TW201223533A - Usage of astragalosides, and pharmaceutical composition comtaining astragalosides - Google Patents

Abstract

A pharmaceutical composition comprises astragalosides for treating aristolochic acid nephropathy which includes symptoms such as tubular atrophy, cell infiltration and interstitial fibrosis.

Description

201223533 VI. Description of the Invention: [Technical Field] The present invention relates to a pharmaceutical composition for treating kidney disease, and more particularly to a medical composition containing scutellaria, which can be used to treat aristolochic acid. Kidney disease. [Prior Art] In 1991, a group of young women in Belgium took a drug from the same weight-loss clinic, and rapid interstitial nephritis occurred. After investigation, it was discovered that it may be aristolochic acid in the drug ingredient. related. Aristolochic acid nephropathy is a rapidly progressing interstitial nephritis that often leads to end stage renal disease and urinary tract cancer. Aristolochic acid (AA) is a common component of Aristolochia plants, including a mixture of 10-nitrophenanthrene-l-acid as the basic skeleton. Aristolochic acid I (AAI) and Aristolochic acid II (AAII) (Figure 1). The boiling point of aristolochic acid I (boiling point, B.P.) is 275-278 ° C, and the onset temperature of decomposition is 281-286. (:; while the aristolochic acid II has a boiling point of 270-273 ° C and a cracking temperature of 270-272 ° C. Because of the high boiling point and pyrolysis temperature, the general method of decoction does not decompose. Acid-induced nephropathy (AAN) is a persistently worsening kidney disease. In terms of clinical characterization, it mainly includes early severe anemia, normal aseptic pyuria, mild renal small & proteinuria. For example: Low molecular weight proteinuria 'Low molecular weight low molecular weight proteins include: α1_microglobulin (α1_ 201223533 microglobulin), · microglobulin (p2_micr〇gl〇b progenitor), Krebs cell protein ( Clara cell protein, retin〇l binding protein, etc. will rise. * In terms of pathological features, the renal area is symmetrically atrophied, mainly due to the structure of the proximal renal tubule and the damage of Wei. The pathological features are extensive renal interstitial fibrosis, combined with tubular atrophy and hollowing, and blebs are formed in the epithelial cells of the proximal S3 segment, accompanied by brush-like edges (4). Rder) disappeared, the interstitial cells were infiltrated, the lesions were most obvious in the cortical surface, and the deeper the cortex was deeper than the human heart and the ball was secreted _ there was fibrosis and irregular thickening, compared with fresh tubules, f The spheroidal phase is less affected. AAN progresses to the end of the kidney money very quickly, so that some drugs to protect the kidneys such as: angiotensin-converting enzyme (angiotensin-converting enzyme her brain rs) or calcium channel blockers (calcium-channel blockers) ) can not delay the deterioration of kidney disease. There is no standard treatment for aristolochic acid nephropathy in the clinic. Only a small, non-aged g(10) small study and a few case reports indicate that class _ (stents) can continue to deteriorate. However, these patients have long-term use of E-alcohol to produce useful and inevitable side effects of drugs, including fractures, abnormal blood sugar levels, deep vein thrombosis, changes in lining and type, which often cause another new problem in treatment. Therefore, finding a safe and effective treatment for aristolochic acid nephropathy is an important issue at present. Disclosure of the Invention An object of the present invention is to provide a pharmaceutical composition containing xanthine saponin, which can safely and effectively treat nephrotic renal lesions. Another object of the present invention is to provide a use of a xanthine saponin using 201223533 to prepare a medicament for treating aristolochic acid nephropathy. The invention utilizes an animal experimental model similar to human aristolochic acid nephropathy, analyzes the nephrotoxic pathogenesis and pathological histomorphology, and explores the type B transforming growth factor (TGF-/S)' hepatocyte growth factor ( HGF), the relationship between matrix metalloproteinase (MMP-9) and this nephropathy, and the efficacy of jaundice and saponin as a therapeutic drug, and hope to find a safe and delaying aristolochic acid nephropathy A therapeutic drug for the deterioration of renal function in patients. An embodiment of the present invention describes a pharmaceutical composition for treating kidney disease, comprising astragaloside as an active ingredient thereof, wherein the nephropathy is induced by aristolochic acid, and the condition includes atrophy of renal tubular epithelial cells and damaged portions of renal tubules. Macrophage infiltration, renal tubular damage, accumulation of transforming growth factor (TGF-million), and tubulointerstitial fibrosis. In particular, the effective use amount of astragaloside is about 10 to 40 mg/kg/day. In one embodiment, the xanthine saponin is contained in a first xanthine concentrate, and the preparation method of the first xanthene concentrate comprises: pulverizing a xanthine slice into a xanthine powder, adding yellow to each liter of distilled water The mixture was mixed at a ratio of 2 〇〇g to form a mixed liquid, and the mixture was heated to boiling for 1 hour, and the filtrate was collected, and the filtrate was concentrated under a reduced pressure concentrator and lyophilized. In particular, the effective amount of the first xanthine concentrate is from 0.5 to 2. g/kg/day. In the embodiment, the scutellaria is contained in a second yellow retort, and the preparation method of the second scutellum concentrate comprises: pulverizing the scutellaria slice into a sassafras powder to 95% per liter Methanol is added to the ratio of 1〇〇〇g of scutellaria powder to form a mixture liquid. After the mixture is placed in the ultrasonic wave shaker for a minute, it is stopped for 5 minutes. After standing for 12 hours, the liquid is collected and then concentrated under reduced pressure. The filtrate was concentrated and cooled; Donggan (4) was obtained. _ Ground, the second scutellaria concentrate has 201223533 effective use of 0.5 to 2.0 g / kg / day. The other embodiment of the present invention - the description of the species - scutellaria sinensis micro-preparation has: the use of the individual to treat aristolochic acid-induced nephropathy, wherein the Astragalus sinensis is present in a jaundice, The kidney disease includes atrophy of the renal epithelial cells, a hyperglycemia (M_phage) / Wenhu, a renal tubular lesion, a growth factor of transforming growth factor (TGF_million), and a tubulointerstitial. Fibrosis. Another embodiment of the invention illustrates the use of a saponin for the preparation of a medicament for treating an individual in need thereof: (4) tubular epithelial cell atrophy, (b) macrophage infiltration at the damaged portion of the renal tubule, ( There is a accumulation of transforming growth factor (TGF-β) in the damaged part of the morphological tubule and (the interstitial fibrosis of the inflammatory kidney tube, wherein the saponin is contained in a scutellaria concentrate, the yellow sputum concentrate is yellow The bismuth powder is prepared by extracting with distilled water or 95% methanol. In the above examples, the accumulation of macrophage infiltration and the accumulation of transforming growth factor (TGF-call) in the damaged portion of the renal tubule are stained by immunofluorescence. And it is observed by a common light-focusing laser scanning microscope. [Embodiment] The present invention "medicine composition for treating ureteric acid nephropathy" is described in detail with reference to the drawings, and the pharmaceutical composition is illustrated by the preferred embodiment. The pharmacological effects of the present invention will enable the industrial use of the present invention to be fully utilized without difficulty according to the contents, drawings and descriptions disclosed below. [Example 1] The efficacy of Astragalus saponin IV in chronic AAN evaluation Astragali Radix is a perennial plant of Fabaceae 201223533. Its medicinal parts are dry roots. Astragalus is also known as Astragalus, Daisy, Dai Dai, Laughing grass, Baiben, Wangsun, rouge, etc. In recent years, more than 100 ingredients have been identified in Astragalus, the main components including flavonoids, Astragalus polysaccharides (APS) and Astragalosides (Fig. 2), and the main/tongue component is Astragalosides. The basic structure of Astragalus membranaceus is 6/6/6/5 four carbon rings, plus side The composition of the chain can be classified as triterpene glycosides 〇

The known saponins of saponins are mainly astragaloside pvhj (aStragal〇side I~VIII), acetaminophen saponin, soyasaponin I, isoflavone saponin, π (is〇astragal〇side 1) Π)Specially, the most representative of Astragai〇side iv, followed by Astragaloside I. The content of Astragalus saponin IV and Astragalus saponin I is the highest in A along the two lines, about 3 to 4 times that of other lines; and the alternative to Astragalus is the isoflavones contained in the ugly households &amp; ) and the content of astragaloside is significantly lower. The experimental animals required for the efficacy evaluation were C3H/He six-week-old male mice, purchased from the National Experimental Animal Center of the Corporation, and the experimental animals were raised by the experimental animal tissues of Taipei Medical University. Ingest food and provide plenty of water. The room temperature of the fostering environment is about to catch, the relative humidity is maintained at 7〇~80%, and the automatic illumination of 12 hours of light and dark is maintained. The experimental drugs include: a xanthine concentrate extracted with distilled water, a xanthine concentrate extracted with methanol, and xanthine saponin IV. 201223533 Before entering the experiment, the average daily drinking water per mouse was about 3.5 mL. Sodium aristolochic acid sodium salt (AAI 63%, AAII 31%) was dissolved in distilled water (dw) as drinking water for experimental and control mice at a dose of 3 〇#g/mL (0.5 mg/kg). /day), normal drinking water was resumed after 56 days of continuous administration, and then administered to the experimental group by oral administration of aqueous.丨mL distilled water extracted aqueous extract of astragalus (hereinafter referred to as AE) 0.5 g/ Kg/day, 1.0 g/kg/day, 2·0 g/kg/day, methanolic extract of astragalus (AM) 0.5 g/kg/day, 1.0 g /kg/day, 2.0 g/kg/day and astragaloside IV (AIV) 10 mg/kg/day, 20 mg/kg/day, 40 mg/kg/day, continued for 14 days. The control group was given the same amount of steamed water, and the mice in the normal group were given steamed water. In order to observe the therapeutic effect of Astragalus sinensis extract and Astragalus saponin IV extracted from steaming water and sterol solution on AAN, the animals in each group were sacrificed 14 days after administration of each group of therapeutic drugs and distilled water, experimental group and control group. In the normal group, each group of animals each had 1 ,, a total of 5 〇. (Fig. 3) In order to establish a model of aristolochic acid nephropathy, mouse urine must be collected to measure the content of urinary protein, N-acetylglucosamine (nag), and the blood of the mice is taken to determine the serum. The contents of urea nitrogen (BUN) and serum creatinine (Scr) were used to evaluate renal function. In addition, the use of microscopic examination of photographic methods to quantify the degree of renal tissue damage 'and the use of immunofluorescence staining to further establish the pathological mechanism of the aristolochic acid nephropathy model, and to explore the type B transforming growth factor (TGFj), Relationship between hepatocyte growth factor (HGF), matrix metalloproteinase (MMP-9) and aristolochic acid nephropathy. In this example, urine protein content is detected by absorbance, and 4-methyl umbrella 201223533 幵v-N-acetyl-β-D-glucosamine (4_me also yiumbeiiifeiyi &gt; T_aeetyl_p_D_gluec) saminide, 4-MU- NAG) acts as a substrate for NAG, which causes NAG in the urine to act to produce 4-methylumbelliferone (4-MU). Because 4-MU emits fluorescence, 4- Fluorescence produced by MU is used as a standard for quantitative determination. The content of BUN and creatinine in serum is determined by an automatic biochemical analyzer. Periodoic Acid Schiff's stain (hereinafter referred to as PAS) Dyeing ··· The prepared left kidney paraffin embedded section (4_5 μπι) is dewaxed first, washed with water for 10~20 seconds, soaked in 0.5% periodic acid solution (perldic acid, ffl04) for about 5 to 10 minutes; distilled water After washing 3 times, stain with Schiff's reagent for 10 to 15 minutes, then directly transfer to freshly prepared sodium bisulfite solution (NaHS03), transfer three times, each time for 3 minutes, rinse with water 3 5 minutes. Contrast staining using Sumu purple The hematoxylin stain solution was subjected to tissue nuclear staining, and the water was rinsed until the specimen was blue. The cells were dehydrated by 70% ethanol, 95% ethanol, 99.5% ethanol, Xylene, etc. Finally, it is covered with a mounting medium for microscopic examination. Quantification of the degree of tissue damage: kidney tissue using microscopy tissue photography, using optical microscope 200 times (eyepiece X objective lens: l〇XX 20X) and digital camera Randomly searched 20 non-repetitive sites to observe the degree of renal tubular and interstitial damage, and assessed renal tubular epithelial cell atrophy, cell infiltration, and interstitial fibrosis (interstitial) by tissue quantification. Fibrosis), scored in 3 stages, the sum was the Tubulointerstitial histological score (THIS), and the average of each group was counted. Immunofluorescence staining ( Immunofluorescence): fresh cold; East slice back to warm (room temperature 25 ° C), moistened with phosphate buffered saline (PBS, pH 7.4) After treatment with 10% normal rabbit serum, the selected specific antibody TGF-β, HGF and MMP-9 were appropriately diluted in PBS and added to each tissue, and the other tissue of the same section was only added to PBS. As a negative control, the reaction was washed three times with PBS. The secondary antibody Tetramethyl Rhodamine Isothiocyanate (TRITC) -labeled rabbit anti-goat IgG was added in a dark environment, followed by continuous washing with PBS 3 times, air-dried, and finally capped with 90% glycerol. Using a confocal microscope, a diode-pumped solid-state laser (DPSS Laser) was used as a light source to observe TGF-β, HGF and MMP-9 in tissues. Performance situation. The data were expressed as mean + SD. The results of urine protein, NAG content and serum BUN, Scr, blood glucose, tissue staining by PAS and immunofluorescence staining were all confirmed by Mann-Whitney. U test) Method statistics. [Example 2] High Performance Liquid Chromatography of Astragalus Saponin IV in Astragalus Concentrate and Evaporative Light Scattering Detector (HPLC-ELSD) Method Analysis Analysis of the Content of Astragalus Saponin IV of Astragalus Concentrate Four is shown. The figure shows (A) standard astragalus saponin IV (1 mg/mL), (B) test product by steaming water extracted xanthine concentrate (100 mg/mL), (C) test product with sterol 201223533 Extracted xanthine concentrate (100 mg/mL). The retention time of the standard xanthine saponin Iv is about 6 minutes. Using this HPLC-ELSD analysis method, the content of xanthine saponin iv contained in distilled water extracted from xanthine concentrate (100 mg/mL) was 427.4 soil 2·9 Wg/g, and its content percentage was 0.0427 ± 0.0003%, equivalent to the percentage of dry roots is 〇.〇〇76±〇.〇〇〇1%; the scutellaria saponin iv contained in the scutellaria extract (100 mg/mL) extracted with sterol The content is 974.6 ± 36.9 yg / g 'the percentage of its content is 0.0975 ± 0.0037%, which is equivalent to the percentage of 3 in the dry root is 0.0153 ± 0.0006%. The AM content is about twice that of AE. [Example 3] Evaluation of the efficacy of steaming your water-extracted Astragalus membranaceus concentrate on chronic aristolochic acid nephropathy When preparing the scutellaria extract concentrated in distilled water, take the astragalus slice iAstragalus membi*anaceus var· Momgholicus, produced in Shanxi, China) crushed it, weighed 1 〇〇〇g of scutellaria powder and added 5 L of distilled water, heated it to boiling for 1 hour and then repeated twice, collecting the filtrate twice, after combining After that, it was concentrated by a vacuum condenser and dried by cooling to obtain 178.13 g of a scutellite concentrate, and the yield was 7.81 〇/〇. The results of urine and blood analysis were as follows: In the analysis of proteinuria, after 7 days of treatment, the treatment group AE 〇.5 g/kg group, AE ! 〇g/kg group, 2 〇 group and group The % hour proteinuria proteinuria was 2.59 ± a38 mg/day, 2 53 ± Q l8 mg/day and 2.66 ± 0.29 mg/day, respectively, compared to the AAN control group of 3 11 201223533 ± 0.21 mg/day. The difference (p < 〇〇 5). After 14 days of drug administration, the proteinuria levels in the treatment group ΑΕ 0.5 g/kg group, ΑΕ [ο _ group, 2 〇 〇 group were 2.64 ± 0.21 mg/day, 249 ± 〇 18 mg/day and 2.71, respectively. There was also a decrease in ±0.31 mg/day, which was statistically significant compared to the control group of 3.58 ± 0.15 mg/day & 〇〇〇 (Figure 5). In the NAG content determination results, after 7 days of administration of the therapeutic drug, the urinary NAG contained in the AE 0.5 g/kg group, the AE 1.0 g/kg group, and the AE 2_0 g/kg group in the treatment group was 2.96 ± 0.30 &quot;M/min/L, 2.86 + 0.41 ^ M/min/L and 2.85 ± 0.37 //M/min/L compared with the control group of 3·4ΐ ± 〇33 //M/min/L The trend is reduced, and there is statistically #&lt; 0.05). After 14 days of treatment, the AE 0.5g/kg group, the AE 1.0 g/kg group, and the AE 2.0 g/kg group were 3.05 ± 0.20 &quot;M/min/L, 2.75 ± 〇.17 /zM/min/L, respectively. And the 2.48 ± 0.20 μΜ/min/L treatment group had a decrease in urinary NAG content, which was statistically different from the control group of 3.47 ± 〇 32 (p &lt; 0.05) (Fig. 6). BUN analysis in serum: 14 days after continuous administration of the therapeutic drug, the AE0.5 g/kg group, the AE 1.0 g/kg group, and the AE 2.0 g/kg group in the treatment group were 27.20 ± 2.05* mg/dL, 24.75, respectively. ± 1.7Pmg/dL and 26.60 ± 2.41* mg/dL both reduced serum BUN' and were statistically significant compared to AAN control group (30.2〇±0.84 mg/dL) (p &lt; 〇〇 5). Control Normal mice were given a serum BUN value of 19.00 + 2.00 mg/dL for 14 days in the treatment group. Scr analysis in serum: 14 days after the administration of the therapeutic drugs, in the AE 0.5g/kg group, AE 1.0 g/kg group, 2.0 g/kg group in the treatment group 12 201223533

The Scr values were 0.28 ± 0.04 mg/dL, 〇·23 ± 0.05 mg/dL and ο υ + 0.05 mg/dL, which decreased blood, : Scr value and had a system compared with 0.36 ± 0.05 mg/dL in the control group. The meaning of the school (p &lt; 0 05). Control normal mice The serum Scr value of the drug group administered in the treatment group was 〇16 ± 〇 〇 7 mg/dL. Histopathological PAS staining: Under the light microscope 2 times, the pathological changes of the kidney tissue of the mice treated with the therapeutic drugs were observed (Fig. 7), and the normal group (A), the control group (B) and the continuous administration of η days were observed. Post-AE 0.5 g/kg (〇, AE 1.0 g/kg (D) and AE 2.0 g/kg (E) treatment group PAS pathological tissue map. Compared with the control group, renal tubular injury can be found in the treatment group Mild, interstitial fibrosis, cell infiltration, and tubular atrophy may be slowed down. Tissue damage stem analysis: PAS-stained tissue was observed under a microscope and scored according to the pathological damage scale. Quantitative results showed that the AE 0.5 g/kg group, AE 1.0 g/kg group and AE 2.0 g/kg group in the treatment group were 5.14 ± 0.61, 3_97 ± 0·31, 4.45 ± 0.54, respectively. There was a statistically significant difference compared with the control group of 6.90 ± 0.79 (p &lt; 0.01) (Figure 8). Immunofluorescence staining and quantitative analysis: 1. TGF-cold immunofluorescence staining in kidney tissue Thereafter, the performance of TGF-yS was observed by a conjugated-focus laser scanning microscope. Compared with the control group, 67.75 ± 8.38 of the renal tissue treatment group AE 0.5 g / kg group, AE 1 .〇g / kg group and AE 2.0 g / kg group were 28.75 ± 5.81, 21.00 ± 1.67, 26.80 ± 2.23, respectively. The fluorescence coloration points were reduced by 58%, 69%, and 60%, respectively. The AE 0.5 g/kg, 1.0 g/kg, and 2.0 g/kg treatment groups were able to detect the fluorescence of renal tubules and renal interstitial sites. 13 201223533 Color point reduction showed that the administration of AE 0.5 g/kg, 1.0 g/kg, 2.0 g/kg reduced the performance of TGF-yS (Fig. 9).

2. After HGF mouse kidney tissue was stained with HGF immunofluorescence, the fluorescence color point of HGF was observed by conjugated focal laser scanning microscope. Compared with the control group, 12.75 ± 1.79, ί, and AE 0.5 g / kg, group, AE 1.0 g / kg group, AE 2.0 g / kg group were 39.00 ± 3.94, 48.40 ± 6.39 and 40.76 ± 5.64, respectively. Fluorescent color points increased by 3.1 times, 3.8 times and 3.2 times, respectively. Fluorescent bright spots in the renal tubules and renal interstitial sites were found in all three groups, indicating that AGF 〇 5 g/kg, 1.0 g/kg, and 2.0 g/kg all increased HGF performance (Fig. 9). 3. MMP-9 Observed the performance of renal tissue after MMP-9 immunofluorescence staining, compared with the control group (5.98 ± 0.93), AE 0.5 g/kg group, AE 1.0 g/kg group and AE2.0g/ The kg group was 19.52 ± 2.94, 19.67 ± 1.31 and 22.25 ± 3.77, respectively, and the fluorescence color points increased by 3 3 times, 33 times and 37 times respectively. The three groups treated the light in the renal tubules and renal interstitial parts. The coloration points were increased by ^, indicating that the administration of AE 0.5 g / kg, U coffee, 2 〇 _ can increase the performance of swollen _9 (Figure 9). [Embodiment 4] Evaluation of the effect of nephropathy on methanol extraction of chronic aristolochic acid in the preparation of methanol-extracted xanthine concentrate, the department took yellow chopped and added 95% transfer ' L ' placed in the super After the stun shock ib minutes, V lag for 5 minutes 'reverse operation three times' after 12 hours of standing liquid, and then add 2012 L L 95% methanol 0.5 L, repeat the above steps, then combine the two filtrates, then decompression The concentrate was concentrated and lyophilized to obtain a scutellaria concentrate 156.72 g 'yield 15.67%. The results of the exhibition fluid and blood analysis were as follows: In the analysis of proteinuria, after treatment with the drug for 7 days, the treatment group was in the AM 0.5 g/kg group, the AM 1.0 g/kg group, and the AM 2.0 g/kg group. The hourly proteinuria content was 2.58 ± 0.29 mg/day, 2.43 ± 0.09 mg/day, and 2_51 ± 0.29 mg/day, which was a decrease from the AAN control group of 3.27 ± 0.21 mg/day and was statistically different. φ&lt;〇〇5). After 14 days of giving the music, the proteinuria content of the AM 0.5 g/kg group, the AM 1.0 g/kg group, and the AM2.0 g/kg group were 2.55 ± 0.23 mg/day, 2.42 ± 0.32 mg/day, respectively. 2_60 ± 0.29 mg/day, there was also a statistically significant difference compared to the control group of 3.58 ± 0.15 mg/clay (Fig. 10). In the NAG content determination results, after 7 days of administration of the therapeutic drug, the urine NAG content of the AM 0.5g/kg group, the AM 1.〇g/kg group, and the AM 2_〇沙吕 group in the treatment group were 3.05, respectively. ± 0.19 &quot;M/min/L, 2.81 ± 0.33 μ M/min/L and 2.84 ± 0.40 //M/min/L, compared with the control group 3.41 + 0 33 /zM/min/L Trends, and there are statistical differences 2 &lt; 0.05). After 14 days of treatment, the 'AM 〇·5 g/kg group, the AM 〇g/kg group and the AM2.0g/kg group were 3.00 ± 0.16 〇 ll /zM/min/L and 2.74 ± 0.23* yM, respectively. The /min/L treatment group had a decrease in urinary NAG content, which was statistically different from the control group of 3.47 ± 〇32 “pounds toe (p&lt;0.05). Value: 14 days after continuous administration of the therapeutic drug, the AM 0.5 g/kg group, the AM force g/kg group and the AM 2 〇 group in the treatment group can be 15 201223533. The BUN value in the Jinqing is reduced. The value is 28 40 ± 〇89 mg/dL,

24.25 ± 1.50 mg/dL and 24.17 ± 2.04 mg/dL, and 30.20 ± 0.84 mg/dL compared with the AAN control group were statistically significant &amp; 0.05. The serum bun value of the control mice administered to the treatment group for 14 days was 19.00 ± 2.00 mg/dL. Creatinine analysis in serum: 14 days after the administration of the therapeutic drugs, the AM 0.5 g/kg group, the AM 1.0 g/kg group and the ΑΜ 2·〇g/kg group in the treatment group all reduced the Scr value in the serum. 〇26 ± 〇〇5mg/dL, 〇22 ± 0.04 mg/dL and 0.22 ± 0.09 mg/dL, compared with 0.36 ± 0.05 mg/dL in the control group, which was statistically significant (p &lt; 〇〇 5). Control normal mice The serum Scr value of the drug in the treatment group for 14 days was 〇 16 ± 〇 〇 7 mg / dL. Histopathological PAS staining: Under the light microscope 2 times, the pathological changes of the kidney tissue of the mice treated with the therapeutic drugs were observed (Fig. 12), and the normal group (A), the control group (B) and the continuous administration were respectively observed. Pathological tissue map of pas in the treatment group of AM 0.5 g/kg (C), AM 1.0 g/kg (D) and AM2.0 g/kg (E). Compared with the control group, the damage of the renal tubules was mild in the treatment group, and the interstitial fibrosis, cell infiltration, and tubular atrophy were slowed down. Quantitative analysis of tissue damage: PAS-stained tissue was observed under a microscope and scored according to the pathological tissue damage scale. The results of tissue damage quantification showed that the degree of tissue damage in the AM 0.5 g/kg group, AM 1.0 g/kg group and AM 2.0 g/kg group in the treatment group all decreased, and the quantitative analysis values were 5.50 ± 0.92, 4.22, respectively. ± 1.15 and 3.97 ± 0.55 were statistically different from the control group of 6.90 ± 0.79 (Figure 13). Immunofluorescence staining and quantitative analysis: 201223533 I. TGF-β After immunofluorescence staining of renal tissue, the performance of TGF-/S was observed by conjugated laser scanning scanning microscope. Compared with the control group, 67 75 ± 8.38 kidney tissue, AM 0.5 g/kg group, AM 1.0 g/kg group and AM 2.0 g/kg group were 20.20 ± 3.83, 12.40 ± 1.52 and 14.40 ± 3.21, respectively. The bright spots of light color were reduced by 70%, 82% and 79% respectively. In the three groups of treatment groups of 0.5 g/kg, 1.0 g/kg, and 2.0 g/kg, the fluorescent color spots of the renal tubules and renal interstitial parts were reduced, indicating that ^0.5§/1^, 1.08^, 2.08 were given. /1^ can reduce the performance of TGF-points (Figure 14).

2. HGF mouse kidney tissue was stained with HGF immunofluorescence. 'Using a coke focused laser scanning microscope to observe the fluorescent color of HGF. The brightness of the 4 eyes was 12.75 ± 1.79 compared with the control group. AM 〇.5 The g/kg group, the Shiyi group and the AM 2.0 g/kg group were 45.52 ± 2.94, 56.54 ± 5.75 and 54.25 ± 3.9 分别, respectively, and their bright spots increased by 3.6 times, 4.4 times and 4.3 times, respectively. The fluorescence intensity of the renal tubules and renal interstitial sites increased in all three groups, indicating that the administration of AM 0·5 g/kg '1·〇g/kg and 2.0 g/kg increased HGF performance (Fig. XIV) ). 3. MMP-9 Observed the performance of renal tissue after MMP-9 immunofluorescence staining, compared with 5.98 ± 0.93 in the control group, and AM 0.5 g/kg group, AM 1.0 g/kg group and application 2·0 The group was 28.50 ± 4.03, 33.17 ± 2.27 and 46.64 ± 6.72, respectively. The fluorescence deposition was increased by 48 times, 56 times and 78 times, respectively. The fluorescence of the three groups in the renal tubule and renal interstitial area The increase in color bright spots showed that the administration of AM 0.5 g/kg, 1.0 g/kg, and 2.0 g/kg could increase the performance of MMP-9 in 201223533 (Fig. 14). [Example 5] Efficacy evaluation of astragaloside IV against chronic aristolochic acid nephropathy In the present example, xanthine saponin IV was a standard. The results of urine and blood analysis were as follows: In the analysis of proteinuria, after treatment for 7 days, the treatment group was treated with AIV 10 mg/kg, AIV 20 mg/kg, and AIV 40 mg/kg for 24 hours. The proteinuria levels were 2.73 ± 〇3〇mg/day, 2 5〇± 〇.44 mg/day and 2.61 ± 0.22 mg/day, respectively, compared with 3.27 ± 0.21 mg/day in the aan control group. The situation was statistically different (p < 0.05). After 14 days of drug administration, the proteinuria levels of AIV 10 mg/kg, AIV 20 mg/kg, and AIV 40 mg/kg were 2.83 ± 0.45 mg/day, 2.51 ± 0·18 mg/day, and 2.56 ± 0.26, respectively. There was also a statistically significant difference (p&lt; 0.05) between mg/day and a decrease of '3.58 ± 0.15 mg/day compared with the control group (Fig. 15). In the NAG content determination results, after 7 days of treatment, the urinary NAG content in the AIV 10 mg/kg group, the AIV 20 mg/kg group, and the AIV 40 mg/kg group in the treatment group was 2.99 ± 0.34, respectively. M/min/L, 2.87 soil 0.25 #M/min/L and 2.87 ± 0.29 yM/min/L, which is more than the pair of households and 3.41 ± 0.33 from M/min/L, and there is a tendency to Statistical difference &&lt; 0.05). After 14 days of treatment, the AIV 10 mg/kg group, the AIV 20 mg/kg group, and the AIV 40 mg/kg group all had lower urinary NAG content, which was 3_15 ± 0.38 #M/min. /L, 2.83 ± 0.30 /z 201223533 M/min/L and 2.90 ± 0.28 &quot;M/min/L, compared with the control group of 3.47 ± 0.32 /zM/min/L, there is a statistical difference φ &lt; 〇 5) (Figure 16). BUN analysis in serum: 14 days after continuous administration of the therapeutic drug, the AIV 10 mg/kg group, the AIV 20 mg/kg group and the AIV 40 mg/kg group in the treatment group all reduced serum BUN values, respectively. They were 272〇±179 mg/dL, 25.67± 1.86 mg/dL and 26.33±1.53 mg/dL, and were statistically significant compared with the AAN control group (30.20 ± 0.84 mg/dL) (p &lt; 0.05) . The serum bun value of the control mice administered to the treatment group for μ days was 19.00 ± 2.00 mg/dL. Scr analysis in serum: 14 days after the administration of the therapeutic drugs, the AIV 10 mg/kg group, the AIV 20 mg/kg group and the AIV 40 mg/kg group in the treatment group all reduced the serum Scr value. 0.28 ± 0.06 mg / dL, 0.23 ± 0.05 mg / dL and 〇 · 24 ± 0.05 mg / dL, compared with 0.36 ± 0.05 mg / dL in the control group, there is a statistical significance φ &lt; 〇〇 5). Control normal mice The serum Scr value of the drug in the treatment group for 14 days was 0.16 ± 0.07 mg/dL. Histopathological PAS staining: The pathological changes of renal tissue in mice treated with therapeutic drugs were observed under light microscope 200 times (Fig. 17). Normal (A), control group (B) and AIV 10 mg after 14 days of continuous administration were observed. Histopathology of PAS in the /kg (C), AIV 20 mg/kg (D) and AIV 40 mg/kg (E) treatment groups. Compared with the control group, the damage of the renal tubules was mild in the treatment group, and the interstitial fibrosis, cell infiltration and tubular atrophy were slowed down. Quantitative analysis of tissue damage: PAS-stained tissue was observed under a microscope and scored according to the pathological tissue damage scale. Quantification of tissue damage found that the AIV 10 mg/kg group, the AIV 20 mg/kg group, and the AIV 40 mg/kg group were 4.73 ± 0.9, 4.52 ± 0.29, and 4.82 ± 0.56, respectively, in the treatment group. The trend of reduction was statistically different from the control group of 6.90 ± 0.79 (ρ &lt; 0·01). (Fig. 18) Immunofluorescence staining and quantitative analysis: 1. TGF-cold After immunofluorescence staining of renal tissues, the performance of TGF-yS was observed by a conjugated-focus laser scanning microscope. Compared with the control group, 67.75 soil 8.38 kidney tissue, AIV 10 mg/kg group, AIV 20 mg/kg group and AIV 40 mg/kg group were 32.67 ± 2.31, 25.60 ± 5.18 and 31.00 soil U7, respectively. The deposition was reduced by 52%, 62% and 54%, respectively. AIV 10 mg/kg, 20 mg/kg, 40 mg/kg treatment groups were found to have reduced fluorescence deposition in the renal tubules and renal interstitial sites, showing AIV 10 mg/kg, 20 mg/kg, 40 mg. /kg can reduce the performance of TGF-yS (Figure 19).

2. The HGF mouse kidney tissue was stained with HGF immunofluorescence, and the fluorescence deposition performance of HGF was observed by a common focal-focus laser scanning microscope. Compared with the control group of 12.75 ± 1.79 kidney tissue, the AIV 10 mg/kg group, the AIV 20 mg/kg group and the AIV 40 mg/kg group were 26.75 ± 2.87, 46.60 ± 7.20 and 42.63 ± 2.04, respectively. The deposition increased by 2.1 times, 3.7 times and 3.3 times, respectively. Increased fluorescence deposition in the renal tubules and renal interstitial sites was observed in all three groups, indicating that AIV 10 mg/kg, 20 mg/kg, and 40 mg/kg increased HGF performance (Fig. 19). III. ] VIMP-9 Observed the renal tissue after MMP-9 immunofluorescence staining, compared with 5.98 ± 0.93 in the control group, AIV 10 mg/kg group, AIV 20 mg/kg 201223533 group and AIV 40 mg/kg. The groups were 17-78 ± 1.20, 21.50 ± 1.12 and 19.98 ± 2.12, respectively. The fluorescence deposition was increased by 3 times, 3.6 times and 3.3 times, respectively. The three groups of treatment group were in the renal tubule and renal interstitial. The increase in photodeposition showed that the administration of AIV 10 mg/kg, 20 mg/kg, and 40 mg/kg increased the performance of MMP-9 (Fig. 19). In summary, the results of 'in the HPLC-ELSD analysis' show that the water extracted from the scutellaria extract contains 0.0427% of the 0.0003% of the xanthine IV, which is equivalent to 0.0076 ± 0.0001% of the jaundice in the dried root. Bitter IV; methanol extracted xanthine concentrate contains 0.0975 ± 0.0037%, equivalent to 0.0153 ± 0.0006% of dried roots containing scutellaria soap iv. In this embodiment, the xanthine concentrate (AE 0.5g/kg, 1.0g/kg, 2.0g/kg) extracted by distilled water and the scutellaria extract concentrate (am 0.5g/kg, 1.0g/kg) , 2.0 g / kg) and xanthine saponin IV (AIV lOmg / kg, 20 mg / kg, 40 mg / kg) as a therapeutic drug. Figure 20 shows the combined results of Astragalus concentrator (AE, AM) and Astragalus saponin in AAN mice. AE (AE 0.5 g/kg, 1.0 g/kg, 2.0 g/kg), AM (AM 0.5 g/kg, 1.0 g/kg, 2.0 g/kg) and AIV (AIV 10 mg/kg, 20 mg) /kg, 40 mg/kg) in the AAN model of mice, can alleviate the kidney damage caused by AA, and have different degrees of improvement for various renal function indicators. In the above examples, it was found that AE, AM and AjV all reduced BUN, Scr and urine protein in mice. According to the quantitative analysis of hpLC_elsd, AE and AM contain only a small amount of AIV, and the amount of ajV administered is lower than that of pure AIV. The situation in which each group's thief function index is improved is similar. 201223533 The examples of the present invention found that administration of AE, AM and AIV to mice can effectively reduce the NAG content in urine. NAG is an enzyme secreted by the proximal convoluted tubules of the kidney. When the renal tubules are damaged, a large amount of NAG is released in the urine, which is a specific index when the renal proximal tubules are damaged. In the microscopic examination of kidney tissue, the results showed that AE, AM, and AIV all reduced tubular damage, interstitial cell infiltration, and fibrosis caused by sputum. It is speculated that AE, AM, and AIV may be anti-inflammatory and anti-fibrous. The effect of the disease is to alleviate the kidney damage caused by AA. The embodiments of the present invention utilize immunofluorescence staining method, and select TGF_cold, HGF and MMP-9 as primary antibodies to investigate the immunopathological mechanism of three indexes in the aaN nephritis model. In the examples of the present invention, it was found under the observation of the conjugated focal microscope that the administration of AE, AM and AIV could reduce the expression of TGF-cold compared with the untreated aan mouse, showing AA The induced renal fibrosis is inhibited. It is speculated that Astragalus can inhibit the activity of Smad protein, reduce the production of fibroblasts and myofibroblasts, reduce the deposition of interstitial cells, and delay the renal fibrosis caused by AA by reducing the expression of TGF-stone. During the process of renal fibrosis, the concentration of TGF-β will gradually increase, the concentration of HGF will gradually decrease, and the decrease of TGF-々 will increase the concentration of HGF and MMP-9. It was shown that AE, AM and AIV can reduce renal fibrosis caused by AA by reducing TGF- /3, increasing the expression of HGF and MMP-9. In addition, in the embodiment of the present invention, in the interstitial fibrosis portion of the quantitative analysis of tissue damage, each treatment group and the control group have a decrease, and the immunofluorescent staining portion can also see the fibrosis index TGF. - The performance of sputum decreased, while the performance of anti-fibrotic factors HGF and MMP-9 increased significantly 22 201223533 plus, so it is speculated that scutellaria concentrate and saponin IV have good anti-fibrotic effect. Based on the above conclusions, it is speculated that AE, AM and AIV can further reduce the activation of TGF-Lu by inhibiting the action of νρμβ, and improve renal function and fibrosis. In the above examples, mice administered with sputum, AM and AIV were all relieved compared with the control group, and statistical differences were observed (p&lt;005). Differences between different doses between groups

Face, AEl.Og AE2.0 2 AE0.5; AM1.0 and AM2.0 2 AM 0.5; AIV20 2 AIV40 $ AIV10, so the dose of 1〇, 施1.0, AIV20 is estimated to have reached the maximum among the groups Efficacy. In the embodiment of the present invention, she observed an adverse reaction. In the proteinuria section, compared with the experimental day G, the 7th day of the experiment, the egg had a slight increase in the self-urine _, and the mice in the sputum treatment group did not significantly increase the force π. It is speculated that it may be that the therapeutic drug reduces the amount of proteinuria. Among the components in which the jaundice has been separated, the above examples only quantify the yellow test (10) 1V', and in addition to the Astragalus IV, there may be other effective knives that can alleviate the kidney damage caused by sputum, so the future It may be considered to quantify other components and assess their efficacy. ▲In summary, ΑΕ, AM and aiv can all improve the kidney function and reduce the damage in mice. It is speculated that the mechanism may be: AE, AM and aiv have anti-inflammatory and anti-fibrotic effects, thereby alleviating the kidney damage caused. The above detailed description of the county's specific examples of the implementation of the Laijia, but the implementation of the above-mentioned implementation of the invention, the equivalent of the implementation of the invention, the equivalent of the implementation of the spirit of the 23,235,235 Included in the scope of this case. [Simple diagram of the figure] Figure 1. Chemical structure of aristolochic acid I, II; Figure 2. Isoflavones in Astragalus and Astragalus membranaceus; Figure 3. Flow of the implementation method of the present invention; Figure 4A Figure 4C, the standard product Astragalus femur IV and the scutellaria extract (AE) extracted with water, and the HPLC-ELSD spectrum of the scutellaria concentrate extracted with sterol; Figure 5 Huang Wei _ analysis of the white content of the model in the Yanyan model; Figure 6. Analysis of the nag content of the jaundice concentrate extracted by steaming water in the nephritis model; Figure VII, the jaundice extracted by the water The pathological changes of the kidney tissue of f-flame lion; Figure VIII, the concentration of jaundice extracted by _water _ quantitative analysis of tissue damage in the model of Lingyan; Figure IX, the amount of fluorescein dyed by _water Analysis; ^ Figure 10, the yellow Wei Wei of the A Miscellaneous _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ The renal tissue pathological changes of the jaundice concentrate extracted by sterol in the nephritis model; Figure 13. The phlegm-concentrating agent of sterol in this nephritis model Quantitative analysis of tissue damage in Figure 14. Figure 14. Quantitative analysis of the immunofluorescence staining of the xanthine concentrate extracted with methanol in this nephritis model; Figure 15. Analysis of the urine protein content of the scutellaria iv in this nephritis model; Figure 16. Analysis of the NAG content of the saponin IV in this nephritis model; Figure 17. The pathological changes of the kidney in the nephritis model of the scutellaria saponin IV; Figure 18. The tissue damage of the scutellaria saponin IV in this nephritis model Quantitative analysis; Figure 19. Quantitative analysis of xanthine saponin IV in this nephritis model by immunofluorescence staining; Figure 20. The combined effect of Astragalus membranaceus (AE, AM) and Astragalus saponin IV on AAN mice. [Main component symbol description] None 25

Claims (1)

201223533 Seven patent application scope: - A pharmaceutical composition for treating kidney disease, including jaundice as its active ingredient 'the towel' is induced by aristolochic acid, and its symptoms include tubular epithelial cell atrophy, kidney small body damage M_(Maen)Phage) Infiltrating and damaged tubules have transforming growth factor (10) tubulointerstitial fibrosis. and 2. The pharmaceutical composition according to claim 1, wherein the Astragalus saponin is contained in a -D-Astragalus concentrate, and the preparation method of the scutellaria concentrate comprises: pulverizing the scutellaria For the scutellaria powder, add the gram-forming mixture of the scutellaria powder in liters of distilled water, and heat the mixture to boiling for 1 hour, collect, and then concentrate the filtrate with a vacuum concentrator. 'And get lyophilized. 3. The pharmaceutical composition according to claim 2, wherein the amount of the first-yellow _ _ 纽 纽 纽 四 〇 〇 〇 〇 〇 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 4. The pharmaceutical composition according to claim i, wherein the scutellaria barbata is contained in - the second scutellaria sizing agent's preparation method of the second yellow scenting agent comprises: pulverizing the scutellaria For the scutellaria powder, add 95% sterol per liter of sterol to the ratio of the yellow #粉幻_ gram of the shaft-age liquid, and place the mixture after the shock of the ultrasonic oscillator to 'stay and collect the filtrate. The filtrate was concentrated by a reduced pressure concentrator and cooled, and dried in the east. 5. If the pharmaceutical composition of the fourth aspect of the invention is applied, the effective amount of the second scutellaria concentrate is 5 to 2 gram per kilogram of biomass per day. 6. The pharmaceutical composition according to claim 1, wherein the kidney 26 201223533 small tube damaged site has a macrophage infiltration and a growth factor of transforming growth factor (TGF_$) is immune to S color, and The pharmaceutical composition according to claim 1, wherein the effective use amount of the saponin is about 1 to 4 mg per kilogram of biological weight per day. 8. The use of a drug for treating kidney disease induced by aristolochic acid in a subject in need thereof, wherein the astragalus saponin is present in a xanthine powder, and the condition of the kidney disease includes kidney Tubuloblastic cell atrophy, damaged tubule cells (Macr〇phage) infiltration, accumulation of transforming growth factor (TGF-call) in renal tubular damage, and tubulointerstitial fibrosis. 9. Use of a xanthine saponin for the preparation of a medicament for treating an individual in need thereof: (a) tubular epithelial cell atrophy, (b) macrophage infiltration in the damaged portion of the renal tubule, (c) renal tubule The damaged part has the accumulation of transforming growth factor (TGF-/3), and (d) tubulointerstitial fibrosis, wherein the xanthine saponin is contained in a yellow sputum concentrate, the yellow sputum concentrate is yellow The mash powder is prepared by extracting with distilled water or 95% sterol. 10. The use according to claim 8 or 9, wherein the condition in which the renal tubular damage is macrophage infiltration and the accumulation of transforming growth factor (TGF_stone) is stained with immunofluorescence and utilized. Conjugated focal laser scanning microscope observation. 27