Preparation method of processed cortex phellodendri product extract for treating diabetic nephropathy

Abstract

The invention belongs to the technical field of traditional Chinese medicine preparation, and particularly relates to a preparation method of a processed cortex phellodendri product extract for treating diabetic nephropathy. The preparation method comprises the following steps: taking 100g of amur corktree bark decoction pieces, adding 40mL of a salty salt aqueous solution, uniformly stirring, moistening for 2 hours, steaming for 0.5-2 hours, and drying to obtain a processed product; and carrying out reflux extraction twice with 70% ethanol, concentrating, and extracting with ethyl acetate to obtain the active extract. The invention optimizes and determines the key process parameters of mixing and steaming the amur corktree bark for the first time, so that the amur corktree bark has the pharmacological activity obviously superior to that of the traditional processed product in the treatment of diabetic nephropathy. Experiments show that the processed product can significantly improve related indexes of diabetic nephropathy model animals, and the ethyl acetate extract of the processed product shows better antioxidation and cell protection effects in high glucose damaged kidney cells. The process is stable and controllable, the obtained extract is high in targeting property, a new strategy is provided for developing an efficient and quality-controllable traditional Chinese medicine preparation for resisting diabetic nephropathy, and the method has important clinical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine preparation, and particularly relates to a method for preparing a processed Phellodendron chinense extract for treating diabetic nephropathy. Background Art

[0002] Phellodendron chinense Schneid is a commonly used Chinese medicinal herb with the effects of clearing heat and dampness, purging fire and detoxifying. It is the dried bark of the Rutaceae tree, Phellodendron chinense Schneid. The current Chinese Pharmacopoeia lists processing specifications for raw Phellodendron, salted Phellodendron, wine-processed Phellodendron, and Phellodendron charcoal. However, there is a significant technical gap in the standardized process for processing Phellodendron using autumn stone. As a traditional Chinese medicine, autumn stone possesses unique preparation techniques and medicinal value. The mainstream autumn stone on the market is salty autumn stone, a processed salt product. It has a salty flavor, cold properties, and is non-toxic. It enters the lung and kidney meridians and can treat ailments such as consumptive disease, cough and hemoptysis, bone steaming and hot flashes, frequent urination, and nocturnal emission. Zhang Zhongyan's "Xiu Shi Zhi Zhi" (Guide to Cultivating Things) from the Qing Dynasty discusses the theory of autumn stone processing, stating that autumn stone processing suppresses yang and nourishes yin, suggesting that processing Chinese medicines using autumn stone can achieve the goal of nourishing yin and reducing internal heat. Steaming is a method of processing Chinese medicines that enhances efficacy, moderates medicinal properties, and provides tonic benefits. By mixing steamed Phellodendron amurense with salty autumn stone, the drug can be guided downward into the kidney, which not only enhances its function of nourishing kidney yin, but also strengthens its effect of purging kidney fire, showing its unique advantages in nourishing kidney yin and purging kidney fire. Therefore, it has important clinical value in the treatment of kidney yin deficiency syndrome. Therefore, using salty autumn stone mixed with steamed Phellodendron amurense can provide a potential solution for the treatment of kidney yin deficiency syndrome in the early and middle stages of diabetic nephropathy.

[0003] The current method for steaming Phellodendron amurense with salty autumn stone is plagued by technical bottlenecks and a lack of process parameters. Existing literature and patents do not clearly define key parameters for steaming Phellodendron amurense with salty autumn stone (such as the amount of auxiliary ingredients added and the duration of steaming). This results in poor process reproducibility and an unknown active site. Previous studies have shown that directly steaming the product has limited kidney-tonifying effects and requires extraction with a specific polar solvent to achieve optimal therapeutic effects.

[0004] Based on the above research, the present invention established for the first time a standardized preparation process for steamed Phellodendron amurense mixed with Xianqiushi and its active extraction method, ensuring the clinical application and efficacy of traditional processing technology through "precision processing-directional extraction", and providing a new solution for the precise treatment of diabetic nephropathy. Summary of the Invention

[0005] The present invention aims to provide a method for preparing an extract of processed Phellodendron chinense for treating diabetic nephropathy. This method clarifies the processing technology and pharmacological efficacy of steamed Phellodendron chinense mixed with Xianqiushi, providing a basis for the development of new drugs. The extract of steamed Phellodendron chinense mixed with Xianqiushi obtained by this method has excellent therapeutic effects on diabetic nephropathy.

[0006] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions.

[0007] A method for preparing a processed extract of Phellodendron amurense for treating diabetic nephropathy comprises the following steps:

[0008] Step 1: Take 100g of pure Huangbai slices, add 40mL of Xianqiushi water and mix well, stir well and let it soak for 2 hours until the medicine is fully absorbed, then use a steamer to steam and dry to obtain the processed Huangbai mixed with Xianqiushi;

[0009] Step 2: Take the above-mentioned processed product of steamed Phellodendron amurense mixed with salty autumn stone, add 70% ethanol solvent for extraction, reflux extraction twice, each time for 1 hour, combine the two filtrates, and concentrate the filtrates until the extract has no alcohol taste to obtain the extract of steamed product of Phellodendron amurense mixed with salty autumn stone; disperse the extract with an appropriate amount of water, extract twice with ethyl acetate, recover the solvent, combine the two extraction solvents, obtain the ethyl acetate extract, and then dissolve it with water to adjust the concentration to 0.13 g / mL to obtain the processed Phellodendron amurense extract.

[0010] Furthermore, in step 1, the mass of the salty autumn stone in the salty autumn stone water is 1 to 3% of the mass of the pure Phellodendron chinense slices.

[0011] Furthermore, in step 1, the steaming time is 0.5 hours to 2 hours.

[0012] Furthermore, in step 2, the mass of 70% ethanol is 5 to 15 times the mass of the processed product.

[0013] The processed extract of Phellodendron amurense can be used in medicines for treating diabetic nephropathy.

[0014] Compared with the prior art, the present invention has the following beneficial effects.

[0015] 1. The current existing technology still lacks clear parameters in the process of mixing salty autumn stone with steamed Phellodendron amurense, which may easily lead to the failure of the preparation to achieve the expected effect. In addition, this technology also highlights the effective polarity of steamed salty autumn stone in treating diabetic nephropathy, and conducts a comparative study on the effects of salty autumn stone mixed with steamed Phellodendron amurense of different polarities in treating diabetic nephropathy. It is found that the effect of the ethyl acetate layer of salty autumn stone mixed with steamed Phellodendron amurense is more prominent, which is more conducive to the treatment of diabetic nephropathy.

[0016] 2. This patent establishes a diabetic nephropathy rat model and a high-glucose injury model for human renal cortical proximal tubule epithelial HK-2 cells. This validates the therapeutic efficacy of steamed Phellodendron amurense mixed with salty autumn stone in treating diabetic nephropathy. It also identifies for the first time the ethyl acetate polarity of processed Phellodendron amurense mixed with salty autumn stone, which significantly enhances the cell protection rate and antioxidant activity of HK-2 human renal cortical proximal tubule epithelial cells. This technology can enhance the medicinal value of processed Phellodendron amurense, clarify the medicinal efficacy of steamed Phellodendron amurense mixed with salty autumn stone, and provide a basis for the further development of Phellodendron amurense as a drug for treating diabetic nephropathy.

[0017] 3. The preparation method and use of the processed product of Xianqiushi mixed with steamed Phellodendron chinense provided by the present invention, the processing technology of Xianqiushi mixed with steamed Phellodendron chinense used has not been recorded in existing research reports. The preparation method has the characteristics of stable process, simple operation and low cost, and the processed product of Xianqiushi mixed with steamed Phellodendron chinense has a good effect in treating diabetic nephropathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the high performance liquid chromatogram of the test sample in group No. 8 (where 1 is phellodendronine, 2 is berberine, and 3 is berberine hydrochloride).

[0019] Figure 2 It is the extract of the processed product of Xianqiushi mixed with steamed Huangbai extracted with ethyl acetate. DETAILED DESCRIPTION

[0020] The present invention will be further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above contents of the present invention fall within the scope of the present invention.

[0021] Example 1.

[0022] 1. Instruments and materials.

[0023] 1.1 Instruments: Agilent 1100 high-performance liquid chromatograph, METTLER AE240 1 / 100,000 analytical balance, CNC ultrasonic cleaner, oven (WGL-125B), blood glucose meter (SN 1QN95S06219), double-person single-sided clean bench (Shanghai Zhicheng), MCO-15AC carbon dioxide constant temperature incubator (Sanyo Co., Japan), microplate reader (Thermo Fisher), TGL-16C high-speed desktop centrifuge, -80°C refrigerator (Qingdao Haier), micropipette (Thermo Fisher).

[0024] 1.2 Reagents: Phellodendron amurense (Batch No. 2302001) was purchased from Anguo Juyaotang Pharmaceutical Co., Ltd.; acetonitrile and methanol were chromatographically graded, water was double-distilled, and all other reagents were analytically graded. Berberine reference substance (Batch No. A1107AS) and phellodendron amurense reference substance (Batch No. M0107AS) were purchased from Dalian Meilun Biotechnology Co., Ltd.; berberine hydrochloride reference substance (Batch No. MUST-16040702) was purchased from Chengdu Mansite Biotechnology Co., Ltd. for content determination, with purities greater than 98%. Xianqiushi (Batch No. 2211001) was purchased from Anguo Juyaotang Pharmaceutical Co., Ltd. Streptozotocin (STZ) (lot B2001), irbesartan dispersible tablets (lot 2310216), BUN kit (lot 24071538N), SCR kit (lot 24071535N), MAU / ALB (lot 24052458N), glucose (lot 24231121003), mannitol (lot 34230821001), 75% medical disinfectant alcohol, CCK8 kit (Mei Lun Biotechnology Co., Ltd.), and MDA kit (lot 2403005) were all purchased from Wuhan Sanying Biotechnology Co., Ltd. HK-2 cell line (Mei Lun Biotechnology Co., Ltd.), DMEM / f12 high-glucose medium (Mei Lun Biotechnology Co., Ltd.), FBS fetal bovine serum, penicillin-streptomycin-amphotericin B mixed triple antibody solution, trypsin, and PBS were all purchased from Mei Lun Biotechnology Co., Ltd. Water was purified, and all other reagents were of analytical grade.

[0025] 2. This embodiment provides a method for preparing an extract of processed Phellodendron amurense for treating diabetic nephropathy, comprising the following steps:

[0026] Take 100g of pure Phellodendron slices, add 40mL of salty autumn stone water and mix well (the amount of salty autumn stone is 3% of the pure Phellodendron slices), and let it sit for 2 hours until the salty autumn stone water is completely absorbed and the salty autumn stone water completely penetrates into the internal tissue of the Phellodendron slices. Place the moistened Phellodendron on a steamer and steam for 1 hour; dry, cool, remove impurities, take an appropriate amount of the above-mentioned salty autumn stone mixed with steamed Phellodendron processed product, add 10 times 70% ethanol for reflux extraction, extract twice, each time for 1 hour, combine the two extracts, and concentrate the filtrate until the extract has no alcohol taste to obtain the Phellodendron salty autumn stone mixed with steamed product extract, disperse the extract with an appropriate amount of water, extract twice with ethyl acetate, recover the solvent, combine the two extraction solvents, obtain the ethyl acetate extract, and then dissolve it with water to adjust the concentration to 0.13g / mL to obtain the extract of the Phellodendron processed product.

[0027] 3. The contents of berberine hydrochloride, berberine and phellodendron pine after steaming with salty autumn stone were used as evaluation indicators, and the amount of auxiliary materials added and the steaming time were selected as investigation factors. The raw phellodendron chinense was moistened with the auxiliary material (salty autumn stone) amount of 0% (steamed), 1%, 2% and 3%. Then the above samples were steamed in the steaming container for 30 minutes, 60 minutes, 90 minutes and 120 minutes respectively, and 16 samples of steamed phellodendron chinense mixed with salty autumn stone at different factor levels were prepared, so as to select the optimal processing technology of steamed phellodendron chinense mixed with salty autumn stone.

[0028] Evaluation indexes of berberine hydrochloride, phellodendrine, and berberine were determined using high-performance liquid chromatography. Chromatographic conditions: Ecosil C18 column (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile-water (containing 0.3% phosphoric acid and 0.3% potassium dihydrogen phosphate); gradient elution: 10%-14% acetonitrile (0-10 min); 14%-25% acetonitrile (10-20 min); 25% acetonitrile (20-45 min); 25%-75% acetonitrile (45-50 min). Detection wavelength: 284 nm; flow rate: 0.8 mL / min; column temperature: 40°C; injection volume: 5 μL.

[0029] Preparation of reference solution: Accurately weigh berberine hydrochloride reference, phellodendronine reference and berberrubine reference respectively and place them in 50 mL volumetric flasks, add chromatographic methanol to dilute to the scale to obtain 0.968 mg / mL berberine hydrochloride reference solution, 0.991 mg / mL phellodendronine reference solution, and 0.667 mg / mL berberrubine reference solution.

[0030] Preparation of test solution: Take each salty autumn stone, mix with the steamed test sample, crush (pass through a No. 4 sieve), accurately weigh 2.0g, place in a 250mL stoppered conical flask, accurately add 100mL of 20% methanol, ultrasonically treat for 40min, cool, shake well, filter, and take the filtrate. The HPLC chromatogram of the test sample in group 8 is shown in Figure 1 The measurement results are shown in Table 1.

[0031] Table 1 Content score of steamed Phellodendron chinense mixed with Xianqiushi

[0032]

[0033] Comprehensive score = [0.4 × (Xi / Xmax) + 0.4 × (Yi / Ymax) + 0.2 × (Zi / Zmax)] × 100%

[0034] In the above formula, X represents the berberine hydrochloride content, Y represents the phellodendronine content, and Z represents the berberine content. The parameters for the three indicators of the comprehensive score of steamed Phellodendron mixed with salty autumn stone were set to 40%, 40%, and 20%, respectively. A visual analysis of the index component content at each factor level revealed that the optimal processing technique for steaming Phellodendron mixed with autumn stone is to take 100g of pure Phellodendron slices, add 40mL of salty autumn stone water (the amount of salty autumn stone is 3% of the pure Phellodendron slices), mix thoroughly, and steam for 2 hours until the salty autumn stone water is completely absorbed and fully penetrates the internal structure of the Phellodendron slices. The steamed Phellodendron slices are then steamed in a steamer for 1 hour. These process parameters are used as the processing technique for steaming Phellodendron mixed with salty autumn stone.

[0035] 4. To further verify the beneficial effects of the present invention, the following experimental case of "treating diabetic nephropathy with Xianqiushi mixed with steamed Phellodendron amurense" is provided.

[0036] 4.1 Determination method

[0037] After one week of adaptive feeding, the rats were randomly divided into a blank group and a model group. All groups, except the blank group, were fed a high-sugar, high-fat diet for four weeks, fasted for 12 hours but not water, and intraperitoneally injected with STZ (35 mg / kg). Blood glucose levels were measured 72 hours later. After another week of feeding, urine was collected for microalbuminuria measurement. A successful DN model was established in rats when blood glucose levels ≥16.7 mmol / L and 24-hour urine MAU / ALB levels ≥20 mg / L. The successfully established rats were randomly divided into six groups, each containing five rats: a model control group, a positive drug group (irbesartan), a low-dose raw Phellodendron amurense group, a high-dose raw Phellodendron amurense group, a low-dose Xianqiu Shizhi-prepared Phellodendron amurense group, and a high-dose Xianqiu Shizhi-prepared Phellodendron amurense group. Drugs were administered orally once daily. The blank and model groups received the same dose of distilled water for four consecutive weeks, with each group receiving 1 mL / 100 g of drug. Rats were weighed weekly, and blood glucose was measured with a glucometer. Twenty-four hours after the last dose, rat urine was collected for 24-hour measurement of MAU / ALB levels. Anesthetized rats were anesthetized with an intraperitoneal injection of urethane solution (20 g / mL). Blood was collected from the abdominal aorta and centrifuged at 3000 rpm for 15 minutes. The supernatant was collected and stored at -80°C until further use. After collection, the urine was centrifuged at 4000 × g for 10 minutes. The supernatant was collected and assayed for MAU / ALB levels according to the kit instructions. Rat serum was used for SCR and BUN assays.

[0038] 4.2 Measurement results

[0039] 4.2.1 Detection of S-Cr levels in rats in each group

[0040] Table 2 S-Cr content in rats in each group (mean ± SD, n = 5)

[0041]

[0042]

[0043] Compared with the blank group, * P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01; the same dose of Xianqiushi mixed with steamed Phellodendron chinense group was compared with raw Phellodendron chinense: a P<0.05, aa P<0.01.

[0044] 4.2.2 Detection of BUN levels in rats in each group

[0045] Table 3 BUN content of rats in each group (mean ± SD, n = 5)

[0046]

[0047] Compared with the blank group, * P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01; the same dose of Xianqiushi mixed with steamed Phellodendron chinense group was compared with raw Phellodendron chinense: a P<0.05, aa P<0.01.

[0048] 4.2.3 Detection of MAU / ALB levels in rats in each group

[0049] Table 4 MAU / ALB content of rats in each group

[0050]

[0051] Compared with the blank group, * P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01; the same dose of Xianqiushi mixed with steamed Phellodendron chinense group was compared with raw Phellodendron chinense: a P<0.05, aa P<0.01.

[0052] Compared with the blank group, the model group showed increased levels of S-Cr, BUN, and MAU / ALB. Compared with the model group, each drug-treated group showed varying degrees of reduction in these levels. The positive group, the low-dose raw Phellodendron chinense group, the high-dose Xianqiushi mixed with steamed Phellodendron chinense group, and the low-dose Xianqiushi mixed with steamed Phellodendron chinense group showed the most significant decreases in S-Cr (P < 0.01); the high-dose Xianqiushi mixed with steamed Phellodendron chinense group and the low-dose Xianqiushi mixed with steamed Phellodendron chinense group showed the most significant decreases in BUN (P < 0.01). The positive drug group, the high-dose Xianqiushi mixed with steamed Phellodendron chinense group, and the low-dose Xianqiushi mixed with steamed Phellodendron chinense group showed the most significant decreases in MAU / ALB (P < 0.01). In summary, the in vivo experiments confirm that the Xianqiushi mixed with steamed Phellodendron chinense in this patent have the effect of treating diabetic nephropathy.

[0053] 5. To further verify that the extracted part of the present invention has the best activity, the following experimental cases of "treating diabetic nephropathy with steamed Phellodendron amurense mixed with Xianqiushi" extracted with solvents of different polarity are provided.

[0054] 5.1 Determination method.

[0055] HK-2 cells were obtained, digested with trypsin, and the cell concentration was adjusted to 1×10 5 , seeded into a 96-well plate, 100 μL of cell suspension was added to each well, and cultured in an incubator for 24 hours. After the cells adhered, 100 μL of drug solution containing high-glucose complete medium was added to the experimental wells, and 100 μL of DMEM / F12 complete medium containing high glucose was added to the model group. A blank well without cells and a high osmotic pressure control group were also set up to eliminate osmotic pressure interference. Three parallel wells were set up for each mass concentration. 100 μL of petroleum ether extract, ethyl acetate extract, and n-butanol extract of raw Phellodendron chinense and steamed Phellodendron chinense mixed with Xianqiushi were added to the high-glucose drug group, respectively, containing high-glucose complete medium and a drug concentration of 100 μg / mL. 100 μL of DMEM / F12 high-glucose complete medium was added to the blank group and model group. Three replicate wells were made in parallel for each drug group. After culturing in the incubator for 48 h, 100 μL of CCK-8 solution was added, and the cells were moved to the incubator and incubated for 2 h. The absorbance (A) value of each well was detected at 450 nm on a microplate reader, and the cell survival rate was calculated. The relative maximum survival rate concentration of the three was used as the later cell administration concentration.

[0056] Cell protection rate = (OD value of drug administration group - OD value of model group) / OD value of model group

[0057] According to the above grouping, HK-2 were placed in 6-well plates and cultured for 48 hours. The MDA content was measured using an enzyme-labeled instrument according to the kit instructions. SPSS 16.0 was used for statistical analysis. The comparison among the groups was conducted by one-way analysis of variance, and the difference was statistically significant when P < 0.05.

[0058] 4.2 Measurement results

[0059] 4.2.1 Effects of Xianqiushi mixed with steamed Phellodendron amurense before and after administration on the cell protection rate of HK-2 cells damaged by high glucose

[0060] Table 5 Differences in cell protection rates of different extraction sites of processed products (mean ± SD, n = 3)

[0061]

[0062]

[0063] The same dose of Xianqiushi mixed with steamed Phellodendron chinense was compared with the raw product: a P<0.05, aa P<0.01

[0064] 4.2.2 Effects of Xianqiushi on malondialdehyde (MDA) content in HK-2 cells before and after steaming Phellodendron amurense

[0065] Table 6 Effects of different drug administration groups on the malondialdehyde (MDA) content in HK-2 cells

[0066]

[0067] Compared with the blank group, * P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01; the same dose of Xianqiushi mixed with steamed Phellodendron chinense group was compared with raw Phellodendron chinense: a P<0.05, aa P<0.01.

[0068] From the experimental results, it can be seen that compared with the model group, the steamed product of Phellodendron chinense mixed with Xianqiushi can significantly increase the cell protection rate and malondialdehyde (MDA) content under high glucose damage (P<0.05 or P<0.01), indicating that steamed Phellodendron chinense mixed with Xianqiushi has a certain effect in treating diabetic nephropathy. In the pharmacodynamic comparison of different polar parts, it was found that the polar part of the ethyl acetate layer has the best therapeutic effect on diabetic nephropathy. In summary, in the preparation process of the extract of steamed Phellodendron chinense mixed with Xianqiushi for treating diabetic nephropathy in this patent, the polar part of ethyl acetate is used for extraction and the diabetic nephropathy is treated.

[0069] Example 2.

[0070] 100g of pure Phellodendron decoction pieces were mixed with 40mL of salty autumnal stone water (the amount of salty autumnal stone was 2% of the pure Phellodendron decoction pieces). After being steamed for 2 hours until the salty autumnal stone water was completely absorbed, the mixture was steamed in a steamer for 1 hour and dried to obtain the salty autumnal stone-steamed Phellodendron. The steamed Phellodendron decoction was extracted with 5 times the amount of 70% ethanol under reflux twice, each time for 1 hour. The mixture was filtered, the filtrates were combined, and the ethanol was recovered under reduced pressure until the alcohol flavor was eliminated to obtain the alcohol extract. The extract was dispersed in an appropriate amount of water and extracted with petroleum ether. The remaining aqueous layer was extracted again with petroleum ether, and the solvent was recovered. The two petroleum ether extraction solutions were combined and then dissolved in water. The concentration was adjusted to 0.13g / mL to obtain the processed Phellodendron decoction. The test results are shown in Tables 5 and 6.

[0071] Example 3.

[0072] 100g of pure Phellodendron decoction pieces were mixed with 40mL of salty autumnal stone water (the amount of salty autumnal stone was 2% of the pure Phellodendron decoction pieces). The mixture was allowed to stand for 2 hours until the salty autumnal stone water was completely absorbed. The mixture was then steamed in a steamer for 1 hour and dried to obtain the salty autumnal stone-steamed Phellodendron decoction pieces. The steamed Phellodendron decoction pieces were then extracted with 5 times the amount of 70% ethanol under reflux twice, each time for 1 hour. The mixture was filtered, the filtrates were combined, and the ethanol was recovered under reduced pressure until the alcohol flavor was eliminated to obtain the alcohol extract. The extract was then dispersed in an appropriate amount of water and extracted with ethyl acetate. The remaining aqueous layer was then extracted with ethyl acetate again. The solvent was recovered, and the two ethyl acetate extracts were combined. The extract was then dissolved in water and the concentration was adjusted to 0.13g / mL to obtain the processed Phellodendron decoction. The test results are shown in Tables 5 and 6.

[0073] Example 4.

[0074] 100g of pure Phellodendron decoction pieces were mixed with 40mL of salty autumnal stone water (the amount of salty autumnal stone was 2% of the pure Phellodendron decoction pieces). After being steamed for 2 hours until the salty autumnal stone water was completely absorbed, the pieces were steamed in a steamer for 1 hour and dried to obtain the salty autumnal stone-steamed Phellodendron decoction pieces. The steamed Phellodendron decoction pieces were then extracted with 5 times the amount of 70% ethanol under reflux twice, each for 1 hour. The filtrates were combined and the ethanol was recovered under reduced pressure until the alcohol flavor was eliminated to obtain the alcohol extract. The extract was dispersed in an appropriate amount of water and extracted with n-butanol. The remaining aqueous layer was extracted again with n-butanol and the solvent was recovered. The two n-butanol extraction solutions were combined and then dissolved in water. The concentration was adjusted to 0.13g / mL to obtain the processed Phellodendron decoction. The test results are shown in Tables 5 and 6.

Claims

1. A method for preparing a processed extract of Phellodendron amurense for treating diabetic nephropathy, characterized in that: The steps include: Step 1: Take the purified pieces of Phellodendron chinense, add Xianqiushi water and mix well, mix well and let it sit for 2 hours until the medicine is fully absorbed, then steam and dry in a steamer to obtain the processed Phellodendron chinense mixed with Xianqiushi; Step 2: Take the above-mentioned processed product of steamed Phellodendron amurense mixed with salty autumn stone, add 70% ethanol solvent for extraction, reflux extraction twice, each time for 1 hour, combine the two filtrates, and concentrate the filtrates until the extract has no alcohol taste to obtain the extract of steamed product of Phellodendron amurense mixed with salty autumn stone; disperse the extract with an appropriate amount of water, extract twice with ethyl acetate, recover the solvent, combine the two extraction solvents, obtain the ethyl acetate extract, and then dissolve it with water to adjust the concentration to 0.13 g / mL to obtain the processed Phellodendron amurense extract.

2. The method for preparing the processed extract of Phellodendron amurense for treating diabetic nephropathy according to claim 1, characterized in that: In step 1, add 40 mL of Xianqiushi water to every 100 g of Phellodendron chinense slices and mix well.

3. The method for preparing the processed extract of Phellodendron amurense for treating diabetic nephropathy according to claim 1, characterized in that: In the step 1, the mass of the salty autumn stone in the salty autumn stone water is 1-3% of the mass of the pure Phellodendron chinense slices.

4. The method for preparing a processed extract of Phellodendron amurense for treating diabetic nephropathy according to claim 1, characterized in that: In step 1, the steaming time is 0.5 hours to 2 hours.

5. The method for preparing the processed extract of Phellodendron amurense for treating diabetic nephropathy according to claim 1, characterized in that: In step 2, the mass of 70% ethanol is 5 to 15 times the mass of the processed product.

6. The method for preparing the processed extract of Phellodendron amurense for treating diabetic nephropathy according to any one of claims 1 to 5, characterized in that: The extract of the processed Phellodendron amurense is used in medicines for treating diabetic nephropathy.

7. The method for preparing the processed extract of Phellodendron amurense for treating diabetic nephropathy according to any one of claims 1 to 5, characterized in that: The extract of the processed product of Phellodendron chinense can improve the levels of S-Cr, BUN, and MAU / ALB in rats with diabetic nephropathy, thereby achieving the effect of treating diabetic nephropathy.

8. The method for preparing the processed extract of Phellodendron amurense for treating diabetic nephropathy according to any one of claims 1 to 5, characterized in that: The extract of the processed product of Phellodendron amurense can improve the cell protection rate and MDA level of renal cells damaged by high sugar, thereby achieving the effect of treating diabetic nephropathy.

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