Application of cortex phellodendri extract for diuresis

Abstract

The invention relates to application of a cortex phellodendri extract, and relates to the field of medicines. The phellodendrine in the cortex phellodendri extract can effectively increase the urination amount and exert the diuresis effect, so that edema can be treated; the amur corktree bark total alkaloids in the amur corktree bark extract can reduce the re-absorption effect of renal tubules and collecting tubes on urine by inhibiting the inflammatory response of a body and the expression of aquaporin in the kidney, so that the urination amount is increased, the diuretic effect is exerted, and the treatment of edema is further facilitated.

Description

Technical Field

[0001] The invention relates to the field of medicines, in particular to an application of a Phellodendron chinense extract for diuresis. Background Art

[0002] Edema is a clinical symptom of multiple organ and system diseases caused by a disorder of water metabolism and imbalance of body fluid balance due to various reasons. The balance of water metabolism is a necessary condition for maintaining normal life activities of the human body, and it has a very close relationship with the lungs, spleen, kidneys, triple energizer, bladder and other internal organs. Only when the waste substances entering the body are excreted through metabolism can the normal physiological activities of various organs be carried out. However, the excretion pathways of various wastes are different. Any dysfunction of any internal organ will affect the normal metabolism of body fluids.

[0003] The slow circulation of body fluid in the body can easily cause symptoms such as edema, difficulty urinating, vomiting, or joint pain and swelling. If things go on like this for a long time, it will lead to severe diseases such as water retention. In recent years, edema is a complication of many diseases, such as heart failure, hypertension, diabetes, etc., and has become a common and frequently-occurring disease for patients to be hospitalized. Diuresis is the key to the treatment of these diseases. At present, the treatment of edema is mainly based on the treatment of the primary disease combined with diuretics such as diuretics and swelling for comprehensive treatment. Diuretics are a commonly used drug in clinical practice. They can directly act on the kidneys, affect the urine production process, promote the excretion of electrolytes and water, and thus effectively relieve the symptoms of edema. However, the diuretics currently used in clinical practice can cause electrolyte disorders, acid-base imbalance, arrhythmias, arteriosclerosis, diabetes, coronary heart disease and other problems, and may also directly damage the kidneys, causing great damage to the kidneys, causing hypokalemia, and even uremia in severe cases. Many studies have shown that Chinese medicine has the advantages of multiple targets and fewer adverse reactions in terms of efficacy. Therefore, it is still of great clinical significance to find a drug that can effectively promote diuresis while minimizing side effects. Summary of the invention

[0004] In view of the above problems, the present invention provides a use of a Phellodendron chinense extract for diuresis in the preparation of a diuretic and / or edema-treating drug, wherein the phellodendron alkaloids in the Phellodendron chinense extract can effectively increase the urine output, exert a diuretic effect, and thus be beneficial to the treatment of edema; the Phellodendron chinense total alkaloids in the Phellodendron chinense extract can inhibit the body's inflammatory response, inhibit the expression of aquaporins in the kidneys, and weaken the reabsorption of urine by the renal tubules and collecting ducts, thereby increasing the urine output, exerting a diuretic effect, and thus being beneficial to the treatment of edema.

[0005] In order to achieve the above-mentioned purpose, the present invention provides an application of a Phellodendron amurense extract for diuresis in the preparation of a diuretic and / or edema-treating drug, wherein the Phellodendron amurense extract comprises phellodendrine and / or Phellodendron amurense total alkaloids, and the Phellodendron amurense total alkaloids are obtained from Phellodendron amurense by percolation extraction.

[0006] In one embodiment, the drug further comprises a pharmaceutically acceptable excipient.

[0007] In one embodiment, the Phellodendron chinense extract inhibits the body's inflammatory response, inhibits the expression of aquaporins in the kidney, and weakens the reabsorption of urine by the renal tubules and collecting ducts.

[0008] In one embodiment, the drug is in the form of oral solution, pills, granules, capsules, tablets, pellets, injections or powders.

[0009] In one embodiment, when the Phellodendron chinense extract includes Phellodendron alkaloids, the dosage of the Phellodendron alkaloids is 0.5-10 mg / kg / d;

[0010] When the Phellodendron chinense extract includes Phellodendron chinense total alkaloids, the dosage of the Phellodendron chinense total alkaloids is 20-60 mg / kg / d;

[0011] When the Phellodendron amurense extract comprises phellodendron alkaloids and total Phellodendron amurense alkaloids, the dosage of the Phellodendron amurense extract is 4-7 mg / kg / d.

[0012] The present invention also provides a method for preparing the total alkaloids of Phellodendron chinense in the Phellodendron chinense extract in the application, which comprises: mixing Phellodendron chinense with acid solution, adding solvent, soaking, performing percolation extraction, collecting percolation solution, concentrating, purifying, and obtaining the total alkaloids of Phellodendron chinense.

[0013] In one embodiment, the purification includes: adding acid to the concentrated Phellodendron chinense total alkaloid extract, standing, removing precipitate, concentrating, standing, adjusting pH, concentrating, drying to obtain a solid, dissolving the solid, filtering, taking the filtrate, drying to obtain a Phellodendron chinense extract.

[0014] In one embodiment, the acid comprises hydrochloric acid.

[0015] In one embodiment, the soaking time is 80-90 hours, and the flow rate of the percolation extraction is 0.8-1.2 L / h.

[0016] The present invention also provides a medicine for diuresis and / or edema treatment, comprising a medicine active ingredient and a pharmaceutically acceptable auxiliary material, wherein the medicine active ingredient comprises the Phellodendron amurense extract used in the application.

[0017] In one embodiment, the active pharmaceutical ingredient also includes an ingredient compatible with the Phellodendron chinense extract.

[0018] The invention also provides the total alkaloids of Phellodendron chinense obtained by the preparation method.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention discloses an application of a Phellodendron chinense extract for diuresis in the preparation of a drug for diuresis and / or treatment of edema. The phellodendron alkaloids in the Phellodendron chinense extract can effectively increase the amount of urine excretion, exert a diuretic effect, and thus be beneficial to the treatment of edema; the Phellodendron chinense total alkaloids in the Phellodendron chinense extract can inhibit the body's inflammatory response, inhibit the expression of aquaporins in the kidneys, and weaken the reabsorption of urine by the renal tubules and collecting ducts, thereby increasing the amount of urine excretion, exerting a diuretic effect, and thus being beneficial to the treatment of edema. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The infrared spectrum of the total alkaloids of Phellodendron chinense in the embodiment;

[0022] Figure 2 It is the standard curve diagram of phellodendrine in the embodiment;

[0023] Figure 3 HPLC comparison spectra in the examples, where A is the standard solution, B is the sample solution, and C is the blank solution;

[0024] Figure 4 The results of the effect of TAP on the serum biochemical indexes of mice in the embodiment are shown in Figure 1, wherein A is the effect of TAP on the ADH level, B is the effect of TAP on the ANP level, C is the effect of TAP on the IL-1β level, and D is the effect of TAP on the TNF-α level; compared with the CON group, #: p<0.05, ##: p<0.01;

[0025] Figure 5 Schematic diagram of kidney pathological morphology in the embodiment, wherein A is the control group, B is the hydrochlorothiazide group, C is the low-dose group of total alkaloids of Phellodendron amurense, D is the high-dose group of total alkaloids of Phellodendron amurense, E is the low-dose group of phellodendron alkaloids, F is the group of phellodendron alkaloids + total alkaloids of Phellodendron amurense, and G is the high-dose group of phellodendron alkaloids;

[0026] Figure 6 The results of the effect of TAP on the expression of inflammatory factor proteins in the examples are shown in Figure 1, where A is the electrophoresis of TLR4, NF-κB, p-NF-κB and NLRP3 proteins, B is the expression of TLR4 and β-acting proteins, C is the expression of NLRP3 and β-acting proteins, and D is the expression of p-NF-κB and NF-κB proteins; compared with the CON group: *p<0.05, **: p<0.01;

[0027] Figure 7The results of the effect of TAP on the expression of aquaporin in the examples are shown in Figure 1, wherein A is the electrophoresis diagram of AQP2 and AQP3 proteins, B is the expression levels of AQP2 and β-acting proteins, and C is the expression levels of AQP3 and β-acting proteins; compared with the CON group, *p<0.05, **p<0.01. DETAILED DESCRIPTION

[0028] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0030] source:

[0031] Phellodendron chinense slices (Baokang Chinese medicine slices processing company), phellodendron alkali standard (Shanghai Maclean Biochemical Technology Co., Ltd.), hydrochloric acid (Tianjin Damao Chemical Reagent Factory), sodium hydroxide (Tianjin Beilian Fine Chemicals Development Co., Ltd.), methanol (analytical grade, Tianjin Ruijinte Chemical Co., Ltd.), methanol (chromatographic grade, Tianjin Biaoshiqi Technology Development Co., Ltd.), ethanol (analytical grade, Tianjin Yongda Chemical Reagent Co., Ltd.), acetonitrile (Simark imported from Germany), phosphoric acid (Tianjin Damao Chemical Reagent Factory), potassium bismuth iodide (Shanghai Maclean Biochemical Technology Co., Ltd.), iodine (Tianjin Shengao Chemical Reagent Co., Ltd.), potassium iodide (Shanghai Yindian Chemical Co., Ltd.), dodecane Sodium benzyl sulfonate (Tianjin Guangfu Technology Development Co., Ltd.), AL204 electronic balance (Mettler-Toledo Co., Ltd.), Milli-Q pure water machine (Millipore Corporation, USA), RE-52A rotary evaporator (Shanghai Yarong Biochemical Instrument Factory), SHB-B circulating water multi-purpose vacuum pump (Zhengzhou Great Wall Science and Technology Industry and Trade Co., Ltd.), LC-20AT high performance liquid chromatograph (Shimadzu International Trade Co., Ltd.), high-speed centrifuge (eppendorf), vortex oscillator (Shanghai Huxi Instrument Co., Ltd.), vacuum decompression drying oven (Shanghai Haixiang), electric constant temperature water bath (Beijing Chang'an Scientific Instrument Factory), Ph detector (Mettler-Toledo Co., Ltd.).

[0032] Unless otherwise specified, the reagents, materials, and equipment used in this example are all commercially available; the experimental methods are all conventional experimental methods in the art unless otherwise specified.

[0033] Example

[0034] 1. Extraction of total alkaloids from Phellodendron amurense.

[0035] 1. Percolation extraction method.

[0036] Weigh 500g of dried Phellodendron amurense accurately, crush it and pass it through a 16-mesh sieve. Put the crushed medicinal silk into a sealed container, add an appropriate amount of hydrochloric acid aqueous solution, stir evenly, moisten the medicinal materials, and place them in a sealed container for 2 hours to allow the medicinal materials to fully expand. Pad the absorbent cotton at the bottom of the percolation cylinder, and load the already moistened medicinal materials into the percolation cylinder in small quantities and multiple times. Each time it is loaded, it must be evenly flattened with moderate tightness. Spread a piece of filter paper with the same thickness as the cylinder on the loaded medicinal materials and press it with stones. Fix the percolation cylinder on the iron frame, add 5L of 5% HCl aqueous solution to the round-bottom flask where the solution is placed, and install the percolation device. Slowly add an appropriate amount of solvent, wait for the gas to be discharged, soak for 82h, and control the flow rate to 1L / h for percolation extraction. Collect 4.5 liters of percolation liquid. Abandon the drug residue, concentrate the collected percolation liquid under reduced pressure at 65℃ water bath until a light yellow powder precipitates, and obtain 3.3L of concentrated total alkaloid extract. The obtained extract was slowly stirred and concentrated hydrochloric acid was added to adjust Ph = 1, and the extract was allowed to stand at room temperature for 14 hours. The yellow precipitate was filtered out, and the mother liquor was concentrated under reduced pressure at 55°C in a water bath until a yellow solid precipitated, and the mother liquor was allowed to stand at room temperature for 14 hours. The operation was repeated 3 times, and the mother liquor was allowed to stand at room temperature for 15 hours. The mother liquor was stirred and dripped with 10% NaOH aqueous solution to adjust Ph = 8, and the mother liquor was concentrated under reduced pressure at 60°C in a water bath and dried to obtain a solid. The obtained solid was dissolved in 100% methanol, and methanol solution was added at a ratio of solid weight: methanol volume = 1g: 10ml to dissolve the solid. This step was repeated until no solid precipitated after dissolving in methanol. Filtered with a 0.45um organic filter membrane to obtain a clear and transparent filtrate, which was dried to obtain 17.7g of purified TAP.

[0037] 2. Qualitative identification of total alkaloids in Phellodendron chinense.

[0038] 2.1 Infrared spectroscopy.

[0039] Take an appropriate amount of evenly ground solid sample, spread it evenly on the upper surface of the ATR window, and press it tightly to make close contact. The wave number range is 4000cm -1 ~400cm -1 , resolution 4cm -1 By analyzing the infrared spectrum of TAP, the characteristic absorption peaks were determined, further confirming the presence of alkaloid structure in TAP.

[0040] 2.2 Reaction of total alkaloids from Phellodendron amurense.

[0041] Take a small amount of total alkaloids from Phellodendron amurense, add methanol to dissolve it, take two test tubes, add 2 ml of total alkaloids solution from Phellodendron amurense to each test tube, 2 drops of acid solution, add iodine-potassium iodide reagent and potassium bismuth iodide reagent in sequence, shake well, let stand and observe, and make 3 parallel samples at the same time.

[0042] 2.3 Determination of extraction rate and content of total alkaloids from Phellodendron amurense.

[0043] 2.3.1TAP extraction rate.

[0044] The extraction rate of total alkaloids of Phellodendron amurense was calculated (extraction rate % = total alkaloids of Phellodendron amurense / mass of Phellodendron amurense × 100%).

[0045] 2.3.2TAP content determination.

[0046] Add 10mL of 0.02 mol / mL hydrochloric acid to the total alkaloid sample of Phellodendron amurense, use phenolphthalein as an indicator, back-titrate with 0.02 mol / mL sodium hydroxide, and calculate the total alkaloid content of Phellodendron amurense as phellodendronine.

[0047] Calculation formula: Total alkaloid content of Phellodendron amurense = C × (V 空白 -V NaOH )×M÷W×100%. Where C is the concentration of NaOH used for titration; V NaOH The volume of NaOH used to titrate the alkaloid with excess hydrochloric acid; V 空白 is the volume of NaOH used to titrate the same volume of hydrochloric acid with the same concentration; M is the relative biological molecular weight of the alkaloid. In this experiment, the molecular weight of phellodendron alkaloid 342 is selected as the representative; W is the sample mass.

[0048] 2.4 Determination of phellodendronine content and methodology study.

[0049] The content of phellodendron alkaloids (PhC) in TAP was determined by high performance liquid chromatography (HPLC). Filler: octadecylsilane bonded silica gel (C 18 , 4.6×250mm, 5μm); column temperature 40℃; flow rate 1mL / min; detection wavelength 284nm; mobile phase A: acetonitrile, mobile phase B: 0.1% phosphoric acid aqueous solution (0.2g of sodium dodecyl sulfate is added for every 100ml), elution condition A:B=30:70.

[0050] (1) Preparation of reference solution: Accurately weigh 5 mg of PhC standard, place in a 50 ml volumetric flask, dilute to volume with methanol, and shake well to make a standard stock solution with a content of 100 μg / mL, filter through a 0.45 μm filter membrane, and store in a refrigerator at 4°C.

[0051] (2) Preparation of sample solution: Accurately weigh TAP, dissolve it in methanol, and filter it through a 0.45 μm filter membrane.

[0052] (3) Negative sample solution: the auxiliary material solution without the raw material.

[0053] 2.4.1 Drawing of standard curve.

[0054] Accurately pipette 0.1ml, 1ml, 2ml, 3ml, 4ml, 5ml, 7ml, 10ml of PhC reference solution into a 10ml volumetric flask, add methanol to make up the volume, perform injection analysis according to the conditions in 2.4, plot a standard curve with the peak area as the ordinate and the concentration as the abscissa, and obtain a regression equation. Calculate the content according to the regression equation.

[0055] 2.4.2 Exclusivity.

[0056] Accurately pipette 10 μL of each of the reference solution, sample solution and negative sample solution and inject them into HPLC, use methanol as the blank control, and draw a chromatogram according to the conditions in 2.4.

[0057] 2.4.3 System adaptability and precision.

[0058] Accurately pipette the PhC standard solution into the liquid chromatograph, inject it three times in a row, and measure the result.

[0059] 2.4.4 Detection limit.

[0060] The PhC reference solution was diluted to a concentration of 0.5 μg / mL, and 1 μL, 2 μL, and 3 μL were injected in sequence until the characteristic peak appeared. The detection limit was obtained based on the peak area.

[0061] 2.4.5 Repeatability.

[0062] Prepare the sample solution according to the steps, in parallel for 6 times, inject the sample according to the method in 2.4, and calculate the relative standard deviation (RSD) value.

[0063] 2.4.6 Stability.

[0064] Accurately pipette 20 mg of the product into a 10 mL volumetric flask, process the sample according to the above determination steps, determine the PhC content at 0 h, 2 h, 4 h, 6 h, 8 h, and 10 h, and calculate the relative standard deviation (RSD) value.

[0065] 2.4.7 Sample recovery rate

[0066] Accurately pipette 9 portions of liquid samples and divide them into 3 groups. Add 50%, 100% and 150% of the test sample components to each group, respectively. After obtaining 9 groups of test sample solutions, determine the PhC content according to method 2.4, calculate the sample recovery rate, and calculate the relative standard deviation (RSD) value.

[0067] 2.5 Statistical processing.

[0068] SPSS 26.0 was used to analyze the data, and the quantitative data were expressed as Mean ± SD. P < 0.05 indicated statistical significance. GraphPad Prism 8.0 was used to make statistical graphs.

[0069] 3. Experimental results.

[0070] 3.1 Qualitative identification experimental results.

[0071] 3.1.1 Infrared spectroscopy.

[0072] The infrared spectrum of total alkaloids of Phellodendron amurense is shown in Figure 1 As shown, the wave number is 1076cm -1 , 1265cm -1 There is an absorption peak at 1636cm, which is inferred to be the characteristic absorption peak of CN stretching vibration of tertiary amine, indicating that there is a tertiary amine structure in the molecule; the wave number is 1636cm -1 There is an absorption peak at 1518cm, which is inferred to be the characteristic absorption peak of carbonyl C=O, indicating that there is a carbonyl structure in the molecule; the wave number is 1518cm -1 There is an absorption peak at 3416cm, which is inferred to be the characteristic absorption peak of the aromatic ring, indicating that there is an aromatic ring structure in the molecule; the wave number is 3416cm -1 There is an absorption peak at , which is inferred to be the characteristic absorption peak of NH. Through the above analysis, it can be determined that the extract (i.e., the total alkaloids of Phellodendron amurense mentioned above) contains alkaloids, and the infrared absorption spectrum results are shown in the following table.

[0073] Table 1 Infrared absorption spectrum results

[0074]

[0075]

[0076] 3.1.2 Alkaloid precipitation reaction.

[0077] The results of the alkaloid qualitative test are shown in the following table. From the experimental results, it can be seen that the total alkaloids of Phellodendron amurense have a precipitation color reaction to the alkaloid qualitative reagent. Therefore, it can be determined that it contains alkaloid compounds.

[0078] Table 2 Qualitative experimental results

[0079]

[0080] 3.2 Extraction rate and content determination results

[0081] 3.2.1 Extraction rate results.

[0082] The percolation extraction method finally obtained 17.7 grams of powder. Substituting it into the formula, the extraction rate of the percolation extraction method was calculated to be 3.5%.

[0083] 3.2.2 TAP content determination results.

[0084] The total alkaloid content was determined by acid-base titration. Weigh TAP 0.02 g accurately, dissolve it in 5 ml methanol, add 10 ml of 0.02 mol / L HCl solution accurately, shake well, add 15 ml of distilled water and an appropriate amount of phenolphthalein indicator, titrate with NaOH solution until it turns pink, and record the data; accurately add hydrochloric acid solution, distilled water and phenolphthalein indicator of the same concentration and volume to a conical flask, titrate with NaOH solution until it turns pink, and record the data as a blank control. NaOH =14.5ml; V 空白 =16.5ml, substitute into the formula, then TAP content = 0.02mol / L×[(16.5mL-14.5mL)]÷1000×342÷0.02×100%≈68.4%.

[0085] 3.3 Determination of phellodendron pine content and methodological research results.

[0086] 3.3.1 Standard curve of phellodendrine.

[0087] The standard curve was drawn with the concentration of the standard as the horizontal axis and the peak area as the vertical axis. The equation of the standard curve was: Y = 10501X + 8643.9 (r = 0.9997). The results are shown in Figure 2 .

[0088] 3.3.2 Determination of phellodendronine content.

[0089] Six samples were tested in parallel according to the above method, and the PhC content was calculated according to the standard curve. The results are shown in the following table. The average PhC content was calculated to be 2.828%.

[0090] Table 3 Content determination

[0091]

[0092] 3.3.3 Exclusivity.

[0093] HPLC analysis was performed according to the method under 2.4, and 10 μL was injected. The results showed that the PhC standard solution had a maximum peak at 19.969 min, and the absorption peak of the sample solution appeared within ±2 min of the absorption peak of the reference solution, indicating that the extraction method can extract PhC. The blank solvent had no absorption peak at the corresponding retention time of the standard and sample, indicating that the method had obvious characteristic absorption peaks and strong specificity. Figure 3 .

[0094] 3.3.4 System adaptability and precision.

[0095] The PhC standard solution was accurately pipetted and injected into the liquid chromatograph for three consecutive times, and the results were measured as shown in the following table. The system adaptability was good, RSD = 0.088%, indicating that the instrument had good precision.

[0096] Table 4 System adaptability test results

[0097]

[0098] 3.3.5 Detection limit.

[0099] The PhC reference solution was diluted to a concentration of about 0.5 μg / mL. No characteristic peak appeared when 1 μL was injected, no characteristic peak appeared when 2 μL was injected, and a characteristic peak appeared when 3 μL was injected. The peak area was 1388, and the detection limit was 0.0015 μg.

[0100] 3.3.6 Repeatability.

[0101] Six parallel sample solutions were injected according to the above method. The peak areas are shown in the following table. The relative standard deviation (RSD) value is 1.86%, which is less than 5%. The method has good repeatability.

[0102] Table 5 TAP repeatability test

[0103]

[0104] 3.3.7 Stability

[0105] Take the sample solution under 2.4.6, place it at room temperature, inject and measure according to the chromatographic conditions, record the peak area and calculate the content. The results show that the standard deviation RSD value of the peak area value is 2.31%, which is less than 3%, indicating that the content of phellodendron chinense in the test solution is stable within 10 hours. As shown in the following table.

[0106] Table 6 Stability test results

[0107]

[0108] 3.3.8 Sample recovery rate.

[0109] 1.0mL of liquid sample was accurately drawn into 9 portions and divided into 3 groups. A standard solution of 0.5μg / ml was prepared. Each group was added with 28μl of standard solution (0.014μg, 50% of the test sample to be tested), 56μl of standard solution (0.028μg, 100% of the test sample to be tested), and 84μl of standard solution (0.042μg, 150% of the test sample to be tested) standard. After obtaining 9 groups of test sample solutions, the content was determined and the recovery rate of the sample was calculated. The results are shown in the following table. The average recovery rate of 50% sample addition was 103.13%, and the relative RSD value was 1.05% <5%. The average recovery rate of 100% sample addition was 100.71%, and the RSD value was 3.07% <5%. The average recovery rate of 150% sample addition was 97.76%, and the RSD value was 1.28% <5%. The recovery rate limits all met 92-105%, indicating that the recovery rate of this method was good.

[0110] Table 7 Content determination sample recovery experimental results

[0111]

[0112] 4. Summary.

[0113] This example uses a variety of technical means such as percolation extraction and HPLC to conduct an in-depth study on the alkaloid components in Phellodendron. Through infrared spectroscopy and alkaloid precipitation reaction identification experiments, the results show that the extract contains alkaloid compounds. The crude alkaloid extract obtained by percolation extraction has a content of 68.4% measured by acid-base titration. After HPLC determination, a characteristic peak was found within the peak time of the reference substance, and the phellodendronine content was 2.828%.

[0114] 2. Study on the diuretic effect of total alkaloids of Phellodendron chinense.

[0115] 1. Experimental materials.

[0116] 1.1 Experimental animals

[0117] 72 SPF ICR male mice, weighing 20±2g, were provided by Liaoning Changsheng Biotechnology Co., Ltd., with health license number SCXK(Liao)-2020-0001SPF, and animal experiment ethics demonstration batch number 2023071101. They were raised at a constant temperature of 20±2℃ and a relative humidity of 45-55%, and formal experiments were carried out after one week of adaptive feeding.

[0118] 1.2 Experimental drugs and reagents.

[0119] The reagents required for this experiment are shown in the following table.

[0120] Table 8 Experimental drugs and reagents

[0121]

[0122]

[0123] 1.3 Experimental instruments.

[0124] The experimental instruments required for this experiment are shown in the following table:

[0125] Table 9 Experimental instruments

[0126]

[0127] 2. Experimental method.

[0128] 2.1 Screening of qualified experimental mice.

[0129] 72 male ICR mice were first placed in mouse cages for 1 week and then in metabolic cages for 2 days. Before the experiment, the abdomen of the mice was gently pressed to make them empty their urine, and then distilled water was given by gavage at a dose of 0.2 mL / 10 g body weight. The mice were immediately placed in metabolic cages (one per cage) and urine was collected for 4 hours. Mice with urine output exceeding 40% of the gavage water volume were qualified experimental animals.

[0130] 2.2 Experimental groups and medication methods.

[0131] 70 mice with qualified urination function in the experiment were randomly divided into 7 groups, namely control group (CON), hydrochlorothiazide positive drug group (HCTZ), total alkaloid high-dose group (TAP-H, 55mg / kg / d), total alkaloid low-dose group (TAP-L, 23mg / kg / d), phellodendron alkaloid low-dose group (PhC-L, 1mg / kg / d), phellodendron alkaloid high-dose group (PhC-H, 8mg / kg / d), phellodendron alkaloid + total alkaloid dosage group (PhC+TAP, 5mg / kg / d). The control group was given an equal volume of normal saline, and the remaining groups were given corresponding drugs by gavage every day for 5 consecutive days. The alkaloids required for gavage were extracted according to the method under "Extraction of total alkaloids of phellodendron alkaloids" in the embodiment.

[0132] 2.3 Urine collection and processing.

[0133] After administration on the 5th day, fasting but not water restriction was performed for 24 hours to eliminate the interference of feces. First, the abdomen of the mouse was gently pressed to drain the remaining urine, and then each group was gavaged with 0.4 ml of saline to create a water load model. After 30 minutes, the control group was given the same volume of saline, and the other groups were gavaged with the corresponding drugs. The mice were immediately placed in metabolic cages, 2 per cage, and urine was drawn once every 2 hours to measure the volume. The urine volume was collected continuously for 8 hours, and the urine volume of each group of animals every 2 hours and the total urine volume of 8 hours were calculated. After the urine was collected, blood was collected from the mouse eyeballs and the mouse was killed by dislocation of the neck. The plasma was allowed to stand for 1 hour, and the supernatant was taken by centrifugation (4°C, 4000r / min, 20min). The processed serum was drawn into a new EP tube, marked, and placed in an ultra-low temperature refrigerator for freezing. The liver, kidney, and spleen of the killed mice were quickly and completely removed, and the residual blood stains around were washed with saline, and the water was absorbed by filter paper, weighed, and marked. In addition, kidneys of 3 mice were randomly selected from each group, fixed with 10% formalin, stored at room temperature, prepared for embedding and sectioning, and stained for renal tissue pathology observation. The remaining tissues were frozen in an ultra-low temperature refrigerator for detection of relevant biochemical indicators.

[0134] 2.4 Organ index determination.

[0135] The liver, kidney and spleen of the mice were removed, rinsed with physiological saline, blotted dry with filter paper and weighed, and the organ index was calculated according to the formula.

[0136] Liver index (%) = liver weight (g) / body weight (g) × 100%

[0137] Renal index (%) = bilateral kidney weight (g) / body weight (g) × 100%

[0138] Spleen index (%) = spleen weight (g) / body weight (g) × 100%

[0139] 2.5ELISA method was used to determine the expression levels of TNF-α, IL-1β, ADH and ANP proteins in serum.

[0140] The serum samples of each group were tested using enzyme-linked immunosorbent assay kits for tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), antidiuretic hormone (ADH), and atrial natriuretic peptide (ANP). The experimental operation was carried out according to the instructions.

[0141] 2.6HE staining was used to observe the pathological changes of renal tissue.

[0142] (1) After fixation with the fixative for 24 h, dehydrate with 75%, 85%, 90%, 95%, and 100% ethanol in sequence, and make the samples transparent with xylene.

[0143] (2) Immerse the tissue in paraffin, remove it after it is soaked, and place it back in paraffin solution for embedding.

[0144] (3) Use a microtome to cut the embedded wax block into thin slices, stick them on anti-shedding slides, and dry them at a constant temperature.

[0145] (4) HE staining: Observe the pathological changes of mouse kidneys under a 200x microscope.

[0146] 2.7Western blot was used to detect the expression of related proteins in renal tissue.

[0147] (1) Extraction of renal tissue protein: 50 mg of renal tissue was added to the lysis buffer at a ratio of 1:9 and fully ground using a homogenizer. After lysis on ice for 1 h, the mixture was centrifuged at 4°C and 12,000 r / min for 10 min, and the supernatant was collected for later use.

[0148] (2) Determination of protein concentration: After diluting the trace protein tissue stock solution, use the BCA kit to determine and calculate the protein concentration.

[0149] (3) Protein quantification: The protein content was set to 40 μg, the sample loading volume was fixed to 10 μL, the volume of protein stock solution required in the sample to be tested was calculated, 1 / 5 of the total sample loading volume of 5× SDS-PAGE protein loading buffer was added, and the remaining part was added with a relative amount of distilled water and mixed.

[0150] (4) Protein denaturation: Place the protein sample prepared above in a 100°C water bath and boil for 10 min. Then take out the sample tube and let it cool. Store it in a -20°C refrigerator for later use.

[0151] (5) Prepare SDS-PAGE gel: Select different ratios of separation gel according to the molecular weight of the target protein, mix thoroughly, and then slowly add it between the glass plates to avoid bubbles. Use anhydrous ethanol solution for liquid sealing and let it stand for about 20 minutes. After a clear solidification line appears, pour out the upper layer of anhydrous ethanol along the edge and use filter paper to absorb the moisture on the edge. Prepare concentrated gel, add the concentrated gel to the separation gel until it is full, and quickly insert the comb vertically. Let it stand until the concentrated gel solidifies and set aside.

[0152] (6) Sample loading: Place the solidified glass gel plate into an electrophoresis tank filled with electrophoresis buffer, gently remove the comb, use a microinjection needle to extract 3 μL of marker and 10 μL of protein sample, and add them into the gel wells.

[0153] (7) Electrophoresis: Use constant voltage electrophoresis, 80 V voltage until the sample reaches the boundary between the concentrated gel and the separation gel (depending on the band situation, you can also run at 20 V voltage for 20 min and then adjust to 80 V), set the voltage to 120 V. End the electrophoresis when the protein sample is 2 cm from the end of the separation gel.

[0154] (8) Membrane washing: Activate the prepared PVDF membrane in methanol for 20 seconds, wash with distilled water for 3 minutes, and then incubate with transfer buffer for 30 minutes.

[0155] (9) Transfer: Soak the sponge and the prepared filter paper completely with transfer buffer, attach the activated PVDF membrane to the gel, avoid bubbles, and mark the front and back. Place the gel and membrane in order, align the positive and negative electrodes, pour in the transfer buffer, place the tank at 4°C with a constant current of 300mA, and transfer for 90 minutes (adjust the transfer time according to the molecular weight of the target protein).

[0156] (10) Blocking: After transfer, block the membrane in 5% skim milk for 1.5 h.

[0157] (11) Incubation with primary antibody: The blocked PVDF membrane was washed with TBS-T and incubated with the corresponding primary antibodies TLR4 (1:1000), NLRP3 (1:500), NF-κB (1:1000), p-NF-κB (1:2000), AQP2 (1:1000) and AQP3 (1:1000) according to the molecular weight of the target protein at 4°C overnight.

[0158] (12) Washing the membrane: After the antibody is recovered, wash the membrane three times with TBS-T, each time for 10 min.

[0159] (13) Add secondary antibody and incubate: Add corresponding secondary antibody (1:4000) and incubate at room temperature for 1 h.

[0160] (14) Imaging and data analysis: The samples were developed with ECL luminescent solution and photographed using a gel imaging device. The protein expression level was calculated based on the grayscale area of ​​the bands and quantitatively analyzed using Image-J software.

[0161] 2.8 Statistical processing.

[0162] SPSS 26.0 was used to analyze the data, and the quantitative data were expressed as Mean ± SD. P < 0.05 indicated statistical significance. GraphPad Prism 8.0 was used to make statistical graphs.

[0163] 3. Experimental results

[0164] 3.1 Effect of TAP on urine volume in mice.

[0165] After continuous administration of the drug for 5 days, the mice were collected for 8 hours of urine. The results showed that compared with the CON group, the 0-2h urine volume and the 8-hour total urine volume in the HCTZ group were significantly different (p<0.05); the 4-6-hour urine volume in the PhC-L group was extremely significantly different (p<0.01); the PhC+TAP group and the PhC-H group had significant differences in the 8-hour total urine volume (p<0.05); the TAP-H group had extremely significant differences in the 8-hour total urine volume (p<0.01). Compared with the CON group, the PhC-L group also increased the urine volume of mice, but the difference was not statistically significant, as shown in the table below.

[0166] Table 10 Mouse urine volume

[0167]

[0168] Note: Compared with CON, * represents significant difference p < 0.05, ** represents extremely significant difference p < 0.01.

[0169] 3.2 Effect of TAP on mouse organ indexes.

[0170] The organ index of normal mice is relatively constant. If edema, atrophy or necrosis occurs, the organ index will also change. As shown in Table 4.4, compared with the control group, the kidney index of the TAP-L group and the PhC-L group was significantly reduced (p < 0.05), indicating that the TAP-L group and the PhC-L group had a certain damage effect on the mouse kidney. There was no significant difference between the other drug-administered groups and the control group, indicating that there was no toxic side effect on the kidney. At the same time, it shows that with the increase of the dosage, the toxicity to the mouse kidney decreased.

[0171] Table 11 Organ Index

[0172]

[0173] Note: Compared with CON, * represents significant difference p < 0.05, ** represents extremely significant difference p < 0.01.

[0174] 3.3 Effect of TAP on the levels of ADH, ANP, IL-1β and TNF-a in mouse serum.

[0175] The expression levels of ADH, ANP, IL-1β and TNF-α in the serum of each group of experimental rats were detected by ELISA. Figure 4As shown: Compared with the CON group, the expression of ADH in the serum of the HCTZ group and each drug-treated group was extremely significantly decreased (p<0.01); the expression of ANP in the serum of the HCTZ group and each drug-treated group was increased, and the expression of ANP in the PhC+TAP group was significantly increased compared with the control group (p<0.05), while the expression of ANP in the other drug-treated groups was not significant.

[0176] IL-1β data showed that compared with the control group, the IL-1β levels in the HCTZ group, TAP-L group, and PhC-L group were significantly reduced (p<0.05), and the PhC-H group was extremely significantly reduced (p<0.01). The levels in the PhC+TAP group and TAP-H group were also reduced, but the differences were not statistically significant (p>0.05). TNF-α data showed that compared with the control group, the TNF-α content in the serum of mice in each drug-treated group was significantly reduced (p<0.05 or p<0.01) except for the PhC+TAP group.

[0177] The results showed that TAP could significantly reduce the levels of ADH, TNF-a, and IL-1β in mouse serum, and significantly increase the level of ANP.

[0178] 3.4 Pathological changes in mouse renal tissue.

[0179] The results of HE staining of the kidneys are as follows Figure 5 As shown in the figure, in the control group, the glomeruli were shrunken, most of the brush borders in the renal tubules disappeared, and there were no obvious lesions in other parts; in the HCTZ group, there was a small amount of inflammatory cell infiltration, and there were no obvious lesions in the rest; in the TAP-L group, there were a large number of inflammatory cells; in the TAP-H group, the PhC-L group, and the PhC+TAP group, there were a small amount of inflammatory cell infiltration in the glomeruli; in the PhC-H group, there were very few inflammatory cells. When TAP drug intervention was given, the glomerular morphology was close to normal and the inflammatory cell infiltration was reduced. The results showed that with the increase of TAP dose, the inflammatory cells decreased.

[0180] 3.5 Effect of TAP on the expression of inflammatory factor proteins.

[0181] The NF-κB signaling pathway is an important cell signaling pathway that is involved in regulating a variety of biological processes, including inflammatory responses. Figure 6), the expression levels of TLR4, NLRP3 and p-NF-κB p65 / NF-κB p65 proteins in renal tissue of mice in control group were higher than those in other groups; compared with those in control group, the expression levels of TLR4, NLRP3 and p-NF-κBp65 / NF-κB p65 proteins in renal tissue of mice in HCTZ group were significantly decreased (p<0.05 or p<0.01), the expression levels of TLR4, NLRP3 and p-NF-κBp65 / NF-κB p65 proteins in renal tissue of each drug-treated group were decreased to varying degrees (p<0.05 or p<0.01), and the levels of individual components decreased but the differences were not statistically significant, indicating that HCTZ and each drug-treated group can inhibit the TLR4 / NF-κB / NLRP3 signaling pathway to varying degrees.

[0182] 3.6 Effect of TAP on the expression of aquaporin.

[0183] Aquaporins (AQPs) are important molecular basis for the kidney to maintain body fluid balance. Aquaporins are specific channel proteins that transport water on cell membranes. The kidney is not only the body's urinary organ, but also the most active organ involved in the process of body fluid transport. It has been found that aquaporins are expressed in different locations of the kidney. In order to study the effect of TAP on mouse aquaporins, the expression of related proteins was detected by Western Blot. The results showed that ( Figure 7 ), the expression levels of AQP2 and AQP3 proteins in the renal tissue of mice in the control group were higher than those in other groups; compared with the control group, the expression levels of AQP2 and AQP3 proteins in the renal tissue of mice in the HCTZ group and each drug-treated group were significantly reduced (p<0.05 or p<0.01). Among them, except for the PhC-L group, the other drug-treated groups had a very significant inhibitory effect on the expression level of AQP3 protein in the renal tissue of mice (p<0.01). In summary, TAP can increase the urine volume of mice by regulating the protein expression of aquaporins.

[0184] 4. Summary.

[0185] This study found that TAP can significantly downregulate the expression of TLR4, NLRP3, p-NF-κB p65, NF-κB p65, AQP2 and AQP3 proteins, indicating that TAP can reduce the expression of inflammatory factors by downregulating the expression of aquaporins and inhibiting the NF-κB inflammatory protein signaling pathway, thereby exerting a diuretic effect. Through the analysis of mouse serum indicators, it was found that TAP can significantly reduce the levels of ADH, IL-1β and TNF-α in serum and increase the level of ANP, indicating that the diuretic effect of TAP may be related to reducing the content of antidiuretic hormone, increasing the level of atrial natriuretic peptide, and inhibiting the inflammatory response in mouse serum and renal tissue.

[0186] The animal selection work for the diuretic activity experiment in this embodiment mainly includes the Aston method and the Kau method. The Kau method is mainly used for the diuretic animal screening of small samples, and the Aston method is mainly suitable for experimental research with a large sample size, a single test index, and a short duration. Animals with a urine ratio of more than 40% are selected for the experiment. The Aston method is more suitable for this experiment, so the Aston method is selected for the diuretic experiment of mice.

[0187] After 5 days of continuous medication, the PhC+TAP and PhC-H groups significantly increased the urine output of mice, and the urine output of the PhC-H group was basically the same as that of the HCTZ group. In the TAP-H group, there was a very significant difference in the total urine output over 8 hours. Moreover, the urine output of each medication group in the first two hours was significantly higher than that in the following hours. The experimental results intuitively confirmed the diuretic effect of the drug, so we continued to explore its diuretic mechanism.

[0188] The organ index of mice is an important biological indicator that can be used to determine the damage of organs. The organ index of normal mice is relatively constant. If the organs of mice are congested, edematous, hypertrophic, atrophic, or necrotic, the organ index will increase or decrease accordingly. According to the organ index, it is possible to preliminarily determine whether the organ is diseased, the degree of disease and the functional status, which can reflect the toxic and side effects of the drug. This study showed that compared with the control group, the kidneys of mice in the TAP-L group and the PhC-L group showed certain damage, and there was no significant difference in the other drug-administered groups, indicating that the renal toxicity of mice decreased with the increase of the dosage.

[0189] TNF-α and IL-1β are very important pro-inflammatory factors. The experimental results showed that the levels of TNF-α and IL-1β in the control group increased significantly, while those in the drug-treated group decreased significantly, indicating that the increase in urine volume in mice is related to the reduction in the expression of inflammatory factors.

[0190] ADH is another important polypeptide hormone that affects fluid balance, also known as arginine vasopressin. Its main function is to increase the permeability of the distal convoluted tubule and collecting duct to water, increase the reabsorption of sodium ions by the thick ascending limb of the loop of Henle, and is a hormone secreted by hypothalamic nerve cells. It reabsorbs water by the renal tubule and reduces the formation of urine. It is a key regulatory hormone for urine concentration and dilution. ANP is a type of peptide hormone, which is mainly released and synthesized by atrial myocytes and has a strong effect of natriuresis, diuresis and antagonism to the RAS system. ANP produces diuretic and natriuretic effects by increasing the glomerular filtration rate, blood flow in the renal medulla and inhibiting the reabsorption of sodium by the proximal convoluted tubule and collecting duct. In addition, ANP can also inhibit the synthesis and release of antidiuretic hormone. The experimental results showed that the diuretic effect of each drug-treated group was equivalent to that of the HCTZ group. Compared with the control group, the expression of ADH in the serum of the HCTZ group and each drug-treated group was significantly reduced, and the expression of ANP in the serum of each group was increased. The decrease in ADH secretion reduces the renal tubular water reabsorption function, hinders the urine concentration process, and increases urine excretion. The increase in ANP content promotes the glomerular filtration rate and the increase in blood flow in the bone marrow of mice, resulting in a decrease in the reabsorption of sodium by the proximal tubules and collecting ducts, which increases urine excretion. At the same time, the increase in ANP can inhibit the secretion and release of ADH, further hindering the concentration of urine and promoting an increase in urine volume in mice.

[0191] HE staining results showed that the glomeruli in the CON group were shrunken, and there were no obvious lesions in other parts. With the increase of drug concentration, the glomerular morphology was close to normal, and the infiltration of inflammatory cells was reduced. This shows that TAP may increase the urine volume of mice by inhibiting inflammatory response.

[0192] NF-κB is widely present in eukaryotic cells. It is an important transcription factor with multidirectional transcriptional regulation, and is involved in a series of processes such as inflammatory response, oxidative stress, cell proliferation and apoptosis. After being stimulated by extracellular inflammatory signals, TLR4 receptor activates IkB kinase (IKK), promotes phosphorylation and degradation of IkBα (NF-κB inhibitory protein), and then activates NF-κB to promote the expression of various inflammatory cytokine genes and activates nuclear entry. After entering the nucleus, NF-κB activates NLRP3, further inducing the secretion of inflammatory factor IL-1β. Therefore, the TLR4 / NF-κB / NLRP3 signaling pathway is an important target for the treatment of inflammation. The experimental results showed that the expression levels of TLR4, NLRP3 and p-NF-κB p65 / NF-κB p65 proteins in the kidney tissue of mice in the CON group were higher than those in other groups; compared with the control group, the expression levels of TLR4, NLRP3 and p-NF-κB p65 / NF-κB p65 proteins in the kidney tissue of mice in the HCTZ group and each drug-treated group were significantly decreased, indicating that the expression levels of p-NF-κB and NLRP3 proteins downstream of TLR4 can be effectively reduced after administration, and the anti-inflammatory effect can be exerted by inhibiting TLR4. It is inferred that it may be by inhibiting the TLR4 / NF-κB / NLRP3 signaling pathway to reduce inflammation and thus increase the urine volume of mice.

[0193] Water transport across membranes plays an important role in maintaining normal cell metabolism. Aquaporins mainly mediate the transport of free water across biological membranes and play an important role in maintaining the stability of the intracellular and extracellular environment. Aquaporins are mainly distributed in the kidneys to maintain the body fluid balance of the kidney. AQP2 plays a key role in the reabsorption of water in the kidneys. AQP2 is expressed in the principal cells of the distal convoluted tubules and collecting ducts and is regulated by ADH. Studies have shown that when ADH levels increase, AQP2 expression can be promoted. ADH can increase the density of AQP2 on the luminal membrane and increase the permeability to water. The expression of AQP2 increases with the continuous increase in ADH levels. Therefore, the decrease in AQP2 expression indirectly indicates that the increase in urine volume is related to the decrease in ADH secretion. At present, there are relatively few reports on AQP3, but many studies have shown that AQP3 can truly play a diuretic function. AQP3-deficient mice can experience severe polyuria. The experimental results showed that the expression levels of AQP2 and AQP3 proteins in the kidney tissue of mice in the CON group were higher than those in other groups; compared with the CON group, the expression levels of AQP2 and AQP3 proteins in the kidney tissue of mice in the HCTZ group and each drug-treated group were significantly reduced. The serum antidiuretic hormone and kidney AQP2 protein content were significantly reduced. It can be inferred that TAP directly affects the expression level of aquaporins in the kidney by regulating the level of antidiuretic hormone to weaken the reabsorption of primary urine by the kidney, and by regulating the expression of AQP2 and AQP3, it weakens the reabsorption capacity of the renal collecting duct to water, thereby increasing urine volume and exerting a diuretic function.

[0194] In summary, the results of this study showed that TAP can inhibit and downregulate the expression of AQP2 and AQP3 proteins, and its mechanism of action may be to weaken the renal collecting duct's ability to reabsorb water, thereby exerting a diuretic function. At the same time, it was found that TAP can slow down the renal inflammatory response and improve renal function damage, and its mechanism may be to inhibit the inflammatory cytokine response and increase urine output by downregulating the TLR4 / NF-κB / NLRP3 signaling pathway.

[0195] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0196] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A use of a Phellodendron chinense extract for diuresis in the preparation of a diuretic and / or edema-treating drug, wherein the Phellodendron chinense extract comprises phellodendronine and / or Phellodendron chinense total alkaloids, wherein the Phellodendron chinense total alkaloids are obtained from Phellodendron chinense by percolation extraction.

2. The use according to claim 1, characterized in that: The drug also includes pharmaceutically acceptable excipients.

3. The use according to claim 1, characterized in that: The Phellodendron chinense extract inhibits the body's inflammatory response, inhibits the expression of aquaporin in the kidney, and weakens the reabsorption of urine by the renal tubules and collecting ducts.

4. The use according to claim 1, characterized in that: The dosage form of the medicine is oral liquid, pill, granule, capsule, tablet, dripping pill, injection or powder.

5. The use according to claim 1, characterized in that: When the Phellodendron chinense extract includes Phellodendron alkaloid, the dosage of the Phellodendron alkaloid is 0.5-10 mg / kg / d; When the Phellodendron chinense extract includes Phellodendron chinense total alkaloids, the dosage of the Phellodendron chinense total alkaloids is 20-60 mg / kg / d; When the Phellodendron amurense extract comprises phellodendron alkaloids and total Phellodendron amurense alkaloids, the dosage of the Phellodendron amurense extract is 4-7 mg / kg / d.

6. A method for preparing total alkaloids of Phellodendron amurense in the Phellodendron amurense extract for use according to any one of claims 1 to 5, characterized in that: The preparation method comprises: mixing the cortex phellodendri with acid solution, adding a solvent, soaking, performing percolation extraction, collecting the percolation solution, concentrating, purifying, and obtaining the cortex phellodendri total alkaloids.

7. The preparation method according to claim 6, characterized in that: The soaking time is 80-90 hours, and the flow rate of the percolation extraction is 0.8-1.2 L / h.

8. A drug for diuresis and / or treatment of edema, characterized in that: The invention comprises a pharmaceutically active ingredient and a pharmaceutically acceptable auxiliary material, wherein the pharmaceutically active ingredient comprises the Phellodendron amurense extract used in any one of claims 1 to 5.

9. The drug according to claim 8, characterized in that The active pharmaceutical ingredients also include ingredients compatible with the Phellodendron chinense extract.

10. The total alkaloids of Phellodendron chinense obtained by the preparation method according to any one of claims 6-7.

Images