the roots of the plants. Therefore, the inventor further tried to extract the active ingredient from the aboveground part of Polygala japonica Houtt. by alcohol extraction. The specific method is as follows.
Preparation Example 3: Components Analysis of Water Extract From The Aboveground Part of Polygala Japonica Houtt.
[0093] The aboveground part of Polygala japonica Houtt. was weighed, in which 10 times the pharmaceutical material amount of deionized water was added, the obtained solution was heated to reflux, extracted 3 times with each time for 3 hours, filtered while hot, and then the water extracts were combined;
[0094] The water extract was concentrated to an extract concentrate with a relative density of 1.1-1.3 g/ml.
[0095] The fingerprint analysis was performed for the deionized water extract concentrate of the aboveground part of Polygala japonica Houtt. by using HPLC.
Preparation Example 4: Components Analysis of Alcohol Extract From The Aboveground Part of Polygala Japonica Houtt
[0096] The aboveground part of Polygala japonica Houtt. was weighed, in which 10 times the pharmaceutical material amount of 50% ethanol was added, the obtained solution was heated to reflux, extracted 3 times with each time for 3 hours, filtered while hot, and then the alcohol extracts were combined;
[0097] The alcohol extract was concentrated to an extract concentrate with a relative density of 1.1-1.3 g/ml.
[0098] The fingerprint analysis was performed for the obtained alcohol extract concentrate of the aboveground part of Polygala japonica Houtt. by using HPLC (see
[0099] In order to confirm the chemical composition of the extract concentrate, we analyzed it by HPLC-MS, in which MS analysis includes positive and negative ions and MS-MS, MS-MS-MS analysis, and compared the analysis results with the existing literatures, and preliminarily confirmed the structure types of four major categories of flavonol compounds, xanthones, glycolipids and saponins. Further, we subdivided flavonol compounds according to the differences in the parent core of flavonol compounds.
[0100] However, due to the complexity and diversity of the spatial structure and connection methods of glycosyls in glycosides, it can be determined whether the connection between multiple glycosides and the parent core of flavonol compounds is a single sugar chain, and the order of glycoside fragmentation in mass spectrometry through HPLC and multi-stage tandem MS, so as to confirm the molecular weight of the terminal sugar in the sugar chain and the basic type of glycogen. However, due to the complexity and diversity of the spatial structure and connection methods of glycosyls in glycosides, it needs to be further identified by other means for the configuration of the hydroxyl group on a certain carbon atom of the sugar ring of the isomer (such as glucose or galactose), the position of the sugar chain linkage between the glycoside and the glycoside (such as a 1-2 linkage, or a 1-4 linkage, or 1-6 linkage), and ? or ? configuration of the sugar configuration. Therefore, only through the structure identified by HPLC-MS-MS, compounds with the same molecular formula may have multiple structural combinations of aglycones in the flavonol compounds and different types of glycosides.
[0101] In order to facilitate the distinction, the present application uses the compound category to identify and distinguish the compounds that may have various combinations of structural units. For example, in the present application, F is used to represent the class of flavonol compounds, and F-Q represents a class of flavonol glycosides in which the parent core of the class of flavonol compounds is quercetin. In the structural determination, 302-162-132 represents that the parent core of aglycone in flavonol is quercetin, to which a glucose (or galactose) is connected, a celose is connected on this glucose (or galactose), wherein 302 is the parent core of quercetin, 162 is the molecular weight of the characteristic peak of glucose (or galactose) fragmentation fragments in mass spectrum, and 132 is the molecular weight of the characteristic peak of celose fragmentation fragments.
[0102] Through HPLC and multi-stage tandem MS analysis, it is preliminarily confirmed that the alcohol extract from the aboveground part of Polygala japonica Hotta. mainly comprises the following components:
TABLE-US-00001 TABLE 1 Component analysis of the alcohol extract from the aboveground parts of Polygala japonica Houtt. Retention time Compound Molecular HPLC/MS category weight Compound structure 1 19.77 F-Q 596 302-162-132 2 *20.344 Xanthones 568 Xanthone III of Polygala japonica Houtt. 3 21.867 F-Q 596 302-162-132 4 22.482 F-Q 464 302-162 5 24.080 F-K 580 286-162-132 6 *30.807 Glycolipids 754 Glycolipid (III-1) 7 32.087 F-7Q 640 316-162-162 8 *34.263 F-7Q-1 640 316-162-162 9 38.077 F-7K 756 300-162-132-162 10 38.813 F-7K 756 300-162-132-162 11 *39.413 F-7K-1 624 300-162-162 12 40.405 F-7Q 610 316-162-132 13 42.477 F-7Q 478 316-162 14 43.145 Saponins 1120 Saponin VIII of Polygala japonica Houtt. 15 *44.852 Glycolipids 768 Glycolipid (III-2) 16 46.718 F-7K 594 300-162-132 17 47.235 F-7Q 786 316-162-162-146 18 48.491 F-74Q 624 330-162-132 19 49.827 F-7K 462 300-162 20 *49.877 F-74Q-1 624 330-294(162 + 132) 21 51.249 Saponins 1164 Saponin 22 53.306 F-7K 462 300-162 23 56.087 F-74Q 492 330-162 24 59.759 F-7K 770 300-162-162-146 25 62.343 Saponins 1398 Saponin XXIX of Polygala japonica Houtt. 26 63.435 Saponins 1252 Saponin X of Polygala japonica Houtt. 27 *65.961 F-74K-1 608 314-162-132 28 67.559 Saponins 1236 Saponin 29 68.042 Saponins 1104 Saponin XXI of Polygala japonica Houtt. 30 68.481 Saponins 872 Saponin 31 69.136 Saponins 1088 Saponin 32 70.406 Saponins 1220 Saponin 33 76.973 Saponins 710 Saponin
[0103] In the above table, the structure of the chemical composition mainly includes the following categories:
(1) Flavonols with Glycosides [0104] 1. A flavonol glycoside compound whose compound category is F-7K. Wherein, the aglycone structure of the compound is Rhamnocitrin with a molecular weight of 300 or 3,4,5-Trihydroxy-7-methoxyflavone, or 7-methoxyl-kaempferol, and the compound has the following general structure:
##STR00019##
[0105] Wherein, R1=glycosyl, which may be selected from the group consisting of OH, OGlc, OGal,OGlcGlc, OGlcGal, OGlcRha, OGalGlc, OGalGal, OGalRha, OGlcGlcApi, OGalGlcApi, OGlcGalApi, and OGalGalApi.
[0106] 2. A flavonol glycoside compound whose compound category is F-7Q. Wherein, the aglycon structure of the compound is Rhamnetin with a molecular weight of 316 or 3,3,4,5-Tetrahydroxy-7-methoxyflavone, or 7-methoxyl-quercetin, and the compound has the following general structure:
##STR00020##
[0107] Wherein, R1=glycosyl, which may be selected from the group consisting of OH, OGlc, OGal, OGlcGlc, OGlcGal, OGlcApi, OGlcRha, OGalGlc, OGalGal, OGalApi, OGalRha, OGlcGlcApi, OGalGlcApi, OGlcGalApi, OGalGalApi, OGalRhaGal, OGalRhaGlc, OGlcRhaGlc, and OGlcRhaGal;
[0108] 3. A flavonol glycoside compound whose compound category is F-74Q. Wherein, the aglycon structure of the compound is Ombuine with a molecular weight of 330 or 3,5,3-Trihydroxy 7,4-dimerhoxyflavone, or 7,4-dimerhoxyl-quercetin, and the compound has the following general structure:
##STR00021##
[0109] Wherein, R1=glycosyl, which may be selected from the group consisting of OH, OGlc, OGal, OGlcApi, and OGalApi. [0110] 4. A flavonol glycoside compound whose compound category is F-K. Wherein, the aglycone structure of the compound is kaempferol with a molecular weight of 286 or 3,4,5,7-Tetrahydroxyflavone, and the compound has the following general structure:
##STR00022##
[0111] Wherein, R may be selected from the group consisting of OH, OGlc, OGal, OGlcRha, and OGalRha. [0112] 5. A flavonol glycoside compound whose compound category is F-Q. Wherein, the aglycone structure is quercetin with a molecular weight of 302 or 3,3,4,5,7-Pentahydroxyflavone, and the compound has the following general structure:
##STR00023##
[0113] Wherein, R may be selected from the group consisting of OH, OGlc, OGal, OGlcRha, and OGalRha.
[0114] 6. A flavonol glycoside compound whose compound category is F-74K. Wherein, the aglycone structure is Ermanin with a molecular weight of 314 or 3,5-dihydroxy 7,4-dimerhoxyflavone, or 7,4-dimerhoxyl-kaempferol, and the compound has the following general structure:
##STR00024##
[0115] Wherein, R=glycosyl, which may be selected from the group consisting of OH, OGal, and OGalApi. [0116] (2) Xanthone compounds of Polygala japonica Houtt.
[0117] It can be confirmed by secondary mass spectrometry that the xanthone compounds of Polygala japonica Houtt. are selected from the following formulas (II-1), (II-2) and (II-3)
##STR00025##
(II-1, polygalaxanthone III)
##STR00026##
(II-2, polygalaxanthone XI)
##STR00027##
(II-3, polygalaxanthone VIII). [0118] (3) Glycolipid compounds
[0119] It can be confirmed by secondary mass spectrometry that the structure of the glycolipid compound is selected from the following formulas (III-1) and (III-2)
##STR00028##
(III-1, 3,6-disinapoyl sucrose)
##STR00029##
(III-2, Tenuifoliside C).
[0120] (4) Saponin compounds
[0121] It can be confirmd by HPLC and multi-stage tandem MS analysis that the alcohol extract from the aboveground part of Polygala japonica Houtt. in the present application includes Polygalasaponin VIII, Polygalasaponin XXI, Polygalasaponin X, Polygalasaponin XXIX and other saponin compounds.
[0122] The alcohol extract from the aboveground part of Polygala japonica Houtt. comprises not only flavonols and xanthones and other target active ingredients, but also saponins, glycolipids and other ingredients. Therefore, the present inventors further tried to further refine the above-mentioned alcohol extract from the aboveground part of Polygala japonica Houtt. by separation methods such as macroporous resin and polyamide resin, so as to separate and enrich the target active ingredients.
Preparation Example 5: Macroporous Resin Refining Treatment of Alcohol Extract from the Aboveground Part Of Polygala Japonica Houtt.
[0123] The extract concentrate obtained in Preparation Example 1 was passed through the D101 macroporous adsorption resin column at a flow rate of 1 times the column bed volume per hour. After the adsorption was completed, it was first washed with 8 times the amount of resin to remove impurities, and then washed with 2-5 times the column bed volume of 0%-25, 25%-50%, 50%-75%, 75%-95% ethanol gradient elution, the elution was performed at a flow rate of 0.5-2 times the column bed volume per hour to obtain the eluent; and the ethanol eluate with different concentrations was concentrated 5-20 times respectively to obtain an eluate concentrate with a relative density of 1.1-1.3 g/ml.
[0124] The fingerprint analysis was performed for the components of the obtained alcohol extract concentrate of the whole herb of Polygala japonica Houtt. and ethanol gradient eluate concentrate through a macroporous adsorption resin column by using HPLC, respectively.
Preparation Example 6: Polyamide Resin Refining Treatment of Water Extract from the Aboveground Part of Polygala Japonica Houtt.
[0125] The extract concentrate obtained in Preparation Example 3 was passed through the polyamide resin column at a flow rate of 0.5-1 times the column bed volume per hour. After the adsorption was completed, it was first washed with 2-8 times the amount of resin to elute and remove impurities, and then washed with 2-5 times column bed volume of 0-25%, 25%-50%, 50%-75%, 75%-95% ethanol gradient elution, the elution was performed at a flow rate of 0.5-2 times column bed volume per hour to obtain the eluent; the above-mentioned ethanol eluate with different concentrations was concentrated 5-20 times respectively to obtain an eluate concentrate with a relative density of 1.1-1.3 g/ml.
[0126] Comparing the separation effect of macroporous resin and polyamide resin, it can be found that polyamide resin can better remove the saponin component of the extract of Polygala japonica Houtt., and the separation and purification effect is better.
[0127] The fingerprint analysis was performed for the components of the obtained extract concentrate of the aboveground part of Polygala japonica Houtt. and ethanol gradient eluate concentrate through a polyamide resin column by using HPLC, respectively.
[0128] It is confirmed by HPLC analysis that the total content of active ingredients (I), (II) and (III) is 50%-90% in the orange-red eluate concentrate of the 0-25% ethanol elution part (as shown in
[0129] The eluate concentrate was dried under reduced pressure at 75? C. and crushed to obtain the enriched active ingredients of the aboveground part of Polygala japonica Houtt., which were used in the drug efficacy comparison experiment.
[0130] The enriched active ingredients of the aboveground part of Polygala japonica Houtt. were analyzed by HPLC-MS, and the MS analysis included positive and negative ions, MS-MS, MS-MS-MS analysis, and the analysis results were compared with the existing literature to confirm the compound structure shown in Table 2 below.
TABLE-US-00002 TABLE 2 Components analysis of water extraction-polyamide resin refined extract of aboveground part of Polygala japonica Houtt. Molecular Retention Compound Molecular formula and No. time(LC) category weight sugar chain Structure confirmation Area % 1 20.090 Xanthones 568 C25H28O15 Polygalaxanthone III 1.74 2 30.779 Glycolipids 754 C34H42O19 3,6-disinapoyl sucrose 2.86 3 32.369 F-7Q 640 C28H32O17 Rhamnetin + Glc (or Gal) + 0.52 316-162-162 Glc (or Gal) Isomers with F-7Q-1 4 34.243 F-7Q-1 640 C28H32O17 Rhamnetin 33.71 316-162-162 3-O-?-D-Glucopyranosyl (1.fwdarw.2)-?-D-galactopyranoside 5 38.100 F-7K 756 C33H40O20 Rhamnocitrin + Glc (or Gal) + 0.50 300-162-132-162 Api + Glc (or Gal) Isomers with Peak No. 6 6 38.802 F-7K 756 C33H40O20 Rhamnocitrin + Glc (or Gal) + 0.12 300-162-132-162 Api + Glc (or Gal) Isomers with Peak No. 5 7 39.472 F-7K-1 624 C28H32O16 Rhamnocitrin 18.97 3-O-?-D-glucopyranosyl (1.fwdarw.2) -?-D- galactopyranoside 8 40.507 F-7Q 610 C27H30O16 Polygalin E or 0.76 316-162-132 Polygalin F 9 42.567 F-7Q 478 C22H22O12 Rhamnetin + Glc (or Gal) 0.42 316-162 10 44.617 Glycolipids 768 C35H44O19 Tenuifoliside C 1.19 11 46.661 F-7K 594 C27H30O15 Rhamnocitrin + Glc (or Gal) + 3.48 300-162-132 Api 12 47.667 F-7K 462 C22H22O11 Rhamnocitrin + Glc (or Gal) 4.62 300-162 13 50.508 F-74Q-1 624 C28H32O16 Polygalin C, 23.60 330-162-132 3,5,3-trihydroxy-7,4- dimethoxyflavone -3-O-?-D- apiofranosyl (1.fwdarw.2)-?-D- galactopyranoside 14 51.535 F-74Q 624 C28H32O16 Polygalin D 0.79 330-162-132 Isomers with Polygalin C 15 56.047 F-74Q 492 C23H24O12 3,5,3-Trihydroxy 7,4- 0.20 330-162 dimerhoxyflavone + Glc (or Gal) 16 62.943 F-7K 770 C34H42O20 Rhamnocitrin + 2Glc (or 1.53 300-162-162-146 Gal) + Rha 17 66.186 F-74K-1 608 C28H32O15 Polygalin B, 4.81 314-(162 + 132) 3,5-Dihydroxy-7,4- dimethoxyflavone- 3-O-?-D-apiofranosyl (1.fwdarw.2)-?-D-galactopyranoside
[0131] Based on the above analysis, it can be confirmed that the main components of the water extraction-polyamide refined extract of Polygala japonica Houtt. comprises flavonol compounds with compound categories of F-7K, F-7Q, F-74Q, F-74K, and the xanthone compound of formula (II-1) (polygalaxanthone III) and glycolipid compounds.
[0132] In Table 2, the compound with a content of 18.97% is identified as F-7K-1, the compound with a content of 33.71% is identified as F-7Q-1, the compound with a content of 23.60% is identified as F-74Q -1, and the compound with a content of 4.81% is identified as compound F-74K-1 (Polygalitol B).
[0133] Wherein the meanings of the above compound category names are exactly the same as those in Table 1.
[0134] It needs to be emphasized that due to the difference in the extracted specific parts (whole herb, rhizome, or stem and leaf) of the plant Polygala japonica Houtt., the difference in the source of origin, and the difference in the preparation of the plant Polygala japonica Houtt. (commercially available dried herbs, fresh plant Polygala japonica Houtt.) as well as the difference in the specific extraction and refining process conditions may lead to different degrees of differences in the structure and content of the active ingredient in the obtained active ingredient extract. In the present invention, preferably the active ingredient extract was obtained from fresh or dried aboveground parts of Polygala japonica Hotta. by water extraction and polyamide column alcohol/water gradient elution.
Preparation Example 7: Separation and Refining of Main Active Compounds
[0135] It can be determined by HPLC-MS-MS whether it is a single sugar chain according to the obvious characteristics when determining the parent core of the flavonol compound and the presence of multiple sugar rings in the molecule. However, there is insufficient basis for discriminating the configuration of the hydroxyl group on a certain carbon atom of a sugar ring with the same molecular weight (such as glucose or galactose). When a sugar chain has more than two sugar rings, there is insufficient basis for determing the linkage position of the sugar (for example, a 1-2 linkage, a 1-4 linkage, or a 1-6 linkage) and ? or ? configuration of the sugar configuration. To this end, the pure compound of the main active ingredient was separated by semi-preparative HPLC, and its specific structure was determined in combination with H NMR, C NMR, and two-dimensional NMR.
[0136] Purification was performed by semi-preparative HPLC. The extract prepared in Example 5 was further separated and purified, and the pure single compounds of the three components with the highest content in the extract were collected respectively, that is, the pure single compounds of compounds F-7Q-1, F-7K-1, and F-74Q-1 were obtained.
[0137] The semi-preparative HPLC instrument and conditions were as follows:
[0138] HPLC test conditions mobile phase: acetonitrile (A), deionized water (B), binary gradient separation;
[0139] Semi-preparative column: 19?250 mm, C18
[0140] Flow rate: 8 mL min-1;
[0141] Detection wavelength: 330 nm;
[0142] Column temperature: room temperature;
[0143] Injection volume: 0.5 mL.
[0144] Chromatogram was recorded for 180 min.
[0145] The structures of compounds F-7Q-1, F-7K-1, and F-74Q-1 were analyzed by NMR and two-dimensional NMR, respectively. The analysis results were as follows: [0146] 1. Confirmation of the structure of compound F-7Q-1
[0147] Molecular weight: 640, sugar chain: 316-162-162,
[0148] The key information related to the two-dimensional NMR of compound F-70-1 was summarized in the following table:
TABLE-US-00003 No. ?H ?C HH COSY HMBC (H.fwdarw.C) 2 156.1 3 133.0 4 177.4 5 160.9 6 6.34 (1H, s) 97.8 C-5, 7, 8, 10 7 164.9 8 6.69 (1H, s) 92.0 C-6, 7, 9, 10 9 156.1 10 104.8 1 119.9 2 7.57 (1H, s) 115.5 C-2, 1, 3, 4 3 145.4 4 150.3 5 6.81 (1H, d, J = 8.4) 115.3 H-6 C-1, 3 6 7.72 (1H, d, J = 8.4) 122.4 H-5 C-2, 2, 4 OCH3 3.86 (3H, s) 56.1 Gal-1 5.69 (1H, d, J = 7.6) 98.4 C-3, 3 2 3.79 (m) 80.9 3 3.59 (m) 73.4 4 3.64 (m) 67.6 5 3.35 (m) 75.9 6 3.26, 3.42 (m) 59.9 Glu-1 4.60 (1H, d, J = 7.7) 104.4 C-2 2 3.09 (m) 74.5 3 3.18 (m) 76.8 4 3.21 (m) 69.5 5 3.23 (m) 76.6 6 3.51, 3.56 (m) 60.6
[0149] Based on the above chromatographic analysis data, it is finally confirmed that the exact spatial structural formula of the main active ingredient compound F-7Q-1 is as follows:
##STR00030##
[0150] The compound name of F-7Q-1 is: Rhamnetin 3O-?-D-glucopyranosyl(1.fwdarw.2)-?-D-galactopyranoside, or Rhamnetin-3-O-(2-O-?-D-glucopyranosyl)-?-D-galactopyranoside. [0151] (2) Confirmation of the structure of compound F-7K-1
[0152] Molecular weight: 624, sugar chain: 300-162-162,
[0153] The key information related to the two-dimensional NMR of compound F-7K-1 was summarized in the following table:
TABLE-US-00004 No. ?H ?C HH COSY HMBC (H.fwdarw.C) 2 156.2 3 133.0 4 177.6 5 161.0 6 6.36 (1H, d, J = 2.2) 97.9 C-5, 7, 8, 10 7 165.0 8 6.74 (1H, d, J = 2.2) 92.2 C-6, 7, 9, 10 9 156.0 10 104.9 1 120.2 2, 6 8.13 (2H, d, J = 8.9) 131.1 H-3, 5 C-2, 2, 4, 6 3, 5 6.89 (2H, d, J = 8.9) 115.5 H-2, 6 C-1, 3, 4, 5 4 161.0 OCH3 3.86 (3H, s) 56.1 C-7 Gal-1 5.70 (1H, d, J = 7.6) 98.3 C-3, 3 2 3.78 (m) 80.5 3 3.58 (m) 73.4 4 3.64 (m) 67.6 5 3.34 (m) 75.9 6 3.27, 3.43 (m) 59.9 Glu-1 4.60 (1H, d, J = 7.8) 104.3 C-2 2 3.08 (m) 74.4 3 3.18 (m) 77.0 4 3.21 (m) 69.7 5 3.34 (m) 76.6 6 3.51, 3.56 (m) 60.8
[0154] Based on the above chromatographic analysis data, it is finally confirmed that the exact spatial structural formula of the main active ingredient compound F-7K-1 is as follows:
##STR00031##
[0155] The compound name of F-7K-1 is: Rhamnocitrin 3O?-D-glucopyranosyl(1.fwdarw.2)-?-D-galactopyranoside, or OR Rhamnocitrin-3O(2O?-D-glucopyranosyl)-?-D-galactopyranoside. [0156] 3. Confirmation of the structure of compound F-74Q-1
[0157] Molecular weight: 624, sugar chain: 330-162-132, identification:
[0158] The key information related to the two-dimensional NMR of compound F-74Q-1 was summarized in the following table:
TABLE-US-00005 No. ?H ?C HH COSY HMBC (H.fwdarw.C) 2 155.7 3 133.8 4 177.6 5 161 6 6.36 (1H, d, J = 2.1) 97.9 C-5, 7, 8, 10 7 165.0 8 6.74 (1H, d, J = 2.1) 92.1 C-6, 7, 9, 10 9 156.2 10 105 1 122.6 2 7.57 (1H, d, J = 2.2) 115.3 C-2, 1, 3 ,4 3 146.1 4 150.2 5 6.98 (1H, d, J = 8.8) 111.2 H-5, 6 C-1, 3, 4 6 7.93 (1H, dd, J = 8.8, 122.2 H-5, 6 C-2, 2, 4 2.2) 7-OCH3 3.87 (3H, s) 56.1 C-7 4-OCH3 3.86 (3H, s) 55.7 C-4 Gal-1 5.62 (1H, d, J = 8.0) 99.0 C-3, 3 2 3.78(m) 74.9 3 3.58(m) 73.8 4 3.64(m) 68.3 5 3.34(m) 75.8 6 3.43, 3.28(m) 60.1 Api-1 5.32 (1H, d, J = 1.0) 108.8 C-2, 2, 3 2 3.80(m) 76.1 3 79.1 4 3.51(m), 3.84(m) 73.9 5 3.46(m), 3.39(m) 64.3
[0159] Based on the above chromatographic analysis data, it is finally confirmed that the exact spatial structural formula of the main active ingredient compound F-74Q-1 is as follows:
##STR00032##
[0160] The compound name of F-74Q-1 is: 3,5,3-trihydroxy-7,4-dimethoxyflavone-3O?-D-apiofranosyl(1.fwdarw.2)-?-galactopyranoside, or Polygalin C, or Polygalitol C.
[0161] Based on the inventors' research, the inventors believe that the isolated compounds F-7Q-1, F-7K-1, and F-74Q-1, as the main component of the Polygala japonica Hotta. extract of the present invention, play a key role for realizing the desired pharmaceutical effect. These compounds have a hydroxyl group as a substituent at the ? position of the carbonyl in the described molecular structure, the hydroxyl group and the ketone carbonyl group act together to more effectively react with calcium ion-containing components of the calculus in the urinary system, thereby more effectively degrading or dissolving the calculus in the urinary system. Therefore, at least one of the compounds F-7Q-1, F-7K-1, and F-74Q-1 can also be used as the main and necessary active ingredient to prepare a corresponding pharmaceutical composition for treating or preventing urolithiasis and urinary tract infections or kidney damage caused by urolithiasis, and as an adjuvant drug after surgical treatment of urolithiasis. The pharmaceutical composition may further comprises pharmaceutically acceptable adjuvants, carriers or excipients.
II. Experimental example of pharmacological activity [0162] 1. Preparation of test samples [0163] {circle around (1)} High-dose group test sample: the water-extracted, 0-25% ethanol gradient eluent by polyamide column prepared in Preparation Example 5 was concentrated and dried to obtain the active ingredient extract. A solution with a density of about 1.2 g/ml was formulated, and the concentration of the effective substance measured by the pharmacopoeia standard and the absorbance method (using rutin as a standard substance to make a standard curve) is about 130.4 mg/ml. [0164] {circle around (2)} Medium-dose group test sample: the high-dose group sample was diluted once to obtain the middle-dose group test sample. [0165] {circle around (3)} Low-dose group test sample: the middle-dose group sample was dilute once to obtain the low-dose group test sample. [0166] {circle around (4)} Positive control group test sample: potassium sodium hydrogen citrate aqueous solution with a concentration of 100 mg/ml, 3 ml per day, equivalent to 300 mg/d. [0167] 2. Animal experiments [0168] 2.1. Experimental animals and feeding conditions
[0169] 36 SD rats were purchased from Shanghai Slack, license number: SCXK (Shanghai) 2017-0005, certificate number: 20170005011248. Drinking water is ultrapure water. The license number for the experimental animal room is SYXK (Zhejiang) 2015-0008.
[0170] Feeding environment: temperature ranges from 20? C. to 25? C., and relative humidity ranges from 40% to 70%. Adaptive feeding was performed for one week before the experiment. [0171] 2.2. Experimental scheme [0172] 2.2.1 Experimental animals
[0173] 36 male SD rats of SPF grade, 6-8 weeks old, 200-250 g. [0174] 2.2.3 Model preparation
[0175] SD rats were fed adaptively for 7 days, and all groups (except the normal group) were administered 1% ethylene glycol (by drinking water)+2% ammonium chloride (by intragastric administration) 2 ml/rat to establish a model for 28 consecutive days. [0176] 2.2.4 Experimental grouping and processing
[0177] 36 male SD rats were randomly divided into 6 groups according to body weight, 6 rats in each group. These are normal group, model group, potassium sodium hydrogen citrate group, traditional Chinese medicine extract (low, medium, high) dose group. During the modeling process, 3 ml of the drug was administered by intragastric administration every day, and the drug included the positive control drug and the purified part through the polyamide column. The animals were euthanized after 4 weeks. [0178] 2.2.5 Kidney
[0179] The kidney tissues were peeled off in vivo, the kidney on one side was stored in a cryopreservation tube at ?80? C. for tissue homogenate to detect Ca2+ concentration, and the kit operation steps were the same as above; the 20 kidney on the other side was fixed in formalin solution for tissue sectioning to perform HE staining. [0180] (1) The steps for making paraffin sections were as follows:
[0181] {circle around (1)} fixation, {circle around (2)} trimming, {circle around (3)} dehydration, {circle around (4)} transparency, {circle around (5)} wax immersion, {circle around (6)} embedding, {circle around (7)} slicing, {circle around (8)} baking slices, {circle around (9)} preservation: packed in boxes and stored at room temperature. [0182] (2) HE staining steps were as follows:
[0183] {circle around (1)} dewaxing and rehydration, {circle around (2)} dyeing, {circle around (3)} dehydration, transparency, mounting,
[0184] {circle around (4)} staining results: the nucleus was blue, the cytoplasm was pink, and the red blood cells were bright red. [0185] 3. Test results [0186] 3.1. Positive test results for calcium oxalate crystals in rat urine
[0187] The results of the urine routine report showed that except the normal group, the calcium oxalate crystals in the urine of the rats in the other groups were all positive. [0188] 3.2. Serum Ca2+ concentration test results
[0189] The difference in Ca2+ concentration in serum was small among groups. Compared with the normal group, the model group and the middle-dose treatment group had significant differences. The Ca2+ concentration in urine had a large difference. Compared with the normal group, the low-dose treatment group and the high-dose treatment group had very significant differences (P<0.01). Compared with the model group, the low-dose treatment group had a very significant difference (P<0.01). Potassium sodium hydrogen citrate drug group had the highest concentration in kidney tissue, and there was a very significant difference (P<0.01) compared with the model group and the normal group; the other groups had no significant difference.
[0190] The CRE levels in serum from high to low were model group>medium dose treatment group>potassium sodium hydrogen citrate drug group>low dose treatment group>high dose treatment group>normal group. Compared with the normal group, the middle-dose treatment group and the high-dose treatment group showed significant differences (P<0.05), and there was no significant difference in the other groups.
[0191] The BUN levels in serum from high to low were model group>low dose treatment group>potassium sodium hydrogen citrate drug group>medium dose treatment group>high dose treatment group>normal group. Compared with normal group, each groups all had very significant differences (P<0.01);
[0192] Compared with the model group, except that the low-dose treatment group had a significant difference (P<0.05), all other groups had very significant differences (P<0.01). [0193] 3.3. Observation results of lesions under HE microscope
[0194] The results consisted of three parts: calcium oxalate crystal aggregation, renal tubule dilation lesions, and chronic renal interstitial inflammatory cell infiltration.
[0195] The characteristics of the animal model were as follows:
[0196] After four weeks, the BUN content in serum of the model animals increased significantly. There was no significant change in blood P, CA content, but the 24-hour urinary OX and CA excretion and renal tissue CA content all increased significantly. It can be observed by naked eyes that the kidneys were enlarged, and the cross-section was pale. The renal cross-section had an obvious friction feeling of fine sand when touched by hand, and the boundary between the renal cortex and renal medulla was unclear. Compared with the normal group, the model group clearly showed: calcium oxalate crystal aggregation, renal tubular epithelial cell swelling, degeneration, necrosis, dilation of the lumen, and chronic renal interstitial inflammatory cell infiltration.
[0197] As shown in
[0198] The Evaluation table for Kidney Lesions was as follows:
TABLE-US-00006 TABLE 3.3.1 Evaluation table for Kidney Lesions Renal tubule Renal interstitial Calcium oxalate dilation inflammatory cell crystal Group lesions infiltration aggregation Normal group ? ? ? Model group +++ ++ +++ Potassium sodium ++ + ++ hydrogen citrate group Low dose group ++ ? ++ Middle dose group + ? + High dose group + ? + Note: No lesion was represented by ?, mild lesion was +, lesion was ++, significant lesion was +++.
[0199] Based on the above-mentioned animal pharmacological test results, it is fully proved that the active ingredient extracts of Polygala japonica Houtt. in the middle dose group and high dose group in the present invention (the compound after the water extract is eluted with 0-25% ethanol by the polyamide resin column) are all significantly better than those of potassium sodium hydrogen citrate of the same quality from three key indicators for the treatment of nephrolithiasis (calcium oxalate crystal aggregation, renal tubule dilation lesions, and chronic renal interstitial inflammatory cell infiltration). Their drug efficacy level has reached the requirements of Chinese drug registration evaluation, which shows that they have excellent potential and market prospects in the treatment of urolithiasis and urinary tract infections or kidney damage caused by urolithiasis and as an adjuvant drug after surgical treatment of urolithiasis.
Beneficial Effect
[0200] The drug of the present invention has significantly better effects than potassium sodium hydrogen citrate in typical test indicators such as calcium oxalate crystal aggregation, renal interstitial inflammatory cell infiltration, and renal tubule dilation lesions, and their drug efficacy level has reached the requirements of Chinese drug registration evaluation, which shows that they have excellent potential and market prospects in the treatment of urolithiasis and urinary tract infections or kidney damage caused by urolithiasis and as an adjuvant drug after surgical treatment of urolithiasis.
[0201] Compared with the most widely used clinical drug potassium sodium hydrogen citrate, since the active ingredients (I)?(III) contained in the extract of Polygala japonica Houtt. of the present invention do not contain sodium and potassium ions, they will not lead to serious side effects such as severe hyperkalemia, arrhythmia, and hypertension similar with potassium sodium hydrogen citrate, and have better safety.
[0202] Compared with other Chinese herbal medicines and drug extracts used for treating diseases related to urolithiasis, the pharmaceutical active ingredient extract of the present invention has simpler components, clearer structure of active ingredients, and more stable and controllable quality.
[0203] In addition, the pharmaceutically active ingredient extract of the present invention is preferably extracted from the aboveground part stems and leaves of the Polygala japonica Houtt., so as to avoid the problem of excessively long growth cycle of pharmaceutical plants caused by the extraction of the whole herb, with lower cost and better environmental protection.
[0204] In summary, the drug of the present invention has clear curative effect on urolithiasis and other related diseases, less side effects (equivalent to or better than the existing mainstream drug for urolithiasis, potassium sodium hydrogen citrate), low cost, simple process, safe and effective, stable and controllable quality, meets modern drug registration requirements, and has excellent medical value and economic value.