Preparation of ethanol extract of aquilaria sinensis and application thereof in preparation of medicine for inhibiting hyperuricemia nephropathy

Abstract

The present application provides a kind of preparation of Agarwood ethanol extract and its application in preparing medicine for inhibiting hyperuricemia nephropathy.The active substances in Agarwood can be effectively extracted in the extraction process, and the active substances in Agarwood can be well preserved, and the effect of Agarwood can be improved.The Agarwood ethanol extract prepared by the present application can be applied to hyperuricemia nephropathy, which can significantly relieve the problems of increased urine volume, kidney enlargement and other problems caused by hyperuricemia, and can improve the kidney function of hyperuricemia, and also has a protective effect on kidney, with good effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of eaglewood, in particular to a preparation method of ethanol extract of Aquilaria sinensis and application thereof in preparation of a medicine for inhibiting hyperuricemic nephropathy. BACKGROUND

[0002] Hyperuricemia (HUA) is a metabolic disease caused by the imbalance between uric acid synthesis and excretion, and its characteristic is that the blood uric acid level in the body is higher than the normal physiological concentration. The blood uric acid level of an adult is higher than 420 μmol / L twice a day, which is usually clinically judged as suffering from hyperuricemia. Hyperuricemic nephropathy (HN) is caused by the deposition of urate crystals in the kidney due to the sustained high uric acid state of the body, which causes uric acid kidney stones, kidney inflammation, renal tubular damage, and renal tubular interstitial fibrosis. At present, HUA has become a global epidemic. According to relevant statistics, its incidence rate is increasing year by year, and the proportion of young people is becoming higher and higher. HUA has become an independent risk factor for many diseases, and if not treated in time, it may further cause gouty nephropathy (GN), chronic kidney disease (CKD), end stage renal disease (ESRD), etc.

[0003] So far, the treatment of HN mainly focuses on drugs that inhibit uric acid production and promote uric acid excretion, such as first-line drugs allopurinol and benzbromarone. However, in actual clinical application, they are limited due to their serious side effects, for example, allopurinol can cause severe hypersensitivity reactions, and about half of the patients reflect that the uric acid cannot be reduced to the target level by using allopurinol alone, and benzbromarone has potential liver toxicity. Therefore, it is extremely feasible and necessary to research and develop a drug that can effectively reduce the blood uric acid level of patients and delay the occurrence and development of hyperuricemic renal damage.

[0004] Eaglewood is a rare traditional Chinese medicine with antibacterial, anti-inflammatory, antioxidant, cough-relieving, sleep-aiding and other pharmacological effects. The derivative series products of eaglewood play an important role in the fields of traditional Chinese medicine, health care and other fields. Aquilaria sinensis is considered to be the highest quality eaglewood and has a broad application prospect. So far, no one has reported the application of ethanol extract of Aquilaria sinensis in the medicine for inhibiting hyperuricemic nephropathy. SUMMARY

[0005] In view of this, the present application provides a preparation method of ethanol extract of Aquilaria sinensis and application thereof in preparation of a medicine for inhibiting hyperuricemic nephropathy.

[0006] The technical scheme of the present application is implemented as follows:

[0007] The application of the ethanol extract of Aquilaria sinensis in the preparation of a medicine for inhibiting hyperuricemia nephropathy.

[0008] The application of the ethanol extract of Aquilaria sinensis in the preparation of a medicine for reducing blood uric acid and delaying hyperuricemia kidney damage.

[0009] Further, the preparation method of the ethanol extract of Aquilaria sinensis comprises the following steps: crushing Aquilaria sinensis raw materials into agarwood powder, then adding an ethanol solution to soak for 60-180 min, and then heating and refluxing extraction for 3-4 times at 50-80 DEG C, filtering, and taking the filtrate to perform vacuum evaporation, thereby obtaining the ethanol extract of Aquilaria sinensis.

[0010] Further, the particle size of the agarwood powder is 40-60 mesh.

[0011] Further, the concentration of the ethanol solution is 90-98 v / v %.

[0012] Further, the concentration of the ethanol solution is 95 v / v %.

[0013] Further, the solid-liquid ratio of the agarwood powder and the ethanol solution is 1:(5-20) g / mL.

[0014] Further, the process of the reflux extraction is as follows: first, reflux extraction is performed at 50-60 DEG C for 60-80 min, then the temperature is increased to 65-70 DEG C for reflux extraction for 60-100 min, and finally the temperature is increased to 75-80 DEG C for reflux extraction for 50-70 min.

[0015] Further, the vacuum evaporation is performed at a temperature of 40-60 DEG C and a vacuum degree of -0.05 to -0.08 MPa for 2-3 h.

[0016] Further, the process of the vacuum evaporation is as follows: vacuum evaporation is performed at a temperature of 40-45 DEG C and a vacuum degree of -0.07 to -0.08 MPa for 1-1.5 h, then the temperature is increased to 50-60 DEG C, the vacuum degree is adjusted to -0.05 to -0.06 MPa, and vacuum evaporation is performed for 1-1.5 h.

[0017] Compared with the prior art, the present application has the following beneficial effects:

[0018] The active substances in the Aquilaria sinensis can be effectively extracted, and the active substances in the Aquilaria sinensis can be well reserved, and the effect of the Aquilaria sinensis is improved. The ethanol extract of the Aquilaria sinensis prepared by the application can be applied to the hyperuricemia nephropathy, and can significantly relieve the problems of increased urine volume and kidney enlargement caused by hyperuricemia, and can improve the kidney function of hyperuricemia, and also has a protective effect on the kidney, and has a good effect. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The urinary volume content of HN mice in each group.

[0020] Figure 2 The kidney index of HN mice in each group.

[0021] Figure 3 The SUA content of HN mice in each group.

[0022] Figure 4 The UUA content of HN mice in each group.

[0023] Figure 5 The SCr content of HN mice in each group.

[0024] Figure 6 The albuminuria content of HN mice in each group.

[0025] Figure 7 The BUN content of HN mice in each group.

[0026] Figure 8 The kidney pathological staining of HN mice in each group.

[0027] Figure 9 The Nlrp3 content of HN mice in each group.

[0028] Figure 10 The IL-1β content of HN mice in each group.

[0029] Figure 11 The Mcp-1 content of HN mice in each group.

[0030] Figure 12 The F4 / 80 content of HN mice in each group.

[0031] Figure 13The content of the renal fibrosis factor Fn1 of the HN mice in each group.

[0032] Figure 14 The content of the renal fibrosis factor Tgf-β1 of the HN mice in each group.

[0033] Figure 15 The content of the renal fibrosis factor Timp-1 of the HN mice in each group.

[0034] Figure 16 The content of the renal fibrosis factor Col4a1 of the HN mice in each group.

[0035] In the figure, NC is the normal control group of mice, HN is the model group of mice, AP is the positive drug AP (allopurinol) group of mice, BBR is the positive drug BBR (benzbromarone) group of mice, QN-L is the low-dose group of mice of ethanol extract of Aquilaria sinensis, QN-M is the medium-dose group of mice of ethanol extract of Aquilaria sinensis, and QN-H is the high-dose group of mice of ethanol extract of Aquilaria sinensis. DETAILED DESCRIPTION

[0036] In order to better understand the technical content of the present application, the following specific examples are provided to further illustrate the present application.

[0037] The experimental methods used in the embodiments of the present application are all conventional methods unless otherwise specified.

[0038] The materials, reagents, etc. used in the embodiments of the present application can be obtained from commercial channels unless otherwise specified.

[0039] Example 1 - Preparation method of ethanol extract of Aquilaria sinensis

[0040] The method comprises the following steps: crushing the raw material of Aquilaria sinensis into 50-mesh agarwood powder, then soaking it in an ethanol solution with a concentration of 90v / v% for 60 min, the ratio of agarwood powder to ethanol solution is 1:5 g / mL, first refluxing for 60 min at 50℃, then refluxing for 100 min at 65℃, and finally refluxing for 70 min at 75℃, filtering, taking the filtrate to vacuum evaporate at a temperature of 40℃ and a vacuum degree of-0.08 MPa for 1.5 h, then vacuum evaporate at a temperature of 60℃ and a vacuum degree of-0.06 MPa for 1 h, to obtain the ethanol extract of Aquilaria sinensis.

[0041] Example 2 - Preparation method of ethanol extract of Aquilaria sinensis

[0042] The preparation method comprises the following steps: crushing Agarwood raw materials into 40-mesh Agarwood powder, then adding 98-v / v % ethanol solution to soak for 180 min, the Agarwood powder and the ethanol solution are in a ratio of 1:15 g / mL, first extracting at 60 ℃ for 80 min, then increasing the temperature to 70 ℃ to extract for 90 min, finally increasing the temperature to 80 ℃ to extract for 60 min, filtering, taking the filtrate to evaporate under vacuum at 45 ℃ and a vacuum degree of -0.07 MPa for 1.5 h, then increasing the temperature to 50 ℃ and adjusting the vacuum degree to -0.05 MPa to evaporate under vacuum for 1.5 h, thereby obtaining the Agarwood ethanol extract.

[0043] Example 3 - Preparation method of Agarwood ethanol extract

[0044] The preparation method comprises the following steps: crushing Agarwood raw materials into 60-mesh Agarwood powder, then adding 95-v / v % ethanol solution to soak for 120 min, the Agarwood powder and the ethanol solution are in a ratio of 1:10 g / mL, first extracting at 60 ℃ for 70 min, then increasing the temperature to 65 ℃ to extract for 80 min, finally increasing the temperature to 75 ℃ to extract for 50 min, filtering, taking the filtrate to evaporate under vacuum at 40 ℃ and a vacuum degree of -0.075 MPa for 1.5 h, then increasing the temperature to 60 ℃ and adjusting the vacuum degree to -0.05 MPa to evaporate under vacuum for 1.5 h, thereby obtaining the Agarwood ethanol extract.

[0045] Example 4 - Preparation method of Agarwood ethanol extract

[0046] The preparation method comprises the following steps: crushing Agarwood raw materials into 50-mesh Agarwood powder, then adding 92-v / v % ethanol solution to soak for 100 min, the Agarwood powder and the ethanol solution are in a ratio of 1:20 g / mL, first extracting at 50 ℃ for 65 min, then increasing the temperature to 70 ℃ to extract for 60 min, finally increasing the temperature to 80 ℃ to extract for 55 min, filtering, taking the filtrate to evaporate under vacuum at 45 ℃ and a vacuum degree of -0.08 MPa for 2 h, thereby obtaining the Agarwood ethanol extract.

[0047] Comparative Example 1

[0048] The difference between the comparative example and Example 3 is that the concentration and the amount of the ethanol solution are different, and the rest is the same as Example 3. The concentration of the ethanol solution in the comparative example is 70-v / v %, and the Agarwood powder and the ethanol solution are in a ratio of 1:30 g / mL.

[0049] Comparative Example 2

[0050] The difference between the comparative example and Example 3 is that the extraction process is different, and the rest is the same as Example 3.

[0051] The preparation method of the ethanol extract of Aquilaria sinensis of the present comparative example comprises the following steps: the raw material of Aquilaria sinensis is crushed into agarwood powder with a mesh size of 60, then 95v / v% ethanol solution is added for soaking for 120 min, the ratio of agarwood powder to ethanol solution is 1:10 g / mL, reflux extraction is carried out at 70℃ for 1 h, ethanol solution is added to keep the ratio of liquid to solid at 1:10 g / mL, and then the extraction is repeated twice, filtration is carried out, all the filtrates are vacuum evaporated at a temperature of 40℃ and a vacuum degree of-0.075 MPa for 1.5 h, then the temperature is increased to 60℃ and the vacuum degree is adjusted to-0.05 MPa for vacuum evaporation for 1.5 h, and thus the ethanol extract of Aquilaria sinensis is obtained.

[0052] Comparative Example 3

[0053] The difference between the present comparative example and Example 3 is that the process of vacuum evaporation is different, and the rest is the same as Example 3.

[0054] The preparation method of the ethanol extract of Aquilaria sinensis of the present comparative example comprises the following steps: the raw material of Aquilaria sinensis is crushed into agarwood powder with a mesh size of 60, then 95v / v% ethanol solution is added for soaking for 120 min, the ratio of agarwood powder to ethanol solution is 1:10 g / mL, reflux extraction is carried out at 70℃ for 1 h, ethanol solution is added to keep the ratio of liquid to solid at 1:10 g / mL, and then the extraction is repeated twice, filtration is carried out, all the filtrates are vacuum evaporated at a temperature of 40℃ and a vacuum degree of-0.075 MPa for 1.5 h, then the temperature is increased to 60℃ and the vacuum degree is adjusted to-0.05 MPa for vacuum evaporation for 1.5 h, and thus the ethanol extract of Aquilaria sinensis is obtained.

[0055] The extraction yield (%) of the ethanol extract of Aquilaria sinensis prepared according to the preparation method of Example 1-4 and Comparative Examples 1-3 is calculated respectively, and the results are shown in Table 1 below.

[0056] Extraction yield (%) = mass of ethanol extract of Aquilaria sinensis / mass of raw material of Aquilaria sinensis x 100%

[0057] Table 1 Extraction yield of ethanol extract of Aquilaria sinensis

[0058] Item Recovery rate (%) Example 1 13.43 Example 2 14.06 Example 3 14.52 Example 4 12.68 Comparative Example 1 12.37 Comparative Example 2 10.41 Comparative Example 3 11.85

[0059] As shown in Table 1 above, through the synergistic effect of each step process of the present application, the active substance components in Aquilaria sinensis can be effectively extracted, and the extraction efficiency is high. Compared with Example 3, the ethanol solution concentration of Comparative Example 1 is not reasonable, the reflux extraction process parameters of Comparative Example 2 are not matched, and the process of vacuum evaporation of Comparative Example 3 is not scientific, which all have an impact on the extraction of the ethanol extract of Aquilaria sinensis. The preparation method of Example 3 of the present application can obtain the ethanol extract of Aquilaria sinensis with a higher yield.

[0060] Experimental process

[0061] The ethanol extract of Aquilaria sinensis obtained from Example 3 was subjected to related experiments for inhibiting hyperuricemia nephropathy, and the specific experimental process was as follows.

[0062] 1. Experimental raw materials

[0063] 1.1 Experimental animals

[0064] Purchased from Guangdong Medical Animal Center, body weight 18-20 g, 6-7 weeks old, SPF level C57BL / 6 male mice 70. Animal production license number: SCXK (Yue) 2022-0002. All animal experiments were carried out in accordance with the relevant provisions of the Chinese Animal Welfare Act, and were approved by the Laboratory Animal Ethics Committee of Guangdong Pharmaceutical University (No. gdpulacspf2022147).

[0065] 1.2 Concentration and preparation of experimental drug raw materials

[0066] Positive drug allopurinol tablets (AP, specification: 0.1 g, batch number: 20220502, Hefei Jiulian Pharmaceutical Co., Ltd.); positive drug benzbromarone tablets (BBR, specification: 50 mg, batch number: 2016612, Germany Herma Pharmaceutical Factory); modeling drug potassium oxonate powder (PO, item number: 156124, Sigma-Aldrich); modeling drug hypoxanthine powder (Hx, item number: H9377, Sigma-Aldrich).

[0067] (1) Preparation of 0.5% CMC-Na solvent

[0068] Weigh 5 g of CMC-Na powder into a 1 L glass bottle, add 1 L of water to the high-temperature sterilization pot, sterilize and dissolve for standby use.

[0069] (2) Preparation of PBS solvent

[0070] Pour a small bottle of PBS powder (item number: GH3308, Beijing Dingguo Changsheng Biotechnology Co., Ltd.) into a 1 L glass bottle, add 1 L of water to the high-temperature sterilization pot, sterilize and dissolve for standby use.

[0071] (3) Preparation of Aquilaria sinensis ethanol extract drug

[0072] The Aquilaria sinensis ethanol extract prepared in Example 3 was mixed with 0.5% CMC-Na, and then configured into experimental sample drug solution concentrations of Aquilaria sinensis ethanol extract low dose (QN-L) 0.005 g / mL, Aquilaria sinensis ethanol extract medium dose (QN-M) 0.01 g / mL and Aquilaria sinensis ethanol extract high dose (QN-H) 0.02 g / mL.

[0073] (4) Preparation of the modeling drug

[0074] ① Preparation of the potassium oxonate modeling drug solution: potassium oxonate was mixed with PBS solvent to prepare a potassium oxonate modeling drug solution with a concentration of 0.02 g / mL;

[0075] ② Preparation of the hypoxanthine modeling drug solution: hypoxanthine was mixed with 0.5% CMC-Na to prepare a hypoxanthine modeling drug solution with a concentration of 0.05 g / mL.

[0076] (5) Preparation of the positive drug

[0077] ① Preparation of the positive drug allopurinol (positive drug AP): allopurinol was mixed with 0.5% CMC-Na to prepare a positive drug allopurinol (positive drug AP) with a concentration of 0.0015 g / mL.

[0078] ② Preparation of the positive drug benzbromarone (positive drug BBR): benzbromarone was mixed with 0.5% CMC-Na to prepare a positive drug benzbromarone (positive drug BBR) with a concentration of 0.002 g / mL.

[0079] 1.3 Animal modeling and experimental pretreatment

[0080] Seventy male mice were randomly divided into 7 groups, 10 mice in each group, namely the normal control group (NC), the model group (HN), allopurinol (positive drug AP), benzbromarone (positive drug BBR), low-dose ethanol extract of Aquilaria sinensis group (QN-L), medium-dose ethanol extract of Aquilaria sinensis group (QN-M), and high-dose ethanol extract of Aquilaria sinensis group (QN-H).

[0081] The body weight of the mice was 30 g per mouse, the drug administration volume was 10 μL / g / d, and the drugs were prepared for each group of 10 mice. Continuous gavage was performed for 3 weeks (w) with 7 days of gavage per week.

[0082] At 9:00 every morning, the mice were given gavage, and the normal group and the model group were given 0.5% CMC-Na, and the other groups were given the corresponding treatment drugs. At 15:00 every afternoon, except for the normal group which was given 0.5% CMC-Na, the rest of the groups were given gavage of the hypoxanthine modeling drug solution (10 μL / g / d) combined with intraperitoneal injection of the potassium oxonate modeling drug solution (10 μL / g / d) to induce hyperuricemia. The same continuous process was performed for 3 weeks with 7 days per week to cause kidney injury in C57BL / 6 mice, and HN mice were obtained. During this period, the state, body weight, and food and water intake of the mice were recorded every day.

[0083] The mice were fasted but not deprived of water on the 20th day of administration, and 24h urine was collected. The 24h urine excretion volume (mL) of each mouse was recorded. The mice were taken blood 1.5h after the completion of modeling. After standing at room temperature for 30min, the blood was centrifuged at 3000r·min -1 for 15min, and serum was separated. After the mice were sacrificed by cervical dislocation, the kidneys were removed, and the cut-off kidneys were removed from the outer envelope and all adipose tissue. The kidney weight of each mouse was recorded. The kidney weight / body weight ratio was calculated as follows: kidney weight (mg) ÷ body weight (g) × 100%. The left kidney was dehydrated and embedded for pathological sectioning, and the right kidney and liver were stored in a -80°C freezer.

[0084] 1.4 Data statistics

[0085] The pathological results of this experiment were analyzed and statistically analyzed using Image J software; the experimental data were statistically analyzed using PrismGraphPad 9.0 software. All results are expressed as x ± SEM, and one-way ANOVA is used for comparison between groups. P<0.05 represents a statistically significant difference between groups. # represents a significant difference between the NC group and the HN group: #p<0.05, ##p<0.01, ###p<0.001, and ####p<0.0001; * represents a significant difference between the treatment group and the HN group: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0086] 1.5 Experimental process and results

[0087] (1) Comparison of urine volume and kidney weight / body weight ratio in HN mice

[0088] From Figure 1 and Figure 2 it can be seen that the collected urine volume and kidney / body weight ratio of each group of mice showed that the ethanol extract of Aquilaria sinensis had a significant effect on relieving the increase in urine volume and kidney enlargement caused by hyperuricemia.

[0089] (2) Determination of blood uric acid and urine uric acid in HN mice

[0090] The determination method of serum uric acid (SUA) and urine uric acid (UUA) is similar. After the experimental animals were taken, the animal blood supernatant and urine supernatant were obtained. Then, the uric acid (UA) detection kit (microplate method) purchased from Nanjing Jiancheng Biological Institute was used for determination. The specific operation is as follows:

[0091] Take 96-well enzyme-coated plates, add 5 μL distilled water to the blank wells, add 5 μL uric acid standard (concentration 400 μmol / L) to the standard wells, and add 5 μL corresponding vortex-mixed serum / urine supernatant sample to the test wells. Set a duplicate well for each of the above to reduce error. Add 250 μL of reagent 1 containing Tris-HCl buffer, peroxidase and uricase to each well, gently shake the 96-well enzyme-coated plate with a plate shaker to mix the reagent, and then incubate at 37°C for 10 minutes before detecting the OD value. Calculate the blood uric acid level / urine uric acid level of the sample according to the following formula. UA concentration (μmol / L) = (OD test-OD blank) / (OD standard-OD blank) calibration standard concentration. The experimental results are as follows Figure 3 and Figure 4 as shown.

[0092] From Figure 3 and Figure 4 it can be seen that the SUA level of the HN group is significantly increased, and the UUA level is significantly decreased, indicating that the hyperuricemia kidney disease model is successfully induced. The ethanol extract of Aquilaria sinensis reduces the SUA content and increases the UUA after administration, and the effect of the high-dose group is more prominent.

[0093] (3) Determination of serum creatinine and 24h urine protein of HN mice

[0094] ① Determination of serum creatinine (SCr) of HN mice

[0095] The collected blood samples were placed at 37°C for 30 min, centrifuged at 3000 rpm for 15 min, and the serum was collected and stored at -80°C. The creatinine level in the mouse serum was determined according to the Nanjing Jiancheng creatinine (CRE) test kit (item number C011-2) instructions, as follows:

[0096]

[0097] 37°C incubation for 5 min, detection of absorbance value A1 at 546 nm wavelength

[0098]

[0099] 37°C incubation for 5 min, detection of absorbance value A2 at 546 nm wavelength

[0100] Calculation formula: ΔA = A2-186 / 246×A1

[0101] Serum creatinine content (μmol / L) = (ΔA test-ΔA blank) / (ΔA standard-ΔA blank)×calibration standard concentration.

[0102] (2) Measurement of 24h albuminuria of HN mice

[0103] The 24h urine of each group of mice was collected and the urine volume was recorded. Appropriate samples were stored at -80°C. The protein level of mouse urine was determined according to the instructions of Nanjing Jiancheng Urine Protein Test Kit (Item No. C035-2), as follows:

[0104] Blank tube Standard tube Test tube Distilled water (mL) 0.05 / / 563 mg / L protein standard solution (mL) / 0.05 / Sample (mL) / / 0.05 CBB application liquid (mL) 3.0 3.0 3.0

[0105] Mix well: stand for 5 min, wavelength 595 nm, light path 1 cm, double distilled water zero, determine the absorbance of each tube.

[0106] Calculation formula: 24h urine protein content = urine protein concentration (mg / mL) x 24h urine volume (mL)

[0107] Urine protein concentration (mg / mL) = standard concentration (563 mg / L) x (measured OD value-blank OD value) ÷ (standard OD value-blank OD value).

[0108] The results of determination of serum creatinine and 24h urine protein of HN mice are as follows Figure 5 and Figure 6

[0109] From Figure 5 and Figure 6 It can be seen that serum creatinine and 24h urine protein, as parameters of glomerular filtration rate, reflect kidney function. The above two indicators of HN model group were significantly up-regulated, and were significantly down-regulated after treatment with A. anomala ethanol extract, indicating that A. anomala ethanol extract can increase the creatinine clearance rate of mouse kidney and glomerular filtration rate, and improve the kidney function of HN mice.

[0110] (4) Measurement of urea nitrogen of HN mice

[0111] Using mouse serum samples, the index was determined according to the instructions of Nanjing Jiancheng Urea Nitrogen (BUN) Test Kit (Item No. C013-2), as follows:

[0112]

[0113] Calculation formula: Urea nitrogen concentration (mmol / L) = (measured OD value-blank OD value) ÷ (standard OD value-blank OD value) x standard concentration (10 mmol / L) x N, wherein N: dilution multiple before sample test. The test results are as shown below Figure 7

[0114] From Figure 7 ​​It can be seen that the urea nitrogen data shows that the BUN of HN mice is significantly increased, and the ethanol extract of Agarwood has a very significant effect on reducing the urea nitrogen level in the blood of hyperuricemia kidney disease mice, indicating that it has a protective effect on the kidney of hyperuricemia kidney disease mice.

[0115] (5) H&E pathological staining of HN mouse kidney

[0116] (1) Fixation of kidney tissue: after cutting the kidney of one side of the mouse longitudinally, half of it was immediately immersed in neutral formalin solution for fixation.

[0117] (2) Tissue dehydration: after fixation, the tissue was placed in an automatic dehydrator and immersed in 70%-80%-90%-95% ethanol for half an hour, anhydrous ethanol I, anhydrous ethanol II for one hour, xylene I, xylene II for half an hour, low melting point paraffin, high melting point paraffin for one hour, and the tissue was embedded.

[0118] (3) Sectioning, spreading and sticking: after embedding the tissue block, the section was cut into a tissue section about 4 μm thick on the sectioning machine. The cut tissue section was attached to a glass slide and placed on a spreading table for spreading. When the tissue section was gradually flattened, the excess distilled water was removed and placed in an oven for drying.

[0119] (4) De-waxing, staining and mounting: de-waxing was performed according to xylene and decreasing concentration of alcohol, and the de-waxed section was immersed in hematoxylin solution for 10 min. The section was taken out and washed with water for 5 min. After the cell nucleus was dyed blue, 1% hydrochloric acid ethanol solution was used for differentiation, i.e. washing with water for 10 min. After washing with distilled water, the section was immersed in 1% eosin solution for 25 s. After removing the floating color with distilled water, dehydrating was performed with increasing concentration of ethanol, xylene was used for transparency, and bean-sized neutral gum was used for mounting.

[0120] (6) Masson pathological staining of HN mouse kidney

[0121] ① The pre-tissue processing operation steps are the same as (5).

[0122] ② De-waxing, staining and mounting: de-waxing was performed according to xylene and decreasing concentration of alcohol, and the de-waxed section was immersed in: a. hematoxylin for 5 min, and then washed with running water for 5 min; b. hydrochloric acid ethanol was used for differentiating the tissue for 1 s, and then washed with running water for 10 min; c. blue liquid was used for blueing for a few seconds, and then washed with running water until the water became light; d. acid magenta was used for staining for 3-5 min, and then washed with water; e. acetic acid working solution (distilled water: acetic acid solution was prepared according to a certain proportion) was used for washing the section; f. after phosphomolybdic acid solution treatment, the phosphomolybdic acid solution on the glass slide was poured off (without water washing); g. benzamine blue staining solution was used for re-staining, and then distilled water was used for removing the floating color; h. dehydrating was performed with increasing concentration of ethanol, xylene was used for transparency, and gum was used for mounting.

[0123] (7) Sirius red pathological staining of kidneys in HN mice

[0124] ① The initial tissue processing steps are the same as in (5); ② After dewaxing, let stand in distilled water for 5 minutes; ③ Stain with Sirius red for 30 minutes; ④ Rinse briefly with running water; ⑤ Dehydrate: 75% ethanol 2s → 85% ethanol 2s → 95% ethanol 2s → 100% ethanol 2s → xylene 2s → xylene 2s; ⑥ Air dry and mount with neutral resin. The staining results are as follows. Figure 8 As shown.

[0125] Depend on Figure 8 The staining results showed that HE staining revealed normal glomerular morphology, orderly tubular arrangement, normal cell morphology, and no obvious inflammatory infiltration in the kidneys of normal mice. In contrast, the HN model group mice exhibited pathological features such as glomerular structural atrophy, widespread tubular dilation and degeneration, disordered arrangement of tubular epithelial cells, vacuolar degeneration and partial detachment of some cells, and significant inflammatory cell infiltration. The ethanol extract of Qinan agarwood significantly improved these pathological features. This invention evaluated collagen deposition in the kidney tissues of mice in each group using Masson staining and Sirius red staining. Pathological results showed that, compared with the normal group, the model group mice induced by PO+Hx showed extensive collagen deposition, mainly expressed in the damaged and dilated tubular interstitium, with the most severe fibrosis. Compared with the model group, treatment with the ethanol extract of Qinan agarwood inhibited abnormal collagen proliferation in hyperuricemic kidneys, thereby alleviating renal fibrosis in hyperuricemic nephropathy mice.

[0126] (8) Detection of gene expression related to kidney inflammation and fibrosis in HN mice

[0127] Quantitative real-time PCR was used to detect the mRNA expression of renal inflammation-related markers: Nlrp3, IL-1β, Mcp-1, F4 / 80, and fibrosis-related factors: Fn1, Tgf-β1, Timp-1, and Col4a1.

[0128] Total RNA was extracted from kidney tissue using the Trizol method and reverse transcribed using the PrimeScript™ gDNA RT kit. Quantitative real-time PCR was performed using SYBR Green SuperMix, and Ct values ​​were measured. The PCR reaction conditions were: 95℃ for 5 min; 95℃ for 10 s; 60℃ for 15 s; 72℃ for 20 s; for 40 cycles. β-actin was used as an internal reference gene, and the relative expression level of each gene mRNA was calculated using the 2-ΔΔCt method (ΔCt = target gene Ct value - internal reference gene Ct value). The primer sequences are shown in the table below. The experimental results are as follows. Figure 9-16 .

[0129]

[0130]

[0131] By Figure 9-16 It can be seen that the fluorescence quantitative PCR detects the kidney inflammation related factors: Nlrp3, IL-1beta, Mcp-1, F4 / 80; fibrosis related factors: Fn1, Tgf-beta1, Timp-1, Col4a1 indexes, and the model group is up-regulated, which proves that the model is successful, and each index appears different degrees of down-regulation after administration of Qinnanshengxi ethanol extract, which indicates that Qinnanshengxi ethanol extract has the effect of improving the kidney inflammation and fibrosis of HN mice.

[0132] In summary, the present application constructs a hyperuricemic nephropathy mouse model by intraperitoneal injection of hypoxanthine modeling liquid (PO, 200mg / kg / d) for 21 consecutive days combined with gavage of potassium oxonate modeling liquid (Hx, 500mg / kg / d), and observes whether Qinnanshengxi ethanol extract has the effect of improving hyperuricemic nephropathy from the serum biochemical level of animals, kidney tissue pathological staining and gene level.

[0133] The size of the kidney of the mouse, the change of the kidney / body weight ratio, the content of uric acid, creatinine and urea nitrogen in the serum, the volume of urine and the content of uric acid and urine protein in the urine supernatant are observed. The experimental results show that Qinnanshengxi can effectively reduce the levels of blood uric acid, blood creatinine, urea nitrogen and 24h urine protein, and significantly increase the urine uric acid. It is indicated that Qinnanshengxi can inhibit the accumulation of uric acid and has the effect of protecting the kidney function of hyperuricemic nephropathy mice. Combined with the staining results of H&E, MASSON and Sirius red, Qinnanshengxi can reduce glomerular atrophy, renal tubular dilation and interstitial damage, improve inflammatory infiltration and collagen deposition. And the q-PCR (quantitative real-time PCR) analysis of inflammatory cytokines and fibrosis cytokines from the mR NA level shows that Qinnanshengxi can down-regulate the expression of inflammatory indexes and fibrosis indexes, and improve the kidney inflammation and fibrosis of HN mice. Therefore, it is considered that Qinnanshengxi can reduce the level of uric acid and has the effect of protecting the kidney function of hyperuricemic nephropathy mice.

[0134] The above merely describes preferred embodiments of the present application and is not intended to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. The use of ethanol extract of Agarwood in the preparation of a drug for inhibiting hyperuricemia nephropathy, characterized in that, The preparation method of the ethanol extract of Aquilaria sinensis includes the following steps: crushing the raw material of Aquilaria sinensis into agarwood powder, then adding ethanol solution to soak for 60-180 min, heating and refluxing for 3-4 times at 50-80 DEG C, filtering, and taking the filtrate to vacuum evaporate, thus obtaining the ethanol extract of Aquilaria sinensis.

2. The application of the ethanol extract of Aquilaria sinensis in preparing a medicine for reducing blood uric acid and delaying kidney damage of hyperuricemia.

3. Use according to claim 1, characterized in that, The particle size of the agarwood powder is 40-60 mesh.

4. Use according to claim 1, characterized in that, The concentration of the ethanol solution is 90-98 v / v %.

5. The use according to claim 1, characterized in that, The ratio of the agarwood powder to the ethanol solution is 1:(5-20) g / mL.

6. Use according to claim 1, characterized in that, The process of the reflux extraction is: first refluxing at 50-60 DEG C for 60-80 min, then increasing the temperature to 65-70 DEG C to reflux for 60-100 min, and finally increasing the temperature to 75-80 DEG C to reflux for 50-70 min.

7. The use according to claim 1, characterized in that, The vacuum evaporation is carried out at a temperature of 40-60 DEG C and a vacuum degree of -0.05 to -0.08 MPa for 2-3 h.

8. Use according to claim 7, characterized in that, The process of the vacuum evaporation is: vacuum evaporation at a temperature of 40-45 DEG C and a vacuum degree of -0.07 to -0.08 MPa for 1-1.5 h, then increasing the temperature to 50-60 DEG C and adjusting the vacuum degree to -0.05 to -0.06 MPa for vacuum evaporation for 1-1.5 h.

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