Application of guaiane type sesquiterpene compound in preparation of medicine for treating renal injury

Abstract

The invention discloses an application of a guaiane type sesquiterpene compound in preparation of a medicine for treating renal injury. The renal injury is one or a combination of more of acute nephritis, chronic nephritis or diabetic nephropathy. The guaiane type sesquiterpene compound is separated from an ixeris sonchifolia hance ethanol extract, the ixeris sonchifolia hance lactone compound and an ixeris sonchifolia hance lactone-containing effective part have relatively strong activity of inhibiting kidney injury factors Kim1, Il1b and Mcp1 on a cellular level, and show the activity of resisting acute / chronic nephritis and diabetic nephropathy on a mouse animal model; the composition has a synergistic effect when being combined with astragaloside. # imgabs0 #

Description

Technical Field

[0001] The invention relates to the application of guaiacyl sesquiterpene compounds in preparing medicines for treating kidney damage, and in particular to the application of guaiacyl sesquiterpene compounds in preparing medicines for treating acute and chronic nephritis or diabetic nephropathy. Background Art

[0002] Kudiezi, also known as Gypsophila, is the whole herb of Ixeris sonchifolia (Bunge) Hance of the Asteraceae family. It mainly grows in the wilderness, hillsides, roadsides, rivers and sparse forests in North my country, Northeast China and East China. It is a perennial herb. According to the "Changbai Mountain Plant Medicinal Records", it has the effects of analgesia, heat clearing, detoxification and swelling. It is mainly used to treat appendicitis, enteritis, lung abscess, carbuncle and sore. Modern pharmacological experiments have shown that Kudiezi has the activities of lowering blood lipids, anti-tumor, anti-cardiovascular and cerebrovascular diseases, sedation and analgesia, and improving microcirculation. Among them, in clinical practice, Kudiezi injection is widely used in cardiovascular and cerebrovascular diseases such as angina pectoris and coronary heart disease. In recent years, with the in-depth research on the chemical composition, pharmacological activity and clinical application of Kudiezi, it was found that it mainly contains compounds such as sesquiterpene lactones, flavonoids, triterpene saponins and adenosine. Currently, lxerin Z, 11,13α-dihydroixerin Z, lxerin Z2, 3-Hydroxydehydroleucodin, 8-Desoxyartelin, Ixerin X, Ixerin Z1, Sonchifoliasolide A and B have been reported to be isolated, among which lxerin Z has the highest content (>0.2%).

[0003]

[0004] Regarding the extraction of sesquiterpene lactone components in bitter dish, the invention patent with application number 201810773862.6 discloses a method for preparing sesquiterpene lactone components in bitter dish, which uses 80-90% ethanol extraction and hot reflux, and then uses macroporous resin and silica gel column to prepare sesquiterpene lactone components in bitter dish. This method can only prepare low-polarity sesquiterpene lactone compounds, and the separation effect of sesquiterpene lactone glycoside compounds with high content in bitter dish is not good. In the article "Separation and Structural Identification of Water-Soluble Components of Bitter Melon Chrysanthemi Indica" published in "Natural Product Research and Development" (2010.22:430-432), it is disclosed that calcium oxide is used to precipitate the liquid, and sodium hydroxide is used to adjust the liquid to neutrality, and then ethanol precipitation is used, and then polyamide column is passed. Although this method solves the problem of removing macromolecular impurities such as protons, it does not improve the retention of effective ingredients, and there are many steps for impurity removal, which aggravates the loss of effective ingredients.

[0005] Kidney damage is generally divided into acute kidney damage and chronic kidney damage, which is a progressive disease caused by changes in kidney structure and function. Acute and chronic kidney damage continues to progress to chronic kidney disease (CKD) and even to end-stage renal disease (ESRD). Diabetic nephropathy (DKD) is the "main force" of chronic kidney disease. DKD has become the second cause of ESKD, second only to glomerulonephritis. The mortality rate of patients with kidney damage is much higher than that of patients with healthy kidneys. The quality of life of patients is reduced, their life span is shortened, and the economic burden is heavy. At present, the common method of clinical treatment of kidney damage is to block the renin-angiotensin (RAS) system with angiotensin-converting enzyme inhibitors or angiotensin receptor blockers. However, RAS blockade is only applicable to CKD patients with obvious proteinuria. The combination of ACE inhibitors with ARBs or renin inhibitors has obvious side effects such as hyperkalemia and a significant increase in the incidence of acute kidney injury. Endothelin 1 receptor antagonist atrasentan and sodium-glucose transporter 2 inhibitor canagliflozin have been shown to slow the progression of CKD in patients with diabetic nephropathy (DKD). However, endothelin 1 receptor antagonists can cause fluid retention and an increased risk of cardiovascular events, sodium-glucose transporter 2 inhibitors are prone to side effects such as urinary tract infections, and CKD will continue to progress and worsen in most patients after treatment. Therefore, it is necessary to develop drugs with fewer side effects and that can delay or even stabilize the progression of CKD. Summary of the invention

[0006] Purpose of the invention: The purpose of the present invention is to provide a guaiacyl sesquiterpene compound that can stably and significantly inhibit kidney damage and increased transcription levels of inflammation-related marker genes for use in the preparation of a drug for the treatment of kidney damage, and also to provide a pharmaceutical composition containing the above compound for use in the preparation of a drug for the treatment of kidney damage.

[0007] Technical solution: The present invention discloses the use of guaiacyl sesquiterpene compounds in the preparation of drugs for treating renal injury.

[0008] Preferably, the renal injury is one or more combinations of acute nephritis, chronic nephritis or diabetic nephropathy.

[0009] Preferably, the guaiacyl sesquiterpene compound is as shown in the general formula I:

[0010]

[0011] wherein R1 and R2 together form a double bond; or R1 is methyl and R2 is hydrogen;

[0012] R3 is H, glucosyl or glucosyl substituted with 2-hydroxy-3-(4-hydroxyphenyl)propionic acid.

[0013] Preferably, the guaiacyl sesquiterpene compound is selected from the following compounds:

[0014]

[0015] Preferably, the guaiacane-type sesquiterpene compound is extracted from Ipomoea australis extract.

[0016] Preferably, the extraction is specifically:

[0017] (1) Crush the dried Kudiezi medicinal material, soak it in aqueous ethanol, heat it under reflux, combine the extracts, cool it to room temperature, and then centrifuge and filter it;

[0018] (2) After concentrating the filtrate, anhydrous ethanol is added to dissolve and elute, and the extract of Ilex erythrorhizon is obtained by filtration and concentration, and the guaiac-type sesquiterpene compounds are obtained after separation by reverse phase HPLC column.

[0019] Preferably, in step (1), after centrifugal filtration, the filtrate is adsorbed using D101 macroporous resin.

[0020] Preferably, after adsorption, 10-40% ethanol is used to dissolve and elute sugars, proteins, inorganic salts or sesquiterpene lactone glycosides.

[0021] Preferably, in step (2), after the concentrated filtrate is added with anhydrous ethanol for dissolution and elution, specifically, after the filtrate is concentrated under reduced pressure to 1 / 5 to 1 / 20, 0.8 to 0.95% of the Kudiezi crude drug amount of anhydrous ethanol is added under stirring.

[0022] Preferably, the Ipomoea australis extract comprises compounds I-1 to I-5, wherein the mass percentage of compound I-1 is 20% to 90%.

[0023] Preferably, the guaiacane-type sesquiterpene compound is used in combination with an astragalus extract to prepare a drug for treating renal injury, and the content of astragaloside IV in the astragalus extract is 10%-98%.

[0024] The present invention also discloses a pharmaceutical composition, comprising a guaiacyl sesquiterpene compound, or a pharmaceutically acceptable salt or isomer thereof, and one or both of a bitter melon extract or an astragalus extract, and a pharmaceutically acceptable carrier, adjuvant or auxiliary material.

[0025] Preferably, the ratio of the guaiacane-type sesquiterpene compound to the astragalus extract is 1:3 to 3:1, preferably 1:3, calculated as the weight ratio of compound I to astragaloside IV.

[0026] The above-mentioned pharmaceutical composition is used in the preparation of medicine for treating kidney damage.

[0027] Principle of the invention: The Chinese herbal medicine Kudiezi is rich in compounds such as sesquiterpene lactones, flavonoids, triterpene saponins and adenosine. Guaiacene-type sesquiterpene compounds are the main sesquiterpene types, among which lxerin Z has the highest content (>0.2%). In the preparation method of guaiacene-type sesquiterpene compounds prepared from the whole herb of Kudiezi, Kudiezi medicinal materials are extracted with ethanol below 20%, adsorbed by macroporous resin, gradient eluted with 10% and 20-50% ethanol, and alcohol precipitation, etc., which can effectively remove sugars, proteins, inorganic salts and non-sesquiterpene small molecules, and obtain active sesquiterpene lactone glycosides with a yield of more than 0.4%. The Kudiezi extract contains guaiacene-type sesquiterpene compounds I-1 to I-5, of which the mass percentage of compound I-1 is 20 to 90%.

[0028] In kidney damage caused by acute inflammation, the guaiacyl sesquiterpene compounds extracted from Kudiezi significantly reduce the levels of serum creatinine and urea nitrogen, which are kidney damage markers, and significantly inhibit the increase in the transcription level of kidney damage and inflammation-related marker genes; at the same time, in kidney damage caused by chronic inflammation, the guaiacyl sesquiterpene compounds extracted from Kudiezi can significantly inhibit the increase in the transcription level of kidney damage, fibrosis, and inflammation-related marker genes. Therefore, the present invention prepares the guaiacyl sesquiterpene compounds from the whole herb of Kudiezi, which is used to prepare drugs for the treatment of acute or chronic nephritis, and has a very high therapeutic effect.

[0029] According to the theory of traditional Chinese medicine, diabetic nephropathy is mostly caused by deficiency of both qi and yin, especially heat toxicity and blood stasis. It is a disease that cannot be cured for a long time. The treatment focuses on invigorating qi, detoxifying and removing blood stasis. Kudiezi cools blood and activates blood circulation, clears heat and removes blood stasis, and Huangqi replenishes qi and raises yang, consolidates the exterior and benefits the body. The two are used together to attack and supplement at the same time. Astragaloside IV, the active ingredient of Huangqi, can improve energy metabolism and improve kidney damage by restoring mitochondrial function, activating fatty acid oxidation and tricarboxylic acid cycle processes; Ixerin Z, the active ingredient of Kudiezi, can improve microvascular inflammation, enhance immunity and reduce the level of kidney inflammation. Therefore, the combination of Ixerin Z and Huangqi can play a synergistic role in the regulation mechanism. The present invention finds the effect of Ixerin Z, the active ingredient of Kudiezi, combined with Huangqi glycoside IV (AS-IV) (dose: 1mg / kg+3mg / kg) on ​​the renal function of diabetic nephropathy mice, and the above-mentioned combination has a significantly stronger effect on reducing the renal function indicators, renal inflammation and fibrosis factors of diabetic nephropathy mice than Ixerin Z alone, achieving an excellent therapeutic effect.

[0030] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The guaiacyl sesquiterpene compound obtained by extracting the bitter disc can effectively improve cell damage in the preparation of drugs for treating acute or chronic nephritis, significantly inhibit the increase in the transcription level of kidney damage and inflammation-related marker genes, and has a strong inhibitory activity on kidney damage factors Kim1, Il1b and Mcp1 at the cellular level; (2) The efficacy of the compound is further greatly improved after combined use with astragaloside IV. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described below.

[0032] Example 1

[0033] Preparation of guaiacyl sesquiterpenoid compounds:

[0034] (1) 200 g of dried Kudiezi medicinal materials from Anguo, Hubei were crushed, soaked in 10 times the amount of 10% ethanol for 1 hour, heated under reflux for 3 times, 30 minutes each time, and the three extracts were combined, cooled to room temperature, and centrifuged and filtered;

[0035] (2) After the filtrate is concentrated, it is dissolved in 100 mL of anhydrous ethanol, filtered, and the filtrate is concentrated to obtain 2.92 g of the bitter dish extract IXE-1, with a yield of 1.46%. After reverse phase HPLC preparative column separation, Ixerin Z (Compound I-1), 11,13α-dihydroixerinZ (Compound I-2), lxerinZ2 (Compound I-3), 3-Hydroxydehydroleucodin (Compound I-4) and 8-Desoxyartelin (Compound I-5) can be obtained.

[0036] Chromatographic analysis conditions:

[0037] Chromatographic column: Agilent Zorbax SB-C18 4.6*250mm; mobile phase: acetonitrile A-0.1% trifluoroacetic acid aqueous solution B, gradient elution (0-15min, 25% A, 75% B; 15-25min, 25%~45% A, 75%~65% B); flow rate 0.8mL / min; detection wavelength 270nm; column temperature 30℃; injection volume 5μL; analysis time 25min, retention time of Ixerin Z is 11.6min.

[0038] Weigh 20.34 mg of compound Ixerin Z (self-made, purity > 99% by HPLC) as reference substance, add ultrapure water to a 10 mL volumetric flask, and draw a standard curve. Measure the peak area at a wavelength of 270 nm, and draw a standard curve with the peak area value as the ordinate and the solution concentration as the abscissa. The regression equation of Ixerin Z is: Y = 8816332.6X + 25409.0 (R 2 =0.9994), and the linear range was 0.06-1.02 mg / mL.

[0039] Chromatographic analysis results:

[0040] The content of Ixerin Z, i.e., guaiacyl sesquiterpene compound I-1, in the sample of Example 1 is 22.7%.

[0041] Example 2

[0042] Preparation of the effective fraction IXE-2 of the Kudiezi plant extract:

[0043] (1) 200 g of dried Kudiezi medicinal material from Anguo, Hubei Province was crushed and soaked in 10% ethanol (10 times the amount) for 1 hour. The mixture was heated under reflux for 3 times, each time for 30 minutes. The three extracts were combined, cooled to room temperature, and centrifuged. The filtrate was slowly added with 1.5 times the amount of D101 macroporous resin.

[0044] (2) After adsorption, sugar, protein and inorganic salts were washed away with 2BV 10% ethanol, and then eluted with 3BV 20% and 30% ethanol in sequence, and finally eluted with 3BV 40% ethanol to obtain sesquiterpene lactone glycosides, a total of 1.25 g of Kudiezi extract IXE-2 was obtained, with a yield of 0.62%.

[0045] In Example 2, 1.25 g of IXE-2 was subjected to reverse phase HPLC preparative column (25%-45% methanol water gradient elution), and HPLC tracking detection. The main component of the 25% methanol fraction was Ixerin Z, which was concentrated under reduced pressure to obtain 158 mg of yellow powder (purity>98%), and the main components of the 35% methanol fraction were 11,13α-dihydroixerinZ and lxerinZ2, which were concentrated under reduced pressure to obtain 47 mg and 29 mg of yellow powder (purity>96%). The main components of the 45% methanol fraction were 3-Hydroxydehydroleucodin and 8-Desoxyartelin, which were concentrated under reduced pressure to obtain 67 mg and 18 mg of yellow powder (purity>96%).

[0046] Ixerin Z: 1HNMR(500MHz,CD3OD)δ6.11(s,1H),5.58(s,1H),5.25(s,1H),3.83(s,1H),3.69(s,1H),3.64(s,2H),3.40(dd,J=22.7,10.4Hz,4H),3.33(dq,J=3.2,1.5Hz,4H),3.07(s,1H),2.65(s,1H),2.46(s,3H),2.43(s,1H),2.30(s,1H),2.28(s,3H),1.45(s,1H).ESI-MS(m / z):[M+H] + =423.1(calcd:423.1)。

[0047] 11,13α-dihydroixerinZ: 1 H NMR(500MHz,D2O)δ5.07(s,1H),3.87(s,1H),3.85(s,1H),3.74(s,1H),3.72-3.68(m,2H),3.67(s,1H),3.50(dd,J=17.4,7.8Hz,3H),2.42(s,2H),2.35(s,3H),2.28(s,1H),2.18(s,3H),2.10(s,1H),1.95(s,1H),1.45(s,1H),1.28(s,3H),1.15(s,2H).ESI-MS(m / z):[M+H] + =425.1(calcd:425.1)。

[0048] lxerinZ2: 1 H NMR(500MHz,DMSO)δ6.86(s,2H),6.57(s,2H),5.97(s,1H),5.49(s,1H),5.29(s,1H),4.28(s,1H),4.06(s,2H),3.77-3.74(m,3H),3.65(s,1H),2.38(s,1H),2.31(s,4H),2.18(s,2H),2.15(d,J=5.9Hz,1H),2.09(s,4H),2.05(s,1H),1.24(s,1H).ESI-MS(m / z):[M+H] + =587.1(calcd:587.1)。

[0049] 3-Hydroxydehydroleucodin: 1H NMR (500MHz, CD3OD) δ6.10(s,1H),5.57(s,1H),3.57(s,1H),3.51(s,1H),3.04(s,1H),2.64(s,1H ),2.46(s,3H),2.42(s,1H),2.27(s,1H),2.15(s,3H),1.38(d,J=50.6Hz,2H).ESI-MS(m / z):[M+H] + =261.1(calcd:261.1).

[0050] 8-Desoxyartelin: 1 H NMR (500MHz, CD3OD) δ3.60(t,J=10.0Hz,1H),3.39(s,1H),2.55(t,J=13.5Hz,1H),2.45(s,3H),2. 36(dt,J=15.7,6.9Hz,2H),2.12(s,3H),2.08-1.98(m,2H),1.35(dd,J=24.7,12.3Hz,2H),1.23(d J=6.9Hz,3H).ESI-MS(m / z):[M+H] + =263.1(calcd:263.1).

[0051] HPLC content analysis showed that the content of Ixerin Z, i.e., guaiacyl sesquiterpene compound I-1, in the extract was 42.95%.

[0052] Example 3

[0053] Preparation of the effective fraction IXE-3 of the Kudiezi plant extract:

[0054] (1) 200 g of dried Kudiezi medicinal material from Anguo, Hubei Province was crushed and soaked in 10% ethanol (10 times the amount) for 1 hour. The mixture was heated under reflux for 3 times, each time for 30 minutes. The three extracts were combined, cooled to room temperature, and centrifuged. The filtrate was slowly added with 1.5 times the amount of D101 macroporous resin.

[0055] (2) After adsorption, sugar, protein and inorganic salt were washed away with 2BV 10% ethanol, and then eluted with 3BV 20% and 30% ethanol in turn, and finally eluted with 3BV 40% ethanol. After vacuum concentration to 1 / 5 of the amount of the raw herb of Kudiezi, anhydrous ethanol of 4 / 5 of the amount of the raw herb of Kudiezi was slowly added under stirring. After standing for 12 hours, the precipitate was discarded by centrifugation, and the filtrate was vacuum concentrated to dryness to obtain 0.94 g of Kudiezi extract IXE-3 with a yield of 0.47%.

[0056] HPLC content analysis showed that the content of Ixerin Z, i.e., guaiacyl sesquiterpene compound I-1, in the extract was 66.17%.

[0057] Example 4

[0058] Preparation of the effective fraction IXE-4 of the Kudiezi plant extract:

[0059] (1) 200 g of dried Kudiezi medicinal material from Anguo, Hubei Province was crushed and soaked in 10% ethanol (10 times the amount) for 1 hour. The mixture was heated under reflux for 3 times, each time for 30 minutes. The three extracts were combined, cooled to room temperature, and centrifuged. The filtrate was slowly added with 1.5 times the amount of D101 macroporous resin.

[0060] (2) After adsorption, sugar, protein and inorganic salt were washed away with 2BV 10% ethanol, and then eluted with 3BV 20% and 30% ethanol in turn, and finally eluted with 3BV 40% ethanol. After vacuum concentration to 1 / 10 of the amount of Kudiezi crude drug, 9 / 10 of the amount of Kudiezi crude drug anhydrous ethanol was slowly added under stirring. After standing for 12 hours, the precipitate was discarded by centrifugation, and the filtrate was vacuum concentrated to dryness to obtain 1.02 g of Kudiezi extract IXE-4 with a yield of 0.51%.

[0061] HPLC content analysis showed that the content of Ixerin Z in the extract was 72.24%.

[0062] Example 5

[0063] Preparation of the effective fraction IXE-5 of the Kudiezi plant extract:

[0064] (1) 200 g of dried Kudiezi medicinal material from Anguo, Hubei Province was crushed and soaked in 10% ethanol (10 times the amount) for 1 hour. The mixture was heated under reflux for 3 times, each time for 30 minutes. The three extracts were combined, cooled to room temperature, and centrifuged. The filtrate was slowly added with 1.5 times the amount of D101 macroporous resin.

[0065] (2) After adsorption, sugar, protein and inorganic salt were washed away with 2BV 10% ethanol, and then eluted with 3BV 20% and 30% ethanol in turn, and finally eluted with 3BV 40% ethanol. After vacuum concentration to 1 / 20 of the amount of Kudiezi crude drug, anhydrous ethanol of 19 / 20 of the Kudiezi crude drug was slowly added under stirring. After standing for 12 hours, the precipitate was discarded by centrifugation, and the filtrate was vacuum concentrated to dryness to obtain 0.92 g of Kudiezi extract IXE-5 with a yield of 0.46%.

[0066] HPLC content analysis showed that the content of Ixerin Z in the extract was 70.98%.

[0067] Example 6

[0068] Preparation of the effective fraction IXE-6 of the Kudiezi plant extract:

[0069] (1) 200 g of dried Kudiezi medicinal material from Anguo, Hubei Province was crushed and soaked in 10% ethanol (10 times the amount) for 1 hour. The mixture was heated under reflux for 3 times, each time for 30 minutes. The three extracts were combined, cooled to room temperature, and centrifuged. The filtrate was slowly added with 1.5 times the amount of D101 macroporous resin.

[0070] (2) After adsorption, sugar, protein and inorganic salt were washed off with 2BV 10% ethanol, and then eluted with 3BV 20%, 30% ethanol and finally 40% ethanol. After vacuum concentration to 1 / 10 of the amount of Kudiezi crude drug, 9 / 10 of the amount of Kudiezi crude drug was slowly added with anhydrous ethanol under stirring. After standing for 12 hours, the precipitate was discarded by centrifugation, and the filtrate was concentrated to dryness under vacuum. The extract was eluted isocratically on a reverse phase HPLC preparative column (25% methanol) to obtain 446 mg of Kudiezi extract IXE-6 with a yield of 0.22%.

[0071] HPLC content analysis showed that the content of Ixerin Z in the extract was 90.24%.

[0072] Example 7

[0073] Preparation of the composition of kudzu lactone and astragaloside IV:

[0074] The Ixerin Z (purity>98%) purified in Examples 1-6 and the astragaloside extract (astragaloside content 10% to 98%) were mixed at a weight ratio of 1:3 to 3:1 to prepare a composition. The specific method is: Ixerin Z was combined with astragaloside Ⅳ at a weight ratio of 1:3 to prepare composition 1; combined with astragaloside Ⅳ at a weight ratio of 1:1 to prepare composition 2; combined with astragaloside Ⅳ at a weight ratio of 3:1 to prepare composition 3, and the following experiment was performed.

[0075] The active sesquiterpene lactone glycoside extract of Ilex oleifera obtained by the method of Examples 1-7 and the composition thereof are ground into fine powder, uniformly mixed with an appropriate amount of starch, granulated in a conventional manner, dried, sieved, and punched into tablets or capsules.

[0076] Determination of the activity of active sesquiterpenoid compounds (I-1 to I-5) in bitter sauce fruit to improve renal tubular epithelial cell (HK2) damage:

[0077] Experimental methods: HK2 cells were stimulated with TGF-β (10 ng / mL) for 48 hours to construct a renal injury cell model, and I-1, I-2, I-3, I-4 and I-5, sesquiterpene lactone compounds from Ipomoea batatas were administered at the same time. qPCR was used to detect the effects of the compounds on the expression of genes related to renal injury and inflammation.

[0078] Table 1. Effects of compounds I-1 to I-5 (1 μM) on the expression of genes related to kidney injury and inflammation in HK2 cells

[0079]

[0080]

[0081] Note: **p<0.01 (compared with TGF-β group)

[0082] The results showed that: as shown in Table 1, compound I-1 had the strongest effect on improving HK2 cell damage stimulated by TGF-β.

[0083] Determination of HK2 cell injury activity of effective parts of Kudiezi plant extract (IXE-1~IXE-6):

[0084] Experimental methods: HK2 cells were stimulated with TGF-β (10 ng / mL) for 48 hours to construct a renal injury cell model. At the same time, different proportions of IEX-1, IEX-2, IEX-3, IEX-4, IEX-5 and IEX-6 were given, and qPCR was used to detect the effects of the compounds on the expression of genes related to renal injury and inflammation.

[0085] Table 2. Effects of effective fractions IEX-1 to IXE-6 (50 μg / mL) on the expression of genes related to kidney injury and inflammation in HK2 cells

[0086] Group Kim1 Il1b Mcp1 TGF-β 1.07±0.36 1.21±0.28 1.23±0.17 IEX-1 0.75±0.18* 0.82±0.13* 0.68±0.21** IEX-2 0.68±0.09** 0.74±0.20** 0.66±0.31** IEX-3 0.45±0.21** 0.51±0.21** 0.44±0.17** IEX-4 0.22±0.13** 0.32±0.12** 0.29±0.087** IEX-5 0.31±0.19** 0.37±0.11** 0.34±0.11** IEX-6 0.19±0.19** 0.29±0.12** 0.25±0.23**

[0087] Note: **p<0.05; **p<0.01 (compared with TGF-β group)

[0088] The results showed that as shown in Table 2, site IEX-4 had the strongest effect on improving HK2 cell damage under TGF-β stimulation.

[0089] Determination of the activity of Ixerin Z (I-1), an active sesquiterpene lactone glycoside from Ixerin Z. erythrorhizon, and its combination in treating acute kidney injury:

[0090] Experimental methods: An acute kidney injury model was established in C57BL / 6J mice by intraperitoneal injection of 250 mg / kg folic acid for 48 hours. Ixerin Z was orally administered to mice at concentrations of 5 and 10 mg / kg / d to investigate the therapeutic effects of different concentrations of Ixerin Z on renal injury.

[0091] Table 3. Expression of creatinine and urea nitrogen in serum of mice orally administered Ixerin Z

[0092] Group Number Creatinine (μM) Urea nitrogen (mM) <![CDATA[NaHCO3]]> 5 57.39±33.21 13.21±1.62 FA 5 506.96±136.97 50.00±2.13 Ixerin Z (5 mg / kg) 5 224.93±30.53** 38.41±0.68** Ixerin Z (10 mg / kg) 5 148.69±25.10** 24.26±2.65**

[0093] Note: *p<0.05; **p<0.01 (compared with FA group)

[0094] Table 4. Relative expression levels of inflammatory genes in the kidneys of mice orally administered Ixerin Z

[0095] Group (n=5) Mcp1 Tnf Il1b Il6 <![CDATA[NaHCO3]]> 1.08±0.07 0.83±0.24 1.00±0.08 0.99±0.32 FA 39.67±4.63 5.30±1.52 3.10±0.53 6.81±3.28 Ixerin Z (5 mg / kg) 8.40±3.75** 3.88±0.75 1.44±0.47** 3.01±0.44* Ixerin Z (10 mg / kg) 4.92±2.42** 1.70±0.38** 0.95±0.20** 2.31±0.54**

[0096] Note: *p<0.05; **p<0.01 (compared with FA group)

[0097] Table 5. Relative expression levels of renal injury genes in mice orally administered Ixerin Z

[0098] Group (n=5) Kim1 Ngal Klotho <![CDATA[NaHCO3]]> 1.36±1.09 0.82±0.26 1.02±0.45 FA 146.85±21.89 114.70±47.58 0.016±0.012 Ixerin Z (5 mg / kg) 60.91±27.06** 23.93±7.16** 0.043±0.020* Ixerin Z (10 mg / kg) 29.74±12.89** 8.23±7.67** 0.12±0.86**

[0099] Note: *p<0.05; **p<0.01 (compared with FA group)

[0100] Table 6. Relative expression levels of inflammatory genes in the kidneys of mice orally administered Ixerin Z combined with astragaloside IV

[0101] Group (n=5) Il6 Ifna Cxcl10 Icam <![CDATA[NaHCO3]]> 1.07±0.18 0.92±0.06 1.09±0.60 1.01±0.24 FA 63.39±11.04 4.10±2.09 62.11±19.62 8.48±2.46 Ixerin Z 33.33±20.41** 1.84±0.50** 26.66±16.52** 5.16±1.92* AS-IV 35.79±6.29** 1.02±0.67** 36.24±6.04** 4.66±1.66* Composition 1 12.55±10.47## 0.77±0.34## 17.31±2.31## 2.69±0.59## Composition 2 40.88±21.68 2.11±0.62 42.10±13.38 12.36±1.22 Composition 3 25.84±9.82 1.98±0.85 37.67±7.09 5.28±1.40

[0102] Note: *p<0.05; **p<0.01 (compared with FA group); ##p<0.01 (compared with Ixerin Z group)

[0103] Table 7. Relative expression levels of chemokine genes in the kidneys of mice orally administered Ixerin Z combined with astragaloside IV

[0104]

[0105]

[0106] Note: *p<0.05; **p<0.01 (compared with FA group); ##p<0.01 (compared with Ixerin Z group)

[0107] Table 8. Relative expression levels of renal injury genes in mice orally administered Ixerin Z combined with astragaloside IV

[0108] Group (n=5) Kim1 Klotho Ngal <![CDATA[NaHCO3]]> 1.01±0.12 1.12±0.29 1.18±0.13 FA 66.23±8.94 0.21±0.06 176.43±14.07 Ixerin Z 40.86±15.25* 0.42±0.07* 98.21±17.99** AS-IV 29.44±6.43** 0.29±0.12* 100.91±19.68** Composition 1 21.26±6.91## 0.63±0.19## 81.65±22.74# Composition 2 18.60±1.57 0.38±0.10 113.53±8.93 Composition 3 20.87±6.15 0.48±0.63 102.04±12.21

[0109] Note: *p<0.05; **p<0.01 (compared with FA group); ##p<0.01 (compared with Ixerin Z group)

[0110] The results showed that, as shown in Tables 3 to 8, after oral administration of different concentrations of Ixerin Z to mice, the levels of serum creatinine and urea nitrogen, markers of kidney injury, decreased significantly compared with the acute kidney injury group; Ixerin Z significantly inhibited the increase in the transcription level of kidney injury and inflammation-related marker genes; after administration of Ixerin Z combined with astragaloside IV (AS-IV), i.e., composition 1, it can be seen that its effect is significantly stronger than that of Ixerin Z alone. The above results show that Ixerin Z has a good effect in treating acute kidney injury, and the efficacy is further improved after combined use with astragaloside IV (Ixerin Z: astragaloside IV = 1:3).

[0111] Determination of the activity of active sesquiterpene lactone glycoside Ixerin Z (I-1) from Ixerinus odoratus, extract IXE-4 and their combination in treating chronic renal injury

[0112] Experimental methods: A chronic renal injury model of C57BL / 6J mice was established by unilateral ureteral ligation surgery. Ixerin Z and IXE-4 were orally administered to mice at a concentration of 5 mg / kg / d to investigate the therapeutic effects of Ixerin Z and IXE-4 on renal injury.

[0113] Table 9. Relative expression levels of inflammatory genes in the kidneys of mice orally administered Ixerin Z and its extracts

[0114] Group (n=5) Mcp1 Tnf Il1b Sham 1.03±0.11 1.02±0.45 1.04±0.10 UUO 23.80±7.42 12.36±1.72 125.32±23.13 Ixerin Z (5 mg / kg) 13.98±8.22* 3.80±1.48** 17.21±6.48** IXE-4 (5 mg / kg) 9.03±4.12* 2.69±1.95** 9.15±4.93**

[0115] Note: *p<0.05; **p<0.01 (compared with UUO group)

[0116] Table 10. Relative expression levels of renal injury genes in mice orally administered Ixerin Z and its extracts

[0117] Group (n=5) Kim1 Ngal Klotho <![CDATA[NaHCO3]]> 0.89±0.19 0.79±0.45 1.11±0.09 FA 97.39±26.70 0.13±1.72 0.030±0.019 Ixerin Z (5 mg / kg) 20.26±14.06** 167.45±72.73** 0.20±0.052** IXE-4 (5 mg / kg) 21.43±17.74** 58.62±24.38** 0.16±0.03**

[0118] Note: *p<0.05; **p<0.01 (compared with UUO group)

[0119] Table 11. Relative expression levels of renal injury genes in mice orally administered Ixerin Z and its extracts

[0120] Group (n=5) Acta2 Col1a1 Col3a1 Sham 1.29±0.43 1.07±0.16 1.11±0.26 UUO 11.88±6.86 16.68±9.58 17.19±4.73 Ixerin Z (5 mg / kg) 3.58±1.42* 6.41±3.26** 10.20±5.01* IXE-4 (5 mg / kg) 4.86±2.09* 5.51±3.21** 3.21±1.18**

[0121] Note: *p<0.05; **p<0.01 (compared with UUO group)

[0122] Table 12. Relative expression levels of renal inflammation and fibrosis genes in mice orally administered Ixerin Z combined with astragaloside IV

[0123]

[0124]

[0125] Note: *p<0.05; **p<0.01 (compared with UUO group); ##p<0.01 (compared with Ixerin Z group)

[0126] The results showed that after oral administration of different concentrations of Ixerin Z or IXE-4 to mice, Ixerin Z significantly inhibited the increase in transcription levels of renal injury, fibrosis, and inflammation-related marker genes compared with the mice in the surgical group. After the administration of Ixerin Z combined with astragaloside IV (AS-IV), that is, composition 1, it can be seen that the therapeutic effect on renal injury is significantly stronger than that of Ixerin Z alone. The above results show that Ixerin Z has a good effect in treating chronic renal injury, and the efficacy is further improved after combined use with astragaloside IV (Ixerin Z: astragaloside IV = 1:3).

[0127] Determination of the activity of Ixerin Z (I-1), an active sesquiterpene lactone glycoside in Ixerinus officinalis, in treating diabetic nephropathy:

[0128] Experimental methods: STZ 50mg / kg was injected intraperitoneally for 5 consecutive days to establish the model of diabetic nephropathy, and HFD was fed for 12 weeks to establish the model of diabetic nephropathy; Ixerin Z was orally administered to mice at concentrations of 1 and 10mg / kg / d for 4 consecutive weeks to investigate the therapeutic effects of different concentrations of Ixerin Z on diabetic nephropathy.

[0129] Table 13. Serum creatinine and urea nitrogen levels in diabetic kidney model mice after oral administration of Ixerin Z

[0130] Group Creatinine (μM) Urea nitrogen (mM) Normal 15.94±3.68 21.57±7.14 Model 26.20±4.75 63.03±10.39 Ixerin Z (1 mg / kg) 21.48±5.10 48.66±13.99 Ixerin Z (10 mg / kg) 16.80±4.35** 43.98±8.51*

[0131] Note: *p<0.05 vs Model group; **p<0.01 vs Model group, n=6

[0132] Table 14. Urine protein levels in diabetic kidney model mice after oral administration of Ixerin Z

[0133] Group Urine protein (mg / mL) Normal 0.45±0.30 Model 2.16±0.52 Ixerin Z (1 mg / kg) 1.42±0.41* Ixerin Z (10 mg / kg) 1.26±0.49**

[0134] Note: *p<0.05 vs Model group; **p<0.01 vs Model group, n=6

[0135] Table 15. Relative expression levels of renal injury genes in diabetic kidney model mice orally administered Ixerin Z

[0136]

[0137]

[0138] Note: *p<0.05 vs Model group; **p<0.01 vs Model group, n=6

[0139] Table 16. Relative expression levels of renal fibrosis factors in diabetic kidney model mice after oral administration of Ixerin Z

[0140] Group α-SMA Vimentin Normal 1.43±0.55 0.81±0.31 Model 3.70±1.52 2.03±0.32 Ixerin Z (1 mg / kg) 1.97±1.32* 1.12±0.80* Ixerin Z (10 mg / kg) 1.89±0.77* 0.72±0.41**

[0141] Note: *p<0.05 vs Model group; **p<0.01 vs Model group, n=6

[0142] The results showed that, as shown in Tables 13 to 16, after oral administration of different concentrations of Ixerin Z to mice, the levels of serum creatinine, urea nitrogen, and urine protein, the markers of overall renal function, decreased significantly compared with the model group; Ixerin Z significantly inhibited the increase in the transcription level of renal tubular injury and fibrosis-related marker genes. Moreover, Ixerin Z has a dose-dependent effect on the improvement of diabetic nephropathy, and the effect of Ixerin Z (10 mg / kg) is more significant.

[0143] Based on the clinical research basis that Kudiezi combined with large doses of Astragalus membranaceus improves the renal function of diabetic patients, the effects of its active ingredient Ixerin Z combined with astragaloside IV (AS-IV) (composition 1, dose: 1 mg / kg+3 mg / kg) on ​​the renal function of diabetic nephropathy mice were further investigated.

[0144] Table 17. Serum creatinine and urea nitrogen levels in diabetic kidney model mice after oral administration of Ixerin Z combined with astragaloside IV

[0145] Group Creatinine (μM) Urea nitrogen (mM) Normal 19.21±2.38 17.33±2.41 Model 39.75±8.13 67.46±4.70 Ixerin Z 26.91±3.31 47.23±6.24 AS-IV 29.93±4.35 47.83±8.23 Composition 1 17.50±2.31* 31.11±4.51**

[0146] Note: *p<0.05 vs Ixerin Z group; **p<0.01 vs Ixerin Z group, n=5

[0147] Table 18. Urinary protein levels in diabetic kidney model mice orally administered Ixerin Z combined with astragaloside IV

[0148]

[0149]

[0150] Note: *p<0.05 vs Ixerin Z group; **p<0.01 vs Ixerin Z group, n=5

[0151] Table 19. Relative expression levels of renal inflammatory factors in mice with diabetic kidney model after oral administration of Ixerin Z combined with astragaloside IV

[0152] Group Tnf Mcp1 Normal 1.52±0.52 1.11±0.12 Model 5.10±0.50 4.11±0.81 Ixerin Z 2.44±0.49 2.39±0.12 AS-IV 2.93±0.77 1.49±0.47 Composition 1 1.18±0.67** 1.12±0.16**

[0153] Note: *p<0.05 vs Ixerin Z group; **p<0.01 vs Ixerin Z group, n=5

[0154] Table 20. Relative expression levels of renal fibrosis factors in mice with diabetic kidney model induced by oral administration of Ixerin Z combined with astragaloside IV

[0155] Group α-SMA Vimentin Normal 1.25±0.52 0.92±0.15 Model 3.28±0.37 5.80±0.75 Ixerin Z 1.79±0.19 3.27±0.54 AS-IV 1.97±0.30 3.26±0.17 Composition 1 1.04±0.27** 1.96±0.04**

[0156] Note: *p<0.05 vs Ixerin Z group; **p<0.01 vs Ixerin Z group, n=5

[0157] The results showed that, as shown in Tables 17 to 20, after 4 weeks of administration of Ixerin Z combined with astragaloside IV (AS-IV), the effect of reducing renal function indicators, renal inflammation and fibrosis factors in diabetic nephropathy mice was significantly stronger than that of Ixerin Z alone. The above results show that Ixerin Z can significantly improve the renal function and renal inflammation of diabetic nephropathy mice. At the same time, from the results of Composition 1, it can be seen that the efficacy of Ixerin Z combined with astragaloside IV (Ixerin Z: astragaloside IV = 1:3) is further improved.

[0158] From the above experiments, it can be seen that the prepared guaiacyl sesquiterpene compounds and the combination with astragaloside IV, after oral administration, significantly inhibited the increase in the transcription level of renal damage and inflammation-related marker genes in renal damage caused by acute inflammation and chronic inflammation, respectively. In particular, the active sesquiterpene lactone glycoside Ixerin Z (Compound I-1) in Kudiezi significantly increased its concentration after a special preparation process, and within a suitable concentration range, oral administration significantly reduced the levels of serum creatinine and urea nitrogen, which are markers of renal damage, and significantly improved the overall renal function and renal inflammatory damage level of diabetic nephropathy mice, achieving an excellent therapeutic effect.

Claims

1. Application of guaiacyl sesquiterpenoid compounds in the preparation of drugs for treating renal injury.

2. The use according to claim 1, characterized in that: The renal injury is one or more combinations of acute nephritis, chronic nephritis or diabetic nephropathy.

3. The use according to claim 1, characterized in that: The guaiacyl sesquiterpene compound is shown in general formula I: wherein R1 and R2 together form a double bond; or R1 is methyl and R2 is hydrogen; R3 is H, glucosyl or glucosyl substituted with 2-hydroxy-3-(4-hydroxyphenyl)propionic acid.

4. The use according to claim 3, characterized in that: The guaiacyl sesquiterpene compound is selected from the following compounds:

5. The use according to claim 1, characterized in that: The guaiacyl sesquiterpene compound is extracted from the extract of Ipomoea australis.

6. The use according to claim 5, characterized in that: The Kusnezoffia oleifera extract comprises compounds I-1 to I-5, wherein compound I-1 accounts for 20% to 90% of the mass of the Kusnezoffia oleifera extract.

7. The use according to claim 1, characterized in that: The guaiacyl sesquiterpene compound is used in combination with an astragalus extract to prepare a medicine for treating kidney damage. The content of astragaloside IV in the astragalus extract is 10%-98%.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the guaiac-type sesquiterpene compound according to claim 1, or a pharmaceutically acceptable salt or isomer thereof, and one or both of the bitter disc extract according to claim 5 or the astragalus extract according to claim 7, and a pharmaceutically acceptable carrier, adjuvant or auxiliary material.

9. The pharmaceutical composition according to claim 8, characterized in that The ratio of the guaiacane-type sesquiterpene compound to the astragalus extract is 1:3 to 3:1 based on the weight ratio of compound I to astragaloside IV.

10. Use of the pharmaceutical composition according to claim 8 in preparing a medicament for treating renal injury.

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