A drug for treating diseases related to urinary system lithiasis and its preparation method

Abstract

The present invention provides a medicinally active extract obtained from the plant Polyspora axillaris ( Polygala japonica Houtt. ), which contains flavonol compounds of the following formula (I) as the first active ingredient, and optionally contains xanthone compounds of the following formula (II) as the second active ingredient and glycolipid compounds of the following formula (III-1) or (III-2) as the third active ingredient. Animal experiments have confirmed that the drug of the present invention is significantly superior to potassium sodium hydrogen citrate in typical test indexes such as calcium oxalate crystal aggregation, renal interstitial inflammatory cell infiltration and renal tubular dilation lesions, indicating that it has extremely good potential and market prospects in the treatment of urinary system lithiasis and urinary tract infections or kidney injuries caused by urinary system lithiasis, and as an adjuvant drug after surgical treatment of urinary system lithiasis. (I) (II) (III).

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry. Specifically, the present invention provides a drug for treating urinary calculus. The drug is an extract of Polygala japonica Houtt. containing flavonol compounds with a specific structure, xanthone, and glycolipid as the main active ingredients. Among them, the extract is preferably an extract of the stems and leaves of Polygala japonica Houtt. The drug of the present invention has significantly better effects than the existing mainstream clinical drug potassium sodium hydrogen citrate in treating urinary calculus, urinary tract infection or kidney injury caused by urinary calculus, and as an adjuvant drug after surgical treatment of urinary calculus, with fewer side effects, and has good medical and economic value. Background Art

[0002] Urinary calculus (referred to as urolithiasis) is one of the earliest diseases discovered by humans. As early as 4800 BC, more than 6800 years ago, urinary stones were found in the El Amrah tomb in Egypt. More than 2000 years ago, Hippocrates noticed kidney abscess caused by kidney stones and also described gout. In ancient Chinese medical books more than two thousand years ago, there were also records of stone strangury and sand strangury. Urolithiasis is not only a human disease but also can be seen in animals.

[0003] Urolithiasis is a general term for diseases caused by urinary calculus, and its stones are formed by the precipitation and aggregation of crystals in urine. The pathogenesis of urinary stones is related to factors that cause supersaturation of salts promoting stone formation in urine (such as excessive excretion of salts, urine acidity, and reduced urine volume), pre-formed nuclei (such as uric acid crystals and other stones), and abnormalities in crystal formation inhibitors. Idiopathic hypercalciuria [urinary calcium > 300 mg / d (> 7.5 mmol / d) in men and > 250 mg / d (6.2 mmol / d) in women] is a genetic disease, seen in 50% of men with calcium stones and 75% of women with calcium stones. Hypocitraturia [urinary citrate < 350 mg / d (< 1820 μmol / d)] alone or in combination with other diseases can promote stone formation because normally, citrate can combine with urinary calcium to form soluble calcium citrate salts.

[0004] For a long time, people have been exploring effective treatments for urolithiasis, including extracorporeal shock wave lithotripsy, ureteroscopy lithotripsy, minimally invasive lithotripsy (stone diameter ≤ 2 cm), open surgical treatment (stone diameter ≥ 2 cm), and drug treatment (stone diameter < 0.6 cm). Among them, extracorporeal shock wave lithotripsy breaks up stones at the narrow openings of the upper, middle, and lower ureters, which will inevitably cause ureteral damage. Impacting stones in the renal pelvis may also cause kidney damage, with clinically obvious hematuria. Ureteroscopic lithotripsy and stone removal can also cause damage to the ureter and kidney, and may result in incomplete stone removal. Minimally invasive lithotripsy and stone removal can cause kidney damage and possible incomplete stone removal. With the development of clinical medicine, open surgery is rarely used to treat kidney stones.

[0005] In clinical practice, Western medicine often uses potassium citrate sodium granules, potassium citrate, thiazide diuretics, magnesium agents, acetylcysteine ​​and other excretion-promoting agents to treat urinary tract stones. The efficacy is not ideal and the toxic and side effects are obvious. Potassium citrate sodium granules (trade name: Youlait) are the first citrate preparations that successfully dissolve and prevent uric acid stones developed by the German Ma Dr. Pharmaceutical Factory (MADAUS AG) in 1965. In 2005, it was recommended by the Urolithology Group of the Urology Society of the Chinese Medical Association as the only legal citrate preparation with independent chemical structure and stone-dissolving effect in China. However, potassium citrate sodium granules need to be taken in a very high effective dose, with a daily dose of 4 bags (2.5 grams per bag, a total of 10 grams of granules), taken three times after meals. Take one bag each in the morning and noon, and two bags in the evening. The granules can be taken with water. One gram of potassium sodium citrate contains 0.172 grams or 4.4 mmol of potassium and 0.1 grams or 4.4 mmol of sodium (equivalent to 0.26 grams of sodium chloride). Taking such a large amount of sodium and potassium ions every day can cause serious diseases such as hyperkalemia, arrhythmia, and hypertension, which severely limits the scope of use of this drug.

[0006] In China, a large number of Chinese herbal medicines and proprietary Chinese medicines treatment regimens have also been explored in clinical and medical research. For example, Chinese patent applications CN103285355A, CN103704591A, CN104083644A, CN105213919A, CN105998861A, CN1653929A, etc. record a series of multi-formula Chinese herbal medicines for the treatment of urinary lithiasis. However, multi-formula Chinese herbal medicines generally have problems such as complex components, primitive pharmaceutical processes, difficult quality control, inaccurate quantitative detection methods, large dosages, imperfect quality control standards, and do not meet modern clinical pharmaceutical standards and medication requirements. In addition, some traditional proprietary Chinese medicines such as Mishitong, Paishi Chongji, Shilintong Tablets, and Relinqing Granules are also common proprietary Chinese medicines used to treat urinary system stones. However, these traditional proprietary Chinese medicines have problems such as complex components, primitive pharmaceutical processes, difficult quality control, inaccurate quantitative detection methods, large dosages, and imperfect quality control standards. And they do not meet modern clinical medication requirements.

[0007] Therefore, it is still an urgent pursuit in the medical field to develop drugs for urinary lithiasis that are low in cost, simple in process, safe and effective, stable and controllable in quality, have definite curative effects, have small side effects (equivalent to or better than the mainstream drug potassium sodium hydrogen citrate for urinary lithiasis), are better absorbed in the body, and meet modern drug registration requirements.

[0008] Polygala japonica Houtt. is a plant of the Polygalaceae family widely distributed in China. It has been widely used medicinally in the Chinese folk, mainly for expectorant cough, dissipating stasis and stopping bleeding, calming the mind and tranquilizing the nerves, detoxifying and detumescence, etc.

[0009] The functions and indications of the traditional Chinese medicine Polygala japonica Houtt. included in the Chinese Pharmacopoeia (Volume I, 2015 Edition) are expectorant cough, promoting blood circulation and detumescence, detoxifying and relieving pain. It is used for cough with profuse phlegm, sore throat; externally for traumatic injury, furuncle and carbuncle, snake and insect bites.

[0010] The prepared proprietary Chinese medicine of Polygala japonica Houtt. included in the Chinese Pharmacopoeia (Volume I, 2015 Edition) is Compound Polygala japonica Houtt. Granules. The prescription is: Polygala japonica Houtt. 150g, Isatis indigotica Fort. 350g, Chrysanthemum indicum L. 200g, Lygodium japonicum (Thunb.) Sw. 250g, Hedyotis diffusa Willd. 250g, Viola philippica Cav. 200g. The functions and indications are clearing heat and relieving sore throat, dissipating stagnation and relieving pain, expectorant cough. It is used for pharyngeal swelling, sore throat, fever, cough caused by wind-heat attacking the lung or phlegm-heat congesting the lung; acute pharyngitis, acute attack of chronic pharyngitis and upper respiratory tract infection with the above syndromes.

[0011] In addition, a large number of patent documents record the extraction of various active ingredients from Polygala japonica Houtt. for medical use. For example, Patent CN1303097C records the saponin compounds of Polygala japonica Houtt., their aglycones, total saponins and total sapogenins, and their effects in treating depression, improving intelligence, sedation, anti-anxiety and hypnosis. Patent CN104004110B records the application of a polysaccharide extracted from Polygala japonica Houtt. in the preparation of drugs and health foods for enhancing the immune function of the body. Patent CN108159126A records the application of a saponin extract of Polygala japonica Houtt. in the preparation of anti-tumor drugs. Patent CN103006793B records the separation and purification process of the anti-inflammatory effective part of Polygala japonica Houtt., and discloses that the total flavonoids and total saponin extracts of Polygala japonica Houtt. are the anti-inflammatory effective parts. Patent CN108948125A, a method for preparing sapogenin of Polygala japonica Houtt., mentions the method for preparing sapogenin of Polygala japonica Houtt. and the method for flavonoids of Polygala japonica Houtt., among which four specific flavonoid molecules are mentioned: the leaves contain kaempferol-3-O-6”-O-(3-hydroxy-3-methyl-glutaryl) glucoside, astragalin, kaempferol 3-(6-acetyl) glucoside, kaempferol 3,7-diglucoside. However, the medicinal activities of the extracted components have not been confirmed. Moreover, the existing public documents do not disclose the use of specific extracts of Polygala japonica Houtt. for the treatment of urinary system lithiasis.

[0012] In addition, the above patents all use the whole herb of Polygala japonica Houtt. and regard saponin compounds as indispensable active ingredients. The quality control detection standard of the compounds of Polygala japonica Houtt. recorded in the Chinese Pharmacopoeia (2015 Edition, Volume I) is: calculated by dry product, the content of polygalasaponin hexose (C53H86O23) shall not be less than 0.60%. The chemical components of Polygala japonica Houtt. include saponins, flavonoids, glycolipids, alkaloids, phenols, tannins, polysaccharides, etc., and the components are very complex. According to the literature search of CNKI, there are more than 100 compounds with determined molecular structures. Therefore, it is very difficult to develop the whole herb of Polygala japonica Houtt. into a natural drug that meets modern medical standards. In fact, at present, only compound preparations of Polygala japonica Houtt. are sold on the market, and even Polygala japonica Houtt. used as a single Chinese herbal medicine is not on the market. Summary of the Invention

[0013] Therefore, the main object of the present invention is to provide a drug for urinary system lithiasis that is low-cost, simple in process, safe and effective, stable and controllable in quality, definite in curative effect, small in side effects (equivalent to or better than the mainstream drug potassium sodium hydrogen citrate for urinary system lithiasis), better absorbed in the body, and meets the requirements of modern drug registration.

[0014] The inventors of the present invention have found through a large number of experimental studies that a medicinal active extract of flavonol compounds with a specific structure of formula (I) is obtained by extracting from the plant Polygala japonica Houtt.

[0015] Specifically, the present invention provides a medicinal active extract obtained from the plant Polygala japonica Houtt., which contains flavonol compounds of the following formula (I) as the first active ingredient.

[0016]

[0017] Wherein,

[0018] R 1 is selected from -OH, -O-Glc, -O-Gal, -O-Api, -ORha, -O-Glc-Glc, -O-Glc-Gal, -O-Glc-Api, -O-Glc-Rha, -O-Gal-Glc, -O-Gal-Gal, -O-Gal-Api, -O-Gal-Rha, -O-Glc-Glc-Api, -O-Gal-Glc-Api, -O-Glc-Gal-Api, -O-Gal-Gal-Api, -O-Gal-Rha-Gal, -O-Gal-Rha-Glc, -O-Glc-Rha-Glc, -O-Glc-Rha-Gal;

[0019] R 2 is a substituent selected from -OH, -O-Me, -O-Glc, -O-Gal, -O-Api, -O-Rha;

[0020] R 3 is a substituent selected from H, OH, -O-Me, -O-Glc, -O-Gal, -O-Api, -O-Rha;

[0021] R 4 is a substituent selected from OH, -O-Me;

[0022] The medicinal active extract optionally contains: xanthone compounds of the following formula (II) as the second active ingredient, and glycolipid compounds of the following formula (III) as the third active ingredient

[0023]

[0024] Wherein, R 5is a substituent selected from -O-Gal, -O-Api, -ORha, -O-Glc-Glc, -O-Glc-Gal, -O-Glc-Api, -O-Glc-Rha, -O-Gal-Glc, -O-Gal-Gal, -O-Gal-Api, -O-Gal-Rha, -O-Api-Glc, -O-Api-Gal, -O-Api-Api, -O-Api-Rha; R 6 is a substituent selected from -OH, -O-Me;

[0025]

[0026] wherein R 7 and R 8 are each independently selected from H, CH 3 ;

[0027] wherein, in the above formulas (I) and (II), R 1 to R 6 as defined, Glc represents glucosyl, Gal represents galactosyl, Api represents apiose, and Rha represents rhamnose.

[0028] In a preferred technical solution, the flavonol compound of the formula (I) structure as the first active ingredient is preferably selected from one or more of the following general formula compounds

[0029]

[0030] wherein, R1 is selected from -OH, -O-Glc, -O-Gal, -O-Glc-Glc, -O-Glc-Gal, -O-Glc-Rha, -O-Gal-Glc, -O-Gal-Gal, -O-Gal-Rha, -O-Glc-Glc-Api, -O-Gal-Glc-Api, -O-Glc-Gal-Api, -O-Gal-Gal-Api

[0031]

[0032] Among them, R1 is selected from -OH, -O-Glc, -O-Gal, -O-Glc-Glc, -O-Glc-Gal, -O-Glc-Api, -O-Glc-Rha, -O-Gal-Glc, -O-Gal-Gal, -O-Gal-Api, -O-Gal-Rha, -O-Glc-Glc-Api, -O-Gal-Glc-Api, -O-Glc-Gal-Api, -O-Gal-Gal-Api, -O-Gal-Rha-Gal, -O-Gal-Rha-Glc, -O-Glc-Rha-Glc, -O-Glc-Rha-Gal

[0033]

[0034] Among them, R1 is selected from -OH, -O-Glc, -O-Gal, -O-Glc-Api, -O-Gal-Api

[0035]

[0036] Among them, R is selected from -OH, -O-Glc, -O-Gal, -O-Glc-Rha, -O-Gal-Rha

[0037]

[0038] Among them, R is selected from -OH, -O-Glc, -O-Gal, -O-Glc-Rha, -O-Gal-Rha

[0039]

[0040] Among them, R is selected from -OH, -O-Gal, -O-Gal-Api;

[0041] The xanthone of formula (II) structure as the second active ingredient is preferably selected from one or more of the following formulas (II-1), (III-2), and (II-3)

[0042]

[0043] The glycolipid compound as the third active ingredient is preferably a compound of the following formula (III-1) or (III-2):

[0044]

[0045]

[0046] In a further preferred technical solution, the first active ingredient in the medicinal active extract of Polygala japonica Houtt. is selected from the flavonol compounds of the above general formulas F-7K, F-7Q, F-74Q, and F-74K.

[0047] In a further preferred technical solution, the first active ingredient in the medicinal active extract of Polygala japonica Houtt. is preferably selected from at least one of the following compounds:

[0048]

[0049] In a further preferred technical solution, in the medicinal active extract of Polygala japonica Houtt., the total content of the flavonol compound of formula (I) as the first active ingredient, and optionally the xanthone of formula (II) and the glycolipid of formula (III) as the second active ingredient, accounts for 30-100% of the total extract of Polygala japonica Houtt. Among them, the component content (%) is the HPLC% content measured by the HPLC integration area normalization method using the industry-standard method.

[0050] In a further preferred technical solution, in the medicinal active extract of Polygala japonica Houtt., 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.

[0051] In a further preferred technical solution, in the medicinal active extract of Polygala japonica Houtt., 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.

[0052] The present invention also provides a preparation method of the medicinal active ingredient extract of Polygala japonica Houtt., including the following steps

[0053] (1) Pretreatment of Polygala japonica Houtt.

[0054] Take the whole herb of Polygala japonica Houtt., or the aerial part of Polygala japonica Houtt., or commercially available Polygala japonica Houtt. medicinal materials, wash, crush, and obtain Polygala japonica Houtt. raw materials;

[0055] (2) Coarse extraction of the effective part of Polygala japonica Houtt.

[0056] Take a certain amount of the Polygala japonica Houtt. raw materials obtained in step (1), heat and reflux with ethanol at a concentration of 20-95% (v / v) about 6-12 times the weight of the Polygala japonica Houtt. raw materials, reflux for 1-3 hours each time, reflux and extract 1-3 times repeatedly, combine the obtained alcohol extracts, filter or centrifuge, and concentrate to obtain the total alcohol extract of Polygala japonica Houtt.;

[0057] Or,

[0058] Take a certain amount of the raw material of Polygala japonica Houtt. obtained in step (1), heat it to boiling with deionized water about 6 - 15 times the weight of the raw material of Polygala japonica Houtt. and keep boiling for 1 - 3 hours, extract repeatedly for 1 - 3 times, combine the obtained water extracts, filter or centrifuge, and concentrate to obtain the total water extract of Polygala japonica Houtt.

[0059] (3) Refinement of the effective part of Polygala japonica Houtt.

[0060] Separate the concentrated solution of the total water extract or total alcohol extract of Polygala japonica Houtt. in step (2) with a macroporous adsorption resin or polyamide resin chromatographic column, elute successively with water / ethanol gradients of different ratios until the eluate is colorless, collect the eluate with a 0 - 95% ethanol gradient, and dry it under reduced pressure to obtain the required medicinal active extract of Polygala japonica Houtt.

[0061] In the above extraction method, preferably, the macroporous resin in step (3) is selected from D101 type, HPD100 type, HPD200 type or AB - 8 type macroporous resin, and the polyamide resin is selected from 100 - 200 mesh polyamide resin from Shanghai Reagent.

[0062] In the above extraction method, preferably, the Polygala japonica Houtt. in step (1) is preferably the stem and leaf part of Polygala japonica Houtt.

[0063] In the above extraction method, preferably, the macroporous resin in step (3) is selected from D101 type or AB - 8 type macroporous resin, and the polyamide resin is selected from 100 - 200 mesh polyamide resin from Shanghai Reagent.

[0064] In the above extraction method, preferably, the gradient elution in step (3) is successively carried out with water, 25% ethanol, 50% ethanol, 75% ethanol, and 95% ethanol until the eluate is colorless.

[0065] The present invention provides the use of the extract of the medicinal active ingredients of Polygala japonica Houtt. in the preparation of a drug, and the drug is used for the treatment or prevention of urinary system lithiasis, and urinary tract infection or kidney injury caused by urinary system lithiasis, and as an adjuvant drug after surgical treatment of urinary system lithiasis.

[0066] The present invention also provides a pharmaceutical composition, which contains at least one selected from the following compounds as an active ingredient:

[0067]

[0068] In a preferred technical solution, the pharmaceutical composition further contains a pharmaceutically acceptable carrier, excipient or auxiliary material.

[0069] The present invention further improves the use of the above-mentioned pharmaceutical composition in the preparation of a drug, and the drug is used for treating or preventing urinary system lithiasis, urinary tract infection or kidney injury caused by urinary system lithiasis, and as an adjuvant drug after surgical treatment of urinary system lithiasis.

[0070] The present invention also provides the use of any one of the following compounds in the preparation of a drug,

[0071]

[0072] and the drug is used for treating or preventing urinary system lithiasis, urinary tract infection or kidney injury caused by urinary system lithiasis, and as an adjuvant drug after surgical treatment of urinary system lithiasis.

[0073] The inventors of the present invention found through a large number of experimental studies that the active extract of the present invention has significantly better effects than the traditional Chinese medicines and plant extracts recorded in the currently known published literatures in treating urinary system lithiasis, urinary tract infection or kidney injury caused by urinary system lithiasis, and as an adjuvant drug after surgical treatment of urinary system lithiasis, and has equivalent or better effects than the known clinical western medicine potassium sodium hydrogen citrate (see the pharmacological examples described later for details). Preliminary studies have shown that the key lies in that the compounds of formula (I) and formula (II) selected as the main active ingredients in the present invention have a hydroxyl substituent at the β-position of the carbonyl group of the flavonoid compound and xanthone. This hydroxyl group acts together with the ketone carbonyl group to more effectively react with the calcium ion-containing stone components in the urinary system, thereby more effectively degrading or dissolving the stones in the urinary system.

[0074] Therefore, in a specific technical solution of the present invention, there is provided the use of any one of the aforementioned compounds of formula (I), formula (II), or a combination of two or more thereof in the preparation of a drug, and the drug is used for treating or preventing urinary system lithiasis, urinary tract infection or kidney injury caused by urinary system lithiasis, and as an adjuvant drug after surgical treatment of urinary system lithiasis.

[0075] In a preferred technical solution of the present invention, there is provided the use of any one of the aforementioned compounds of formula (I-1) to (I-4) and / or formula (II-1) to (II-3), or a combination of two or more thereof in the preparation of a drug, and the drug is used for treating or preventing urinary system lithiasis, urinary tract infection or kidney injury caused by urinary system lithiasis, and as an adjuvant drug after surgical treatment of urinary system lithiasis.

[0076] The active extract of Polygala japonica Houtt. of the present invention can also be prepared into various dosage forms by conventional pharmaceutical methods, such as gastrointestinal dosage forms like capsules, tablets, pills, oral liquids, granules, tinctures, sustained-release agents, etc., and parenteral dosage forms like injections and topical preparations.

[0077] Description of the Drawings

[0078] Figure 1 It is the HPLC analysis chromatogram of the ethanol extract of the whole herb of Polygala japonica Houtt. of the present invention.

[0079] Figure 2 It is the HPLC analysis chromatogram of the ethanol extract of the aerial part of Polygala japonica Houtt. of the present invention.

[0080] Figure 3 It is the HPLC analysis chromatogram of the effective part obtained after gradient elution of the aerial part of Polygala japonica Houtt. of the present invention with polyamide resin.

[0081] Figure 4 It is the C-H correlation two-dimensional nuclear magnetic resonance spectrum of compound F-7Q-1 of the present invention.

[0082] Figure 5 It is the C-H long-range two-dimensional nuclear magnetic resonance correlation spectrum of compound F-7Q-1 of the present invention.

[0083] Figure 6 It is the C-H correlation two-dimensional nuclear magnetic resonance spectrum of compound F-7K-1 of the present invention.

[0084] Figure 7 It is the C-H long-range correlation two-dimensional nuclear magnetic resonance spectrum of compound F-7K-1 of the present invention.

[0085] Figure 8 It is the C-H correlation two-dimensional nuclear magnetic resonance spectrum of compound F-74Q-1 of the present invention.

[0086] Figure 9 It is the C-H long-range correlation two-dimensional nuclear magnetic resonance spectrum of compound F-74Q-1 of the present invention.

[0087] Figure 10 It is the observation result under HE microscopy of the animal experiment on renal tubular dilation lesion of the drug of the present invention (normal group).

[0088] Figure 11 It is the observation result under HE microscopy of the animal experiment on renal tubular dilation lesion of the drug of the present invention (model group).

[0089] Figure 12 It is the observation result under HE microscopy of the animal experiment on renal tubular dilation lesion of the drug of the present invention (potassium sodium hydrogen citrate drug group).

[0090] Figure 13Observation results of the animal experiment on renal tubular dilation lesions of the drug of the present invention under HE microscopy (low-dose group)

[0091] Figure 14 Observation results of the animal experiment on renal tubular dilation lesions of the drug of the present invention under HE microscopy (medium-dose group)

[0092] Figure 15 Observation results of the animal experiment on renal tubular dilation lesions of the drug of the present invention under HE microscopy (high-dose group) Example

[0093] The embodiments of the present invention will be described in detail below. The examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention and should not be construed as a limitation of the present invention.

[0094] I. Preparation examples

[0095] Preparation example 1: Analysis of the ethanol extract components of the whole herb of Polygala japonica

[0096] Weigh the whole herb of Polygala japonica, add 10 times the amount of the medicinal material of 75% ethanol, heat under reflux, extract 3 times, 3 hours each time, filter while it is hot, and then combine the ethanol extracts;

[0097] Concentrate the ethanol extract to an extraction concentrate with a relative density of 1.1 - 1.3 g / mL.

[0098] Perform fingerprint analysis on the obtained extraction concentrate of the ethanol extract of the whole herb of Polygala japonica using HPLC.

[0099] HPLC test conditions Mobile phase: acetonitrile (A), 0.1% formic acid aqueous solution (B), binary gradient separation;

[0100] Flow rate: 0.8 mL·min-1;

[0101] Detection wavelength: 330 nm;

[0102] Column temperature: 20 °C;

[0103] Injection volume: 20 μL.

[0104] Record the chromatogram for 90 min.

[0105] Preparation example 2: Analysis of the water extract components of the whole herb of Polygala japonica

[0106] Weigh the whole herb of Polygala japonica, add 10 times the amount of the medicinal material of deionized water, heat under reflux, extract 2 times, 3 hours each time, filter while it is hot, and then combine the water extracts. The extract is a dark brownish-yellow liquid.

[0107] Concentrate the water extract to an extraction concentrate with a relative density of 1.1 - 1.3 g / mL.

[0108] The obtained water-extracted concentrated solution of the whole herb of Glehnia littoralis was analyzed by HPLC for fingerprint analysis (see the attached figure of the specification for details). Figure 1 ), it can be basically determined that the extract mainly contains dozens of compounds of four major components and dozens of compounds of other types. In the HPLC spectrum, the peak time of xanthone is between 12-25 minutes, the peak time of flavonol compounds is between 18-68 minutes, the peak time of glycolipids is between 15-45 minutes, and the peak time of saponin is between 42-85 minutes.

[0109] Comparing the components obtained by water extraction and alcohol extraction, it can be found that due to the high polarity of water, the content of impurities with low polarity such as chlorophyll in the extract obtained by water extraction will be relatively small, while the content of high polarity tannin components will be relatively large. The extract is dark brown. The content of low polar components (such as chlorophyll, etc.) in the alcohol extract is relatively high, while the content of high polar components (such as tannin, etc.) is significantly reduced. The alcohol extract appears greenish, but turns brown after cooling and standing.

[0110] The whole plant extraction consumes a lot of medicinal plants. Considering that most of the commercially available melon seeds gold medicinal materials are above-ground parts, it is also beneficial to protect medicinal plant resources to select only the above-ground parts and retain the plant roots. Therefore, the inventor further tried to use alcohol extraction to extract the active ingredients from the above-ground parts of melon seeds gold. The specific method is as follows.

[0111] Preparation Example 3: Analysis of water extract components of the aerial part of Glehnia littoralis

[0112] Weigh the aerial part of the Glehnia littoralis, add 10 times the amount of deionized water as the medicinal material, heat and reflux, extract 3 times, 3 hours each time, filter while hot, and then combine the water extracts;

[0113] The water extract is concentrated to an extract concentrate having a relative density of 1.1-1.3 g / ml.

[0114] The fingerprint spectrum of the deionized water extract concentrate of the aerial part of Glehnia littoralis was analyzed by HPLC.

[0115] Preparation Example 4: Analysis of alcohol-extracted components of the aerial part of Glehnia littoralis

[0116] Weigh the aerial part of the golden osmanthus plant, add 50% ethanol 10 times the amount of the medicinal material, heat and reflux, extract 3 times, 3 hours each time, filter while hot, and then combine the alcohol extracts;

[0117] The alcohol extract is concentrated to an extract concentrate having a relative density of 1.1-1.3 g / ml.

[0118] Fingerprint analysis was performed on the concentrated ethanol extract of the aerial parts of *Polygala japonica* Houtt. using HPLC.

[0119] (The results are shown in the appendix Figure 2 ).

[0120] To confirm the chemical composition of this concentrated extract, we performed HPLC-MS analysis, where the MS analysis included positive and negative ions as well as MS-MS and MS-MS-MS analyses. The analysis results were compared with existing literature, and the structural types of four major types of compounds, namely flavonols, xanthones, glycolipids, and saponins, were preliminarily confirmed. Further, we subdivided the flavonols according to the differences in the flavonol compound nuclei.

[0121] However, due to the complexity and diversity of the spatial structure and connection mode of sugars in glycosides. Through HPLC and tandem MS, we can determine whether the multiple glycosides are connected to the flavonol compound nucleus by a single sugar chain, and the order of glycoside fragmentation in the mass spectrum to confirm the molecular weight of the sugar at the end of the sugar chain and the basic type of glycone. However, due to the complexity and diversity of the spatial structure and connection mode of sugars in glycosides. For the configuration of the hydroxyl group on a certain carbon atom of the sugar ring of isomers (such as glucose or galactose), the position of the sugar chain connection between glycosides (such as 1-2 connection, 1-4 connection, or 1-6 connection), and the α or β configuration of the sugar, other means are needed for further identification. Therefore, there is a possibility of multiple structural combinations of flavonol aglycones and different types of glycosides for compounds with the same molecular formula identified only by HPLC-MS-MS.

[0122] For the sake of easy distinction, this application uses compound categories to identify and distinguish compounds with possible multiple structural unit combinations. For example, in this application, F is used to represent the major category of flavonol compounds, F-Q represents a class of flavonol glycosides with quercetin as the nucleus of the flavonol compound major category. In the structure determination, 302-162-132 represents that the flavonol aglycone nucleus is quercetin connected to a glucose (or galactose), and on this glucose (or galactose) is connected a apiose. 302 is the quercetin nucleus, 162 is the molecular weight of the characteristic peak of the fragmentation of glucose (or galactose) in the mass spectrum, and 132 is the molecular weight of the characteristic peak of the fragmentation of apiose.

[0123] Through HPLC and tandem MS analysis, it was preliminarily confirmed that the ethanol extract of the aerial parts of *Polygala japonica* Houtt. mainly contains the following components:

[0124] Table 1: Component analysis of the ethanol extract of the aerial parts of *Polygala japonica* Houtt.

[0125]

[0126]

[0127] In the above table, the chemical component structures mainly include the following categories:

[0128] (1) Flavonols with glycosides

[0129] 1. The compound category is F-7K flavonol glycoside compounds. Among them, 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. The compound has the following general formula structure:

[0130]

[0131] Among them, R1 = glycosyl, and can be selected from -OH, -O-Glc, -O-Gal, -O-Glc-Glc, -O-Glc-Gal, -O-Glc-Rha, -O-Gal-Glc, -O-Gal-Gal, -O-Gal-Rha, -O-Glc-Glc-Api, -O-Gal-Glc-Api, -O-Glc-Gal-Api, -O-Gal-Gal-Api,

[0132] 2. The compound category is F-7Q flavonol glycoside compounds. Among them, the aglycone structure of the compound is Rhamnetin with a molecular weight of 316, or 3,3',4',5-Tetrahydroxy-7-methoxyflavone, or 7-methoxyl-quercetin. The compound has the following general formula structure:

[0133]

[0134] Among them, R1 = glycosyl, and can be selected from -OH, -O-Glc, -O-Gal, -O-Glc-Glc, -O-Glc-Gal, -O-Glc-Api, -O-Glc-Rha, -O-Gal-Glc, -O-Gal-Gal, -O-Gal-Api, -O-Gal-Rha, -O-Glc-Glc-Api, -O-Gal-Glc-Api, -O-Glc-Gal-Api, -O-Gal-Gal-Api, -O-Gal-Rha-Gal, -O-Gal-Rha-Glc, -O-Glc-Rha-Glc, -O-Glc-Rha-Gal;

[0135] 3. Flavonol glycoside compounds with the compound class of F-74Q. Among them, the aglycone 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. The compound has the following general formula structure:

[0136]

[0137] Among them, R1 is a glycosyl group, and can be selected from -OH, -O-Glc, -O-Gal, -O-Glc-Api, -O-Gal-Api,

[0138] 4. Flavonol glycoside compounds with the compound class of F-K. Among them, the aglycone structure of the compound is kaempferol with a molecular weight of 286 or 3,4',5,7-Tetrahydroxyflavone. The compound has the following general formula structure:

[0139]

[0140] Among them, R can be selected from -OH, -O-Glc, -O-Gal, -O-Glc-Rha, -O-Gal-Rha,

[0141] 5. Flavonol glycoside compounds with the compound class of F-Q. Among them, the aglycone structure is quercetin with a molecular weight of 302 or 3,3',4',5,7-Pentahydroxyflavone. The compound has the following general formula structure:

[0142]

[0143] Among them, R can be selected from -OH, -O-Glc, -O-Gal, -O-Glc-Rha, -O-Gal-Rha

[0144] 6. Flavonol glycoside compounds with the compound class of F-74K. Among them, the aglycone structure is Ermanin with a molecular weight of 314 or 3,5-dihydroxy 7,4′-dimerhoxyflavone, or 7,4′-dimerhoxyl-kaempferol. The compound has the following general formula structure:

[0145]

[0146] Among them, R = glycosyl group, and can be selected from -OH, -O-Gal, -O-Gal-Api.

[0147] (2) Polygalaxanthone (Xanthone) Compounds

[0148] The polygalaxanthone (xanthone) compounds can be confirmed by secondary mass spectrometry to be selected from the following formulas (II-1), (II-2), and (II-3)

[0149]

[0150]

[0151] (3) Glycolipid Compounds

[0152] The structures of the glycolipid compounds can be confirmed by secondary mass spectrometry to be selected from the following formulas (III-1) and (III-2)

[0153]

[0154] (4) Saponin Compounds

[0155] Through HPLC and multi-stage tandem MS analysis, it can be confirmed that the ethanol extract of the aerial parts of Polygala japonica in this application contains saponin compounds such as Polygalasaponin VIII, Polygalasaponin XXI, Polygalasaponin X, and Polygalasaponin XXIX.

[0156] In addition to the target active ingredients such as flavonols and xanthones, the ethanol extract of the aerial parts of Polygala japonica also contains components such as saponins and glycolipids. Therefore, the inventor further attempts to further refine the ethanol extract of the aerial parts of Polygala japonica through separation methods such as macroporous resin and polyamide resin to separate and enrich the target active ingredients.

[0157] Preparation Example 5: Macroporous Resin Refinement Treatment of Ethanol Extract of Aerial Parts of Polygala japonica

[0158] The extraction and concentration solution obtained in Preparation Example 1 was passed through a D101 macroporous adsorption resin column at a flow rate of 1 column bed volume per hour. After adsorption, first, 8 times the resin volume of water was used to wash and remove impurities, and then gradient elution was carried out with 2 - 5 column bed volumes of 0% - 25%, 25% - 50%, 50% - 75%, and 75% - 95% ethanol respectively. The elution flow rate was carried out at 0.5 - 2 column bed volumes per hour to obtain an eluate; and the eluates of different concentrations of ethanol were respectively concentrated 5 - 20 times to obtain an elution concentrate with a relative density of 1.1 - 1.3 g / ml.

[0159] HPLC was used to perform fingerprint analysis on the components of the ethanol extraction and concentration solution of the whole herb of Polygala japonica obtained and the ethanol gradient elution concentrate of the macroporous adsorption resin column respectively.

[0160] Preparation Example 6: Refinement of the Aqueous Extract from the Aerial Parts of *Polygala japonica* Houtt. with Polyamide Resin

[0161] The extraction and concentration solution obtained in Preparation Example 3 was passed through a polyamide resin column at a flow rate of 0.5 - 1 column bed volume per hour. After adsorption, impurities were removed by washing with 2 - 8 resin volumes of water first, and then gradient elution was carried out with 0 - 25%, 25% - 50%, 50% - 75%, and 75% - 95% ethanol at 2 - 5 column bed volumes respectively. The elution was carried out at a flow rate of 0.5 - 2 column bed volumes per hour to obtain the eluate; the eluates with different ethanol concentrations were concentrated 5 - 20 times respectively to obtain elution concentrated solutions with a relative density of 1.1 - 1.3 g / mL.

[0162] By comparing the separation effects of macroporous resin and polyamide resin, it can be found that polyamide resin can better remove the saponin components in the *Polygala japonica* Houtt. extract, and the separation and refinement effect is better.

[0163] HPLC was used to analyze the fingerprint of the extraction and concentration solution from the aerial parts of *Polygala japonica* Houtt. after ethanol gradient elution through a polyamide resin column.

[0164] Through HPLC analysis, it was determined that the total content of the active ingredients (I), (II), and (III) in the orange-red elution concentrated solution eluted with 0 - 25% ethanol was 50% - 90%. (As shown in the attached Figure 3 instructions).

[0165] This elution concentrated solution was dried under reduced pressure at 75 °C and pulverized to obtain the enriched active ingredients from the aerial parts of *Polygala japonica* Houtt., which were used for the pharmacodynamic comparison experiment.

[0166] The enriched active ingredients from the aerial parts of *Polygala japonica* Houtt. were analyzed by HPLC-MS, and the MS analysis included positive ions, negative ions, MS-MS, and MS-MS-MS analyses. The analysis results were compared with the existing literature to confirm the compound structures in Table 2 below.

[0167] Table 2: Component Analysis of the Aqueous Extract - Polyamide Resin Refined Extract from the Aerial Parts of *Polygala japonica* Houtt.

[0168]

[0169]

[0170] Based on the above analysis, it can be confirmed that the main components of the aqueous extract - polyamide refined extract from the aerial parts of *Polygala japonica* Houtt. include flavonol compounds with compound categories of F-7K, F-7Q, F-74Q, and F-74K, as well as the xanthone compound (polygalaxanthone Ⅲ) of formula (II-1) and glycolipid compounds.

[0171] Among them, the compound with a content ratio of 18.97% in Table 2 is labeled as F-7K-1, the compound with a content ratio of 33.71% is labeled as F-7Q-1, the compound with a content ratio of 23.60% is labeled as F-74Q-1, and the compound with a content ratio of 4.81% is labeled as compound F-74K-1 (polygalitol B).

[0172] Among them, the meaning of the above compound category names is exactly the same as that in Table 1.

[0173] It should be emphasized that due to differences in the specific parts of the Polygala japonica plant being extracted (whole herb, rhizome, or stem and leaves), differences in the origin of the plant, differences in the preparation of the Polygala japonica plant (commercially available dried herbs, fresh Polygala japonica plants), and differences in the specific extraction and purification process conditions, there may be varying degrees of differences in the structure and content of the active ingredients in the obtained active ingredient extracts. The present invention preferably uses an active ingredient extract obtained by water extraction and polyamide column ethanol / water gradient elution of fresh or dried aerial parts of Polygala japonica.

[0174] Preparation Example 7: Separation and Purification of Main Active Compounds

[0175] HPLC-MS-MS can determine the parent nucleus of flavonol compounds and whether it is a single sugar chain based on obvious characteristics when there are 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 whether it is glucose or galactose). When the linkage position of sugars with more than two sugar rings on a sugar chain (such as 1-2 linkage, 1-4 linkage, or 1-6 linkage) and the configuration of the sugar, α or β configuration, the discrimination basis is insufficient. Therefore, the pure compound of the main active ingredient is separated by semi-preparative HPLC, and its specific structure is determined by combining 1H NMR, 13C NMR, and two-dimensional NMR.

[0176] The extract prepared in Example 5 was further separated and purified by semi-preparative HPLC, and pure single compounds of the three components with the highest content in the extract were separately collected, that is, pure single compounds of compounds F-7Q-1, F-7K-1, and F-74Q-1 were obtained.

[0177] The semi-preparative HPLC instrument and conditions are as follows:

[0178] HPLC test conditions Mobile phase: acetonitrile (A), deionized water (B), binary gradient separation;

[0179] Semi-preparative column: 19×250 mm, C18

[0180] Flow rate: 8 mL·min-1;

[0181] Detection wavelength: 330 nm;

[0182] Column temperature: room temperature;

[0183] Sample injection volume: 0.5 mL.

[0184] Record the chromatogram for 180 min.

[0185] Structural analysis of compounds F-7Q-1, F-7K-1, and F-74Q-1 was performed by nuclear magnetic resonance and two-dimensional nuclear magnetic resonance respectively. The analysis results are as follows:

[0186] 1. Structure confirmation of compound F-7Q-1

[0187] Molecular weight: 640, sugar chain: 316 - 162 - 162,

[0188] The key information of two-dimensional nuclear magnetic resonance correlation of compound F-7Q-1 is summarized in the following table:

[0189]

[0190]

[0191] Based on the above spectroscopic analysis data, the exact spatial structure formula of the main active ingredient compound F-7Q-1 is finally confirmed as follows:

[0192]

[0193] The compound name of F-7Q-1 is: Rhamnetin 3-O-β-D-glucopyranosyl(1→2)-β-D-galactopyranoside, or Rhamnetin-3-O-(2″-O-β-D-glucopyranosyl)-β-D-galactopyranoside

[0194] (2) Structure confirmation of compound F-7K-1

[0195] Molecular weight: 624, sugar chain: 300 - 162 - 162,

[0196] The key information of two-dimensional nuclear magnetic resonance correlation of compound F-7K-1 is summarized in the following table:

[0197]

[0198]

[0199] Based on the above spectroscopic analysis data, the exact spatial structure formula of the main active ingredient compound F-7K-1 is finally confirmed as follows:

[0200]

[0201] The chemical name of compound F-7K-1 is: Rhamnocitrin 3-O-β-D-glucopyranosyl(1→2)-β-D-galactopyranoside, or Rhamnocitrin-3-O-(2″-O-β-D-glucopyranosyl)-β-D-galactopyranoside

[0202] 3. Structure confirmation of compound F-74Q-1

[0203] Molecular weight: 624, sugar chain: 330-162-132, identification:

[0204] The key information of two-dimensional nuclear magnetic resonance correlation of compound F-74Q-1 is summarized in the following table:

[0205]

[0206]

[0207] Based on the above spectroscopic analysis data, the exact spatial structure formula of the main active ingredient compound F-74Q-1 is finally confirmed as follows:

[0208]

[0209] The chemical name of compound F-74Q-1: 3,5,3’-trihydroxy-7,4’-dimethoxyflavone-3-O-β-D-apiofranosyl(1→2)-β-D-galactopyranoside, or Polygalin C, or Polygala alcohol C

[0210] 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 components of the Polygala japonica Houtt. extract of the present invention, play a key role in achieving the desired medicinal effects. These compounds have a hydroxyl substituent at the β-position of the carbonyl group in the molecular structure, and the cooperation of this hydroxyl group with the ketone carbonyl group can more effectively react with the calcium ion-containing stone components in the urinary system, thereby more effectively degrading or dissolving the urinary stones. 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 essential active ingredient to prepare the corresponding pharmaceutical composition for treating or preventing urinary lithiasis, urinary tract infections or kidney injuries caused by urinary lithiasis, and as an adjuvant drug after surgical treatment of urinary lithiasis. The said pharmaceutical composition may further contain pharmaceutically acceptable excipients, carriers or excipients.

[0211] II. Pharmacological Activity Experimental Examples

[0212] 1. Preparation of Test Samples

[0213] ① Test sample for high-dose group: Take the active ingredient extract obtained by concentrating and drying the water extract and the 0-25% ethanol gradient eluate of the polyamide column prepared in Preparation Example 5. Prepare a solution with a density of about 1.2 g / ml, and the effective substance concentration measured by the absorbance method (using rutin as the reference substance to make a standard curve) according to the pharmacopoeia standard is about 130.4 mg / ml.

[0214] ② Test sample for medium-dose group: Dilute the high-dose group sample by one-fold to obtain the test sample for the medium-dose group.

[0215] ③ Test sample for low-dose group: Dilute the medium-dose group sample by one-fold to obtain the test sample for the low-dose group.

[0216] ④ Test sample for positive control group: An aqueous solution of potassium sodium hydrogen citrate with a concentration of 100 mg / ml, 3 ml per day, equivalent to 300 mg / d.

[0217] 2. Animal Experiments

[0218] 2.1 Experimental Animals and Breeding Conditions

[0219] 36 SD rats were purchased from Shanghai Slake, license number: SCXK (Shanghai) 2017-0005, certificate number: 20170005011248. The drinking water was ultrapure water. The license number for the experimental animal house was SYXK (Zhejiang) 2015-0008.

[0220] Breeding environment: Temperature range 20-25°C, relative humidity range 40-70%. Adaptively feed for one week before the experiment.

[0221] 2.2. Experimental protocol

[0222] 2.2.1 Experimental animals

[0223] Thirty-six SPF-grade male SD rats, 6 - 8 weeks old, weighing 200 - 250 g.

[0224] 2.2.3 Model preparation

[0225] After 7 days of adaptive feeding of SD rats, except for the normal group, the remaining groups were given modeling drugs by drinking 1% ethylene glycol and gavage with 2% ammonium chloride at 2 ml / rat for 28 consecutive days.

[0226] 2.2.4 Experimental grouping and treatment

[0227] Thirty-six male SD rats were randomly divided into 6 groups of 6 rats each according to body weight. They were divided into a normal group, a model group, a potassium sodium hydrogen citrate group, and low, medium, and high dose groups of traditional Chinese medicine extract. During the modeling process, drugs were continuously administered by gavage at 3 ml per day. The drugs included positive control drugs and the part purified by polyamide column. The animals were euthanized after 4 weeks.

[0228] 2.2.5 Kidneys

[0229] Kidney tissues were dissected in vivo. One side was placed in a cryotube and stored at -80 °C for detecting the Ca2+ concentration in tissue homogenate. The operation steps of the kit were the same as above; the other side was fixed in formalin solution for HE staining of tissue sections.

[0230] (1) The steps for making paraffin sections are as follows:

[0231] ① Fixation, ② Trimming, ③ Dehydration, ④ Clearing, ⑤ Wax infiltration, ⑥ Embedding, ⑦ Sectioning,

[0232] ⑧ Baking the sections, ⑨ Preservation: Packing in boxes and storing at room temperature.

[0233] (2) The steps for HE staining are as follows:

[0234] ① Deparaffinization and rehydration, ② Staining, ③ Dehydration, clearing, and mounting,

[0235] ④ Staining results: The cell nuclei are blue, the cytoplasm is pink, and the red blood cells are brightly red.

[0236] 3. Test results

[0237] 3.1. Positive test results of calcium oxalate crystals in rat urine

[0238] The results of the urine routine report showed that calcium oxalate crystals in the urine of rats in all groups except the normal group were positive.

[0239] 3.2. Test results of serum Ca2+ concentration

[0240] The Ca2+ concentration in serum showed little difference among groups. Compared with the normal group, there were significant differences between the model group and the medium-dose treatment group. There were large differences in the Ca2+ concentration in urine. Compared with the normal group, there were extremely significant differences between the low-dose treatment group and the high-dose treatment group (P<0.01). Compared with the model group, there was an extremely significant difference in the low-dose treatment group (P<0.01). The concentration of potassium sodium hydrogen citrate in the renal tissue was the highest, and there were extremely significant differences compared with both the model group and the normal group (P<0.01). There were no significant differences in the other groups.

[0241] The CRE levels in serum were in the order of model group > medium-dose treatment group > potassium sodium hydrogen citrate group > low-dose treatment group > high-dose treatment group > normal group from high to low. Compared with the normal group, there were significant differences between the medium-dose treatment group and the high-dose treatment group (P<0.05), and there were no significant differences in the other groups.

[0242] The BUN levels in serum were in the order of model group > low-dose treatment group > potassium sodium hydrogen citrate group > medium-dose treatment group > high-dose treatment group > normal group from high to low. Compared with the normal group, there were extremely significant differences in all groups (P<0.01);

[0243] Compared with the model group, except for the low-dose treatment group with a significant difference (P<0.05), the other groups had extremely significant differences (P<0.01).

[0244] 3.3. Observation results of lesions under HE microscopy

[0245] The results included three parts: calcium oxalate crystal aggregation, renal tubular dilation lesions, and chronic inflammatory cell infiltration in the renal interstitium

[0246] The characteristics of this animal model were as follows:

[0247] At four weeks, the serum BUN content of the model animals increased significantly. The blood P and CA contents showed no obvious changes, while the 24-hour urinary OX and CA excretion amounts and the CA content in the renal tissue all increased significantly. Macroscopically, the kidneys of the model animals were enlarged, the cut surface was pale, and there was an obvious fine sand friction feeling when touching the renal section with the hand. The boundary between the renal cortex and the renal medulla was unclear. Compared with the normal group, in the model group, it was obvious that there were calcium oxalate crystal aggregations, swelling, degeneration, necrosis, and lumen dilation of renal tubular epithelial cells, and chronic inflammatory cell infiltration in the renal interstitium.

[0248] As shown in the attached instructions Figures 10 to 15As shown, under HE microscopy, it can be seen that compared with the normal group, the renal tubules in the model group were significantly dilated, and a large amount of brownish-yellow calcium oxalate crystals were observed, with inflammatory cell infiltration in the local renal interstitium; compared with the model group, the dilatation lesions of the renal tubules in the medium- and high-dose groups improved, the brownish-yellow calcium oxalate crystals were significantly reduced, and there was no obvious inflammatory cell infiltration in the renal interstitium; the dilatation lesions of the renal tubules in the low-dose group improved to some extent, there was no obvious inflammatory cell infiltration in the renal interstitium, and the brownish-yellow calcium oxalate crystals were not significantly reduced; in the potassium sodium hydrogen citrate group, the dilatation lesions of the renal tubules improved, and occasional inflammatory cell infiltration in the renal interstitium was seen, and the brownish-yellow calcium oxalate crystals were not significantly reduced.

[0249] The evaluation form of kidney lesions is as follows:

[0250] Table 3: Evaluation form of kidney lesions

[0251]

[0252] Note: No lesion is indicated as -, mild lesion as +, lesion as ++, and significant lesion as +++.

[0253] Based on the above results of animal pharmacological experiments, it is fully proved that the effective substances of Polygala japonica Houtt. in the medium-dose group and high-dose group of the present invention (compounds eluted from the polyamide resin column with 0-25% ethanol from the water extract) are significantly superior to potassium sodium hydrogen citrate of the same quality in the three key indicators for the treatment of kidney stones (aggregation of calcium oxalate crystals, dilatation lesions of renal tubules, and infiltration of chronic inflammatory cells in the renal interstitium). Its pharmacodynamic level meets the requirements of drug registration and review in China, indicating that it has extremely good potential and market prospects in the treatment of urinary system lithiasis, urinary tract infection or kidney injury caused by urinary system lithiasis, and as an adjuvant drug after surgical treatment of urinary system lithiasis.

[0254] Beneficial effects

[0255] The effects of the drug of the present invention on typical test indicators such as aggregation of calcium oxalate crystals, infiltration of inflammatory cells in the renal interstitium, and dilatation lesions of renal tubules are significantly superior to potassium sodium hydrogen citrate. Its pharmacodynamic level meets the requirements of drug review in China, indicating that it has extremely good potential and market prospects in the treatment of urinary system lithiasis, urinary tract infection or kidney injury caused by urinary system lithiasis, and as an adjuvant drug after surgical treatment of urinary system lithiasis.

[0256] Compared with the currently 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, it will not cause serious side effects such as severe hyperkalemia, arrhythmia, and hypertension similar to potassium sodium hydrogen citrate, and has better safety.

[0257] Compared with other Chinese herbal medicines and drug extracts for treating diseases related to urinary system lithiasis, the components of the medicinal active extract of the present invention are simpler, the structures of the active ingredients are clearer, and the quality is more stable and controllable.

[0258] In addition, the medicinal active extract of the present invention is preferably extracted from the aerial stems and leaves of Polygala japonica Houtt., avoiding the problem of too long growth cycle of medicinal plants caused by extracting the whole herb, with lower cost and better environmental protection.

[0259] To sum up, the drug of the present invention has definite curative effect on diseases related to urinary system lithiasis, etc., has small side effects (equivalent to or better than the mainstream drug potassium sodium hydrogen citrate for urinary system lithiasis in the existing), has low cost, simple process, is safe and effective, has stable and controllable quality, and meets the requirements of modern drug registration for drugs for urinary system lithiasis, and has excellent medical value and economic value.

Claims

1. Use of the active medicinal extract of Polygala japonica Houtt. in the preparation of a medicament, wherein the medicament is used for treating or preventing urinary calculi, urinary tract infection or kidney injury caused by urinary calculi, and as an adjuvant medicament after surgical treatment of urinary calculi, wherein the active medicinal extract of Polygala japonica Houtt. is obtained by a method comprising the following steps: (1) Pretreatment of Polygala japonica Houtt. Take the whole herb of Polygala japonica Houtt. or the aerial part of Polygala japonica Houtt., or commercially available Polygala japonica Houtt. medicinal materials, wash, pulverize to obtain Polygala japonica Houtt. raw materials; (2) Coarse extraction of the effective part of Polygala japonica Houtt. Take the Polygala japonica Houtt. raw materials obtained in step (1), heat and reflux with ethanol at a concentration of 20-95% (v / v) 6-12 times the weight of the Polygala japonica Houtt. raw materials for 1-3 hours each time, reflux and extract 1-3 times repeatedly, combine the obtained ethanol extracts, concentrate to obtain the total ethanol extract of Polygala japonica Houtt.; Or, Take the Polygala japonica Houtt. raw materials obtained in step (1), heat deionized water at 6-15 times the weight of the Polygala japonica Houtt. raw materials to boiling and keep boiling for 1-3 hours, extract 1-3 times repeatedly, combine the obtained water extracts, concentrate to obtain the total water extract of Polygala japonica Houtt.; (3) Refinement of the effective part of Polygala japonica Houtt. Filter or centrifuge the total water extract or total ethanol extract of Polygala japonica Houtt. in step (2), after concentrating the filtrate or supernatant, separate it with a polyamide resin chromatographic column, elute successively with water / ethanol gradients of different ratios until the eluate is colorless, collect the 0-25% ethanol eluate, and evaporate to dryness under reduced pressure to obtain the required active medicinal extract of Polygala japonica Houtt.

2. The use according to claim 1, wherein, the active medicinal extract of Polygala japonica Houtt. contains at least one of the following compounds:

3. The use according to claim 1 or 2, wherein in step (1), the Polygala japonica Houtt. raw materials are the stem and leaf parts of Polygala japonica Houtt.

4. The use according to claim 1 or 2, wherein in step (3), the polyamide resin is 100-200 mesh Shanghai Test polyamide resin.

5. The use according to claim 1 or 2, wherein in step (3), the gradient elution is successively eluting with water, 25% ethanol, 50% ethanol, 75% ethanol, 95% ethanol until the eluate is colorless.

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